Proteomic analysis of the venom from the scorpion Mesobuthus martensii.

PubMed

Xu, Xiaobo; Duan, Zhigui; Di, Zhiyong; He, Yawen; Li, Jianglin; Li, Zhongjie; Xie, Chunliang; Zeng, Xiongzhi; Cao, Zhijian; Wu, Yingliang; Liang, Songping; Li, Wenxin

2014-06-25

The scorpion Mesobuthus martensii is the most populous species in eastern Asian countries, and several toxic components have been identified from their venoms. Nevertheless, a complete proteomic profile of the venom of M. martensii is still not available. In this study, the venom of M. martensii was analyzed by comprehensive proteomic approaches. 153 fractions were isolated from the M. martensii venom by 2-DE, SDS-PAGE and RP-HPLC. The ESI-Q-TOF MS results of all fractions were used to search the scorpion genomic and transcriptomic databases. Totally, 227 non-redundant protein sequences were unambiguously identified, composed of 134 previously known and 93 previously unknown proteins. Among 134 previously known proteins, 115 proteins were firstly confirmed from the M. martensii crude venom and 19 toxins were confirmed once again, involving 43 typical toxins, 7 atypical toxins, 12 venom enzymes and 72 cell associated proteins. In typical toxins, 7 novel-toxin sequences were identified, including 3 Na(+)-channel toxins, 3K(+)-channel toxins and 1 no-annotation toxin. These results increased 230% (115/50) venom components compared with previous studies from the M. martensii venom, especially 50% (24/48) typical toxins. Additionally, a mass fingerprint obtained by MALDI-TOF MS indicated that the scorpion venom contained more than 200 different molecular mass components. This work firstly gave a systematic investigation of the M. martensii venom by combined proteomics strategy coupled with genomics and transcriptomics. A large number of protein components were unambiguously identified from the venom of M. martensii, most of which were confirmed for the first time. We also contributed 7 novel-toxin sequences and 93 protein sequences previously unknown to be part of the venom, for which we assigned potential biological functions. Besides, we obtained a mass fingerprint of the M. martensii venom. Together, our study not only provides the most comprehensive catalog of the molecular diversity of the M. martensii venom at the proteomic level, but also enriches the composition information of scorpion venom. Copyright © 2014 Elsevier B.V. All rights reserved.

Comprehensive Analysis of Proteomic Differences between Escherichia coli K-12 and B Strains Using Multiplexed Isobaric Tandem Mass Tag (TMT) Labeling.

PubMed

Han, Mee-Jung

2017-11-28

The Escherichia coli K-12 and B strains are among the most frequently used bacterial hosts for scientific research and biotechnological applications. However, omics analyses have revealed that E. coli K-12 and B exhibit notably different genotypic and phenotypic attributes, even though they were derived from the same ancestor. In a previous study, we identified a limited number of proteins from the two strains using two-dimensional gel electrophoresis and tandem mass spectrometry (MS/MS). In this study, an in-depth analysis of the physiological behavior of the E. coli K-12 and B strains at the proteomic level was performed using six-plex isobaric tandem mass tag-based quantitative MS. Additionally, the best lysis buffer for increasing the efficiency of protein extraction was selected from three tested buffers prior to the quantitative proteomic analysis. This study identifies the largest number of proteins in the two E. coli strains reported to date and is the first to show the dynamics of these proteins. Notable differences in proteins associated with key cellular properties, including some metabolic pathways, the biosynthesis and degradation of amino acids, membrane integrity, cellular tolerance, and motility, were found between the two representative strains. Compared with previous studies, these proteomic results provide a more holistic view of the overall state of E. coli cells based on a single proteomic study and reveal significant insights into why the two strains show distinct phenotypes. Additionally, the resulting data provide in-depth information that will help fine-tune processes in the future.

The Escherichia coli Peripheral Inner Membrane Proteome*

PubMed Central

Papanastasiou, Malvina; Orfanoudaki, Georgia; Koukaki, Marina; Kountourakis, Nikos; Sardis, Marios Frantzeskos; Aivaliotis, Michalis; Karamanou, Spyridoula; Economou, Anastassios

2013-01-01

Biological membranes are essential for cell viability. Their functional characteristics strongly depend on their protein content, which consists of transmembrane (integral) and peripherally associated membrane proteins. Both integral and peripheral inner membrane proteins mediate a plethora of biological processes. Whereas transmembrane proteins have characteristic hydrophobic stretches and can be predicted using bioinformatics approaches, peripheral inner membrane proteins are hydrophilic, exist in equilibria with soluble pools, and carry no discernible membrane targeting signals. We experimentally determined the cytoplasmic peripheral inner membrane proteome of the model organism Escherichia coli using a multidisciplinary approach. Initially, we extensively re-annotated the theoretical proteome regarding subcellular localization using literature searches, manual curation, and multi-combinatorial bioinformatics searches of the available databases. Next we used sequential biochemical fractionations coupled to direct identification of individual proteins and protein complexes using high resolution mass spectrometry. We determined that the proposed cytoplasmic peripheral inner membrane proteome occupies a previously unsuspected ∼19% of the basic E. coli BL21(DE3) proteome, and the detected peripheral inner membrane proteome occupies ∼25% of the estimated expressed proteome of this cell grown in LB medium to mid-log phase. This value might increase when fleeting interactions, not studied here, are taken into account. Several proteins previously regarded as exclusively cytoplasmic bind membranes avidly. Many of these proteins are organized in functional or/and structural oligomeric complexes that bind to the membrane with multiple interactions. Identified proteins cover the full spectrum of biological activities, and more than half of them are essential. Our data suggest that the cytoplasmic proteome displays remarkably dynamic and extensive communication with biological membrane surfaces that we are only beginning to decipher. PMID:23230279

Deep Coverage Proteomics Identifies More Low-Abundance Missing Proteins in Human Testis Tissue with Q-Exactive HF Mass Spectrometer.

PubMed

Wei, Wei; Luo, Weijia; Wu, Feilin; Peng, Xuehui; Zhang, Yao; Zhang, Manli; Zhao, Yan; Su, Na; Qi, YingZi; Chen, Lingsheng; Zhang, Yangjun; Wen, Bo; He, Fuchu; Xu, Ping

2016-11-04

Since 2012, missing proteins (MPs) investigation has been one of the critical missions of Chromosome-Centric Human Proteome Project (C-HPP) through various biochemical strategies. On the basis of our previous testis MPs study, faster scanning and higher resolution mass-spectrometry-based proteomics might be conducive to MPs exploration, especially for low-abundance proteins. In this study, Q-Exactive HF (HF) was used to survey proteins from the same testis tissues separated by two separating methods (tricine- and glycine-SDS-PAGE), as previously described. A total of 8526 proteins were identified, of which more low-abundance proteins were uniquely detected in HF data but not in our previous LTQ Orbitrap Velos (Velos) reanalysis data. Further transcriptomics analysis showed that these uniquely identified proteins by HF also had lower expression at the mRNA level. Of the 81 total identified MPs, 74 and 39 proteins were listed as MPs in HF and Velos data sets, respectively. Among the above MPs, 47 proteins (43 neXtProt PE2 and 4 PE3) were ranked as confirmed MPs after verifying with the stringent spectra match and isobaric and single amino acid variants filtering. Functional investigation of these 47 MPs revealed that 11 MPs were testis-specific proteins and 7 MPs were involved in spermatogenesis process. Therefore, we concluded that higher scanning speed and resolution of HF might be factors for improving the low-abundance MP identification in future C-HPP studies. All mass-spectrometry data from this study have been deposited in the ProteomeXchange with identifier PXD004092.

Characterization of individual mouse cerebrospinal fluid proteomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, Jeffrey S.; Angel, Thomas E.; Chavkin, Charles

2014-03-20

Analysis of cerebrospinal fluid (CSF) offers key insight into the status of the central nervous system. Characterization of murine CSF proteomes can provide a valuable resource for studying central nervous system injury and disease in animal models. However, the small volume of CSF in mice has thus far limited individual mouse proteome characterization. Through non-terminal CSF extractions in C57Bl/6 mice and high-resolution liquid chromatography-mass spectrometry analysis of individual murine samples, we report the most comprehensive proteome characterization of individual murine CSF to date. Utilizing stringent protein inclusion criteria that required the identification of at least two unique peptides (1% falsemore » discovery rate at the peptide level) we identified a total of 566 unique proteins, including 128 proteins from three individual CSF samples that have been previously identified in brain tissue. Our methods and analysis provide a mechanism for individual murine CSF proteome analysis.« less

Proteomic biomarkers for ovarian cancer risk in women with polycystic ovary syndrome: a systematic review and biomarker database integration.

PubMed

Galazis, Nicolas; Olaleye, Olalekan; Haoula, Zeina; Layfield, Robert; Atiomo, William

2012-12-01

To review and identify possible biomarkers for ovarian cancer (OC) in women with polycystic ovary syndrome (PCOS). Systematic literature searches of MEDLINE, EMBASE, and Cochrane using the search terms "proteomics," "proteomic," and "ovarian cancer" or "ovarian carcinoma." Proteomic biomarkers for OC were then integrated with an updated previously published database of all proteomic biomarkers identified to date in patients with PCOS. Academic department of obstetrics and gynecology in the United Kingdom. A total of 180 women identified in the six studies. Tissue samples from women with OC vs. tissue samples from women without OC. Proteomic biomarkers, proteomic technique used, and methodologic quality score. A panel of six biomarkers was overexpressed both in women with OC and in women with PCOS. These biomarkers include calreticulin, fibrinogen-γ, superoxide dismutase, vimentin, malate dehydrogenase, and lamin B2. These biomarkers could help improve our understanding of the links between PCOS and OC and could potentially be used to identify subgroups of women with PCOS at increased risk of OC. More studies are required to further evaluate the role these biomarkers play in women with PCOS and OC. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Comparison of methods for profiling O-glycosylation: Human Proteome Organisation Human Disease Glycomics/Proteome Initiative multi-institutional study of IgA1.

PubMed

Wada, Yoshinao; Dell, Anne; Haslam, Stuart M; Tissot, Bérangère; Canis, Kévin; Azadi, Parastoo; Bäckström, Malin; Costello, Catherine E; Hansson, Gunnar C; Hiki, Yoshiyuki; Ishihara, Mayumi; Ito, Hiromi; Kakehi, Kazuaki; Karlsson, Niclas; Hayes, Catherine E; Kato, Koichi; Kawasaki, Nana; Khoo, Kay-Hooi; Kobayashi, Kunihiko; Kolarich, Daniel; Kondo, Akihiro; Lebrilla, Carlito; Nakano, Miyako; Narimatsu, Hisashi; Novak, Jan; Novotny, Milos V; Ohno, Erina; Packer, Nicolle H; Palaima, Elizabeth; Renfrow, Matthew B; Tajiri, Michiko; Thomsson, Kristina A; Yagi, Hirokazu; Yu, Shin-Yi; Taniguchi, Naoyuki

2010-04-01

The Human Proteome Organisation Human Disease Glycomics/Proteome Initiative recently coordinated a multi-institutional study that evaluated methodologies that are widely used for defining the N-glycan content in glycoproteins. The study convincingly endorsed mass spectrometry as the technique of choice for glycomic profiling in the discovery phase of diagnostic research. The present study reports the extension of the Human Disease Glycomics/Proteome Initiative's activities to an assessment of the methodologies currently used for O-glycan analysis. Three samples of IgA1 isolated from the serum of patients with multiple myeloma were distributed to 15 laboratories worldwide for O-glycomics analysis. A variety of mass spectrometric and chromatographic procedures representative of current methodologies were used. Similar to the previous N-glycan study, the results convincingly confirmed the pre-eminent performance of MS for O-glycan profiling. Two general strategies were found to give the most reliable data, namely direct MS analysis of mixtures of permethylated reduced glycans in the positive ion mode and analysis of native reduced glycans in the negative ion mode using LC-MS approaches. In addition, mass spectrometric methodologies to analyze O-glycopeptides were also successful.

Systematic Characterization of the Murine Mitochondrial Proteome Using Functionally Validated Cardiac Mitochondria

PubMed Central

Zhang, Jun; Li, Xiaohai; Mueller, Michael; Wang, Yueju; Zong, Chenggong; Deng, Ning; Vondriska, Thomas M.; Liem, David A.; Yang, Jeong-In; Korge, Paavo; Honda, Henry; Weiss, James N.; Apweiler, Rolf; Ping, Peipei

2009-01-01

Mitochondria play essential roles in cardiac pathophysiology and the murine model has been extensively used to investigate cardiovascular diseases. In the present study, we characterized murine cardiac mitochondria using an LC/MS/MS approach. We extracted and purified cardiac mitochondria; validated their functionality to ensure the final preparation contains necessary components to sustain their normal function; and subjected these validated organelles to LC/MS/MS-based protein identification. A total of 940 distinct proteins were identified from murine cardiac mitochondria, among which, 480 proteins were not previously identified by major proteomic profiling studies. The 940 proteins consist of functional clusters known to support oxidative phosphorylation, metabolism and biogenesis. In addition, there are several other clusters--including proteolysis, protein folding, and reduction/oxidation signaling-which ostensibly represent previously under-appreciated tasks of cardiac mitochondria. Moreover, many identified proteins were found to occupy other subcellular locations, including cytoplasm, ER, and golgi, in addition to their presence in the mitochondria. These results provide a comprehensive picture of the murine cardiac mitochondrial proteome and underscore tissue- and species-specification. Moreover, the use of functionally intact mitochondria insures that the proteomic observations in this organelle are relevant to its normal biology and facilitates decoding the interplay between mitochondria and other organelles. PMID:18348319

Evaluation of empirical rule of linearly correlated peptide selection (ERLPS) for proteotypic peptide-based quantitative proteomics.

PubMed

Liu, Kehui; Zhang, Jiyang; Fu, Bin; Xie, Hongwei; Wang, Yingchun; Qian, Xiaohong

2014-07-01

Precise protein quantification is essential in comparative proteomics. Currently, quantification bias is inevitable when using proteotypic peptide-based quantitative proteomics strategy for the differences in peptides measurability. To improve quantification accuracy, we proposed an "empirical rule for linearly correlated peptide selection (ERLPS)" in quantitative proteomics in our previous work. However, a systematic evaluation on general application of ERLPS in quantitative proteomics under diverse experimental conditions needs to be conducted. In this study, the practice workflow of ERLPS was explicitly illustrated; different experimental variables, such as, different MS systems, sample complexities, sample preparations, elution gradients, matrix effects, loading amounts, and other factors were comprehensively investigated to evaluate the applicability, reproducibility, and transferability of ERPLS. The results demonstrated that ERLPS was highly reproducible and transferable within appropriate loading amounts and linearly correlated response peptides should be selected for each specific experiment. ERLPS was used to proteome samples from yeast to mouse and human, and in quantitative methods from label-free to O18/O16-labeled and SILAC analysis, and enabled accurate measurements for all proteotypic peptide-based quantitative proteomics over a large dynamic range. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Proteomics offers insight to the mechanism behind Pisum sativum L. response to pea seed-borne mosaic virus (PSbMV).

PubMed

Cerna, Hana; Černý, Martin; Habánová, Hana; Šafářová, Dana; Abushamsiya, Kifah; Navrátil, Milan; Brzobohatý, Břetislav

2017-02-05

Pea seed-borne mosaic virus (PSbMV) significantly reduces yields in a broad spectra of legumes. The eukaryotic translation initiation factor has been shown to confer resistance to this pathogen, thus implying that translation and proteome dynamics play a role in resistance. This study presents the results of a proteome-wide analysis of Pisum sativum L. response to PSbMV infection. LC-MS profiling of two contrasting pea cultivars, resistant (B99) and susceptible (Raman) to PSbMV infection, detected >2300 proteins, 116 of which responded to PSbMV ten and/or twenty days post-inoculation. These differentially abundant proteins are involved in number of processes that have previously been reported in the plant-pathogen response, including protein and amino acid metabolism, stress signaling, redox homeostasis, carbohydrate metabolism, and lipid metabolism. We complemented our proteome-wide analysis work with targeted analyses of free amino acids and selected small molecules, fatty acid profiling, and enzyme activity assays. Data from these additional experiments support our findings and validate the biological relevance of the observed proteome changes. We found surprising similarities in the resistant and susceptible cultivars, which implies that a seemingly unaffected plant, with no detectable levels of PSbMV, actively suppresses viral replication. Plant resistance to PSbMV is connected to translation initiation factors, yet the processes involved are still poorly understood at the proteome level. To the best of our knowledge, this is the first survey of the global proteomic response to PSbMV in plants. The combination of label-free LC-MS profiling and two contrasting cultivars (resistant and susceptible) provided highly sensitive snapshots of protein abundance in response to PSbMV infection. PSbMV is a member of the largest family of plant viruses and our results are in accordance with previously characterized potyvirus-responsive proteomes. Hence, the results of this study can further extend our knowledge about these pathogens. We also show that even though no viral replication is detected in the PSbMV-resistant cultivar B99, it is still significantly affected by PSbMV inoculation. Copyright © 2016 Elsevier B.V. All rights reserved.

Shotgun proteomics of plant plasma membrane and microdomain proteins using nano-LC-MS/MS.

PubMed

Takahashi, Daisuke; Li, Bin; Nakayama, Takato; Kawamura, Yukio; Uemura, Matsuo

2014-01-01

Shotgun proteomics allows the comprehensive analysis of proteins extracted from plant cells, subcellular organelles, and membranes. Previously, two-dimensional gel electrophoresis-based proteomics was used for mass spectrometric analysis of plasma membrane proteins. In order to get comprehensive proteome profiles of the plasma membrane including highly hydrophobic proteins with a number of transmembrane domains, a mass spectrometry-based shotgun proteomics method using nano-LC-MS/MS for proteins from the plasma membrane proteins and plasma membrane microdomain fraction is described. The results obtained are easily applicable to label-free protein semiquantification.

Proteomic analysis of three gonad types of swamp eel reveals genes differentially expressed during sex reversal.

PubMed

Sheng, Yue; Zhao, Wei; Song, Ying; Li, Zhigang; Luo, Majing; Lei, Quan; Cheng, Hanhua; Zhou, Rongjia

2015-05-18

A variety of mechanisms are engaged in sex determination in vertebrates. The teleost fish swamp eel undergoes sex reversal naturally and is an ideal model for vertebrate sexual development. However, the importance of proteome-wide scanning for gonad reversal was not previously determined. We report a 2-D electrophoresis analysis of three gonad types of proteomes during sex reversal. MS/MS analysis revealed a group of differentially expressed proteins during ovary to ovotestis to testis transformation. Cbx3 is up-regulated during gonad reversal and is likely to have a role in spermatogenesis. Rab37 is down-regulated during the reversal and is mainly associated with oogenesis. Both Cbx3 and Rab37 are linked up in a protein network. These datasets in gonadal proteomes provide a new resource for further studies in gonadal development.

Effects of High-Pressure Treatment on the Muscle Proteome of Hake by Bottom-Up Proteomics.

PubMed

Carrera, Mónica; Fidalgo, Liliana G; Saraiva, Jorge A; Aubourg, Santiago P

2018-05-02

A bottom-up proteomics approach was applied for the study of the effects of high-pressure (HP) treatment on the muscle proteome of fish. The performance of the approach was established for a previous HP treatment (150-450 MPa for 2 min) on frozen (up to 5 months at -10 °C) European hake ( Merluccius merluccius). Concerning possible protein biomarkers of quality changes, a significant degradation after applying a pressure ≥430 MPa could be observed for phosphoglycerate mutase-1, enolase, creatine kinase, fructose bisphosphate aldolase, triosephosphate isomerase, and nucleoside diphosphate kinase; contrary, electrophoretic bands assigned to tropomyosin, glyceraldehyde-3-phosphate dehydrogenase, and beta parvalbumin increased their intensity after applying a pressure ≥430 MPa. This repository of potential protein biomarkers may be very useful for further HP investigations related to fish quality.

Placental Proteomics: A Shortcut to Biological Insight

PubMed Central

Robinson, John M.; Vandré, Dale D.; Ackerman, William E.

2012-01-01

Proteomics analysis of biological samples has the potential to identify novel protein expression patterns and/or changes in protein expression patterns in different developmental or disease states. An important component of successful proteomics research, at least in its present form, is to reduce the complexity of the sample if it is derived from cells or tissues. One method to simplify complex tissues is to focus on a specific, highly purified sub-proteome. Using this approach we have developed methods to prepare highly enriched fractions of the apical plasma membrane of the syncytiotrophoblast. Through proteomics analysis of this fraction we have identified over five hundred proteins several of which were previously not known to reside in the syncytiotrophoblast. Herein, we focus on two of these, dysferlin and myoferlin. These proteins, largely known from studies of skeletal muscle, may not have been found in the human placenta were it not for discovery-based proteomics analysis. This new knowledge, acquired through a discovery-driven approach, can now be applied for the generation of hypothesis-based experimentation. Thus discovery-based and hypothesis-based research are complimentary approaches that when coupled together can hasten scientific discoveries. PMID:19070895

Forensic proteomics for the evaluation of the post-mortem decay in bones.

PubMed

Procopio, Noemi; Williams, Anna; Chamberlain, Andrew T; Buckley, Michael

2018-04-15

Current methods for evaluation the of post-mortem interval (PMI) of skeletal remains suffer from poor accuracy due to the great number of variables that affect the diagenetic process and to the lack of specific guidelines to address this issue. During decomposition, proteins can undergo cumulative decay over the time, resulting in a decrease in the range and abundance of proteins present (i.e., the proteome) in different tissues as well as in an increase of post-translational modifications occurring in these proteins. In this study, we investigate the applicability of bone proteomic analyses to simulated forensic contexts, looking for specific biomarkers that may help the estimation of PMI, as well as evaluate a previously discovered marker for the estimation of biological age. We noticed a reduction of particular plasma and muscle proteins with increasing PMIs, as well as an increased deamidation of biglycan, a protein with a role in modulating bone growth and mineralization. We also corroborated our previous results regarding the use of fetuin-A as a potential biomarker for the estimation of age-at-death, demonstrating the applicability and the great potential that proteomics may have towards forensic sciences. The estimation of the post-mortem interval has a key role in forensic investigations, however nowadays it still suffers from poor reliability, especially when body tissues are heavily decomposed. Here we propose for the first time the application of bone proteomics to the estimation of the time elapsed since death and found several new potential biomarkers to address this, demonstrating the applicability of proteomic analyses to forensic sciences. Copyright © 2018. Published by Elsevier B.V.

CPTAC researchers report first large-scale integrated proteomic and genomic analysis of a human cancer | Office of Cancer Clinical Proteomics Research

Cancer.gov

Investigators from the National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium (CPTAC) who comprehensively analyzed 95 human colorectal tumor samples, have determined how gene alterations identified in previous analyses of the same samples are expressed at the protein level. The integration of proteomic and genomic data, or proteogenomics, provides a more comprehensive view of the biological features that drive cancer than genomic analysis alone and may help identify the most important targets for cancer detection and intervention.

Integrated proteomic and genomic analysis of colorectal cancer

Cancer.gov

Investigators who analyzed 95 human colorectal tumor samples have determined how gene alterations identified in previous analyses of the same samples are expressed at the protein level. The integration of proteomic and genomic data, or proteogenomics, pro

A comprehensive proteomics study on platelet concentrates: Platelet proteome, storage time and Mirasol pathogen reduction technology.

PubMed

Salunkhe, Vishal; De Cuyper, Iris M; Papadopoulos, Petros; van der Meer, Pieter F; Daal, Brunette B; Villa-Fajardo, María; de Korte, Dirk; van den Berg, Timo K; Gutiérrez, Laura

2018-03-19

Platelet concentrates (PCs) represent a blood transfusion product with a major concern for safety as their storage temperature (20-24°C) allows bacterial growth, and their maximum storage time period (less than a week) precludes complete microbiological testing. Pathogen inactivation technologies (PITs) provide an additional layer of safety to the blood transfusion products from known and unknown pathogens such as bacteria, viruses, and parasites. In this context, PITs, such as Mirasol Pathogen Reduction Technology (PRT), have been developed and are implemented in many countries. However, several studies have shown in vitro that Mirasol PRT induces a certain level of platelet shape change, hyperactivation, basal degranulation, and increased oxidative damage during storage. It has been suggested that Mirasol PRT might accelerate what has been described as the platelet storage lesion (PSL), but supportive molecular signatures have not been obtained. We aimed at dissecting the influence of both variables, that is, Mirasol PRT and storage time, at the proteome level. We present comprehensive proteomics data analysis of Control PCs and PCs treated with Mirasol PRT at storage days 1, 2, 6, and 8. Our workflow was set to perform proteomics analysis using a gel-free and label-free quantification (LFQ) approach. Semi-quantification was based on LFQ signal intensities of identified proteins using MaxQuant/Perseus software platform. Data are available via ProteomeXchange with identifier PXD008119. We identified marginal differences between Mirasol PRT and Control PCs during storage. However, those significant changes at the proteome level were specifically related to the functional aspects previously described to affect platelets upon Mirasol PRT. In addition, the effect of Mirasol PRT on the platelet proteome appeared not to be exclusively due to an accelerated or enhanced PSL. In summary, semi-quantitative proteomics allows to discern between proteome changes due to Mirasol PRT or PSL, and proves to be a methodology suitable to phenotype platelets in an unbiased manner, in various physiological contexts.

Tear film proteome in age-related macular degeneration.

PubMed

Winiarczyk, Mateusz; Kaarniranta, Kai; Winiarczyk, Stanisław; Adaszek, Łukasz; Winiarczyk, Dagmara; Mackiewicz, Jerzy

2018-06-01

Age-related macular degeneration (AMD) is the main reason for blindness in elderly people in the developed countries. Current screening protocols have limitations in detecting the early signs of retinal degeneration. Therefore, it would be desirable to find novel biomarkers for early detection of AMD. Development of novel biomarkers would help in the prevention, diagnostics, and treatment of AMD. Proteomic analysis of tear film has shown promise in this research area. If an optimal set of biomarkers could be obtained from accessible body fluids, it would represent a reliable way to monitor disease progression and response to novel therapies. Tear films were collected on Schirmer strips from a total of 22 patients (8 with wet AMD, 6 with dry AMD, and 8 control individuals). 2D electrophoresis was used to separate tear film proteins prior to their identification with matrix-assisted laser desorption/ionization time of flight spectrometer (MALDI-TOF/TOF) and matching with functional databases. A total of 342 proteins were identified. Most of them were previously described in various proteomic studies concerning AMD. Shootin-1, histatin-3, fidgetin-like protein 1, SRC kinase signaling inhibitor, Graves disease carrier protein, actin cytoplasmic 1, prolactin-inducible protein 1, and protein S100-A7A were upregulated in the tear film samples isolated from AMD patients and were not previously linked with this disease in any proteomic analysis. The upregulated proteins supplement our current knowledge of AMD pathogenesis, providing evidence that certain specific proteins are expressed into the tear film in AMD. As far we are aware, this is the first study to have undertaken a comprehensive in-depth analysis of the human tear film proteome in AMD patients.

Respiratory Mucosal Proteome Quantification in Human Influenza Infections.

PubMed

Marion, Tony; Elbahesh, Husni; Thomas, Paul G; DeVincenzo, John P; Webby, Richard; Schughart, Klaus

2016-01-01

Respiratory influenza virus infections represent a serious threat to human health. Underlying medical conditions and genetic make-up predispose some influenza patients to more severe forms of disease. To date, only a few studies have been performed in patients to correlate a selected group of cytokines and chemokines with influenza infection. Therefore, we evaluated the potential of a novel multiplex micro-proteomics technology, SOMAscan, to quantify proteins in the respiratory mucosa of influenza A and B infected individuals. The analysis included but was not limited to quantification of cytokines and chemokines detected in previous studies. SOMAscan quantified more than 1,000 secreted proteins in small nasal wash volumes from infected and healthy individuals. Our results illustrate the utility of micro-proteomic technology for analysis of proteins in small volumes of respiratory mucosal samples. Furthermore, when we compared nasal wash samples from influenza-infected patients with viral load ≥ 2(8) and increased IL-6 and CXCL10 to healthy controls, we identified 162 differentially-expressed proteins between the two groups. This number greatly exceeds the number of DEPs identified in previous studies in human influenza patients. Most of the identified proteins were associated with the host immune response to infection, and changes in protein levels of 151 of the DEPs were significantly correlated with viral load. Most important, SOMAscan identified differentially expressed proteins heretofore not associated with respiratory influenza infection in humans. Our study is the first report for the use of SOMAscan to screen nasal secretions. It establishes a precedent for micro-proteomic quantification of proteins that reflect ongoing response to respiratory infection.

Respiratory Mucosal Proteome Quantification in Human Influenza Infections

PubMed Central

Marion, Tony; Elbahesh, Husni; Thomas, Paul G.; DeVincenzo, John P.; Webby, Richard; Schughart, Klaus

2016-01-01

Respiratory influenza virus infections represent a serious threat to human health. Underlying medical conditions and genetic make-up predispose some influenza patients to more severe forms of disease. To date, only a few studies have been performed in patients to correlate a selected group of cytokines and chemokines with influenza infection. Therefore, we evaluated the potential of a novel multiplex micro-proteomics technology, SOMAscan, to quantify proteins in the respiratory mucosa of influenza A and B infected individuals. The analysis included but was not limited to quantification of cytokines and chemokines detected in previous studies. SOMAscan quantified more than 1,000 secreted proteins in small nasal wash volumes from infected and healthy individuals. Our results illustrate the utility of micro-proteomic technology for analysis of proteins in small volumes of respiratory mucosal samples. Furthermore, when we compared nasal wash samples from influenza-infected patients with viral load ≥ 28 and increased IL-6 and CXCL10 to healthy controls, we identified 162 differentially-expressed proteins between the two groups. This number greatly exceeds the number of DEPs identified in previous studies in human influenza patients. Most of the identified proteins were associated with the host immune response to infection, and changes in protein levels of 151 of the DEPs were significantly correlated with viral load. Most important, SOMAscan identified differentially expressed proteins heretofore not associated with respiratory influenza infection in humans. Our study is the first report for the use of SOMAscan to screen nasal secretions. It establishes a precedent for micro-proteomic quantification of proteins that reflect ongoing response to respiratory infection. PMID:27088501

Diatom Proteomics Reveals Unique Acclimation Strategies to Mitigate Fe Limitation

PubMed Central

Nunn, Brook L.; Faux, Jessica F.; Hippmann, Anna A.; Maldonado, Maria T.; Harvey, H. Rodger; Goodlett, David R.; Boyd, Philip W.; Strzepek, Robert F.

2013-01-01

Phytoplankton growth rates are limited by the supply of iron (Fe) in approximately one third of the open ocean, with major implications for carbon dioxide sequestration and carbon (C) biogeochemistry. To date, understanding how alteration of Fe supply changes phytoplankton physiology has focused on traditional metrics such as growth rate, elemental composition, and biophysical measurements such as photosynthetic competence (Fv/Fm). Researchers have subsequently employed transcriptomics to probe relationships between changes in Fe supply and phytoplankton physiology. Recently, studies have investigated longer-term (i.e. following acclimation) responses of phytoplankton to various Fe conditions. In the present study, the coastal diatom, Thalassiosira pseudonana, was acclimated (10 generations) to either low or high Fe conditions, i.e. Fe-limiting and Fe-replete. Quantitative proteomics and a newly developed proteomic profiling technique that identifies low abundance proteins were employed to examine the full complement of expressed proteins and consequently the metabolic pathways utilized by the diatom under the two Fe conditions. A total of 1850 proteins were confidently identified, nearly tripling previous identifications made from differential expression in diatoms. Given sufficient time to acclimate to Fe limitation, T. pseudonana up-regulates proteins involved in pathways associated with intracellular protein recycling, thereby decreasing dependence on extracellular nitrogen (N), C and Fe. The relative increase in the abundance of photorespiration and pentose phosphate pathway proteins reveal novel metabolic shifts, which create substrates that could support other well-established physiological responses, such as heavily silicified frustules observed for Fe-limited diatoms. Here, we discovered that proteins and hence pathways observed to be down-regulated in short-term Fe starvation studies are constitutively expressed when T. pseudonana is acclimated (i.e., nitrate and nitrite transporters, Photosystem II and Photosystem I complexes). Acclimation of the diatom to the desired Fe conditions and the comprehensive proteomic approach provides a more robust interpretation of this dynamic proteome than previous studies. PMID:24146769

Determination of burn patient outcome by large-scale quantitative discovery proteomics

PubMed Central

Finnerty, Celeste C.; Jeschke, Marc G.; Qian, Wei-Jun; Kaushal, Amit; Xiao, Wenzhong; Liu, Tao; Gritsenko, Marina A.; Moore, Ronald J.; Camp, David G.; Moldawer, Lyle L.; Elson, Constance; Schoenfeld, David; Gamelli, Richard; Gibran, Nicole; Klein, Matthew; Arnoldo, Brett; Remick, Daniel; Smith, Richard D.; Davis, Ronald; Tompkins, Ronald G.; Herndon, David N.

2013-01-01

Objective Emerging proteomics techniques can be used to establish proteomic outcome signatures and to identify candidate biomarkers for survival following traumatic injury. We applied high-resolution liquid chromatography-mass spectrometry (LC-MS) and multiplex cytokine analysis to profile the plasma proteome of survivors and non-survivors of massive burn injury to determine the proteomic survival signature following a major burn injury. Design Proteomic discovery study. Setting Five burn hospitals across the U.S. Patients Thirty-two burn patients (16 non-survivors and 16 survivors), 19–89 years of age, were admitted within 96 h of injury to the participating hospitals with burns covering >20% of the total body surface area and required at least one surgical intervention. Interventions None. Measurements and Main Results We found differences in circulating levels of 43 proteins involved in the acute phase response, hepatic signaling, the complement cascade, inflammation, and insulin resistance. Thirty-two of the proteins identified were not previously known to play a role in the response to burn. IL-4, IL-8, GM-CSF, MCP-1, and β2-microglobulin correlated well with survival and may serve as clinical biomarkers. Conclusions These results demonstrate the utility of these techniques for establishing proteomic survival signatures and for use as a discovery tool to identify candidate biomarkers for survival. This is the first clinical application of a high-throughput, large-scale LC-MS-based quantitative plasma proteomic approach for biomarker discovery for the prediction of patient outcome following burn, trauma or critical illness. PMID:23507713

Transcriptome and proteomic analysis of mango (Mangifera indica Linn) fruits.

PubMed

Wu, Hong-xia; Jia, Hui-min; Ma, Xiao-wei; Wang, Song-biao; Yao, Quan-sheng; Xu, Wen-tian; Zhou, Yi-gang; Gao, Zhong-shan; Zhan, Ru-lin

2014-06-13

Here we used Illumina RNA-seq technology for transcriptome sequencing of a mixed fruit sample from 'Zill' mango (Mangifera indica Linn) fruit pericarp and pulp during the development and ripening stages. RNA-seq generated 68,419,722 sequence reads that were assembled into 54,207 transcripts with a mean length of 858bp, including 26,413 clusters and 27,794 singletons. A total of 42,515(78.43%) transcripts were annotated using public protein databases, with a cut-off E-value above 10(-5), of which 35,198 and 14,619 transcripts were assigned to gene ontology terms and clusters of orthologous groups respectively. Functional annotation against the Kyoto Encyclopedia of Genes and Genomes database identified 23,741(43.79%) transcripts which were mapped to 128 pathways. These pathways revealed many previously unknown transcripts. We also applied mass spectrometry-based transcriptome data to characterize the proteome of ripe fruit. LC-MS/MS analysis of the mango fruit proteome was using tandem mass spectrometry (MS/MS) in an LTQ Orbitrap Velos (Thermo) coupled online to the HPLC. This approach enabled the identification of 7536 peptides that matched 2754 proteins. Our study provides a comprehensive sequence for a systemic view of transcriptome during mango fruit development and the most comprehensive fruit proteome to date, which are useful for further genomics research and proteomic studies. Our study provides a comprehensive sequence for a systemic view of both the transcriptome and proteome of mango fruit, and a valuable reference for further research on gene expression and protein identification. This article is part of a Special Issue entitled: Proteomics of non-model organisms. Copyright © 2014 Elsevier B.V. All rights reserved.

Proteomics of the Lysosome

PubMed Central

Lübke, Torben; Lobel, Peter; Sleat, David

2009-01-01

Defects in lysosomal function have been associated with numerous monogenic human diseases typically classified as lysosomal storage diseases. However, there is increasing evidence that lysosomal proteins are also involved in more widespread human diseases including cancer and Alzheimer disease. Thus, there is a continuing interest in understanding the cellular functions of the lysosome and an emerging approach to this is the identification of its constituent proteins by proteomic analyses. To date, the mammalian lysosome has been shown to contain ~ 60 soluble luminal proteins and ~25 transmembrane proteins. However, recent proteomic studies based upon affinity purification of soluble components or subcellular fractionation to obtain both soluble and membrane components suggest that there may be many more of both classes of protein resident within this organelle than previously appreciated. Discovery of such proteins has important implications for understanding the function and the dynamics of the lysosome but can also lead the way towards the discovery of the genetic basis for human diseases of hitherto unknown etiology. Here, we describe current approaches to lysosomal proteomics and data interpretation and review the new lysosomal proteins that have recently emerged from such studies. PMID:18977398

In-depth proteomic analysis of a mollusc shell: acid-soluble and acid-insoluble matrix of the limpet Lottia gigantea

PubMed Central

2012-01-01

Background Invertebrate biominerals are characterized by their extraordinary functionality and physical properties, such as strength, stiffness and toughness that by far exceed those of the pure mineral component of such composites. This is attributed to the organic matrix, secreted by specialized cells, which pervades and envelops the mineral crystals. Despite the obvious importance of the protein fraction of the organic matrix, only few in-depth proteomic studies have been performed due to the lack of comprehensive protein sequence databases. The recent public release of the gastropod Lottia gigantea genome sequence and the associated protein sequence database provides for the first time the opportunity to do a state-of-the-art proteomic in-depth analysis of the organic matrix of a mollusc shell. Results Using three different sodium hypochlorite washing protocols before shell demineralization, a total of 569 proteins were identified in Lottia gigantea shell matrix. Of these, 311 were assembled in a consensus proteome comprising identifications contained in all proteomes irrespective of shell cleaning procedure. Some of these proteins were similar in amino acid sequence, amino acid composition, or domain structure to proteins identified previously in different bivalve or gastropod shells, such as BMSP, dermatopontin, nacrein, perlustrin, perlucin, or Pif. In addition there were dozens of previously uncharacterized proteins, many containing repeated short linear motifs or homorepeats. Such proteins may play a role in shell matrix construction or control of mineralization processes. Conclusions The organic matrix of Lottia gigantea shells is a complex mixture of proteins comprising possible homologs of some previously characterized mollusc shell proteins, but also many novel proteins with a possible function in biomineralization as framework building blocks or as regulatory components. We hope that this data set, the most comprehensive available at present, will provide a platform for the further exploration of biomineralization processes in molluscs. PMID:22540284

Extracellular proteome analysis of Leptospira interrogans serovar Lai.

PubMed

Zeng, Lingbing; Zhang, Yunyi; Zhu, Yongzhang; Yin, Haidi; Zhuang, Xuran; Zhu, Weinan; Guo, Xiaokui; Qin, Jinhong

2013-10-01

Abstract Leptospirosis is one of the most important zoonoses. Leptospira interrogans serovar Lai is a pathogenic spirochete that is responsible for leptospirosis. Extracellular proteins play an important role in the pathogenicity of this bacterium. In this study, L. interrogans serovar Lai was grown in protein-free medium; the supernatant was collected and subsequently analyzed as the extracellular proteome. A total of 66 proteins with more than two unique peptides were detected by MS/MS, and 33 of these were predicted to be extracellular proteins by a combination of bioinformatics analyses, including Psortb, cello, SoSuiGramN and SignalP. Comparisons of the transcriptional levels of these 33 genes between in vivo and in vitro conditions revealed that 15 genes were upregulated and two genes were downregulated in vivo compared to in vitro. A BLAST search for the components of secretion system at the genomic and proteomic levels revealed the presence of the complete type I secretion system and type II secretion system in this strain. Moreover, this strain also exhibits complete Sec translocase and Tat translocase systems. The extracellular proteome analysis of L. interrogans will supplement the previously generated whole proteome data and provide more information for studying the functions of specific proteins in the infection process and for selecting candidate molecules for vaccines or diagnostic tools for leptospirosis.

Comparative proteomic analysis of two pathogenic Tritrichomonas foetus genotypes: there is more to the proteome than meets the eye.

PubMed

Stroud, Leah J; Šlapeta, Jan; Padula, Matthew P; Druery, Dylan; Tsiotsioras, George; Coorssen, Jens R; Stack, Colin M

2017-03-01

Certain clinical isolates of Tritrichomonas foetus infect the urogenital tract of cattle while others infect the gastrointestinal tract of cats. Previous studies have identified subtle genetic differences between these isolates with the term "genotype" adopted to reflect host origin. The aim of this work was to seek evidence of host-specific adaptation and to clarify the relationship between T. foetus genotypes. To do this we characterised the proteomes of both genotypes using two-dimensional gel electrophoresis (2DE) coupled with LC-MS/MS. Our comparative analysis of the data revealed that both genotypes exhibited largely similar proteoform profiles; however differentiation was possible with 24 spots identified as having a four-fold or greater change. Deeper analysis using 2DE zymography and protease-specific fluorogenic substrates revealed marked differences in cysteine protease (CP) expression profiles between the two genotypes. These variances in CP activities could also account for the pathogenic and histopathological differences previously observed between T. foetus genotypes in cross-infection studies. Our findings highlight the importance of CPs as major determinants of parasite virulence and provide a foundation for future host-parasite interaction studies, with direct implications for the development of vaccines or drugs targeting T. foetus. Copyright © 2017 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

Serum proteomic analysis of extracorporeal shock wave therapy-enhanced diabetic wound healing in a streptozotocin-induced diabetes model.

PubMed

Yang, Ming-Yu; Chiang, Yuan-Cheng; Huang, Yu-Ting; Chen, Chien-Chang; Wang, Feng-Sheng; Wang, Ching-Jen; Kuo, Yur-Ren

2014-01-01

Previous studies have demonstrated that extracorporeal shock wave therapy has a significant positive effect on accelerating diabetic wound healing. However, the systemic effect after therapy is still unclear. This study investigated the plasma protein expression in the extracorporeal shock wave therapy group and diabetic controls using proteomic study. A dorsal skin defect (6 × 5 cm) in a streptozotocin-induced diabetic Wistar rat model was used. Diabetic rats receiving either no therapy or extracorporeal shock wave therapy after wounding were analyzed. The spots of interest were subjected to in-gel trypsin digestion and matrix-assisted laser desorption ionization time-of-flight mass spectrometry to elucidate the peptide mass fingerprints. The mass spectrometric characteristics of the identified proteins, including their theoretical isoelectric points, molecular weights, sequence coverage, and Mascot score, were analyzed. Protein expression was validated using immunohistochemical analysis of topical periwounding tissues. The proteomic study revealed that at days 3 and 10 after therapy rats had significantly higher abundance of haptoglobin and significantly lower levels of the vitamin D-binding protein precursor as compared with the diabetic controls. Immunohistochemical staining of topical periwounding tissue also revealed significant upregulation of haptoglobin and downregulation of vitamin D-binding protein expression in the extracorporeal shock wave therapy group, which was consistent with the systemic proteome study. Proteome analyses demonstrated an upregulation of haptoglobin and a downregulation of vitamin D-binding protein in extracorporeal shock wave therapy-enhanced diabetic wound healing.

MEERCAT: Multiplexed Efficient Cell Free Expression of Recombinant QconCATs For Large Scale Absolute Proteome Quantification*

PubMed Central

Takemori, Nobuaki; Takemori, Ayako; Tanaka, Yuki; Endo, Yaeta; Hurst, Jane L.; Gómez-Baena, Guadalupe; Harman, Victoria M.; Beynon, Robert J.

2017-01-01

A major challenge in proteomics is the absolute accurate quantification of large numbers of proteins. QconCATs, artificial proteins that are concatenations of multiple standard peptides, are well established as an efficient means to generate standards for proteome quantification. Previously, QconCATs have been expressed in bacteria, but we now describe QconCAT expression in a robust, cell-free system. The new expression approach rescues QconCATs that previously were unable to be expressed in bacteria and can reduce the incidence of proteolytic damage to QconCATs. Moreover, it is possible to cosynthesize QconCATs in a highly-multiplexed translation reaction, coexpressing tens or hundreds of QconCATs simultaneously. By obviating bacterial culture and through the gain of high level multiplexing, it is now possible to generate tens of thousands of standard peptides in a matter of weeks, rendering absolute quantification of a complex proteome highly achievable in a reproducible, broadly deployable system. PMID:29055021

Comparative proteomic study on Brassica hexaploid and its parents provides new insights into the effects of polyploidization.

PubMed

Shen, Yanyue; Zhang, Yu; Zou, Jun; Meng, Jinling; Wang, Jianbo

2015-01-01

Polyploidy has played an important role in promoting plant evolution through genomic merging and doubling. Although genomic and transcriptomic changes have been observed in polyploids, the effects of polyploidization on proteomic divergence are poorly understood. In this study, we reported quantitative analysis of proteomic changes in leaves of Brassica hexaploid and its parents using isobaric tags for relative and absolute quantitation (iTRAQ) coupled with mass spectrometry. A total of 2044 reproducible proteins were quantified by at least two unique peptides. We detected 452 proteins differentially expressed between Brassica hexaploid and its parents, and 100 proteins were non-additively expressed in Brassica hexaploid, which suggested a trend of non-additive protein regulation following genomic merger and doubling. Functional categories of cellular component biogenesis, immune system process, and response to stimulus, were significantly enriched in non-additive proteins, probably providing a driving force for variation and adaptation in allopolyploids. In particular, majority of the total 452 differentially expressed proteins showed expression level dominance of one parental expression, and there was an expression level dominance bias toward the tetraploid progenitor. In addition, the percentage of differentially expressed proteins that matched previously reported differentially genes were relatively low. This study aimed to get new insights into the effects of polyploidization on proteomic divergence. Using iTRAQ LC-MS/MS technology, we identified 452 differentially expressed proteins between allopolyploid and its parents which involved in response to stimulus, multi-organism process, and immune system process, much more than previous studies using 2-DE coupled with mass spectrometry technology. Therefore, our manuscript represents the most comprehensive analysis of protein profiles in allopolyploid and its parents, which will lead to a better understanding of novelty and plasticity of the allopolyploid genomes. Copyright © 2014 Elsevier B.V. All rights reserved.

Affordable proteomics: the two-hybrid systems.

PubMed

Gillespie, Marc

2003-06-01

Numerous proteomic methodologies exist, but most require a heavy investment in expertise and technology. This puts these approaches out of reach for many laboratories and small companies, rarely allowing proteomics to be used as a pilot approach for biomarker or target identification. Two proteomic approaches, 2D gel electrophoresis and the two-hybrid systems, are currently available to most researchers. The two-hybrid systems, though accommodating to large-scale experiments, were originally designed as practical screens, that by comparison to current proteomics tools were small-scale, affordable and technically feasible. The screens rapidly generated data, identifying protein interactions that were previously uncharacterized. The foundation for a two-hybrid proteomic investigation can be purchased as separate kits from a number of companies. The true power of the technique lies not in its affordability, but rather in its portability. The two-hybrid system puts proteomics back into laboratories where the output of the screens can be evaluated by researchers with experience in the particular fields of basic research, cancer biology, toxicology or drug development.

A DIGE proteomic analysis for high-intensity exercise-trained rat skeletal muscle.

PubMed

Yamaguchi, Wataru; Fujimoto, Eri; Higuchi, Mitsuru; Tabata, Izumi

2010-09-01

Exercise training induces various adaptations in skeletal muscles. However, the mechanisms remain unclear. In this study, we conducted 2D-DIGE proteomic analysis, which has not yet been used for elucidating adaptations of skeletal muscle after high-intensity exercise training (HIT). For 5 days, rats performed HIT, which consisted of 14 20-s swimming exercise bouts carrying a weight (14% of the body weight), and 10-s pause between bouts. The 2D-DIGE analysis was conducted on epitrochlearis muscles excised 18 h after the final training exercise. Proteomic profiling revealed that out of 800 detected and matched spots, 13 proteins exhibited changed expression by HIT compared with sedentary rats. All proteins were identified by MALDI-TOF/MS. Furthermore, using western immunoblot analyses, significantly changed expressions of NDUFS1 and parvalbumin (PV) were validated in relation to HIT. In conclusion, the proteomic 2D-DIGE analysis following HIT-identified expressions of NDUFS1 and PV, previously unknown to have functions related to exercise-training adaptations.

A Comparative Quantitative Proteomic Study Identifies New Proteins Relevant for Sulfur Oxidation in the Purple Sulfur Bacterium Allochromatium vinosum

PubMed Central

Weissgerber, Thomas; Sylvester, Marc; Kröninger, Lena

2014-01-01

In the present study, we compared the proteome response of Allochromatium vinosum when growing photoautotrophically in the presence of sulfide, thiosulfate, and elemental sulfur with the proteome response when the organism was growing photoheterotrophically on malate. Applying tandem mass tag analysis as well as two-dimensional (2D) PAGE, we detected 1,955 of the 3,302 predicted proteins by identification of at least two peptides (59.2%) and quantified 1,848 of the identified proteins. Altered relative protein amounts (≥1.5-fold) were observed for 385 proteins, corresponding to 20.8% of the quantified A. vinosum proteome. A significant number of the proteins exhibiting strongly enhanced relative protein levels in the presence of reduced sulfur compounds are well documented essential players during oxidative sulfur metabolism, e.g., the dissimilatory sulfite reductase DsrAB. Changes in protein levels generally matched those observed for the respective relative mRNA levels in a previous study and allowed identification of new genes/proteins participating in oxidative sulfur metabolism. One gene cluster (hyd; Alvin_2036-Alvin_2040) and one hypothetical protein (Alvin_2107) exhibiting strong responses on both the transcriptome and proteome levels were chosen for gene inactivation and phenotypic analyses of the respective mutant strains, which verified the importance of the so-called Isp hydrogenase supercomplex for efficient oxidation of sulfide and a crucial role of Alvin_2107 for the oxidation of sulfur stored in sulfur globules to sulfite. In addition, we analyzed the sulfur globule proteome and identified a new sulfur globule protein (SgpD; Alvin_2515). PMID:24487535

Proteome regulation during Olea europaea fruit development.

PubMed

Bianco, Linda; Alagna, Fiammetta; Baldoni, Luciana; Finnie, Christine; Svensson, Birte; Perrotta, Gaetano

2013-01-01

Widespread in the Mediterranean basin, Olea europaea trees are gaining worldwide popularity for the nutritional and cancer-protective properties of the oil, mechanically extracted from ripe fruits. Fruit development is a physiological process with remarkable impact on the modulation of the biosynthesis of compounds affecting the quality of the drupes as well as the final composition of the olive oil. Proteomics offers the possibility to dig deeper into the major changes during fruit development, including the important phase of ripening, and to classify temporal patterns of protein accumulation occurring during these complex physiological processes. In this work, we started monitoring the proteome variations associated with olive fruit development by using comparative proteomics coupled to mass spectrometry. Proteins extracted from drupes at three different developmental stages were separated on 2-DE and subjected to image analysis. 247 protein spots were revealed as differentially accumulated. Proteins were identified from a total of 121 spots and discussed in relation to olive drupe metabolic changes occurring during fruit development. In order to evaluate if changes observed at the protein level were consistent with changes of mRNAs, proteomic data produced in the present work were compared with transcriptomic data elaborated during previous studies. This study identifies a number of proteins responsible for quality traits of cv. Coratina, with particular regard to proteins associated to the metabolism of fatty acids, phenolic and aroma compounds. Proteins involved in fruit photosynthesis have been also identified and their pivotal contribution in oleogenesis has been discussed. To date, this study represents the first characterization of the olive fruit proteome during development, providing new insights into fruit metabolism and oil accumulation process.

Identification of new intrinsic proteins in Arabidopsis plasma membrane proteome.

PubMed

Marmagne, Anne; Rouet, Marie-Aude; Ferro, Myriam; Rolland, Norbert; Alcon, Carine; Joyard, Jacques; Garin, Jérome; Barbier-Brygoo, Hélène; Ephritikhine, Geneviève

2004-07-01

Identification and characterization of anion channel genes in plants represent a goal for a better understanding of their central role in cell signaling, osmoregulation, nutrition, and metabolism. Though channel activities have been well characterized in plasma membrane by electrophysiology, the corresponding molecular entities are little documented. Indeed, the hydrophobic protein equipment of plant plasma membrane still remains largely unknown, though several proteomic approaches have been reported. To identify new putative transport systems, we developed a new proteomic strategy based on mass spectrometry analyses of a plasma membrane fraction enriched in hydrophobic proteins. We produced from Arabidopsis cell suspensions a highly purified plasma membrane fraction and characterized it in detail by immunological and enzymatic tests. Using complementary methods for the extraction of hydrophobic proteins and mass spectrometry analyses on mono-dimensional gels, about 100 proteins have been identified, 95% of which had never been found in previous proteomic studies. The inventory of the plasma membrane proteome generated by this approach contains numerous plasma membrane integral proteins, one-third displaying at least four transmembrane segments. The plasma membrane localization was confirmed for several proteins, therefore validating such proteomic strategy. An in silico analysis shows a correlation between the putative functions of the identified proteins and the expected roles for plasma membrane in transport, signaling, cellular traffic, and metabolism. This analysis also reveals 10 proteins that display structural properties compatible with transport functions and will constitute interesting targets for further functional studies.

Quantitative Proteomics Uncovers Novel Factors Involved in Developmental Differentiation of Trypanosoma brucei

PubMed Central

Dejung, Mario; Subota, Ines; Bucerius, Ferdinand; Dindar, Gülcin; Freiwald, Anja; Engstler, Markus; Boshart, Michael; Butter, Falk; Janzen, Christian J.

2016-01-01

Developmental differentiation is a universal biological process that allows cells to adapt to different environments to perform specific functions. African trypanosomes progress through a tightly regulated life cycle in order to survive in different host environments when they shuttle between an insect vector and a vertebrate host. Transcriptomics has been useful to gain insight into RNA changes during stage transitions; however, RNA levels are only a moderate proxy for protein abundance in trypanosomes. We quantified 4270 protein groups during stage differentiation from the mammalian-infective to the insect form and provide classification for their expression profiles during development. Our label-free quantitative proteomics study revealed previously unknown components of the differentiation machinery that are involved in essential biological processes such as signaling, posttranslational protein modifications, trafficking and nuclear transport. Furthermore, guided by our proteomic survey, we identified the cause of the previously observed differentiation impairment in the histone methyltransferase DOT1B knock-out strain as it is required for accurate karyokinesis in the first cell division during differentiation. This epigenetic regulator is likely involved in essential chromatin restructuring during developmental differentiation, which might also be important for differentiation in higher eukaryotic cells. Our proteome dataset will serve as a resource for detailed investigations of cell differentiation to shed more light on the molecular mechanisms of this process in trypanosomes and other eukaryotes. PMID:26910529

The Multinational Arabidopsis Steering Subcommittee for Proteomics Assembles the Largest Proteome Database Resource for Plant Systems Biology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weckwerth, Wolfram; Baginsky, Sacha; Van Wijk, Klass

2009-12-01

In the past 10 years, we have witnessed remarkable advances in the field of plant molecular biology. The rapid development of proteomic technologies and the speed with which these techniques have been applied to the field have altered our perception of how we can analyze proteins in complex systems. At nearly the same time, the availability of the complete genome for the model plant Arabidopsis thaliana was released; this effort provides an unsurpassed resource for the identification of proteins when researchers use MS to analyze plant samples. Recognizing the growth in this area, the Multinational Arabidopsis Steering Committee (MASC) establishedmore » a subcommittee for A. thaliana proteomics in 2006 with the objective of consolidating databases, technique standards, and experimentally validated candidate genes and functions. Since the establishment of the Multinational Arabidopsis Steering Subcommittee for Proteomics (MASCP), many new approaches and resources have become available. Recently, the subcommittee established a webpage to consolidate this information (www.masc-proteomics.org). It includes links to plant proteomic databases, general information about proteomic techniques, meeting information, a summary of proteomic standards, and other relevant resources. Altogether, this website provides a useful resource for the Arabidopsis proteomics community. In the future, the website will host discussions and investigate the cross-linking of databases. The subcommittee members have extensive experience in arabidopsis proteomics and collectively have produced some of the most extensive proteomics data sets for this model plant (Table S1 in the Supporting Information has a list of resources). The largest collection of proteomics data from a single study in A. thaliana was assembled into an accessible database (AtProteome; http://fgcz-atproteome.unizh.ch/index.php) and was recently published by the Baginsky lab.1 The database provides links to major Arabidopsis online resources, and raw data have been deposited in PRIDE and PRIDE BioMart. Included in this database is an Arabidopsis proteome map that provides evidence for the expression of {approx}50% of all predicted gene models, including several alternative gene models that are not represented in The Arabidopsis Information Resource (TAIR) protein database. A set of organ-specific biomarkers is provided, as well as organ-specific proteotypic peptides for 4105 proteins that can be used to facilitate targeted quantitative proteomic surveys. In the future, the AtProteome database will be linked to additional existing resources developed by MASCP members, such as PPDB, ProMEX, and SUBA. The most comprehensive study on the Arabidopsis chloroplast proteome, which includes information on chloroplast sorting signals, posttranslational modifications (PTMs), and protein abundances (analyzed by high-accuracy MS [Orbitrap]), was recently published by the van Wijk lab.2 These and previous data are available via the plant proteome database (PPDB; http://ppdb.tc.cornell.edu) for A. thaliana and maize. PPDB provides genome-wide experimental and functional characterization of the A. thaliana and maize proteomes, including PTMs and subcellular localization information, with an emphasis on leaf and plastid proteins. Maize and Arabidopsis proteome entries are directly linked via internal BLAST alignments within PPDB. Direct links for each protein to TAIR, SUBA, ProMEX, and other resources are also provided.« less

Proteomic analyses for profiling regulated proteins/enzymes by Fucus vesiculosus fucoidan in B16 melanoma cells: A combination of enzyme kinetics functional study.

PubMed

Wang, Zhi-Jiang; Zheng, Li; Yang, Jun-Mo; Kang, Yani; Park, Yong-Doo

2018-06-01

Fucoidans are complex sulfated polysaccharides that have a wide range of biological activities. Previously, we reported the various effects of Fucus vesiculosus fucoidan on tyrosinase and B16 melanoma cells. In this study, to identify fucoidan-targeted proteins in B16 melanoma cells, we performed a proteomics study and integrated enzyme kinetics. We detected 19 candidate proteins dysregulated by fucoidan treatment. Among the probed proteins, the enzyme kinetics of two candidate enzymes, namely lactate dehydrogenase (LDH) as an upregulated protein and superoxide dismutase (SOD) as a downregulated enzyme, were determined. The enzyme kinetics results showed that Fucus vesiculosus fucoidan significantly inhibited LDH catalytic function while it did not affect SOD activity even at a high dose, while only slightly decreased activity (up to 10%) at a low dose. Based on our previous and present observations, fucoidan could inhibit B16 melanoma cells growth via regulating proteins/enzymes expression levels such as LDH and SOD known as cell survival biomarkers. Interestingly, both expression level and enzyme catalytic activity of LDH were regulated by fucoidan, which could directly induce the apoptotic effect on B16 melanoma cells along with SOD downregulation. This study highlights how combining proteomics with enzyme kinetics can yield valuable insights into fucoidan targets. Copyright © 2018 Elsevier B.V. All rights reserved.

Transcriptomic analysis of Arabidopsis developing stems: a close-up on cell wall genes

PubMed Central

Minic, Zoran; Jamet, Elisabeth; San-Clemente, Hélène; Pelletier, Sandra; Renou, Jean-Pierre; Rihouey, Christophe; Okinyo, Denis PO; Proux, Caroline; Lerouge, Patrice; Jouanin, Lise

2009-01-01

Background Different strategies (genetics, biochemistry, and proteomics) can be used to study proteins involved in cell biogenesis. The availability of the complete sequences of several plant genomes allowed the development of transcriptomic studies. Although the expression patterns of some Arabidopsis thaliana genes involved in cell wall biogenesis were identified at different physiological stages, detailed microarray analysis of plant cell wall genes has not been performed on any plant tissues. Using transcriptomic and bioinformatic tools, we studied the regulation of cell wall genes in Arabidopsis stems, i.e. genes encoding proteins involved in cell wall biogenesis and genes encoding secreted proteins. Results Transcriptomic analyses of stems were performed at three different developmental stages, i.e., young stems, intermediate stage, and mature stems. Many genes involved in the synthesis of cell wall components such as polysaccharides and monolignols were identified. A total of 345 genes encoding predicted secreted proteins with moderate or high level of transcripts were analyzed in details. The encoded proteins were distributed into 8 classes, based on the presence of predicted functional domains. Proteins acting on carbohydrates and proteins of unknown function constituted the two most abundant classes. Other proteins were proteases, oxido-reductases, proteins with interacting domains, proteins involved in signalling, and structural proteins. Particularly high levels of expression were established for genes encoding pectin methylesterases, germin-like proteins, arabinogalactan proteins, fasciclin-like arabinogalactan proteins, and structural proteins. Finally, the results of this transcriptomic analyses were compared with those obtained through a cell wall proteomic analysis from the same material. Only a small proportion of genes identified by previous proteomic analyses were identified by transcriptomics. Conversely, only a few proteins encoded by genes having moderate or high level of transcripts were identified by proteomics. Conclusion Analysis of the genes predicted to encode cell wall proteins revealed that about 345 genes had moderate or high levels of transcripts. Among them, we identified many new genes possibly involved in cell wall biogenesis. The discrepancies observed between results of this transcriptomic study and a previous proteomic study on the same material revealed post-transcriptional mechanisms of regulation of expression of genes encoding cell wall proteins. PMID:19149885

Urinary Collagen Fragments Are Significantly Altered in Diabetes: A Link to Pathophysiology

PubMed Central

Argilés, Àngel; Cerna, Marie; Delles, Christian; Dominiczak, Anna F.; Gayrard, Nathalie; Iphöfer, Alexander; Jänsch, Lothar; Jerums, George; Medek, Karel; Mischak, Harald; Navis, Gerjan J.; Roob, Johannes M.; Rossing, Kasper; Rossing, Peter; Rychlík, Ivan; Schiffer, Eric; Schmieder, Roland E.; Wascher, Thomas C.; Winklhofer-Roob, Brigitte M.; Zimmerli, Lukas U.; Zürbig, Petra; Snell-Bergeon, Janet K.

2010-01-01

Background The pathogenesis of diabetes mellitus (DM) is variable, comprising different inflammatory and immune responses. Proteome analysis holds the promise of delivering insight into the pathophysiological changes associated with diabetes. Recently, we identified and validated urinary proteomics biomarkers for diabetes. Based on these initial findings, we aimed to further validate urinary proteomics biomarkers specific for diabetes in general, and particularity associated with either type 1 (T1D) or type 2 diabetes (T2D). Methodology/Principal Findings Therefore, the low-molecular-weight urinary proteome of 902 subjects from 10 different centers, 315 controls and 587 patients with T1D (n = 299) or T2D (n = 288), was analyzed using capillary-electrophoresis mass-spectrometry. The 261 urinary biomarkers (100 were sequenced) previously discovered in 205 subjects were validated in an additional 697 subjects to distinguish DM subjects (n = 382) from control subjects (n = 315) with 94% (95% CI: 92–95) accuracy in this study. To identify biomarkers that differentiate T1D from T2D, a subset of normoalbuminuric patients with T1D (n = 68) and T2D (n = 42) was employed, enabling identification of 131 biomarker candidates (40 were sequenced) differentially regulated between T1D and T2D. These biomarkers distinguished T1D from T2D in an independent validation set of normoalbuminuric patients (n = 108) with 88% (95% CI: 81–94%) accuracy, and in patients with impaired renal function (n = 369) with 85% (95% CI: 81–88%) accuracy. Specific collagen fragments were associated with diabetes and type of diabetes indicating changes in collagen turnover and extracellular matrix as one hallmark of the molecular pathophysiology of diabetes. Additional biomarkers including inflammatory processes and pro-thrombotic alterations were observed. Conclusions/Significance These findings, based on the largest proteomic study performed to date on subjects with DM, validate the previously described biomarkers for DM, and pinpoint differences in the urinary proteome of T1D and T2D, indicating significant differences in extracellular matrix remodeling. PMID:20927192

Proteobionics: biomimetics in proteomics.

PubMed

Sommer, Andrei P; Gheorghiu, Eleonora

2006-03-01

Proteomics was established 10 years ago by the analysis of microbial genomes via their protein complement or proteome. Bionics is an ancient art, which converts structures optimized by nature into advanced technical products. Previously, we analyzed survival modalities in nanobacteria and converted the interplay between survival-oriented protein functions and nanoscale mineral shells into models for advanced drug delivery. Exploiting protein functions observed in nature to design biomedical products and therapies could be named proteobionics. Here, we present examples for this new branch of nanoproteomics.

Characterization of the canine urinary proteome.

PubMed

Brandt, Laura E; Ehrhart, E J; Scherman, Hataichanok; Olver, Christine S; Bohn, Andrea A; Prenni, Jessica E

2014-06-01

Urine is an attractive biofluid for biomarker discovery as it is easy and minimally invasive to obtain. While numerous studies have focused on the characterization of human urine, much less research has focused on canine urine. The objectives of this study were to characterize the universal canine urinary proteome (both soluble and exosomal), to determine the overlap between the canine proteome and a representative human urinary proteome study, to generate a resource for future canine studies, and to determine the suitability of the dog as a large animal model for human diseases. The soluble and exosomal fractions of normal canine urine were characterized using liquid chromatography tandem mass spectrometry (LC-MS/MS). Biological Networks Gene Ontology (BiNGO) software was utilized to assign the canine urinary proteome to respective Gene Ontology categories, such as Cellular Component, Molecular Function, and Biological Process. Over 500 proteins were confidently identified in normal canine urine. Gene Ontology analysis revealed that exosomal proteins were largely derived from an intracellular location, while soluble proteins included both extracellular and membrane proteins. Exosome proteins were assigned to metabolic processes and localization, while soluble proteins were primarily annotated to specific localization processes. Several proteins identified in normal canine urine have previously been identified in human urine where these proteins are related to various extrarenal and renal diseases. The results of this study illustrate the potential of the dog as an animal model for human disease states and provide the framework for future studies of canine renal diseases. © 2014 American Society for Veterinary Clinical Pathology and European Society for Veterinary Clinical Pathology.

iProphet: Multi-level Integrative Analysis of Shotgun Proteomic Data Improves Peptide and Protein Identification Rates and Error Estimates*

PubMed Central

Shteynberg, David; Deutsch, Eric W.; Lam, Henry; Eng, Jimmy K.; Sun, Zhi; Tasman, Natalie; Mendoza, Luis; Moritz, Robert L.; Aebersold, Ruedi; Nesvizhskii, Alexey I.

2011-01-01

The combination of tandem mass spectrometry and sequence database searching is the method of choice for the identification of peptides and the mapping of proteomes. Over the last several years, the volume of data generated in proteomic studies has increased dramatically, which challenges the computational approaches previously developed for these data. Furthermore, a multitude of search engines have been developed that identify different, overlapping subsets of the sample peptides from a particular set of tandem mass spectrometry spectra. We present iProphet, the new addition to the widely used open-source suite of proteomic data analysis tools Trans-Proteomics Pipeline. Applied in tandem with PeptideProphet, it provides more accurate representation of the multilevel nature of shotgun proteomic data. iProphet combines the evidence from multiple identifications of the same peptide sequences across different spectra, experiments, precursor ion charge states, and modified states. It also allows accurate and effective integration of the results from multiple database search engines applied to the same data. The use of iProphet in the Trans-Proteomics Pipeline increases the number of correctly identified peptides at a constant false discovery rate as compared with both PeptideProphet and another state-of-the-art tool Percolator. As the main outcome, iProphet permits the calculation of accurate posterior probabilities and false discovery rate estimates at the level of sequence identical peptide identifications, which in turn leads to more accurate probability estimates at the protein level. Fully integrated with the Trans-Proteomics Pipeline, it supports all commonly used MS instruments, search engines, and computer platforms. The performance of iProphet is demonstrated on two publicly available data sets: data from a human whole cell lysate proteome profiling experiment representative of typical proteomic data sets, and from a set of Streptococcus pyogenes experiments more representative of organism-specific composite data sets. PMID:21876204

The Brain Proteome of the Ubiquitin Ligase Peli1 Knock-Out Mouse during Experimental Autoimmune Encephalomyelitis.

PubMed

Lereim, Ragnhild Reehorst; Oveland, Eystein; Xiao, Yichuan; Torkildsen, Øivind; Wergeland, Stig; Myhr, Kjell-Morten; Sun, Shao-Cong; Berven, Frode S

2016-09-01

The ubiquitin ligase Peli1 has previously been suggested as a potential treatment target in multiple sclerosis. In the multiple sclerosis disease model, experimental autoimmune encephalomyelitis, Peli1 knock-out led to less activated microglia and less inflammation in the central nervous system. Despite being important in microglia, Peli1 expression has also been detected in glial and neuronal cells. In the present study the overall brain proteomes of Peli1 knock-out mice and wild-type mice were compared prior to experimental autoimmune encephalomyelitis induction, at onset of the disease and at disease peak. Brain samples from the frontal hemisphere, peripheral from the extensive inflammatory foci, were analyzed using TMT-labeling of sample pools, and the discovered proteins were verified in individual mice using label-free proteomics. The greatest proteomic differences between Peli1 knock-out and wild-type mice were observed at the disease peak. In Peli1 knock-out a higher degree of antigen presentation, increased activity of adaptive and innate immune cells and alterations to proteins involved in iron metabolism were observed during experimental autoimmune encephalomyelitis. These results unravel global effects to the brain proteome when abrogating Peli1 expression, underlining the importance of Peli1 as a regulator of the immune response also peripheral to inflammatory foci during experimental autoimmune encephalomyelitis. The proteomics data is available in PRIDE with accession PXD003710.

Proteomics of drug resistance in Candida glabrata biofilms.

PubMed

Seneviratne, C Jayampath; Wang, Yu; Jin, Lijian; Abiko, Y; Samaranayake, Lakshman P

2010-04-01

Candida glabrata is a fungal pathogen that causes a variety of mucosal and systemic infections among compromised patient populations with higher mortality rates. Previous studies have shown that biofilm mode of the growth of the fungus is highly resistant to antifungal agents compared with the free-floating or planktonic mode of growth. Therefore, in the present study, we used 2-D DIGE to evaluate the differential proteomic profiles of C. glabrata under planktonic and biofilm modes of growth. Candida glabrata biofilms were developed on polystyrene surfaces and age-matched planktonic cultures were obtained in parallel. Initially, biofilm architecture, viability, and antifungal susceptibility were evaluated. Differentially expressed proteins more than 1.5-fold in DIGE analysis were subjected to MS/MS. The transcriptomic regulation of these biomarkers was evaluated by quantitative real-time PCR. Candida glabrata biofilms were highly resistant to the antifungals and biocides compared with the planktonic mode of growth. Candida glabrata biofilm proteome when compared with its planktonic proteome showed upregulation of stress response proteins, while glycolysis enzymes were downregulated. Similar trend could be observed at transcriptomic level. In conclusion, C. glabrata biofilms possess higher amount of stress response proteins, which may potentially contribute to the higher antifungal resistance seen in C. glabrata biofilms.

Snake venomics of Lachesis muta rhombeata and genus-wide antivenomics assessment of the paraspecific immunoreactivity of two antivenoms evidence the high compositional and immunological conservation across Lachesis.

PubMed

Pla, Davinia; Sanz, Libia; Molina-Sánchez, Pedro; Zorita, Virginia; Madrigal, Marvin; Flores-Díaz, Marietta; Alape-Girón, Alberto; Núñez, Vitelbina; Andrés, Vicente; Gutiérrez, José María; Calvete, Juan J

2013-08-26

We report the proteomic analysis of the Atlantic bushmaster, Lachesis muta rhombeata, from Brazil. Along with previous characterization of the venom proteomes of L. stenophrys (Costa Rica), L. melanocephala (Costa Rica), L. acrochorda (Colombia), and L. muta muta (Bolivia), the present study provides the first overview of the composition and distribution of venom proteins across this wide-ranging genus, and highlights the remarkable similar compositional and pharmacological profiles across Lachesis venoms. The paraspecificity of two antivenoms, produced at Instituto Vital Brazil (Brazil) and Instituto Clodomiro Picado (Costa Rica) using different conspecific taxa in the immunization mixtures, was assessed using genus-wide comparative antivenomics. This study confirms that the proteomic similarity among Lachesis sp. venoms is mirrored in their high immunological conservation across the genus. The clinical and therapeutic consequences of genus-wide venomics and antivenomics investigations of Lachesis venoms are discussed. The proteomics characterization of L. m. rhombeata venom completes the overview of Lachesis venom proteomes and confirms the remarkable toxin profile conservation across the five clades of this wide-ranging genus. Genus-wide antivenomics showed that two antivenoms, produced against L. stenophrys or L. m. rhombeata, exhibit paraspecificity towards all other congeneric venoms. Our venomics study shows that, despite the broad geographic distribution of the genus, monospecific antivenoms may achieve clinical coverage for any Lachesis sp. envenoming. Copyright © 2013 Elsevier B.V. All rights reserved.

Proteome Regulation during Olea europaea Fruit Development

PubMed Central

Bianco, Linda; Alagna, Fiammetta; Baldoni, Luciana; Finnie, Christine; Svensson, Birte; Perrotta, Gaetano

2013-01-01

Background Widespread in the Mediterranean basin, Olea europaea trees are gaining worldwide popularity for the nutritional and cancer-protective properties of the oil, mechanically extracted from ripe fruits. Fruit development is a physiological process with remarkable impact on the modulation of the biosynthesis of compounds affecting the quality of the drupes as well as the final composition of the olive oil. Proteomics offers the possibility to dig deeper into the major changes during fruit development, including the important phase of ripening, and to classify temporal patterns of protein accumulation occurring during these complex physiological processes. Methodology/Principal Findings In this work, we started monitoring the proteome variations associated with olive fruit development by using comparative proteomics coupled to mass spectrometry. Proteins extracted from drupes at three different developmental stages were separated on 2-DE and subjected to image analysis. 247 protein spots were revealed as differentially accumulated. Proteins were identified from a total of 121 spots and discussed in relation to olive drupe metabolic changes occurring during fruit development. In order to evaluate if changes observed at the protein level were consistent with changes of mRNAs, proteomic data produced in the present work were compared with transcriptomic data elaborated during previous studies. Conclusions/Significance This study identifies a number of proteins responsible for quality traits of cv. Coratina, with particular regard to proteins associated to the metabolism of fatty acids, phenolic and aroma compounds. Proteins involved in fruit photosynthesis have been also identified and their pivotal contribution in oleogenesis has been discussed. To date, this study represents the first characterization of the olive fruit proteome during development, providing new insights into fruit metabolism and oil accumulation process. PMID:23349718

Phenobarbital Induces Alterations in the Proteome of Hepatocytes and Mesenchymal Cells of Rat Livers

PubMed Central

Klepeisz, Philip; Sagmeister, Sandra; Haudek-Prinz, Verena; Pichlbauer, Melanie; Grasl-Kraupp, Bettina; Gerner, Christopher

2013-01-01

Preceding studies on the mode of action of non-genotoxic hepatocarcinogens (NGCs) have concentrated on alterations induced in hepatocytes (HCs). A potential role of non-parenchymal liver cells (NPCs) in NGC-driven hepatocarcinogenesis has been largely neglected so far. The aim of this study is to characterize NGC-induced alterations in the proteome profiles of HCs as well as NPCs. We chose the prototypic NGC phenobarbital (PB) which was applied to male rats for a period of 14 days. The livers of PB-treated rats were perfused by collagenase and the cell suspensions obtained were subjected to density gradient centrifugation to separate HCs from NPCs. In addition, HCs and NPC isolated from untreated animals were treated with PB in vitro. Proteome profiling was done by CHIP-HPLC and ion trap mass spectrometry. Proteome analyses of the in vivo experiments showed many of the PB effects previously described in HCs by other methods, e.g. induction of phase I and phase II drug metabolising enzymes. In NPCs proteins related to inflammation and immune regulation such as PAI-1 and S100-A10, ADP-ribosyl cyclase 1 and to cell migration such as kinesin-1 heavy chain, myosin regulatory light chain RLC-A and dihydropyrimidinase-related protein 1 were found to be induced, indicating major PB effects on these cells. Remarkably, in vitro treatment of HCs and NPCs with PB hardly reproduced the proteome alterations observed in vivo, indicating differences of NGC induced responses of cells at culture conditions compared to the intact organism. To conclude, the present study clearly demonstrated that PB induces proteome alterations not only in HCs but also in NPCs. Thus, any profound molecular understanding on the mode of action of NGCs has to consider effects on cells of the hepatic mesenchyme. PMID:24204595

Feasibility of investigating differential proteomic expression in depression: implications for biomarker development in mood disorders

PubMed Central

Frye, M A; Nassan, M; Jenkins, G D; Kung, S; Veldic, M; Palmer, B A; Feeder, S E; Tye, S J; Choi, D S; Biernacka, J M

2015-01-01

The objective of this study was to determine whether proteomic profiling in serum samples can be utilized in identifying and differentiating mood disorders. A consecutive sample of patients with a confirmed diagnosis of unipolar (UP n=52) or bipolar depression (BP-I n=46, BP-II n=49) and controls (n=141) were recruited. A 7.5-ml blood sample was drawn for proteomic multiplex profiling of 320 proteins utilizing the Myriad RBM Discovery Multi-Analyte Profiling platform. After correcting for multiple testing and adjusting for covariates, growth differentiation factor 15 (GDF-15), hemopexin (HPX), hepsin (HPN), matrix metalloproteinase-7 (MMP-7), retinol-binding protein 4 (RBP-4) and transthyretin (TTR) all showed statistically significant differences among groups. In a series of three post hoc analyses correcting for multiple testing, MMP-7 was significantly different in mood disorder (BP-I+BP-II+UP) vs controls, MMP-7, GDF-15, HPN were significantly different in bipolar cases (BP-I+BP-II) vs controls, and GDF-15, HPX, HPN, RBP-4 and TTR proteins were all significantly different in BP-I vs controls. Good diagnostic accuracy (ROC-AUC⩾0.8) was obtained most notably for GDF-15, RBP-4 and TTR when comparing BP-I vs controls. While based on a small sample not adjusted for medication state, this discovery sample with a conservative method of correction suggests feasibility in using proteomic panels to assist in identifying and distinguishing mood disorders, in particular bipolar I disorder. Replication studies for confirmation, consideration of state vs trait serial assays to delineate proteomic expression of bipolar depression vs previous mania, and utility studies to assess proteomic expression profiling as an advanced decision making tool or companion diagnostic are encouraged. PMID:26645624

Affinity Proteomics in the mountains: Alpbach 2015.

PubMed

Taussig, Michael J

2016-09-25

The 2015 Alpbach Workshop on Affinity Proteomics, organised by the EU AFFINOMICS consortium, was the 7th workshop in this series. As in previous years, the focus of the event was the current state of affinity methods for proteome analysis, including complementarity with mass spectrometry, progress in recombinant binder production methods, alternatives to classical antibodies as affinity reagents, analysis of proteome targets, industry focus on biomarkers, and diagnostic and clinical applications. The combination of excellent science with Austrian mountain scenery and winter sports engender an atmosphere that makes this series of workshops exceptional. The articles in this Special Issue represent a cross-section of the presentations at the 2015 meeting. Copyright © 2016 Elsevier B.V. All rights reserved.

Draft de novo transcriptome assembly and proteome characterization of the electric lobe of Tetronarce californica: a molecular tool for the study of cholinergic neurotransmission in the electric organ.

PubMed

Stavrianakou, Maria; Perez, Ricardo; Wu, Cheng; Sachs, Matthew S; Aramayo, Rodolfo; Harlow, Mark

2017-08-14

The electric organ of Tetronarce californica (an electric ray formerly known as Torpedo californica) is a classic preparation for biochemical studies of cholinergic neurotransmission. To broaden the usefulness of this preparation, we have performed a transcriptome assembly of the presynaptic component of the electric organ (the electric lobe). We combined our assembled transcriptome with a previous transcriptome of the postsynaptic electric organ, to define a MetaProteome containing pre- and post-synaptic components of the electric organ. Sequencing yielded 102 million paired-end 100 bp reads. De novo Trinity assembly was performed at Kmer 25 (default) and Kmers 27, 29, and 31. Trinity, generated around 103,000 transcripts, and 78,000 genes per assembly. Assemblies were evaluated based on the number of bases/transcripts assembled, RSEM-EVAL scores and informational content and completeness. We found that different assemblies scored differently according to the evaluation criteria used, and that while each individual assembly contained unique information, much of the assembly information was shared by all assemblies. To generate the presynaptic transcriptome (electric lobe), while capturing all information, assemblies were first clustered and then combined with postsynaptic transcripts (electric organ) downloaded from NCBI. The completness of the resulting clustered predicted MetaProteome was rigorously evaluated by comparing its information against the predicted proteomes from Homo sapiens, Callorhinchus milli, and the Transporter Classification Database (TCDB). In summary, we obtained a MetaProteome containing 92%, 88.5%, and 66% of the expected set of ultra-conserved sequences (i.e., BUSCOs), expected to be found for Eukaryotes, Metazoa, and Vertebrata, respectively. We cross-annotated the conserved set of proteins shared between the T. californica MetaProteome and the proteomes of H. sapiens and C. milli, using the H. sapiens genome as a reference. This information was used to predict the position in human pathways of the conserved members of the T. californica MetaProteome. We found proteins not detected before in T. californica, corresponding to processes involved in synaptic vesicle biology. Finally, we identified 42 transporter proteins in TCDB that were detected by the T. californica MetaProteome (electric fish) and not selected by a control proteome consisting of the combined proteomes of 12 widely diverse non-electric fishes by Reverse-Blast-Hit Blast. Combined, the information provided here is not only a unique tool for the study of cholinergic neurotransmission, but it is also a starting point for understanding the evolution of early vertebrates.

Proteomic Profiling of Cranial (Superior) Cervical Ganglia Reveals Beta-Amyloid and Ubiquitin Proteasome System Perturbations in an Equine Multiple System Neuropathy.

PubMed

McGorum, Bruce C; Pirie, R Scott; Eaton, Samantha L; Keen, John A; Cumyn, Elizabeth M; Arnott, Danielle M; Chen, Wenzhang; Lamont, Douglas J; Graham, Laura C; Llavero Hurtado, Maica; Pemberton, Alan; Wishart, Thomas M

2015-11-01

Equine grass sickness (EGS) is an acute, predominantly fatal, multiple system neuropathy of grazing horses with reported incidence rates of ∼2%. An apparently identical disease occurs in multiple species, including but not limited to cats, dogs, and rabbits. Although the precise etiology remains unclear, ultrastructural findings have suggested that the primary lesion lies in the glycoprotein biosynthetic pathway of specific neuronal populations. The goal of this study was therefore to identify the molecular processes underpinning neurodegeneration in EGS. Here, we use a bottom-up approach beginning with the application of modern proteomic tools to the analysis of cranial (superior) cervical ganglion (CCG, a consistently affected tissue) from EGS-affected patients and appropriate control cases postmortem. In what appears to be the proteomic application of modern proteomic tools to equine neuronal tissues and/or to an inherent neurodegenerative disease of large animals (not a model of human disease), we identified 2,311 proteins in CCG extracts, with 320 proteins increased and 186 decreased by greater than 20% relative to controls. Further examination of selected proteomic candidates by quantitative fluorescent Western blotting (QFWB) and subcellular expression profiling by immunohistochemistry highlighted a previously unreported dysregulation in proteins commonly associated with protein misfolding/aggregation responses seen in a myriad of human neurodegenerative conditions, including but not limited to amyloid precursor protein (APP), microtubule associated protein (Tau), and multiple components of the ubiquitin proteasome system (UPS). Differentially expressed proteins eligible for in silico pathway analysis clustered predominantly into the following biofunctions: (1) diseases and disorders, including; neurological disease and skeletal and muscular disorders and (2) molecular and cellular functions, including cellular assembly and organization, cell-to-cell signaling and interaction (including epinephrine, dopamine, and adrenergic signaling and receptor function), and small molecule biochemistry. Interestingly, while the biofunctions identified in this study may represent pathways underpinning EGS-induced neurodegeneration, this is also the first demonstration of potential molecular conservation (including previously unreported dysregulation of the UPS and APP) spanning the degenerative cascades from an apparently unrelated condition of large animals, to small animal models with altered neuronal vulnerability, and human neurological conditions. Importantly, this study highlights the feasibility and benefits of applying modern proteomic techniques to veterinary investigations of neurodegenerative processes in diseases of large animals. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Cantharidin, a protein phosphatase inhibitor, strongly upregulates detoxification enzymes in the Arabidopsis proteome

USDA-ARS?s Scientific Manuscript database

Cantharidin is a potent natural herbicide. This work was conducted to probe its mode of action. We previously published its effect on transcription of plant genes (mRNA production) with transcriptomic methods. This paper follows up and looks at cantharidin effects translation of mRNA using proteom...

iTRAQ-based quantitative proteomic analysis reveals proteomic changes in three fenoxaprop-P-ethyl-resistant Beckmannia syzigachne biotypes with differing ACCase mutations.

PubMed

Pan, Lang; Zhang, Jian; Wang, Junzhi; Yu, Qin; Bai, Lianyang; Dong, Liyao

2017-05-08

American sloughgrass (Beckmannia syzigachne Steud.) is a weed widely distributed in wheat fields of China. In recent years, the evolution of herbicide (fenoxaprop-P-ethyl)-resistant populations has decreased the susceptibility of B. syzigachne. This study compared 4 B. syzigachne populations (3 resistant and 1 susceptible) using iTRAQ to characterize fenoxaprop-P-ethyl resistance in B. syzigachne at the proteomic level. Through searching the UniProt database, 3104 protein species were identified from 13,335 unique peptides. Approximately 2834 protein species were assigned to 23 functional classifications provided by the COG database. Among these, 2299 protein species were assigned to 125 predicted pathways. The resistant biotype contained 8 protein species that changed in abundance relative to the susceptible biotype; they were involved in photosynthesis, oxidative phosphorylation, and fatty acid biosynthesis pathways. In contrast to previous studies comparing only 1 resistant and 1 susceptible population, our use of 3 fenoxaprop-resistant B. syzigachne populations with different genetic backgrounds minimized irrelevant differential expression and eliminated false positives. Therefore, we could more confidently link the differentially expressed proteins to herbicide resistance. Proteomic analysis demonstrated that fenoxaprop-P-ethyl resistance is associated with photosynthetic capacity, a connection that might be related to the target-site mutations in resistant B. syzigachne. This is the first large-scale proteomics study examining herbicide stress responses in different B. syzigachne biotypes. This study has biological relevance because it is the first to employ proteomic analysis for understanding the mechanisms underlying Beckmannia syzigachne herbicide resistance. The plant is a major weed in China and negatively affects crop yield, but has developed considerable resistance to the most common herbicide, fenoxaprop-P-ethyl. Through comparisons of resistant and sensitive biotypes, our study identified multiple proteins (involved in photosynthesis, oxidative phosphorylation, and fatty acid biosynthesis) that are putatively linked to B. syzigachne herbicide response. This large-scale proteomics study, sorely lacking in weed science, contributes valuable data that can be applied to more fine-tuned analyses on the functions of specific proteins in herbicide resistance. Copyright © 2017 Elsevier B.V. All rights reserved.

Inconsistencies in the red blood cell membrane proteome analysis: generation of a database for research and diagnostic applications

PubMed Central

Hegedűs, Tamás; Chaubey, Pururawa Mayank; Várady, György; Szabó, Edit; Sarankó, Hajnalka; Hofstetter, Lia; Roschitzki, Bernd; Sarkadi, Balázs

2015-01-01

Based on recent results, the determination of the easily accessible red blood cell (RBC) membrane proteins may provide new diagnostic possibilities for assessing mutations, polymorphisms or regulatory alterations in diseases. However, the analysis of the current mass spectrometry-based proteomics datasets and other major databases indicates inconsistencies—the results show large scattering and only a limited overlap for the identified RBC membrane proteins. Here, we applied membrane-specific proteomics studies in human RBC, compared these results with the data in the literature, and generated a comprehensive and expandable database using all available data sources. The integrated web database now refers to proteomic, genetic and medical databases as well, and contains an unexpected large number of validated membrane proteins previously thought to be specific for other tissues and/or related to major human diseases. Since the determination of protein expression in RBC provides a method to indicate pathological alterations, our database should facilitate the development of RBC membrane biomarker platforms and provide a unique resource to aid related further research and diagnostics. Database URL: http://rbcc.hegelab.org PMID:26078478

Coupling Capillary Zone Electrophoresis to a Q Exactive HF Mass Spectrometer for Top-down Proteomics: 580 Proteoform Identifications from Yeast.

PubMed

Zhao, Yimeng; Sun, Liangliang; Zhu, Guijie; Dovichi, Norman J

2016-10-07

We used reversed-phase liquid chromatography to separate the yeast proteome into 23 fractions. These fractions were then analyzed using capillary zone electrophoresis (CZE) coupled to a Q-Exactive HF mass spectrometer using an electrokinetically pumped sheath flow interface. The parameters of the mass spectrometer were first optimized for top-down proteomics using a mixture of seven model proteins; we observed that intact protein mode with a trapping pressure of 0.2 and normalized collision energy of 20% produced the highest intact protein signals and most protein identifications. Then, we applied the optimized parameters for analysis of the fractionated yeast proteome. From this, 580 proteoforms and 180 protein groups were identified via database searching of the MS/MS spectra. This number of proteoform identifications is two times larger than that of previous CZE-MS/MS studies. An additional 3,243 protein species were detected based on the parent ion spectra. Post-translational modifications including N-terminal acetylation, signal peptide removal, and oxidation were identified.

Integrating cell biology and proteomic approaches in plants.

PubMed

Takáč, Tomáš; Šamajová, Olga; Šamaj, Jozef

2017-10-03

Significant improvements of protein extraction, separation, mass spectrometry and bioinformatics nurtured advancements of proteomics during the past years. The usefulness of proteomics in the investigation of biological problems can be enhanced by integration with other experimental methods from cell biology, genetics, biochemistry, pharmacology, molecular biology and other omics approaches including transcriptomics and metabolomics. This review aims to summarize current trends integrating cell biology and proteomics in plant science. Cell biology approaches are most frequently used in proteomic studies investigating subcellular and developmental proteomes, however, they were also employed in proteomic studies exploring abiotic and biotic stress responses, vesicular transport, cytoskeleton and protein posttranslational modifications. They are used either for detailed cellular or ultrastructural characterization of the object subjected to proteomic study, validation of proteomic results or to expand proteomic data. In this respect, a broad spectrum of methods is employed to support proteomic studies including ultrastructural electron microscopy studies, histochemical staining, immunochemical localization, in vivo imaging of fluorescently tagged proteins and visualization of protein-protein interactions. Thus, cell biological observations on fixed or living cell compartments, cells, tissues and organs are feasible, and in some cases fundamental for the validation and complementation of proteomic data. Validation of proteomic data by independent experimental methods requires development of new complementary approaches. Benefits of cell biology methods and techniques are not sufficiently highlighted in current proteomic studies. This encouraged us to review most popular cell biology methods used in proteomic studies and to evaluate their relevance and potential for proteomic data validation and enrichment of purely proteomic analyses. We also provide examples of representative studies combining proteomic and cell biology methods for various purposes. Integrating cell biology approaches with proteomic ones allow validation and better interpretation of proteomic data. Moreover, cell biology methods remarkably extend the knowledge provided by proteomic studies and might be fundamental for the functional complementation of proteomic data. This review article summarizes current literature linking proteomics with cell biology. Copyright © 2017 Elsevier B.V. All rights reserved.

Label-free protein profiling of formalin-fixed paraffin-embedded (FFPE) heart tissue reveals immediate mitochondrial impairment after ionising radiation.

PubMed

Azimzadeh, Omid; Scherthan, Harry; Yentrapalli, Ramesh; Barjaktarovic, Zarko; Ueffing, Marius; Conrad, Marcus; Neff, Frauke; Calzada-Wack, Julia; Aubele, Michaela; Buske, Christian; Atkinson, Michael J; Hauck, Stefanie M; Tapio, Soile

2012-04-18

Qualitative proteome profiling of formalin-fixed, paraffin-embedded (FFPE) tissue is advancing the field of clinical proteomics. However, quantitative proteome analysis of FFPE tissue is hampered by the lack of an efficient labelling method. The usage of conventional protein labelling on FFPE tissue has turned out to be inefficient. Classical labelling targets lysine residues that are blocked by the formalin treatment. The aim of this study was to establish a quantitative proteomics analysis of FFPE tissue by combining the label-free approach with optimised protein extraction and separation conditions. As a model system we used FFPE heart tissue of control and exposed C57BL/6 mice after total body irradiation using a gamma ray dose of 3 gray. We identified 32 deregulated proteins (p≤0.05) in irradiated hearts 24h after the exposure. The proteomics data were further evaluated and validated by bioinformatics and immunoblotting investigation. In good agreement with our previous results using fresh-frozen tissue, the analysis indicated radiation-induced alterations in three main biological pathways: respiratory chain, lipid metabolism and pyruvate metabolism. The label-free approach enables the quantitative measurement of radiation-induced alterations in FFPE tissue and facilitates retrospective biomarker identification using clinical archives. Copyright © 2012 Elsevier B.V. All rights reserved.

Data-Driven Approach To Determine Popular Proteins for Targeted Proteomics Translation of Six Organ Systems.

PubMed

Lam, Maggie P Y; Venkatraman, Vidya; Xing, Yi; Lau, Edward; Cao, Quan; Ng, Dominic C M; Su, Andrew I; Ge, Junbo; Van Eyk, Jennifer E; Ping, Peipei

2016-11-04

Amidst the proteomes of human tissues lie subsets of proteins that are closely involved in conserved pathophysiological processes. Much of biomedical research concerns interrogating disease signature proteins and defining their roles in disease mechanisms. With advances in proteomics technologies, it is now feasible to develop targeted proteomics assays that can accurately quantify protein abundance as well as their post-translational modifications; however, with rapidly accumulating number of studies implicating proteins in diseases, current resources are insufficient to target every protein without judiciously prioritizing the proteins with high significance and impact for assay development. We describe here a data science method to prioritize and expedite assay development on high-impact proteins across research fields by leveraging the biomedical literature record to rank and normalize proteins that are popularly and preferentially published by biomedical researchers. We demonstrate this method by finding priority proteins across six major physiological systems (cardiovascular, cerebral, hepatic, renal, pulmonary, and intestinal). The described method is data-driven and builds upon the collective knowledge of previous publications referenced on PubMed to lend objectivity to target selection. The method and resulting popular protein lists may also be useful for exploring biological processes associated with various physiological systems and research topics, in addition to benefiting ongoing efforts to facilitate the broad translation of proteomics technologies.

Proteomics study of extracellular fibrinolytic proteases from Bacillus licheniformis RO3 and Bacillus pumilus 2.g isolated from Indonesian fermented food

NASA Astrophysics Data System (ADS)

Nur Afifah, Diana; Rustanti, Ninik; Anjani, Gemala; Syah, Dahrul; Yanti; Suhartono, Maggy T.

2017-02-01

This paper presents the proteomics study which includes separation, identification and characterization of proteins. The experiment on Indonesian fermented food such as extracellular fibrinolytic protease from Bacillus licheniformis RO3 and Bacillus pumilus 2.g isolated from red oncom and tempeh gembus was conducted. The experimental works comprise the following steps: (1) a combination of one- and two-dimensional electrophoresis analysis, (2) mass spectrometry analysis using MALDI-TOF-MS and (3) investigation using protein database. The result suggested that there were new two protein fractions of B. licheniformis RO3 and three protein fractions of B. pumilus 2.g. These result has not been previously reported.

Proteomic Approaches and Identification of Novel Therapeutic Targets for Alcoholism

PubMed Central

Gorini, Giorgio; Adron Harris, R; Dayne Mayfield, R

2014-01-01

Recent studies have shown that gene regulation is far more complex than previously believed and does not completely explain changes at the protein level. Therefore, the direct study of the proteome, considerably different in both complexity and dynamicity to the genome/transcriptome, has provided unique insights to an increasing number of researchers. During the past decade, extraordinary advances in proteomic techniques have changed the way we can analyze the composition, regulation, and function of protein complexes and pathways underlying altered neurobiological conditions. When combined with complementary approaches, these advances provide the contextual information for decoding large data sets into meaningful biologically adaptive processes. Neuroproteomics offers potential breakthroughs in the field of alcohol research by leading to a deeper understanding of how alcohol globally affects protein structure, function, interactions, and networks. The wealth of information gained from these advances can help pinpoint relevant biomarkers for early diagnosis and improved prognosis of alcoholism and identify future pharmacological targets for the treatment of this addiction. PMID:23900301

Plant subcellular proteomics: Application for exploring optimal cell function in soybean.

PubMed

Wang, Xin; Komatsu, Setsuko

2016-06-30

Plants have evolved complicated responses to developmental changes and stressful environmental conditions. Subcellular proteomics has the potential to elucidate localized cellular responses and investigate communications among subcellular compartments during plant development and in response to biotic and abiotic stresses. Soybean, which is a valuable legume crop rich in protein and vegetable oil, can grow in several climatic zones; however, the growth and yield of soybean are markedly decreased under stresses. To date, numerous proteomic studies have been performed in soybean to examine the specific protein profiles of cell wall, plasma membrane, nucleus, mitochondrion, chloroplast, and endoplasmic reticulum. In this review, methods for the purification and purity assessment of subcellular organelles from soybean are summarized. In addition, the findings from subcellular proteomic analyses of soybean during development and under stresses, particularly flooding stress, are presented and the proteins regulated among subcellular compartments are discussed. Continued advances in subcellular proteomics are expected to greatly contribute to the understanding of the responses and interactions that occur within and among subcellular compartments during development and under stressful environmental conditions. Subcellular proteomics has the potential to investigate the cellular events and interactions among subcellular compartments in response to development and stresses in plants. Soybean could grow in several climatic zones; however, the growth and yield of soybean are markedly decreased under stresses. Numerous proteomics of cell wall, plasma membrane, nucleus, mitochondrion, chloroplast, and endoplasmic reticulum was carried out to investigate the respecting proteins and their functions in soybean during development or under stresses. In this review, methods of subcellular-organelle enrichment and purity assessment are summarized. In addition, previous findings of subcellular proteomics are presented, and functional proteins regulated among different subcellular are discussed. Subcellular proteomics contributes greatly to uncovering responses and interactions among subcellular compartments during development and under stressful environmental conditions in soybean. Copyright © 2016 Elsevier B.V. All rights reserved.

Proteomic analysis of single mammalian cells enabled by microfluidic nanodroplet sample preparation and ultrasensitive nanoLC-MS.

PubMed

Zhu, Ying; Clair, Geremy; Chrisler, William; Shen, Yufeng; Zhao, Rui; Shukla, Anil; Moore, Ronald; Misra, Ravi; Pryhuber, Gloria; Smith, Richard; Ansong, Charles; Kelly, Ryan T

2018-05-24

We report on the quantitative proteomic analysis of single mammalian cells. Fluorescence-activated cell sorting was employed to deposit cells into a newly developed nanodroplet sample processing chip, after which samples were analysed by ultrasensitive nanoLC-MS. An average of ~670 protein groups were confidently identified from single HeLa cells, which is a far greater level of proteome coverage for single cells than has been previously reported. We demonstrate that the single cell proteomics platform can be used to differentiate cell types from enzyme-dissociated human lung primary cells and identify specific protein markers for epithelial and mesenchymal cells. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Beyond plant defense: insights on the potential of salicylic and methylsalicylic acid to contain growth of the phytopathogen Botrytis cinerea

PubMed Central

Dieryckx, Cindy; Gaudin, Vanessa; Dupuy, Jean-William; Bonneu, Marc; Girard, Vincent; Job, Dominique

2015-01-01

Using Botrytis cinerea we confirmed in the present work several previous studies showing that salicylic acid, a main plant hormone, inhibits fungal growth in vitro. Such an inhibitory effect was also observed for the two salicylic acid derivatives, methylsalicylic and acetylsalicylic acid. In marked contrast, 5-sulfosalicylic acid was totally inactive. Comparative proteomics from treated vs. control mycelia showed that both the intracellular and extracellular proteomes were affected in the presence of salicylic acid or methylsalicylic acid. These data suggest several mechanisms that could potentially account for the observed fungal growth inhibition, notably pH regulation, metal homeostasis, mitochondrial respiration, ROS accumulation and cell wall remodeling. The present observations support a role played by the phytohormone SA and derivatives in directly containing the pathogen. Data are available via ProteomeXchange with identifier PXD002873. PMID:26528317

Proteomic analysis of the response to cell cycle arrests in human myeloid leukemia cells.

PubMed

Ly, Tony; Endo, Aki; Lamond, Angus I

2015-01-02

Previously, we analyzed protein abundance changes across a 'minimally perturbed' cell cycle by using centrifugal elutriation to differentially enrich distinct cell cycle phases in human NB4 cells (Ly et al., 2014). In this study, we compare data from elutriated cells with NB4 cells arrested at comparable phases using serum starvation, hydroxyurea, or RO-3306. While elutriated and arrested cells have similar patterns of DNA content and cyclin expression, a large fraction of the proteome changes detected in arrested cells are found to reflect arrest-specific responses (i.e., starvation, DNA damage, CDK1 inhibition), rather than physiological cell cycle regulation. For example, we show most cells arrested in G2 by CDK1 inhibition express abnormally high levels of replication and origin licensing factors and are likely poised for genome re-replication. The protein data are available in the Encyclopedia of Proteome Dynamics (

Systems biology definition of the core proteome of metabolism and expression is consistent with high-throughput data.

PubMed

Yang, Laurence; Tan, Justin; O'Brien, Edward J; Monk, Jonathan M; Kim, Donghyuk; Li, Howard J; Charusanti, Pep; Ebrahim, Ali; Lloyd, Colton J; Yurkovich, James T; Du, Bin; Dräger, Andreas; Thomas, Alex; Sun, Yuekai; Saunders, Michael A; Palsson, Bernhard O

2015-08-25

Finding the minimal set of gene functions needed to sustain life is of both fundamental and practical importance. Minimal gene lists have been proposed by using comparative genomics-based core proteome definitions. A definition of a core proteome that is supported by empirical data, is understood at the systems-level, and provides a basis for computing essential cell functions is lacking. Here, we use a systems biology-based genome-scale model of metabolism and expression to define a functional core proteome consisting of 356 gene products, accounting for 44% of the Escherichia coli proteome by mass based on proteomics data. This systems biology core proteome includes 212 genes not found in previous comparative genomics-based core proteome definitions, accounts for 65% of known essential genes in E. coli, and has 78% gene function overlap with minimal genomes (Buchnera aphidicola and Mycoplasma genitalium). Based on transcriptomics data across environmental and genetic backgrounds, the systems biology core proteome is significantly enriched in nondifferentially expressed genes and depleted in differentially expressed genes. Compared with the noncore, core gene expression levels are also similar across genetic backgrounds (two times higher Spearman rank correlation) and exhibit significantly more complex transcriptional and posttranscriptional regulatory features (40% more transcription start sites per gene, 22% longer 5'UTR). Thus, genome-scale systems biology approaches rigorously identify a functional core proteome needed to support growth. This framework, validated by using high-throughput datasets, facilitates a mechanistic understanding of systems-level core proteome function through in silico models; it de facto defines a paleome.

Developmental distribution of the plasma membrane-enriched proteome in the maize primary root growth zone.

PubMed

Zhang, Zhe; Voothuluru, Priyamvada; Yamaguchi, Mineo; Sharp, Robert E; Peck, Scott C

2013-01-01

Within the growth zone of the maize primary root, there are well-defined patterns of spatial and temporal organization of cell division and elongation. However, the processes underlying this organization remain poorly understood. To gain additional insights into the differences amongst the defined regions, we performed a proteomic analysis focusing on fractions enriched for plasma membrane (PM) proteins. The PM is the interface between the plant cell and the apoplast and/or extracellular space. As such, it is a key structure involved in the exchange of nutrients and other molecules as well as in the integration of signals that regulate growth and development. Despite the important functions of PM-localized proteins in mediating these processes, a full understanding of dynamic changes in PM proteomes is often impeded by low relative concentrations relative to total proteins. Using a relatively simple strategy of treating microsomal fractions with Brij-58 detergent to enrich for PM proteins, we compared the developmental distribution of proteins within the root growth zone which revealed a number of previously known as well as novel proteins with interesting patterns of abundance. For instance, the quantitative proteomic analysis detected a gradient of PM aquaporin proteins similar to that previously reported using immunoblot analyses, confirming the veracity of this strategy. Cellulose synthases increased in abundance with increasing distance from the root apex, consistent with expected locations of cell wall deposition. The similar distribution pattern for Brittle-stalk-2-like protein implicates that this protein may also have cell wall related functions. These results show that the simplified PM enrichment method previously demonstrated in Arabidopsis can be successfully applied to completely unrelated plant tissues and provide insights into differences in the PM proteome throughout growth and development zones of the maize primary root.

Proteomic analysis of human aqueous humor using multidimensional protein identification technology

PubMed Central

Richardson, Matthew R.; Price, Marianne O.; Price, Francis W.; Pardo, Jennifer C.; Grandin, Juan C.; You, Jinsam; Wang, Mu

2009-01-01

Aqueous humor (AH) supports avascular tissues in the anterior segment of the eye, maintains intraocular pressure, and potentially influences the pathogenesis of ocular diseases. Nevertheless, the AH proteome is still poorly defined despite several previous efforts, which were hindered by interfering high abundance proteins, inadequate animal models, and limited proteomic technologies. To facilitate future investigations into AH function, the AH proteome was extensively characterized using an advanced proteomic approach. Samples from patients undergoing cataract surgery were pooled and depleted of interfering abundant proteins and thereby divided into two fractions: albumin-bound and albumin-depleted. Multidimensional Protein Identification Technology (MudPIT) was utilized for each fraction; this incorporates strong cation exchange chromatography to reduce sample complexity before reversed-phase liquid chromatography and tandem mass spectrometric analysis. Twelve proteins had multi-peptide, high confidence identifications in the albumin-bound fraction and 50 proteins had multi-peptide, high confidence identifications in the albumin-depleted fraction. Gene ontological analyses were performed to determine which cellular components and functions were enriched. Many proteins were previously identified in the AH and for several their potential role in the AH has been investigated; however, the majority of identified proteins were novel and only speculative roles can be suggested. The AH was abundant in anti-oxidant and immunoregulatory proteins as well as anti-angiogenic proteins, which may be involved in maintaining the avascular tissues. This is the first known report to extensively characterize and describe the human AH proteome and lays the foundation for future work regarding its function in homeostatic and pathologic states. PMID:20019884

Proteomics Analysis of the Causative Agent of Typhoid Fever

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ansong, Charles; Yoon, Hyunjin; Norbeck, Angela D.

2008-02-01

Typhoid fever is a potentially fatal disease caused by the bacterial pathogen Salmonella enterica serovar Typhi (S. typhi). S. typhi infection is a complex process that involves numerous bacterially-encoded virulence determinants, and these are thought to confer both stringent human host specificity and a high mortality rate. In the present study we used a liquid chromatography-mass spectrometry (LC-MS) based proteomics strategy to investigate the proteome of logarithmic, stationary phase, and low pH/low magnesium (MgM) S. typhi cultures. This represents the first large scale comprehensive characterization of the S. typhi proteome. Our analysis identified a total of 2066 S. typhi proteins.more » In an effort to identify putative S. typhi-specific virulence factors, we then compared our S. typhi results to those obtained in a previously published study of the S. typhimurium proteome under similar conditions (Adkins J.N. et al (2006) Mol Cell Prot). Comparative proteomic analysis of S. typhi (strain Ty2) and S. typhimurium (strains LT2 and 14028) revealed a subset of highly expressed proteins unique to S. typhi that were exclusively detected under conditions that mimic the infective state in macrophage cells. These proteins included CdtB, HlyE, and a conserved protein encoded by t1476. The differential expression of selected proteins was confirmed by Western blot analysis. Taken together with the current literature, our observations suggest that this subset of proteins may play a role in S. typhi pathogenesis and human host specificity. In addition, we observed products of the biotin (bio) operon displayed a higher abundance in the more virulent strains S. typhi-Ty2 and S. typhimurium-14028 compared to the virulence attenuated S. typhimurium strain LT2, suggesting bio proteins may contribute to Salmonella pathogenesis.« less

Chronic Cocaine Use Causes Changes in the Striatal Proteome Depending on the Endogenous Expression of Pleiotrophin.

PubMed

Vicente-Rodríguez, Marta; Herradón, Gonzalo; Ferrer-Alcón, Marcel; Uribarri, María; Pérez-García, Carmen

2015-07-20

The neurotrophic factor pleiotrophin (PTN) is upregulated in different brain areas after the administration of different drugs of abuse, including psychostimulants. PTN has been shown to prevent cocaine-induced cytotoxicity in NG108-15 and PC12 cells. We previously demonstrated that specific phosphoproteins related to neurodegeneration processes are differentially regulated in the mouse striatum by a single cocaine (15 mg/kg) administration depending on the endogenous expression of PTN. Since neurodegenerative processes are usually observed in patients exposed to toxicants for longer duration, we have now performed a striatal proteomic study using samples enriched in phosphorylated proteins from PTN knockout (PTN-/-) mice, from mice with transgenic PTN overexpression (PTN-Tg) in the brain, and from wild type (WT) mice after a chronic treatment with cocaine (15 mg/kg/day for 7 days). We have successfully identified 23 proteins significantly affected by chronic cocaine exposure, genotype, or both. Most of these proteins, including peroxiredoxin-6 (PRDX6), triosephosphate isomerase (TPI1), ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), and annexins A5 (ANXA5) and A7 (ANXA7), may be of significant importance because they were previously identified in proteomic studies in animals treated with psychostimulants and/or because they are related to neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease. The data support a protective role of PTN against chronic cocaine-induced neural alterations.

Defining the human deubiquitinating enzyme interaction landscape.

PubMed

Sowa, Mathew E; Bennett, Eric J; Gygi, Steven P; Harper, J Wade

2009-07-23

Deubiquitinating enzymes (Dubs) function to remove covalently attached ubiquitin from proteins, thereby controlling substrate activity and/or abundance. For most Dubs, their functions, targets, and regulation are poorly understood. To systematically investigate Dub function, we initiated a global proteomic analysis of Dubs and their associated protein complexes. This was accomplished through the development of a software platform called CompPASS, which uses unbiased metrics to assign confidence measurements to interactions from parallel nonreciprocal proteomic data sets. We identified 774 candidate interacting proteins associated with 75 Dubs. Using Gene Ontology, interactome topology classification, subcellular localization, and functional studies, we link Dubs to diverse processes, including protein turnover, transcription, RNA processing, DNA damage, and endoplasmic reticulum-associated degradation. This work provides the first glimpse into the Dub interaction landscape, places previously unstudied Dubs within putative biological pathways, and identifies previously unknown interactions and protein complexes involved in this increasingly important arm of the ubiquitin-proteasome pathway.

Defining the Human Deubiquitinating Enzyme Interaction Landscape

PubMed Central

Sowa, Mathew E.; Bennett, Eric J.; Gygi, Steven P.; Harper, J. Wade

2009-01-01

Summary Deubiquitinating enzymes (Dubs) function to remove covalently attached ubiquitin from proteins, thereby controlling substrate activity and/or abundance. For most Dubs, their functions, targets, and regulation are poorly understood. To systematically investigate Dub function, we initiated a global proteomic analysis of Dubs and their associated protein complexes. This was accomplished through the development of a software platform, called CompPASS, which uses unbiased metrics to assign confidence measurements to interactions from parallel non-reciprocal proteomic datasets. We identified 774 candidate interacting proteins associated with 75 Dubs. Using Gene Ontology, interactome topology classification, sub-cellular localization and functional studies, we link Dubs to diverse processes, including protein turnover, transcription, RNA processing, DNA damage, and endoplasmic reticulum-associated degradation. This work provides the first glimpse into the Dub interaction landscape, places previously unstudied Dubs within putative biological pathways, and identifies previously unknown interactions and protein complexes involved in this increasingly important arm of the ubiquitin-proteasome pathway. PMID:19615732

Improved prediction of peptide detectability for targeted proteomics using a rank-based algorithm and organism-specific data.

PubMed

Qeli, Ermir; Omasits, Ulrich; Goetze, Sandra; Stekhoven, Daniel J; Frey, Juerg E; Basler, Konrad; Wollscheid, Bernd; Brunner, Erich; Ahrens, Christian H

2014-08-28

The in silico prediction of the best-observable "proteotypic" peptides in mass spectrometry-based workflows is a challenging problem. Being able to accurately predict such peptides would enable the informed selection of proteotypic peptides for targeted quantification of previously observed and non-observed proteins for any organism, with a significant impact for clinical proteomics and systems biology studies. Current prediction algorithms rely on physicochemical parameters in combination with positive and negative training sets to identify those peptide properties that most profoundly affect their general detectability. Here we present PeptideRank, an approach that uses learning to rank algorithm for peptide detectability prediction from shotgun proteomics data, and that eliminates the need to select a negative dataset for the training step. A large number of different peptide properties are used to train ranking models in order to predict a ranking of the best-observable peptides within a protein. Empirical evaluation with rank accuracy metrics showed that PeptideRank complements existing prediction algorithms. Our results indicate that the best performance is achieved when it is trained on organism-specific shotgun proteomics data, and that PeptideRank is most accurate for short to medium-sized and abundant proteins, without any loss in prediction accuracy for the important class of membrane proteins. Targeted proteomics approaches have been gaining a lot of momentum and hold immense potential for systems biology studies and clinical proteomics. However, since only very few complete proteomes have been reported to date, for a considerable fraction of a proteome there is no experimental proteomics evidence that would allow to guide the selection of the best-suited proteotypic peptides (PTPs), i.e. peptides that are specific to a given proteoform and that are repeatedly observed in a mass spectrometer. We describe a novel, rank-based approach for the prediction of the best-suited PTPs for targeted proteomics applications. By building on methods developed in the field of information retrieval (e.g. web search engines like Google's PageRank), we circumvent the delicate step of selecting positive and negative training sets and at the same time also more closely reflect the experimentalist´s need for selecting e.g. the 5 most promising peptides for targeting a protein of interest. This approach allows to predict PTPs for not yet observed proteins or for organisms without prior experimental proteomics data such as many non-model organisms. Copyright © 2014 Elsevier B.V. All rights reserved.

Application of Large-Scale Aptamer-Based Proteomic Profiling to Planned Myocardial Infarctions.

PubMed

Jacob, Jaison; Ngo, Debby; Finkel, Nancy; Pitts, Rebecca; Gleim, Scott; Benson, Mark D; Keyes, Michelle J; Farrell, Laurie A; Morgan, Thomas; Jennings, Lori L; Gerszten, Robert E

2018-03-20

Emerging proteomic technologies using novel affinity-based reagents allow for efficient multiplexing with high-sample throughput. To identify early biomarkers of myocardial injury, we recently applied an aptamer-based proteomic profiling platform that measures 1129 proteins to samples from patients undergoing septal alcohol ablation for hypertrophic cardiomyopathy, a human model of planned myocardial injury. Here, we examined the scalability of this approach using a markedly expanded platform to study a far broader range of human proteins in the context of myocardial injury. We applied a highly multiplexed, expanded proteomic technique that uses single-stranded DNA aptamers to assay 4783 human proteins (4137 distinct human gene targets) to derivation and validation cohorts of planned myocardial injury, individuals with spontaneous myocardial infarction, and at-risk controls. We found 376 target proteins that significantly changed in the blood after planned myocardial injury in a derivation cohort (n=20; P <1.05E-05, 1-way repeated measures analysis of variance, Bonferroni threshold). Two hundred forty-seven of these proteins were validated in an independent planned myocardial injury cohort (n=15; P <1.33E-04, 1-way repeated measures analysis of variance); >90% were directionally consistent and reached nominal significance in the validation cohort. Among the validated proteins that were increased within 1 hour after planned myocardial injury, 29 were also elevated in patients with spontaneous myocardial infarction (n=63; P <6.17E-04). Many of the novel markers identified in our study are intracellular proteins not previously identified in the peripheral circulation or have functional roles relevant to myocardial injury. For example, the cardiac LIM protein, cysteine- and glycine-rich protein 3, is thought to mediate cardiac mechanotransduction and stress responses, whereas the mitochondrial ATP synthase F 0 subunit component is a vasoactive peptide on its release from cells. Last, we performed aptamer-affinity enrichment coupled with mass spectrometry to technically verify aptamer specificity for a subset of the new biomarkers. Our results demonstrate the feasibility of large-scale aptamer multiplexing at a level that has not previously been reported and with sample throughput that greatly exceeds other existing proteomic methods. The expanded aptamer-based proteomic platform provides a unique opportunity for biomarker and pathway discovery after myocardial injury. © 2017 American Heart Association, Inc.

Quantitative Dynamics of Proteome, Acetylome, and Succinylome during Stem-Cell Differentiation into Hepatocyte-like Cells.

PubMed

Liu, Zekun; Zhang, Qing-Bin; Bu, Chen; Wang, Dawei; Yu, Kai; Gan, Zhixue; Chang, Jianfeng; Cheng, Zhongyi; Liu, Zexian

2018-06-21

Stem-cell differentiation is a complex biological process controlled by a series of functional protein clusters and signaling transductions, especially metabolism-related pathways. Although previous studies have quantified the proteome and phosphoproteome for stem-cell differentiation, the investigation of acylation-mediated regulation is still absent. In this study, we quantitatively profiled the proteome, acetylome, and succinylome in pluripotent human embryonic stem cells (hESCs) and differentiated hepatocyte-like cells (HLCs). In total, 3843 proteins, 185 acetylation sites in 103 proteins, and 602 succinylation sites in 391 proteins were quantified. The quantitative proteome showed that in differentiated HLCs the TGF-β, JAK-STAT, and RAS signaling pathways were activated, whereas ECM-related processes such as sulfates and leucine degradation were depressed. Interestingly, it was observed that the acetylation and succinylation were more intensive in hESCs, whereas protein processing in endoplasmic reticulum and the carbon metabolic pathways were especially highly succinylated. Because the metabolism patterns in pluripotent hESCs and the differentiated HLCs were different, we proposed that the dynamic acylations, especially succinylation, might regulate the Warburg-like effect and TCA cycle during differentiation. Taken together, we systematically profiled the protein and acylation levels of regulation in pluripotent hESCs and differentiated HLCs, and the results indicated the important roles of acylation in pluripotency maintenance and differentiation.

A genome-wide structure-based survey of nucleotide binding proteins in M. tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhagavat, Raghu; Kim, Heung -Bok; Kim, Chang -Yub

Nucleoside tri-phosphates (NTP) form an important class of small molecule ligands that participate in, and are essential to a large number of biological processes. Here, we seek to identify the NTP binding proteome (NTPome) in M. tuberculosis (M.tb), a deadly pathogen. Identifying the NTPome is useful not only for gaining functional insights of the individual proteins but also for identifying useful drug targets. From an earlier study, we had structural models of M.tb at a proteome scale from which a set of 13,858 small molecule binding pockets were identified. We use a set of NTP binding sub-structural motifs derived frommore » a previous study and scan the M.tb pocketome, and find that 1,768 proteins or 43% of the proteome can theoretically bind NTP ligands. Using an experimental proteomics approach involving dye-ligand affinity chromatography, we confirm NTP binding to 47 different proteins, of which 4 are hypothetical proteins. Our analysis also provides the precise list of binding site residues in each case, and the probable ligand binding pose. In conclusion, as the list includes a number of known and potential drug targets, the identification of NTP binding can directly facilitate structure-based drug design of these targets.« less

Integrative Analysis of Subcellular Quantitative Proteomics Studies Reveals Functional Cytoskeleton Membrane-Lipid Raft Interactions in Cancer.

PubMed

Shah, Anup D; Inder, Kerry L; Shah, Alok K; Cristino, Alexandre S; McKie, Arthur B; Gabra, Hani; Davis, Melissa J; Hill, Michelle M

2016-10-07

Lipid rafts are dynamic membrane microdomains that orchestrate molecular interactions and are implicated in cancer development. To understand the functions of lipid rafts in cancer, we performed an integrated analysis of quantitative lipid raft proteomics data sets modeling progression in breast cancer, melanoma, and renal cell carcinoma. This analysis revealed that cancer development is associated with increased membrane raft-cytoskeleton interactions, with ∼40% of elevated lipid raft proteins being cytoskeletal components. Previous studies suggest a potential functional role for the raft-cytoskeleton in the action of the putative tumor suppressors PTRF/Cavin-1 and Merlin. To extend the observation, we examined lipid raft proteome modulation by an unrelated tumor suppressor opioid binding protein cell-adhesion molecule (OPCML) in ovarian cancer SKOV3 cells. In agreement with the other model systems, quantitative proteomics revealed that 39% of OPCML-depleted lipid raft proteins are cytoskeletal components, with microfilaments and intermediate filaments specifically down-regulated. Furthermore, protein-protein interaction network and simulation analysis showed significantly higher interactions among cancer raft proteins compared with general human raft proteins. Collectively, these results suggest increased cytoskeleton-mediated stabilization of lipid raft domains with greater molecular interactions as a common, functional, and reversible feature of cancer cells.

Statistical Analysis of Variation in the Human Plasma Proteome

DOE PAGES

Corzett, Todd H.; Fodor, Imola K.; Choi, Megan W.; ...

2010-01-01

Quantifying the variation in the human plasma proteome is an essential prerequisite for disease-specific biomarker detection. We report here on the longitudinal and individual variation in human plasma characterized by two-dimensional difference gel electrophoresis (2-D DIGE) using plasma samples from eleven healthy subjects collected three times over a two week period. Fixed-effects modeling was used to remove dye and gel variability. Mixed-effects modeling was then used to quantitate the sources of proteomic variation. The subject-to-subject variation represented the largest variance component, while the time-within-subject variation was comparable to the experimental variation found in a previous technical variability study where onemore » human plasma sample was processed eight times in parallel and each was then analyzed by 2-D DIGE in triplicate. Here, 21 protein spots had larger than 50% CV, suggesting that these proteins may not be appropriate as biomarkers and should be carefully scrutinized in future studies. Seventy-eight protein spots showing differential protein levels between different individuals or individual collections were identified by mass spectrometry and further characterized using hierarchical clustering. The results present a first step toward understanding the complexity of longitudinal and individual variation in the human plasma proteome, and provide a baseline for improved biomarker discovery.« less

Statistical analysis of variation in the human plasma proteome.

PubMed

Corzett, Todd H; Fodor, Imola K; Choi, Megan W; Walsworth, Vicki L; Turteltaub, Kenneth W; McCutchen-Maloney, Sandra L; Chromy, Brett A

2010-01-01

Quantifying the variation in the human plasma proteome is an essential prerequisite for disease-specific biomarker detection. We report here on the longitudinal and individual variation in human plasma characterized by two-dimensional difference gel electrophoresis (2-D DIGE) using plasma samples from eleven healthy subjects collected three times over a two week period. Fixed-effects modeling was used to remove dye and gel variability. Mixed-effects modeling was then used to quantitate the sources of proteomic variation. The subject-to-subject variation represented the largest variance component, while the time-within-subject variation was comparable to the experimental variation found in a previous technical variability study where one human plasma sample was processed eight times in parallel and each was then analyzed by 2-D DIGE in triplicate. Here, 21 protein spots had larger than 50% CV, suggesting that these proteins may not be appropriate as biomarkers and should be carefully scrutinized in future studies. Seventy-eight protein spots showing differential protein levels between different individuals or individual collections were identified by mass spectrometry and further characterized using hierarchical clustering. The results present a first step toward understanding the complexity of longitudinal and individual variation in the human plasma proteome, and provide a baseline for improved biomarker discovery.

A genome-wide structure-based survey of nucleotide binding proteins in M. tuberculosis

DOE PAGES

Bhagavat, Raghu; Kim, Heung -Bok; Kim, Chang -Yub; ...

2017-10-02

Nucleoside tri-phosphates (NTP) form an important class of small molecule ligands that participate in, and are essential to a large number of biological processes. Here, we seek to identify the NTP binding proteome (NTPome) in M. tuberculosis (M.tb), a deadly pathogen. Identifying the NTPome is useful not only for gaining functional insights of the individual proteins but also for identifying useful drug targets. From an earlier study, we had structural models of M.tb at a proteome scale from which a set of 13,858 small molecule binding pockets were identified. We use a set of NTP binding sub-structural motifs derived frommore » a previous study and scan the M.tb pocketome, and find that 1,768 proteins or 43% of the proteome can theoretically bind NTP ligands. Using an experimental proteomics approach involving dye-ligand affinity chromatography, we confirm NTP binding to 47 different proteins, of which 4 are hypothetical proteins. Our analysis also provides the precise list of binding site residues in each case, and the probable ligand binding pose. In conclusion, as the list includes a number of known and potential drug targets, the identification of NTP binding can directly facilitate structure-based drug design of these targets.« less

iAB-RBC-283: A proteomically derived knowledge-base of erythrocyte metabolism that can be used to simulate its physiological and patho-physiological states.

PubMed

Bordbar, Aarash; Jamshidi, Neema; Palsson, Bernhard O

2011-07-12

The development of high-throughput technologies capable of whole cell measurements of genes, proteins, and metabolites has led to the emergence of systems biology. Integrated analysis of the resulting omic data sets has proved to be hard to achieve. Metabolic network reconstructions enable complex relationships amongst molecular components to be represented formally in a biologically relevant manner while respecting physical constraints. In silico models derived from such reconstructions can then be queried or interrogated through mathematical simulations. Proteomic profiling studies of the mature human erythrocyte have shown more proteins present related to metabolic function than previously thought; however the significance and the causal consequences of these findings have not been explored. Erythrocyte proteomic data was used to reconstruct the most expansive description of erythrocyte metabolism to date, following extensive manual curation, assessment of the literature, and functional testing. The reconstruction contains 281 enzymes representing functions from glycolysis to cofactor and amino acid metabolism. Such a comprehensive view of erythrocyte metabolism implicates the erythrocyte as a potential biomarker for different diseases as well as a 'cell-based' drug-screening tool. The analysis shows that 94 erythrocyte enzymes are implicated in morbid single nucleotide polymorphisms, representing 142 pathologies. In addition, over 230 FDA-approved and experimental pharmaceuticals have enzymatic targets in the erythrocyte. The advancement of proteomic technologies and increased generation of high-throughput proteomic data have created the need for a means to analyze these data in a coherent manner. Network reconstructions provide a systematic means to integrate and analyze proteomic data in a biologically meaning manner. Analysis of the red cell proteome has revealed an unexpected level of complexity in the functional capabilities of human erythrocyte metabolism.

Spectrum-to-Spectrum Searching Using a Proteome-wide Spectral Library*

PubMed Central

Yen, Chia-Yu; Houel, Stephane; Ahn, Natalie G.; Old, William M.

2011-01-01

The unambiguous assignment of tandem mass spectra (MS/MS) to peptide sequences remains a key unsolved problem in proteomics. Spectral library search strategies have emerged as a promising alternative for peptide identification, in which MS/MS spectra are directly compared against a reference library of confidently assigned spectra. Two problems relate to library size. First, reference spectral libraries are limited to rediscovery of previously identified peptides and are not applicable to new peptides, because of their incomplete coverage of the human proteome. Second, problems arise when searching a spectral library the size of the entire human proteome. We observed that traditional dot product scoring methods do not scale well with spectral library size, showing reduction in sensitivity when library size is increased. We show that this problem can be addressed by optimizing scoring metrics for spectrum-to-spectrum searches with large spectral libraries. MS/MS spectra for the 1.3 million predicted tryptic peptides in the human proteome are simulated using a kinetic fragmentation model (MassAnalyzer version2.1) to create a proteome-wide simulated spectral library. Searches of the simulated library increase MS/MS assignments by 24% compared with Mascot, when using probabilistic and rank based scoring methods. The proteome-wide coverage of the simulated library leads to 11% increase in unique peptide assignments, compared with parallel searches of a reference spectral library. Further improvement is attained when reference spectra and simulated spectra are combined into a hybrid spectral library, yielding 52% increased MS/MS assignments compared with Mascot searches. Our study demonstrates the advantages of using probabilistic and rank based scores to improve performance of spectrum-to-spectrum search strategies. PMID:21532008

The Functional Network of the Arabidopsis Plastoglobule Proteome Based on Quantitative Proteomics and Genome-Wide Coexpression Analysis1[C][W][OA

PubMed Central

Lundquist, Peter K.; Poliakov, Anton; Bhuiyan, Nazmul H.; Zybailov, Boris; Sun, Qi; van Wijk, Klaas J.

2012-01-01

Plastoglobules (PGs) in chloroplasts are thylakoid-associated monolayer lipoprotein particles containing prenyl and neutral lipids and several dozen proteins mostly with unknown functions. An integrated view of the role of the PG is lacking. Here, we better define the PG proteome and provide a conceptual framework for further studies. The PG proteome from Arabidopsis (Arabidopsis thaliana) leaf chloroplasts was determined by mass spectrometry of isolated PGs and quantitative comparison with the proteomes of unfractionated leaves, thylakoids, and stroma. Scanning electron microscopy showed the purity and size distribution of the isolated PGs. Compared with previous PG proteome analyses, we excluded several proteins and identified six new PG proteins, including an M48 metallopeptidase and two Absence of bc1 complex (ABC1) atypical kinases, confirmed by immunoblotting. This refined PG proteome consisted of 30 proteins, including six ABC1 kinases and seven fibrillins together comprising more than 70% of the PG protein mass. Other fibrillins were located predominantly in the stroma or thylakoid and not in PGs; we discovered that this partitioning can be predicted by their isoelectric point and hydrophobicity. A genome-wide coexpression network for the PG genes was then constructed from mRNA expression data. This revealed a modular network with four distinct modules that each contained at least one ABC1K and/or fibrillin gene. Each module showed clear enrichment in specific functions, including chlorophyll degradation/senescence, isoprenoid biosynthesis, plastid proteolysis, and redox regulators and phosphoregulators of electron flow. We propose a new testable model for the PGs, in which sets of genes are associated with specific PG functions. PMID:22274653

QCloud: A cloud-based quality control system for mass spectrometry-based proteomics laboratories

PubMed Central

Chiva, Cristina; Olivella, Roger; Borràs, Eva; Espadas, Guadalupe; Pastor, Olga; Solé, Amanda

2018-01-01

The increasing number of biomedical and translational applications in mass spectrometry-based proteomics poses new analytical challenges and raises the need for automated quality control systems. Despite previous efforts to set standard file formats, data processing workflows and key evaluation parameters for quality control, automated quality control systems are not yet widespread among proteomics laboratories, which limits the acquisition of high-quality results, inter-laboratory comparisons and the assessment of variability of instrumental platforms. Here we present QCloud, a cloud-based system to support proteomics laboratories in daily quality assessment using a user-friendly interface, easy setup, automated data processing and archiving, and unbiased instrument evaluation. QCloud supports the most common targeted and untargeted proteomics workflows, it accepts data formats from different vendors and it enables the annotation of acquired data and reporting incidences. A complete version of the QCloud system has successfully been developed and it is now open to the proteomics community (http://qcloud.crg.eu). QCloud system is an open source project, publicly available under a Creative Commons License Attribution-ShareAlike 4.0. PMID:29324744

A year (2014-2015) of plants in Proteomics journal. Progress in wet and dry methodologies, moving from protein catalogs, and the view of classic plant biochemists.

PubMed

Sanchez-Lucas, Rosa; Mehta, Angela; Valledor, Luis; Cabello-Hurtado, Francisco; Romero-Rodrıguez, M Cristina; Simova-Stoilova, Lyudmila; Demir, Sekvan; Rodriguez-de-Francisco, Luis E; Maldonado-Alconada, Ana M; Jorrin-Prieto, Ana L; Jorrín-Novo, Jesus V

2016-03-01

The present review is an update of the previous one published in Proteomics 2015 Reviews special issue [Jorrin-Novo, J. V. et al., Proteomics 2015, 15, 1089-1112] covering the July 2014-2015 period. It has been written on the bases of the publications that appeared in Proteomics journal during that period and the most relevant ones that have been published in other high-impact journals. Methodological advances and the contribution of the field to the knowledge of plant biology processes and its translation to agroforestry and environmental sectors will be discussed. This review has been organized in four blocks, with a starting general introduction (literature survey) followed by sections focusing on the methodology (in vitro, in vivo, wet, and dry), proteomics integration with other approaches (systems biology and proteogenomics), biological information, and knowledge (cell communication, receptors, and signaling), ending with a brief mention of some other biological and translational topics to which proteomics has made some contribution. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comparison of Different Protein Extraction Methods for Gel-Based Proteomic Analysis of Ganoderma spp.

PubMed

Al-Obaidi, Jameel R; Saidi, Noor Baity; Usuldin, Siti Rokhiyah Ahmad; Hussin, Siti Nahdatul Isnaini Said; Yusoff, Noornabeela Md; Idris, Abu Seman

2016-04-01

Ganoderma species are a group of fungi that have the ability to degrade lignin polymers and cause severe diseases such as stem and root rot and can infect economically important plants and perennial crops such as oil palm, especially in tropical countries such as Malaysia. Unfortunately, very little is known about the complex interplay between oil palm and Ganoderma in the pathogenesis of the diseases. Proteomic technologies are simple yet powerful tools in comparing protein profile and have been widely used to study plant-fungus interaction. A critical step to perform a good proteome research is to establish a method that gives the best quality and a wide coverage of total proteins. Despite the availability of various protein extraction protocols from pathogenic fungi in the literature, no single extraction method was found suitable for all types of pathogenic fungi. To develop an optimized protein extraction protocol for 2-DE gel analysis of Ganoderma spp., three previously reported protein extraction protocols were compared: trichloroacetic acid, sucrose and phenol/ammonium acetate in methanol. The third method was found to give the most reproducible gels and highest protein concentration. Using the later method, a total of 10 protein spots (5 from each species) were successfully identified. Hence, the results from this study propose phenol/ammonium acetate in methanol as the most effective protein extraction method for 2-DE proteomic studies of Ganoderma spp.

Screening of missing proteins in the human liver proteome by improved MRM-approach-based targeted proteomics.

PubMed

Chen, Chen; Liu, Xiaohui; Zheng, Weimin; Zhang, Lei; Yao, Jun; Yang, Pengyuan

2014-04-04

To completely annotate the human genome, the task of identifying and characterizing proteins that currently lack mass spectrometry (MS) evidence is inevitable and urgent. In this study, as the first effort to screen missing proteins in large scale, we developed an approach based on SDS-PAGE followed by liquid chromatography-multiple reaction monitoring (LC-MRM), for screening of those missing proteins with only a single peptide hit in the previous liver proteome data set. Proteins extracted from normal human liver were separated in SDS-PAGE and digested in split gel slice, and the resulting digests were then subjected to LC-schedule MRM analysis. The MRM assays were developed through synthesized crude peptides for target peptides. In total, the expressions of 57 target proteins were confirmed from 185 MRM assays in normal human liver tissues. Among the proved 57 one-hit wonders, 50 proteins are of the minimally redundant set in the PeptideAtlas database, 7 proteins even have none MS-based information previously in various biological processes. We conclude that our SDS-PAGE-MRM workflow can be a powerful approach to screen missing or poorly characterized proteins in different samples and to provide their quantity if detected. The MRM raw data have been uploaded to ISB/SRM Atlas/PASSEL (PXD000648).

Firing the Sting: Chemically Induced Discharge of Cnidae Reveals Novel Proteins and Peptides from Box Jellyfish (Chironex fleckeri) Venom

PubMed Central

Jouiaei, Mahdokht; Casewell, Nicholas R.; Yanagihara, Angel A.; Nouwens, Amanda; Cribb, Bronwen W.; Whitehead, Darryl; Jackson, Timothy N. W.; Ali, Syed A.; Wagstaff, Simon C.; Koludarov, Ivan; Alewood, Paul; Hansen, Jay; Fry, Bryan G.

2015-01-01

Cnidarian venom research has lagged behind other toxinological fields due to technical difficulties in recovery of the complex venom from the microscopic nematocysts. Here we report a newly developed rapid, repeatable and cost effective technique of venom preparation, using ethanol to induce nematocyst discharge and to recover venom contents in one step. Our model species was the Australian box jellyfish (Chironex fleckeri), which has a notable impact on public health. By utilizing scanning electron microscopy and light microscopy, we examined nematocyst external morphology before and after ethanol treatment and verified nematocyst discharge. Further, to investigate nematocyst content or “venom” recovery, we utilized both top-down and bottom-up transcriptomics–proteomics approaches and compared the proteome profile of this new ethanol recovery based method to a previously reported high activity and recovery protocol, based upon density purified intact cnidae and pressure induced disruption. In addition to recovering previously characterized box jellyfish toxins, including CfTX-A/B and CfTX-1, we recovered putative metalloproteases and novel expression of a small serine protease inhibitor. This study not only reveals a much more complex toxin profile of Australian box jellyfish venom but also suggests that ethanol extraction method could augment future cnidarian venom proteomics research efforts. PMID:25793725

Proteomic analysis of knock-down HLA-G in invasion of human trophoblast cell line JEG-3

PubMed Central

Liu, Haiyan; Liu, Xueyuan; Jin, Hong; Yang, Fengying; Gu, Weirong; Li, Xiaotian

2013-01-01

Previous studies showed that aberrant HLA-G expression in trophoblast cells plays important roles in trophoblast invasion; however, the mechanisms remain to be explored. In this study, we found that suppressed HLA-G expression could dramatically decrease the mRNA and protein expression levels of matrix metalloproteinase 2 and matrix metalloproteinase 9, and in the proteome assay, there were 3 identified proteins namely, prefoldin 1, eukaryotic translation elongation factor 2 and malate dehydrogenase 2, which were verified by Western blot and known to be associated with invasion, cell cycle and cell metabolism, respectively. Collectively, our study indicated a potential involvement of HLA-G in autocrine networks that may regulate prefoldin, MMPs and trophoblast invasion at the maternal-fetal interface in human pregnancy. PMID:24228107

Quantitative proteomics-based analysis supports a significant role of GTG proteins in regulation of ABA response in Arabidopsis roots.

PubMed

Alvarez, Sophie; Roy Choudhury, Swarup; Hicks, Leslie M; Pandey, Sona

2013-03-01

Abscisic acid (ABA) is proposed to be perceived by multiple receptors in plants. We have previously reported on the role of two GPCR-type G-proteins (GTG proteins) as plasma membrane-localized ABA receptors in Arabidopsis thaliana. However, due to the presence of multiple transmembrane domains, detailed structural and biochemical characterization of GTG proteins remains limited. Since ABA induces substantial changes in the proteome of plants, a labeling LC-based quantitative proteomics approach was applied to elucidate the global effects and possible downstream targets of GTG1/GTG2 proteins. Quantitative differences in protein abundance between wild-type and gtg1gtg2 were analyzed for evaluation of the effect of ABA on the root proteome and its dependence on the presence of functional GTG1/GTG2 proteins. The results presented in this study reveal the most comprehensive ABA-responsive root proteome reported to date in Arabidopsis. Notably, the majority of ABA-responsive proteins required the presence of GTG proteins, supporting their key role in ABA signaling. These observations were further confirmed by additional experiments. Overall, comparison of the ABA-dependent protein abundance changes in wild-type versus gtg1gtg2 provides clues to their possible links with some of the well-established effectors of the ABA signaling pathways and their role in mediating phytohormone cross-talk.

Analysis of Intrinsic Peptide Detectability via Integrated Label-Free and SRM-Based Absolute Quantitative Proteomics.

PubMed

Jarnuczak, Andrew F; Lee, Dave C H; Lawless, Craig; Holman, Stephen W; Eyers, Claire E; Hubbard, Simon J

2016-09-02

Quantitative mass spectrometry-based proteomics of complex biological samples remains challenging in part due to the variability and charge competition arising during electrospray ionization (ESI) of peptides and the subsequent transfer and detection of ions. These issues preclude direct quantification from signal intensity alone in the absence of a standard. A deeper understanding of the governing principles of peptide ionization and exploitation of the inherent ionization and detection parameters of individual peptides is thus of great value. Here, using the yeast proteome as a model system, we establish the concept of peptide F-factor as a measure of detectability, closely related to ionization efficiency. F-factor is calculated by normalizing peptide precursor ion intensity by absolute abundance of the parent protein. We investigated F-factor characteristics in different shotgun proteomics experiments, including across multiple ESI-based LC-MS platforms. We show that F-factors mirror previously observed physicochemical predictors as peptide detectability but demonstrate a nonlinear relationship between hydrophobicity and peptide detectability. Similarly, we use F-factors to show how peptide ion coelution adversely affects detectability and ionization. We suggest that F-factors have great utility for understanding peptide detectability and gas-phase ion chemistry in complex peptide mixtures, selection of surrogate peptides in targeted MS studies, and for calibration of peptide ion signal in label-free workflows. Data are available via ProteomeXchange with identifier PXD003472.

A comprehensive and scalable database search system for metaproteomics.

PubMed

Chatterjee, Sandip; Stupp, Gregory S; Park, Sung Kyu Robin; Ducom, Jean-Christophe; Yates, John R; Su, Andrew I; Wolan, Dennis W

2016-08-16

Mass spectrometry-based shotgun proteomics experiments rely on accurate matching of experimental spectra against a database of protein sequences. Existing computational analysis methods are limited in the size of their sequence databases, which severely restricts the proteomic sequencing depth and functional analysis of highly complex samples. The growing amount of public high-throughput sequencing data will only exacerbate this problem. We designed a broadly applicable metaproteomic analysis method (ComPIL) that addresses protein database size limitations. Our approach to overcome this significant limitation in metaproteomics was to design a scalable set of sequence databases assembled for optimal library querying speeds. ComPIL was integrated with a modified version of the search engine ProLuCID (termed "Blazmass") to permit rapid matching of experimental spectra. Proof-of-principle analysis of human HEK293 lysate with a ComPIL database derived from high-quality genomic libraries was able to detect nearly all of the same peptides as a search with a human database (~500x fewer peptides in the database), with a small reduction in sensitivity. We were also able to detect proteins from the adenovirus used to immortalize these cells. We applied our method to a set of healthy human gut microbiome proteomic samples and showed a substantial increase in the number of identified peptides and proteins compared to previous metaproteomic analyses, while retaining a high degree of protein identification accuracy and allowing for a more in-depth characterization of the functional landscape of the samples. The combination of ComPIL with Blazmass allows proteomic searches to be performed with database sizes much larger than previously possible. These large database searches can be applied to complex meta-samples with unknown composition or proteomic samples where unexpected proteins may be identified. The protein database, proteomic search engine, and the proteomic data files for the 5 microbiome samples characterized and discussed herein are open source and available for use and additional analysis.

Neurobiological Signatures of Alcohol Dependence Revealed by Protein Profiling

PubMed Central

Gorini, Giorgio; Roberts, Amanda J.; Mayfield, R. Dayne

2013-01-01

Alcohol abuse causes dramatic neuroadaptations in the brain, which contribute to tolerance, dependence, and behavioral modifications. Previous proteomic studies in human alcoholics and animal models have identified candidate alcoholism-related proteins. However, recent evidences suggest that alcohol dependence is caused by changes in co-regulation that are invisible to single protein-based analysis. Here, we analyze global proteomics data to integrate differential expression, co-expression networks, and gene annotations to unveil key neurobiological rearrangements associated with the transition to alcohol dependence modeled by a Chronic Intermittent Ethanol (CIE), two-bottle choice (2BC) paradigm. We analyzed cerebral cortices (CTX) and midbrains (MB) from male C57BL/6J mice subjected to a CIE, 2BC paradigm, which induces heavy drinking and represents one of the best available animal models for alcohol dependence and relapse drinking. CIE induced significant changes in protein levels in dependent mice compared with their non-dependent controls. Multiple protein isoforms showed region-specific differential regulation as a result of post-translational modifications. Our integrative analysis identified modules of co-expressed proteins that were highly correlated with CIE treatment. We found that modules most related to the effects of CIE treatment coordinate molecular imbalances in endocytic- and energy-related pathways, with specific proteins involved, such as dynamin-1. The qRT-PCR experiments validated both differential and co-expression analyses, and the correspondence among our data and previous genomic and proteomic studies in humans and rodents substantiates our findings. The changes identified above may play a key role in the escalation of ethanol consumption associated with dependence. Our approach to alcohol addiction will advance knowledge of brain remodeling mechanisms and adaptive changes in response to drug abuse, contribute to understanding of organizational principles of CTX and MB proteomes, and define potential new molecular targets for treating alcohol addiction. The integrative analysis employed here highlight the advantages of systems approaches in studying the neurobiology of alcohol addiction. PMID:24358215

Proteomic Signatures of Acquired Letrozole Resistance in Breast Cancer: Suppressed Estrogen Signaling and Increased Cell Motility and Invasiveness*

PubMed Central

Tilghman, Syreeta L.; Townley, Ian; Zhong, Qiu; Carriere, Patrick P.; Zou, Jin; Llopis, Shawn D.; Preyan, Lynez C.; Williams, Christopher C.; Skripnikova, Elena; Bratton, Melyssa R.; Zhang, Qiang; Wang, Guangdi

2013-01-01

Aromatase inhibitors, such as letrozole, have become the first-line treatment for postmenopausal women with estrogen-dependent breast cancer. However, acquired resistance remains a major clinical obstacle. Previous studies demonstrated constitutive activation of the MAPK signaling, overexpression of HER2, and down-regulation of aromatase and ERα in letrozole-resistant breast cancer cells. Given the complex signaling network involved in letrozole-refractory breast cancer and the lack of effective treatment for hormone resistance, further investigation of aromatase inhibitor resistance by a novel systems biology approach may reveal previously unconsidered molecular changes that could be utilized as therapeutic targets. This study was undertaken to characterize for the first time global proteomic alterations occurring in a letrozole-resistant cell line. A quantitative proteomic analysis of the whole cell lysates of LTLT-Ca (resistant) versus AC-1 cells (sensitive) was performed to identify significant protein expression changes. A total of 1743 proteins were identified and quantified, of which 411 were significantly up-regulated and 452 significantly down-regulated (p < 0.05, fold change > 1.20). Bioinformatics analysis revealed that acquired letrozole resistance is associated with a hormone-independent, more aggressive phenotype. LTLT-Ca cells exhibited 84% and 138% increase in migration and invasion compared with the control cells. The ROCK inhibitor partially abrogated the enhanced migration and invasion of the letrozole-resistant cells. Flow cytometric analyses also demonstrated an increase in vimentin and twist expression in letrozole-resistance cells, suggesting an onset of epithelial to mesenchymal transition (EMT). Moreover, targeted gene expression arrays confirmed a 28-fold and sixfold up-regulation of EGFR and HER2, respectively, whereas ERα and pS2 were dramatically reduced by 28-fold and 1100-fold, respectively. Taken together, our study revealed global proteomic signatures of a letrozole-resistant cell line associated with hormone independence, enhanced cell motility, EMT and the potential values of several altered proteins as novel prognostic markers or therapeutic targets for letrozole resistant breast cancer. PMID:23704778

Proteomic analysis reveals differential accumulation of small heat shock proteins and late embryogenesis abundant proteins between ABA-deficient mutant vp5 seeds and wild-type Vp5 seeds in maize

PubMed Central

Wu, Xiaolin; Gong, Fangping; Yang, Le; Hu, Xiuli; Tai, Fuju; Wang, Wei

2014-01-01

ABA is a major plant hormone that plays important roles during many phases of plant life cycle, including seed development, maturity and dormancy, and especially the acquisition of desiccation tolerance. Understanding of the molecular basis of ABA-mediated plant response to stress is of interest not only in basic research on plant adaptation but also in applied research on plant productivity. Maize mutant viviparous-5 (vp5), deficient in ABA biosynthesis in seeds, is a useful material for studying ABA-mediated response in maize. Due to carotenoid deficiency, vp5 endosperm is white, compared to yellow Vp5 endosperm. However, the background difference at proteome level between vp5 and Vp5 seeds is unclear. This study aimed to characterize proteome alterations of maize vp5 seeds and to identify ABA-dependent proteins during seed maturation. We compared the embryo and endosperm proteomes of vp5 and Vp5 seeds by gel-based proteomics. Up to 46 protein spots, most in embryos, were found to be differentially accumulated between vp5 and Vp5. The identified proteins included small heat shock proteins (sHSPs), late embryogenesis abundant (LEA) proteins, stress proteins, storage proteins and enzymes among others. However, EMB564, the most abundant LEA protein in maize embryo, accumulated in comparable levels between vp5 and Vp5 embryos, which contrasted to previously characterized, greatly lowered expression of emb564 mRNA in vp5 embryos. Moreover, LEA proteins and sHSPs displayed differential accumulations in vp5 embryos: six out of eight identified LEA proteins decreased while nine sHSPs increased in abundance. Finally, we discussed the possible causes of global proteome alterations, especially the observed differential accumulation of identified LEA proteins and sHSPs in vp5 embryos. The data derived from this study provides new insight into ABA-dependent proteins and ABA-mediated response during maize seed maturation. PMID:25653661

Proteogenomic characterization of human colon and rectal cancer

PubMed Central

Zhang, Bing; Wang, Jing; Wang, Xiaojing; Zhu, Jing; Liu, Qi; Shi, Zhiao; Chambers, Matthew C.; Zimmerman, Lisa J.; Shaddox, Kent F.; Kim, Sangtae; Davies, Sherri R.; Wang, Sean; Wang, Pei; Kinsinger, Christopher R.; Rivers, Robert C.; Rodriguez, Henry; Townsend, R. Reid; Ellis, Matthew J.C.; Carr, Steven A.; Tabb, David L.; Coffey, Robert J.; Slebos, Robbert J.C.; Liebler, Daniel C.

2014-01-01

Summary We analyzed proteomes of colon and rectal tumors previously characterized by the Cancer Genome Atlas (TCGA) and performed integrated proteogenomic analyses. Somatic variants displayed reduced protein abundance compared to germline variants. mRNA transcript abundance did not reliably predict protein abundance differences between tumors. Proteomics identified five proteomic subtypes in the TCGA cohort, two of which overlapped with the TCGA “MSI/CIMP” transcriptomic subtype, but had distinct mutation, methylation, and protein expression patterns associated with different clinical outcomes. Although copy number alterations showed strong cis- and trans-effects on mRNA abundance, relatively few of these extend to the protein level. Thus, proteomics data enabled prioritization of candidate driver genes. The chromosome 20q amplicon was associated with the largest global changes at both mRNA and protein levels; proteomics data highlighted potential 20q candidates including HNF4A, TOMM34 and SRC. Integrated proteogenomic analysis provides functional context to interpret genomic abnormalities and affords a new paradigm for understanding cancer biology. PMID:25043054

[MALDI-TOF and SELDI-TOF analysis: "tandem" techniques to identify potential biomarker in fibromyalgia].

PubMed

Giacomelli, C; Bazzichi, L; Giusti, L; Ciregia, F; Baldini, C; Da Valle, Y; De Feo, F; Sernissi, F; Rossi, A; Bombardieri, S; Lucacchini, A

2011-11-09

Fibromyalgia (FM) is characterized by the presence of chronic widespread pain throughout the musculoskeletal system and diffuse tenderness. Unfortunately, no laboratory tests have been appropriately validated for FM and correlated with the subsets and activity. The aim of this study was to apply a proteomic technique in saliva of FM patients: the Surface Enhance Laser Desorption/Ionization Time-of-Flight (SELDI-TOF). For this study, 57 FM patients and 35 HC patients were enrolled. The proteomic analysis of saliva was carried out using SELDI-TOF. The analysis was performed using different chip arrays with different characteristics of binding. The statistical analysis was performed using cluster analysis and the difference between two groups was underlined using Student’s t-test. Spectra analysis highlighted the presence of several peaks differently expressed in FM patients compared with controls. The preliminary results obtained by SELDI-TOF analysis were compared with those obtained in our previous study performed on whole saliva of FM patients by using electrophoresis. The m/z of two peaks, increased in FM patients, seem to overlap well with the molecular weight of calgranulin A and C and Rho GDP-dissociation inhibitor 2, which we had found up-regulated in our previous study. These preliminary results showed the possibility of identifying potential salivary biomarker through salivary proteomic analysis with MALDI-TOF and SELDI-TOF in FM patients. The peaks observed allow us to focus on some of the particular pathogenic aspects of FM, the oxidative stress which contradistinguishes this condition, the involvement of proteins related to the cytoskeletal arrangements, and central sensibilization.

Subnanogram proteomics: impact of LC column selection, MS instrumentation and data analysis strategy on proteome coverage for trace samples

DOE PAGES

Zhu, Ying; Zhao, Rui; Piehowski, Paul D.; ...

2017-09-01

One of the greatest challenges for mass spectrometry (MS)-based proteomics is the limited ability to analyze small samples. Here in this study, we investigate the relative contributions of liquid chromatography (LC), MS instrumentation and data analysis methods with the aim of improving proteome coverage for sample sizes ranging from 0.5 ng to 50 ng. We show that the LC separations utilizing 30-μm-i.d. columns increase signal intensity by >3-fold relative to those using 75-μm-i.d. columns, leading to 32% increase in peptide identifications. The Orbitrap Fusion Lumos MS significantly boosted both sensitivity and sequencing speed relative to earlier generation Orbitraps (e.g., LTQ-Orbitrap),more » leading to a ~3-fold increase in peptide identifications and 1.7-fold increase in identified protein groups for 2 ng tryptic digests of the bacterium S. oneidensis. The Match Between Runs algorithm of open-source MaxQuant software further increased proteome coverage by ~95% for 0.5 ng samples and by ~42% for 2 ng samples. Using the best combination of the above variables, we were able to identify >3,000 proteins from 10 ng tryptic digests from both HeLa and THP-1 mammalian cell lines. We also identified >950 proteins from subnanogram archaeal/bacterial cocultures. Finally, the present ultrasensitive LC-MS platform achieves a level of proteome coverage not previously realized for ultra-small sample loadings, and is expected to facilitate the analysis of subnanogram samples, including single mammalian cells.« less

Cellular response of human neuroblastoma cells to α-synuclein fibrils, the main constituent of Lewy bodies.

PubMed

Pieri, Laura; Chafey, Philippe; Le Gall, Morgane; Clary, Guilhem; Melki, Ronald; Redeker, Virginie

2016-01-01

α-Synuclein (α-Syn) fibrils are the main constituent of Lewy bodies and a neuropathological hallmark of Parkinson's disease (PD). The propagation of α-Syn assemblies from cell to cell suggests that they are involved in PD progression. We previously showed that α-Syn fibrils are toxic because of their ability to bind and permeabilize cell membranes. Here, we document the cellular response in terms of proteome changes of SH-SY5Y cells exposed to exogenous α-Syn fibrils. We compare the proteomes of cells of neuronal origin exposed or not either to oligomeric or fibrillar α-Syn using two dimensional differential in-gel electrophoresis (2D-DIGE) and mass spectrometry. Only α-Syn fibrils induce significant changes in the proteome of SH-SY5Y cells. In addition to proteins associated to apoptosis and toxicity, or proteins previously linked to neurodegenerative diseases, we report an overexpression of proteins involved in intracellular vesicle trafficking. We also report a remarkable increase in fibrillar α-Syn heterogeneity, mainly due to C-terminal truncations. Our results show that cells of neuronal origin adapt their proteome to exogenous α-Syn fibrils and actively modify those assemblies. Cells of neuronal origin adapt their proteome to exogenous toxic α-Syn fibrils and actively modify those assemblies. Our results bring insights into the cellular response and clearance events the cells implement to face the propagation of α-Syn assemblies associated to pathology.

Proteome and metabolome profiling of cytokinin action in Arabidopsis identifying both distinct and similar responses to cytokinin down- and up-regulation.

PubMed

Černý, Martin; Kuklová, Alena; Hoehenwarter, Wolfgang; Fragner, Lena; Novák, Ondrej; Rotková, Gabriela; Jedelsky, Petr L; Žáková, Katerina; Šmehilová, Mária; Strnad, Miroslav; Weckwerth, Wolfram; Brzobohaty, Bretislav

2013-11-01

In plants, numerous developmental processes are controlled by cytokinin (CK) levels and their ratios to levels of other hormones. While molecular mechanisms underlying the regulatory roles of CKs have been intensely researched, proteomic and metabolomic responses to CK deficiency are unknown. Transgenic Arabidopsis seedlings carrying inducible barley cytokinin oxidase/dehydrogenase (CaMV35S>GR>HvCKX2) and agrobacterial isopentenyl transferase (CaMV35S>GR>ipt) constructs were profiled to elucidate proteome- and metabolome-wide responses to down- and up-regulation of CK levels, respectively. Proteome profiling identified >1100 proteins, 155 of which responded to HvCKX2 and/or ipt activation, mostly involved in growth, development, and/or hormone and light signalling. The metabolome profiling covered 79 metabolites, 33 of which responded to HvCKX2 and/or ipt activation, mostly amino acids, carbohydrates, and organic acids. Comparison of the data sets obtained from activated CaMV35S>GR>HvCKX2 and CaMV35S>GR>ipt plants revealed unexpectedly extensive overlaps. Integration of the proteomic and metabolomic data sets revealed: (i) novel components of molecular circuits involved in CK action (e.g. ribosomal proteins); (ii) previously unrecognized links to redox regulation and stress hormone signalling networks; and (iii) CK content markers. The striking overlaps in profiles observed in CK-deficient and CK-overproducing seedlings might explain surprising previously reported similarities between plants with down- and up-regulated CK levels.

Study of cellular oncometabolism via multidimensional protein identification technology.

PubMed

Aukim-Hastie, Claire; Garbis, Spiros D

2014-01-01

Cellular proteomics is becoming a widespread clinical application, matching the definition of bench-to-bedside translation. Among various fields of investigation, this approach can be applied to the study of the metabolic alterations that accompany oncogenesis and tumor progression, which are globally referred to as oncometabolism. Here, we describe a multidimensional protein identification technology (MuDPIT)-based strategy that can be employed to study the cellular proteome of malignant cells and tissues. This method has previously been shown to be compatible with the reproducible, in-depth analysis of up to a thousand proteins in clinical samples. The possibility to employ this technique to study clinical specimens demonstrates its robustness. MuDPIT is advantageous as compared to other approaches because it is direct, highly sensitive, and reproducible, it provides high resolution with ultra-high mass accuracy, it allows for relative quantifications, and it is compatible with multiplexing (thus limiting costs).This method enables the direct assessment of the proteomic profile of neoplastic cells and tissues and could be employed in the near future as a high-throughput, rapid, quantitative, and cost-effective screening platform for clinical samples. © 2014 Elsevier Inc. All rights reserved.

Oocyte spindle proteomics analysis leading to rescue of chromosome congression defects in cloned embryos

PubMed Central

Duan, Xunbao; Zhong, Zhisheng; Potireddy, Santhi; Moncada, Camilo; Merali, Salim; Latham, Keith E.

2015-01-01

Embryos produced by somatic cell nuclear transfer (SCNT) display low term developmental potential. This is associated with deficiencies in spindle composition prior to activation and at early mitotic divisions, including failure to assemble certain proteins on the spindle. The protein-deficient spindles are accompanied by chromosome congression defects prior to activation and during the first mitotic divisions of the embryo. The molecular basis for these deficiencies and how they might be avoided are unknown. Proteomic analyses of spindles isolated from normal metaphase II (MII) stage oocytes and SCNT constructs, along with a systematic immunofluorescent survey of known spindle-associated proteins were undertaken. This was the first proteomics study of mammalian oocyte spindles. The study revealed four proteins as being deficient in spindles of SCNT embryos in addition to those previously identified; these were clathrin heavy chain (CLTC), aurora B kinase, dynactin 4, and casein kinase 1 alpha. Due to substantial reduction in CLTC abundance after spindle removal, we undertook functional studies to explore the importance of CLTC in oocyte spindle function and in chromosome congression defects of cloned embryos. Using siRNA knockdown we demonstrated an essential role for CLTC in chromosome congression during oocyte maturation. We also demonstrated rescue of chromosome congression defects in SCNT embryos at the first mitosis using CLTC mRNA injection. These studies are the first to employ proteomics analyses coupled to functional interventions to rescue a specific molecular defect in cloned embryos. PMID:20883044

"Omics" of Selenium Biology: A Prospective Study of Plasma Proteome Network Before and After Selenized-Yeast Supplementation in Healthy Men.

PubMed

Sinha, Indu; Karagoz, Kubra; Fogle, Rachel L; Hollenbeak, Christopher S; Zea, Arnold H; Arga, Kazim Y; Stanley, Anne E; Hawkes, Wayne C; Sinha, Raghu

2016-04-01

Low selenium levels have been linked to a higher incidence of cancer and other diseases, including Keshan, Chagas, and Kashin-Beck, and insulin resistance. Additionally, muscle and cardiovascular disorders, immune dysfunction, cancer, neurological disorders, and endocrine function have been associated with mutations in genes encoding for selenoproteins. Selenium biology is complex, and a systems biology approach to study global metabolomics, genomics, and/or proteomics may provide important clues to examining selenium-responsive markers in circulation. In the current investigation, we applied a global proteomics approach on plasma samples collected from a previously conducted, double-blinded placebo controlled clinical study, where men were supplemented with selenized-yeast (Se-Yeast; 300 μg/day, 3.8 μmol/day) or placebo-yeast for 48 weeks. Proteomic analysis was performed by iTRAQ on 8 plasma samples from each arm at baseline and 48 weeks. A total of 161 plasma proteins were identified in both arms. Twenty-two proteins were significantly altered following Se-Yeast supplementation and thirteen proteins were significantly changed after placebo-yeast supplementation in healthy men. The differentially expressed proteins were involved in complement and coagulation pathways, immune functions, lipid metabolism, and insulin resistance. Reconstruction and analysis of protein-protein interaction network around selected proteins revealed several hub proteins. One of the interactions suggested by our analysis, PHLD-APOA4, which is involved in insulin resistance, was subsequently validated by Western blot analysis. Our systems approach illustrates a viable platform for investigating responsive proteomic profile in 'before and after' condition following Se-Yeast supplementation. The nature of proteins identified suggests that selenium may play an important role in complement and coagulation pathways, and insulin resistance.

Concurrent Label-Free Mass Spectrometric Analysis of Dystrophin Isoform Dp427 and the Myofibrosis Marker Collagen in Crude Extracts from mdx-4cv Skeletal Muscles

PubMed Central

Murphy, Sandra; Zweyer, Margit; Mundegar, Rustam R.; Henry, Michael; Meleady, Paula; Swandulla, Dieter; Ohlendieck, Kay

2015-01-01

The full-length dystrophin protein isoform of 427 kDa (Dp427), the absence of which represents the principal abnormality in X-linked muscular dystrophy, is difficult to identify and characterize by routine proteomic screening approaches of crude tissue extracts. This is probably related to its large molecular size, its close association with the sarcolemmal membrane, and its existence within a heterogeneous glycoprotein complex. Here, we used a careful extraction procedure to isolate the total protein repertoire from normal versus dystrophic mdx-4cv skeletal muscles, in conjunction with label-free mass spectrometry, and successfully identified Dp427 by proteomic means. In contrast to a considerable number of previous comparative studies of the total skeletal muscle proteome, using whole tissue proteomics we show here for the first time that the reduced expression of this membrane cytoskeletal protein is the most significant alteration in dystrophinopathy. This agrees with the pathobiochemical concept that the almost complete absence of dystrophin is the main defect in Duchenne muscular dystrophy and that the mdx-4cv mouse model of dystrophinopathy exhibits only very few revertant fibers. Significant increases in collagens and associated fibrotic marker proteins, such as fibronectin, biglycan, asporin, decorin, prolargin, mimecan, and lumican were identified in dystrophin-deficient muscles. The up-regulation of collagen in mdx-4cv muscles was confirmed by immunofluorescence microscopy and immunoblotting. Thus, this is the first mass spectrometric study of crude tissue extracts that puts the proteomic identification of dystrophin in its proper pathophysiological context. PMID:28248273

Proteome analysis identifies L1CAM/CD171 and DPP4/CD26 as novel markers of human skin mast cells.

PubMed

Gschwandtner, M; Paulitschke, V; Mildner, M; Brunner, P M; Hacker, S; Eisenwort, G; Sperr, W R; Valent, P; Gerner, C; Tschachler, E

2017-01-01

The function of skin mast cells has been well documented in IgE-mediated allergic reactions, whereas other mast cell functions are poorly defined. This study aimed at identifying novel mast cell proteins by proteome analysis of primary human skin mast cells. The proteome of skin mast cells was compared to other cell types and analyzed using bioinformatics. The expression and function of two proteins hitherto not described in skin mast cells was investigated in isolated mast cells as well as in mast cells in situ. Within the mast cell proteome, we identified 49 highly expressed proteins previously not described in mast cells; 21 of these proteins were found to be selectively expressed in mast cells. Two proteins, the neural cell adhesion molecule L1 and dipeptidyl peptidase 4, were further studied. L1 was found to be highly expressed in mast cells in normal, psoriasis, and mastocytosis skin. Dipeptidyl peptidase 4 was found to be expressed in mast cells in normal, psoriasis, and mastocytosis skin as well as in bone marrow mast cells in patients with systemic mastocytosis. In normal skin, mast cells were identified as a major source of dipeptidyl peptidase 4 and we also found that skin mast cells and fibroblasts secrete an active form of this enzyme. In a systematic proteomics approach we identified two novel mast cell proteins potentially relevant to skin homeostasis: neural cell adhesion molecule L1 and dipeptidyl peptidase 4. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Proteomic Characterization of Dermal Interstitial Fluid Extracted Using a Novel Microneedle-Assisted Technique.

PubMed

Tran, Bao Quoc; Miller, Philip R; Taylor, Robert M; Boyd, Gabrielle; Mach, Phillip M; Rosenzweig, C Nicole; Baca, Justin T; Polsky, Ronen; Glaros, Trevor

2018-01-05

As wearable fitness devices have gained commercial acceptance, interest in real-time monitoring of an individual's physiological status using noninvasive techniques has grown. Microneedles have been proposed as a minimally invasive technique for sampling the dermal interstitial fluid (ISF) for clinical monitoring and diagnosis, but little is known about its composition. In this study, a novel microneedle array was used to collect dermal ISF from three healthy human donors and compared with matching serum and plasma samples. Using a shotgun quantitative proteomic approach, 407 proteins were quantified with at least one unique peptide, and of those, 135 proteins were differently expressed at least 2-fold. Collectively, these proteins tended to originate from the cytoplasm, membrane bound vesicles, and extracellular vesicular exosomes. Proteomic analysis confirmed previously published work that indicates that ISF is highly similar to both plasma and serum. In this study, less than one percent of proteins were uniquely identified in ISF. Taken together, ISF could serve as a minimally invasive alternative for blood-derived fluids with potential for real-time monitoring applications.

[Advances in the study of the nucleolus].

PubMed

Feng, Jin-Mei; Sun, Jun; Wen, Jian-Fan

2012-12-01

As the most prominent sub-nuclear compartment in the interphase nucleus and the site of ribosome biogenesis, the nucleolus synthesizes and processes rRNA and also assembles ribosomal subunits. Though several lines of research in recent years have indicated that the nucleolus might have additional functions-such as the assembling of signal recognition particles, the processing of mRNA, tRNA and telomerase activities, and regulating the cell cycle-proteomic analyses of the nucleolus in three representative eukaryotic species has shown that a plethora of proteins either have no association with ribosome biogenesis or are of presently unknown function. This phenomenon further indicates that the composition and function of the nucleolus is far more complicated than previously thought. Meanwhile, the available nucleolar proteome databases has provided new approaches and led to remarkable progress in understanding the nucleolus. Here, we have summarized recent advances in the study of the nucleolus, including new discoveries of its structure, function, genomics/proteomics as well as its origin and evolution. Moreover, we highlight several of the important unresolved issues in this field.

Strigolactone-regulated proteins revealed by iTRAQ-based quantitative proteomics in Arabidopsis.

PubMed

Li, Zhou; Czarnecki, Olaf; Chourey, Karuna; Yang, Jun; Tuskan, Gerald A; Hurst, Gregory B; Pan, Chongle; Chen, Jin-Gui

2014-03-07

Strigolactones (SLs) are a new class of plant hormones. In addition to acting as a key inhibitor of shoot branching, SLs stimulate seed germination of root parasitic plants and promote hyphal branching and root colonization of symbiotic arbuscular mycorrhizal fungi. They also regulate many other aspects of plant growth and development. At the transcription level, SL-regulated genes have been reported. However, nothing is known about the proteome regulated by this new class of plant hormones. A quantitative proteomics approach using an isobaric chemical labeling reagent, iTRAQ, to identify the proteome regulated by SLs in Arabidopsis seedlings is presented. It was found that SLs regulate the expression of about three dozen proteins that have not been previously assigned to SL pathways. These findings provide a new tool to investigate the molecular mechanism of action of SLs.

CPTAC Releases Cancer Proteome Confirmatory Colon Study Data | Office of Cancer Clinical Proteomics Research

Cancer.gov

The National Cancer Institute (NCI) Clinical Proteomic Tumor Analysis Consortium (CPTAC) announces the release of the cancer proteome confirmatory colon study data. The goal of the study is to analyze the proteomes of approximately 100 confirmatory colon tumor patients, which includes tumor and adjacent normal samples, with liquid chromatography-tandem mass spectrometry (LC-MS/MS) global proteomic and phosphoproteomic profiling.

Mining for Microbial Gems: Integrating Proteomics in the Postgenomic Natural Product Discovery Pipeline.

PubMed

Du, Chao; van Wezel, Gilles P

2018-04-30

Natural products (NPs) are a major source of compounds for medical, agricultural, and biotechnological industries. Many of these compounds are of microbial origin, and, in particular, from Actinobacteria or filamentous fungi. To successfully identify novel compounds that correlate to a bioactivity of interest, or discover new enzymes with desired functions, systematic multiomics approaches have been developed over the years. Bioinformatics tools harness the rapidly expanding wealth of genome sequence information, revealing previously unsuspected biosynthetic diversity. Varying growth conditions or application of elicitors are applied to activate cryptic biosynthetic gene clusters, and metabolomics provide detailed insights into the NPs they specify. Combining these technologies with proteomics-based approaches to profile the biosynthetic enzymes provides scientists with insights into the full biosynthetic potential of microorganisms. The proteomics approaches include enrichment strategies such as employing activity-based probes designed by chemical biology, as well as unbiased (quantitative) proteomics methods. In this review, the opportunities and challenges in microbial NP research are discussed, and, in particular, the application of proteomics to link biosynthetic enzymes to the molecules they produce, and vice versa. © 2018 The Authors. Proteomics Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Proteomic analysis of synaptoneurosomes highlights the relevant role of local translation in the hippocampus.

PubMed

Benito, Itziar; Casañas, Juan José; Montesinos, María Luz

2018-06-19

Several proteomic analyses have been performed on synaptic fractions isolated from cortex or even total brain, resulting in preparations with a high synaptic heterogeneity and complexity. Synaptoneurosomes (SNs) are subcellular membranous elements that contain sealed pre- and post-synaptic components. They are obtained by subcellular fractionation of brain homogenates and serve as a suitable model to study many aspects of the synapse physiology. Here we report the proteomic content of SNs isolated from hippocampus of adult mice, a brain region involved in memory that presents lower synaptic heterogeneity than cortex. Interestingly, in addition to pre- and post-synaptic proteins, we found that proteins involved in RNA binding and translation were overrepresented in our preparation. These results validate the protocol we previously reported for SNs isolation, and, as reported by other authors, highlight the relevance of local synaptic translation for hippocampal physiology. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Proteomic analysis of Chlorella vulgaris: Potential targets for enhanced lipid accumulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guarnieri, Michael T.; Nag, Ambarish; Yang, Shihui

2013-11-01

Oleaginous microalgae are capable of producing large quantities of fatty acids and triacylglycerides. As such, they are promising feedstocks for the production of biofuels and bioproducts. Genetic strain-engineering strategies offer a means to accelerate the commercialization of algal biofuels by improving the rate and total accumulation of microalgal lipids. However, the industrial potential of these organisms remains to be met, largely due to the incomplete knowledgebase surrounding the mechanisms governing the induction of algal lipid biosynthesis. Such strategies require further elucidation of genes and gene products controlling algal lipid accumulation. In this study, we have set out to examine thesemore » mechanisms and identify novel strain-engineering targets in the oleaginous microalga, Chlorella vulgaris. Comparative shotgun proteomic analyses have identified a number of novel targets, including previously unidentified transcription factors and proteins involved in cell signaling and cell cycle regulation. These results lay the foundation for strain-improvement strategies and demonstrate the power of translational proteomic analysis.« less

Proteomic analysis of the response to cell cycle arrests in human myeloid leukemia cells

PubMed Central

Ly, Tony; Endo, Aki; Lamond, Angus I

2015-01-01

Abstract Previously, we analyzed protein abundance changes across a ‘minimally perturbed’ cell cycle by using centrifugal elutriation to differentially enrich distinct cell cycle phases in human NB4 cells (Ly et al., 2014). In this study, we compare data from elutriated cells with NB4 cells arrested at comparable phases using serum starvation, hydroxyurea, or RO-3306. While elutriated and arrested cells have similar patterns of DNA content and cyclin expression, a large fraction of the proteome changes detected in arrested cells are found to reflect arrest-specific responses (i.e., starvation, DNA damage, CDK1 inhibition), rather than physiological cell cycle regulation. For example, we show most cells arrested in G2 by CDK1 inhibition express abnormally high levels of replication and origin licensing factors and are likely poised for genome re-replication. The protein data are available in the Encyclopedia of Proteome Dynamics (http://www.peptracker.com/epd/), an online, searchable resource. DOI: http://dx.doi.org/10.7554/eLife.04534.001 PMID:25555159

Proteome analysis of bell pepper (Capsicum annuum L.) chromoplasts.

PubMed

Siddique, Muhammad Asim; Grossmann, Jonas; Gruissem, Wilhelm; Baginsky, Sacha

2006-12-01

We report a comprehensive proteome analysis of chromoplasts from bell pepper (Capsicum annuum L.). The combination of a novel strategy for database-independent detection of proteins from tandem mass spectrometry (MS/MS) data with standard database searches allowed us to identify 151 proteins with a high level of confidence. These include several well-known plastid proteins but also novel proteins that were not previously reported from other plastid proteome studies. The majority of the identified proteins are active in plastid carbohydrate and amino acid metabolism. Among the most abundant individual proteins are capsanthin/capsorubin synthase and fibrillin, which are involved in the synthesis and storage of carotenoids that accumulate to high levels in chromoplasts. The relative abundances of the identified chromoplast proteins differ remarkably compared with their abundances in other plastid types, suggesting a chromoplast-specific metabolic network. Our results provide an overview of the major metabolic pathways active in chromoplasts and extend existing knowledge about prevalent metabolic activities of different plastid types.

Simulated linear test applied to quantitative proteomics.

PubMed

Pham, T V; Jimenez, C R

2016-09-01

Omics studies aim to find significant changes due to biological or functional perturbation. However, gene and protein expression profiling experiments contain inherent technical variation. In discovery proteomics studies where the number of samples is typically small, technical variation plays an important role because it contributes considerably to the observed variation. Previous methods place both technical and biological variations in tightly integrated mathematical models that are difficult to adapt for different technological platforms. Our aim is to derive a statistical framework that allows the inclusion of a wide range of technical variability. We introduce a new method called the simulated linear test, or the s-test, that is easy to implement and easy to adapt for different models of technical variation. It generates virtual data points from the observed values according to a pre-defined technical distribution and subsequently employs linear modeling for significance analysis. We demonstrate the flexibility of the proposed approach by deriving a new significance test for quantitative discovery proteomics for which missing values have been a major issue for traditional methods such as the t-test. We evaluate the result on two label-free (phospho) proteomics datasets based on ion-intensity quantitation. Available at http://www.oncoproteomics.nl/software/stest.html : t.pham@vumc.nl. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Quantitative proteomics analysis using 2D-PAGE to investigate the effects of cigarette smoke and aerosol of a prototypic modified risk tobacco product on the lung proteome in C57BL/6 mice.

PubMed

Elamin, Ashraf; Titz, Bjoern; Dijon, Sophie; Merg, Celine; Geertz, Marcel; Schneider, Thomas; Martin, Florian; Schlage, Walter K; Frentzel, Stefan; Talamo, Fabio; Phillips, Blaine; Veljkovic, Emilija; Ivanov, Nikolai V; Vanscheeuwijck, Patrick; Peitsch, Manuel C; Hoeng, Julia

2016-08-11

Smoking is associated with several serious diseases, such as lung cancer and chronic obstructive pulmonary disease (COPD). Within our systems toxicology framework, we are assessing whether potential modified risk tobacco products (MRTP) can reduce smoking-related health risks compared to conventional cigarettes. In this article, we evaluated to what extent 2D-PAGE/MALDI MS/MS (2D-PAGE) can complement the iTRAQ LC-MS/MS results from a previously reported mouse inhalation study, in which we assessed a prototypic MRTP (pMRTP). Selected differentially expressed proteins identified by both LC-MS/MS and 2D-PAGE approaches were further verified using reverse-phase protein microarrays. LC-MS/MS captured the effects of cigarette smoke (CS) on the lung proteome more comprehensively than 2D-PAGE. However, an integrated analysis of both proteomics data sets showed that 2D-PAGE data complement the LC-MS/MS results by supporting the overall trend of lower effects of pMRTP aerosol than CS on the lung proteome. Biological effects of CS exposure supported by both methods included increases in immune-related, surfactant metabolism, proteasome, and actin cytoskeleton protein clusters. Overall, while 2D-PAGE has its value, especially as a complementary method for the analysis of effects on intact proteins, LC-MS/MS approaches will likely be the method of choice for proteome analysis in systems toxicology investigations. Quantitative proteomics is anticipated to play a growing role within systems toxicology assessment frameworks in the future. To further understand how different proteomics technologies can contribute to toxicity assessment, we conducted a quantitative proteomics analysis using 2D-PAGE and isobaric tag-based LC-MS/MS approaches and compared the results produced from the 2 approaches. Using a prototypic modified risk tobacco product (pMRTP) as our test item, we show compared with cigarette smoke, how 2D-PAGE results can complement and support LC-MS/MS data, demonstrating the much lower effects of pMRTP aerosol than cigarette smoke on the mouse lung proteome. The combined analysis of 2D-PAGE and LC-MS/MS data identified an effect of cigarette smoke on the proteasome and actin cytoskeleton in the lung. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Comparative proteomic profiling of the choline transporter-like1 (CHER1) mutant provides insights into plasmodesmata composition of fully developed Arabidopsis thaliana leaves.

PubMed

Kraner, Max E; Müller, Carmen; Sonnewald, Uwe

2017-11-01

In plants, intercellular communication and exchange are highly dependent on cell wall bridging structures between adhering cells, so-called plasmodesmata (PD). In our previous genetic screen for PD-deficient Arabidopsis mutants, we described choline transporter-like 1 (CHER1) being important for PD genesis and maturation. Leaves of cher1 mutant plants have up to 10 times less PD, which do not develop to complex structures. Here we utilize the T-DNA insertion mutant cher1-4 and report a deep comparative proteomic workflow for the identification of cell-wall-embedded PD-associated proteins. Analyzing triplicates of cell-wall-enriched fractions in depth by fractionation and quantitative high-resolution mass spectrometry, we compared > 5000 proteins obtained from fully developed leaves. Comparative data analysis and subsequent filtering generated a list of 61 proteins being significantly more abundant in Col-0. This list was enriched for previously described PD-associated proteins. To validate PD association of so far uncharacterized proteins, subcellular localization analyses were carried out by confocal laser-scanning microscopy. This study confirmed the association of PD for three out of four selected candidates, indicating that the comparative approach indeed allowed identification of so far undescribed PD-associated proteins. Performing comparative cell wall proteomics of Nicotiana benthamiana tissue, we observed an increase in abundance of these three selected candidates during sink to source transition. Taken together, our comparative proteomic approach revealed a valuable data set of potential PD-associated proteins, which can be used as a resource to unravel the molecular composition of complex PD and to investigate their function in cell-to-cell communication. © 2017 The Authors. The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.

Characterization and Comprehensive Proteome Profiling of Exosomes Secreted by Hepatocytes

PubMed Central

Conde-Vancells, Javier; Rodriguez-Suarez, Eva; Embade, Nieves; Gil, David; Matthiesen, Rune; Valle, Mikel; Elortza, Felix; Lu, Shelly C.; Mato, Jose M.; Falcon-Perez, Juan M.

2009-01-01

Synopsis Exosomes constitute a discrete population of nanometer-sized (30-150 nm) vesicles formed in endocytic compartments and released to the extracellular environment by different cell types. In this work we demonstrated by electron microscopic, western blotting and proteomic analyses that primary hepatocytes secrete exosome-like vesicles containing proteins involved in metabolizing lipoproteins, endogenous compounds as well as xenobiotics. These new findings contribute to improve our knowledge about biology's hepatocyte and may have important diagnostic, prognosis and therapeutic implications in liver diseases Exosomes represent a discrete population of vesicles that are secreted from various cell types to the extracellular media. Their protein and lipid composition are a consequence of sorting events at the level of the multivesicular body, a central organelle which integrates endocytic and secretory pathways. Characterization of exosomes from different biological samples has shown the presence of common as well as cell-type specific proteins. Remarkably, the protein content of the exosomes is modified upon pathological or stress conditions. Hepatocytes play a central role in the body response to stress metabolizing potentially harmful endogenous substances as well as xenobiotics. In the present study we described and characterized for first time exosome secretion in non-tumoral hepatocytes, and using a systematic proteomic approach, we establish the first extensive proteome of a hepatocyte-derived exosome population which should be useful in furthering our understanding of the hepatic function and in the identification of components that may serve as biomarkers for hepatic alterations. Our analysis identifies a significant number of proteins previously described among exosomes derived from others cell types as well as proteins involved in metabolizing lipoproteins, endogenous compounds and xenobiotics, not previously described in exosomes. Furthermore, we demonstrated that exosomal membrane proteins can constitute an interesting tool to express non-exosomal proteins into exosomes with therapeutic purposes. PMID:19367702

Data Independent Acquisition analysis in ProHits 4.0.

PubMed

Liu, Guomin; Knight, James D R; Zhang, Jian Ping; Tsou, Chih-Chiang; Wang, Jian; Lambert, Jean-Philippe; Larsen, Brett; Tyers, Mike; Raught, Brian; Bandeira, Nuno; Nesvizhskii, Alexey I; Choi, Hyungwon; Gingras, Anne-Claude

2016-10-21

Affinity purification coupled with mass spectrometry (AP-MS) is a powerful technique for the identification and quantification of physical interactions. AP-MS requires careful experimental design, appropriate control selection and quantitative workflows to successfully identify bona fide interactors amongst a large background of contaminants. We previously introduced ProHits, a Laboratory Information Management System for interaction proteomics, which tracks all samples in a mass spectrometry facility, initiates database searches and provides visualization tools for spectral counting-based AP-MS approaches. More recently, we implemented Significance Analysis of INTeractome (SAINT) within ProHits to provide scoring of interactions based on spectral counts. Here, we provide an update to ProHits to support Data Independent Acquisition (DIA) with identification software (DIA-Umpire and MSPLIT-DIA), quantification tools (through DIA-Umpire, or externally via targeted extraction), and assessment of quantitative enrichment (through mapDIA) and scoring of interactions (through SAINT-intensity). With additional improvements, notably support of the iProphet pipeline, facilitated deposition into ProteomeXchange repositories and enhanced export and viewing functions, ProHits 4.0 offers a comprehensive suite of tools to facilitate affinity proteomics studies. It remains challenging to score, annotate and analyze proteomics data in a transparent manner. ProHits was previously introduced as a LIMS to enable storing, tracking and analysis of standard AP-MS data. In this revised version, we expand ProHits to include integration with a number of identification and quantification tools based on Data-Independent Acquisition (DIA). ProHits 4.0 also facilitates data deposition into public repositories, and the transfer of data to new visualization tools. Copyright © 2016 Elsevier B.V. All rights reserved.

Isolation and Proteomic Characterization of the Mouse Sperm Acrosomal Matrix*

PubMed Central

Guyonnet, Benoit; Zabet-Moghaddam, Masoud; SanFrancisco, Susan; Cornwall, Gail A.

2012-01-01

A critical step during fertilization is the sperm acrosome reaction in which the acrosome releases its contents allowing the spermatozoa to penetrate the egg investments. The sperm acrosomal contents are composed of both soluble material and an insoluble material called the acrosomal matrix (AM). The AM is thought to provide a stable structure from which associated proteins are differentially released during fertilization. Because of its important role during fertilization, efforts have been put toward isolating the AM for biochemical study and to date AM have been isolated from hamster, guinea pig, and bull spermatozoa. However, attempts to isolate AM from mouse spermatozoa, the species in which fertilization is well-studied, have been unsuccessful possibly because of the small size of the mouse sperm acrosome and/or its fusiform shape. Herein we describe a procedure for the isolation of the AM from caput and cauda mouse epididymal spermatozoa. We further carried out a proteomic analysis of the isolated AM from both sperm populations and identified 501 new proteins previously not detected by proteomics in mouse spermatozoa. A comparison of the AM proteome from caput and cauda spermatozoa showed that the AM undergoes maturational changes during epididymal transit similar to other sperm domains. Together, our studies suggest the AM to be a dynamic and functional structure carrying out a variety of biological processes as implied by the presence of a diverse group of proteins including proteases, chaperones, hydrolases, transporters, enzyme modulators, transferases, cytoskeletal proteins, and others. PMID:22707618

Responses of Nannochloropsis oceanica IMET1 to Long-Term Nitrogen Starvation and Recovery1[C][W][OA

PubMed Central

Dong, Hong-Po; Williams, Ernest; Wang, Da-zhi; Xie, Zhang-Xian; Hsia, Ru-ching; Jenck, Alizée; Halden, Rolf; Li, Jing; Chen, Feng; Place, Allen R.

2013-01-01

The Nannochloropsis genus contains oleaginous microalgae that have served as model systems for developing renewable biodiesel. Recent genomic and transcriptomic studies on Nannochloropsis species have provided insights into the regulation of lipid production in response to nitrogen stress. Previous studies have focused on the responses of Nannochloropsis species to short-term nitrogen stress, but the effect of long-term nitrogen deprivation remains largely unknown. In this study, physiological and proteomic approaches were combined to understand the mechanisms by which Nannochloropsis oceanica IMET1 is able to endure long-term nitrate deprivation and its ability to recover homeostasis when nitrogen is amended. Changes of the proteome during chronic nitrogen starvation espoused the physiological changes observed, and there was a general trend toward recycling nitrogen and storage of lipids. This was evidenced by a global down-regulation of protein expression, a retained expression of proteins involved in glycolysis and the synthesis of fatty acids, as well as an up-regulation of enzymes used in nitrogen scavenging and protein turnover. Also, lipid accumulation and autophagy of plastids may play a key role in maintaining cell vitality. Following the addition of nitrogen, there were proteomic changes and metabolic changes observed within 24 h, which resulted in a return of the culture to steady state within 4 d. These results demonstrate the ability of N. oceanica IMET1 to recover from long periods of nitrate deprivation without apparent detriment to the culture and provide proteomic markers for genetic modification. PMID:23637339

Identification and Characterization of Soluble Factors Involved in Delayed Effects of Low Dose Radiation. Final report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baulch, Janet

2013-09-11

This is a 'glue grant' that was part of a DOE Low Dose project entitled 'Identification and Characterization of Soluble Factors Involved in Delayed Effects of Low Dose Radiation'. This collaborative program has involved Drs. David L. Springer from Pacific Northwest National Laboratory (PNNL), John H. Miller from Washington State University, Tri-cities (WSU) and William F. Morgan then from the University of Maryland, Baltimore (UMB). In July 2008, Dr. Morgan moved to PNNL and Dr. Janet E. Baulch became PI for this project at University of Maryland. In November of 2008, a one year extension with no new funds wasmore » requested to complete the proteomic analyses. The project stemmed from studies in the Morgan laboratory demonstrating that genomically unstable cells secret a soluble factor or factors into the culture medium, that cause cytogenetic aberrations and apoptosis in normal parental GM10115 cells. The purpose of this project was to identify the death inducing effect (DIE) factor or factors, estimate their relative abundance, identify the cell signaling pathways involved and finally recapitulate DIE in normal cells by exogenous manipulation of putative DIE factors in culture medium. As reported in detail in the previous progress report, analysis of culture medium from the parental cell line, and stable and unstable clones demonstrated inconsistent proteomic profiles as relate to candidate DIE factors. While the proposed proteomic analyses did not provide information that would allow DIE factors to be identified, the analyses provided another important set of observations. Proteomic analysis suggested that proteins associated with the cellular response to oxidative stress and mitochondrial function were elevated in the medium from unstable clones in a manner consistent with mitochondrial dysfunction. These findings correlate with previous studies of these clones that demonstrated functional differences between the mitochondria of stable and unstable clones. These mitochondrial abnormalities in the unstable clones contributes to oxidative stress.« less

A chemical proteomics approach for global analysis of lysine monomethylome profiling.

PubMed

Wu, Zhixiang; Cheng, Zhongyi; Sun, Mingwei; Wan, Xuelian; Liu, Ping; He, Tieming; Tan, Minjia; Zhao, Yingming

2015-02-01

Methylation of lysine residues on histone proteins is known to play an important role in chromatin structure and function. However, non-histone protein substrates of this modification remain largely unknown. An effective approach for system-wide analysis of protein lysine methylation, particularly lysine monomethylation, is lacking. Here we describe a chemical proteomics approach for global screening for monomethyllysine substrates, involving chemical propionylation of monomethylated lysine, affinity enrichment of the modified monomethylated peptides, and HPLC/MS/MS analysis. Using this approach, we identified with high confidence 446 lysine monomethylation sites in 398 proteins, including three previously unknown histone monomethylation marks, representing the largest data set of protein lysine monomethylation described to date. Our data not only confirms previously discovered lysine methylation substrates in the nucleus and spliceosome, but also reveals new substrates associated with diverse biological processes. This method hence offers a powerful approach for dynamic study of protein lysine monomethylation under diverse cellular conditions and in human diseases. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A Proteome-wide Fission Yeast Interactome Reveals Network Evolution Principles from Yeasts to Human.

PubMed

Vo, Tommy V; Das, Jishnu; Meyer, Michael J; Cordero, Nicolas A; Akturk, Nurten; Wei, Xiaomu; Fair, Benjamin J; Degatano, Andrew G; Fragoza, Robert; Liu, Lisa G; Matsuyama, Akihisa; Trickey, Michelle; Horibata, Sachi; Grimson, Andrew; Yamano, Hiroyuki; Yoshida, Minoru; Roth, Frederick P; Pleiss, Jeffrey A; Xia, Yu; Yu, Haiyuan

2016-01-14

Here, we present FissionNet, a proteome-wide binary protein interactome for S. pombe, comprising 2,278 high-quality interactions, of which ∼ 50% were previously not reported in any species. FissionNet unravels previously unreported interactions implicated in processes such as gene silencing and pre-mRNA splicing. We developed a rigorous network comparison framework that accounts for assay sensitivity and specificity, revealing extensive species-specific network rewiring between fission yeast, budding yeast, and human. Surprisingly, although genes are better conserved between the yeasts, S. pombe interactions are significantly better conserved in human than in S. cerevisiae. Our framework also reveals that different modes of gene duplication influence the extent to which paralogous proteins are functionally repurposed. Finally, cross-species interactome mapping demonstrates that coevolution of interacting proteins is remarkably prevalent, a result with important implications for studying human disease in model organisms. Overall, FissionNet is a valuable resource for understanding protein functions and their evolution. Copyright © 2016 Elsevier Inc. All rights reserved.

Arabidopsis proteome responses to the smoke-derived growth regulator karrikin.

PubMed

Baldrianová, Jana; Černý, Martin; Novák, Jan; Jedelský, Petr L; Divíšková, Eva; Brzobohatý, Břetislav

2015-04-29

Karrikins are butenolide plant growth regulators in smoke from burning plant material that have proven ability to promote germination and seedling photomorphogenesis. However, the molecular mechanisms underlying these processes are unclear. Here we provide the first proteome-wide analysis of early responses to karrikin in plants (Arabidopsis seedlings). Image analysis of two-dimensionally separated proteins, Rubisco-depleted proteomes and phosphoproteomes, together with LC-MS profiling, detected >1900 proteins, 113 of which responded to karrikin treatment. All the differentially abundant proteins (except HSP70-3) are novel karrikin-responders, and most are involved in photosynthesis, carbohydrate metabolism, redox homeostasis, transcription control, proteosynthesis, protein transport and processing, or protein degradation. Our data provide functionally complementary information to previous identifications of karrikin-responsive genes and evidence for a novel karrikin signalling pathway originating in chloroplasts. We present an updated model of karrikin signalling that integrates proteomic data and is supported by growth response observations. Karrikin has shown promising potential in agricultural applications, yet this process is poorly understood at the molecular level. To the best of our knowledge, this is the first survey of early global proteomic responses to karrikin in plants (Arabidopsis seedlings). The combination of label-free LC-MS profiling and 2-DE analyses provided highly sensitive snapshots of protein abundance and quantitative information on proteoform-level changes. These results present evidence of proteasome-independent karrikin signalling pathways and provide novel targets for detailed mechanistic studies using, e.g., mutants and transgenic plants. Copyright © 2015. Published by Elsevier B.V.

Proteomic identification of fat-browning markers in cultured white adipocytes treated with curcumin.

PubMed

Kim, Sang Woo; Choi, Jae Heon; Mukherjee, Rajib; Hwang, Ki-Chul; Yun, Jong Won

2016-04-01

We previously reported that curcumin induces browning of primary white adipocytes via enhanced expression of brown adipocyte-specific genes. In this study, we attempted to identify target proteins responsible for this fat-browning effect by analyzing proteomic changes in cultured white adipocytes in response to curcumin treatment. To elucidate the role of curcumin in fat-browning, we conducted comparative proteomic analysis of primary adipocytes between control and curcumin-treated cells using two-dimensional electrophoresis combined with MALDI-TOF-MS. We also investigated fatty acid metabolic targets, mitochondrial biogenesis, and fat-browning-associated proteins using combined proteomic and network analyses. Proteomic analysis revealed that 58 protein spots from a total of 325 matched spots showed differential expression between control and curcumin-treated adipocytes. Using network analysis, most of the identified proteins were proven to be involved in various metabolic and cellular processes based on the PANTHER classification system. One of the most striking findings is that hormone-sensitive lipase (HSL) was highly correlated with main browning markers based on the STRING database. HSL and two browning markers (UCP1, PGC-1α) were co-immunoprecipitated with these markers, suggesting that HSL possibly plays a role in fat-browning of white adipocytes. Our results suggest that curcumin increased HSL levels and other browning-specific markers, suggesting its possible role in augmentation of lipolysis and suppression of lipogenesis by trans-differentiation from white adipocytes into brown adipocytes (beige).

A flexible statistical model for alignment of label-free proteomics data – incorporating ion mobility and product ion information

PubMed Central

2013-01-01

Background The goal of many proteomics experiments is to determine the abundance of proteins in biological samples, and the variation thereof in various physiological conditions. High-throughput quantitative proteomics, specifically label-free LC-MS/MS, allows rapid measurement of thousands of proteins, enabling large-scale studies of various biological systems. Prior to analyzing these information-rich datasets, raw data must undergo several computational processing steps. We present a method to address one of the essential steps in proteomics data processing - the matching of peptide measurements across samples. Results We describe a novel method for label-free proteomics data alignment with the ability to incorporate previously unused aspects of the data, particularly ion mobility drift times and product ion information. We compare the results of our alignment method to PEPPeR and OpenMS, and compare alignment accuracy achieved by different versions of our method utilizing various data characteristics. Our method results in increased match recall rates and similar or improved mismatch rates compared to PEPPeR and OpenMS feature-based alignment. We also show that the inclusion of drift time and product ion information results in higher recall rates and more confident matches, without increases in error rates. Conclusions Based on the results presented here, we argue that the incorporation of ion mobility drift time and product ion information are worthy pursuits. Alignment methods should be flexible enough to utilize all available data, particularly with recent advancements in experimental separation methods. PMID:24341404

A flexible statistical model for alignment of label-free proteomics data--incorporating ion mobility and product ion information.

PubMed

Benjamin, Ashlee M; Thompson, J Will; Soderblom, Erik J; Geromanos, Scott J; Henao, Ricardo; Kraus, Virginia B; Moseley, M Arthur; Lucas, Joseph E

2013-12-16

The goal of many proteomics experiments is to determine the abundance of proteins in biological samples, and the variation thereof in various physiological conditions. High-throughput quantitative proteomics, specifically label-free LC-MS/MS, allows rapid measurement of thousands of proteins, enabling large-scale studies of various biological systems. Prior to analyzing these information-rich datasets, raw data must undergo several computational processing steps. We present a method to address one of the essential steps in proteomics data processing--the matching of peptide measurements across samples. We describe a novel method for label-free proteomics data alignment with the ability to incorporate previously unused aspects of the data, particularly ion mobility drift times and product ion information. We compare the results of our alignment method to PEPPeR and OpenMS, and compare alignment accuracy achieved by different versions of our method utilizing various data characteristics. Our method results in increased match recall rates and similar or improved mismatch rates compared to PEPPeR and OpenMS feature-based alignment. We also show that the inclusion of drift time and product ion information results in higher recall rates and more confident matches, without increases in error rates. Based on the results presented here, we argue that the incorporation of ion mobility drift time and product ion information are worthy pursuits. Alignment methods should be flexible enough to utilize all available data, particularly with recent advancements in experimental separation methods.

Proteomic analysis of bovine nucleolus.

PubMed

Patel, Amrutlal K; Olson, Doug; Tikoo, Suresh K

2010-09-01

Nucleolus is the most prominent subnuclear structure, which performs a wide variety of functions in the eukaryotic cellular processes. In order to understand the structural and functional role of the nucleoli in bovine cells, we analyzed the proteomic composition of the bovine nucleoli. The nucleoli were isolated from Madin Darby bovine kidney cells and subjected to proteomic analysis by LC-MS/MS after fractionation by SDS-PAGE and strong cation exchange chromatography. Analysis of the data using the Mascot database search and the GPM database search identified 311 proteins in the bovine nucleoli, which contained 22 proteins previously not identified in the proteomic analysis of human nucleoli. Analysis of the identified proteins using the GoMiner software suggested that the bovine nucleoli contained proteins involved in ribosomal biogenesis, cell cycle control, transcriptional, translational and post-translational regulation, transport, and structural organization. Copyright © 2010 Beijing Genomics Institute. Published by Elsevier Ltd. All rights reserved.

Integrated proteogenomic characterization of human high grade serous ovarian cancer

PubMed Central

Zhang, Bai; McDermott, Jason E; Zhou, Jian-Ying; Petyuk, Vladislav A; Chen, Li; Ray, Debjit; Sun, Shisheng; Yang, Feng; Chen, Lijun; Wang, Jing; Shah, Punit; Cha, Seong Won; Aiyetan, Paul; Woo, Sunghee; Tian, Yuan; Gritsenko, Marina A; Clauss, Therese R; Choi, Caitlin; Monroe, Matthew E; Thomas, Stefani; Nie, Song; Wu, Chaochao; Moore, Ronald J; Yu, Kun-Hsing; Tabb, David L; Fenyö, David; Bafna, Vineet; Wang, Yue; Rodriguez, Henry; Boja, Emily S; Hiltke, Tara; Rivers, Robert C; Sokoll, Lori; Zhu, Heng; Shih, Ie-Ming; Cope, Leslie; Pandey, Akhilesh; Zhang, Bing; Snyder, Michael P; Levine, Douglas A; Smith, Richard D

2016-01-01

SUMMARY To provide a detailed analysis of the molecular components and underlying mechanisms associated with ovarian cancer, we performed a comprehensive mass spectrometry-based proteomic characterization of 174 ovarian tumors previously analyzed by The Cancer Genome Atlas (TCGA), of which 169 were high-grade serous carcinomas (HGSC). Integrating our proteomic measurements with the genomic data yielded a number of insights into disease such as how different copy number alternations influence the proteome, the proteins associated with chromosomal instability, the sets of signaling pathways that diverse genome rearrangements converge on, as well as the ones most associated with short overall survival. Specific protein acetylations associated with homologous recombination deficiency suggest a potential means for stratifying patients for therapy. In addition to providing a valuable resource, these findings provide a view of how the somatic genome drives the cancer proteome and associations between protein and post-translational modification levels and clinical outcomes in HGSC. PMID:27372738

Polyphemus, Odysseus and the ovine milk proteome.

PubMed

Cunsolo, Vincenzo; Fasoli, Elisa; Di Francesco, Antonella; Saletti, Rosaria; Muccilli, Vera; Gallina, Serafina; Righetti, Pier Giorgio; Foti, Salvatore

2017-01-30

In the last years the amount of ovine milk production, mainly used to formulate a wide range of different and exclusive dairy products often categorized as gourmet food, has been progressively increasing. Taking also into account that sheep milk (SM) also appears to be potentially less allergenic than cow's one, an in-depth information about its protein composition is essential to improve the comprehension of its potential benefits for human consumption. The present work reports the results of an in-depth characterization of SM whey proteome, carried out by coupling the CPLL technology with SDS-PAGE and high resolution UPLC-nESI MS/MS analysis. This approach allowed the identification of 718 different protein components, 644 of which are from unique genes. Particularly, this identification has expanded literature data about sheep whey proteome by 193 novel proteins previously undetected, many of which are involved in the defence/immunity mechanisms or in the nutrient delivery system. A comparative analysis of SM proteome known to date with cow's milk proteome, evidenced that while about 29% of SM proteins are also present in CM, 71% of the identified components appear to be unique of SM proteome and include a heterogeneous group of components which seem to have health-promoting benefits. The data have been deposited to the ProteomeXchange with identifier . Copyright © 2016 Elsevier B.V. All rights reserved.

Urinary Proteomic Biomarkers for Diagnosis and Risk Stratification of Autosomal Dominant Polycystic Kidney Disease: A Multicentric Study

PubMed Central

Kistler, Andreas D.; Serra, Andreas L.; Siwy, Justyna; Poster, Diane; Krauer, Fabienne; Torres, Vicente E.; Mrug, Michal; Grantham, Jared J.; Bae, Kyongtae T.; Bost, James E.; Mullen, William; Wüthrich, Rudolf P.; Mischak, Harald; Chapman, Arlene B.

2013-01-01

Treatment options for autosomal dominant polycystic kidney disease (ADPKD) will likely become available in the near future, hence reliable diagnostic and prognostic biomarkers for the disease are strongly needed. Here, we aimed to define urinary proteomic patterns in ADPKD patients, which aid diagnosis and risk stratification. By capillary electrophoresis online coupled to mass spectrometry (CE-MS), we compared the urinary peptidome of 41 ADPKD patients to 189 healthy controls and identified 657 peptides with significantly altered excretion, of which 209 could be sequenced using tandem mass spectrometry. A support-vector-machine based diagnostic biomarker model based on the 142 most consistent peptide markers achieved a diagnostic sensitivity of 84.5% and specificity of 94.2% in an independent validation cohort, consisting of 251 ADPKD patients from five different centers and 86 healthy controls. The proteomic alterations in ADPKD included, but were not limited to markers previously associated with acute kidney injury (AKI). The diagnostic biomarker model was highly specific for ADPKD when tested in a cohort consisting of 481 patients with a variety of renal and extrarenal diseases, including AKI. Similar to ultrasound, sensitivity and specificity of the diagnostic score depended on patient age and genotype. We were furthermore able to identify biomarkers for disease severity and progression. A proteomic severity score was developed to predict height adjusted total kidney volume (htTKV) based on proteomic analysis of 134 ADPKD patients and showed a correlation of r = 0.415 (p<0.0001) with htTKV in an independent validation cohort consisting of 158 ADPKD patients. In conclusion, the performance of peptidomic biomarker scores is superior to any other biochemical markers of ADPKD and the proteomic biomarker patterns are a promising tool for prognostic evaluation of ADPKD. PMID:23326375

The Skeleton Forming Proteome of an Early Branching Metazoan: A Molecular Survey of the Biomineralization Components Employed by the Coralline Sponge Vaceletia Sp.

PubMed Central

Wörheide, Gert; Jackson, Daniel John

2015-01-01

The ability to construct a mineralized skeleton was a major innovation for the Metazoa during their evolution in the late Precambrian/early Cambrian. Porifera (sponges) hold an informative position for efforts aimed at unraveling the origins of this ability because they are widely regarded to be the earliest branching metazoans, and are among the first multi-cellular animals to display the ability to biomineralize in the fossil record. Very few biomineralization associated proteins have been identified in sponges so far, with no transcriptome or proteome scale surveys yet available. In order to understand what genetic repertoire may have been present in the last common ancestor of the Metazoa (LCAM), and that may have contributed to the evolution of the ability to biocalcify, we have studied the skeletal proteome of the coralline demosponge Vaceletia sp. and compare this to other metazoan biomineralizing proteomes. We bring some spatial resolution to this analysis by dividing Vaceletia’s aragonitic calcium carbonate skeleton into “head” and “stalk” regions. With our approach we were able to identify 40 proteins from both the head and stalk regions, with many of these sharing some similarity to previously identified gene products from other organisms. Among these proteins are known biomineralization compounds, such as carbonic anhydrase, spherulin, extracellular matrix proteins and very acidic proteins. This report provides the first proteome scale analysis of a calcified poriferan skeletal proteome, and its composition clearly demonstrates that the LCAM contributed several key enzymes and matrix proteins to its descendants that supported the metazoan ability to biocalcify. However, lineage specific evolution is also likely to have contributed significantly to the ability of disparate metazoan lineages to biocalcify. PMID:26536128

The nuclear proteome of Trypanosoma brucei

PubMed Central

Goos, Carina; Dejung, Mario; Janzen, Christian J.; Butter, Falk

2017-01-01

Trypanosoma brucei is a protozoan flagellate that is transmitted by tsetse flies into the mammalian bloodstream. The parasite has a huge impact on human health both directly by causing African sleeping sickness and indirectly, by infecting domestic cattle. The biology of trypanosomes involves some highly unusual, nuclear-localised processes. These include polycistronic transcription without classical promoters initiated from regions defined by histone variants, trans-splicing of all transcripts to the exon of a spliced leader RNA, transcription of some very abundant proteins by RNA polymerase I and antigenic variation, a switch in expression of the cell surface protein variants that allows the parasite to resist the immune system of its mammalian host. Here, we provide the nuclear proteome of procyclic Trypanosoma brucei, the stage that resides within the tsetse fly midgut. We have performed quantitative label-free mass spectrometry to score 764 significantly nuclear enriched proteins in comparison to whole cell lysates. A comparison with proteomes of several experimentally characterised nuclear and non-nuclear structures and pathways confirmed the high quality of the dataset: the proteome contains about 80% of all nuclear proteins and less than 2% false positives. Using motif enrichment, we found the amino acid sequence KRxR present in a large number of nuclear proteins. KRxR is a sub-motif of a classical eukaryotic monopartite nuclear localisation signal and could be responsible for nuclear localization of proteins in Kinetoplastida species. As a proof of principle, we have confirmed the nuclear localisation of six proteins with previously unknown localisation by expressing eYFP fusion proteins. While proteome data of several T. brucei organelles have been published, our nuclear proteome closes an important gap in knowledge to study trypanosome biology, in particular nuclear-related processes. PMID:28727848

Surveying alignment-free features for Ortholog detection in related yeast proteomes by using supervised big data classifiers.

PubMed

Galpert, Deborah; Fernández, Alberto; Herrera, Francisco; Antunes, Agostinho; Molina-Ruiz, Reinaldo; Agüero-Chapin, Guillermin

2018-05-03

The development of new ortholog detection algorithms and the improvement of existing ones are of major importance in functional genomics. We have previously introduced a successful supervised pairwise ortholog classification approach implemented in a big data platform that considered several pairwise protein features and the low ortholog pair ratios found between two annotated proteomes (Galpert, D et al., BioMed Research International, 2015). The supervised models were built and tested using a Saccharomycete yeast benchmark dataset proposed by Salichos and Rokas (2011). Despite several pairwise protein features being combined in a supervised big data approach; they all, to some extent were alignment-based features and the proposed algorithms were evaluated on a unique test set. Here, we aim to evaluate the impact of alignment-free features on the performance of supervised models implemented in the Spark big data platform for pairwise ortholog detection in several related yeast proteomes. The Spark Random Forest and Decision Trees with oversampling and undersampling techniques, and built with only alignment-based similarity measures or combined with several alignment-free pairwise protein features showed the highest classification performance for ortholog detection in three yeast proteome pairs. Although such supervised approaches outperformed traditional methods, there were no significant differences between the exclusive use of alignment-based similarity measures and their combination with alignment-free features, even within the twilight zone of the studied proteomes. Just when alignment-based and alignment-free features were combined in Spark Decision Trees with imbalance management, a higher success rate (98.71%) within the twilight zone could be achieved for a yeast proteome pair that underwent a whole genome duplication. The feature selection study showed that alignment-based features were top-ranked for the best classifiers while the runners-up were alignment-free features related to amino acid composition. The incorporation of alignment-free features in supervised big data models did not significantly improve ortholog detection in yeast proteomes regarding the classification qualities achieved with just alignment-based similarity measures. However, the similarity of their classification performance to that of traditional ortholog detection methods encourages the evaluation of other alignment-free protein pair descriptors in future research.

Functional insights from proteome-wide structural modeling of Treponema pallidum subspecies pallidum, the causative agent of syphilis.

PubMed

Houston, Simon; Lithgow, Karen Vivien; Osbak, Kara Krista; Kenyon, Chris Richard; Cameron, Caroline E

2018-05-16

Syphilis continues to be a major global health threat with 11 million new infections each year, and a global burden of 36 million cases. The causative agent of syphilis, Treponema pallidum subspecies pallidum, is a highly virulent bacterium, however the molecular mechanisms underlying T. pallidum pathogenesis remain to be definitively identified. This is due to the fact that T. pallidum is currently uncultivatable, inherently fragile and thus difficult to work with, and phylogenetically distinct with no conventional virulence factor homologs found in other pathogens. In fact, approximately 30% of its predicted protein-coding genes have no known orthologs or assigned functions. Here we employed a structural bioinformatics approach using Phyre2-based tertiary structure modeling to improve our understanding of T. pallidum protein function on a proteome-wide scale. Phyre2-based tertiary structure modeling generated high-confidence predictions for 80% of the T. pallidum proteome (780/978 predicted proteins). Tertiary structure modeling also inferred the same function as primary structure-based annotations from genome sequencing pipelines for 525/605 proteins (87%), which represents 54% (525/978) of all T. pallidum proteins. Of the 175 T. pallidum proteins modeled with high confidence that were not assigned functions in the previously annotated published proteome, 167 (95%) were able to be assigned predicted functions. Twenty-one of the 175 hypothetical proteins modeled with high confidence were also predicted to exhibit significant structural similarity with proteins experimentally confirmed to be required for virulence in other pathogens. Phyre2-based structural modeling is a powerful bioinformatics tool that has provided insight into the potential structure and function of the majority of T. pallidum proteins and helped validate the primary structure-based annotation of more than 50% of all T. pallidum proteins with high confidence. This work represents the first T. pallidum proteome-wide structural modeling study and is one of few studies to apply this approach for the functional annotation of a whole proteome.

A Review: Proteomics in Retinal Artery Occlusion, Retinal Vein Occlusion, Diabetic Retinopathy and Acquired Macular Disorders.

PubMed

Cehofski, Lasse Jørgensen; Honoré, Bent; Vorum, Henrik

2017-04-28

Retinal artery occlusion (RAO), retinal vein occlusion (RVO), diabetic retinopathy (DR) and age-related macular degeneration (AMD) are frequent ocular diseases with potentially sight-threatening outcomes. In the present review we discuss major findings of proteomic studies of RAO, RVO, DR and AMD, including an overview of ocular proteome changes associated with anti-vascular endothelial growth factor (VEGF) treatments. Despite the severe outcomes of RAO, the proteome of the disease remains largely unstudied. There is also limited knowledge about the proteome of RVO, but proteomic studies suggest that RVO is associated with remodeling of the extracellular matrix and adhesion processes. Proteomic studies of DR have resulted in the identification of potential therapeutic targets such as carbonic anhydrase-I. Proliferative diabetic retinopathy is the most intensively studied stage of DR. Proteomic studies have established VEGF, pigment epithelium-derived factor (PEDF) and complement components as key factors associated with AMD. The aim of this review is to highlight the major milestones in proteomics in RAO, RVO, DR and AMD. Through large-scale protein analyses, proteomics is bringing new important insights into these complex pathological conditions.

Label-free proteomic analysis to confirm the predicted proteome of Corynebacterium pseudotuberculosis under nitrosative stress mediated by nitric oxide.

PubMed

Silva, Wanderson M; Carvalho, Rodrigo D; Soares, Siomar C; Bastos, Isabela Fs; Folador, Edson L; Souza, Gustavo Hmf; Le Loir, Yves; Miyoshi, Anderson; Silva, Artur; Azevedo, Vasco

2014-12-04

Corynebacterium pseudotuberculosis biovar ovis is a facultative intracellular pathogen, and the etiological agent of caseous lymphadenitis in small ruminants. During the infection process, the bacterium is subjected to several stress conditions, including nitrosative stress, which is caused by nitric oxide (NO). In silico analysis of the genome of C. pseudotuberculosis ovis 1002 predicted several genes that could influence the resistance of this pathogen to nitrosative stress. Here, we applied high-throughput proteomics using high definition mass spectrometry to characterize the functional genome of C. pseudotuberculosis ovis 1002 in the presence of NO-donor Diethylenetriamine/nitric oxide adduct (DETA/NO), with the aim of identifying proteins involved in nitrosative stress resistance. We characterized 835 proteins, representing approximately 41% of the predicted proteome of C. pseudotuberculosis ovis 1002, following exposure to nitrosative stress. In total, 102 proteins were exclusive to the proteome of DETA/NO-induced cells, and a further 58 proteins were differentially regulated between the DETA/NO and control conditions. An interactomic analysis of the differential proteome of C. pseudotuberculosis in response to nitrosative stress was also performed. Our proteomic data set suggested the activation of both a general stress response and a specific nitrosative stress response, as well as changes in proteins involved in cellular metabolism, detoxification, transcriptional regulation, and DNA synthesis and repair. Our proteomic analysis validated previously-determined in silico data for C. pseudotuberculosis ovis 1002. In addition, proteomic screening performed in the presence of NO enabled the identification of a set of factors that can influence the resistance and survival of C. pseudotuberculosis during exposure to nitrosative stress.

Proteomic approach toward molecular backgrounds of drug resistance of osteosarcoma cells in spheroid culture system.

PubMed

Arai, Kazuya; Sakamoto, Ruriko; Kubota, Daisuke; Kondo, Tadashi

2013-08-01

Chemoresistance is one of the most critical prognostic factors in osteosarcoma, and elucidation of the molecular backgrounds of chemoresistance may lead to better clinical outcomes. Spheroid cells resemble in vivo cells and are considered an in vitro model for the drug discovery. We found that spheroid cells displayed more chemoresistance than conventional monolayer cells across 11 osteosarcoma cell lines. To investigate the molecular mechanisms underlying the resistance to chemotherapy, we examined the proteomic differences between the monolayer and spheroid cells by 2D-DIGE. Of the 4762 protein species observed, we further investigated 435 species with annotated mass spectra in the public proteome database, Genome Medicine Database of Japan Proteomics. Among the 435 protein species, we found that 17 species exhibited expression level differences when the cells formed spheroids in more than five cell lines and four species out of these 17 were associated with spheroid-formation associated resistance to doxorubicin. We confirmed the upregulation of cathepsin D in spheroid cells by western blotting. Cathepsin D has been implicated in chemoresistance of various malignancies but has not previously been implemented in osteosarcoma. Our study suggested that the spheroid system may be a useful tool to reveal the molecular backgrounds of chemoresistance in osteosarcoma. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Proteomics Analysis of Molecular Risk Factors in the Ocular Hypertensive Human Retina

PubMed Central

Yang, Xiangjun; Hondur, Gözde; Li, Ming; Cai, Jian; Klein, Jon B.; Kuehn, Markus H.; Tezel, Gülgün

2015-01-01

Purpose To better understand ocular hypertension–induced early molecular alterations that may determine the initiation of neurodegeneration in human glaucoma, this study analyzed retinal proteomic alterations in the ocular hypertensive human retina. Methods Retina samples were obtained from six human donors with ocular hypertension (without glaucomatous injury) and six age- and sex-matched normotensive controls. Retinal proteins were analyzed by two-dimensional LC-MS/MS (liquid chromatography and linear ion trap mass spectrometry) using oxygen isotope labeling for relative quantification of protein expression. Proteomics data were validated by Western blot and immunohistochemical analyses of selected proteins. Results Out of over 2000 retinal proteins quantified, hundreds exhibited over 2-fold increased or decreased expression in ocular hypertensive samples relative to normotensive controls. Bioinformatics linked the proteomics datasets to various pathways important for maintenance of cellular homeostasis in the ocular hypertensive retina. Upregulated proteins included various heat shock proteins, ubiquitin proteasome pathway components, antioxidants, and DNA repair enzymes, while many proteins involved in mitochondrial oxidative phosphorylation exhibited downregulation in the ocular hypertensive retina. Despite the altered protein expression reflecting intrinsic adaptive/protective responses against mitochondrial energy failure, oxidative stress, and unfolded proteins, no alterations suggestive of an ongoing cell death process or neuroinflammation were detectable. Conclusions This study provides information about ocular hypertension–related molecular risk factors for glaucoma development. Molecular alterations detected in the ocular hypertensive human retina as opposed to previously detected alterations in human donor retinas with clinically manifest glaucoma suggest that proteome alterations determine the individual threshold to tolerate the ocular hypertension–induced tissue stress or convert to glaucomatous neurodegeneration when intrinsic adaptive/protective responses are overwhelmed. PMID:26348630

Dynamic Response of Mycobacterium vanbaalenii PYR-1 to BP Deepwater Horizon Crude Oil

PubMed Central

Kim, Seong-Jae; Kweon, Ohgew; Sutherland, John B.; Kim, Hyun-Lee; Jones, Richard C.; Burback, Brian L.; Graves, Steven W.; Psurny, Edward

2015-01-01

We investigated the response of the hydrocarbon-degrading Mycobacterium vanbaalenii PYR-1 to crude oil from the BP Deepwater Horizon (DWH) spill, using substrate depletion, genomic, and proteome analyses. M. vanbaalenii PYR-1 cultures were incubated with BP DWH crude oil, and proteomes and degradation of alkanes and polycyclic aromatic hydrocarbons (PAHs) were analyzed at four time points over 30 days. Gas chromatography-mass spectrometry (GC-MS) analysis showed a chain length-dependent pattern of alkane degradation, with C12 and C13 being degraded at the highest rate, although alkanes up to C28 were degraded. Whereas phenanthrene and pyrene were completely degraded, a significantly smaller amount of fluoranthene was degraded. Proteome analysis identified 3,948 proteins, with 876 and 1,859 proteins up- and downregulated, respectively. We observed dynamic changes in protein expression during BP crude oil incubation, including transcriptional factors and transporters potentially involved in adaptation to crude oil. The proteome also provided a molecular basis for the metabolism of the aliphatic and aromatic hydrocarbon components in the BP DWH crude oil, which included upregulation of AlkB alkane hydroxylase and an expression pattern of PAH-metabolizing enzymes different from those in previous proteome expression studies of strain PYR-1 incubated with pure or mixed PAHs, particularly the ring-hydroxylating oxygenase (RHO) responsible for the initial oxidation of aromatic hydrocarbons. Based on these results, a comprehensive cellular response of M. vanbaalenii PYR-1 to BP crude oil was proposed. This study increases our fundamental understanding of the impact of crude oil on the cellular response of bacteria and provides data needed for development of practical bioremediation applications. PMID:25888169

Dynamic changes in the date palm fruit proteome during development and ripening

PubMed Central

Marondedze, Claudius; Gehring, Christoph; Thomas, Ludivine

2014-01-01

Date palm (Phoenix dactylifera) is an economically important fruit tree in the Middle East and North Africa and is characterized by large cultivar diversity, making it a good model for studies on fruit development and other important traits. Here in gel comparative proteomics combined with tandem mass spectrometry were used to study date fruit development and ripening. Total proteins were extracted using a phenol-based protocol. A total of 189 protein spots were differentially regulated (p≤0.05). The identified proteins were classified into 14 functional categories. The categories with the most proteins were ‘disease and defense’ (16.5%) and ‘metabolism’ (15.4%). Twenty-nine proteins have not previously been identified in other fleshy fruits and 64 showed contrasting expression patterns in other fruits. Abundance of most proteins with a role in abiotic stress responses increased during ripening with the exception of heat shock proteins. Proteins with a role in anthocyanin biosynthesis, glycolysis, tricarboxylic acid cycle and cell wall degradation were upregulated particularly from the onset of ripening and during ripening. In contrast, expression of pentose phosphate- and photosynthesis-related proteins decreased during fruit maturation. Although date palm is considered a climacteric species, the analysis revealed downregulation of two enzymes involved in ethylene biosynthesis, suggesting an ethylene-independent ripening of ‘Barhi’ fruits. In summary, this proteomics study provides insights into physiological processes during date fruit development and ripening at the systems level and offers a reference proteome for the study of regulatory mechanisms that can inform molecular and biotechnological approaches to further improvements of horticultural traits including fruit quality and yield. PMID:26504545

Biomolecular Profiling of Jet Fuel Toxicity Using Proteomics

DTIC Science & Technology

2006-02-28

pulmonary alveolar type II cells and macrophages, and human epidermal keratinocytes in various exposure models. Results strongly suggest an injurious effect ...of exposure on all cells studied. In both pulmonary and skin cells, the protein profiles of JP-8 effect corroborates previous histological findings...potential intervention by Substance P (SP) in the pulmonary effects of JP-8 exposure , studies incorporating SP treatment along with JP-8 exposure

Morphology and proteome characterization of the salivary glands of the western chinch bug, Blissus occiduus (Hemiptera: Blissidae)

USDA-ARS?s Scientific Manuscript database

The western chinch bug, Blissus occiduus Barber, is a serious pest of buffalograss due to physical and chemical damage caused during the feeding process. Although previous work has investigated the feeding behaviors of chinch bugs in the Blissus complex, no study to date has explored salivary gland ...

A Review: Proteomics in Retinal Artery Occlusion, Retinal Vein Occlusion, Diabetic Retinopathy and Acquired Macular Disorders

PubMed Central

Cehofski, Lasse Jørgensen; Honoré, Bent; Vorum, Henrik

2017-01-01

Retinal artery occlusion (RAO), retinal vein occlusion (RVO), diabetic retinopathy (DR) and age-related macular degeneration (AMD) are frequent ocular diseases with potentially sight-threatening outcomes. In the present review we discuss major findings of proteomic studies of RAO, RVO, DR and AMD, including an overview of ocular proteome changes associated with anti-vascular endothelial growth factor (VEGF) treatments. Despite the severe outcomes of RAO, the proteome of the disease remains largely unstudied. There is also limited knowledge about the proteome of RVO, but proteomic studies suggest that RVO is associated with remodeling of the extracellular matrix and adhesion processes. Proteomic studies of DR have resulted in the identification of potential therapeutic targets such as carbonic anhydrase-I. Proliferative diabetic retinopathy is the most intensively studied stage of DR. Proteomic studies have established VEGF, pigment epithelium-derived factor (PEDF) and complement components as key factors associated with AMD. The aim of this review is to highlight the major milestones in proteomics in RAO, RVO, DR and AMD. Through large-scale protein analyses, proteomics is bringing new important insights into these complex pathological conditions. PMID:28452939

The developmental proteome of Drosophila melanogaster

PubMed Central

Casas-Vila, Nuria; Bluhm, Alina; Sayols, Sergi; Dinges, Nadja; Dejung, Mario; Altenhein, Tina; Kappei, Dennis; Altenhein, Benjamin; Roignant, Jean-Yves; Butter, Falk

2017-01-01

Drosophila melanogaster is a widely used genetic model organism in developmental biology. While this model organism has been intensively studied at the RNA level, a comprehensive proteomic study covering the complete life cycle is still missing. Here, we apply label-free quantitative proteomics to explore proteome remodeling across Drosophila’s life cycle, resulting in 7952 proteins, and provide a high temporal-resolved embryogenesis proteome of 5458 proteins. Our proteome data enabled us to monitor isoform-specific expression of 34 genes during development, to identify the pseudogene Cyp9f3Ψ as a protein-coding gene, and to obtain evidence of 268 small proteins. Moreover, the comparison with available transcriptomic data uncovered examples of poor correlation between mRNA and protein, underscoring the importance of proteomics to study developmental progression. Data integration of our embryogenesis proteome with tissue-specific data revealed spatial and temporal information for further functional studies of yet uncharacterized proteins. Overall, our high resolution proteomes provide a powerful resource and can be explored in detail in our interactive web interface. PMID:28381612

Quantitative proteomics of bronchoalveolar lavage fluid in lung adenocarcinoma.

PubMed

Almatroodi, Saleh A; McDonald, Christine F; Collins, Allison L; Darby, Ian A; Pouniotis, Dodie S

2015-01-01

The most commonly reported primary lung cancer subtype is adenocarcinoma, which is associated with a poor prognosis and short survival. Proteomic studies on human body fluids such as bronchoalveolar lavage fluid (BALF) have become essential methods for biomarker discovery, examination of tumor pathways and investigation of potential treatments. This study used quantitative proteomics to investigate the up-regulation of novel proteins in BALF from patients with primary lung adenocarcinoma in order to identify potential biomarkers. BALF samples from individuals with and without primary lung adenocarcinoma were analyzed using liquid chromatography-mass spectrometry. One thousand and one hundred proteins were identified, 33 of which were found to be consistently overexpressed in all lung adenocarcinoma samples compared to non-cancer controls. A number of overexpressed proteins have been previously shown to be related to lung cancer progression including S100-A8, annexin A1, annexin A2, thymidine phosphorylase and transglutaminase 2. The overexpression of a number of specific proteins in BALF from patients with primary lung adenocarcinoma may be used as a potential biomarker for lung adenocarcinoma. Copyright© 2015, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.

Enhancement of Environmental Hazard Degradation in the Presence of Lignin: a Proteomics Study

DOE PAGES

Sun, Su; Xie, Shangxian; Cheng, Yanbing; ...

2017-09-12

Proteomics studies of fungal systems have progressed dramatically based on the availability of more fungal genome sequences in recent years. Different proteomics strategies have been applied toward characterization of fungal proteome and revealed important gene functions and proteome dynamics. Presented here is the application of shot-gun proteomic technology to study the bio-remediation of environmental hazards by white-rot fungus. Lignin, a naturally abundant component of the plant biomass, is discovered to promote the degradation of Azo dye by white-rot fungus Irpex lacteus CD2 in the lignin/dye/fungus system. Shotgun proteomics technique was used to understand degradation mechanism at the protein level formore » the lignin/dye/fungus system. Our proteomics study can identify about two thousand proteins (one third of the predicted white-rot fungal proteome) in a single experiment, as one of the most powerful proteomics platforms to study the fungal system to date. The study shows a significant enrichment of oxidoreduction functional category under the dye/lignin combined treatment. An in vitro validation is performed and supports our hypothesis that the synergy of Fenton reaction and manganese peroxidase might play an important role in DR5B dye degradation. The results could guide the development of effective bioremediation strategies and efficient lignocellulosic biomass conversion.« less

Enhancement of Environmental Hazard Degradation in the Presence of Lignin: a Proteomics Study.

PubMed

Sun, Su; Xie, Shangxian; Cheng, Yanbing; Yu, Hongbo; Zhao, Honglu; Li, Muzi; Li, Xiaotong; Zhang, Xiaoyu; Yuan, Joshua S; Dai, Susie Y

2017-09-12

Proteomics studies of fungal systems have progressed dramatically based on the availability of more fungal genome sequences in recent years. Different proteomics strategies have been applied toward characterization of fungal proteome and revealed important gene functions and proteome dynamics. Presented here is the application of shot-gun proteomic technology to study the bio-remediation of environmental hazards by white-rot fungus. Lignin, a naturally abundant component of the plant biomass, is discovered to promote the degradation of Azo dye by white-rot fungus Irpex lacteus CD2 in the lignin/dye/fungus system. Shotgun proteomics technique was used to understand degradation mechanism at the protein level for the lignin/dye/fungus system. Our proteomics study can identify about two thousand proteins (one third of the predicted white-rot fungal proteome) in a single experiment, as one of the most powerful proteomics platforms to study the fungal system to date. The study shows a significant enrichment of oxidoreduction functional category under the dye/lignin combined treatment. An in vitro validation is performed and supports our hypothesis that the synergy of Fenton reaction and manganese peroxidase might play an important role in DR5B dye degradation. The results could guide the development of effective bioremediation strategies and efficient lignocellulosic biomass conversion.

Enhancement of Environmental Hazard Degradation in the Presence of Lignin: a Proteomics Study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Su; Xie, Shangxian; Cheng, Yanbing

Proteomics studies of fungal systems have progressed dramatically based on the availability of more fungal genome sequences in recent years. Different proteomics strategies have been applied toward characterization of fungal proteome and revealed important gene functions and proteome dynamics. Presented here is the application of shot-gun proteomic technology to study the bio-remediation of environmental hazards by white-rot fungus. Lignin, a naturally abundant component of the plant biomass, is discovered to promote the degradation of Azo dye by white-rot fungus Irpex lacteus CD2 in the lignin/dye/fungus system. Shotgun proteomics technique was used to understand degradation mechanism at the protein level formore » the lignin/dye/fungus system. Our proteomics study can identify about two thousand proteins (one third of the predicted white-rot fungal proteome) in a single experiment, as one of the most powerful proteomics platforms to study the fungal system to date. The study shows a significant enrichment of oxidoreduction functional category under the dye/lignin combined treatment. An in vitro validation is performed and supports our hypothesis that the synergy of Fenton reaction and manganese peroxidase might play an important role in DR5B dye degradation. The results could guide the development of effective bioremediation strategies and efficient lignocellulosic biomass conversion.« less

Search for sarcoidosis candidate genes by integration of data from genomic, transcriptomic and proteomic studies.

PubMed

Maver, Ales; Medica, Igor; Peterlin, Borut

2009-12-01

The search for gene candidates in multifactorial diseases such as sarcoidosis can be based on the integration of linkage association data, gene expression data, and protein profile data from genomic, transcriptomic and proteomic studies, respectively. In this study we performed a literature-based search for studies reporting such data, followed by integration of collected information. Different databases were examined--Medline, HugGE Navigator, ArrayExpress and Gene Expression Omnibus (GEO). Candidate genes were defined as genes which were reported in at least 2 different types of omics studies. Genes previously investigated in sarcoidosis were excluded from further analyses. We identified 177 genes associated with sarcoidosis as potential new candidate genes. Subsequently, 9 gene candidates identified to overlap in 2 different types of studies (genomic, transcriptomic and/or proteomic) were consistently reported in at least 3 studies: SERPINB1, FABP4, S100A8, HBEGF, IL7R, LRIG1, PTPN23, DPM2 and NUP214. These genes are involved in regulation of immune response, cellular proliferation, apoptosis, inhibition of protease activity, lipid metabolism. Exact biological functions of HBEGF, LRIG1, PTPN23, DPM2 and NUP214 remain to be completely elucidated. We propose 9 candidate genes: SERPINB1, FABP4, S100A8, HBEGF, IL7R, LRIG1, PTPN23, DPM2 and NUP214, as genes with high potential for association with sarcoidosis.

A systematic evaluation of normalization methods in quantitative label-free proteomics.

PubMed

Välikangas, Tommi; Suomi, Tomi; Elo, Laura L

2018-01-01

To date, mass spectrometry (MS) data remain inherently biased as a result of reasons ranging from sample handling to differences caused by the instrumentation. Normalization is the process that aims to account for the bias and make samples more comparable. The selection of a proper normalization method is a pivotal task for the reliability of the downstream analysis and results. Many normalization methods commonly used in proteomics have been adapted from the DNA microarray techniques. Previous studies comparing normalization methods in proteomics have focused mainly on intragroup variation. In this study, several popular and widely used normalization methods representing different strategies in normalization are evaluated using three spike-in and one experimental mouse label-free proteomic data sets. The normalization methods are evaluated in terms of their ability to reduce variation between technical replicates, their effect on differential expression analysis and their effect on the estimation of logarithmic fold changes. Additionally, we examined whether normalizing the whole data globally or in segments for the differential expression analysis has an effect on the performance of the normalization methods. We found that variance stabilization normalization (Vsn) reduced variation the most between technical replicates in all examined data sets. Vsn also performed consistently well in the differential expression analysis. Linear regression normalization and local regression normalization performed also systematically well. Finally, we discuss the choice of a normalization method and some qualities of a suitable normalization method in the light of the results of our evaluation. © The Author 2016. Published by Oxford University Press.

Proteome-wide characterization of sugarbeet seed vigor and its tissue specific expression

PubMed Central

Catusse, Julie; Strub, Jean-Marc; Job, Claudette; Van Dorsselaer, Alain; Job, Dominique

2008-01-01

Proteomic analysis of mature sugarbeet seeds led to the identification of 759 proteins and their specific tissue expression in root, cotyledons, and perisperm. In particular, the proteome of the perispermic storage tissue found in many seeds of the Caryophyllales is described here. The data allowed us to reconstruct in detail the metabolism of the seeds toward recapitulating facets of seed development and provided insights into complex behaviors such as germination. The seed appears to be well prepared to mobilize the major classes of reserves (the proteins, triglycerides, phytate, and starch) during germination, indicating that the preparation of the seed for germination is mainly achieved during its maturation on the mother plant. Furthermore, the data revealed several pathways that can contribute to seed vigor, an important agronomic trait defined as the potential to produce vigorous seedlings, such as glycine betaine accumulation in seeds. This study also identified several proteins that, to our knowledge, have not previously been described in seeds. For example, the data revealed that the sugarbeet seed can initiate translation either through the traditional cap-dependent mechanism or by a cap-independent process. The study of the tissue specificity of the seed proteome demonstrated a compartmentalization of metabolic activity between the roots, cotyledons, and perisperm, indicating a division of metabolic tasks between the various tissues. Furthermore, the perisperm, although it is known as a dead tissue, appears to be very active biochemically, playing multiple roles in distributing sugars and various metabolites to other tissues of the embryo. PMID:18635686

A proteomic and ultrastructural characterization of Aspergillus fumigatus' conidia adaptation at different culture ages.

PubMed

Anjo, Sandra I; Figueiredo, Francisco; Fernandes, Rui; Manadas, Bruno; Oliveira, Manuela

2017-05-24

The airborne fungus Aspergillus fumigatus is one of the most common agents of human fungal infections with a remarkable impact on public health. However, A. fumigatus conidia atmospheric resistance and longevity mechanisms are still unknown. Therefore, in this work, the processes underlying conidial adaptation were studied by a time course evaluation of the proteomics and ultrastructural changes of A. fumigatus' conidia at three time-points selected according to relevant changes previously established in conidial survival rates. The proteomics characterization revealed that conidia change from a highly active metabolic to a dormant state, culminating in cell autolysis as revealed by the increased levels of hydrolytic enzymes. Structural characterization corroborates the proteomics data, with noticeable changes observed in mitochondria, nucleus and plasma membrane ultrastructure, accompanied by the formation of autophagic vacuoles. These changes are consistent with both apoptotic and autophagic processes, and indicate that the changes in protein levels may anticipate those in cell morphology. The findings presented in this work not only clarify the processes underlying conidial adaptation to nutrient limiting conditions but can also be exploited for improving infection control strategies and in the development of new therapeutical drugs. Additionally, the present study was deposited in a public database and thus, it may also be a valuable dataset to be used by the scientific community as a tool to understand and identified other potential targets associated with conidia resistance. Copyright © 2017. Published by Elsevier B.V.

2D-Difference Gel Electrophoretic Proteomic Analysis of a Cell Culture Model of Alveolar Rhabdomyosarcoma

PubMed Central

Pressey, Joseph G.; Pressey, Christine S.; Robinson, Gloria; Herring, Richie; Wilson, Landon; Kelly, David R.; Kim, Helen

2011-01-01

To evaluate the consequences of expression of the protein encoded by PAX3-FOXO1 (P3F) in the pediatric malignancy alveolar rhabdomyosarcoma (A-RMS), we developed and evaluated a genetically defined in vitro model of A-RMS tumorigenesis. The expression of P3F in cooperation with simian virus 40 (SV40) Large-T (LT) antigen in murine C3H10T1/2 fibroblasts led to robust malignant transformation. Using 2 dimensional difference gel electrophoresis (2D-DIGE) we compared proteomes from lysates from cells that express P3F + LT versus from cells that express LT alone. Analysis of 2D gel spot patterns by DeCyder™ image analysis software indicated 93 spots that were different in abundance. Peptide mass fingerprint analysis of the 93 spots by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis identified 37 non-redundant proteins. 2D DIGE analysis of cell culture media conditioned by cells transduced by P3F + LT versus by LT alone found 29 spots in the P3F + LT cells leading to the identification of 11 non-redundant proteins. A substantial number of proteins with potential roles in tumorigenesis and myogenesis were detected, most of which have not been identified in previous wide-scale expression studies of RMS experimental models or tumors. We validated the 2D gel image analysis findings by western blot analysis and immunohistochemistry (IHC). Thus, the 2D DIGE proteomics methodology described here provided an important discovery approach to the study of RMS biology and complements the findings of previous mRNA expression studies. PMID:21110518

Step-by-step strategy for protein enrichment and proteome characterisation of extracellular polymeric substances in wastewater treatment systems.

PubMed

Silva, Ana F; Carvalho, Gilda; Soares, Renata; Coelho, Ana V; Barreto Crespo, M Teresa

2012-08-01

Extracellular polymeric substances (EPS) are keys in biomass aggregation and settleability in wastewater treatment systems. In membrane bioreactors (MBR), EPS are an important factor as they are considered to be largely responsible for membrane fouling. Proteins were shown to be the major component of EPS produced by activated sludge and to be correlated with the properties of the sludge, like settling, hydrophobicity and cell aggregation. Previous EPS proteomic studies of activated sludge revealed several problems, like the interference of other EPS molecules in protein analysis. In this study, a successful strategy was outlined to identify the proteins from soluble and bound EPS extracted from activated sludge of a lab-scale MBR. EPS samples were first subjected to pre-concentration through lyophilisation, centrifugal ultrafiltration or concentration with a dialysis membrane coated by a highly absorbent powder of polyacrylate-polyalcohol, preceded or not by a dialysis step. The highest protein concentration factors were achieved with the highly absorbent powder method without previous dialysis step. Four protein precipitation methods were then tested: acetone, trichloroacetic acid (TCA), perchloric acid and a commercial kit. Protein profiles were compared in 4-12 % sodium dodecyl sulphate polyacrylamide gel electrophoresis gels. Both acetone and TCA should be applied for the highest coverage for soluble EPS proteins, whereas TCA was the best method for bound EPS proteins. All visible bands of selected profiles were subjected to mass spectrometry analysis. A high number of proteins (25-32 for soluble EPS and 17 for bound EPS) were identified. As a conclusion of this study, a workflow is proposed for the successful proteome characterisation of soluble and bound EPS from activated sludge samples.

2D-difference gel electrophoretic proteomic analysis of a cell culture model of alveolar rhabdomyosarcoma.

PubMed

Pressey, Joseph G; Pressey, Christine S; Robinson, Gloria; Herring, Richie; Wilson, Landon; Kelly, David R; Kim, Helen

2011-02-04

To evaluate the consequences of expression of the protein encoded by PAX3-FOXO1 (P3F) in the pediatric malignancy alveolar rhabdomyosarcoma (A-RMS), we developed and evaluated a genetically defined in vitro model of A-RMS tumorigenesis. The expression of P3F in cooperation with simian virus 40 (SV40) Large-T (LT) antigen in murine C3H10T1/2 fibroblasts led to robust malignant transformation. Using 2-dimensional-difference gel electrophoresis (2D-DIGE), we compared proteomes from lysates from cells that express P3F + LT versus from cells that express LT alone. Analysis of 2D gel spot patterns by DeCyder image analysis software indicated 93 spots that were different in abundance. Peptide mass fingerprint analysis of the 93 spots by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis identified 37 nonredundant proteins. 2D-DIGE analysis of cell culture media conditioned by cells transduced by P3F + LT versus by LT alone found 29 spots in the P3F + LT cells leading to the identification of 11 nonredundant proteins. A substantial number of proteins with potential roles in tumorigenesis and myogenesis were detected, most of which have not been identified in previous wide-scale expression studies of RMS experimental models or tumors. We validated the 2D gel image analysis findings by Western blot analysis and immunohistochemistry (IHC). Thus, the 2D-DIGE proteomics methodology described here provided an important discovery approach to the study of RMS biology and complements the findings of previous mRNA expression studies.

Liquid-phase-based separation systems for depletion, prefractionation and enrichment of proteins in biological fluids and matrices for in-depth proteomics analysis – An update covering the period 2011-2014

PubMed Central

Puangpila, Chanida; Mayadunne, Erandi; Rassi, Ziad El

2015-01-01

This review article expands on the previous one (S. Selvaraju and Z. El Rassi, Electrophoresis 2012, 33, 74-88) by reviewing pertinent literature in the period extending from early 2011 to present. As the previous review article, the present one is concerned with proteomic sample preparation (e.g., depletion of high abundance proteins, reduction of the protein dynamic concentration range, enrichment of a particular sub-proteome), and the subsequent chromatographic and/or electrophoretic pre-fractionation prior to peptide separation and identification by LC-MS/MS. This review article is distinguished from its second version published in Electrophoresis 2012, 33, 74-88 by expanding on capturing/enriching sub-phosphoproteomes by immobilized metal affinity chromatography and metal oxide affinity chromatography. Seventy-seven papers published in the period extending from mid 2011 to the present have been reviewed. By no means this review article is exhaustive, given the fact that its aim is to give a concise treatment of the latest developments in the field. PMID:25287967

NHS-Esters As Versatile Reactivity-Based Probes for Mapping Proteome-Wide Ligandable Hotspots.

PubMed

Ward, Carl C; Kleinman, Jordan I; Nomura, Daniel K

2017-06-16

Most of the proteome is considered undruggable, oftentimes hindering translational efforts for drug discovery. Identifying previously unknown druggable hotspots in proteins would enable strategies for pharmacologically interrogating these sites with small molecules. Activity-based protein profiling (ABPP) has arisen as a powerful chemoproteomic strategy that uses reactivity-based chemical probes to map reactive, functional, and ligandable hotspots in complex proteomes, which has enabled inhibitor discovery against various therapeutic protein targets. Here, we report an alkyne-functionalized N-hydroxysuccinimide-ester (NHS-ester) as a versatile reactivity-based probe for mapping the reactivity of a wide range of nucleophilic ligandable hotspots, including lysines, serines, threonines, and tyrosines, encompassing active sites, allosteric sites, post-translational modification sites, protein interaction sites, and previously uncharacterized potential binding sites. Surprisingly, we also show that fragment-based NHS-ester ligands can be made to confer selectivity for specific lysine hotspots on specific targets including Dpyd, Aldh2, and Gstt1. We thus put forth NHS-esters as promising reactivity-based probes and chemical scaffolds for covalent ligand discovery.

Novel "omics" approach for study of low-abundance, low-molecular-weight components of a complex biological tissue: regional differences between chorionic and basal plates of the human placenta.

PubMed

Kedia, Komal; Nichols, Caitlin A; Thulin, Craig D; Graves, Steven W

2015-11-01

Tissue proteomics has relied heavily on two-dimensional gel electrophoresis, for protein separation and quantification, then single protein isolation, trypsin digestion, and mass spectrometric protein identification. Such methods are predominantly used for study of high-abundance, full-length proteins. Tissue peptidomics has recently been developed but is still used to study the most highly abundant species, often resulting in observation and identification of dozens of peptides only. Tissue lipidomics is likewise new, and reported studies are limited. We have developed an "omics" approach that enables over 7,000 low-molecular-weight, low-abundance species to be surveyed and have applied this to human placental tissue. Because the placenta is believed to be involved in complications of pregnancy, its proteomic evaluation is of substantial interest. In previous research on the placental proteome, abundant, high-molecular-weight proteins have been studied. Application of large-scale, global proteomics or peptidomics to the placenta have been limited, and would be challenging owing to the anatomic complexity and broad concentration range of proteins in this tissue. In our approach, involving protein depletion, capillary liquid chromatography, and tandem mass spectrometry, we attempted to identify molecular differences between two regions of the same placenta with only slightly different cellular composition. Our analysis revealed 16 species with statistically significant differences between the two regions. Tandem mass spectrometry enabled successful sequencing, or otherwise enabled chemical characterization, of twelve of these. The successful discovery and identification of regional differences between the expression of low-abundance, low-molecular weight biomolecules reveals the potential of our approach.

Proteomics and transcriptomics analyses of Arabidopsis floral buds uncover important functions of ARABIDOPSIS SKP1-LIKE1

DOE PAGES

Lu, Dihong; Ni, Weimin; Stanley, Bruce A.; ...

2016-03-03

The ARABIDOPSIS SKP1-LIKE1 (ASK1) protein functions as a subunit of SKP1-CUL1-F-box (SCF) E3 ubiquitin ligases. Previous genetic studies showed that ASK1 plays important roles in Arabidopsis flower development and male meiosis. However, the molecular impact of ASK1-containing SCF E3 ubiquitin ligases (ASK1-E3s) on the floral proteome and transcriptome is unknown. Here we identified proteins that are potentially regulated by ASK1-E3s by comparing floral bud proteomes of wild-type and the ask1 mutant plants. More than 200 proteins were detected in the ask1 mutant but not in wild-type and >300 were detected at higher levels in the ask1 mutant than in wild-type,more » but their RNA levels were not significantly different between wild-type and ask1 floral buds as shown by transcriptomics analysis, suggesting that they are likely regulated at the protein level by ASK1-E3s. Integrated analyses of floral proteomics and transcriptomics of ask1 and wild-type uncovered several potential aspects of ASK1-E3 functions, including regulation of transcription regulators, kinases, peptidases, and ribosomal proteins, with implications on possible mechanisms of ASK1-E3 functions in floral development. In conclusion, our results suggested that ASK1-E3s play important roles in Arabidopsis protein degradation during flower development. This study opens up new possibilities for further functional studies of these candidate E3 substrates.« less

Proteomics and transcriptomics analyses of Arabidopsis floral buds uncover important functions of ARABIDOPSIS SKP1-LIKE1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, Dihong; Ni, Weimin; Stanley, Bruce A.

The ARABIDOPSIS SKP1-LIKE1 (ASK1) protein functions as a subunit of SKP1-CUL1-F-box (SCF) E3 ubiquitin ligases. Previous genetic studies showed that ASK1 plays important roles in Arabidopsis flower development and male meiosis. However, the molecular impact of ASK1-containing SCF E3 ubiquitin ligases (ASK1-E3s) on the floral proteome and transcriptome is unknown. Here we identified proteins that are potentially regulated by ASK1-E3s by comparing floral bud proteomes of wild-type and the ask1 mutant plants. More than 200 proteins were detected in the ask1 mutant but not in wild-type and >300 were detected at higher levels in the ask1 mutant than in wild-type,more » but their RNA levels were not significantly different between wild-type and ask1 floral buds as shown by transcriptomics analysis, suggesting that they are likely regulated at the protein level by ASK1-E3s. Integrated analyses of floral proteomics and transcriptomics of ask1 and wild-type uncovered several potential aspects of ASK1-E3 functions, including regulation of transcription regulators, kinases, peptidases, and ribosomal proteins, with implications on possible mechanisms of ASK1-E3 functions in floral development. In conclusion, our results suggested that ASK1-E3s play important roles in Arabidopsis protein degradation during flower development. This study opens up new possibilities for further functional studies of these candidate E3 substrates.« less

Proteomic and phosphoproteomic analysis of polyethylene glycol-induced osmotic stress in root tips of common bean (Phaseolus vulgaris L.)

PubMed Central

Horst, Walter Johannes

2013-01-01

Previous studies have shown that polyethylene glycol (PEG)-induced osmotic stress (OS) reduces cell-wall (CW) porosity and limits aluminium (Al) uptake by root tips of common bean (Phaseolus vulgaris L.). A subsequent transcriptomic study suggested that genes related to CW processes are involved in adjustment to OS. In this study, a proteomic and phosphoproteomic approach was applied to identify OS-induced protein regulation to further improve our understanding of how OS affects Al accumulation. Analysis of total soluble proteins in root tips indicated that, in total, 22 proteins were differentially regulated by OS; these proteins were functionally categorized. Seventy-seven per- cent of the total expressed proteins were involved in metabolic pathways, particularly of carbohydrate and amino acid metabolism. An analysis of the apoplastic proteome revealed that OS reduced the level of five proteins and increased that of seven proteins. Investigation of the total soluble phosphoproteome suggested that dehydrin responded to OS with an enhanced phosphorylation state without a change in abundance. A cellular immunolocalization analysis indicated that dehydrin was localized mainly in the CW. This suggests that dehydrin may play a major protective role in the OS-induced physical breakdown of the CW structure and thus maintenance of the reversibility of CW extensibility during recovery from OS. The proteomic and phosphoproteomic analyses provided novel insights into the complex mechanisms of OS-induced reduction of Al accumulation in the root tips of common bean and highlight a key role for modification of CW structure. PMID:24123251

Comparative genomic and proteomic analyses of Clostridium acetobutylicum Rh8 and its parent strain DSM 1731 revealed new understandings on butanol tolerance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bao, Guanhui; University of Chinese Academy of Sciences, Beijing; Dong, Hongjun

Highlights: • Genomes of a butanol tolerant strain and its parent strain were deciphered. • Comparative genomic and proteomic was applied to understand butanol tolerance. • None differentially expressed proteins have mutations in its corresponding genes. • Mutations in ribosome might be responsible for the global difference of proteomics. - Abstract: Clostridium acetobutylicum strain Rh8 is a butanol-tolerant mutant which can tolerate up to 19 g/L butanol, 46% higher than that of its parent strain DSM 1731. We previously performed comparative cytoplasm- and membrane-proteomic analyses to understand the mechanism underlying the improved butanol tolerance of strain Rh8. In this work,more » we further extended this comparison to the genomic level. Compared with the genome of the parent strain DSM 1731, two insertion sites, four deletion sites, and 67 single nucleotide variations (SNVs) are distributed throughout the genome of strain Rh8. Among the 67 SNVs, 16 SNVs are located in the predicted promoters and intergenic regions; while 29 SNVs are located in the coding sequence, affecting a total of 21 proteins involved in transport, cell structure, DNA replication, and protein translation. The remaining 22 SNVs are located in the ribosomal genes, affecting a total of 12 rRNA genes in different operons. Analysis of previous comparative proteomic data indicated that none of the differentially expressed proteins have mutations in its corresponding genes. Rchange Algorithms analysis indicated that the mutations occurred in the ribosomal genes might change the ribosome RNA thermodynamic characteristics, thus affect the translation strength of these proteins. Take together, the improved butanol tolerance of C. acetobutylicum strain Rh8 might be acquired through regulating the translational process to achieve different expression strength of genes involved in butanol tolerance.« less

Mastitomics, the integrated omics of bovine milk in an experimental model of Streptococcus uberis mastitis: 2. Label-free relative quantitative proteomics.

PubMed

Mudaliar, Manikhandan; Tassi, Riccardo; Thomas, Funmilola C; McNeilly, Tom N; Weidt, Stefan K; McLaughlin, Mark; Wilson, David; Burchmore, Richard; Herzyk, Pawel; Eckersall, P David; Zadoks, Ruth N

2016-08-16

Mastitis, inflammation of the mammary gland, is the most common and costly disease of dairy cattle in the western world. It is primarily caused by bacteria, with Streptococcus uberis as one of the most prevalent causative agents. To characterize the proteome during Streptococcus uberis mastitis, an experimentally induced model of intramammary infection was used. Milk whey samples obtained from 6 cows at 6 time points were processed using label-free relative quantitative proteomics. This proteomic analysis complements clinical, bacteriological and immunological studies as well as peptidomic and metabolomic analysis of the same challenge model. A total of 2552 non-redundant bovine peptides were identified, and from these, 570 bovine proteins were quantified. Hierarchical cluster analysis and principal component analysis showed clear clustering of results by stage of infection, with similarities between pre-infection and resolution stages (0 and 312 h post challenge), early infection stages (36 and 42 h post challenge) and late infection stages (57 and 81 h post challenge). Ingenuity pathway analysis identified upregulation of acute phase protein pathways over the course of infection, with dominance of different acute phase proteins at different time points based on differential expression analysis. Antimicrobial peptides, notably cathelicidins and peptidoglycan recognition protein, were upregulated at all time points post challenge and peaked at 57 h, which coincided with 10 000-fold decrease in average bacterial counts. The integration of clinical, bacteriological, immunological and quantitative proteomics and other-omic data provides a more detailed systems level view of the host response to mastitis than has been achieved previously.

Integrated genomics and proteomics of the Torpedo californica electric organ: concordance with the mammalian neuromuscular junction

PubMed Central

2011-01-01

Background During development, the branchial mesoderm of Torpedo californica transdifferentiates into an electric organ capable of generating high voltage discharges to stun fish. The organ contains a high density of cholinergic synapses and has served as a biochemical model for the membrane specialization of myofibers, the neuromuscular junction (NMJ). We studied the genome and proteome of the electric organ to gain insight into its composition, to determine if there is concordance with skeletal muscle and the NMJ, and to identify novel synaptic proteins. Results Of 435 proteins identified, 300 mapped to Torpedo cDNA sequences with ≥2 peptides. We identified 14 uncharacterized proteins in the electric organ that are known to play a role in acetylcholine receptor clustering or signal transduction. In addition, two human open reading frames, C1orf123 and C6orf130, showed high sequence similarity to electric organ proteins. Our profile lists several proteins that are highly expressed in skeletal muscle or are muscle specific. Synaptic proteins such as acetylcholinesterase, acetylcholine receptor subunits, and rapsyn were present in the electric organ proteome but absent in the skeletal muscle proteome. Conclusions Our integrated genomic and proteomic analysis supports research describing a muscle-like profile of the organ. We show that it is a repository of NMJ proteins but we present limitations on its use as a comprehensive model of the NMJ. Finally, we identified several proteins that may become candidates for signaling proteins not previously characterized as components of the NMJ. PMID:21798097

The application of proteomics in different aspects of hepatocellular carcinoma research.

PubMed

Xing, Xiaohua; Liang, Dong; Huang, Yao; Zeng, Yongyi; Han, Xiao; Liu, Xiaolong; Liu, Jingfeng

2016-08-11

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors, which is causing the second leading cancer-related death worldwide. With the significant advances of high-throughput protein analysis techniques, the proteomics offered an extremely useful and versatile analytical platform for biomedical researches. In recent years, different proteomic strategies have been widely applied in the various aspects of HCC studies, ranging from screening the early diagnostic and prognostic biomarkers to in-depth investigating the underlying molecular mechanisms. In this review, we would like to systematically summarize the current applications of proteomics in hepatocellular carcinoma study, and discuss the challenges of applying proteomics in study clinical samples, as well as discuss the possible application of proteomics in precision medicine. In this review, we have systematically summarized the current applications of proteomics in hepatocellular carcinoma study, ranging from screening biomarkers to in-depth investigating the underlying molecular mechanisms. In addition, we have discussed the challenges of applying proteomics in study clinical samples, as well as the possible applications of proteomics in precision medicine. We believe that this review would help readers to be better familiar with the recent progresses of clinical proteomics, especially in the field of hepatocellular carcinoma research. Copyright © 2016 Elsevier B.V. All rights reserved.

Effect of MK-801 and Clozapine on the Proteome of Cultured Human Oligodendrocytes

PubMed Central

Cassoli, Juliana S.; Iwata, Keiko; Steiner, Johann; Guest, Paul C.; Turck, Christoph W.; Nascimento, Juliana M.; Martins-de-Souza, Daniel

2016-01-01

Separate lines of evidence have demonstrated the involvement of N-methyl-D-aspartate (NMDA) receptor and oligodendrocyte dysfunctions in schizophrenia. Here, we have carried out shotgun mass spectrometry proteome analysis of oligodendrocytes treated with the NMDA receptor antagonist MK-801 to gain potential insights into these effects at the molecular level. The MK-801 treatment led to alterations in the levels of 68 proteins, which are associated with seven distinct biological processes. Most of these proteins are involved in energy metabolism and many have been found to be dysregulated in previous proteomic studies of post-mortem brain tissues from schizophrenia patients. Finally, addition of the antipsychotic clozapine to MK-801-treated oligodendrocyte cultures resulted in changes in the levels of 45 proteins and treatment with clozapine alone altered 122 proteins and many of these showed opposite changes to the MK-801 effects. Therefore, these proteins and the associated energy metabolism pathways should be explored as potential biomarkers of antipsychotic efficacy. In conclusion, MK-801 treatment of oligodendrocytes may provide a useful model for testing the efficacy of novel treatment approaches. PMID:26973466

Proteomic Biomarkers for Acute Interstitial Lung Disease in Gefitinib-Treated Japanese Lung Cancer Patients

PubMed Central

Kawakami, Takao; Nagasaka, Keiko; Takami, Sachiko; Wada, Kazuya; Tu, Hsiao-Kun; Otsuji, Makiko; Kyono, Yutaka; Dobashi, Tae; Komatsu, Yasuhiko; Kihara, Makoto; Akimoto, Shingo; Peers, Ian S.; South, Marie C.; Higenbottam, Tim; Fukuoka, Masahiro; Nakata, Koichiro; Ohe, Yuichiro; Kudoh, Shoji; Clausen, Ib Groth; Nishimura, Toshihide; Marko-Varga, György; Kato, Harubumi

2011-01-01

Interstitial lung disease (ILD) events have been reported in Japanese non-small-cell lung cancer (NSCLC) patients receiving EGFR tyrosine kinase inhibitors. We investigated proteomic biomarkers for mechanistic insights and improved prediction of ILD. Blood plasma was collected from 43 gefitinib-treated NSCLC patients developing acute ILD (confirmed by blinded diagnostic review) and 123 randomly selected controls in a nested case-control study within a pharmacoepidemiological cohort study in Japan. We generated ∼7 million tandem mass spectrometry (MS/MS) measurements with extensive quality control and validation, producing one of the largest proteomic lung cancer datasets to date, incorporating rigorous study design, phenotype definition, and evaluation of sample processing. After alignment, scaling, and measurement batch adjustment, we identified 41 peptide peaks representing 29 proteins best predicting ILD. Multivariate peptide, protein, and pathway modeling achieved ILD prediction comparable to previously identified clinical variables; combining the two provided some improvement. The acute phase response pathway was strongly represented (17 of 29 proteins, p = 1.0×10−25), suggesting a key role with potential utility as a marker for increased risk of acute ILD events. Validation by Western blotting showed correlation for identified proteins, confirming that robust results can be generated from an MS/MS platform implementing strict quality control. PMID:21799770

Differential expression proteomics of human colorectal cancer based on a syngeneic cellular model for the progression of adenoma to carcinoma.

PubMed

Roth, Udo; Razawi, Hanieh; Hommer, Julia; Engelmann, Katja; Schwientek, Tilo; Müller, Stefan; Baldus, Stephan E; Patsos, Georgios; Corfield, Anthony P; Paraskeva, Christos; Hanisch, Franz-Georg

2010-01-01

This is the first differential expression proteomics study on a human syngeneic cellular in vitro progression model of the colorectal adenoma-to-carcinoma sequence, the anchorage-dependent non-tumorigenic adenoma derived cell line AA/C1 and the derived anchorage-independent and tumorigenic carcinoma cell line AA/C1/SB10C. The study is based on quantitative 2-DE and is complemented by Western blot validation. Excluding redundancies due to proteolysis and post-translational modified isoforms of over 2000 protein spots, 13 proteins were revealed as regulated with statistical variance being within the 95th confidence level and were identified by peptide mass fingerprinting in MALDI MS. Progression-associated proteins belong to the functional complexes of anaerobic glycolysis/gluconeogenesis, steroid biosynthesis, prostaglandin biosynthesis, the regulation and maintenance of the cytoskeleton, protein biosynthesis and degradation, the regulation of apoptosis or other functions. Partial but significant overlap was revealed with previous proteomics and transcriptomics studies in colorectal carcinoma. Among upregulated proteins we identified 3-HMG-CoA synthase, protein phosphatase 1, prostaglandin E synthase 2, villin 1, annexin A1, triosephosphate isomerase, phosphoserine aminotransferase 1, fumarylacetoacetate hydrolase and pyrroline-5-carboxylate reductase 1 (PYCR1), while glucose-regulated protein 78, cathepsin D, lamin A/C and quinolate phosphoribosyltransferase were downregulated.

Proteomic Dissection of Seed Germination and Seedling Establishment in Brassica napus

PubMed Central

Gu, Jianwei; Chao, Hongbo; Gan, Lu; Guo, Liangxing; Zhang, Kai; Li, Yonghong; Wang, Hao; Raboanatahiry, Nadia; Li, Maoteng

2016-01-01

The success of seed germination and establishment of a normal seedling are key determinants of plant species propagation. At present, only a few studies have focused on the genetic control of seed germination by using a proteomic approach in Brassica napus. In the present study, the protein expression pattern of seed germination was investigated using differential fluorescence two-dimensional gel electrophoresis in B. napus. One hundred and thirteen differentially expressed proteins (DEPs) that were mainly involved in storage (23.4%), energy metabolism (18.9%), protein metabolism (16.2%), defense/disease (12.6%), seed maturation (11.7%), carbohydrate metabolism (4.5%), lipid metabolism (4.5%), amino acids metabolism (3.6%), cell growth/division (3.6%), and some unclear functions (2.7%) were observed by proteomic analysis. Seventeen genes corresponding to 11 DEPs were identified within or near the associated linkage disequilibrium regions related to seed germination and vigor quantitative traits reported in B. napus in previous studies. The expression pattern of proteins showed that heterotrophic metabolism could be activated in the process of seed germination and that the onset of defense mechanisms might start during seed germination. These findings will help generate a more in-depth understanding of the mobilization of seed storage reserves and regulation mechanisms of the germination process in B. napus. PMID:27822216

Proteomic Identification of the Galectin-1-Involved Molecular Pathways in Urinary Bladder Urothelial Carcinoma.

PubMed

Li, Chien-Feng; Shen, Kun-Hung; Chien, Lan-Hsiang; Huang, Cheng-Hao; Wu, Ting-Feng; He, Hong-Lin

2018-04-19

Among various heterogeneous types of bladder tumors, urothelial carcinoma is the most prevalent lesion. Some of the urinary bladder urothelial carcinomas (UBUCs) develop local recurrence and may cause distal invasion. Galectin-1 de-regulation significantly affects cell transformation, cell proliferation, angiogenesis, and cell invasiveness. In continuation of our previous investigation on the role of galectin-1 in UBUC tumorigenesis, in this study, proteomics strategies were implemented in order to find more galectin-1-associated signaling pathways. The results of this study showed that galectin-1 knockdown could induce 15 down-regulated proteins and two up-regulated proteins in T24 cells. These de-regulated proteins might participate in lipid/amino acid/energy metabolism, cytoskeleton, cell proliferation, cell-cell interaction, cell apoptosis, metastasis, and protein degradation. The aforementioned dys-regulated proteins were confirmed by western immunoblotting. Proteomics results were further translated to prognostic markers by analyses of biopsy samples. Results of cohort studies demonstrated that over-expressions of glutamine synthetase, alcohol dehydrogenase (NADP⁺), fatty acid binding protein 4, and toll interacting protein in clinical specimens were all significantly associated with galectin-1 up-regulation. Univariate analyses showed that de-regulations of glutamine synthetase and fatty acid binding protein 4 in clinical samples were respectively linked to disease-specific survival and metastasis-free survival.

Proteomic Analysis of Mouse Hypothalamus under Simulated Microgravity

PubMed Central

Sarkar, Poonam; Sarkar, Shubhashish; Ramesh, Vani; Kim, Helen; Barnes, Stephen; Kulkarni, Anil; Hall, Joseph C.; Wilson, Bobby L.; Thomas, Renard L.; Pellis, Neal R.

2009-01-01

Exposure to altered microgravity during space travel induces changes in the brain and these are reflected in many of the physical behavior seen in the astronauts. The vulnerability of the brain to microgravity stress has been reviewed and reported. Identifying microgravity-induced changes in the brain proteome may aid in understanding the impact of the microgravity environment on brain function. In our previous study we have reported changes in specific proteins under simulated microgravity in the hippocampus using proteomics approach. In the present study the profiling of the hypothalamus region in the brain was studied as a step towards exploring the effect of microgravity in this region of the brain. Hypothalamus is the critical region in the brain that strictly controls the pituitary gland that in turn is responsible for the secretion of important hormones. Here we report a 2-dimensional gel electrophoretic analysis of the mouse hypothalamus in response to simulated microgravity. Lowered glutathione and differences in abundance expression of seven proteins were detected in the hypothalamus of mice exposed to microgravity. These changes included decreased superoxide dismutase-2 (SOD-2) and increased malate dehydrogenase and peroxiredoxin-6, reflecting reduction of the antioxidant system in the hypothalamus. Taken together the results reported here indicate that oxidative imbalance occurred in the hypothalamus in response to simulated microgravity. PMID:18473167

Highly efficient peptide separations in proteomics. Part 2: bi- and multidimensional liquid-based separation techniques.

PubMed

Sandra, Koen; Moshir, Mahan; D'hondt, Filip; Tuytten, Robin; Verleysen, Katleen; Kas, Koen; François, Isabelle; Sandra, Pat

2009-04-15

Multidimensional liquid-based separation techniques are described for maximizing the resolution of the enormous number of peptides generated upon tryptic digestion of proteomes, and hence, reduce the spatial and temporal complexity of the sample to a level that allows successful mass spectrometric analysis. This review complements the previous contribution on unidimensional high performance liquid chromatography (HPLC). Both chromatography and electrophoresis will be discussed albeit with reversed-phase HPLC (RPLC) as the final separation dimension prior to MS analysis.

Evolutionary Proteomics Uncovers Ancient Associations of Cilia with Signaling Pathways.

PubMed

Sigg, Monika Abedin; Menchen, Tabea; Lee, Chanjae; Johnson, Jeffery; Jungnickel, Melissa K; Choksi, Semil P; Garcia, Galo; Busengdal, Henriette; Dougherty, Gerard W; Pennekamp, Petra; Werner, Claudius; Rentzsch, Fabian; Florman, Harvey M; Krogan, Nevan; Wallingford, John B; Omran, Heymut; Reiter, Jeremy F

2017-12-18

Cilia are organelles specialized for movement and signaling. To infer when during evolution signaling pathways became associated with cilia, we characterized the proteomes of cilia from sea urchins, sea anemones, and choanoflagellates. We identified 437 high-confidence ciliary candidate proteins conserved in mammals and discovered that Hedgehog and G-protein-coupled receptor pathways were linked to cilia before the origin of bilateria and transient receptor potential (TRP) channels before the origin of animals. We demonstrated that candidates not previously implicated in ciliary biology localized to cilia and further investigated ENKUR, a TRP channel-interacting protein identified in the cilia of all three organisms. ENKUR localizes to motile cilia and is required for patterning the left-right axis in vertebrates. Moreover, mutation of ENKUR causes situs inversus in humans. Thus, proteomic profiling of cilia from diverse eukaryotes defines a conserved ciliary proteome, reveals ancient connections to signaling, and uncovers a ciliary protein that underlies development and human disease. Copyright © 2017 Elsevier Inc. All rights reserved.

Mspire-Simulator: LC-MS shotgun proteomic simulator for creating realistic gold standard data.

PubMed

Noyce, Andrew B; Smith, Rob; Dalgleish, James; Taylor, Ryan M; Erb, K C; Okuda, Nozomu; Prince, John T

2013-12-06

The most important step in any quantitative proteomic pipeline is feature detection (aka peak picking). However, generating quality hand-annotated data sets to validate the algorithms, especially for lower abundance peaks, is nearly impossible. An alternative for creating gold standard data is to simulate it with features closely mimicking real data. We present Mspire-Simulator, a free, open-source shotgun proteomic simulator that goes beyond previous simulation attempts by generating LC-MS features with realistic m/z and intensity variance along with other noise components. It also includes machine-learned models for retention time and peak intensity prediction and a genetic algorithm to custom fit model parameters for experimental data sets. We show that these methods are applicable to data from three different mass spectrometers, including two fundamentally different types, and show visually and analytically that simulated peaks are nearly indistinguishable from actual data. Researchers can use simulated data to rigorously test quantitation software, and proteomic researchers may benefit from overlaying simulated data on actual data sets.

An integrated native mass spectrometry and top-down proteomics method that connects sequence to structure and function of macromolecular complexes

NASA Astrophysics Data System (ADS)

Li, Huilin; Nguyen, Hong Hanh; Ogorzalek Loo, Rachel R.; Campuzano, Iain D. G.; Loo, Joseph A.

2018-02-01

Mass spectrometry (MS) has become a crucial technique for the analysis of protein complexes. Native MS has traditionally examined protein subunit arrangements, while proteomics MS has focused on sequence identification. These two techniques are usually performed separately without taking advantage of the synergies between them. Here we describe the development of an integrated native MS and top-down proteomics method using Fourier-transform ion cyclotron resonance (FTICR) to analyse macromolecular protein complexes in a single experiment. We address previous concerns of employing FTICR MS to measure large macromolecular complexes by demonstrating the detection of complexes up to 1.8 MDa, and we demonstrate the efficacy of this technique for direct acquirement of sequence to higher-order structural information with several large complexes. We then summarize the unique functionalities of different activation/dissociation techniques. The platform expands the ability of MS to integrate proteomics and structural biology to provide insights into protein structure, function and regulation.

Integrated Proteogenomic Characterization of Human High-Grade Serous Ovarian Cancer.

PubMed

Zhang, Hui; Liu, Tao; Zhang, Zhen; Payne, Samuel H; Zhang, Bai; McDermott, Jason E; Zhou, Jian-Ying; Petyuk, Vladislav A; Chen, Li; Ray, Debjit; Sun, Shisheng; Yang, Feng; Chen, Lijun; Wang, Jing; Shah, Punit; Cha, Seong Won; Aiyetan, Paul; Woo, Sunghee; Tian, Yuan; Gritsenko, Marina A; Clauss, Therese R; Choi, Caitlin; Monroe, Matthew E; Thomas, Stefani; Nie, Song; Wu, Chaochao; Moore, Ronald J; Yu, Kun-Hsing; Tabb, David L; Fenyö, David; Bafna, Vineet; Wang, Yue; Rodriguez, Henry; Boja, Emily S; Hiltke, Tara; Rivers, Robert C; Sokoll, Lori; Zhu, Heng; Shih, Ie-Ming; Cope, Leslie; Pandey, Akhilesh; Zhang, Bing; Snyder, Michael P; Levine, Douglas A; Smith, Richard D; Chan, Daniel W; Rodland, Karin D

2016-07-28

To provide a detailed analysis of the molecular components and underlying mechanisms associated with ovarian cancer, we performed a comprehensive mass-spectrometry-based proteomic characterization of 174 ovarian tumors previously analyzed by The Cancer Genome Atlas (TCGA), of which 169 were high-grade serous carcinomas (HGSCs). Integrating our proteomic measurements with the genomic data yielded a number of insights into disease, such as how different copy-number alternations influence the proteome, the proteins associated with chromosomal instability, the sets of signaling pathways that diverse genome rearrangements converge on, and the ones most associated with short overall survival. Specific protein acetylations associated with homologous recombination deficiency suggest a potential means for stratifying patients for therapy. In addition to providing a valuable resource, these findings provide a view of how the somatic genome drives the cancer proteome and associations between protein and post-translational modification levels and clinical outcomes in HGSC. VIDEO ABSTRACT. Copyright © 2016 Elsevier Inc. All rights reserved.

Transcriptional and Proteomic Profiling of Aspergillus flavipes in Response to Sulfur Starvation.

PubMed

El-Sayed, Ashraf S A; Yassin, Marwa A; Ali, Gul Shad

2015-01-01

Aspergillus flavipes has received considerable interest due to its potential to produce therapeutic enzymes involved in sulfur amino acid metabolism. In natural habitats, A. flavipes survives under sulfur limitations by mobilizing endogenous and exogenous sulfur to operate diverse cellular processes. Sulfur limitation affects virulence and pathogenicity, and modulates proteome of sulfur assimilating enzymes of several fungi. However, there are no previous reports aimed at exploring effects of sulfur limitation on the regulation of A. flavipes sulfur metabolism enzymes at the transcriptional, post-transcriptional and proteomic levels. In this report, we show that sulfur limitation affects morphological and physiological responses of A. flavipes. Transcription and enzymatic activities of several key sulfur metabolism genes, ATP-sulfurylase, sulfite reductase, methionine permease, cysteine synthase, cystathionine β- and γ-lyase, glutathione reductase and glutathione peroxidase were increased under sulfur starvation conditions. A 50 kDa protein band was strongly induced by sulfur starvation, and the proteomic analyses of this protein band using LC-MS/MS revealed similarity to many proteins involved in the sulfur metabolism pathway.

Top-Down Targeted Proteomics Reveals Decrease in Myosin Regulatory Light-Chain Phosphorylation That Contributes to Sarcopenic Muscle Dysfunction.

PubMed

Gregorich, Zachery R; Peng, Ying; Cai, Wenxuan; Jin, Yutong; Wei, Liming; Chen, Albert J; McKiernan, Susan H; Aiken, Judd M; Moss, Richard L; Diffee, Gary M; Ge, Ying

2016-08-05

Sarcopenia, the loss of skeletal muscle mass and function with advancing age, is a significant cause of disability and loss of independence in the elderly and thus, represents a formidable challenge for the aging population. Nevertheless, the molecular mechanism(s) underlying sarcopenia-associated muscle dysfunction remain poorly understood. In this study, we employed an integrated approach combining top-down targeted proteomics with mechanical measurements to dissect the molecular mechanism(s) in age-related muscle dysfunction. Top-down targeted proteomic analysis uncovered a progressive age-related decline in the phosphorylation of myosin regulatory light chain (RLC), a critical protein involved in the modulation of muscle contractility, in the skeletal muscle of aging rats. Top-down tandem mass spectrometry analysis identified a previously unreported bis-phosphorylated proteoform of fast skeletal RLC and localized the sites of decreasing phosphorylation to Ser14/15. Of these sites, Ser14 phosphorylation represents a previously unidentified site of phosphorylation in RLC from fast-twitch skeletal muscle. Subsequent mechanical analysis of single fast-twitch fibers isolated from the muscles of rats of different ages revealed that the observed decline in RLC phosphorylation can account for age-related decreases in the contractile properties of sarcopenic fast-twitch muscles. These results strongly support a role for decreasing RLC phosphorylation in sarcopenia-associated muscle dysfunction and suggest that therapeutic modulation of RLC phosphorylation may represent a new avenue for the treatment of sarcopenia.

2016 update of the PRIDE database and its related tools

PubMed Central

Vizcaíno, Juan Antonio; Csordas, Attila; del-Toro, Noemi; Dianes, José A.; Griss, Johannes; Lavidas, Ilias; Mayer, Gerhard; Perez-Riverol, Yasset; Reisinger, Florian; Ternent, Tobias; Xu, Qing-Wei; Wang, Rui; Hermjakob, Henning

2016-01-01

The PRoteomics IDEntifications (PRIDE) database is one of the world-leading data repositories of mass spectrometry (MS)-based proteomics data. Since the beginning of 2014, PRIDE Archive (http://www.ebi.ac.uk/pride/archive/) is the new PRIDE archival system, replacing the original PRIDE database. Here we summarize the developments in PRIDE resources and related tools since the previous update manuscript in the Database Issue in 2013. PRIDE Archive constitutes a complete redevelopment of the original PRIDE, comprising a new storage backend, data submission system and web interface, among other components. PRIDE Archive supports the most-widely used PSI (Proteomics Standards Initiative) data standard formats (mzML and mzIdentML) and implements the data requirements and guidelines of the ProteomeXchange Consortium. The wide adoption of ProteomeXchange within the community has triggered an unprecedented increase in the number of submitted data sets (around 150 data sets per month). We outline some statistics on the current PRIDE Archive data contents. We also report on the status of the PRIDE related stand-alone tools: PRIDE Inspector, PRIDE Converter 2 and the ProteomeXchange submission tool. Finally, we will give a brief update on the resources under development ‘PRIDE Cluster’ and ‘PRIDE Proteomes’, which provide a complementary view and quality-scored information of the peptide and protein identification data available in PRIDE Archive. PMID:26527722

Advances in targeted proteomics and applications to biomedical research

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Tujin; Song, Ehwang; Nie, Song

Targeted proteomics technique has emerged as a powerful protein quantification tool in systems biology, biomedical research, and increasing for clinical applications. The most widely used targeted proteomics approach, selected reaction monitoring (SRM), also known as multiple reaction monitoring (MRM), can be used for quantification of cellular signaling networks and preclinical verification of candidate protein biomarkers. As an extension to our previous review on advances in SRM sensitivity (Shi et al., Proteomics, 12, 1074–1092, 2012) herein we review recent advances in the method and technology for further enhancing SRM sensitivity (from 2012 to present), and highlighting its broad biomedical applications inmore » human bodily fluids, tissue and cell lines. Furthermore, we also review two recently introduced targeted proteomics approaches, parallel reaction monitoring (PRM) and data-independent acquisition (DIA) with targeted data extraction on fast scanning high-resolution accurate-mass (HR/AM) instruments. Such HR/AM targeted quantification with monitoring all target product ions addresses SRM limitations effectively in specificity and multiplexing; whereas when compared to SRM, PRM and DIA are still in the infancy with a limited number of applications. Thus, for HR/AM targeted quantification we focus our discussion on method development, data processing and analysis, and its advantages and limitations in targeted proteomics. Finally, general perspectives on the potential of achieving both high sensitivity and high sample throughput for large-scale quantification of hundreds of target proteins are discussed.« less

Proteomic insights into floral biology.

PubMed

Li, Xiaobai; Jackson, Aaron; Xie, Ming; Wu, Dianxing; Tsai, Wen-Chieh; Zhang, Sheng

2016-08-01

The flower is the most important biological structure for ensuring angiosperms reproductive success. Not only does the flower contain critical reproductive organs, but the wide variation in morphology, color, and scent has evolved to entice specialized pollinators, and arguably mankind in many cases, to ensure the successful propagation of its species. Recent proteomic approaches have identified protein candidates related to these flower traits, which has shed light on a number of previously unknown mechanisms underlying these traits. This review article provides a comprehensive overview of the latest advances in proteomic research in floral biology according to the order of flower structure, from corolla to male and female reproductive organs. It summarizes mainstream proteomic methods for plant research and recent improvements on two dimensional gel electrophoresis and gel-free workflows for both peptide level and protein level analysis. The recent advances in sequencing technologies provide a new paradigm for the ever-increasing genome and transcriptome information on many organisms. It is now possible to integrate genomic and transcriptomic data with proteomic results for large-scale protein characterization, so that a global understanding of the complex molecular networks in flower biology can be readily achieved. This article is part of a Special Issue entitled: Plant Proteomics--a bridge between fundamental processes and crop production, edited by Dr. Hans-Peter Mock. Copyright © 2016 Elsevier B.V. All rights reserved.

CPTAC Releases Cancer Proteome Confirmatory Breast Study Data | Office of Cancer Clinical Proteomics Research

Cancer.gov

An estimated 252,710 new cases of female breast cancer, accounting for 15% of all new cancer cases, occurred in 2017. To better understand proteogenomic abnormalities in breast cancer, the National Cancer Institute (NCI) Clinical Proteomic Tumor Analysis Consortium (CPTAC) announces the release of the cancer proteome confirmatory breast study data. The goal of the study was to comprehensively characterize the proteome and phosphoproteome on approximately 100 prospectively collected breast tumor and adjacent normal tissues.

Directed proteomic analysis of the human nucleolus.

PubMed

Andersen, Jens S; Lyon, Carol E; Fox, Archa H; Leung, Anthony K L; Lam, Yun Wah; Steen, Hanno; Mann, Matthias; Lamond, Angus I

2002-01-08

The nucleolus is a subnuclear organelle containing the ribosomal RNA gene clusters and ribosome biogenesis factors. Recent studies suggest it may also have roles in RNA transport, RNA modification, and cell cycle regulation. Despite over 150 years of research into nucleoli, many aspects of their structure and function remain uncharacterized. We report a proteomic analysis of human nucleoli. Using a combination of mass spectrometry (MS) and sequence database searches, including online analysis of the draft human genome sequence, 271 proteins were identified. Over 30% of the nucleolar proteins were encoded by novel or uncharacterized genes, while the known proteins included several unexpected factors with no previously known nucleolar functions. MS analysis of nucleoli isolated from HeLa cells in which transcription had been inhibited showed that a subset of proteins was enriched. These data highlight the dynamic nature of the nucleolar proteome and show that proteins can either associate with nucleoli transiently or accumulate only under specific metabolic conditions. This extensive proteomic analysis shows that nucleoli have a surprisingly large protein complexity. The many novel factors and separate classes of proteins identified support the view that the nucleolus may perform additional functions beyond its known role in ribosome subunit biogenesis. The data also show that the protein composition of nucleoli is not static and can alter significantly in response to the metabolic state of the cell.

Quantitative Phospho-proteomic Analysis of TNFα/NFκB Signaling Reveals a Role for RIPK1 Phosphorylation in Suppressing Necrotic Cell Death.

PubMed

Mohideen, Firaz; Paulo, Joao A; Ordureau, Alban; Gygi, Steve P; Harper, J Wade

2017-07-01

TNFα is a potent inducer of inflammation due to its ability to promote gene expression, in part via the NFκB pathway. Moreover, in some contexts, TNFα promotes Caspase-dependent apoptosis or RIPK1/RIPK3/MLKL-dependent necrosis. Engagement of the TNF Receptor Signaling Complex (TNF-RSC), which contains multiple kinase activities, promotes phosphorylation of several downstream components, including TAK1, IKKα/IKKβ, IκBα, and NFκB. However, immediate downstream phosphorylation events occurring in response to TNFα signaling are poorly understood at a proteome-wide level. Here we use Tandem Mass Tagging-based proteomics to quantitatively characterize acute TNFα-mediated alterations in the proteome and phosphoproteome with or without inhibition of the cIAP-dependent survival arm of the pathway with a SMAC mimetic. We identify and quantify over 8,000 phosphorylated peptides, among which are numerous known sites in the TNF-RSC, NFκB, and MAP kinase signaling systems, as well as numerous previously unrecognized phosphorylation events. Functional analysis of S320 phosphorylation in RIPK1 demonstrates a role for this event in suppressing its kinase activity, association with CASPASE-8 and FADD proteins, and subsequent necrotic cell death during inflammatory TNFα stimulation. This study provides a resource for further elucidation of TNFα-dependent signaling pathways. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization of the bovine milk proteome in early-lactation Holstein and Jersey breeds of dairy cows.

PubMed

Tacoma, Rinske; Fields, Julia; Ebenstein, David B; Lam, Ying-Wai; Greenwood, Sabrina L

2016-01-01

Milk is a highly nutritious natural product that provides not only a rich source of amino acids to the consumer but also hundreds of bioactive peptides and proteins known to elicit health-benefitting activities. We investigated the milk protein profile produced by Holstein and Jersey dairy cows maintained under the same diet, management and environmental conditions using proteomic approaches that optimize protein extraction and characterization of the low abundance proteins within the skim milk fraction of bovine milk. In total, 935 low abundance proteins were identified. Gene ontology classified all proteins identified into various cellular localization and function categories. A total of 43 low abundance proteins were differentially expressed between the two dairy breeds. Bioactive proteins involved in host-defense, including lactotransferrin (P=0.0026) and complement C2 protein (P=0.0001), were differentially expressed by the two breeds, whereas others such as osteopontin (P=0.1788) and lactoperoxidase (P=0.2973) were not. This work is the first to outline the protein profile produced by two important breeds of dairy cattle maintained under the same diet, environment and management conditions in order to observe likely true breed differences. This research now allows us to better understand and contrast further research examining the bovine proteome that includes these different breeds. Within the last decade, the amount of research characterizing the bovine milk proteome has increased due to growing interest in the bioactive proteins that are present in milk. Proteomic analysis of low abundance whey proteins has mainly focused on human breast milk; however, previous research has highlighted the presence of bioactive proteins in bovine milk. Recent publications outlining the cross-reactivity of bovine bioactive proteins on human biological function highlight the need for further investigation into the bovine milk proteome. The rationale behind this study is to characterize and compare the low abundance protein profile in the skim milk fraction produced from Holstein and Jersey breeds of dairy cattle, which are two major dairy cattle breeds in the USA. A combination of fractionation strategies was used to efficiently enrich the low abundance proteins from bovine skim milk for proteomic profiling. A total of 935 low abundance proteins were identified and compared between the two bovine breeds. The results from this study provide insight into breed differences and similarities in the milk proteome profile produced by two breeds of dairy cattle. Copyright © 2015 Elsevier B.V. All rights reserved.

Development of Proteomics-Based Fungicides: New Strategies for Environmentally Friendly Control of Fungal Plant Diseases

PubMed Central

Acero, Francisco Javier Fernández; Carbú, María; El-Akhal, Mohamed Rabie; Garrido, Carlos; González-Rodríguez, Victoria E.; Cantoral, Jesús M.

2011-01-01

Proteomics has become one of the most relevant high-throughput technologies. Several approaches have been used for studying, for example, tumor development, biomarker discovery, or microbiology. In this “post-genomic” era, the relevance of these studies has been highlighted as the phenotypes determined by the proteins and not by the genotypes encoding them that is responsible for the final phenotypes. One of the most interesting outcomes of these technologies is the design of new drugs, due to the discovery of new disease factors that may be candidates for new therapeutic targets. To our knowledge, no commercial fungicides have been developed from targeted molecular research, this review will shed some light on future prospects. We will summarize previous research efforts and discuss future innovations, focused on the fight against one of the main agents causing a devastating crops disease, fungal phytopathogens. PMID:21340014

IgE recognition of chimeric isoforms of the honeybee (Apis mellifera) venom allergen Api m 10 evaluated by protein array technology.

PubMed

Van Vaerenbergh, Matthias; De Smet, Lina; Rafei-Shamsabadi, David; Blank, Simon; Spillner, Edzard; Ebo, Didier G; Devreese, Bart; Jakob, Thilo; de Graaf, Dirk C

2015-02-01

Api m 10 has recently been established as novel major allergen that is recognized by more than 60% of honeybee venom (HBV) allergic patients. Previous studies suggest Api m 10 protein heterogeneity which may have implications for diagnosis and immunotherapy of HBV allergy. In the present study, RT-PCR revealed the expression of at least nine additional Api m 10 transcript isoforms by the venom glands. Two distinct mechanisms are responsible for the generation of these isoforms: while the previously known variant 2 is produced by an alternative splicing event, novel identified isoforms are intragenic chimeric transcripts. To the best of our knowledge, this is the first report of the identification of chimeric transcripts generated by the honeybee. By a retrospective proteomic analysis we found evidence for the presence of several of these isoforms in the venom proteome. Additionally, we analyzed IgE reactivity to different isoforms by protein array technology using sera from HBV allergic patients, which revealed that IgE recognition of Api m 10 is both isoform- and patient-specific. While it was previously demonstrated that the majority of HBV allergic patients display IgE reactivity to variant 2, our study also shows that some patients lacking IgE antibodies for variant 2 display IgE reactivity to two of the novel identified Api m 10 variants, i.e. variants 3 and 4. Copyright © 2014 Elsevier Ltd. All rights reserved.

Applications of mass spectrometry for quantitative protein analysis in formalin-fixed paraffin-embedded tissues

PubMed Central

Steiner, Carine; Ducret, Axel; Tille, Jean-Christophe; Thomas, Marlene; McKee, Thomas A; Rubbia-Brandt, Laura A; Scherl, Alexander; Lescuyer, Pierre; Cutler, Paul

2014-01-01

Proteomic analysis of tissues has advanced in recent years as instruments and methodologies have evolved. The ability to retrieve peptides from formalin-fixed paraffin-embedded tissues followed by shotgun or targeted proteomic analysis is offering new opportunities in biomedical research. In particular, access to large collections of clinically annotated samples should enable the detailed analysis of pathologically relevant tissues in a manner previously considered unfeasible. In this paper, we review the current status of proteomic analysis of formalin-fixed paraffin-embedded tissues with a particular focus on targeted approaches and the potential for this technique to be used in clinical research and clinical diagnosis. We also discuss the limitations and perspectives of the technique, particularly with regard to application in clinical diagnosis and drug discovery. PMID:24339433

Integration of gel-based proteome data with pProRep.

PubMed

Laukens, Kris; Matthiesen, Rune; Lemière, Filip; Esmans, Eddy; Onckelen, Harry Van; Jensen, Ole Nørregaard; Witters, Erwin

2006-11-15

pProRep is a web application integrating electrophoretic and mass spectral data from proteome analyses into a relational database. The graphical web-interface allows users to upload, analyse and share experimental proteome data. It offers researchers the possibility to query all previously analysed datasets and can visualize selected features, such as the presence of a certain set of ions in a peptide mass spectrum, on the level of the two-dimensional gel. The pProRep package and instructions for its use can be downloaded from http://www.ptools.ua.ac.be/pProRep. The application requires a web server that runs PHP 5 (http://www.php.net) and MySQL. Some (non-essential) extensions need additional freely available libraries: details are described in the installation instructions.

Chemical-Biological Properties of Zinc Sensors TSQ and Zinquin: Formation of Sensor-Zn-Protein Adducts versus Zn(Sensor)2 Complexes.

PubMed

Nowakowski, Andrew B; Meeusen, Jeffrey W; Menden, Heather; Tomasiewicz, Henry; Petering, David H

2015-12-21

Fluorescent zinc sensors are the most commonly used tool to study the intracellular mobile zinc status within cellular systems. Previously, we have shown that the quinoline-based sensors Zinquin and 6-methoxy-8-p-toluenesulfonamido-quinoline (TSQ) predominantly form ternary adducts with members of the Zn-proteome. Here, the chemistries of these sensors are further characterized, including how Zn(sensor)2 complexes may react in an intracellular environment. We demonstrate that these sensors are typically used in higher concentrations than needed to obtain maximum signal. Exposing cells to either Zn(Zinquin)2 or Zn(TSQ)2 resulted in efficient cellular uptake and the formation of sensor-Zn-protein adducts as evidenced by both a fluorescence spectral shift toward that of ternary adducts and the localization of the fluorescence signal within the proteome after gel filtration of cellular lysates. Likewise, reacting Zn(sensor)2 with the Zn-proteome from LLC-PK1 cells resulted in the formation of sensor-Zn-protein ternary adducts that could be inhibited by first saturating the Zn- proteome with excess sensor. Further, a native SDS-PAGE analysis of the Zn-proteome reacted with either the sensor or the Zn(sensor)2 complex revealed that both reactions result in the formation of a similar set of sensor-Zn-protein fluorescent products. The results of this experiment also demonstrated that TSQ and Zinquin react with different members of the Zn-proteome. Reactions with the model apo-Zn-protein bovine serum albumin showed that both Zn(TSQ)2 and Zn(Zinquin)2 reacted to form ternary adducts with its apo-Zn-binding site. Moreover, incubating Zn(sensor)2 complexes with non-zinc binding proteins failed to elicit a spectral shift in the fluorescence spectrum, supporting the premise that blue-shifted emission spectra are due to sensor-Zn-protein ternary adducts. It was concluded that Zn(sensors)2 species do not play a significant role in the overall reaction between these sensors and intact cells. In turn, this study further supports the formation of sensor-Zn-protein adducts as the principal observed fluorescent product during experiments employing these two sensors.

Remodeling Cildb, a popular database for cilia and links for ciliopathies

PubMed Central

2014-01-01

Background New generation technologies in cell and molecular biology generate large amounts of data hard to exploit for individual proteins. This is particularly true for ciliary and centrosomal research. Cildb is a multi–species knowledgebase gathering high throughput studies, which allows advanced searches to identify proteins involved in centrosome, basal body or cilia biogenesis, composition and function. Combined to localization of genetic diseases on human chromosomes given by OMIM links, candidate ciliopathy proteins can be compiled through Cildb searches. Methods Othology between recent versions of the whole proteomes was computed using Inparanoid and ciliary high throughput studies were remapped on these recent versions. Results Due to constant evolution of the ciliary and centrosomal field, Cildb has been recently upgraded twice, with new species whole proteomes and new ciliary studies, and the latter version displays a novel BioMart interface, much more intuitive than the previous ones. Conclusions This already popular database is designed now for easier use and is up to date in regard to high throughput ciliary studies. PMID:25422781

Examining hemodialyzer membrane performance using proteomic technologies

PubMed Central

Bonomini, Mario; Pieroni, Luisa; Di Liberato, Lorenzo; Sirolli, Vittorio; Urbani, Andrea

2018-01-01

The success and the quality of hemodialysis therapy are mainly related to both clearance and biocompatibility properties of the artificial membrane packed in the hemodialyzer. Performance of a membrane is strongly influenced by its interaction with the plasma protein repertoire during the extracorporeal procedure. Recognition that a number of medium–high molecular weight solutes, including proteins and protein-bound molecules, are potentially toxic has prompted the development of more permeable membranes. Such membrane engineering, however, may cause loss of vital proteins, with membrane removal being nonspecific. In addition, plasma proteins can be adsorbed onto the membrane surface upon blood contact during dialysis. Adsorption can contribute to the removal of toxic compounds and governs the biocompatibility of a membrane, since surface-adsorbed proteins may trigger a variety of biologic blood pathways with pathophysiologic consequences. Over the last years, use of proteomic approaches has allowed polypeptide spectrum involved in the process of hemodialysis, a key issue previously hampered by lack of suitable technology, to be assessed in an unbiased manner and in its full complexity. Proteomics has been successfully applied to identify and quantify proteins in complex mixtures such as dialysis outflow fluid and fluid desorbed from dialysis membrane containing adsorbed proteins. The identified proteins can also be characterized by their involvement in metabolic and signaling pathways, molecular networks, and biologic processes through application of bioinformatics tools. Proteomics may thus provide an actual functional definition as to the effect of a membrane material on plasma proteins during hemodialysis. Here, we review the results of proteomic studies on the performance of hemodialysis membranes, as evaluated in terms of solute removal efficiency and blood–membrane interactions. The evidence collected indicates that the information provided by proteomic investigations yields improved molecular and functional knowledge and may lead to the development of more efficient membranes for the potential benefit of the patient. PMID:29296087

Proteomic Stable Isotope Probing Reveals Biosynthesis Dynamics of Slow Growing Methane Based Microbial Communities

DOE PAGES

Marlow, Jeffery; Skennerton, Connor T.; Li, Zhou; ...

2016-04-29

Marine methane seep habitats represent an important control on the global flux of methane between the subsurface and water column reservoirs. Meta-omics studies have begun to outline community-wide metabolic potential, but expression patterns of proteins that enact sulfate-mediated anaerobic methane oxidation in seeps are poorly characterized. Proteomic stable isotope probing (proteomic SIP) offers an additional layer of information for characterizing phylogenetically specific, functionally relevant activity in mixed microbial communities. Here we applied proteomic SIP to 15NH4+ and CH4 amended seep sediment microcosms in an attempt to track the protein synthesis of slow-growing, low-energy microbial systems. Across all samples, 3495 proteinsmore » were identified, 21% of which were 15N-labeled. We observed active synthesis (15N enrichment) of all proteins believed to be involved in sulfate reduction and reverse methanogenesis including methylenetetrahydromethanopterin reductase (Mer). The abundance and phylogenetic range of methyl-coenzyme M reductase (Mcr) orthologs produced during incubation experiments suggests that seeps provide sufficient niches for multiple organisms performing analogous metabolisms. Twenty-eight previously unreported post-translational modifications of McrA were measured, indicating dynamic enzymatic machinery and offering a dimension of functional diversity beyond gene-dictated sequence. RNA polymerase associated with putative sulfur-oxidizing Epsilonproteobacteria and aerobic Gammaproteobacteria were more abundant among pre-incubation proteins, suggesting diminished metabolic activity in long-term anoxic, sulfidic experimental incubations. Twenty-six proteins of unknown function were detected in all proteomic experiments and actively expressed in labeled experiments, suggesting that they play important roles in methane seep ecosystems. The addition of stable isotope probing to environmental proteomics experiments provides a mechanism to begin to assess the degree to which diagnostic meatbolic proteins are long-lived or acively synthesized in complex, slow-growing microbial communities. Our work here demonstrates that sediment-hosted microbial assemblages in marine methane seeps are dynamic, heterogeneous systems with broad functional diversity.« less

Examining hemodialyzer membrane performance using proteomic technologies.

PubMed

Bonomini, Mario; Pieroni, Luisa; Di Liberato, Lorenzo; Sirolli, Vittorio; Urbani, Andrea

2018-01-01

The success and the quality of hemodialysis therapy are mainly related to both clearance and biocompatibility properties of the artificial membrane packed in the hemodialyzer. Performance of a membrane is strongly influenced by its interaction with the plasma protein repertoire during the extracorporeal procedure. Recognition that a number of medium-high molecular weight solutes, including proteins and protein-bound molecules, are potentially toxic has prompted the development of more permeable membranes. Such membrane engineering, however, may cause loss of vital proteins, with membrane removal being nonspecific. In addition, plasma proteins can be adsorbed onto the membrane surface upon blood contact during dialysis. Adsorption can contribute to the removal of toxic compounds and governs the biocompatibility of a membrane, since surface-adsorbed proteins may trigger a variety of biologic blood pathways with pathophysiologic consequences. Over the last years, use of proteomic approaches has allowed polypeptide spectrum involved in the process of hemodialysis, a key issue previously hampered by lack of suitable technology, to be assessed in an unbiased manner and in its full complexity. Proteomics has been successfully applied to identify and quantify proteins in complex mixtures such as dialysis outflow fluid and fluid desorbed from dialysis membrane containing adsorbed proteins. The identified proteins can also be characterized by their involvement in metabolic and signaling pathways, molecular networks, and biologic processes through application of bioinformatics tools. Proteomics may thus provide an actual functional definition as to the effect of a membrane material on plasma proteins during hemodialysis. Here, we review the results of proteomic studies on the performance of hemodialysis membranes, as evaluated in terms of solute removal efficiency and blood-membrane interactions. The evidence collected indicates that the information provided by proteomic investigations yields improved molecular and functional knowledge and may lead to the development of more efficient membranes for the potential benefit of the patient.

Proteomic Stable Isotope Probing Reveals Biosynthesis Dynamics of Slow Growing Methane Based Microbial Communities

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marlow, Jeffery; Skennerton, Connor T.; Li, Zhou

Marine methane seep habitats represent an important control on the global flux of methane between the subsurface and water column reservoirs. Meta-omics studies have begun to outline community-wide metabolic potential, but expression patterns of proteins that enact sulfate-mediated anaerobic methane oxidation in seeps are poorly characterized. Proteomic stable isotope probing (proteomic SIP) offers an additional layer of information for characterizing phylogenetically specific, functionally relevant activity in mixed microbial communities. Here we applied proteomic SIP to 15NH4+ and CH4 amended seep sediment microcosms in an attempt to track the protein synthesis of slow-growing, low-energy microbial systems. Across all samples, 3495 proteinsmore » were identified, 21% of which were 15N-labeled. We observed active synthesis (15N enrichment) of all proteins believed to be involved in sulfate reduction and reverse methanogenesis including methylenetetrahydromethanopterin reductase (Mer). The abundance and phylogenetic range of methyl-coenzyme M reductase (Mcr) orthologs produced during incubation experiments suggests that seeps provide sufficient niches for multiple organisms performing analogous metabolisms. Twenty-eight previously unreported post-translational modifications of McrA were measured, indicating dynamic enzymatic machinery and offering a dimension of functional diversity beyond gene-dictated sequence. RNA polymerase associated with putative sulfur-oxidizing Epsilonproteobacteria and aerobic Gammaproteobacteria were more abundant among pre-incubation proteins, suggesting diminished metabolic activity in long-term anoxic, sulfidic experimental incubations. Twenty-six proteins of unknown function were detected in all proteomic experiments and actively expressed in labeled experiments, suggesting that they play important roles in methane seep ecosystems. The addition of stable isotope probing to environmental proteomics experiments provides a mechanism to begin to assess the degree to which diagnostic meatbolic proteins are long-lived or acively synthesized in complex, slow-growing microbial communities. Our work here demonstrates that sediment-hosted microbial assemblages in marine methane seeps are dynamic, heterogeneous systems with broad functional diversity.« less

Recent Advance in Applications of Proteomics Technologies on Traditional Chinese Medicine Research

PubMed Central

Zhu, Fangshi; Liu, Xuan; Li, Qi; Su, Shi-bing

2015-01-01

Proteomics technology, a major component of system biology, has gained comprehensive attention in the area of medical diagnosis, drug development, and mechanism research. On the holistic and systemic theory, proteomics has a convergence with traditional Chinese medicine (TCM). In this review, we discussed the applications of proteomic technologies in diseases-TCM syndrome combination researches. We also introduced the proteomic studies on the in vivo and in vitro effects and underlying mechanisms of TCM treatments using Chinese herbal medicine (CHM), Chinese herbal formula (CHF), and acupuncture. Furthermore, the combined studies of proteomics with other “-omics” technologies in TCM were also discussed. In summary, this report presents an overview of the recent advances in the application of proteomic technologies in TCM studies and sheds a light on the future global and further research on TCM. PMID:26557869

Phage-Induced Expression of CRISPR-Associated Proteins is Revealed by Shotgun Proteomics in Streptococcus thermophilus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Young, Jacque C; Dill, Brian; Pan, Chongle

The CRISPR/Cas system, comprised of clustered regularly interspaced short palindromic repeats along with their associated (Cas) proteins, protects bacteria and archaea from viral predation and invading nucleic acids. While the mechanism of action for this acquired immunity is currently under investigation, the response of Cas protein expression to phage infection has yet to be elucidated. In this study, we employed shotgun proteomics to measure the global proteome expression in a model system for studying the CRISPR/Cas response: infection of S. thermophilus DGCC7710 with phage 2972. Host and viral proteins were simultaneously measured following inoculation at two different multiplicities of infectionmore » and across various time points using two-dimensional liquid chromatography tandem mass spectroscopy. Thirty-seven out of forty predicted viral proteins were detected, including all proteins of the structural virome and viral effector proteins. In total, 1,013 of 2,079 predicted S. thermophilus proteins were detected, facilitating the monitoring of host protein synthesis changes in response to virus infection. Importantly, Cas proteins from all four CRISPR loci in the S. thermophilus DGCC7710 genome were detected, including loci previously thought to be inactive. Many Cas proteins were found to be constitutively expressed, but several demonstrated increased abundance during peak infection, including the Cas9 proteins from the CRISPR1 and CRISPR3 loci, which are key players in the interference phase of the CRISPR/Cas response. Altogether, these results provide novel insights into the proteomic response of S. thermophilus, specifically CRISPR-associated proteins, upon phage 2972 infection.« less

Proteome | Office of Cancer Clinical Proteomics Research

Cancer.gov

A proteome is the entire complement of proteins, including modifications made to a particular set of proteins, produced by an organism or a cellular system. This will vary with time and distinct requirements such as growth conditions and stresses, and thus is highly dynamic and spatial. Proteomics is the study of the proteome.

Systematic Evaluation of the Use of Human Plasma and Serum for Mass-Spectrometry-Based Shotgun Proteomics.

PubMed

Lan, Jiayi; Núñez Galindo, Antonio; Doecke, James; Fowler, Christopher; Martins, Ralph N; Rainey-Smith, Stephanie R; Cominetti, Ornella; Dayon, Loïc

2018-04-06

Over the last two decades, EDTA-plasma has been used as the preferred sample matrix for human blood proteomic profiling. Serum has also been employed widely. Only a few studies have assessed the difference and relevance of the proteome profiles obtained from plasma samples, such as EDTA-plasma or lithium-heparin-plasma, and serum. A more complete evaluation of the use of EDTA-plasma, heparin-plasma, and serum would greatly expand the comprehensiveness of shotgun proteomics of blood samples. In this study, we evaluated the use of heparin-plasma with respect to EDTA-plasma and serum to profile blood proteomes using a scalable automated proteomic pipeline (ASAP 2 ). The use of plasma and serum for mass-spectrometry-based shotgun proteomics was first tested with commercial pooled samples. The proteome coverage consistency and the quantitative performance were compared. Furthermore, protein measurements in EDTA-plasma and heparin-plasma samples were comparatively studied using matched sample pairs from 20 individuals from the Australian Imaging, Biomarkers and Lifestyle (AIBL) Study. We identified 442 proteins in common between EDTA-plasma and heparin-plasma samples. Overall agreement of the relative protein quantification between the sample pairs demonstrated that shotgun proteomics using workflows such as the ASAP 2 is suitable in analyzing heparin-plasma and that such sample type may be considered in large-scale clinical research studies. Moreover, the partial proteome coverage overlaps (e.g., ∼70%) showed that measures from heparin-plasma could be complementary to those obtained from EDTA-plasma.

Low temperature conditioning of garlic (Allium sativum L.) “seed” cloves induces alterations in sprouts proteome

PubMed Central

Dufoo-Hurtado, Miguel D.; Huerta-Ocampo, José Á.; Barrera-Pacheco, Alberto; Barba de la Rosa, Ana P.; Mercado-Silva, Edmundo M.

2015-01-01

Low-temperature conditioning of garlic “seed” cloves substitutes the initial climatic requirements of the crop and accelerates the cycle. We have reported that “seed” bulbs from “Coreano” variety conditioned at 5°C for 5 weeks reduces growth and plant weight as well as the crop yields and increases the synthesis of phenolic compounds and anthocyanins. Therefore, this treatment suggests a cold stress. Plant acclimation to stress is associated with deep changes in proteome composition. Since proteins are directly involved in plant stress response, proteomics studies can significantly contribute to unravel the possible relationships between protein abundance and plant stress acclimation. The aim of this work was to study the changes in the protein profiles of garlic “seed” cloves subjected to conditioning at low-temperature using proteomics approach. Two sets of garlic bulbs were used, one set was stored at room temperature (23°C), and the other was conditioned at low temperature (5°C) for 5 weeks. Total soluble proteins were extracted from sprouts of cloves and separated by two-dimensional gel electrophoresis. Protein spots showing statistically significant changes in abundance were analyzed by LC-ESI-MS/MS and identified by database search analysis using the Mascot search engine. The results revealed that low-temperature conditioning of garlic “seed” cloves causes alterations in the accumulation of proteins involved in different physiological processes such as cellular growth, antioxidative/oxidative state, macromolecules transport, protein folding and transcription regulation process. The metabolic pathways affected include protein biosynthesis and quality control system, photosynthesis, photorespiration, energy production, and carbohydrate and nucleotide metabolism. These processes can work cooperatively to establish a new cellular homeostasis that might be related with the physiological and biochemical changes observed in previous studies. PMID:26029231

Identification of proteins capable of metal reduction from the proteome of the Gram-positive bacterium Desulfotomaculum reducens MI-1 using an NADH-based activity assay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Otwell, Annie E.; Sherwood, Roberts; Zhang, Sheng

Metal reduction capability has been found in numerous species of environmentally abundant Gram-positive bacteria. However, understanding of microbial metal reduction is based almost solely on studies of Gram-negative organisms. In this study, we focus on Desulfotomaculum reducens MI-1, a Gram-positive metal reducer whose genome lacks genes with similarity to any characterized metal reductase. D. reducens has been shown to reduce not only Fe(III), but also the environmentally important contaminants U(VI) and Cr(VI). By extracting, separating, and analyzing the functional proteome of D. reducens, using a ferrozine-based assay in order to screen for chelated Fe(III)-NTA reduction with NADH as electron donor,more » we have identified proteins not previously characterized as iron reductases. Their function was confirmed by heterologous expression in E. coli. These are the protein NADH:flavin oxidoreductase (Dred_2421) and a protein complex composed of oxidoreductase FAD/NAD(P)-binding subunit (Dred_1685) and dihydroorotate dehydrogenase 1B (Dred_1686). Dred_2421 was identified in the soluble proteome and is predicted to be a cytoplasmic protein. Dred_1685 and Dred_1686 were identified in both the soluble as well as the insoluble (presumably membrane) protein fraction, suggesting a type of membrane-association, although PSORTb predicts both proteins are cytoplasmic. Furthermore, we show that these proteins have the capability to reduce soluble Cr(VI) and U(VI) with NADH as electron donor. This study is the first functional proteomic analysis of D. reducens, and one of the first analyses of metal and radionuclide reduction in an environmentally relevant Gram-positive bacterium.« less

Serum Proteome Profiles in Stricturing Crohn’s Disease: A pilot study.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Townsend, Peter; Zhang, Qibin; Shapiro, Jason

Background: Crohn’s disease (CD) is a form of inflammatory bowel disease (IBD) with different described behaviors, including stricture. At present, there are no laboratory studies that can differentiate stricturing CD from other phenotypes of IBD. We performed a pilot study to examine differences in the proteome among patients with stricturing Crohn’s disease, non-stricturing Crohn’s disease, and ulcerative colitis (UC). Methods: Serum samples were selected from the Ocean State Crohn’s and Colitis Area Registry (OSCCAR), an established cohort of patients with IBD. Crohn’s disease patients with surgically-resected stricture were matched with similar patients with Crohn’s disease without known stricture, and withmore » UC. Serum samples from each patient were digested and analyzed using liquid chromatography-mass spectrometry to characterize the proteome. Statistical analyses were performed to identify peptides and proteins that can differentiate CD with stricture. Results: Samples from 9 patients in each group (27 total patients) were analyzed. Baseline demographic characteristics were similar among the three groups. We quantified 7668 peptides and 897 proteins for analysis. ROC analysis identified a subset of peptides with an area under the curve greater than 0.9, indicating greater separation potential. Partial least squares discriminant analysis was able to distinguish among the three groups with up to 70% accuracy by peptides, and up to 80% accuracy by proteins. We identified the significantly different proteins and peptides, and determined their function based on previously published literature. Conclusions: The serum of patients with stricturing CD, non-stricturing CD, and UC are distinguishable via proteomic analysis. Some of the proteins that differentiate the stricturing phenotype have been implicated in complement activation, fibrinolytic pathways, and lymphocyte adhesion.« less

Low temperature conditioning of garlic (Allium sativum L.) "seed" cloves induces alterations in sprouts proteome.

PubMed

Dufoo-Hurtado, Miguel D; Huerta-Ocampo, José Á; Barrera-Pacheco, Alberto; Barba de la Rosa, Ana P; Mercado-Silva, Edmundo M

2015-01-01

Low-temperature conditioning of garlic "seed" cloves substitutes the initial climatic requirements of the crop and accelerates the cycle. We have reported that "seed" bulbs from "Coreano" variety conditioned at 5°C for 5 weeks reduces growth and plant weight as well as the crop yields and increases the synthesis of phenolic compounds and anthocyanins. Therefore, this treatment suggests a cold stress. Plant acclimation to stress is associated with deep changes in proteome composition. Since proteins are directly involved in plant stress response, proteomics studies can significantly contribute to unravel the possible relationships between protein abundance and plant stress acclimation. The aim of this work was to study the changes in the protein profiles of garlic "seed" cloves subjected to conditioning at low-temperature using proteomics approach. Two sets of garlic bulbs were used, one set was stored at room temperature (23°C), and the other was conditioned at low temperature (5°C) for 5 weeks. Total soluble proteins were extracted from sprouts of cloves and separated by two-dimensional gel electrophoresis. Protein spots showing statistically significant changes in abundance were analyzed by LC-ESI-MS/MS and identified by database search analysis using the Mascot search engine. The results revealed that low-temperature conditioning of garlic "seed" cloves causes alterations in the accumulation of proteins involved in different physiological processes such as cellular growth, antioxidative/oxidative state, macromolecules transport, protein folding and transcription regulation process. The metabolic pathways affected include protein biosynthesis and quality control system, photosynthesis, photorespiration, energy production, and carbohydrate and nucleotide metabolism. These processes can work cooperatively to establish a new cellular homeostasis that might be related with the physiological and biochemical changes observed in previous studies.

Epsilon-Q: An Automated Analyzer Interface for Mass Spectral Library Search and Label-Free Protein Quantification.

PubMed

Cho, Jin-Young; Lee, Hyoung-Joo; Jeong, Seul-Ki; Paik, Young-Ki

2017-12-01

Mass spectrometry (MS) is a widely used proteome analysis tool for biomedical science. In an MS-based bottom-up proteomic approach to protein identification, sequence database (DB) searching has been routinely used because of its simplicity and convenience. However, searching a sequence DB with multiple variable modification options can increase processing time, false-positive errors in large and complicated MS data sets. Spectral library searching is an alternative solution, avoiding the limitations of sequence DB searching and allowing the detection of more peptides with high sensitivity. Unfortunately, this technique has less proteome coverage, resulting in limitations in the detection of novel and whole peptide sequences in biological samples. To solve these problems, we previously developed the "Combo-Spec Search" method, which uses manually multiple references and simulated spectral library searching to analyze whole proteomes in a biological sample. In this study, we have developed a new analytical interface tool called "Epsilon-Q" to enhance the functions of both the Combo-Spec Search method and label-free protein quantification. Epsilon-Q performs automatically multiple spectral library searching, class-specific false-discovery rate control, and result integration. It has a user-friendly graphical interface and demonstrates good performance in identifying and quantifying proteins by supporting standard MS data formats and spectrum-to-spectrum matching powered by SpectraST. Furthermore, when the Epsilon-Q interface is combined with the Combo-Spec search method, called the Epsilon-Q system, it shows a synergistic function by outperforming other sequence DB search engines for identifying and quantifying low-abundance proteins in biological samples. The Epsilon-Q system can be a versatile tool for comparative proteome analysis based on multiple spectral libraries and label-free quantification.

Proteomic diversity of high-density lipoprotein explains its association with clinical outcome in patients with heart failure.

PubMed

Emmens, Johanna Elisabeth; Jones, Donald J L; Cao, Thong H; Chan, Daniel C S; Romaine, Simon P R; Quinn, Paulene A; Anker, Stefan D; Cleland, John G; Dickstein, Kenneth; Filippatos, Gerasimos; Hillege, Hans L; Lang, Chim C; Ponikowski, Piotr; Samani, Nilesh J; van Veldhuisen, Dirk J; Zannad, Faiz; Zwinderman, Aeilko H; Metra, Marco; de Boer, Rudolf A; Voors, Adriaan A; Ng, Leong L

2018-02-01

Previously, low high-density lipoprotein (HDL) cholesterol was found to be one of the strongest predictors of mortality and/or heart failure (HF) hospitalisation in patients with HF. We therefore performed in-depth investigation of the multifunctional HDL proteome to reveal underlying pathophysiological mechanisms explaining the association between HDL and clinical outcome. We selected a cohort of 90 HF patients with 1:1 cardiovascular death/survivor ratio from BIOSTAT-CHF. A novel optimised protocol for selective enrichment of lipoproteins was used to prepare plasma. Enriched lipoprotein content of samples was analysed using high resolution nanoscale liquid chromatography-mass spectrometry-based proteomics, utilising a label free approach. Within the HDL proteome, 49 proteins significantly differed between deaths and survivors. An optimised model of 12 proteins predicted death with 76% accuracy (Nagelkerke R 2 =0.37, P < 0.001). The strongest contributors to this model were filamin-A (related to crosslinking of actin filaments) [odds ratio (OR) 0.31, 95% confidence interval (CI) 0.15-0.61, P = 0.001] and pulmonary surfactant-associated protein B (related to alveolar capillary membrane function) (OR 2.50, 95% CI 1.57-3.98, P < 0.001). The model predicted mortality with an area under the curve of 0.82 (95% CI 0.77-0.87, P < 0.001). Internal cross validation resulted in 73.3 ± 7.2% accuracy. This study shows marked differences in composition of the HDL proteome between HF survivors and deaths. The strongest differences were seen in proteins reflecting crosslinking of actin filaments and alveolar capillary membrane function, posing potential pathophysiological mechanisms underlying the association between HDL and clinical outcome in HF. © 2017 The Authors. European Journal of Heart Failure © 2017 European Society of Cardiology.

Proteomic Analysis of the Soybean Symbiosome Identifies New Symbiotic Proteins*

PubMed Central

Clarke, Victoria C.; Loughlin, Patrick C.; Gavrin, Aleksandr; Chen, Chi; Brear, Ella M.; Day, David A.; Smith, Penelope M.C.

2015-01-01

Legumes form a symbiosis with rhizobia in which the plant provides an energy source to the rhizobia bacteria that it uses to fix atmospheric nitrogen. This nitrogen is provided to the legume plant, allowing it to grow without the addition of nitrogen fertilizer. As part of the symbiosis, the bacteria in the infected cells of a new root organ, the nodule, are surrounded by a plant-derived membrane, the symbiosome membrane, which becomes the interface between the symbionts. Fractions containing the symbiosome membrane (SM) and material from the lumen of the symbiosome (peribacteroid space or PBS) were isolated from soybean root nodules and analyzed using nongel proteomic techniques. Bicarbonate stripping and chloroform-methanol extraction of isolated SM were used to reduce complexity of the samples and enrich for hydrophobic integral membrane proteins. One hundred and ninety-seven proteins were identified as components of the SM, with an additional fifteen proteins identified from peripheral membrane and PBS protein fractions. Proteins involved in a range of cellular processes such as metabolism, protein folding and degradation, membrane trafficking, and solute transport were identified. These included a number of proteins previously localized to the SM, such as aquaglyceroporin nodulin 26, sulfate transporters, remorin, and Rab7 homologs. Among the proteome were a number of putative transporters for compounds such as sulfate, calcium, hydrogen ions, peptide/dicarboxylate, and nitrate, as well as transporters for which the substrate is not easy to predict. Analysis of the promoter activity for six genes encoding putative SM proteins showed nodule specific expression, with five showing expression only in infected cells. Localization of two proteins was confirmed using GFP-fusion experiments. The data have been deposited to the ProteomeXchange with identifier PXD001132. This proteome will provide a rich resource for the study of the legume-rhizobium symbiosis. PMID:25724908

Proteomes and Ubiquitylomes Analysis Reveals the Involvement of Ubiquitination in Protein Degradation in Petunias1

PubMed Central

Liu, Juanxu; Wei, Qian; Wang, Rongmin; Yang, Weiyuan; Ma, Yueyue; Chen, Guoju

2017-01-01

Petal senescence is a complex programmed process. It has been demonstrated previously that treatment with ethylene, a plant hormone involved in senescence, can extensively alter transcriptome and proteome profiles in plants. However, little is known regarding the impact of ethylene on posttranslational modification (PTM) or the association between PTM and the proteome. Protein degradation is one of the hallmarks of senescence, and ubiquitination, a major PTM in eukaryotes, plays important roles in protein degradation. In this study, we first obtained reference petunia (Petunia hybrida) transcriptome data via RNA sequencing. Next, we quantitatively investigated the petunia proteome and ubiquitylome and the association between them in petunia corollas following ethylene treatment. In total, 51,799 unigenes, 3,606 proteins, and 2,270 ubiquitination sites were quantified 16 h after ethylene treatment. Treatment with ethylene resulted in 14,448 down-regulated and 6,303 up-regulated unigenes (absolute log2 fold change > 1 and false discovery rate < 0.001), 284 down-regulated and 233 up-regulated proteins, and 320 up-regulated and 127 down-regulated ubiquitination sites using a 1.5-fold threshold (P < 0.05), indicating that global ubiquitination levels increase during ethylene-mediated corolla senescence in petunia. Several putative ubiquitin ligases were up-regulated at the protein and transcription levels. Our results showed that the global proteome and ubiquitylome were negatively correlated and that ubiquitination could be involved in the degradation of proteins during ethylene-mediated corolla senescence in petunia. Ethylene regulates hormone signaling transduction pathways at both the protein and ubiquitination levels in petunia corollas. In addition, our results revealed that ethylene increases the ubiquitination levels of proteins involved in endoplasmic reticulum-associated degradation. PMID:27810942

Complete Proteome of a Quinolone-Resistant Salmonella Typhimurium Phage Type DT104B Clinical Strain

PubMed Central

Correia, Susana; Nunes-Miranda, Júlio D.; Pinto, Luís; Santos, Hugo M.; de Toro, María; Sáenz, Yolanda; Torres, Carmen; Capelo, José Luis; Poeta, Patrícia; Igrejas, Gilberto

2014-01-01

Salmonellosis is one of the most common and widely distributed foodborne diseases. The emergence of Salmonella strains that are resistant to a variety of antimicrobials is a serious global public health concern. Salmonella enterica serovar Typhimurium definitive phage type 104 (DT104) is one of these emerging epidemic multidrug resistant strains. Here we collate information from the diverse and comprehensive range of experiments on Salmonella proteomes that have been published. We then present a new study of the proteome of the quinolone-resistant Se20 strain (phage type DT104B), recovered after ciprofloxacin treatment and compared it to the proteome of reference strain SL1344. A total of 186 and 219 protein spots were recovered from Se20 and SL1344 protein extracts, respectively, after two-dimensional gel electrophoresis. The signatures of 94% of the protein spots were successfully identified through matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS). Three antimicrobial resistance related proteins, whose genes were previously detected by polymerase chain reaction (PCR), were identified in the clinical strain. The presence of these proteins, dihydropteroate synthase type-2 (sul2 gene), aminoglycoside resistance protein A (strA gene) and aminoglycoside 6'-N-acetyltransferase type Ib-cr4 (aac(6')-Ib-cr4 gene), was confirmed in the DT104B clinical strain. The aac(6')-Ib-cr4 gene is responsible for plasmid-mediated aminoglycoside and quinolone resistance. This is a preliminary analysis of the proteome of these two S. Typhimurium strains and further work is being developed to better understand how antimicrobial resistance is developing in this pathogen. PMID:25196519

Effects of Alanyl-Glutamine Treatment on the Peritoneal Dialysis Effluent Proteome Reveal Pathomechanism-Associated Molecular Signatures*

PubMed Central

Herzog, Rebecca; Boehm, Michael; Unterwurzacher, Markus; Wagner, Anja; Parapatics, Katja; Májek, Peter; Mueller, André C.; Lichtenauer, Anton; Bennett, Keiryn L.; Alper, Seth L.; Vychytil, Andreas; Aufricht, Christoph; Kratochwill, Klaus

2018-01-01

Peritoneal dialysis (PD) is a modality of renal replacement therapy in which the high volumes of available PD effluent (PDE) represents a rich source of biomarkers for monitoring disease and therapy. Although this information could help guide the management of PD patients, little is known about the potential of PDE to define pathomechanism-associated molecular signatures in PD. We therefore subjected PDE to a high-performance multiplex proteomic analysis after depletion of highly-abundant plasma proteins and enrichment of low-abundance proteins. A combination of label-free and isobaric labeling strategies was applied to PDE samples from PD patients (n = 20) treated in an open-label, randomized, two-period, cross-over clinical trial with standard PD fluid or with a novel PD fluid supplemented with alanyl-glutamine (AlaGln). With this workflow we identified 2506 unique proteins in the PDE proteome, greatly increasing coverage beyond the 171 previously-reported proteins. The proteins identified range from high abundance plasma proteins to low abundance cellular proteins, and are linked to larger numbers of biological processes and pathways, some of which are novel for PDE. Interestingly, proteins linked to membrane remodeling and fibrosis are overrepresented in PDE compared with plasma, whereas the proteins underrepresented in PDE suggest decreases in host defense, immune-competence and response to stress. Treatment with AlaGln-supplemented PD fluid is associated with reduced activity of membrane injury-associated mechanisms and with restoration of biological processes involved in stress responses and host defense. Our study represents the first application of the PDE proteome in a randomized controlled prospective clinical trial of PD. This novel proteomic workflow allowed detection of low abundance biomarkers to define pathomechanism-associated molecular signatures in PD and their alterations by a novel therapeutic intervention. PMID:29208752

Exploring Biological and Geological Age-related Changes through Variations in Intra- and Intertooth Proteomes of Ancient Dentine.

PubMed

Procopio, Noemi; Chamberlain, Andrew T; Buckley, Michael

2018-03-02

Proteomic analyses are becoming more widely used in archeology not only due to the greater preservation of proteins in ancient specimens than DNA but also because they can offer different information, particularly relating to compositional preservation and potentially a means to estimate biological and geological age. However, it remains unclear to what extent different burial environments impact these aspects of proteome decay. Teeth have to date been much less studied than bone but are ideal to explore how proteins decay with time due to the negligible turnover that occurs in dentine relative to bone. We investigated the proteome variability and deamidation levels of different sections of molar teeth from archeological bovine mandibles as well as their mandibular bone. We obtained a greater yield of proteins from the crown of the teeth but did not find differences between the different molars analyzed within each mandible. We also obtained the best variety of protein from a well-preserved mandible that was not the youngest one in terms of chronological age, showing the influence of the preservation conditions on the final proteomic outcome. Intriguingly, we also noticed an increase in abundance levels of fetuin-A in biologically younger mandibles as reported previously, but the opposite trend in tooth dentine. Interestingly, we observed higher glutamine deamidation levels in teeth from the geologically oldest mandible despite it being the biologically youngest specimen, showing that the archeological age strongly impacts on the level of deamidations observed, much more so than biological aging. This indicates that the glutamine deamidation ratio of selected peptides may act as a good predictor of the relative geochronological age of archeological specimens.

Temporal proteomic analysis reveals defects in small-intestinal development of porcine fetuses with intrauterine growth restriction.

PubMed

Wang, Xiaoqiu; Lin, Gang; Liu, Chuang; Feng, Cuiping; Zhou, Huaijun; Wang, Taiji; Li, Defa; Wu, Guoyao; Wang, Junjun

2014-07-01

The fetus/neonate with intrauterine growth restriction (IUGR) has a high perinatal mortality and morbidity rate, as well as reduced efficiency for nutrients utilization. Our previous studies showed alterations of intestinal proteome in IUGR piglets both at birth and during the nursing period. Considering the potential long-term impacts of fetal programming and substantial increases in amounts of amniotic fluid nutrients from mid-gestation in pigs, the present study involved IUGR porcine fetuses from days 60 to 110 of gestation (mid to late gestation). We identified 59 differentially expressed proteins in the fetal small intestine that are related to intestinal growth, development and reprogramming. Our results further indicated increased abundances of proteins and enzymes associated with oxidative stress, apoptosis and protein degradation, as well as decreased abundances of proteins that are required for maintenance of cell structure and motility, absorption and transport of nutrients, energy metabolism, and protein synthesis in the fetal gut. Moreover, IUGR from middle to late gestation was associated with reduced expression of intestinal proteins that participate in regulation of gene expression and signal transduction. Collectively, these findings provide the first evidence for altered proteomes in the small intestine of IUGR fetuses, thereby predisposing the gut to metabolic defects during gestation and neonatal periods. Copyright © 2014 Elsevier Inc. All rights reserved.

Genomic and Proteomic Dissection of the Ubiquitous Plant Pathogen, Armillaria mellea: Toward a New Infection Model System

PubMed Central

2013-01-01

Armillaria mellea is a major plant pathogen. Yet, no large-scale “-omics” data are available to enable new studies, and limited experimental models are available to investigate basidiomycete pathogenicity. Here we reveal that the A. mellea genome comprises 58.35 Mb, contains 14473 gene models, of average length 1575 bp (4.72 introns/gene). Tandem mass spectrometry identified 921 mycelial (n = 629 unique) and secreted (n = 183 unique) proteins. Almost 100 mycelial proteins were either species-specific or previously unidentified at the protein level. A number of proteins (n = 111) was detected in both mycelia and culture supernatant extracts. Signal sequence occurrence was 4-fold greater for secreted (50.2%) compared to mycelial (12%) proteins. Analyses revealed a rich reservoir of carbohydrate degrading enzymes, laccases, and lignin peroxidases in the A. mellea proteome, reminiscent of both basidiomycete and ascomycete glycodegradative arsenals. We discovered that A. mellea exhibits a specific killing effect against Candida albicans during coculture. Proteomic investigation of this interaction revealed the unique expression of defensive and potentially offensive A. mellea proteins (n = 30). Overall, our data reveal new insights into the origin of basidiomycete virulence and we present a new model system for further studies aimed at deciphering fungal pathogenic mechanisms. PMID:23656496

Regulation of Lipid Metabolism by Dicer Revealed through SILAC Mice

PubMed Central

Huang, Tai-Chung; Saharabuddhe, Nandini A.; Kim, Min-Sik; Getnet, Derese; Yang, Yi; Peterson, Jonathan M.; Ghosh, Bidyut; Chaerkady, Raghothama; Leach, Steven D.; Marchionni, Luigi; Wong, G. William; Pandey, Akhilesh

2012-01-01

Dicer is a ribonuclease whose major role is to generate mature microRNAs although additional functions have been proposed. Deletion of Dicer leads to embryonic lethality in mice. To study the role of Dicer in adults, we generated mice in which administration of tamoxifen induces deletion of Dicer. Surprisingly, disruption of Dicer in adult mice induced lipid accumulation in the small intestine. To dissect the underlying mechanisms, we carried out miRNA, mRNA and proteomic profiling of small intestine. The proteomic analysis was done using mice metabolically labeled with heavy lysine (SILAC mice) for an in vivo readout. We identified 646 proteins of which 80 were upregulated >2-fold and 75 were downregulated. Consistent with the accumulation of lipids, Dicer disruption caused a marked decrease of microsomal triglyceride transfer protein, long-chain fatty acyl-CoA ligase 5, fatty acid binding protein, and very-long-chain fatty acyl-CoA dehydrogenase, among others. We validated these results using multiple reaction monitoring (MRM) experiments by targeting proteotypic peptides. Our data reveal a previously unappreciated role of Dicer in lipid metabolism. These studies demonstrate a systems biology approach by integrating mouse models, metabolic labeling, gene expression profiling and quantitative proteomics can be a powerful tool for understanding complex biological systems. PMID:22313051

Analysis of the Human Prostate-Specific Proteome Defined by Transcriptomics and Antibody-Based Profiling Identifies TMEM79 and ACOXL as Two Putative, Diagnostic Markers in Prostate Cancer

PubMed Central

O'Hurley, Gillian; Busch, Christer; Fagerberg, Linn; Hallström, Björn M.; Stadler, Charlotte; Tolf, Anna; Lundberg, Emma; Schwenk, Jochen M.; Jirström, Karin; Bjartell, Anders; Gallagher, William M.; Uhlén, Mathias; Pontén, Fredrik

2015-01-01

To better understand prostate function and disease, it is important to define and explore the molecular constituents that signify the prostate gland. The aim of this study was to define the prostate specific transcriptome and proteome, in comparison to 26 other human tissues. Deep sequencing of mRNA (RNA-seq) and immunohistochemistry-based protein profiling were combined to identify prostate specific gene expression patterns and to explore tissue biomarkers for potential clinical use in prostate cancer diagnostics. We identified 203 genes with elevated expression in the prostate, 22 of which showed more than five-fold higher expression levels compared to all other tissue types. In addition to previously well-known proteins we identified two poorly characterized proteins, TMEM79 and ACOXL, with potential to differentiate between benign and cancerous prostatic glands in tissue biopsies. In conclusion, we have applied a genome-wide analysis to identify the prostate specific proteome using transcriptomics and antibody-based protein profiling to identify genes with elevated expression in the prostate. Our data provides a starting point for further functional studies to explore the molecular repertoire of normal and diseased prostate including potential prostate cancer markers such as TMEM79 and ACOXL. PMID:26237329

Investigation of the Cell Surface Proteome of Human Periodontal Ligament Stem Cells

PubMed Central

Xiong, Jimin; Menicanin, Danijela; Marino, Victor

2016-01-01

The present study examined the cell surface proteome of human periodontal ligament stem cells (PDLSC) compared to human fibroblasts. Cell surface proteins were prelabelled with CyDye before processing to extract the membrane lysates, which were separated using 2D electrophoresis. Selected differentially expressed protein “spots” were identified using Mass spectrometry. Four proteins were selected for validation: CD73, CD90, Annexin A2, and sphingosine kinase 1 previously associated with mesenchymal stem cells. Flow cytometric analysis found that CD73 and CD90 were highly expressed by human PDLSC and gingival fibroblasts but not by keratinocytes, indicating that these antigens could be used as potential markers for distinguishing between mesenchymal cells and epithelial cell populations. Annexin A2 was also found to be expressed at low copy number on the cell surface of human PDLSC and gingival fibroblasts, while human keratinocytes lacked any cell surface expression of Annexin A2. In contrast, sphingosine kinase 1 expression was detected in all the cell types examined using immunocytochemical analysis. These proteomic studies form the foundation to further define the cell surface protein expression profile of PDLSC in order to better characterise this cell population and help develop novel strategies for the purification of this stem cell population. PMID:27579043

Investigation of the Cell Surface Proteome of Human Periodontal Ligament Stem Cells.

PubMed

Xiong, Jimin; Menicanin, Danijela; Zilm, Peter S; Marino, Victor; Bartold, P Mark; Gronthos, Stan

2016-01-01

The present study examined the cell surface proteome of human periodontal ligament stem cells (PDLSC) compared to human fibroblasts. Cell surface proteins were prelabelled with CyDye before processing to extract the membrane lysates, which were separated using 2D electrophoresis. Selected differentially expressed protein "spots" were identified using Mass spectrometry. Four proteins were selected for validation: CD73, CD90, Annexin A2, and sphingosine kinase 1 previously associated with mesenchymal stem cells. Flow cytometric analysis found that CD73 and CD90 were highly expressed by human PDLSC and gingival fibroblasts but not by keratinocytes, indicating that these antigens could be used as potential markers for distinguishing between mesenchymal cells and epithelial cell populations. Annexin A2 was also found to be expressed at low copy number on the cell surface of human PDLSC and gingival fibroblasts, while human keratinocytes lacked any cell surface expression of Annexin A2. In contrast, sphingosine kinase 1 expression was detected in all the cell types examined using immunocytochemical analysis. These proteomic studies form the foundation to further define the cell surface protein expression profile of PDLSC in order to better characterise this cell population and help develop novel strategies for the purification of this stem cell population.

Comparative proteomics reveal the mechanism of Tween80 enhanced phenanthrene biodegradation by Sphingomonas sp. GY2B.

PubMed

Liu, Shasha; Guo, Chuling; Dang, Zhi; Liang, Xujun

2017-03-01

Previous study concerning the effects of surfactants on phenanthrene biodegradation focused on observing the changes of cell characteristics of Sphingomonas sp. GY2B. However, the impact of surfactants on the expression of bacterial proteins, controlling phenanthrene transport and catabolism, remains obscure. To overcome the knowledge gap, comparative proteomic approaches were used to investigate protein expressions of Sphingomonas sp. GY2B during phenanthrene biodegradation in the presence and absence of a nonionic surfactant, Tween80. A total of 23 up-regulated and 19 down-regulated proteins were detected upon Tween80 treatment. Tween80 could regulate ion transport (e.g. H + ) in cell membrane to provide driving force (ATP) for the transmembrane transport of phenanthrene thus increasing its uptake and biodegradation by GY2B. Moreover, Tween80 probably increased GY2B vitality and growth by inducing the expression of peptidylprolyl isomerase to stabilize cell membrane, increasing the abundances of proteins involved in intracellular metabolic pathways (e.g. TCA cycle), as well as decreasing the abundances of translation/transcription-related proteins and cysteine desulfurase, thereby facilitating phenanthrene biodegradation. This study may facilitate a better understanding of the mechanisms that regulate surfactants-enhanced biodegradation of PAHs at the proteomic level. Copyright © 2016 Elsevier Inc. All rights reserved.

Proteomic analysis of the organic matrix of the abalone Haliotis asinina calcified shell

PubMed Central

2010-01-01

Background The formation of the molluscan shell is regulated to a large extent by a matrix of extracellular macromolecules that are secreted by the shell forming tissue, the mantle. This so called "calcifying matrix" is a complex mixture of proteins and glycoproteins that is assembled and occluded within the mineral phase during the calcification process. While the importance of the calcifying matrix to shell formation has long been appreciated, most of its protein components remain uncharacterised. Results Recent expressed sequence tag (EST) investigations of the mantle tissue from the tropical abalone (Haliotis asinina) provide an opportunity to further characterise the proteins in the shell by a proteomic approach. In this study, we have identified a total of 14 proteins from distinct calcified layers of the shell. Only two of these proteins have been previously characterised from abalone shells. Among the novel proteins are several glutamine- and methionine-rich motifs and hydrophobic glycine-, alanine- and acidic aspartate-rich domains. In addition, two of the new proteins contained Kunitz-like and WAP (whey acidic protein) protease inhibitor domains. Conclusion This is one of the first comprehensive proteomic study of a molluscan shell, and should provide a platform for further characterization of matrix protein functions and interactions. PMID:21050442

Quantitative proteome profile of water deficit stress responses in eastern cottonwood ( Populus deltoides) leaves

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abraham, Paul E.; Garcia, Benjamin J.; Gunter, Lee E.

Drought stress is a recurring feature of world climate and the single most important factor influencing agricultural yield worldwide. Plants display highly variable, species-specific responses to drought and these responses are multifaceted, requiring physiological and morphological changes influenced by genetic and molecular mechanisms. Moreover, the reproducibility of water deficit studies is very cumbersome, which significantly impedes research on drought tolerance, because how a plant responds is highly influenced by the timing, duration, and intensity of the water deficit. Despite progress in the identification of drought-related mechanisms in many plants, the molecular basis of drought resistance remains to be fully understoodmore » in trees, particularly in poplar species because their wide geographic distribution results in varying tolerances to drought. Herein, we aimed to better understand this complex phenomenon in eastern cottonwood ( Populus deltoides) by performing a detailed contrast of the proteome changes between two different water deficit experiments to identify functional intersections and divergences in proteome responses. We investigated plants subjected to cyclic water deficit and compared these responses to plants subjected to prolonged acute water deficit. In total, we identified 108,012 peptide sequences across both experiments that provided insight into the quantitative state of 22,737 Populus gene models and 8,199 functional protein groups in response to drought. Together, these datasets provide the most comprehensive insight into proteome drought responses in poplar to date and a direct proteome comparison between short period dehydration shock and cyclic, post-drought re-watering. Altogether, this investigation provides novel insights into drought avoidance mechanisms that are distinct from progressive drought stress. Additionally, we identified proteins that have been associated as drought-relevant in previous studies. Importantly, we highlight the RD26 transcription factor as a gene regulated at both the transcript and protein level, regardless of species and drought condition, and, thus, represents a key, universal drought marker for Populus species.« less

Search for Allergens from the Pollen Proteome of Sunflower (Helianthus annuus L.): A Major Sensitizer for Respiratory Allergy Patients.

PubMed

Ghosh, Nandini; Sircar, Gaurab; Saha, Bodhisattwa; Pandey, Naren; Gupta Bhattacharya, Swati

2015-01-01

Respiratory allergy triggered by pollen allergens is increasing at an alarming rate worldwide. Sunflower pollen is thought to be an important source of inhalant allergens. Present study aims to identify the prevalence of sunflower pollinosis among the Indian allergic population and characterizes the pollen allergens using immuno-proteomic tools. Clinico-immunological tests were performed to understand the prevalence of sensitivity towards sunflower pollen among the atopic population. Sera from selected sunflower positive patients were used as probe to detect the IgE-reactive proteins from the one and two dimensional electrophoretic separated proteome of sunflower pollen. The antigenic nature of the sugar moiety of the glycoallergens was studied by meta-periodate modification of IgE-immunoblot. Finally, these allergens were identified by mass-spectrometry. Prevalence of sunflower pollen sensitization was observed among 21% of the pollen allergic population and associated with elevated level of specific IgE and histamine in the sera of these patients. Immunoscreening of sunflower pollen proteome with patient sera detected seven IgE-reactive proteins with varying molecular weight and pI. Hierarchical clustering of 2D-immunoblot data highlighted three allergens characterized by a more frequent immuno-reactivity and increased levels of IgE antibodies in the sera of susceptible patients. These allergens were considered as the major allergens of sunflower pollen and were found to have their glycan moiety critical for inducing IgE response. Homology driven search of MS/MS data of these IgE-reactive proteins identified seven previously unreported allergens from sunflower pollen. Three major allergenic proteins were identified as two pectate lyases and a cysteine protease. Novelty of the present report is the identification of a panel of seven sunflower pollen allergens for the first time at immuno-biochemical and proteomic level, which substantiated the clinical evidence of sunflower allergy. Further purification and recombinant expression of these allergens will improve component-resolved diagnosis and therapy of pollen allergy.

Quantitative proteome profile of water deficit stress responses in eastern cottonwood ( Populus deltoides) leaves

DOE PAGES

Abraham, Paul E.; Garcia, Benjamin J.; Gunter, Lee E.; ...

2018-02-15

Drought stress is a recurring feature of world climate and the single most important factor influencing agricultural yield worldwide. Plants display highly variable, species-specific responses to drought and these responses are multifaceted, requiring physiological and morphological changes influenced by genetic and molecular mechanisms. Moreover, the reproducibility of water deficit studies is very cumbersome, which significantly impedes research on drought tolerance, because how a plant responds is highly influenced by the timing, duration, and intensity of the water deficit. Despite progress in the identification of drought-related mechanisms in many plants, the molecular basis of drought resistance remains to be fully understoodmore » in trees, particularly in poplar species because their wide geographic distribution results in varying tolerances to drought. Herein, we aimed to better understand this complex phenomenon in eastern cottonwood ( Populus deltoides) by performing a detailed contrast of the proteome changes between two different water deficit experiments to identify functional intersections and divergences in proteome responses. We investigated plants subjected to cyclic water deficit and compared these responses to plants subjected to prolonged acute water deficit. In total, we identified 108,012 peptide sequences across both experiments that provided insight into the quantitative state of 22,737 Populus gene models and 8,199 functional protein groups in response to drought. Together, these datasets provide the most comprehensive insight into proteome drought responses in poplar to date and a direct proteome comparison between short period dehydration shock and cyclic, post-drought re-watering. Altogether, this investigation provides novel insights into drought avoidance mechanisms that are distinct from progressive drought stress. Additionally, we identified proteins that have been associated as drought-relevant in previous studies. Importantly, we highlight the RD26 transcription factor as a gene regulated at both the transcript and protein level, regardless of species and drought condition, and, thus, represents a key, universal drought marker for Populus species.« less

Deciphering the molecular mechanisms underlying sea urchin reversible adhesion: A quantitative proteomics approach.

PubMed

Lebesgue, Nicolas; da Costa, Gonçalo; Ribeiro, Raquel Mesquita; Ribeiro-Silva, Cristina; Martins, Gabriel G; Matranga, Valeria; Scholten, Arjen; Cordeiro, Carlos; Heck, Albert J R; Santos, Romana

2016-04-14

Marine bioadhesives have unmatched performances in wet environments, being an inspiration for biomedical applications. In sea urchins specialized adhesive organs, tube feet, mediate reversible adhesion, being composed by a disc, producing adhesive and de-adhesive secretions, and a motile stem. After tube foot detachment, the secreted adhesive remains bound to the substratum as a footprint. Sea urchin adhesive is composed by proteins and sugars, but so far only one protein, Nectin, was shown to be over-expressed as a transcript in tube feet discs, suggesting its involvement in sea urchin adhesion. Here we use high-resolution quantitative mass-spectrometry to perform the first study combining the analysis of the differential proteome of an adhesive organ, with the proteome of its secreted adhesive. This strategy allowed us to identify 163 highly over-expressed disc proteins, specifically involved in sea urchin reversible adhesion; to find that 70% of the secreted adhesive components fall within five protein groups, involved in exocytosis and microbial protection; and to provide evidences that Nectin is not only highly expressed in tube feet discs but is an actual component of the adhesive. These results give an unprecedented insight into the molecular mechanisms underlying sea urchin adhesion, and opening new doors to develop wet-reliable, reversible, and ecological biomimetic adhesives. Sea urchins attach strongly but in a reversible manner to substratum, being a valuable source of inspiration for industrial and biomedical applications. Yet, the molecular mechanisms governing reversible adhesion are still poorly studied delaying the engineering of biomimetic adhesives. We used the latest mass spectrometry techniques to analyze the differential proteome of an adhesive organ and the proteome of its secreted adhesive, allowing us to uncover the key players in sea urchin reversible adhesion. We demonstrate, that Nectin, a protein previously pointed out as potentially involved in sea urchin adhesion, is not only highly expressed in tube feet discs, but is a genuine component of the secreted adhesive. Copyright © 2016 Elsevier B.V. All rights reserved.

Improving mass measurement accuracy in mass spectrometry based proteomics by combining open source tools for chromatographic alignment and internal calibration.

PubMed

Palmblad, Magnus; van der Burgt, Yuri E M; Dalebout, Hans; Derks, Rico J E; Schoenmaker, Bart; Deelder, André M

2009-05-02

Accurate mass determination enhances peptide identification in mass spectrometry based proteomics. We here describe the combination of two previously published open source software tools to improve mass measurement accuracy in Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS). The first program, msalign, aligns one MS/MS dataset with one FTICRMS dataset. The second software, recal2, uses peptides identified from the MS/MS data for automated internal calibration of the FTICR spectra, resulting in sub-ppm mass measurement errors.

Comparative Proteomics of Human and Macaque Milk Reveals Species-Specific Nutrition during Postnatal Development.

PubMed

Beck, Kristen L; Weber, Darren; Phinney, Brett S; Smilowitz, Jennifer T; Hinde, Katie; Lönnerdal, Bo; Korf, Ian; Lemay, Danielle G

2015-05-01

Milk has been well established as the optimal nutrition source for infants, yet there is still much to be understood about its molecular composition. Therefore, our objective was to develop and compare comprehensive milk proteomes for human and rhesus macaques to highlight differences in neonatal nutrition. We developed a milk proteomics technique that overcomes previous technical barriers including pervasive post-translational modifications and limited sample volume. We identified 1606 and 518 proteins in human and macaque milk, respectively. During analysis of detected protein orthologs, we identified 88 differentially abundant proteins. Of these, 93% exhibited increased abundance in human milk relative to macaque and include lactoferrin, polymeric immunoglobulin receptor, alpha-1 antichymotrypsin, vitamin D-binding protein, and haptocorrin. Furthermore, proteins more abundant in human milk compared with macaque are associated with development of the gastrointestinal tract, the immune system, and the brain. Overall, our novel proteomics method reveals the first comprehensive macaque milk proteome and 524 newly identified human milk proteins. The differentially abundant proteins observed are consistent with the perspective that human infants, compared with nonhuman primates, are born at a slightly earlier stage of somatic development and require additional support through higher quantities of specific proteins to nurture human infant maturation.

Proteomic Analysis of Pathogenic Fungi Reveals Highly Expressed Conserved Cell Wall Proteins

PubMed Central

Champer, Jackson; Ito, James I.; Clemons, Karl V.; Stevens, David A.; Kalkum, Markus

2016-01-01

We are presenting a quantitative proteomics tally of the most commonly expressed conserved fungal proteins of the cytosol, the cell wall, and the secretome. It was our goal to identify fungi-typical proteins that do not share significant homology with human proteins. Such fungal proteins are of interest to the development of vaccines or drug targets. Protein samples were derived from 13 fungal species, cultured in rich or in minimal media; these included clinical isolates of Aspergillus, Candida, Mucor, Cryptococcus, and Coccidioides species. Proteomes were analyzed by quantitative MSE (Mass Spectrometry—Elevated Collision Energy). Several thousand proteins were identified and quantified in total across all fractions and culture conditions. The 42 most abundant proteins identified in fungal cell walls or supernatants shared no to very little homology with human proteins. In contrast, all but five of the 50 most abundant cytosolic proteins had human homologs with sequence identity averaging 59%. Proteomic comparisons of the secreted or surface localized fungal proteins highlighted conserved homologs of the Aspergillus fumigatus proteins 1,3-β-glucanosyltransferases (Bgt1, Gel1-4), Crf1, Ecm33, EglC, and others. The fact that Crf1 and Gel1 were previously shown to be promising vaccine candidates, underlines the value of the proteomics data presented here. PMID:26878023

Suberoylanilide hydroxamic acid treatment reveals crosstalks among proteome, ubiquitylome and acetylome in non-small cell lung cancer A549 cell line.

PubMed

Wu, Quan; Cheng, Zhongyi; Zhu, Jun; Xu, Weiqing; Peng, Xiaojun; Chen, Chuangbin; Li, Wenting; Wang, Fengsong; Cao, Lejie; Yi, Xingling; Wu, Zhiwei; Li, Jing; Fan, Pingsheng

2015-03-31

Suberoylanilide hydroxamic acid (SAHA) is a well-known histone deacetylase (HDAC) inhibitor and has been used as practical therapy for breast cancer and non-small cell lung cancer (NSCLC). It is previously demonstrated that SAHA treatment could extensively change the profile of acetylome and proteome in cancer cells. However, little is known about the impact of SAHA on other protein modifications and the crosstalks among different modifications and proteome, hindering the deep understanding of SAHA-mediated cancer therapy. In this work, by using SILAC technique, antibody-based affinity enrichment and high-resolution LC-MS/MS analysis, we investigated quantitative proteome, acetylome and ubiquitylome as well as crosstalks among the three datasets in A549 cells toward SAHA treatment. In total, 2968 proteins, 1099 acetylation sites and 1012 ubiquitination sites were quantified in response to SAHA treatment, respectively. With the aid of intensive bioinformatics, we revealed that the proteome and ubiquitylome were negatively related upon SAHA treatment. Moreover, the impact of SAHA on acetylome resulted in 258 up-regulated and 99 down-regulated acetylation sites at the threshold of 1.5 folds. Finally, we identified 55 common sites with both acetylation and ubiquitination, among which ubiquitination level in 43 sites (78.2%) was positive related to acetylation level.

Proteome analysis to assess physiological changes in Escherichia coli grown under glucose-limited fed-batch conditions.

PubMed

Raman, Babu; Nandakumar, M P; Muthuvijayan, Vignesh; Marten, Mark R

2005-11-05

Proteome analysis was used to compare global protein expression changes in Escherichia coli fermentation between exponential and glucose-limited fed-batch phase. Two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry were used to separate and identify 49 proteins showing >2-fold difference in expression. Proteins upregulated during exponential phase include ribonucleotide biosynthesis enzymes and ribosomal recycling factor. Proteins upregulated during fed-batch phase include those involved in high-affinity glucose uptake, transport and degradation of alternate carbon sources and TCA cycle, suggesting an enhanced role of the cycle under glucose- and energy-limited conditions. We report the upregulation of several putative proteins (ytfQ, ygiS, ynaF, yggX, yfeX), not identified in any previous study under carbon-limited conditions. Copyright (c) 2005 Wiley Periodicals, Inc.

SILAC-based proteomics of human primary endothelial cell morphogenesis unveils tumor angiogenic markers.

PubMed

Zanivan, Sara; Maione, Federica; Hein, Marco Y; Hernández-Fernaud, Juan Ramon; Ostasiewicz, Pawel; Giraudo, Enrico; Mann, Matthias

2013-12-01

Proteomics has been successfully used for cell culture on dishes, but more complex cellular systems have proven to be challenging and so far poorly approached with proteomics. Because of the complexity of the angiogenic program, we still do not have a complete understanding of the molecular mechanisms involved in this process, and there have been no in depth quantitative proteomic studies. Plating endothelial cells on matrigel recapitulates aspects of vessel growth, and here we investigate this mechanism by using a spike-in SILAC quantitative proteomic approach. By comparing proteomic changes in primary human endothelial cells morphogenesis on matrigel to general adhesion mechanisms in cells spreading on culture dish, we pinpoint pathways and proteins modulated by endothelial cells. The cell-extracellular matrix adhesion proteome depends on the adhesion substrate, and a detailed proteomic profile of the extracellular matrix secreted by endothelial cells identified CLEC14A as a matrix component, which binds to MMRN2. We verify deregulated levels of these proteins during tumor angiogenesis in models of multistage carcinogenesis. This is the most in depth quantitative proteomic study of endothelial cell morphogenesis, which shows the potential of applying high accuracy quantitative proteomics to in vitro models of vessel growth to shed new light on mechanisms that accompany pathological angiogenesis. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD000359.

Clinical veterinary proteomics: Techniques and approaches to decipher the animal plasma proteome.

PubMed

Ghodasara, P; Sadowski, P; Satake, N; Kopp, S; Mills, P C

2017-12-01

Over the last two decades, technological advancements in the field of proteomics have advanced our understanding of the complex biological systems of living organisms. Techniques based on mass spectrometry (MS) have emerged as powerful tools to contextualise existing genomic information and to create quantitative protein profiles from plasma, tissues or cell lines of various species. Proteomic approaches have been used increasingly in veterinary science to investigate biological processes responsible for growth, reproduction and pathological events. However, the adoption of proteomic approaches by veterinary investigators lags behind that of researchers in the human medical field. Furthermore, in contrast to human proteomics studies, interpretation of veterinary proteomic data is difficult due to the limited protein databases available for many animal species. This review article examines the current use of advanced proteomics techniques for evaluation of animal health and welfare and covers the current status of clinical veterinary proteomics research, including successful protein identification and data interpretation studies. It includes a description of an emerging tool, sequential window acquisition of all theoretical fragment ion mass spectra (SWATH-MS), available on selected mass spectrometry instruments. This newly developed data acquisition technique combines advantages of discovery and targeted proteomics approaches, and thus has the potential to advance the veterinary proteomics field by enhancing identification and reproducibility of proteomics data. Copyright © 2017 Elsevier Ltd. All rights reserved.

Nipple aspirate fluid-A liquid biopsy for diagnosing breast health.

PubMed

Shaheed, Sadr-Ul; Tait, Catherine; Kyriacou, Kyriacos; Mullarkey, Joanne; Burrill, Wayne; Patterson, Laurence H; Linforth, Richard; Salhab, Mohamed; Sutton, Chris W

2017-09-01

Nipple secretions are protein-rich and a potential source of breast cancer biomarkers for breast cancer screening. Previous studies of specific proteins have shown limited correlation with clinicopathological features. Our aim, in this pilot study, was to investigate the intra- and interpatient protein composition of nipple secretions and the implications for their use as liquid biopsies. Matched pairs of nipple discharge/nipple aspirate fluid (NAF, n = 15) were characterized for physicochemical properties and SDS-PAGE. Four pairs were selected for semiquantitative proteomic profiling and trypsin-digested peptides analyzed using 2D-LC Orbitrap Fusion MS. The resulting data were subject to bioinformatics analysis and statistical evaluation for functional significance. A total of 1990 unique proteins were identified many of which are established cancer-associated markers. Matched pairs shared the greatest similarity (average Pearson correlation coefficient of 0.94), but significant variations between individuals were observed. This was the most complete proteomic study of nipple discharge/nipple aspirate fluid to date providing a valuable source for biomarker discovery. The high level of milk proteins in healthy volunteer samples compared to the cancer patients was associated with galactorrhoea. Using matched pairs increased confidence in patient-specific protein levels but changes relating to cancer stage require investigation of a larger cohort. © 2017 The Authors. PROTEOMICS-Clinical Applications published by WILEY-VCH Verlag GmbH & Co. KGaA.

Genomic, Proteomic, and Metabolite Characterization of Gemfibrozil-Degrading Organism Bacillus sp. GeD10.

PubMed

Kjeldal, Henrik; Zhou, Nicolette A; Wissenbach, Dirk K; von Bergen, Martin; Gough, Heidi L; Nielsen, Jeppe L

2016-01-19

Gemfibrozil is a widely used hypolipidemic and triglyceride lowering drug. Excess of the drug is excreted and discharged into the environment primarily via wastewater treatment plant effluents. Bacillus sp. GeD10, a gemfibrozil-degrader, was previously isolated from activated sludge. It is the first identified bacterium capable of degrading gemfibrozil. Gemfibrozil degradation by Bacillus sp. GeD10 was here studied through genome sequencing, quantitative proteomics and metabolite analysis. From the bacterial proteome of Bacillus sp. GeD10 1974 proteins were quantified, of which 284 proteins were found to be overabundant by more than 2-fold (FDR corrected p-value ≤0.032, fold change (log2) ≥ 1) in response to gemfibrozil exposure. Metabolomic analysis identified two hydroxylated intermediates as well as a glucuronidated hydroxyl-metabolite of gemfibrozil. Overall, gemfibrozil exposure in Bacillus sp. GeD10 increased the abundance of several enzymes potentially involved in gemfibrozil degradation as well as resulted in the production of several gemfibrozil metabolites. The potential catabolic pathway/modification included ring-hydroxylation preparing the substrate for subsequent ring cleavage by a meta-cleaving enzyme. The identified genes may allow for monitoring of potential gemfibrozil-degrading organisms in situ and increase the understanding of microbial processing of trace level contaminants. This study represents the first omics study on a gemfibrozil-degrading bacterium.

The Aotus nancymaae erythrocyte proteome and its importance for biomedical research.

PubMed

Moreno-Pérez, D A; García-Valiente, R; Ibarrola, N; Muro, A; Patarroyo, M A

2017-01-30

The Aotus nancymaae species has been of great importance in researching the biology and pathogenesis of malaria, particularly for studying Plasmodium molecules for including them in effective vaccines against such microorganism. In spite of the forgoing, there has been no report to date describing the biology of parasite target cells in primates or their biomedical importance. This study was thus designed to analyse A. nancymaae erythrocyte protein composition using MS data collected during a previous study aimed at characterising the Plasmodium vivax proteome and published in the pertinent literature. Most peptides identified were similar to those belonging to 1189 Homo sapiens molecules; >95% of them had orthologues in New World primates. GO terms revealed a correlation between categories having the greatest amount of proteins and vital cell function. Integral membrane molecules were also identified which could be possible receptors facilitating interaction with Plasmodium species. The A. nancymaae erythrocyte proteome is described here for the first time, as a starting point for more in-depth/extensive studies. The data reported represents a source of invaluable information for laboratories interested in carrying out basic and applied biomedical investigation studies which involve using this primate. An understanding of the proteomics characteristics of A. nancymaae erythrocytes represents a fascinating area for research regarding the study of the pathogenesis of malaria since these are the main target for Plasmodium invasion. However, and even though Aotus is one of the non-human primate models considered most appropriate for biomedical research, knowledge of its proteome, particularly its erythrocytes, remains unknown. According to the above and bearing in mind the lack of information about the A. nancymaae species genome and transcriptome, this study involved a search for primate proteins for comparing their MS/MS spectra with the available information for Homo sapiens. The great similarity found between the primate's molecules and those for humans supported the use of the monkeys or their cells for continuing assays involved in studying malaria. Integral membrane receptors used by Plasmodium for invading cells were also found; this required timely characterisation for evaluating their therapeutic role. The list of erythrocyte protein composition reported here represents a useful source of basic knowledge for advancing biomedical investigation in this field. Copyright © 2016 Elsevier B.V. All rights reserved.

PrePhyloPro: phylogenetic profile-based prediction of whole proteome linkages

PubMed Central

Niu, Yulong; Liu, Chengcheng; Moghimyfiroozabad, Shayan; Yang, Yi

2017-01-01

Direct and indirect functional links between proteins as well as their interactions as part of larger protein complexes or common signaling pathways may be predicted by analyzing the correlation of their evolutionary patterns. Based on phylogenetic profiling, here we present a highly scalable and time-efficient computational framework for predicting linkages within the whole human proteome. We have validated this method through analysis of 3,697 human pathways and molecular complexes and a comparison of our results with the prediction outcomes of previously published co-occurrency model-based and normalization methods. Here we also introduce PrePhyloPro, a web-based software that uses our method for accurately predicting proteome-wide linkages. We present data on interactions of human mitochondrial proteins, verifying the performance of this software. PrePhyloPro is freely available at http://prephylopro.org/phyloprofile/. PMID:28875072

Venom-gland transcriptomics and venom proteomics of the black-back scorpion (Hadrurus spadix) reveal detectability challenges and an unexplored realm of animal toxin diversity.

PubMed

Rokyta, Darin R; Ward, Micaiah J

2017-03-15

The order Scorpiones is one of the most ancient and diverse lineages of venomous animals, having originated approximately 430 million years ago and diversified into 14 extant families. Although partial venom characterizations have been described for numerous scorpion species, we provided the first quantitative transcriptome/proteome comparison for a scorpion species using single-animal approaches. We sequenced the venom-gland transcriptomes of a male and female black-back scorpion (Hadrurus spadix) from the family Caraboctonidae using the Illumina sequencing platform and conducted independent quantitative mass-spectrometry analyses of their venoms. We identified 79 proteomically confirmed venom proteins, an additional 69 transcripts with homology to toxins from other species, and 596 nontoxin proteins expressed at high levels in the venom glands. The venom of H. spadix was rich in antimicrobial peptides, K + -channel toxins, and several classes of peptidases. However, the most diverse and one of the most abundant classes of putative toxins could not be assigned even a tentative functional role on the basis of homology, indicating that this venom contained a wealth of previously unexplored animal toxin diversity. We found good agreement between both transcriptomic and proteomic abundances across individuals, but transcriptomic and proteomic abundandances differed substantially within each individual. Small peptide toxins such as K + -channel toxins and antimicrobial peptides proved challenging to detect proteomically, at least in part due to the significant proteolytic processing involved in their maturation. In addition, we found a significant tendency for our proteomic approach to overestimate the abundances of large putative toxins and underestimate the abundances of smaller toxins. Copyright © 2017 Elsevier Ltd. All rights reserved.

CPTAC Releases Cancer Proteome Confirmatory Ovarian Study Data | Office of Cancer Clinical Proteomics Research

Cancer.gov

A catalogue of molecular aberrations that cause ovarian cancer is critical for developing and deploying diagnostics and therapies that will improve patients’ lives. Because a comprehensive molecular view of cancer is important for ultimately guiding treatment, the National Cancer Institute (NCI) Clinical Proteomic Tumor Analysis Consortium (CPTAC) has released the cancer proteome confirmatory ovarian study data sets.

A Targeted MRM Approach for Tempo-Spatial Proteomics Analyses.

PubMed

Moradian, Annie; Porras-Yakushi, Tanya R; Sweredoski, Michael J; Hess, Sonja

2016-01-01

When deciding to perform a quantitative proteomics analysis, selectivity, sensitivity, and reproducibility are important criteria to consider. The use of multiple reaction monitoring (MRM) has emerged as a powerful proteomics technique in that regard since it avoids many of the problems typically observed in discovery-based analyses. A prerequisite for such a targeted approach is that the protein targets are known, either as a result of previous global proteomics experiments or because a specific hypothesis is to be tested. When guidelines that have been established in the pharmaceutical industry many decades ago are taken into account, setting up an MRM assay is relatively straightforward. Typically, proteotypic peptides with favorable mass spectrometric properties are synthesized with a heavy isotope for each protein that is to be monitored. Retention times and calibration curves are determined using triple-quadrupole mass spectrometers. The use of iRT peptide standards is both recommended and fully integrated into the bioinformatics pipeline. Digested biological samples are mixed with the heavy and iRT standards and quantified. Here we present a generic protocol for the development of an MRM assay.

Alterations in brain cerebral cortex proteome of rabies-infected cat.

PubMed

Kasempimolporn, Songsri; Lumlertdacha, Boonlert; Chulasugandha, Pannipa; Boonchang, Supatsorn; Sitprija, Visith

2014-07-01

Comparative proteome analysis using brain cerebral cortex tissues from cats and dogs infected with/without rabies virus were conducted using both two-dimensional gel-electrophoresis (2-DE) and 2-D fluorescence difference gel- electrophoresis (2D-DIGE) methods. The 2-DE gel images of all samples revealed >1,000 protein spots in each gel. Quantitative intensity analysis revealed the same overall protein pattern in certain regions of the gel, but the rabies-infected brains exhibited more protein spots than the non-infected controls. From approximately 880 protein spots detected by 2D-DIGE, 65 protein spots were increased and 46 were decreased. Eight of these protein spots were randomly selected and annotated by reference to previous known proteome data of rabid dog brains. They were similarly altered in both of the rabies-infected cats and dogs. A more detailed comparison of changes in proteomic profiles of brains between rabid cats and dogs should shed some light on the pathophysiological mechanism of rabies in domestic animals, as most rabies cases have been traceable to or believed to have originated from rabid dogs.

Efficient Exploitation of Separation Space in Two-Dimensional Liquid Chromatography System for Comprehensive and Efficient Proteomic Analyses.

PubMed

Lee, Hangyeore; Mun, Dong-Gi; So, Jeong Eun; Bae, Jingi; Kim, Hokeun; Masselon, Christophe; Lee, Sang-Won

2016-12-06

Proteomics aims to achieve complete profiling of the protein content and protein modifications in cells, tissues, and biofluids and to quantitatively determine changes in their abundances. This information serves to elucidate cellular processes and signaling pathways and to identify candidate protein biomarkers and/or therapeutic targets. Analyses must therefore be both comprehensive and efficient. Here, we present a novel online two-dimensional reverse-phase/reverse-phase liquid chromatography separation platform, which is based on a newly developed online noncontiguous fractionating and concatenating device (NCFC fractionator). In bottom-up proteomics analyses of a complex proteome, this system provided significantly improved exploitation of the separation space of the two RPs, considerably increasing the numbers of peptides identified compared to a contiguous 2D-RP/RPLC method. The fully automated online 2D-NCFC-RP/RPLC system bypassed a number of labor-intensive manual processes required with the previously described offline 2D-NCFC RP/RPLC method, and thus, it offers minimal sample loss in a context of highly reproducible 2D-RP/RPLC experiments.

NeuCode Labeling in Nematodes: Proteomic and Phosphoproteomic Impact of Ascaroside Treatment in Caenorhabditis elegans*

PubMed Central

Rhoads, Timothy W.; Prasad, Aman; Kwiecien, Nicholas W.; Merrill, Anna E.; Zawack, Kelson; Westphall, Michael S.; Schroeder, Frank C.; Kimble, Judith; Coon, Joshua J.

2015-01-01

The nematode Caenorhabditis elegans is an important model organism for biomedical research. We previously described NeuCode stable isotope labeling by amino acids in cell culture (SILAC), a method for accurate proteome quantification with potential for multiplexing beyond the limits of traditional stable isotope labeling by amino acids in cell culture. Here we apply NeuCode SILAC to profile the proteomic and phosphoproteomic response of C. elegans to two potent members of the ascaroside family of nematode pheromones. By consuming labeled E. coli as part of their diet, C. elegans nematodes quickly and easily incorporate the NeuCode heavy lysine isotopologues by the young adult stage. Using this approach, we report, at high confidence, one of the largest proteomic and phosphoproteomic data sets to date in C. elegans: 6596 proteins at a false discovery rate ≤ 1% and 6620 phosphorylation isoforms with localization probability ≥75%. Our data reveal a post-translational signature of pheromone sensing that includes many conserved proteins implicated in longevity and response to stress. PMID:26392051

Transcriptional and Proteomic Profiling of Aspergillus flavipes in Response to Sulfur Starvation

PubMed Central

El-Sayed, Ashraf S. A.; Yassin, Marwa A.; Ali, Gul Shad

2015-01-01

Aspergillus flavipes has received considerable interest due to its potential to produce therapeutic enzymes involved in sulfur amino acid metabolism. In natural habitats, A. flavipes survives under sulfur limitations by mobilizing endogenous and exogenous sulfur to operate diverse cellular processes. Sulfur limitation affects virulence and pathogenicity, and modulates proteome of sulfur assimilating enzymes of several fungi. However, there are no previous reports aimed at exploring effects of sulfur limitation on the regulation of A. flavipes sulfur metabolism enzymes at the transcriptional, post-transcriptional and proteomic levels. In this report, we show that sulfur limitation affects morphological and physiological responses of A. flavipes. Transcription and enzymatic activities of several key sulfur metabolism genes, ATP-sulfurylase, sulfite reductase, methionine permease, cysteine synthase, cystathionine β- and γ-lyase, glutathione reductase and glutathione peroxidase were increased under sulfur starvation conditions. A 50 kDa protein band was strongly induced by sulfur starvation, and the proteomic analyses of this protein band using LC-MS/MS revealed similarity to many proteins involved in the sulfur metabolism pathway. PMID:26633307

Proteomic Analysis of the Multimeric Nuclear Egress Complex of Human Cytomegalovirus*

PubMed Central

Milbradt, Jens; Kraut, Alexandra; Hutterer, Corina; Sonntag, Eric; Schmeiser, Cathrin; Ferro, Myriam; Wagner, Sabrina; Lenac, Tihana; Claus, Claudia; Pinkert, Sandra; Hamilton, Stuart T.; Rawlinson, William D.; Sticht, Heinrich; Couté, Yohann; Marschall, Manfred

2014-01-01

Herpesviral capsids are assembled in the host cell nucleus before being translocated into the cytoplasm for further maturation. The crossing of the nuclear envelope represents a major event that requires the formation of the nuclear egress complex (NEC). Previous studies demonstrated that human cytomegalovirus (HCMV) proteins pUL50 and pUL53, as well as their homologs in all members of Herpesviridae, interact with each other at the nuclear envelope and form the heterodimeric core of the NEC. In order to characterize further the viral and cellular protein content of the multimeric NEC, the native complex was isolated from HCMV-infected human primary fibroblasts at various time points and analyzed using quantitative proteomics. Previously postulated components of the HCMV-specific NEC, as well as novel potential NEC-associated proteins such as emerin, were identified. In this regard, interaction and colocalization between emerin and pUL50 were confirmed by coimmunoprecipitation and confocal microscopy analyses, respectively. A functional validation of viral and cellular NEC constituents was achieved through siRNA-mediated knockdown experiments. The important role of emerin in NEC functionality was demonstrated by a reduction of viral replication when emerin expression was down-regulated. Moreover, under such conditions, reduced production of viral proteins and deregulation of viral late cytoplasmic maturation were observed. Combined, these data prove the functional importance of emerin as an NEC component, associated with pUL50, pUL53, pUL97, p32/gC1qR, and further regulatory proteins. Summarized, our findings provide the first proteomics-based characterization and functional validation of the HCMV-specific multimeric NEC. PMID:24969177

Application of proteomics to ecology and population biology.

PubMed

Karr, T L

2008-02-01

Proteomics is a relatively new scientific discipline that merges protein biochemistry, genome biology and bioinformatics to determine the spatial and temporal expression of proteins in cells, tissues and whole organisms. There has been very little application of proteomics to the fields of behavioral genetics, evolution, ecology and population dynamics, and has only recently been effectively applied to the closely allied fields of molecular evolution and genetics. However, there exists considerable potential for proteomics to impact in areas related to functional ecology; this review will introduce the general concepts and methodologies that define the field of proteomics and compare and contrast the advantages and disadvantages with other methods. Examples of how proteomics can aid, complement and indeed extend the study of functional ecology will be discussed including the main tool of ecological studies, population genetics with an emphasis on metapopulation structure analysis. Because proteomic analyses provide a direct measure of gene expression, it obviates some of the limitations associated with other genomic approaches, such as microarray and EST analyses. Likewise, in conjunction with associated bioinformatics and molecular evolutionary tools, proteomics can provide the foundation of a systems-level integration approach that can enhance ecological studies. It can be envisioned that proteomics will provide important new information on issues specific to metapopulation biology and adaptive processes in nature. A specific example of the application of proteomics to sperm ageing is provided to illustrate the potential utility of the approach.

Differential proteomic responses of selectively bred and wild-type Sydney rock oyster populations exposed to elevated CO2.

PubMed

Thompson, E L; O'Connor, W; Parker, L; Ross, P; Raftos, D A

2015-03-01

Previous work suggests that larvae from Sydney rock oysters that have been selectively bred for fast growth and disease resistance are more resilient to the impacts of ocean acidification than nonselected, wild-type oysters. In this study, we used proteomics to investigate the molecular differences between oyster populations in adult Sydney rock oysters and to identify whether these form the basis for observations seen in larvae. Adult oysters from a selective breeding line (B2) and nonselected wild types (WT) were exposed for 4 weeks to elevated pCO2 (856 μatm) before their proteomes were compared to those of oysters held under ambient conditions (375 μatm pCO2 ). Exposure to elevated pCO2 resulted in substantial changes in the proteomes of oysters from both the selectively bred and wild-type populations. When biological functions were assigned, these differential proteins fell into five broad, potentially interrelated categories of subcellular functions, in both oyster populations. These functional categories were energy production, cellular stress responses, the cytoskeleton, protein synthesis and cell signalling. In the wild-type population, proteins were predominantly upregulated. However, unexpectedly, these cellular systems were downregulated in the selectively bred oyster population, indicating cellular dysfunction. We argue that this reflects a trade-off, whereby an adaptive capacity for enhanced mitochondrial energy production in the selectively bred population may help to protect larvae from the effects of elevated CO2 , whilst being deleterious to adult oysters. © 2015 John Wiley & Sons Ltd.

Analysis of 3-MCPD- and 3-MCPD dipalmitate-induced proteomic changes in rat liver.

PubMed

Braeuning, Albert; Sawada, Stefanie; Oberemm, Axel; Lampen, Alfonso

2015-12-01

3-Monochloropropane-1,2-diol (3-MCPD) and 3-MCPD fatty acid esters are process contaminants in foodstuff which are generated during thermal treatment. Long-term exposure to 3-MCPD or 3-MCPD esters causes toxicity especially in kidney and testis. 3-MCPD esters are efficiently hydrolyzed in the gastrointestinal tract, suggesting that their toxicity is mediated by free 3-MCPD. Combined exposure to free 3-MCPD and 3-MCPD released from 3-MCPD esters might lead to dietary consumption above the tolerable daily intake of 2 μg/kg body weight/day. Suspected mechanisms of 3-MCPD toxicity include the inhibition of glycolysis and oxidative stress. Here, a comparative proteomic approach was followed to analyze the effects of 3-MCPD or 3-MCPD dipalmitate in livers from rats exposed to 10 mg/kg body weight 3-MCPD, an equimolar dose of 3-MCPD dipalmitate, or a 4-fold lower dose of the ester during a 28-day repeated-dose feeding study. Early cellular changes were monitored in the absence of overt toxicity. A comprehensive view of 3-MCPD- or 3-MCPD dipalmitate-triggered proteomic changes in rat liver links to previously proposed mechanisms of toxicity and substantially extends our knowledge on molecular hepatic effects of 3-MCPD. Organ-independent marker proteins altered upon 3-MCPD exposure, for example DJ-1/PARK7, were identified by comparison of the proteomic patterns of kidney, testis and liver. Copyright © 2015 Elsevier Ltd. All rights reserved.

The Escherichia coli Proteome: Past, Present, and Future Prospects†

PubMed Central

Han, Mee-Jung; Lee, Sang Yup

2006-01-01

Proteomics has emerged as an indispensable methodology for large-scale protein analysis in functional genomics. The Escherichia coli proteome has been extensively studied and is well defined in terms of biochemical, biological, and biotechnological data. Even before the entire E. coli proteome was fully elucidated, the largest available data set had been integrated to decipher regulatory circuits and metabolic pathways, providing valuable insights into global cellular physiology and the development of metabolic and cellular engineering strategies. With the recent advent of advanced proteomic technologies, the E. coli proteome has been used for the validation of new technologies and methodologies such as sample prefractionation, protein enrichment, two-dimensional gel electrophoresis, protein detection, mass spectrometry (MS), combinatorial assays with n-dimensional chromatographies and MS, and image analysis software. These important technologies will not only provide a great amount of additional information on the E. coli proteome but also synergistically contribute to other proteomic studies. Here, we review the past development and current status of E. coli proteome research in terms of its biological, biotechnological, and methodological significance and suggest future prospects. PMID:16760308

The Challenge of Human Spermatozoa Proteome: A Systematic Review.

PubMed

Gilany, Kambiz; Minai-Tehrani, Arash; Amini, Mehdi; Agharezaee, Niloofar; Arjmand, Babak

2017-01-01

Currently, there are 20,197 human protein-coding genes in the most expertly curated database (UniProtKB/Swiss-Pro). Big efforts have been made by the international consortium, the Chromosome-Centric Human Proteome Project (C-HPP) and independent researchers, to map human proteome. In brief, anno 2017 the human proteome was outlined. The male factor contributes to 50% of infertility in couples. However, there are limited human spermatozoa proteomic studies. Firstly, the development of the mapping of the human spermatozoa was analyzed. The human spermatozoa have been used as a model for missing proteins. It has been shown that human spermatozoa are excellent sources for finding missing proteins. Y chromosome proteome mapping is led by Iran. However, it seems that it is extremely challenging to map the human spermatozoa Y chromosome proteins based on current mass spectrometry-based proteomics technology. Post-translation modifications (PTMs) of human spermatozoa proteome are the most unexplored area and currently the exact role of PTMs in male infertility is unknown. Additionally, the clinical human spermatozoa proteomic analysis, anno 2017 was done in this study.

Proteomic analysis of protein phosphatase Z1 from Candida albicans

PubMed Central

Pfliegler, Walter P.; Petrényi, Katalin; Boros, Enikő; Pócsi, István; Tőzsér, József; Dombrádi, Viktor

2017-01-01

Protein phosphatase Z is a “novel type” fungus specific serine/threonine protein phosphatase. Previously our research group identified the CaPPZ1 gene in the opportunistic pathogen Candida albicans and reported that the gene deletion had several important physiological consequences. In order to reveal the protein targets and the associated mechanisms behind the functions of the phosphatase a proteomic method was adopted for the comparison of the cappz1 deletion mutant and the genetically matching QMY23 control strain. Proteins extracted from the control and deletion mutant strains were separated by two-dimensional gel electrophoresis and the protein spots were stained with RuBPS and Pro-Q Diamond in order to visualize the total proteome and the phosphoproteome, respectively. The alterations in spot intensities were determined by densitometry and were analysed with the Delta2D (Decodon) software. Spots showing significantly different intensities between the mutant and control strains were excised from the gels and were digested with trypsin. The resulting peptides were identified by LC-MS/MS mass spectrometry. As many as 15 protein spots were found that exhibited significant changes in their intensity upon the deletion of the phosphatase and 20 phosphoproteins were identified in which the level of phosphorylation was modified significantly in the mutant. In agreement with previous findings we found that the affected proteins function in protein synthesis, oxidative stress response, regulation of morphology and metabolism. Among these proteins we identified two potential CaPpz1 substrates (Eft2 and Rpp0) that may regulate the elongation step of translation. RT-qPCR experiments revealed that the expression of the genes coding for the affected proteins was not altered significantly. Thus, the absence of CaPpz1 exerted its effects via protein synthesis/degradation and phosphorylation/dephosphorylation. In addition, our proteomics data strongly suggested a role for CaPpz1 in biofilm formation, was confirmed experimentally. Thus our unbiased proteomic approach lead to the discovery of a novel function for this phosphatase in C. albicans. PMID:28837603

Proteomic analysis of mare follicular fluid during late follicle development

PubMed Central

2011-01-01

Background Follicular fluid accumulates into the antrum of follicle from the early stage of follicle development. Studies on its components may contribute to a better understanding of the mechanisms underlying follicular development and oocyte quality. With this objective, we performed a proteomic analysis of mare follicular fluid. First, we hypothesized that proteins in follicular fluid may differ from those in the serum, and also may change during follicle development. Second, we used four different approaches of Immunodepletion and one enrichment method, in order to overcome the masking effect of high-abundance proteins present in the follicular fluid, and to identify those present in lower abundance. Finally, we compared our results with previous studies performed in mono-ovulant (human) and poly-ovulant (porcine and canine) species in an attempt to identify common and/or species-specific proteins. Methods Follicular fluid samples were collected from ovaries at three different stages of follicle development (early dominant, late dominant and preovulatory). Blood samples were also collected at each time. The proteomic analysis was carried out on crude, depleted and enriched follicular fluid by 2D-PAGE, 1D-PAGE and mass spectrometry. Results Total of 459 protein spots were visualized by 2D-PAGE of crude mare follicular fluid, with no difference among the three physiological stages. Thirty proteins were observed as differentially expressed between serum and follicular fluid. Enrichment method was found to be the most powerful method for detection and identification of low-abundance proteins from follicular fluid. Actually, we were able to identify 18 proteins in the crude follicular fluid, and as many as 113 in the enriched follicular fluid. Inhibins and a few other proteins involved in reproduction could only be identified after enrichment of follicular fluid, demonstrating the power of the method used. The comparison of proteins found in mare follicular fluid with proteins previously identified in human, porcine and canine follicular fluids, led to the identification of 12 common proteins and of several species-specific proteins. Conclusions This study provides the first description of mare follicular fluid proteome during the late follicle development stages. We identified several proteins from crude, depleted and enriched follicular fluid. Our results demonstrate that the enrichment method, combined with 2D-PAGE and mass spectrometry, can be successfully used to visualize and further identify the low-abundance proteins in the follicular fluid. PMID:21923925

Proteomic analysis of mare follicular fluid during late follicle development.

PubMed

Fahiminiya, Somayyeh; Labas, Valérie; Roche, Stéphane; Dacheux, Jean-Louis; Gérard, Nadine

2011-09-17

Follicular fluid accumulates into the antrum of follicle from the early stage of follicle development. Studies on its components may contribute to a better understanding of the mechanisms underlying follicular development and oocyte quality. With this objective, we performed a proteomic analysis of mare follicular fluid. First, we hypothesized that proteins in follicular fluid may differ from those in the serum, and also may change during follicle development. Second, we used four different approaches of Immunodepletion and one enrichment method, in order to overcome the masking effect of high-abundance proteins present in the follicular fluid, and to identify those present in lower abundance. Finally, we compared our results with previous studies performed in mono-ovulant (human) and poly-ovulant (porcine and canine) species in an attempt to identify common and/or species-specific proteins. Follicular fluid samples were collected from ovaries at three different stages of follicle development (early dominant, late dominant and preovulatory). Blood samples were also collected at each time. The proteomic analysis was carried out on crude, depleted and enriched follicular fluid by 2D-PAGE, 1D-PAGE and mass spectrometry. Total of 459 protein spots were visualized by 2D-PAGE of crude mare follicular fluid, with no difference among the three physiological stages. Thirty proteins were observed as differentially expressed between serum and follicular fluid. Enrichment method was found to be the most powerful method for detection and identification of low-abundance proteins from follicular fluid. Actually, we were able to identify 18 proteins in the crude follicular fluid, and as many as 113 in the enriched follicular fluid. Inhibins and a few other proteins involved in reproduction could only be identified after enrichment of follicular fluid, demonstrating the power of the method used. The comparison of proteins found in mare follicular fluid with proteins previously identified in human, porcine and canine follicular fluids, led to the identification of 12 common proteins and of several species-specific proteins. This study provides the first description of mare follicular fluid proteome during the late follicle development stages. We identified several proteins from crude, depleted and enriched follicular fluid. Our results demonstrate that the enrichment method, combined with 2D-PAGE and mass spectrometry, can be successfully used to visualize and further identify the low-abundance proteins in the follicular fluid.

The path to enlightenment: making sense of genomic and proteomic information.

PubMed

Maurer, Martin H

2004-05-01

Whereas genomics describes the study of genome, mainly represented by its gene expression on the DNA or RNA level, the term proteomics denotes the study of the proteome, which is the protein complement encoded by the genome. In recent years, the number of proteomic experiments increased tremendously. While all fields of proteomics have made major technological advances, the biggest step was seen in bioinformatics. Biological information management relies on sequence and structure databases and powerful software tools to translate experimental results into meaningful biological hypotheses and answers. In this resource article, I provide a collection of databases and software available on the Internet that are useful to interpret genomic and proteomic data. The article is a toolbox for researchers who have genomic or proteomic datasets and need to put their findings into a biological context.

Birth of plant proteomics in India: a new horizon.

PubMed

Narula, Kanika; Pandey, Aarti; Gayali, Saurabh; Chakraborty, Niranjan; Chakraborty, Subhra

2015-09-08

In the post-genomic era, proteomics is acknowledged as the next frontier for biological research. Although India has a long and distinguished tradition in protein research, the initiation of proteomics studies was a new horizon. Protein research witnessed enormous progress in protein separation, high-resolution refinements, biochemical identification of the proteins, protein-protein interaction, and structure-function analysis. Plant proteomics research, in India, began its journey on investigation of the proteome profiling, complexity analysis, protein trafficking, and biochemical modeling. The research article by Bhushan et al. in 2006 marked the birth of the plant proteomics research in India. Since then plant proteomics studies expanded progressively and are now being carried out in various institutions spread across the country. The compilation presented here seeks to trace the history of development in the area during the past decade based on publications till date. In this review, we emphasize on outcomes of the field providing prospects on proteomic pathway analyses. Finally, we discuss the connotation of strategies and the potential that would provide the framework of plant proteome research. The past decades have seen rapidly growing number of sequenced plant genomes and associated genomic resources. To keep pace with this increasing body of data, India is in the provisional phase of proteomics research to develop a comparative hub for plant proteomes and protein families, but it requires a strong impetus from intellectuals, entrepreneurs, and government agencies. Here, we aim to provide an overview of past, present and future of Indian plant proteomics, which would serve as an evaluation platform for those seeking to incorporate proteomics into their research programs. This article is part of a Special Issue entitled: Proteomics in India. Copyright © 2015 Elsevier B.V. All rights reserved.

Combining systems pharmacology, transcriptomics, proteomics, and metabolomics to dissect the therapeutic mechanism of Chinese herbal Bufei Jianpi formula for application to COPD

PubMed Central

Zhao, Peng; Yang, Liping; Li, Jiansheng; Li, Ya; Tian, Yange; Li, Suyun

2016-01-01

Bufei Jianpi formula (BJF) has long been used as a therapeutic agent in the treatment of COPD. Systems pharmacology identified 145 active compounds and 175 potential targets of BJF in a previous study. Additionally, BJF was previously shown to effectively prevent COPD and its comorbidities, such as ventricular hypertrophy, by inhibition of inflammatory cytokine production, matrix metalloproteinases expression, and other cytokine production, in vivo. However, the system-level mechanism of BJF for the treatment of COPD is still unclear. The aim of this study was to gain insight into its system-level mechanisms by integrating transcriptomics, proteomics, and metabolomics together with systems pharmacology datasets. Using molecular function, pathway, and network analyses, the genes and proteins regulated in COPD rats and BJF-treated rats could be mainly attributed to oxidoreductase activity, antioxidant activity, focal adhesion, tight junction, or adherens junction. Furthermore, a comprehensive analysis of systems pharmacology, transcript, protein, and metabolite datasets is performed. The results showed that a number of genes, proteins, metabolites regulated in BJF-treated rats and potential target proteins of BJF were involved in lipid metabolism, cell junction, oxidative stress, and inflammatory response, which might be the system-level therapeutic mechanism of BJF treatment. PMID:27042044

Neural Stem Cells (NSCs) and Proteomics*

PubMed Central

Shoemaker, Lorelei D.; Kornblum, Harley I.

2016-01-01

Neural stem cells (NSCs) can self-renew and give rise to the major cell types of the CNS. Studies of NSCs include the investigation of primary, CNS-derived cells as well as animal and human embryonic stem cell (ESC)-derived and induced pluripotent stem cell (iPSC)-derived sources. NSCs provide a means with which to study normal neural development, neurodegeneration, and neurological disease and are clinically relevant sources for cellular repair to the damaged and diseased CNS. Proteomics studies of NSCs have the potential to delineate molecules and pathways critical for NSC biology and the means by which NSCs can participate in neural repair. In this review, we provide a background to NSC biology, including the means to obtain them and the caveats to these processes. We then focus on advances in the proteomic interrogation of NSCs. This includes the analysis of posttranslational modifications (PTMs); approaches to analyzing different proteomic compartments, such the secretome; as well as approaches to analyzing temporal differences in the proteome to elucidate mechanisms of differentiation. We also discuss some of the methods that will undoubtedly be useful in the investigation of NSCs but which have not yet been applied to the field. While many proteomics studies of NSCs have largely catalogued the proteome or posttranslational modifications of specific cellular states, without delving into specific functions, some have led to understandings of functional processes or identified markers that could not have been identified via other means. Many challenges remain in the field, including the precise identification and standardization of NSCs used for proteomic analyses, as well as how to translate fundamental proteomics studies to functional biology. The next level of investigation will require interdisciplinary approaches, combining the skills of those interested in the biochemistry of proteomics with those interested in modulating NSC function. PMID:26494823

Micro-proteomics with iterative data analysis: Proteome analysis in C. elegans at the single worm level.

PubMed

Bensaddek, Dalila; Narayan, Vikram; Nicolas, Armel; Murillo, Alejandro Brenes; Gartner, Anton; Kenyon, Cynthia J; Lamond, Angus I

2016-02-01

Proteomics studies typically analyze proteins at a population level, using extracts prepared from tens of thousands to millions of cells. The resulting measurements correspond to average values across the cell population and can mask considerable variation in protein expression and function between individual cells or organisms. Here, we report the development of micro-proteomics for the analysis of Caenorhabditis elegans, a eukaryote composed of 959 somatic cells and ∼1500 germ cells, measuring the worm proteome at a single organism level to a depth of ∼3000 proteins. This includes detection of proteins across a wide dynamic range of expression levels (>6 orders of magnitude), including many chromatin-associated factors involved in chromosome structure and gene regulation. We apply the micro-proteomics workflow to measure the global proteome response to heat-shock in individual nematodes. This shows variation between individual animals in the magnitude of proteome response following heat-shock, including variable induction of heat-shock proteins. The micro-proteomics pipeline thus facilitates the investigation of stochastic variation in protein expression between individuals within an isogenic population of C. elegans. All data described in this study are available online via the Encyclopedia of Proteome Dynamics (http://www.peptracker.com/epd), an open access, searchable database resource. © 2015 The Authors. PROTEOMICS Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Functional study of miR-27a in human hepatic stellate cells by proteomic analysis: comprehensive view and a role in myogenic tans-differentiation.

PubMed

Ji, Yuhua; Zhang, Jinsheng; Wang, Wenwen; Ji, Juling

2014-01-01

We previous reported that miR-27a regulates lipid metabolism and cell proliferation during hepatic stellate cells (HSCs) activation. To further explore the biological function and underlying mechanisms of miR-27a in HSCs, global protein expression affected by overexpression of miR-27a in HSCs was analyzed by a cleavable isotope-coded affinity tags (cICAT) based comparative proteomic approach. In the present study, 1267 non-redundant proteins were identified with unique accession numbers (score ≥1.3, i.e. confidence ≥95%), among which 1171 were quantified and 149 proteins (12.72%) were differentially expressed with a differential expression ratio of 1.5. We found that up-regulated proteins by miR-27a mainly participate in cell proliferation and myogenesis, while down-regulated proteins were the key enzymes involved in de novo lipid synthesis. The expression of a group of six miR-27a regulated proteins was validated and the function of one miR-27a regulated protein was further validated. The results not only delineated the underlying mechanism of miR-27a in modulating fat metabolism and cell proliferation, but also revealed a novel role of miR-27a in promoting myogenic tans-differentiation during HSCs activation. This study also exemplified proteomics strategy as a powerful tool for the functional study of miRNA.

A Description of the Clinical Proteomic Tumor Analysis Consortium (CPTAC) Common Data Analysis Pipeline

PubMed Central

Rudnick, Paul A.; Markey, Sanford P.; Roth, Jeri; Mirokhin, Yuri; Yan, Xinjian; Tchekhovskoi, Dmitrii V.; Edwards, Nathan J.; Thangudu, Ratna R.; Ketchum, Karen A.; Kinsinger, Christopher R.; Mesri, Mehdi; Rodriguez, Henry; Stein, Stephen E.

2016-01-01

The Clinical Proteomic Tumor Analysis Consortium (CPTAC) has produced large proteomics datasets from the mass spectrometric interrogation of tumor samples previously analyzed by The Cancer Genome Atlas (TCGA) program. The availability of the genomic and proteomic data is enabling proteogenomic study for both reference (i.e., contained in major sequence databases) and non-reference markers of cancer. The CPTAC labs have focused on colon, breast, and ovarian tissues in the first round of analyses; spectra from these datasets were produced from 2D LC-MS/MS analyses and represent deep coverage. To reduce the variability introduced by disparate data analysis platforms (e.g., software packages, versions, parameters, sequence databases, etc.), the CPTAC Common Data Analysis Platform (CDAP) was created. The CDAP produces both peptide-spectrum-match (PSM) reports and gene-level reports. The pipeline processes raw mass spectrometry data according to the following: (1) Peak-picking and quantitative data extraction, (2) database searching, (3) gene-based protein parsimony, and (4) false discovery rate (FDR)-based filtering. The pipeline also produces localization scores for the phosphopeptide enrichment studies using the PhosphoRS program. Quantitative information for each of the datasets is specific to the sample processing, with PSM and protein reports containing the spectrum-level or gene-level (“rolled-up”) precursor peak areas and spectral counts for label-free or reporter ion log-ratios for 4plex iTRAQ™. The reports are available in simple tab-delimited formats and, for the PSM-reports, in mzIdentML. The goal of the CDAP is to provide standard, uniform reports for all of the CPTAC data, enabling comparisons between different samples and cancer types as well as across the major ‘omics fields. PMID:26860878

Proteomics-driven Antigen Discovery for Development of Vaccines Against Gonorrhea*

PubMed Central

Zielke, Ryszard A.; Wierzbicki, Igor H.; Baarda, Benjamin I.; Gafken, Philip R.; Soge, Olusegun O.; Holmes, King K.; Jerse, Ann E.; Unemo, Magnus

2016-01-01

Expanding efforts to develop preventive gonorrhea vaccines is critical because of the dire possibility of untreatable gonococcal infections. Reverse vaccinology, which includes genome and proteome mining, has proven very successful in the discovery of vaccine candidates against many pathogenic bacteria. However, progress with this approach for a gonorrhea vaccine remains in its infancy. Accordingly, we applied a comprehensive proteomic platform—isobaric tagging for absolute quantification coupled with two-dimensional liquid chromatography and mass spectrometry—to identify potential gonococcal vaccine antigens. Our previous analyses focused on cell envelopes and naturally released membrane vesicles derived from four different Neisseria gonorrhoeae strains. Here, we extended these studies to identify cell envelope proteins of N. gonorrhoeae that are ubiquitously expressed and specifically induced by physiologically relevant environmental stimuli: oxygen availability, iron deprivation, and the presence of human serum. Together, these studies enabled the identification of numerous potential gonorrhea vaccine targets. Initial characterization of five novel vaccine candidate antigens that were ubiquitously expressed under these different growth conditions demonstrated that homologs of BamA (NGO1801), LptD (NGO1715), and TamA (NGO1956), and two uncharacterized proteins, NGO2054 and NGO2139, were surface exposed, secreted via naturally released membrane vesicles, and elicited bactericidal antibodies that cross-reacted with a panel of temporally and geographically diverse isolates. In addition, analysis of polymorphisms at the nucleotide and amino acid levels showed that these vaccine candidates are highly conserved among N. gonorrhoeae strains. Finally, depletion of BamA caused a loss of N. gonorrhoeae viability, suggesting it may be an essential target. Together, our data strongly support the use of proteomics-driven discovery of potential vaccine targets as a sound approach for identifying promising gonococcal antigens. PMID:27141096

Contrasting Physiological and Proteomic Adaptations to Iron and/or Copper Limitation in Two Strains of the Same Open Ocean Diatom Thalassiosira oceanica

NASA Astrophysics Data System (ADS)

Schuback, N.; Hippmann, A.; Maldonado, M. T.; Allen, A. E.; McCrow, J.; Foster, L. J.; Green, B. R.; Alami, M.

2016-02-01

Iron plays a significant role in controlling marine primary productivity. Despite that extremely low dissolved iron (Fe) concentrations are found in Fe-limited regions, some phytoplankton are able to survive and thrive. Two strains of the model oceanic diatom Thalassiosira oceanica, TO 1003 and TO 1005, have both been used in previous studies to characterize adaptations to iron limitation. These studies have shown that T. oceanica has lowered its Fe requirements and increased its Fe acquisition efficiency compared to coastal counterparts. Both strategies may impose a higher cellular copper (Cu) demand. However, the underlying biochemical adaptations in these oceanic diatoms remain unknown. Recently, the genome, as well as the first proteomic and transcriptomic analyses of T. oceanica 1005 grown under different Fe levels, were published. To further our understanding of the interplay between Fe- and Cu- physiology in open ocean diatoms, we examined an array of physiological responses to varying degrees of Fe-, Cu- and Fe/Cu co-limitation in both strains. We also determined the differential expression of proteins using stable isotope labeling and LC-MS/MS proteomic analysis. The two strains, TO 1003 and TO 1005, need markedly different metal concentrations in the media. TO1003 requires 30% less Cu to sustain its optimal growth and less than 1/10th of the minimum Cu that is needed by TO 1005 to survive. In contrast, TO 1005 is able to grow with less Fe available in the media. The physiological and proteomic responses of these two strains when acclimated to low Fe and/or Cu concentrations will be presented. The evolutionary implications will be discussed.

A Description of the Clinical Proteomic Tumor Analysis Consortium (CPTAC) Common Data Analysis Pipeline.

PubMed

Rudnick, Paul A; Markey, Sanford P; Roth, Jeri; Mirokhin, Yuri; Yan, Xinjian; Tchekhovskoi, Dmitrii V; Edwards, Nathan J; Thangudu, Ratna R; Ketchum, Karen A; Kinsinger, Christopher R; Mesri, Mehdi; Rodriguez, Henry; Stein, Stephen E

2016-03-04

The Clinical Proteomic Tumor Analysis Consortium (CPTAC) has produced large proteomics data sets from the mass spectrometric interrogation of tumor samples previously analyzed by The Cancer Genome Atlas (TCGA) program. The availability of the genomic and proteomic data is enabling proteogenomic study for both reference (i.e., contained in major sequence databases) and nonreference markers of cancer. The CPTAC laboratories have focused on colon, breast, and ovarian tissues in the first round of analyses; spectra from these data sets were produced from 2D liquid chromatography-tandem mass spectrometry analyses and represent deep coverage. To reduce the variability introduced by disparate data analysis platforms (e.g., software packages, versions, parameters, sequence databases, etc.), the CPTAC Common Data Analysis Platform (CDAP) was created. The CDAP produces both peptide-spectrum-match (PSM) reports and gene-level reports. The pipeline processes raw mass spectrometry data according to the following: (1) peak-picking and quantitative data extraction, (2) database searching, (3) gene-based protein parsimony, and (4) false-discovery rate-based filtering. The pipeline also produces localization scores for the phosphopeptide enrichment studies using the PhosphoRS program. Quantitative information for each of the data sets is specific to the sample processing, with PSM and protein reports containing the spectrum-level or gene-level ("rolled-up") precursor peak areas and spectral counts for label-free or reporter ion log-ratios for 4plex iTRAQ. The reports are available in simple tab-delimited formats and, for the PSM-reports, in mzIdentML. The goal of the CDAP is to provide standard, uniform reports for all of the CPTAC data to enable comparisons between different samples and cancer types as well as across the major omics fields.

Understanding global changes of the liver proteome during murine schistosomiasis using a label-free shotgun approach.

PubMed

Campos, Jonatan Marques; Neves, Leandro Xavier; de Paiva, Nívia Carolina Nogueira; de Oliveira E Castro, Renata Alves; Casé, Ana Helena; Carneiro, Cláudia Martins; Andrade, Milton Hércules Guerra; Castro-Borges, William

2017-01-16

Schistosomiasis is an endemic disease affecting over 207 million people worldwide caused by helminth parasites of the genus Schistosoma. In Brazil the disease is responsible for the loss of up to 800 lives annually, resulting from the desabilitating effects of this chronic condition. In this study, we infected Balb/c mice with Schistosoma mansoni and analysed global changes in the proteomic profile of soluble liver proteins. Our shotgun analyses revealed predominance of up-regulation of proteins at 5weeks of infection, coinciding with the onset of egg laying, and a remarkable down-regulation of liver constituents at 7weeks, when severe tissue damage is installed. Representatives of glycolytic enzymes and stress response (in particular at the endoplasmic reticulum) were among the most differentially expressed molecules found in the infected liver. Collectively, our data contribute over 70 molecules not previously reported to be found at altered levels in murine schistosomiasis to further exploration of their potential as biomarkers of the disease. Moreover, understanding their intricate interaction using bioinformatics approach can potentially bring clarity to unknown mechanisms linked to the establishment of this condition in the vertebrate host. To our knowledge, this study refers to the first shotgun proteomic analysis to provide an inventory of the global changes in the liver soluble proteome caused by Schistosoma mansoni in the Balb/c model. It also innovates by yielding data on quantification of the identified molecules as a manner to clarify and give insights into the underlying mechanisms for establishment of Schistosomiasis, a neglected tropical disease with historical prevalence in Brazil. Copyright © 2016 Elsevier B.V. All rights reserved.

Protein differences between human trapezius and vastus lateralis muscles determined with a proteomic approach.

PubMed

Hadrévi, Jenny; Hellström, Fredrik; Kieselbach, Thomas; Malm, Christer; Pedrosa-Domellöf, Fatima

2011-08-10

The trapezius muscle is a neck muscle that is susceptible to chronic pain conditions associated with repetitive tasks, commonly referred to as chronic work-related myalgia, hence making the trapezius a muscle of clinical interest. To provide a basis for further investigations of the proteomic traits of the trapezius muscle in disease, two-dimensional difference gel electrophoresis (2D-DIGE) was performed on the healthy trapezius using vastus lateralis as a reference. To obtain as much information as possible from the vast proteomic data set, both one-way ANOVA, with and without false discovery rate (FDR) correlation, and partial least square projection to latent structures with discriminant analysis (PLS-DA) were combined to compare the outcome of the analysis. The trapezius and vastus lateralis showed significant differences in metabolic, contractile and regulatory proteins, with different results depending on choice of statistical approach and pre-processing technique. Using the standard method, FDR correlated one-way ANOVA, 42 protein spots differed significantly in abundance between the two muscles. Complementary analysis using immunohistochemistry and western blot confirmed the results from the 2D-DIGE analysis. The proteomic approach used in the present study combining 2D-DIGE and multivariate modelling provided a more comprehensive comparison of the protein profiles of the human trapezius and vastus lateralis muscle, than previously possible to obtain with immunohistochemistry or SDS-PAGE alone. Although 2D-DIGE has inherent limitations it is particularly useful to comprehensively screen for important structural and metabolic proteins, and appears to be a promising tool for future studies of patients suffering from chronic work related myalgia or other muscle diseases.

Integrated redox proteomics and metabolomics of mitochondria to identify mechanisms of cd toxicity.

PubMed

Go, Young-Mi; Roede, James R; Orr, Michael; Liang, Yongliang; Jones, Dean P

2014-05-01

Cadmium (Cd) exposure contributes to human diseases affecting liver, kidney, lung, and other organ systems, but mechanisms underlying the pleotropic nature of these toxicities are poorly understood. Cd accumulates in humans from dietary, environmental (including cigarette smoke), and occupational sources, and has a twenty-year biologic half-life. Our previous mouse and cell studies showed that environmental low-dose Cd exposure altered protein redox states resulting in stimulation of inflammatory signaling and disruption of the actin cytoskeleton system, suggesting that Cd could impact multiple mechanisms of disease. In the current study, we investigated the effects of acute Cd exposure on the redox proteome and metabolome of mouse liver mitochondria to gain insight into associated toxicological mechanisms and functions. We analyzed redox states of liver mitochondrial proteins by redox proteomics using isotope coded affinity tag (ICAT) combined mass spectrometry. Redox ICAT identified 2687 cysteine-containing peptides (peptidyl Cys) of which 1667 peptidyl Cys (657 proteins) were detected in both control and Cd-exposed samples. Of these, 46% (1247 peptidyl Cys, 547 proteins) were oxidized by Cd more than 1.5-fold relative to controls. Bioinformatics analysis using MetaCore software showed that Cd affected 86 pathways, including 24 Cys in proteins functioning in branched chain amino acid (BCAA) and 14 Cys in proteins functioning in fatty acid (acylcarnitine/carnitine) metabolism. Consistently, high-resolution metabolomics data showed that Cd treatment altered levels of BCAA and carnitine metabolites. Together, these results show that mitochondrial protein redox and metabolites are targets in Cd-induced hepatotoxicity. The results further indicate that redox proteomics and metabolomics can be used in an integrated systems approach to investigate complex disease mechanisms.

Early Targets of miR-34a in Neuroblastoma*

PubMed Central

De Antonellis, Pasqualino; Carotenuto, Marianeve; Vandenbussche, Jonathan; De Vita, Gennaro; Ferrucci, Veronica; Medaglia, Chiara; Boffa, Iolanda; Galiero, Alessandra; Di Somma, Sarah; Magliulo, Daniela; Aiese, Nadia; Alonzi, Alessandro; Spano, Daniela; Liguori, Lucia; Chiarolla, Cristina; Verrico, Antonio; Schulte, Johannes H.; Mestdagh, Pieter; Vandesompele, Jo; Gevaert, Kris; Zollo, Massimo

2014-01-01

Several genes encoding for proteins involved in proliferation, invasion, and apoptosis are known to be direct miR-34a targets. Here, we used proteomics to screen for targets of miR-34a in neuroblastoma (NBL), a childhood cancer that originates from precursor cells of the sympathetic nervous system. We examined the effect of miR-34a overexpression using a tetracycline inducible system in two NBL cell lines (SHEP and SH-SY5Y) at early time points of expression (6, 12, and 24 h). Proteome analysis using post-metabolic labeling led to the identification of 2,082 proteins, and among these 186 were regulated (112 proteins down-regulated and 74 up-regulated). Prediction of miR-34a targets via bioinformatics showed that 32 transcripts held miR-34a seed sequences in their 3′-UTR. By combining the proteomics data with Kaplan Meier gene-expression studies, we identified seven new gene products (ALG13, TIMM13, TGM2, ABCF2, CTCF, Ki67, and LYAR) that were correlated with worse clinical outcomes. These were further validated in vitro by 3′-UTR seed sequence regulation. In addition, Michigan Molecular Interactions searches indicated that together these proteins affect signaling pathways that regulate cell cycle and proliferation, focal adhesions, and other cellular properties that overall enhance tumor progression (including signaling pathways such as TGF-β, WNT, MAPK, and FAK). In conclusion, proteome analysis has here identified early targets of miR-34a with relevance to NBL tumorigenesis. Along with the results of previous studies, our data strongly suggest miR-34a as a useful tool for improving the chance of therapeutic success with NBL. PMID:24912852

Research Resource: A Dual Proteomic Approach Identifies Regulated Islet Proteins During β-Cell Mass Expansion In Vivo.

PubMed

Horn, Signe; Kirkegaard, Jeannette S; Hoelper, Soraya; Seymour, Philip A; Rescan, Claude; Nielsen, Jens H; Madsen, Ole D; Jensen, Jan N; Krüger, Marcus; Grønborg, Mads; Ahnfelt-Rønne, Jonas

2016-01-01

Diabetes is characterized by insulin insufficiency due to a relative paucity of functional β-cell mass. Thus, strategies for increasing β-cell mass in situ are sought-after for therapeutic purposes. Pregnancy is a physiological state capable of inducing robust β-cell mass expansion, however, the mechanisms driving this expansion are not fully understood. Thus, the aim of this study was to characterize pregnancy-induced changes in the islet proteome at the peak of β-cell proliferation in mice. Islets from pregnant and nonpregnant littermates were compared via 2 proteomic strategies. In vivo pulsed stable isotope labeling of amino acids in cell culture was used to monitor de novo protein synthesis during the first 14.5 days of pregnancy. In parallel, protein abundance was determined using ex vivo dimethyl labelling at gestational day 14.5. Comparison of the 2 datasets revealed 170 islet proteins to be up regulated as a response to pregnancy. These included several proteins, not previously associated with pregnancy-induced islet expansion, such as CLIC1, STMN1, MCM6, PPIB, NEDD4, and HLTF. Confirming the validity of our approach, we also identified proteins encoded by genes known to be associated with pregnancy-induced islet expansion, such as CHGB, IGFBP5, MATN2, EHHADH, IVD, and BMP1. Bioinformatic analyses demonstrated enrichment and activation of the biological functions: "protein synthesis" and "proliferation," and predicted the transcription factors HNF4α, MYC, MYCN, E2F1, NFE2L2, and HNF1α as upstream regulators of the observed expressional changes. As the first characterization of the islet-proteome during pregnancy, this study provides novel insight into the mechanisms involved in promoting pregnancy-induced β-cell mass expansion and function.

Proteomic and Bioinformatic Profile of Primary Human Oral Epithelial Cells

PubMed Central

Ghosh, Santosh K.; Yohannes, Elizabeth; Bebek, Gurkan; Weinberg, Aaron; Jiang, Bin; Willard, Belinda; Chance, Mark R.; Kinter, Michael T.; McCormick, Thomas S.

2012-01-01

Wounding of the oral mucosa occurs frequently in a highly septic environment. Remarkably, these wounds heal quickly and the oral cavity, for the most part, remains healthy. Deciphering the normal human oral epithelial cell (NHOEC) proteome is critical for understanding the mechanism(s) of protection elicited when the mucosal barrier is intact, as well as when it is breached. Combining 2D gel electrophoresis with shotgun proteomics resulted in identification of 1662 NHOEC proteins. Proteome annotations were performed based on protein classes, molecular functions, disease association and membership in canonical and metabolic signaling pathways. Comparing the NHOEC proteome with a database of innate immunity-relevant interactions (InnateDB) identified 64 common proteins associated with innate immunity. Comparison with published salivary proteomes revealed that 738/1662 NHOEC proteins were common, suggesting that significant numbers of salivary proteins are of epithelial origin. Gene ontology analysis showed similarities in the distributions of NHOEC and saliva proteomes with regard to biological processes, and molecular functions. We also assessed the inter-individual variability of the NHOEC proteome and observed it to be comparable with other primary cells. The baseline proteome described in this study should serve as a resource for proteome studies of the oral mucosa, especially in relation to disease processes. PMID:23035736

Comparative proteome analysis of Actinoplanes sp. SE50/110 grown with maltose or glucose shows minor differences for acarbose biosynthesis proteins but major differences for saccharide transporters.

PubMed

Wendler, Sergej; Otto, Andreas; Ortseifen, Vera; Bonn, Florian; Neshat, Armin; Schneiker-Bekel, Susanne; Wolf, Timo; Zemke, Till; Wehmeier, Udo F; Hecker, Michael; Kalinowski, Jörn; Becher, Dörte; Pühler, Alfred

2016-01-10

Actinoplanes sp. SE50/110 is known for the production of the α-glucosidase inhibitor and anti-diabetic drug acarbose. Acarbose (acarviosyl-maltose) is produced as the major product when the bacterium is grown in medium with maltose, while acarviosyl-glucose is the major product when glucose is the sole carbon source in the medium. In this study, a state-of-the-art proteomics approach was applied combining subcellular fractionation, in vivo metabolic labeling and shotgun mass spectrometry to analyze differences in the proteome of Actinoplanes sp. SE50/110 cultures grown in minimal medium containing either maltose or glucose as the sole carbon source. To study proteins in distinct subcellular locations, a cytosolic, an enriched membrane, a membrane shaving and an extracellular fraction were included in the analysis. Altogether, quantitative proteome data was obtained for 2497 proteins representing about 30% of the ca. 8270 predicted proteins of Actinoplanes sp. SE50/110. When comparing protein quantities of maltose- to glucose-grown cultures, differences were observed for saccharide transport and metabolism proteins, whereas differences for acarbose biosynthesis gene cluster proteins were almost absent. The maltose-inducible α-glucosidase/maltase MalL as well as the ABC-type saccharide transporters AglEFG, MalEFG and MstEAF had significantly higher quantities in the maltose growth condition. The only highly abundant saccharide transporter in the glucose condition was the monosaccharide transporter MstEAF, which may indicate that MstEAF is the major glucose importer. Taken all findings together, the previously observed formation of acarviosyl-maltose and acarviosyl-glucose is more closely connected to the transport of saccharides than to a differential expression of the acarbose gene cluster. Diabetes is a global pandemic accounting for about 11% of the worldwide healthcare expenditures (>600 billion US dollars) and is projected to affect 592 million people by 2035 (www.idf.org). Whether Actinoplanes sp. SE50/110 produces type 2 diabetes drug acarbose (acarviosyl-maltose) or another acarviose metabolite such as acarviosyl-glucose as the major product depends on the offered carbon source. The differences observed in this proteome in this study suggest that the differences in the formation of acarviosyl-maltose and acarviosyl-glucose are more closely connected to the transport of saccharides than to a differential expression of the acarbose gene cluster. In addition, the present study provides a comprehensive overview of the proteome of Actinoplanes sp. SE50/110. Copyright © 2015 Elsevier B.V. All rights reserved.

Lipidomics reveals dramatic lipid compositional changes in the maturing postnatal lung

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dautel, Sydney E.; Kyle, Jennifer E.; Clair, Geremy

Lung immaturity is a major cause of morbidity and mortality in premature infants. Understanding the molecular mechanisms driving normal lung development could provide insights on how to ameliorate disrupted development. While transcriptomic and proteomic analyses of normal lung development have been previously reported, characterization of changes in the lipidome is lacking. Lipids play significant roles in the lung, such as dipalmitoylcholine in pulmonary surfactant; however, many of the roles of specific lipid species in normal lung development, as well as in disease states, are not well defined. In this study, we used liquid chromatography-mass spectrometry (LC-MS/MS) to investigate the murinemore » lipidome during normal postnatal lung development. Lipidomics analysis of lungs from post-natal day 7, day 14 and 6-8 week mice (adult) identified 928 unique lipids across 21 lipid subclasses, with dramatic alterations in the lipidome across developmental stages. Our data confirmed previously recognized aspects of post-natal lung development and revealed several insights, including in sphingolipid-mediated apoptosis, inflammation and energy storage/usage. Complementary proteomics, metabolomics and chemical imaging corroborated these observations. Finally, this multi-omic view provides a unique resource and deeper insight into normal pulmonary development.« less

Lipidomics reveals dramatic lipid compositional changes in the maturing postnatal lung

DOE PAGES

Dautel, Sydney E.; Kyle, Jennifer E.; Clair, Geremy; ...

2017-02-01

Lung immaturity is a major cause of morbidity and mortality in premature infants. Understanding the molecular mechanisms driving normal lung development could provide insights on how to ameliorate disrupted development. While transcriptomic and proteomic analyses of normal lung development have been previously reported, characterization of changes in the lipidome is lacking. Lipids play significant roles in the lung, such as dipalmitoylcholine in pulmonary surfactant; however, many of the roles of specific lipid species in normal lung development, as well as in disease states, are not well defined. In this study, we used liquid chromatography-mass spectrometry (LC-MS/MS) to investigate the murinemore » lipidome during normal postnatal lung development. Lipidomics analysis of lungs from post-natal day 7, day 14 and 6-8 week mice (adult) identified 928 unique lipids across 21 lipid subclasses, with dramatic alterations in the lipidome across developmental stages. Our data confirmed previously recognized aspects of post-natal lung development and revealed several insights, including in sphingolipid-mediated apoptosis, inflammation and energy storage/usage. Complementary proteomics, metabolomics and chemical imaging corroborated these observations. Finally, this multi-omic view provides a unique resource and deeper insight into normal pulmonary development.« less

Proteogenomic characterization of human colon and rectal cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Bing; Wang, Jing; Wang, Xiaojing

2014-09-18

We analyzed proteomes of colon and rectal tumors previously characterized by the Cancer Genome Atlas (TCGA) and performed integrated proteogenomic analyses. Protein sequence variants encoded by somatic genomic variations displayed reduced expression compared to protein variants encoded by germline variations. mRNA transcript abundance did not reliably predict protein expression differences between tumors. Proteomics identified five protein expression subtypes, two of which were associated with the TCGA "MSI/CIMP" transcriptional subtype, but had distinct mutation and methylation patterns and associated with different clinical outcomes. Although CNAs showed strong cis- and trans-effects on mRNA expression, relatively few of these extend to the proteinmore » level. Thus, proteomics data enabled prioritization of candidate driver genes. Our analyses identified HNF4A, a novel candidate driver gene in tumors with chromosome 20q amplifications. Integrated proteogenomic analysis provides functional context to interpret genomic abnormalities and affords novel insights into cancer biology.« less

Novel phage group infecting Lactobacillus delbrueckii subsp. lactis, as revealed by genomic and proteomic analysis of bacteriophage Ldl1.

PubMed

Casey, Eoghan; Mahony, Jennifer; Neve, Horst; Noben, Jean-Paul; Dal Bello, Fabio; van Sinderen, Douwe

2015-02-01

Ldl1 is a virulent phage infecting the dairy starter Lactobacillus delbrueckii subsp. lactis LdlS. Electron microscopy analysis revealed that this phage exhibits a large head and a long tail and bears little resemblance to other characterized phages infecting Lactobacillus delbrueckii. In vitro propagation of this phage revealed a latent period of 30 to 40 min and a burst size of 59.9 +/- 1.9 phage particles. Comparative genomic and proteomic analyses showed remarkable similarity between the genome of Ldl1 and that of Lactobacillus plantarum phage ATCC 8014-B2. The genomic and proteomic characteristics of Ldl1 demonstrate that this phage does not belong to any of the four previously recognized L. delbrueckii phage groups, necessitating the creation of a new group, called group e, thus adding to the knowledge on the diversity of phages targeting strains of this industrially important lactic acid bacterial species.

Top-down proteomics reveals a unique protein S-thiolation switch in Salmonella Typimurium in response to infection-like conditions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ansong, Charles; Wu, Si; Meng, Da

Characterization of the mature protein complement in cells is crucial for a better understanding of cellular processes on a systems-wide scale. Bottom-up proteomic approaches often lead to loss of critical information about an endogenous protein’s actual state due to post translational modifications (PTMs) and other processes. Top-down approaches that involve analysis of the intact protein can address this concern but present significant analytical challenges related to the separation quality needed, measurement sensitivity, and speed that result in low throughput and limited coverage. Here we used single-dimension ultra high pressure liquid chromatography mass spectrometry to investigate the comprehensive ‘intact’ proteome ofmore » the Gram negative bacterial pathogen Salmonella Typhimurium. Top-down proteomics analysis revealed 563 unique proteins including 1665 proteoforms generated by PTMs, representing the largest microbial top-down dataset reported to date. Our analysis not only confirmed several previously recognized aspects of Salmonella biology and bacterial PTMs in general, but also revealed several novel biological insights. Of particular interest was differential utilization of the protein S-thiolation forms S-glutathionylation and S-cysteinylation in response to infection-like conditions versus basal conditions, which was corroborated by changes in corresponding biosynthetic pathways. This differential utilization highlights underlying metabolic mechanisms that modulate changes in cellular signaling, and represents to our knowledge the first report of S-cysteinylation in Gram negative bacteria. The demonstrated utility of our simple proteome-wide intact protein level measurement strategy for gaining biological insight should promote broader adoption and applications of top-down proteomics approaches.« less

Accurate proteome-wide protein quantification from high-resolution 15N mass spectra

PubMed Central

2011-01-01

In quantitative mass spectrometry-based proteomics, the metabolic incorporation of a single source of 15N-labeled nitrogen has many advantages over using stable isotope-labeled amino acids. However, the lack of a robust computational framework for analyzing the resulting spectra has impeded wide use of this approach. We have addressed this challenge by introducing a new computational methodology for analyzing 15N spectra in which quantification is integrated with identification. Application of this method to an Escherichia coli growth transition reveals significant improvement in quantification accuracy over previous methods. PMID:22182234

Proteomic analysis of protein deposits on worn daily wear silicone hydrogel contact lenses

PubMed Central

Wei, Xiaojia; Aliwarga, Yulina; Carnt, Nicole A.; Garrett, Qian; Willcox, Mark D.P.

2008-01-01

Purpose Previous studies have demonstrated deposition of tear proteins onto worn contact lenses. In this study, we used proteomic techniques to analyze the protein deposits extracted from worn daily wear silicone hydrogel contact lenses in combination with different lens care solutions. Methods Worn lenses were collected and protein deposits extracted using urea and surfactant. Protein extracts were desalted, concentrated, and then separated using one-dimensional gel electrophoresis. Individual protein components in extracts were identified using liquid chromatography combined with tandem mass spectrometry (LC-MS-MS) after trypsin digestion. Results One-dimensional gel electrophoresis revealed that lysozyme and other small proteins (around 20 kDa) were the most abundant proteins in the extracts. LC-MS-MS revealed a wide array of proteins in lens extracts with lysozyme and lipocalin 1 being the most commonly identified in deposit extracts. Conclusions Worn contact lenses deposit a wide array of proteins from tear film and other sources. Protein deposit profiles varied and were specific for each contact lens material. PMID:18989384

The quantitative proteomes of human-induced pluripotent stem cells and embryonic stem cells

PubMed Central

Munoz, Javier; Low, Teck Y; Kok, Yee J; Chin, Angela; Frese, Christian K; Ding, Vanessa; Choo, Andre; Heck, Albert J R

2011-01-01

Assessing relevant molecular differences between human-induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) is important, given that such differences may impact their potential therapeutic use. Controversy surrounds recent gene expression studies comparing hiPSCs and hESCs. Here, we present an in-depth quantitative mass spectrometry-based analysis of hESCs, two different hiPSCs and their precursor fibroblast cell lines. Our comparisons confirmed the high similarity of hESCs and hiPSCS at the proteome level as 97.8% of the proteins were found unchanged. Nevertheless, a small group of 58 proteins, mainly related to metabolism, antigen processing and cell adhesion, was found significantly differentially expressed between hiPSCs and hESCs. A comparison of the regulated proteins with previously published transcriptomic studies showed a low overlap, highlighting the emerging notion that differences between both pluripotent cell lines rather reflect experimental conditions than a recurrent molecular signature. PMID:22108792

Quantitative SUMO proteomics reveals the modulation of several PML nuclear body associated proteins and an anti-senescence function of UBC9.

PubMed

McManus, Francis P; Bourdeau, Véronique; Acevedo, Mariana; Lopes-Paciencia, Stéphane; Mignacca, Lian; Lamoliatte, Frédéric; Rojas Pino, John W; Ferbeyre, Gerardo; Thibault, Pierre

2018-05-17

Several regulators of SUMOylation have been previously linked to senescence but most targets of this modification in senescent cells remain unidentified. Using a two-step purification of a modified SUMO3, we profiled the SUMO proteome of senescent cells in a site-specific manner. We identified 25 SUMO sites on 23 proteins that were significantly regulated during senescence. Of note, most of these proteins were PML nuclear body (PML-NB) associated, which correlates with the increased number and size of PML-NBs observed in senescent cells. Interestingly, the sole SUMO E2 enzyme, UBC9, was more SUMOylated during senescence on its Lys-49. Functional studies of a UBC9 mutant at Lys-49 showed a decreased association to PML-NBs and the loss of UBC9's ability to delay senescence. We thus propose both pro- and anti-senescence functions of protein SUMOylation.

Advances in targeted proteomics and applications to biomedical research

PubMed Central

Shi, Tujin; Song, Ehwang; Nie, Song; Rodland, Karin D.; Liu, Tao; Qian, Wei-Jun; Smith, Richard D.

2016-01-01

Targeted proteomics technique has emerged as a powerful protein quantification tool in systems biology, biomedical research, and increasing for clinical applications. The most widely used targeted proteomics approach, selected reaction monitoring (SRM), also known as multiple reaction monitoring (MRM), can be used for quantification of cellular signaling networks and preclinical verification of candidate protein biomarkers. As an extension to our previous review on advances in SRM sensitivity herein we review recent advances in the method and technology for further enhancing SRM sensitivity (from 2012 to present), and highlighting its broad biomedical applications in human bodily fluids, tissue and cell lines. Furthermore, we also review two recently introduced targeted proteomics approaches, parallel reaction monitoring (PRM) and data-independent acquisition (DIA) with targeted data extraction on fast scanning high-resolution accurate-mass (HR/AM) instruments. Such HR/AM targeted quantification with monitoring all target product ions addresses SRM limitations effectively in specificity and multiplexing; whereas when compared to SRM, PRM and DIA are still in the infancy with a limited number of applications. Thus, for HR/AM targeted quantification we focus our discussion on method development, data processing and analysis, and its advantages and limitations in targeted proteomics. Finally, general perspectives on the potential of achieving both high sensitivity and high sample throughput for large-scale quantification of hundreds of target proteins are discussed. PMID:27302376

Proteome Analysis of Cytoplasmatic and Plastidic β-Carotene Lipid Droplets in Dunaliella bardawil1[OPEN

PubMed Central

Davidi, Lital; Levin, Yishai; Ben-Dor, Shifra; Pick, Uri

2015-01-01

The halotolerant green alga Dunaliella bardawil is unique in that it accumulates under stress two types of lipid droplets: cytoplasmatic lipid droplets (CLD) and β-carotene-rich (βC) plastoglobuli. Recently, we isolated and analyzed the lipid and pigment compositions of these lipid droplets. Here, we describe their proteome analysis. A contamination filter and an enrichment filter were utilized to define core proteins. A proteome database of Dunaliella salina/D. bardawil was constructed to aid the identification of lipid droplet proteins. A total of 124 and 42 core proteins were identified in βC-plastoglobuli and CLD, respectively, with only eight common proteins. Dunaliella spp. CLD resemble cytoplasmic droplets from Chlamydomonas reinhardtii and contain major lipid droplet-associated protein and enzymes involved in lipid and sterol metabolism. The βC-plastoglobuli proteome resembles the C. reinhardtii eyespot and Arabidopsis (Arabidopsis thaliana) plastoglobule proteomes and contains carotene-globule-associated protein, plastid-lipid-associated protein-fibrillins, SOUL heme-binding proteins, phytyl ester synthases, β-carotene biosynthesis enzymes, and proteins involved in membrane remodeling/lipid droplet biogenesis: VESICLE-INDUCING PLASTID PROTEIN1, synaptotagmin, and the eyespot assembly proteins EYE3 and SOUL3. Based on these and previous results, we propose models for the biogenesis of βC-plastoglobuli and the biosynthesis of β-carotene within βC-plastoglobuli and hypothesize that βC-plastoglobuli evolved from eyespot lipid droplets. PMID:25404729

The Hemolymph Proteome of Fed and Starved Drosophila Larvae

PubMed Central

Goetze, Sandra; Ahrens, Christian H.; Omasits, Ulrich; Marty, Florian; Simigdala, Nikiana; Meyer, Imke; Wollscheid, Bernd; Brunner, Erich; Hafen, Ernst; Lehner, Christian F.

2013-01-01

The co-operation of specialized organ systems in complex multicellular organisms depends on effective chemical communication. Thus, body fluids (like blood, lymph or intraspinal fluid) contain myriads of signaling mediators apart from metabolites. Moreover, these fluids are also of crucial importance for immune and wound responses. Compositional analyses of human body fluids are therefore of paramount diagnostic importance. Further improving their comprehensiveness should increase our understanding of inter-organ communication. In arthropods, which have trachea for gas exchange and an open circulatory system, the single dominating interstitial fluid is the hemolymph. Accordingly, a detailed analysis of hemolymph composition should provide an especially comprehensive picture of chemical communication and defense in animals. Therefore we used an extensive protein fractionation workflow in combination with a discovery-driven proteomic approach to map out the detectable protein composition of hemolymph isolated from Drosophila larvae. Combined mass spectrometric analysis revealed more than 700 proteins extending far beyond the previously known Drosophila hemolymph proteome. Moreover, by comparing hemolymph isolated from either fed or starved larvae, we provide initial provisional insights concerning compositional changes in response to nutritional state. Storage proteins in particular were observed to be strongly reduced by starvation. Our hemolymph proteome catalog provides a rich basis for data mining, as exemplified by our identification of potential novel cytokines, as well as for future quantitative analyses by targeted proteomics. PMID:23840627

Learning from Heterogeneous Data Sources: An Application in Spatial Proteomics

PubMed Central

Breckels, Lisa M.; Holden, Sean B.; Wojnar, David; Mulvey, Claire M.; Christoforou, Andy; Groen, Arnoud; Trotter, Matthew W. B.; Kohlbacher, Oliver; Lilley, Kathryn S.; Gatto, Laurent

2016-01-01

Sub-cellular localisation of proteins is an essential post-translational regulatory mechanism that can be assayed using high-throughput mass spectrometry (MS). These MS-based spatial proteomics experiments enable us to pinpoint the sub-cellular distribution of thousands of proteins in a specific system under controlled conditions. Recent advances in high-throughput MS methods have yielded a plethora of experimental spatial proteomics data for the cell biology community. Yet, there are many third-party data sources, such as immunofluorescence microscopy or protein annotations and sequences, which represent a rich and vast source of complementary information. We present a unique transfer learning classification framework that utilises a nearest-neighbour or support vector machine system, to integrate heterogeneous data sources to considerably improve on the quantity and quality of sub-cellular protein assignment. We demonstrate the utility of our algorithms through evaluation of five experimental datasets, from four different species in conjunction with four different auxiliary data sources to classify proteins to tens of sub-cellular compartments with high generalisation accuracy. We further apply the method to an experiment on pluripotent mouse embryonic stem cells to classify a set of previously unknown proteins, and validate our findings against a recent high resolution map of the mouse stem cell proteome. The methodology is distributed as part of the open-source Bioconductor pRoloc suite for spatial proteomics data analysis. PMID:27175778

The hemolymph proteome of fed and starved Drosophila larvae.

PubMed

Handke, Björn; Poernbacher, Ingrid; Goetze, Sandra; Ahrens, Christian H; Omasits, Ulrich; Marty, Florian; Simigdala, Nikiana; Meyer, Imke; Wollscheid, Bernd; Brunner, Erich; Hafen, Ernst; Lehner, Christian F

2013-01-01

The co-operation of specialized organ systems in complex multicellular organisms depends on effective chemical communication. Thus, body fluids (like blood, lymph or intraspinal fluid) contain myriads of signaling mediators apart from metabolites. Moreover, these fluids are also of crucial importance for immune and wound responses. Compositional analyses of human body fluids are therefore of paramount diagnostic importance. Further improving their comprehensiveness should increase our understanding of inter-organ communication. In arthropods, which have trachea for gas exchange and an open circulatory system, the single dominating interstitial fluid is the hemolymph. Accordingly, a detailed analysis of hemolymph composition should provide an especially comprehensive picture of chemical communication and defense in animals. Therefore we used an extensive protein fractionation workflow in combination with a discovery-driven proteomic approach to map out the detectable protein composition of hemolymph isolated from Drosophila larvae. Combined mass spectrometric analysis revealed more than 700 proteins extending far beyond the previously known Drosophila hemolymph proteome. Moreover, by comparing hemolymph isolated from either fed or starved larvae, we provide initial provisional insights concerning compositional changes in response to nutritional state. Storage proteins in particular were observed to be strongly reduced by starvation. Our hemolymph proteome catalog provides a rich basis for data mining, as exemplified by our identification of potential novel cytokines, as well as for future quantitative analyses by targeted proteomics.

Identification of Maturation-Specific Proteins by Single-Cell Proteomics of Human Oocytes

PubMed Central

Virant-Klun, Irma; Leicht, Stefan; Hughes, Christopher; Krijgsveld, Jeroen

2016-01-01

Oocytes undergo a range of complex processes via oogenesis, maturation, fertilization, and early embryonic development, eventually giving rise to a fully functioning organism. To understand proteome composition and diversity during maturation of human oocytes, here we have addressed crucial aspects of oocyte collection and proteome analysis, resulting in the first proteome and secretome maps of human oocytes. Starting from 100 oocytes collected via a novel serum-free hanging drop culture system, we identified 2,154 proteins, whose function indicate that oocytes are largely resting cells with a proteome that is tailored for homeostasis, cellular attachment, and interaction with its environment via secretory factors. In addition, we have identified 158 oocyte-enriched proteins (such as ECAT1, PIWIL3, NLRP7)1 not observed in high-coverage proteomics studies of other human cell lines or tissues. Exploiting SP3, a novel technology for proteomic sample preparation using magnetic beads, we scaled down proteome analysis to single cells. Despite the low protein content of only ∼100 ng per cell, we consistently identified ∼450 proteins from individual oocytes. When comparing individual oocytes at the germinal vesicle (GV) and metaphase II (MII) stage, we found that the Tudor and KH domain-containing protein (TDRKH) is preferentially expressed in immature oocytes, while Wee2, PCNA, and DNMT1 were enriched in mature cells, collectively indicating that maintenance of genome integrity is crucial during oocyte maturation. This study demonstrates that an innovative proteomics workflow facilitates analysis of single human oocytes to investigate human oocyte biology and preimplantation development. The approach presented here paves the way for quantitative proteomics in other quantity-limited tissues and cell types. Data associated with this study are available via ProteomeXchange with identifier PXD004142. PMID:27215607

Identification of Maturation-Specific Proteins by Single-Cell Proteomics of Human Oocytes.

PubMed

Virant-Klun, Irma; Leicht, Stefan; Hughes, Christopher; Krijgsveld, Jeroen

2016-08-01

Oocytes undergo a range of complex processes via oogenesis, maturation, fertilization, and early embryonic development, eventually giving rise to a fully functioning organism. To understand proteome composition and diversity during maturation of human oocytes, here we have addressed crucial aspects of oocyte collection and proteome analysis, resulting in the first proteome and secretome maps of human oocytes. Starting from 100 oocytes collected via a novel serum-free hanging drop culture system, we identified 2,154 proteins, whose function indicate that oocytes are largely resting cells with a proteome that is tailored for homeostasis, cellular attachment, and interaction with its environment via secretory factors. In addition, we have identified 158 oocyte-enriched proteins (such as ECAT1, PIWIL3, NLRP7)(1) not observed in high-coverage proteomics studies of other human cell lines or tissues. Exploiting SP3, a novel technology for proteomic sample preparation using magnetic beads, we scaled down proteome analysis to single cells. Despite the low protein content of only ∼100 ng per cell, we consistently identified ∼450 proteins from individual oocytes. When comparing individual oocytes at the germinal vesicle (GV) and metaphase II (MII) stage, we found that the Tudor and KH domain-containing protein (TDRKH) is preferentially expressed in immature oocytes, while Wee2, PCNA, and DNMT1 were enriched in mature cells, collectively indicating that maintenance of genome integrity is crucial during oocyte maturation. This study demonstrates that an innovative proteomics workflow facilitates analysis of single human oocytes to investigate human oocyte biology and preimplantation development. The approach presented here paves the way for quantitative proteomics in other quantity-limited tissues and cell types. Data associated with this study are available via ProteomeXchange with identifier PXD004142. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

We Are Not Alone: The iMOP Initiative and Its Roles in a Biology- and Disease-Driven Human Proteome Project.

PubMed

Tholey, Andreas; Taylor, Nicolas L; Heazlewood, Joshua L; Bendixen, Emøke

2017-12-01

Mapping of the human proteome has advanced significantly in recent years and will provide a knowledge base to accelerate our understanding of how proteins and protein networks can affect human health and disease. However, providing solutions to human health challenges will likely fail if insights are exclusively based on studies of human samples and human proteomes. In recent years, it has become evident that human health depends on an integrated understanding of the many species that make human life possible. These include the commensal microorganisms that are essential to human life, pathogens, and food species as well as the classic model organisms that enable studies of biological mechanisms. The Human Proteome Organization (HUPO) initiative on multiorganism proteomes (iMOP) works to support proteome research undertaken on nonhuman species that remain widely under-studied compared with the progress in human proteome research. This perspective argues the need for further research on multiple species that impact human life. We also present an update on recent progress in model organisms, microbiota, and food species, address the emerging problem of antibiotics resistance, and outline how iMOP activities could lead to a more inclusive approach for the human proteome project (HPP) to better support proteome research aimed at improving human health and furthering knowledge on human biology.

Statistical Model to Analyze Quantitative Proteomics Data Obtained by 18O/16O Labeling and Linear Ion Trap Mass Spectrometry

PubMed Central

Jorge, Inmaculada; Navarro, Pedro; Martínez-Acedo, Pablo; Núñez, Estefanía; Serrano, Horacio; Alfranca, Arántzazu; Redondo, Juan Miguel; Vázquez, Jesús

2009-01-01

Statistical models for the analysis of protein expression changes by stable isotope labeling are still poorly developed, particularly for data obtained by 16O/18O labeling. Besides large scale test experiments to validate the null hypothesis are lacking. Although the study of mechanisms underlying biological actions promoted by vascular endothelial growth factor (VEGF) on endothelial cells is of considerable interest, quantitative proteomics studies on this subject are scarce and have been performed after exposing cells to the factor for long periods of time. In this work we present the largest quantitative proteomics study to date on the short term effects of VEGF on human umbilical vein endothelial cells by 18O/16O labeling. Current statistical models based on normality and variance homogeneity were found unsuitable to describe the null hypothesis in a large scale test experiment performed on these cells, producing false expression changes. A random effects model was developed including four different sources of variance at the spectrum-fitting, scan, peptide, and protein levels. With the new model the number of outliers at scan and peptide levels was negligible in three large scale experiments, and only one false protein expression change was observed in the test experiment among more than 1000 proteins. The new model allowed the detection of significant protein expression changes upon VEGF stimulation for 4 and 8 h. The consistency of the changes observed at 4 h was confirmed by a replica at a smaller scale and further validated by Western blot analysis of some proteins. Most of the observed changes have not been described previously and are consistent with a pattern of protein expression that dynamically changes over time following the evolution of the angiogenic response. With this statistical model the 18O labeling approach emerges as a very promising and robust alternative to perform quantitative proteomics studies at a depth of several thousand proteins. PMID:19181660

Comprehensive proteome analysis of Actinoplanes sp. SE50/110 highlighting the location of proteins encoded by the acarbose and the pyochelin biosynthesis gene cluster.

PubMed

Wendler, Sergej; Otto, Andreas; Ortseifen, Vera; Bonn, Florian; Neshat, Armin; Schneiker-Bekel, Susanne; Walter, Frederik; Wolf, Timo; Zemke, Till; Wehmeier, Udo F; Hecker, Michael; Kalinowski, Jörn; Becher, Dörte; Pühler, Alfred

2015-07-01

Acarbose is an α-glucosidase inhibitor produced by Actinoplanes sp. SE50/110 that is medically important due to its application in the treatment of type2 diabetes. In this work, a comprehensive proteome analysis of Actinoplanes sp. SE50/110 was carried out to determine the location of proteins of the acarbose (acb) and the putative pyochelin (pch) biosynthesis gene cluster. Therefore, a comprehensive state-of-the-art proteomics approach combining subcellular fractionation, shotgun proteomics and spectral counting to assess the relative abundance of proteins within fractions was applied. The analysis of four different proteome fractions (cytosolic, enriched membrane, membrane shaving and extracellular fraction) resulted in the identification of 1582 of the 8270 predicted proteins. All 22 Acb-proteins and 21 of the 23 Pch-proteins were detected. Predicted membrane-associated, integral membrane or extracellular proteins of the pch and the acb gene cluster were found among the most abundant proteins in corresponding fractions. Intracellular biosynthetic proteins of both gene clusters were not only detected in the cytosolic, but also in the enriched membrane fraction, indicating that the biosynthesis of acarbose and putative pyochelin metabolites takes place at the inner membrane. Actinoplanes sp. SE50/110 is a natural producer of the α-glucosidase inhibitor acarbose, a bacterial secondary metabolite that is used as a drug for the treatment of type 2 diabetes, a disease which is a global pandemic that currently affects 387 million people and accounts for 11% of worldwide healthcare expenditures (www.idf.org). The work presented here is the first comprehensive investigation of protein localization and abundance in Actinoplanes sp. SE50/110 and provides an extensive source of information for the selection of genes for future mutational analysis and other hypothesis driven experiments. The conclusion that acarbose or pyochelin family siderophores are synthesized at the inner side of the cytoplasmic membrane determined from this work, indicates that studying corresponding intermediates will be challenging. In addition to previous studies on the genome and transcriptome, the work presented here demonstrates that the next omic level, the proteome, is now accessible for detailed physiological analysis of Actinoplanes sp. SE50/110, as well as mutants derived from this and related species. Copyright © 2015 Elsevier B.V. All rights reserved.

Characterization of a nonhuman primate model for the study of testicular peritubular cells - comparison with human testicular cells.

PubMed

Schmid, N; Stöckl, J B; Flenkenthaler, F; Dietrich, K-G; Schwarzer, J U; Köhn, F-M; Drummer, C; Fröhlich, T; Arnold, G J; Behr, R; Mayerhofer, A

2018-05-29

Are monkey testicular peritubular cells (MKTPCs) from the common marmoset monkey (Callithrix jacchus) a suitable translational model for the study of human testicular peritubular cells (HTPCs)? MKTPCs can be isolated and propagated in vitro, retain characteristic markers for testicular peritubular cells and their proteome strongly (correlation coefficient of 0.78) overlaps with the proteome of HTPCs. Smooth-muscle-like peritubular cells form the wall of seminiferous tubules, transport sperm, are immunologically active, secrete a plethora of factors and may contribute to the spermatogonial stem cell niche. Mechanistic studies are hampered by heterogeneity of human samples. We established a culture method for MKTPCs and characterized these cells from 6 young adult animals (2-3 years). To examine whether they qualify as a translational model we also examined HTPCs from 7 men and compared the proteomes of both groups. We used explant cultures to obtain MKTPCs, which express smooth muscle markers (calponin (CNN1), smooth muscle actin (ACTA2)), lack FSH-receptors (FSHR) and LH-receptors (LHCGR), but possess androgen receptors (AR). MKTPCs can be passaged at least up to 8 times, without discernable phenotypic changes. Mass-spectrometry-based analyses of the MKTPC and HTPC proteomes were performed. We established a method for isolation and cultivation of MKTPCs, and provide a comprehensive analysis of their protein repertoire. The results let us conclude that MKTPCs are suitable as a nonhuman primate model to study peritubular cell functions. List of identified proteins in MKTPCs by LC-MS/MS is accessible at the ProteomeXchange (identifier PXD009394). This is an in vitro cellular non-human primate model used to provide a window into the role of these cells in the human testis. Previous studies with HTPCs from patients revealed a degree of heterogeneity, possibly due to age, lifestyle and medical history of the individual human donors. We anticipate that the new translational model, derived from young health nonhuman primates, may allow us to circumvent these issues and may lead to a better understanding of the role of peritubular cells. This work was supported by grants from the DFG (MA 1080/27-1; AR 362/9-1; BE 2296/8-1). The authors declare no competing financial interests.

Proteomic alterations in root tips of Arabidopsis thaliana seedlings under altered gravity conditions

NASA Astrophysics Data System (ADS)

Zheng, H. Q.; Wang, H.

Gravity has a profound influence on plant growth and development Removed the influence of gravitational acceleration by spaceflight caused a wide range of cellular changes in plant Whole seedling that germinated and grown on clinostats showed the absent of gravitropism At the cellular level clinostat treatment has specific effects on plant cells such as induce alterations in cell wall composition increase production of heat-soluble proteins impact on the cellular energy metabolism facilitate a uniform distribution of plastids amyloplasts and increase number and volume of nucleoli A number of recent studies have shown that the exposure of Arabidopsis seedlings and callus cells to gravity stimulation hyper g-forces or clinostat rotation induces alterations in gene expression In our previous study the proteome of the Arabidopsis thaliana callus cells were separated by high resolution two-dimensional electrophoresis 2-DE Image analysis revealed that 80 protein spots showed quantitative and qualitative variations after exposure to clinostat rotation treatment We report here a systematic proteomic approach to investigate the altered gravity responsive proteins in root tip of Arabidopsis thaliana cv Landsberg erecta Three-day-old seedlings were exposed for 12h to a horizontal clinostat rotation H simulated weightlessness altered g-forces by centrifugation 7g hypergravity a vertical clinostat rotation V clinostat control or a stationary control grown conditions Total proteins of roots were extracted

Proteome Analysis for Downstream Targets of Oncogenic KRAS - the Potential Participation of CLIC4 in Carcinogenesis in the Lung

PubMed Central

Okudela, Koji; Katayama, Akira; Woo, Tetsukan; Mitsui, Hideaki; Suzuki, Takehisa; Tateishi, Yoko; Umeda, Shigeaki; Tajiri, Michihiko; Masuda, Munetaka; Nagahara, Noriyuki; Kitamura, Hitoshi; Ohashi, Kenichi

2014-01-01

This study investigated the proteome modulated by oncogenic KRAS in immortalized airway epithelial cells. Chloride intracellular channel protein 4 (CLIC4), S100 proteins (S100A2 and S100A11), tropomyosin 2, cathepsin L1, integrinsα3, eukaryotic elongation factor 1, vimentin, and others were discriminated. We here focused on CLIC4 to investigate its potential involvement in carcinogenesis in the lung because previous studies suggested that some chloride channels and chloride channel regulators could function as tumor suppressors. CILC4 protein levels were reduced in some lung cancer cell lines. The restoration of CLIC4 in lung cancer cell lines in which CLIC4 expression was reduced attenuated their growth activity. The immunohistochemical expression of the CLIC4 protein was weaker in primary lung cancer cells than in non-tumorous airway epithelial cells and was occasionally undetectable in some tumors. CLIC4 protein levels were significantly lower in a subtype of mucinous ADC than in others, and were also significantly lower in KRAS-mutated ADC than in EGFR-mutated ADC. These results suggest that the alteration in CLIC4 could be involved in restrictedly the development of a specific fraction of lung adenocarcinomas. The potential benefit of the proteome modulated by oncogenic KRAS to lung cancer research has been demonstrated. PMID:24503901

Exposure to Sound Vibrations Lead to Transcriptomic, Proteomic and Hormonal Changes in Arabidopsis

PubMed Central

Ghosh, Ritesh; Mishra, Ratnesh Chandra; Choi, Bosung; Kwon, Young Sang; Bae, Dong Won; Park, Soo-Chul; Jeong, Mi-Jeong; Bae, Hanhong

2016-01-01

Sound vibration (SV) is considered as an external mechanical force that modulates plant growth and development like other mechanical stimuli (e.g., wind, rain, touch and vibration). A number of previous and recent studies reported developmental responses in plants tailored against SV of varied frequencies. This strongly suggests the existence of sophisticated molecular mechanisms for SV perception and signal transduction. Despite this there exists a huge gap in our understanding regarding the SV-mediated molecular alterations, which is a prerequisite to gain insight into SV-mediated plant development. Herein, we investigated the global gene expression changes in Arabidopsis thaliana upon treatment with five different single frequencies of SV at constant amplitude for 1 h. As a next step, we also studied the SV-mediated proteomic changes in Arabidopsis. Data suggested that like other stimuli, SV also activated signature cellular events, for example, scavenging of reactive oxygen species (ROS), alteration of primary metabolism, and hormonal signaling. Phytohormonal analysis indicated that SV-mediated responses were, in part, modulated by specific alterations in phytohormone levels; especially salicylic acid (SA). Notably, several touch regulated genes were also up-regulated by SV treatment suggesting a possible molecular crosstalk among the two mechanical stimuli, sound and touch. Overall, these results provide a molecular basis to SV triggered global transcriptomic, proteomic and hormonal changes in plant. PMID:27665921

Proteomic Analysis of Interactions between a Deep-Sea Thermophilic Bacteriophage and Its Host at High Temperature ▿ †

PubMed Central

Wei, Dahai; Zhang, Xiaobo

2010-01-01

The virus-host interaction is essential to understanding the role that viruses play in ecological and geochemical processes in deep-sea vent ecosystems. Virus-induced changes in cellular gene expression and host physiology have been studied extensively. However, the molecular mechanism of interaction between a bacteriophage and its host at high temperature remains poorly understood. In the present study, the virus-induced gene expression profile of Geobacillus sp. E263, a thermophile isolated from a deep-sea hydrothermal ecosystem, was characterized. Based on proteomic analysis and random arbitrarily primed PCR (RAP-PCR) of Geobacillus sp. E263 cultured under non-bacteriophage GVE2 infection and GVE2 infection conditions, there were two types of protein/gene profiles in response to GVE2 infection. Twenty differentially expressed genes and proteins were revealed that could be grouped into 3 different categories based on cellular function, suggesting a coordinated response to infection. These differentially expressed genes and proteins were further confirmed by Northern blot analysis. To characterize the host proteins in response to virus infection, aspartate aminotransferase (AST) was inactivated to construct the AST mutant of Geobacillus sp. E263. The results showed that the AST protein was essential in virus infection. Thus, transcriptional and proteomic analyses and functional analysis revealed previously unknown host responses to deep-sea thermophilic virus infection. PMID:20015994

Detection of cow's milk proteins and minor components in human milk using proteomics techniques.

PubMed

Coscia, A; Orrù, S; Di Nicola, P; Giuliani, F; Varalda, A; Peila, C; Fabris, C; Conti, A; Bertino, E

2012-10-01

Cow's milk proteins (CMPs) are the best characterized food allergens. The aim of this study was to investigate cow's milk allergens in human colostrum of term and preterm newborns' mothers, and other minor protein components by proteomics techniques, more sensitive than other techniques used in the past. Sixty-two term and 11 preterm colostrum samples were collected, subjected to a treatment able to increase the concentration of the most diluted proteins and simultaneously to reduce the concentration of the proteins present at high concentration (Proteominer Treatment), and subsequently subjected to the steps of proteomic techniques. The most relevant finding in this study was the detection of the intact bovine alpha-S1-casein in human colostrum, then bovine alpha-1-casein could be considered the cow's milk allergen that is readily secreted in human milk and could be a cause of sensitization to cow's milk in exclusively breastfed predisposed infants. Another interesting result was the detection, at very low concentrations, of proteins previously not described in human milk (galectin-7, the different isoforms of the 14-3-3 protein and the serum amyloid P-component), probably involved in the regulation of the normal cell growth, in the pro-apoptotic function and in the regulation of tissue homeostasis. Further investigations are needed to understand if these families of proteins have specific biological activity in human milk.

Proteomics of the Human Placenta: Promises and Realities

PubMed Central

Robinson, J.M.; Ackerman, W.E.; Kniss, D.A.; Takizawa, T.; Vandré, D.D.

2015-01-01

Proteomics is an area of study that sets as its ultimate goal the global analysis of all of the proteins expressed in a biological system of interest. However, technical limitations currently hamper proteome-wide analyses of complex systems. In a more practical sense, a desired outcome of proteomics research is the translation of large protein data sets into formats that provide meaningful information regarding clinical conditions (e.g., biomarkers to serve as diagnostic and/or prognostic indicators of disease). Herein, we discuss placental proteomics by describing existing studies, pointing out their strengths and weaknesses. In so doing, we strive to inform investigators interested in this area of research about the current gap between hyperbolic promises and realities. Additionally, we discuss the utility of proteomics in discovery-based research, particularly as regards the capacity to unearth novel insights into placental biology. Importantly, when considering under studied systems such as the human placenta and diseases associated with abnormalities in placental function, proteomics can serve as a robust ‘shortcut’ to obtaining information unlikely to be garnered using traditional approaches. PMID:18222537

Exploring the Arabidopsis proteome: influence of protein solubilization buffers on proteome coverage.

PubMed

Marondedze, Claudius; Wong, Aloysius; Groen, Arnoud; Serrano, Natalia; Jankovic, Boris; Lilley, Kathryn; Gehring, Christoph; Thomas, Ludivine

2014-12-31

The study of proteomes provides new insights into stimulus-specific responses of protein synthesis and turnover, and the role of post-translational modifications at the systems level. Due to the diverse chemical nature of proteins and shortcomings in the analytical techniques used in their study, only a partial display of the proteome is achieved in any study, and this holds particularly true for plant proteomes. Here we show that different solubilization and separation methods have profound effects on the resulting proteome. In particular, we observed that the type of detergents employed in the solubilization buffer preferentially enriches proteins in different functional categories. These include proteins with a role in signaling, transport, response to temperature stimuli and metabolism. This data may offer a functional bias on comparative analysis studies. In order to obtain a broader coverage, we propose a two-step solubilization protocol with first a detergent-free buffer and then a second step utilizing a combination of two detergents to solubilize proteins.

Exploring the Arabidopsis Proteome: Influence of Protein Solubilization Buffers on Proteome Coverage

PubMed Central

Marondedze, Claudius; Wong, Aloysius; Groen, Arnoud; Serrano, Natalia; Jankovic, Boris; Lilley, Kathryn; Gehring, Christoph; Thomas, Ludivine

2014-01-01

The study of proteomes provides new insights into stimulus-specific responses of protein synthesis and turnover, and the role of post-translational modifications at the systems level. Due to the diverse chemical nature of proteins and shortcomings in the analytical techniques used in their study, only a partial display of the proteome is achieved in any study, and this holds particularly true for plant proteomes. Here we show that different solubilization and separation methods have profound effects on the resulting proteome. In particular, we observed that the type of detergents employed in the solubilization buffer preferentially enriches proteins in different functional categories. These include proteins with a role in signaling, transport, response to temperature stimuli and metabolism. This data may offer a functional bias on comparative analysis studies. In order to obtain a broader coverage, we propose a two-step solubilization protocol with first a detergent-free buffer and then a second step utilizing a combination of two detergents to solubilize proteins. PMID:25561235

[Application progress of proteomic in pharmacological study of Chinese medicinal formulae].

PubMed

Liu, Yu-Qian; Zhan, Shu-Yu; Ruan, Yu-Er; Zuo, Zhi-Yan; Ji, Xiao-Ming; Wang, Shuai-Jie; Ding, Bao-Yue

2017-10-01

Chinese medicinal formulae are the important means of clinical treatment in traditional Chinese medicine. It is urgent to use modern advanced scientific and technological means to reveal the complicated mechanism of Chinese medicinal formulae because they have the function characteristics of multiple components, multiple targets and integrated regulation. The systematic and comprehensive research model of proteomic is in line with the function characteristics of Chinese medicinal formulae, and proteomic has been widely used in the study of pharmacological mechanism of Chinese medicinal formulae. The recent applications of proteomic in pharmacological study of Chinese medicinal formulae in anti-cardiovascular and cerebrovascular diseases, anti-liver disease, antidiabetic, anticancer, anti-rheumatoid arthritis and other diseases were reviewed in this paper, and then the future development direction of proteomic in pharmacological study of Chinese medicinal formulae was put forward. This review is to provide the ideas and method for proteomic research on function mechanism of Chinese medicinal formulae. Copyright© by the Chinese Pharmaceutical Association.

Analysis of the early heterocyst Cys-proteome in the multicellular cyanobacterium Nostoc punctiforme reveals novel insights into the division of labor within diazotrophic filaments.

PubMed

Sandh, Gustaf; Ramström, Margareta; Stensjö, Karin

2014-12-04

In the filamentous cyanobacterium Nostoc punctiforme ATCC 29133, removal of combined nitrogen induces the differentiation of heterocysts, a cell-type specialized in N2 fixation. The differentiation involves genomic, structural and metabolic adaptations. In cyanobacteria, changes in the availability of carbon and nitrogen have also been linked to redox regulated posttranslational modifications of protein bound thiol groups. We have here employed a thiol targeting strategy to relatively quantify the putative redox proteome in heterocysts as compared to N2-fixing filaments, 24 hours after combined nitrogen depletion. The aim of the study was to expand the coverage of the cell-type specific proteome and metabolic landscape of heterocysts. Here we report the first cell-type specific proteome of newly formed heterocysts, compared to N2-fixing filaments, using the cysteine-specific selective ICAT methodology. The data set defined a good quantitative accuracy of the ICAT reagent in complex protein samples. The relative abundance levels of 511 proteins were determined and 74% showed a cell-type specific differential abundance. The majority of the identified proteins have not previously been quantified at the cell-type specific level. We have in addition analyzed the cell-type specific differential abundance of a large section of proteins quantified in both newly formed and steady-state diazotrophic cultures in N. punctiforme. The results describe a wide distribution of members of the putative redox regulated Cys-proteome in the central metabolism of both vegetative cells and heterocysts of N. punctiforme. The data set broadens our understanding of heterocysts and describes novel proteins involved in heterocyst physiology, including signaling and regulatory proteins as well as a large number of proteins with unknown function. Significant differences in cell-type specific abundance levels were present in the cell-type specific proteomes of newly formed diazotrophic filaments as compared to steady-state cultures. Therefore we conclude that by using our approach we are able to analyze a synchronized fraction of newly formed heterocysts, which enabled a better detection of proteins involved in the heterocyst specific physiology.

Characterisation of the Manduca sexta sperm proteome: Genetic novelty underlying sperm composition in Lepidoptera.

PubMed

Whittington, Emma; Zhao, Qian; Borziak, Kirill; Walters, James R; Dorus, Steve

2015-07-01

The application of mass spectrometry based proteomics to sperm biology has greatly accelerated progress in understanding the molecular composition and function of spermatozoa. To date, these approaches have been largely restricted to model organisms, all of which produce a single sperm morph capable of oocyte fertilisation. Here we apply high-throughput mass spectrometry proteomic analysis to characterise sperm composition in Manduca sexta, the tobacco hornworm moth, which produce heteromorphic sperm, including one fertilisation competent (eupyrene) and one incompetent (apyrene) sperm type. This resulted in the high confidence identification of 896 proteins from a co-mixed sample of both sperm types, of which 167 are encoded by genes with strict one-to-one orthology in Drosophila melanogaster. Importantly, over half (55.1%) of these orthologous proteins have previously been identified in the D. melanogaster sperm proteome and exhibit significant conservation in quantitative protein abundance in sperm between the two species. Despite the complex nature of gene expression across spermatogenic stages, a significant correlation was also observed between sperm protein abundance and testis gene expression. Lepidopteran-specific sperm proteins (e.g., proteins with no homology to proteins in non-Lepidopteran taxa) were present in significantly greater abundance on average than those with homology outside the Lepidoptera. Given the disproportionate production of apyrene sperm (96% of all mature sperm in Manduca) relative to eupyrene sperm, these evolutionarily novel and highly abundant proteins are candidates for possessing apyrene-specific functions. Lastly, comparative genomic analyses of testis-expressed, ovary-expressed and sperm genes identified a concentration of novel sperm proteins shared amongst Lepidoptera of potential relevance to the evolutionary origin of heteromorphic spermatogenesis. As the first published Lepidopteran sperm proteome, this whole-cell proteomic characterisation will facilitate future evolutionary genetic and developmental studies of heteromorphic sperm production and parasperm function. Furthermore, the analyses presented here provide useful annotation information regarding sex-biased gene expression, novel Lepidopteran genes and gene function in the male gamete to complement the newly sequenced and annotated Manduca genome. Copyright © 2015 Elsevier Ltd. All rights reserved.

Proteomics of industrial fungi: trends and insights for biotechnology.

PubMed

de Oliveira, José Miguel P Ferreira; de Graaff, Leo H

2011-01-01

Filamentous fungi are widely known for their industrial applications, namely, the production of food-processing enzymes and metabolites such as antibiotics and organic acids. In the past decade, the full genome sequencing of filamentous fungi increased the potential to predict encoded proteins enormously, namely, hydrolytic enzymes or proteins involved in the biosynthesis of metabolites of interest. The integration of genome sequence information with possible phenotypes requires, however, the knowledge of all the proteins in the cell in a system-wise manner, given by proteomics. This review summarises the progress of proteomics and its importance for the study of biotechnological processes in filamentous fungi. A major step forward in proteomics was to couple protein separation with high-resolution mass spectrometry, allowing accurate protein quantification. Despite the fact that most fungal proteomic studies have been focused on proteins from mycelial extracts, many proteins are related to processes which are compartmentalised in the fungal cell, e.g. β-lactam antibiotic production in the microbody. For the study of such processes, a targeted approach is required, e.g. by organelle proteomics. Typical workflows for sample preparation in fungal organelle proteomics are discussed, including homogenisation and sub-cellular fractionation. Finally, examples are presented of fungal organelle proteomic studies, which have enlarged the knowledge on areas of interest to biotechnology, such as protein secretion, energy production or antibiotic biosynthesis.

Proteomics of industrial fungi: trends and insights for biotechnology

PubMed Central

de Oliveira, José Miguel P. Ferreira

2010-01-01

Filamentous fungi are widely known for their industrial applications, namely, the production of food-processing enzymes and metabolites such as antibiotics and organic acids. In the past decade, the full genome sequencing of filamentous fungi increased the potential to predict encoded proteins enormously, namely, hydrolytic enzymes or proteins involved in the biosynthesis of metabolites of interest. The integration of genome sequence information with possible phenotypes requires, however, the knowledge of all the proteins in the cell in a system-wise manner, given by proteomics. This review summarises the progress of proteomics and its importance for the study of biotechnological processes in filamentous fungi. A major step forward in proteomics was to couple protein separation with high-resolution mass spectrometry, allowing accurate protein quantification. Despite the fact that most fungal proteomic studies have been focused on proteins from mycelial extracts, many proteins are related to processes which are compartmentalised in the fungal cell, e.g. β-lactam antibiotic production in the microbody. For the study of such processes, a targeted approach is required, e.g. by organelle proteomics. Typical workflows for sample preparation in fungal organelle proteomics are discussed, including homogenisation and sub-cellular fractionation. Finally, examples are presented of fungal organelle proteomic studies, which have enlarged the knowledge on areas of interest to biotechnology, such as protein secretion, energy production or antibiotic biosynthesis. PMID:20922379

Proteomics and circadian rhythms: It’s all about signaling!

PubMed Central

Mauvoisin, Daniel; Dayon, Loïc; Gachon, Frédéric; Kussmann, Martin

2014-01-01

1. Abstract Proteomic technologies using mass spectrometry (MS) offer new perspectives in circadian biology, in particular the possibility to study posttranslational modifications (PTMs). To date, only very few studies have been carried out to decipher the rhythmicity of protein expression in mammals with large-scale proteomics. Although signaling has been shown to be of high relevance, comprehensive characterization studies of PTMs are even more rare. This review aims at describing the actual landscape of circadian proteomics and the opportunities and challenges appearing on the horizon. Emphasis was given to signaling processes for their role in metabolic heath as regulated by circadian clocks and environmental factors. Those signaling processes are expected to be better and more deeply characterized in the coming years with proteomics. PMID:25103677

Lime powder treatment reduces urinary excretion of total protein and transferrin but increases uromodulin excretion in patients with urolithiasis.

PubMed

Tosukhowong, Piyaratana; Kulpradit, Pimsuda; Chaiyarit, Sakdithep; Ungjareonwattana, Wattanachai; Kalpongnukul, Nuttiya; Ratchanon, Supoj; Thongboonkerd, Visith

2018-06-01

Our previous study has shown that lime powder (LP) had an inhibitory effect against calcium oxalate stone formation. However, the precise mechanisms underlying such beneficial effect remained unclear. Our present study thus aimed to address the effect of LP on excretory level and compositions of urinary proteins using a proteomics approach. From a total of 80 calcium oxalate stone formers recruited into our 2-year randomized clinical trial of LP effect, 10 patients with comparable age and clinical parameters were selected for this proteomic study. 24-h urine specimens were collected from all subjects, at baseline (before) and after LP treatment for 6 months, and then subjected to quantitative proteomics analysis and subsequent validation by ELISA. Total urinary protein excretion was significantly decreased by LP treatment, but unaffected by placebo. Nanoflow liquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS) followed by quantitative analysis revealed 17 proteins whose levels were significantly altered (16 decreased and 1 increased) exclusively by LP treatment. Among these, the decrease of transferrin and increase of uromodulin were validated by ELISA. Moreover, there was a significant correlation between microalbuminuria and urinary transferrin level by Pearson's correlation test. In summary, LP treatment caused significant reduction in total urinary protein excretion and changes in urinary protein compositions that could be linked to stone inhibitory effects and might be relevant mechanisms responsible for the beneficial effects of LP to prevent kidney stone formation and recurrence.

Quantitative Proteomic Analysis Reveals Populus cathayana Females Are More Sensitive and Respond More Sophisticatedly to Iron Deficiency than Males.

PubMed

Zhang, Sheng; Zhang, Yunxiang; Cao, Yanchun; Lei, Yanbao; Jiang, Hao

2016-03-04

Previous studies have shown that there are significant sexual differences in the morphological and physiological responses of Populus cathayana Rehder to nitrogen and phosphorus deficiencies, but little is known about the sex-specific differences in responses to iron deficiency. In this study, the effects of iron deficiency on the morphology, physiology, and proteome of P. cathayana males and females were investigated. The results showed that iron deficiency (25 days) significantly decreased height growth, photosynthetic rate, chlorophyll content, and tissue iron concentration in both sexes. A comparison between the sexes indicated that iron-deficient males had less height inhibition and photosynthesis system II or chloroplast ultrastructural damage than iron-deficient females. iTRAQ-based quantitative proteomic analysis revealed that 144 and 68 proteins were decreased in abundance (e.g., proteins involved in photosynthesis, carbohydrate and energy metabolism, and gene expression regulation) and 78 and 39 proteins were increased in abundance (e.g., proteins involved in amino acid metabolism and stress response) according to the criterion of ratio ≥1.5 in females and males, respectively. A comparison between the sexes indicated that iron-deficient females exhibited a greater change in the proteins involved in photosynthesis, carbon and energy metabolism, the redox system, and stress responsive proteins. This study reveals females are more sensitive and have a more sophisticated response to iron deficiency compared with males and provides new insights into differential sexual responses to nutrient deficiency.

Time-course human urine proteomics in space-flight simulation experiments.

PubMed

Binder, Hans; Wirth, Henry; Arakelyan, Arsen; Lembcke, Kathrin; Tiys, Evgeny S; Ivanisenko, Vladimir A; Kolchanov, Nikolay A; Kononikhin, Alexey; Popov, Igor; Nikolaev, Evgeny N; Pastushkova, Lyudmila; Larina, Irina M

2014-01-01

Long-term space travel simulation experiments enabled to discover different aspects of human metabolism such as the complexity of NaCl salt balance. Detailed proteomics data were collected during the Mars105 isolation experiment enabling a deeper insight into the molecular processes involved. We studied the abundance of about two thousand proteins extracted from urine samples of six volunteers collected weekly during a 105-day isolation experiment under controlled dietary conditions including progressive reduction of salt consumption. Machine learning using Self Organizing maps (SOM) in combination with different analysis tools was applied to describe the time trajectories of protein abundance in urine. The method enables a personalized and intuitive view on the physiological state of the volunteers. The abundance of more than one half of the proteins measured clearly changes in the course of the experiment. The trajectory splits roughly into three time ranges, an early (week 1-6), an intermediate (week 7-11) and a late one (week 12-15). Regulatory modes associated with distinct biological processes were identified using previous knowledge by applying enrichment and pathway flow analysis. Early protein activation modes can be related to immune response and inflammatory processes, activation at intermediate times to developmental and proliferative processes and late activations to stress and responses to chemicals. The protein abundance profiles support previous results about alternative mechanisms of salt storage in an osmotically inactive form. We hypothesize that reduced NaCl consumption of about 6 g/day presumably will reduce or even prevent the activation of inflammatory processes observed in the early time range of isolation. SOM machine learning in combination with analysis methods of class discovery and functional annotation enable the straightforward analysis of complex proteomics data sets generated by means of mass spectrometry.

A Proteomic and Lipidomic Characterization of Bradyrhizobium diazoefficiens Membranes Under Microaerobic Conditions

NASA Astrophysics Data System (ADS)

Tookmanian, E. M.; Neubauer, C.; Newman, D. K.

2016-12-01

Hopanoids are a class of sterol-like molecules found in modern bacterial membranes. Remarkably, they can leave behind carbon skeletons (hopanes) that persist for millions of years. Previously, hopanes were thought to be biomarkers of cyanobacteria, and thus, indirectly, the evolution of oxygen. As our understanding of the biosynthetic pathway of hopanoids has improved, we have learned that oxygen is not required for hopanoid biosynthesis and that many different bacteria have the genetic potential to synthesize hopanoids. These facts motivate a deeper understanding of the distribution and role(s) of hopanoids in bacteria. Bioinformatic approaches revealed that a subgroup of bacteria that synthesize hopanoids have symbiotic relationships with plants. These symbioses often take the form of root nodules, which have a unique microenvironment including microaerobic conditions to promote nitrogen fixation. We utilized the legume symbiont Bradyrhizobium diazoefficiens to investigate the molecular composition of membranes through lipidomic and proteomic studies. A B. diazoefficiens mutant lacking the C-2 hopanoid methylase (ΔhpnP) was previously shown to have a growth defect compared to wildtype under microaerobic conditions, whereas a mutant unable to synthesize C35 hopanoids (ΔhpnH) failed to grow entirely. Because these different hopanoid classes impact the fitness of this organism under nodule-like growth conditions, we sought to determine how these classes affect the rest of the membrane. Here, we present how the presence or absence of specific hopanoid classes alters the membrane proteome and lipidome of B. diazoefficiens; this information provides clues regarding their cellular function. By constraining the roles hopanoids play in modern niches, we hope to identify conserved biochemical functions that will advance our interpretations of the hopane rock record.

Quantitative Clinical Chemistry Proteomics (qCCP) using mass spectrometry: general characteristics and application.

PubMed

Lehmann, Sylvain; Hoofnagle, Andrew; Hochstrasser, Denis; Brede, Cato; Glueckmann, Matthias; Cocho, José A; Ceglarek, Uta; Lenz, Christof; Vialaret, Jérôme; Scherl, Alexander; Hirtz, Christophe

2013-05-01

Proteomics studies typically aim to exhaustively detect peptides/proteins in a given biological sample. Over the past decade, the number of publications using proteomics methodologies has exploded. This was made possible due to the availability of high-quality genomic data and many technological advances in the fields of microfluidics and mass spectrometry. Proteomics in biomedical research was initially used in 'functional' studies for the identification of proteins involved in pathophysiological processes, complexes and networks. Improved sensitivity of instrumentation facilitated the analysis of even more complex sample types, including human biological fluids. It is at that point the field of clinical proteomics was born, and its fundamental aim was the discovery and (ideally) validation of biomarkers for the diagnosis, prognosis, or therapeutic monitoring of disease. Eventually, it was recognized that the technologies used in clinical proteomics studies [particularly liquid chromatography-tandem mass spectrometry (LC-MS/MS)] could represent an alternative to classical immunochemical assays. Prior to deploying MS in the measurement of peptides/proteins in the clinical laboratory, it seems likely that traditional proteomics workflows and data management systems will need to adapt to the clinical environment and meet in vitro diagnostic (IVD) regulatory constraints. This defines a new field, as reviewed in this article, that we have termed quantitative Clinical Chemistry Proteomics (qCCP).

Proteomics Analysis of the Nucleolus in Adenovirus-infected Cells

PubMed Central

Lam, Yun W.; Evans, Vanessa C.; Heesom, Kate J.; Lamond, Angus I.; Matthews, David A.

2010-01-01

Adenoviruses replicate primarily in the host cell nucleus, and it is well established that adenovirus infection affects the structure and function of host cell nucleoli in addition to coding for a number of nucleolar targeted viral proteins. Here we used unbiased proteomics methods, including high throughput mass spectrometry coupled with stable isotope labeling by amino acids in cell culture (SILAC) and traditional two-dimensional gel electrophoresis, to identify quantitative changes in the protein composition of the nucleolus during adenovirus infection. Two-dimensional gel analysis revealed changes in six proteins. By contrast, SILAC-based approaches identified 351 proteins with 24 proteins showing at least a 2-fold change after infection. Of those, four were previously reported to have aberrant localization and/or functional relevance during adenovirus infection. In total, 15 proteins identified as changing in amount by proteomics methods were examined in infected cells using confocal microscopy. Eleven of these proteins showed altered patterns of localization in adenovirus-infected cells. Comparing our data with the effects of actinomycin D on the nucleolar proteome revealed that adenovirus infection apparently specifically targets a relatively small subset of nucleolar antigens at the time point examined. PMID:19812395

Proteomics analysis of the nucleolus in adenovirus-infected cells.

PubMed

Lam, Yun W; Evans, Vanessa C; Heesom, Kate J; Lamond, Angus I; Matthews, David A

2010-01-01

Adenoviruses replicate primarily in the host cell nucleus, and it is well established that adenovirus infection affects the structure and function of host cell nucleoli in addition to coding for a number of nucleolar targeted viral proteins. Here we used unbiased proteomics methods, including high throughput mass spectrometry coupled with stable isotope labeling by amino acids in cell culture (SILAC) and traditional two-dimensional gel electrophoresis, to identify quantitative changes in the protein composition of the nucleolus during adenovirus infection. Two-dimensional gel analysis revealed changes in six proteins. By contrast, SILAC-based approaches identified 351 proteins with 24 proteins showing at least a 2-fold change after infection. Of those, four were previously reported to have aberrant localization and/or functional relevance during adenovirus infection. In total, 15 proteins identified as changing in amount by proteomics methods were examined in infected cells using confocal microscopy. Eleven of these proteins showed altered patterns of localization in adenovirus-infected cells. Comparing our data with the effects of actinomycin D on the nucleolar proteome revealed that adenovirus infection apparently specifically targets a relatively small subset of nucleolar antigens at the time point examined.

Quantitative Analysis of Proteome Modulations in Alveolar Epithelial Type II Cells in Response to Pulmonary Aspergillus fumigatus Infection.

PubMed

Seddigh, Pegah; Bracht, Thilo; Molinier-Frenkel, Válerie; Castellano, Flavia; Kniemeyer, Olaf; Schuster, Marc; Weski, Juliane; Hasenberg, Anja; Kraus, Andreas; Poschet, Gernot; Hager, Thomas; Theegarten, Dirk; Opitz, Christiane A; Brakhage, Axel A; Sitek, Barbara; Hasenberg, Mike; Gunzer, Matthias

2017-12-01

The ubiquitous mold Aspergillus fumigatus threatens immunosuppressed patients as inducer of lethal invasive aspergillosis. A. fumigatus conidia are airborne and reach the alveoli, where they encounter alveolar epithelial cells (AEC). Previous studies reported the importance of the surfactant-producing AEC II during A. fumigatus infection via in vitro experiments using cell lines. We established a negative isolation protocol yielding untouched primary murine AEC II with a purity >90%, allowing ex vivo analyses of the cells, which encountered the mold in vivo By label-free proteome analysis of AEC II isolated from mice 24h after A. fumigatus or mock infection we quantified 2256 proteins and found 154 proteins to be significantly differentially abundant between both groups (ANOVA p value ≤ 0.01, ratio of means ≥1.5 or ≤0.67, quantified with ≥2 peptides). Most of these proteins were higher abundant in the infected condition and reflected a comprehensive activation of AEC II on interaction with A. fumigatus This was especially represented by proteins related to oxidative phosphorylation, hence energy production. However, the most strongly induced protein was the l-amino acid oxidase (LAAO) Interleukin 4 induced 1 (IL4I1) with a 42.9 fold higher abundance (ANOVA p value 2.91 -10 ). IL4I1 has previously been found in B cells, macrophages, dendritic cells and rare neurons. Increased IL4I1 abundance in AEC II was confirmed by qPCR, Western blot and immunohistology. Furthermore, A. fumigatus infected lungs showed high levels of IL4I1 metabolic products. Importantly, higher IL4I1 abundance was also confirmed in lung tissue from human aspergilloma. Because LAAO are key enzymes for bactericidal product generation, AEC II might actively participate in pathogen defense. We provide insights into proteome changes of primary AEC II thereby opening new avenues to analyze the molecular changes of this central lung cell on infectious threats. Data are available via ProteomeXchange with identifier PXD005834. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Unique proteomic signature for radiation sensitive patients; a comparative study between normo-sensitive and radiation sensitive breast cancer patients.

PubMed

Skiöld, Sara; Azimzadeh, Omid; Merl-Pham, Juliane; Naslund, Ingemar; Wersall, Peter; Lidbrink, Elisabet; Tapio, Soile; Harms-Ringdahl, Mats; Haghdoost, Siamak

2015-06-01

Radiation therapy is a cornerstone of modern cancer treatment. Understanding the mechanisms behind normal tissue sensitivity is essential in order to minimize adverse side effects and yet to prevent local cancer reoccurrence. The aim of this study was to identify biomarkers of radiation sensitivity to enable personalized cancer treatment. To investigate the mechanisms behind radiation sensitivity a pilot study was made where eight radiation-sensitive and nine normo-sensitive patients were selected from a cohort of 2914 breast cancer patients, based on acute tissue reactions after radiation therapy. Whole blood was sampled and irradiated in vitro with 0, 1, or 150 mGy followed by 3 h incubation at 37°C. The leukocytes of the two groups were isolated, pooled and protein expression profiles were investigated using isotope-coded protein labeling method (ICPL). First, leukocytes from the in vitro irradiated whole blood from normo-sensitive and extremely sensitive patients were compared to the non-irradiated controls. To validate this first study a second ICPL analysis comparing only the non-irradiated samples was conducted. Both approaches showed unique proteomic signatures separating the two groups at the basal level and after doses of 1 and 150 mGy. Pathway analyses of both proteomic approaches suggest that oxidative stress response, coagulation properties and acute phase response are hallmarks of radiation sensitivity supporting our previous study on oxidative stress response. This investigation provides unique characteristics of radiation sensitivity essential for individualized radiation therapy. Copyright © 2015 Elsevier B.V. All rights reserved.

Genomics and proteomics in liver fibrosis and cirrhosis

PubMed Central

2012-01-01

Genomics and proteomics have become increasingly important in biomedical science in the past decade, as they provide an opportunity for hypothesis-free experiments that can yield major insights not previously foreseen when scientific and clinical questions are based only on hypothesis-driven approaches. Use of these tools, therefore, opens new avenues for uncovering physiological and pathological pathways. Liver fibrosis is a complex disease provoked by a range of chronic injuries to the liver, among which are viral hepatitis, (non-) alcoholic steatohepatitis and autoimmune disorders. Some chronic liver patients will never develop fibrosis or cirrhosis, whereas others rapidly progress towards cirrhosis in a few years. This variety can be caused by disease-related factors (for example, viral genotype) or host-factors (genetic/epigenetic). It is vital to establish accurate tools to identify those patients at highest risk for disease severity or progression in order to determine who are in need of immediate therapies. Moreover, there is an urgent imperative to identify non-invasive markers that can accurately distinguish mild and intermediate stages of fibrosis. Ideally, biomarkers can be used to predict disease progression and treatment response, but these studies will take many years due to the requirement for lengthy follow-up periods to assess outcomes. Current genomic and proteomic research provides many candidate biomarkers, but independent validation of these biomarkers is lacking, and reproducibility is still a key concern. Thus, great opportunities and challenges lie ahead in the field of genomics and proteomics, which, if successful, could transform the diagnosis and treatment of chronic fibrosing liver diseases. PMID:22214245

Proteomic analysis of corneal endothelial cell-descemet membrane tissues reveals influence of insulin dependence and disease severity in type 2 diabetes mellitus.

PubMed

Skeie, Jessica M; Aldrich, Benjamin T; Goldstein, Andrew S; Schmidt, Gregory A; Reed, Cynthia R; Greiner, Mark A

2018-01-01

The objective of this study was to characterize the proteome of the corneal endothelial cell layer and its basement membrane (Descemet membrane) in humans with various severities of type II diabetes mellitus compared to controls, and identify differentially expressed proteins across a range of diabetic disease severities that may influence corneal endothelial cell health. Endothelium-Descemet membrane complex tissues were peeled from transplant suitable donor corneas. Protein fractions were isolated from each sample and subjected to multidimensional liquid chromatography and tandem mass spectrometry. Peptide spectra were matched to the human proteome, assigned gene ontology, and grouped into protein signaling pathways unique to each of the disease states. We identified an average of 12,472 unique proteins in each of the endothelium-Descemet membrane complex tissue samples. There were 2,409 differentially expressed protein isoforms that included previously known risk factors for type II diabetes mellitus related to metabolic processes, oxidative stress, and inflammation. Gene ontology analysis demonstrated that diabetes progression has many protein footprints related to metabolic processes, binding, and catalysis. The most represented pathways involved in diabetes progression included mitochondrial dysfunction, cell-cell junction structure, and protein synthesis regulation. This proteomic dataset identifies novel corneal endothelial cell and Descemet membrane protein expression in various stages of diabetic disease. These findings give insight into the mechanisms involved in diabetes progression relevant to the corneal endothelium and its basement membrane, prioritize new pathways for therapeutic targeting, and provide insight into potential biomarkers for determining the health of this tissue.

Linking the proteins--elucidation of proteome-scale networks using mass spectrometry.

PubMed

Pflieger, Delphine; Gonnet, Florence; de la Fuente van Bentem, Sergio; Hirt, Heribert; de la Fuente, Alberto

2011-01-01

Proteomes are intricate. Typically, thousands of proteins interact through physical association and post-translational modifications (PTMs) to give rise to the emergent functions of cells. Understanding these functions requires one to study proteomes as "systems" rather than collections of individual protein molecules. The abstraction of the interacting proteome to "protein networks" has recently gained much attention, as networks are effective representations, that lose specific molecular details, but provide the ability to see the proteome as a whole. Mostly two aspects of the proteome have been represented by network models: proteome-wide physical protein-protein-binding interactions organized into Protein Interaction Networks (PINs), and proteome-wide PTM relations organized into Protein Signaling Networks (PSNs). Mass spectrometry (MS) techniques have been shown to be essential to reveal both of these aspects on a proteome-wide scale. Techniques such as affinity purification followed by MS have been used to elucidate protein-protein interactions, and MS-based quantitative phosphoproteomics is critical to understand the structure and dynamics of signaling through the proteome. We here review the current state-of-the-art MS-based analytical pipelines for the purpose to characterize proteome-scale networks. Copyright © 2010 Wiley Periodicals, Inc.

Pre-fractionation strategies to resolve pea (Pisum sativum) sub-proteomes

PubMed Central

Meisrimler, Claudia-Nicole; Menckhoff, Ljiljana; Kukavica, Biljana M.; Lüthje, Sabine

2015-01-01

Legumes are important crop plants and pea (Pisum sativum L.) has been investigated as a model with respect to several physiological aspects. The sequencing of the pea genome has not been completed. Therefore, proteomic approaches are currently limited. Nevertheless, the increasing numbers of available EST-databases as well as the high homology of the pea and medicago genome (Medicago truncatula Gaertner) allow the successful identification of proteins. Due to the un-sequenced pea genome, pre-fractionation approaches have been used in pea proteomic surveys in the past. Aside from a number of selective proteome studies on crude extracts and the chloroplast, few studies have targeted other components such as the pea secretome, an important sub-proteome of interest due to its role in abiotic and biotic stress processes. The secretome itself can be further divided into different sub-proteomes (plasma membrane, apoplast, cell wall proteins). Cell fractionation in combination with different gel-electrophoresis, chromatography methods and protein identification by mass spectrometry are important partners to gain insight into pea sub-proteomes, post-translational modifications and protein functions. Overall, pea proteomics needs to link numerous existing physiological and biochemical data to gain further insight into adaptation processes, which play important roles in field applications. Future developments and directions in pea proteomics are discussed. PMID:26539198

A Proteomics View of the Molecular Mechanisms and Biomarkers of Glaucomatous Neurodegeneration

PubMed Central

Tezel, Gülgün

2013-01-01

Despite improving understanding of glaucoma, key molecular players of neurodegeneration that can be targeted for treatment of glaucoma, or molecular biomarkers that can be useful for clinical testing, remain unclear. Proteomics technology offers a powerful toolbox to accomplish these important goals of the glaucoma research and is increasingly being applied to identify molecular mechanisms and biomarkers of glaucoma. Recent studies of glaucoma using proteomics analysis techniques have resulted in the lists of differentially expressed proteins in human glaucoma and animal models. The global analysis of protein expression in glaucoma has been followed by cell-specific proteome analysis of retinal ganglion cells and astrocytes. The proteomics data have also guided targeted studies to identify post-translational modifications and protein-protein interactions during glaucomatous neurodegeneration. In addition, recent applications of proteomics have provided a number of potential biomarker candidates. Proteomics technology holds great promise to move glaucoma research forward toward new treatment strategies and biomarker discovery. By reviewing the major proteomics approaches and their applications in the field of glaucoma, this article highlights the power of proteomics in translational and clinical research related to glaucoma and also provides a framework for future research to functionally test the importance of specific molecular pathways and validate candidate biomarkers. PMID:23396249

Neural Stem Cells (NSCs) and Proteomics.

PubMed

Shoemaker, Lorelei D; Kornblum, Harley I

2016-02-01

Neural stem cells (NSCs) can self-renew and give rise to the major cell types of the CNS. Studies of NSCs include the investigation of primary, CNS-derived cells as well as animal and human embryonic stem cell (ESC)-derived and induced pluripotent stem cell (iPSC)-derived sources. NSCs provide a means with which to study normal neural development, neurodegeneration, and neurological disease and are clinically relevant sources for cellular repair to the damaged and diseased CNS. Proteomics studies of NSCs have the potential to delineate molecules and pathways critical for NSC biology and the means by which NSCs can participate in neural repair. In this review, we provide a background to NSC biology, including the means to obtain them and the caveats to these processes. We then focus on advances in the proteomic interrogation of NSCs. This includes the analysis of posttranslational modifications (PTMs); approaches to analyzing different proteomic compartments, such the secretome; as well as approaches to analyzing temporal differences in the proteome to elucidate mechanisms of differentiation. We also discuss some of the methods that will undoubtedly be useful in the investigation of NSCs but which have not yet been applied to the field. While many proteomics studies of NSCs have largely catalogued the proteome or posttranslational modifications of specific cellular states, without delving into specific functions, some have led to understandings of functional processes or identified markers that could not have been identified via other means. Many challenges remain in the field, including the precise identification and standardization of NSCs used for proteomic analyses, as well as how to translate fundamental proteomics studies to functional biology. The next level of investigation will require interdisciplinary approaches, combining the skills of those interested in the biochemistry of proteomics with those interested in modulating NSC function. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Screening Novel Molecular Targets of Metformin in Breast Cancer by Proteomic Approach

PubMed Central

Al-Zaidan, Lobna; El Ruz, Rasha Abu; Malki, Ahmed M.

2017-01-01

Metformin is a commonly prescribed antihyperglycemic drug, and has been investigated in vivo and in vitro for its effect to improve the comorbidity of diabetes and various types of cancers. Several studies investigated the therapeutic mechanisms of metformin on cancer cells, but the exact mechanism of metformin’s effect on the proteomic pathways of cancer cells is yet to be further investigated. The main objective of our research line is to discover safe and alternative therapeutic options for breast cancer, we aimed in this study to design a novel “bottom up proteomics workflow” in which proteins were first broken into peptides to reveal their identity, then the proteomes were precisely evaluated using spectrometry analysis. In our study, metformin suppressed cell proliferation and induced apoptosis in human breast carcinoma cell line MCF-7 with minimal toxicity to normal breast epithelial cells MCF-10. Metformin induced apoptosis by arresting cells in G1 phase as evaluated by flow cytometric analysis. Moreover, The G1 phase arrest for the MCF-7 has been confirmed by increased expression levels of p21 and reduction in cyclin D1 level. Additionally, metformin increased the expression levels of p53, Bax, Bad while it reduced expression levels of Akt, Bcl-2, and Mdm2. The study employed a serviceable strategy that investigates metformin-dependent changes in the proteome using a literature-derived network. The protein extracts of the treated and untreated cell lines were analyzed employing proteomic approaches; the findings conveyed a proposed mechanism of the effectual tactics of metformin on breast cancer cells. Metformin proposed an antibreast cancer effect through the examination of the proteomic pathways upon the MCF-7 and MCF-10A exposure to the drug. Our findings proposed prolific proteomic changes that revealed the therapeutic mechanisms of metformin on breast cancer cells upon their exposure. In conclusion, the reported proteomic pathways lead to increase the understanding of breast cancer prognosis and permit future studies to examine the effect of metformin on the proteomic pathways against other types of cancers. Finally, it suggests the possibility to develop further therapeutic generations of metformin with increased anticancer effect through targeting specific proteomes. PMID:29085821

Proteomics: a new approach to the study of disease.

PubMed

Chambers, G; Lawrie, L; Cash, P; Murray, G I

2000-11-01

The global analysis of cellular proteins has recently been termed proteomics and is a key area of research that is developing in the post-genome era. Proteomics uses a combination of sophisticated techniques including two-dimensional (2D) gel electrophoresis, image analysis, mass spectrometry, amino acid sequencing, and bio-informatics to resolve comprehensively, to quantify, and to characterize proteins. The application of proteomics provides major opportunities to elucidate disease mechanisms and to identify new diagnostic markers and therapeutic targets. This review aims to explain briefly the background to proteomics and then to outline proteomic techniques. Applications to the study of human disease conditions ranging from cancer to infectious diseases are reviewed. Finally, possible future advances are briefly considered, especially those which may lead to faster sample throughput and increased sensitivity for the detection of individual proteins. Copyright 2000 John Wiley & Sons, Ltd.

Bioinformatic analysis of the nucleolus.

PubMed

Leung, Anthony K L; Andersen, Jens S; Mann, Matthias; Lamond, Angus I

2003-12-15

The nucleolus is a plurifunctional, nuclear organelle, which is responsible for ribosome biogenesis and many other functions in eukaryotes, including RNA processing, viral replication and tumour suppression. Our knowledge of the human nucleolar proteome has been expanded dramatically by the two recent MS studies on isolated nucleoli from HeLa cells [Andersen, Lyon, Fox, Leung, Lam, Steen, Mann and Lamond (2002) Curr. Biol. 12, 1-11; Scherl, Coute, Deon, Calle, Kindbeiter, Sanchez, Greco, Hochstrasser and Diaz (2002) Mol. Biol. Cell 13, 4100-4109]. Nearly 400 proteins were identified within the nucleolar proteome so far in humans. Approx. 12% of the identified proteins were previously shown to be nucleolar in human cells and, as expected, nearly all of the known housekeeping proteins required for ribosome biogenesis were identified in these analyses. Surprisingly, approx. 30% represented either novel or uncharacterized proteins. This review focuses on how to apply the derived knowledge of this newly recognized nucleolar proteome, such as their amino acid/peptide composition and their homologies across species, to explore the function and dynamics of the nucleolus, and suggests ways to identify, in silico, possible functions of the novel/uncharacterized proteins and potential interaction networks within the human nucleolus, or between the nucleolus and other nuclear organelles, by drawing resources from the public domain.

Hydrogen Sulfide and Reactive Sulfur Species Impact Proteome S-Sulfhydration and Global Virulence Regulation in Staphylococcus aureus.

PubMed

Peng, Hui; Zhang, Yixiang; Palmer, Lauren D; Kehl-Fie, Thomas E; Skaar, Eric P; Trinidad, Jonathan C; Giedroc, David P

2017-10-13

Hydrogen sulfide (H 2 S) is thought to protect bacteria from oxidative stress, but a comprehensive understanding of its function in bacteria is largely unexplored. In this study, we show that the human pathogen Staphylococcus aureus (S. aureus) harbors significant effector molecules of H 2 S signaling, reactive sulfur species (RSS), as low molecular weight persulfides of bacillithiol, coenzyme A, and cysteine, and significant inorganic polysulfide species. We find that proteome S-sulfhydration, a post-translational modification (PTM) in H 2 S signaling, is widespread in S. aureus. RSS levels modulate the expression of secreted virulence factors and the cytotoxicity of the secretome, consistent with an S-sulfhydration-dependent inhibition of DNA binding by MgrA, a global virulence regulator. Two previously uncharacterized thioredoxin-like proteins, denoted TrxP and TrxQ, are S-sulfhydrated in sulfide-stressed cells and are capable of reducing protein hydrodisulfides, suggesting that this PTM is potentially regulatory in S. aureus. In conclusion, our results reveal that S. aureus harbors a pool of proteome- and metabolite-derived RSS capable of impacting protein activities and gene regulation and that H 2 S signaling can be sensed by global regulators to affect the expression of virulence factors.

A novel spectral library workflow to enhance protein identifications.

PubMed

Li, Haomin; Zong, Nobel C; Liang, Xiangbo; Kim, Allen K; Choi, Jeong Ho; Deng, Ning; Zelaya, Ivette; Lam, Maggie; Duan, Huilong; Ping, Peipei

2013-04-09

The innovations in mass spectrometry-based investigations in proteome biology enable systematic characterization of molecular details in pathophysiological phenotypes. However, the process of delineating large-scale raw proteomic datasets into a biological context requires high-throughput data acquisition and processing. A spectral library search engine makes use of previously annotated experimental spectra as references for subsequent spectral analyses. This workflow delivers many advantages, including elevated analytical efficiency and specificity as well as reduced demands in computational capacity. In this study, we created a spectral matching engine to address challenges commonly associated with a library search workflow. Particularly, an improved sliding dot product algorithm, that is robust to systematic drifts of mass measurement in spectra, is introduced. Furthermore, a noise management protocol distinguishes spectra correlation attributed from noise and peptide fragments. It enables elevated separation between target spectral matches and false matches, thereby suppressing the possibility of propagating inaccurate peptide annotations from library spectra to query spectra. Moreover, preservation of original spectra also accommodates user contributions to further enhance the quality of the library. Collectively, this search engine supports reproducible data analyses using curated references, thereby broadening the accessibility of proteomics resources to biomedical investigators. This article is part of a Special Issue entitled: From protein structures to clinical applications. Copyright © 2013 Elsevier B.V. All rights reserved.

Differential quantitative proteomics of Porphyromonas gingivalis by linear ion trap mass spectrometry: non-label methods comparison, q-values and LOWESS curve fitting

PubMed Central

Xia, Qiangwei; Wang, Tiansong; Park, Yoonsuk; Lamont, Richard J.; Hackett, Murray

2009-01-01

Differential analysis of whole cell proteomes by mass spectrometry has largely been applied using various forms of stable isotope labeling. While metabolic stable isotope labeling has been the method of choice, it is often not possible to apply such an approach. Four different label free ways of calculating expression ratios in a classic “two-state” experiment are compared: signal intensity at the peptide level, signal intensity at the protein level, spectral counting at the peptide level, and spectral counting at the protein level. The quantitative data were mined from a dataset of 1245 qualitatively identified proteins, about 56% of the protein encoding open reading frames from Porphyromonas gingivalis, a Gram-negative intracellular pathogen being studied under extracellular and intracellular conditions. Two different control populations were compared against P. gingivalis internalized within a model human target cell line. The q-value statistic, a measure of false discovery rate previously applied to transcription microarrays, was applied to proteomics data. For spectral counting, the most logically consistent estimate of random error came from applying the locally weighted scatter plot smoothing procedure (LOWESS) to the most extreme ratios generated from a control technical replicate, thus setting upper and lower bounds for the region of experimentally observed random error. PMID:19337574

The Succinated Proteome of FH-Mutant Tumours

PubMed Central

Yang, Ming; Ternette, Nicola; Su, Huizhong; Dabiri, Raliat; Kessler, Benedikt M.; Adam, Julie; Teh, Bin Tean; Pollard, Patrick J.

2014-01-01

Inherited mutations in the Krebs cycle enzyme fumarate hydratase (FH) predispose to hereditary leiomyomatosis and renal cell cancer (HLRCC). Loss of FH activity in HLRCC tumours causes accumulation of the Krebs cycle intermediate fumarate to high levels, which may act as an oncometabolite through various, but not necessarily mutually exclusive, mechanisms. One such mechanism, succination, is an irreversible non-enzymatic modification of cysteine residues by fumarate, to form S-(2-succino)cysteine (2SC). Previous studies have demonstrated that succination of proteins including glyceraldehyde 3-phosphate dehydrogenase (GAPDH), kelch-like ECH-associated protein 1 (KEAP1) and mitochondrial aconitase (ACO2) can have profound effects on cellular metabolism. Furthermore, immunostaining for 2SC is a sensitive and specific biomarker for HLRCC tumours. Here, we performed a proteomic screen on an FH-mutant tumour and two HLRCC-derived cancer cell lines and identified 60 proteins where one or more cysteine residues were succinated; 10 of which were succinated at cysteine residues either predicted, or experimentally proven, to be functionally significant. Bioinformatic enrichment analyses identified most succinated targets to be involved in redox signaling. To our knowledge, this is the first proteomic-based succination screen performed in human tumours and cancer-derived cells and has identified novel 2SC targets that may be relevant to the pathogenesis of HLRCC. PMID:25105836

Comparative Proteomics Provides Insights into Metabolic Responses in Rat Liver to Isolated Soy and Meat Proteins.

PubMed

Song, Shangxin; Hooiveld, Guido J; Zhang, Wei; Li, Mengjie; Zhao, Fan; Zhu, Jing; Xu, Xinglian; Muller, Michael; Li, Chunbao; Zhou, Guanghong

2016-04-01

It has been reported that isolated dietary soy and meat proteins have distinct effects on physiology and liver gene expression, but the impact on protein expression responses are unknown. Because these may differ from gene expression responses, we investigated dietary protein-induced changes in liver proteome. Rats were fed for 1 week semisynthetic diets that differed only regarding protein source; casein (reference) was fully replaced by isolated soy, chicken, fish, or pork protein. Changes in liver proteome were measured by iTRAQ labeling and LC-ESI-MS/MS. A robust set totaling 1437 unique proteins was identified and subjected to differential protein analysis and biological interpretation. Compared with casein, all other protein sources reduced the abundance of proteins involved in fatty acid metabolism and Pparα signaling pathway. All dietary proteins, except chicken, increased oxidoreductive transformation reactions but reduced energy and essential amino acid metabolic pathways. Only soy protein increased the metabolism of sulfur-containing and nonessential amino acids. Soy and fish proteins increased translation and mRNA processing, whereas only chicken protein increased TCA cycle but reduced immune responses. These findings were partially in line with previously reported transcriptome results. This study further shows the distinct effects of soy and meat proteins on liver metabolism in rats.

PNAC: a protein nucleolar association classifier

PubMed Central

2011-01-01

Background Although primarily known as the site of ribosome subunit production, the nucleolus is involved in numerous and diverse cellular processes. Recent large-scale proteomics projects have identified thousands of human proteins that associate with the nucleolus. However, in most cases, we know neither the fraction of each protein pool that is nucleolus-associated nor whether their association is permanent or conditional. Results To describe the dynamic localisation of proteins in the nucleolus, we investigated the extent of nucleolar association of proteins by first collating an extensively curated literature-derived dataset. This dataset then served to train a probabilistic predictor which integrates gene and protein characteristics. Unlike most previous experimental and computational studies of the nucleolar proteome that produce large static lists of nucleolar proteins regardless of their extent of nucleolar association, our predictor models the fluidity of the nucleolus by considering different classes of nucleolar-associated proteins. The new method predicts all human proteins as either nucleolar-enriched, nucleolar-nucleoplasmic, nucleolar-cytoplasmic or non-nucleolar. Leave-one-out cross validation tests reveal sensitivity values for these four classes ranging from 0.72 to 0.90 and positive predictive values ranging from 0.63 to 0.94. The overall accuracy of the classifier was measured to be 0.85 on an independent literature-based test set and 0.74 using a large independent quantitative proteomics dataset. While the three nucleolar-association groups display vastly different Gene Ontology biological process signatures and evolutionary characteristics, they collectively represent the most well characterised nucleolar functions. Conclusions Our proteome-wide classification of nucleolar association provides a novel representation of the dynamic content of the nucleolus. This model of nucleolar localisation thus increases the coverage while providing accurate and specific annotations of the nucleolar proteome. It will be instrumental in better understanding the central role of the nucleolus in the cell and its interaction with other subcellular compartments. PMID:21272300

Proteomic characterization of the subpellicular cytoskeleton of Toxoplasma gondii tachyzoites.

PubMed

Gómez de León, Carmen T; Díaz Martín, Rubén Darío; Mendoza Hernández, Guillermo; González Pozos, Sirenia; Ambrosio, Javier R; Mondragón Flores, Ricardo

2014-12-05

Toxoplasma, the causative agent of toxoplasmosis in animals and humans, has a subpellicular cytoskeleton that is involved in motility, cell shape and invasion. Knowledge of components of the cytoskeleton is necessary to understand the invasion mechanisms as well as for the identification of possible therapeutic targets. To date, most cytoskeletal components of Toxoplasma remain unidentified due mainly to the lack of reproducible methods for their isolation. Based on the successful isolation of the cytoskeleton, it was possible to report for the first time, the proteomic characterization of the subpellicular cytoskeleton of Toxoplasma formed by 95 cytoskeletal proteins through proteomic analysis by tandem mass spectrometry of one dimension SDS PAGE. By bioinformatic analysis of the data, proteins were classified as: 18 conventional cytoskeletal proteins; 10 inner membrane complex proteins, including 7 with alveolin repeats; 5 new proteins with alveolin like repeats; 37 proteins associated with other organelles and 25 novel proteins of unknown function. One of the alveolin like proteins not previously described in Toxoplasma named TgArticulin was partially characterized with a specific monoclonal antibody. Presence of TgArticulin was exclusively associated with the cytoskeleton fraction with a cortical distribution. Functions for the several molecules identified are proposed. This manuscript describes, for the first time, the proteome of the subpellicular cytoskeleton of Toxoplasma gondii. The importance of this study is related to the role of the cytoskeleton in the highly invasive capability of a parasite that causes abortion, blindness, and death by encephalitis in immunocompromised patients. Proteomic characterization of the cytoskeleton of T. gondii tachyzoites was possible by the development of a successful procedure for the isolation of the subpellicular cytoskeleton. Knowledge of the composition of the cytoskeleton of Toxoplasma is fundamental for the understanding of the motility and host cell invasion mechanisms, and for the future design and development of toxoplasmicidal drugs with effects against specific components of the cytoskeleton of this parasite that are absent in mammal host cells. Copyright © 2014 Elsevier B.V. All rights reserved.

iTRAQ-based proteome profiling of Saccharomyces cerevisiae and cryotolerant species Saccharomyces uvarum and Saccharomyces kudriavzevii during low-temperature wine fermentation.

PubMed

García-Ríos, Estéfani; Querol, Amparo; Guillamón, José Manuel

2016-09-02

Temperature is one of the most important parameters to affect the duration and rate of alcoholic fermentation and final wine quality. Some species of the Saccharomyces genus have shown better adaptation at low temperature than Saccharomyces cerevisiae, which was the case of cryotolerant yeasts Saccharomyces uvarum and Saccharomyces kudriavzevii. In an attempt to detect inter-specific metabolic differences, we characterized the proteomic landscape of these cryotolerant species grown at 12°C and 28°C, which we compared with the proteome of S. cerevisiae (poorly adapted at low temperature). Our results showed that the main differences among the proteomic profiling of the three Saccharomyces strains grown at 12°C and 28°C lay in translation, glycolysis and amino acid metabolism. Our data corroborate previous transcriptomic results, which suggest that S. kudriavzevii is better adapted to grow at low temperature as a result of enhanced more efficient translation. Fitter amino acid biosynthetic pathways can also be mechanisms that better explain biomass yield in cryotolerant strains. Yet even at low temperature, S. cerevisiae is the most fermentative competitive species. A higher concentration of glycolytic and alcoholic fermentation enzymes in the S. cerevisiae strain might explain such greater fermentation activity. Temperature is one of the main relevant environmental variables that microorganisms have to cope with and it is also a key factor in some industrial processes that involve microorganisms. However, we are still far from understanding the molecular and physiological mechanisms of adaptation at low temperatures. The results obtained in this study provided a global atlas of the proteome changes triggered by temperature in three different species of the genus Saccharomyces with different degree of cryotolerance. These results would facilitate a better understanding of mechanisms for how yeast could adapt at the low temperature of growth. Copyright © 2016 Elsevier B.V. All rights reserved.

Colonization State Influences the Hemocyte Proteome in a Beneficial Squid–Vibrio Symbiosis*

PubMed Central

Schleicher, Tyler R.; VerBerkmoes, Nathan C.; Shah, Manesh; Nyholm, Spencer V.

2014-01-01

The squid Euprymna scolopes and the luminescent bacterium Vibrio fischeri form a highly specific beneficial light organ symbiosis. Not only does the host have to select V. fischeri from the environment, but it must also prevent subsequent colonization by non-symbiotic microorganisms. Host macrophage-like hemocytes are believed to play a role in mediating the symbiosis with V. fischeri. Previous studies have shown that the colonization state of the light organ influences the host's hemocyte response to the symbiont. To further understand the molecular mechanisms behind this process, we used two quantitative mass-spectrometry-based proteomic techniques, isobaric tags for relative and absolute quantification (iTRAQ) and label-free spectral counting, to compare and quantify the adult hemocyte proteomes from colonized (sym) and uncolonized (antibiotic-treated/cured) squid. Overall, iTRAQ allowed for the quantification of 1,024 proteins with two or more peptides. Thirty-seven unique proteins were determined to be significantly different between sym and cured hemocytes (p value < 0.05), with 20 more abundant proteins and 17 less abundant in sym hemocytes. The label-free approach resulted in 1,241 proteins that were identified in all replicates. Of 185 unique proteins present at significantly different amounts in sym hemocytes (as determined by spectral counting), 92 were more abundant and 93 were less abundant. Comparisons between iTRAQ and spectral counting revealed that 30 of the 37 proteins quantified via iTRAQ exhibited trends similar to those identified by the label-free method. Both proteomic techniques mutually identified 16 proteins that were significantly different between the two groups of hemocytes (p value < 0.05). The presence of V. fischeri in the host light organ influenced the abundance of proteins associated with the cytoskeleton, adhesion, lysosomes, proteolysis, and the innate immune response. These data provide evidence that colonization by V. fischeri alters the hemocyte proteome and reveals proteins that may be important for maintaining host–symbiont specificity. PMID:25038065

Linking a compound-heterozygous Parkin mutant (Q311R and A371T) to Parkinson's disease by using proteomic and molecular approaches.

PubMed

Ozgul, Sinem; Kasap, Murat; Akpinar, Gurler; Kanli, Aylin; Güzel, Nil; Karaosmanoglu, Kübra; Baykal, Ahmet Tarik; Iseri, Pervin

2015-01-01

Parkin is an E3-protein ubiquitin ligase, which plays an important role as a scavenger in cell metabolism. Since the discovery of the link between Parkin and Parkinson's disease, Parkin was placed in the center of Parkinson's disease research. Previously, we isolated a mutant form of the Parkin protein (Q311R and A371T) from a Parkinson's disease patient. In this study, we aimed at characterizing this mutant Parkin protein by using biochemical and proteomic approaches. We used neuroblastoma cells (SH-SY5Y) as our model and created two inducible cell lines that expressed the wild type and the mutant Parkin proteins. We first investigated the effect of expressing both the wild type and the mutant Parkin proteins on the overall proteome by using 2D-DIGE approach. The experiments yielded the identification of 22 differentially regulated proteins, of which 13 were regulated in the mutant Parkin expressing cells. Classification of the identified proteins based on biological process and molecular function revealed that the majority of the regulated proteins belonged to protein folding and energy metabolism. Ingenuity Pathway Analysis predicted the presence of a link between the regulated proteins of the mutant Parkin expressing cells and Parkinson's disease. We also performed biochemical characterization studies on the wild type and the mutant Parkin proteins to make sense out of the differences observed at the proteome level. Both proteins displayed biological activity, had similar stabilities and localized similarly to the cytoplasm and the nucleus in SH-SY5Y cells. The mutant protein, however, was cut by a protease and subjected to a post-translational modification. The observed differences at the proteome level might be due to the differences in processing of the mutant Parkin protein. Overall, we were able to create a possible link between a pair of Parkin mutations to its pertinent disease by using 2D-DIGE in combination with biochemical and molecular approaches. Copyright © 2015 Elsevier Ltd. All rights reserved.

Targeting human pathogenic bacteria by siderophores: A proteomics review.

PubMed

Ferreira, Daniela; Seca, Ana M L; C G A, Diana; Silva, Artur M S

2016-08-11

Human bacterial infections are still a major public health problem throughout the world. Therefore it is fundamental to understand how pathogenic bacteria interact with their human host and to develop more advanced drugs or vaccines in response to the increasing bacterial resistance. Since iron is essential to bacterial survival and growth inside the host tissues, these microorganisms have developed highly efficient iron-acquisition systems; the most common one involves the secretion of iron chelators into the extracellular environment, known as siderophores, and the corresponding siderophore-membrane receptors or transporters responsible for the iron uptake. In the past few decades, several biochemical methods and genetic screens have been employed to track down and identify these iron-scavenging molecules. However, compared with the previous "static" approaches, proteomic identification is revealing far more molecules through full protein mapping and becoming more rapid and selective, leading the scientific and medical community to consider standardizing proteomic tools for clinical biomarker detection of bacterial infectious diseases. In this review, we focus on human pathogenic Gram-negative bacteria and discuss the importance of siderophores in their virulence and the available proteomic strategies to identify siderophore-related proteins and their expression level under different growth conditions. The promising use of siderophore antibiotics to overcome bacterial resistance and the future of proteomics in the routine clinical care are also mentioned. Proteomic strategies to identify siderophore-related proteins and their expression level can be helpful to control and/or find a cure of infectious deseases especially if related with multidrug resistance. Siderophores are low-molecular-weight compounds produced by bacteria which can become clinical biomarkers and/or antibiotics used mainly in "Trojan horse" type strategies. Due to the above mention we think that the promising use of siderophore to overcome bacterial resistance and the future of proteomics in the routine clinical care is a hot topic that should be discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

A Minimalistic Resource Allocation Model to Explain Ubiquitous Increase in Protein Expression with Growth Rate

PubMed Central

Keren, Leeat; Segal, Eran; Milo, Ron

2016-01-01

Most proteins show changes in level across growth conditions. Many of these changes seem to be coordinated with the specific growth rate rather than the growth environment or the protein function. Although cellular growth rates, gene expression levels and gene regulation have been at the center of biological research for decades, there are only a few models giving a base line prediction of the dependence of the proteome fraction occupied by a gene with the specific growth rate. We present a simple model that predicts a widely coordinated increase in the fraction of many proteins out of the proteome, proportionally with the growth rate. The model reveals how passive redistribution of resources, due to active regulation of only a few proteins, can have proteome wide effects that are quantitatively predictable. Our model provides a potential explanation for why and how such a coordinated response of a large fraction of the proteome to the specific growth rate arises under different environmental conditions. The simplicity of our model can also be useful by serving as a baseline null hypothesis in the search for active regulation. We exemplify the usage of the model by analyzing the relationship between growth rate and proteome composition for the model microorganism E.coli as reflected in recent proteomics data sets spanning various growth conditions. We find that the fraction out of the proteome of a large number of proteins, and from different cellular processes, increases proportionally with the growth rate. Notably, ribosomal proteins, which have been previously reported to increase in fraction with growth rate, are only a small part of this group of proteins. We suggest that, although the fractions of many proteins change with the growth rate, such changes may be partially driven by a global effect, not necessarily requiring specific cellular control mechanisms. PMID:27073913

Proteome analysis of the triton-insoluble erythrocyte membrane skeleton.

PubMed

Basu, Avik; Harper, Sandra; Pesciotta, Esther N; Speicher, Kaye D; Chakrabarti, Abhijit; Speicher, David W

2015-10-14

Erythrocyte shape and membrane integrity is imparted by the membrane skeleton, which can be isolated as a Triton X-100 insoluble structure that retains the biconcave shape of intact erythrocytes, indicating isolation of essentially intact membrane skeletons. These erythrocyte "Triton Skeletons" have been studied morphologically and biochemically, but unbiased proteome analysis of this substructure of the membrane has not been reported. In this study, different extraction buffers and in-depth proteome analyses were used to more fully define the protein composition of this functionally critical macromolecular complex. As expected, the major, well-characterized membrane skeleton proteins and their associated membrane anchors were recovered in good yield. But surprisingly, a substantial number of additional proteins that are not considered in erythrocyte membrane skeleton models were recovered in high yields, including myosin-9, lipid raft proteins (stomatin, flotillin1 and 2), multiple chaperone proteins (HSPs, protein disulfide isomerase and calnexin), and several other proteins. These results show that the membrane skeleton is substantially more complex than previous biochemical studies indicated, and it apparently has localized regions with unique protein compositions and functions. This comprehensive catalog of the membrane skeleton should lead to new insights into erythrocyte membrane biology and pathogenic mutations that perturb membrane stability. Biological significance Current models of erythrocyte membranes describe fairly simple homogenous structures that are incomplete. Proteome analysis of the erythrocyte membrane skeleton shows that it is quite complex and includes a substantial number of proteins whose roles and locations in the membrane are not well defined. Further elucidation of interactions involving these proteins and definition of microdomains in the membrane that contain these proteins should yield novel insights into how the membrane skeleton produces the normal biconcave erythrocyte shape and how it is perturbed in pathological conditions that destabilize the membrane. Copyright © 2015 Elsevier B.V. All rights reserved.

Comparative bioinformatics analyses and profiling of lysosome-related organelle proteomes

NASA Astrophysics Data System (ADS)

Hu, Zhang-Zhi; Valencia, Julio C.; Huang, Hongzhan; Chi, An; Shabanowitz, Jeffrey; Hearing, Vincent J.; Appella, Ettore; Wu, Cathy

2007-01-01

Complete and accurate profiling of cellular organelle proteomes, while challenging, is important for the understanding of detailed cellular processes at the organelle level. Mass spectrometry technologies coupled with bioinformatics analysis provide an effective approach for protein identification and functional interpretation of organelle proteomes. In this study, we have compiled human organelle reference datasets from large-scale proteomic studies and protein databases for seven lysosome-related organelles (LROs), as well as the endoplasmic reticulum and mitochondria, for comparative organelle proteome analysis. Heterogeneous sources of human organelle proteins and rodent homologs are mapped to human UniProtKB protein entries based on ID and/or peptide mappings, followed by functional annotation and categorization using the iProXpress proteomic expression analysis system. Cataloging organelle proteomes allows close examination of both shared and unique proteins among various LROs and reveals their functional relevance. The proteomic comparisons show that LROs are a closely related family of organelles. The shared proteins indicate the dynamic and hybrid nature of LROs, while the unique transmembrane proteins may represent additional candidate marker proteins for LROs. This comparative analysis, therefore, provides a basis for hypothesis formulation and experimental validation of organelle proteins and their functional roles.

Inter-lab proteomics: data mining in collaborative projects on the basis of the HUPO brain proteome project's pilot studies.

PubMed

Hamacher, Michael; Gröttrup, Bernd; Eisenacher, Martin; Marcus, Katrin; Park, Young Mok; Meyer, Helmut E; Kwon, Kyung-Hoon; Stephan, Christian

2011-01-01

Several projects were initiated by the Human Proteome Organisation (HUPO) focusing on the proteome analysis of distinct human organs. The initiative dedicated to the brain, its development and correlated diseases is the HUPO Brain Proteome Project (HUPO BPP). An objective data submission, storage, and reprocessing strategy have been established with the help of the results gained in a pilot study phase and within subsequent studies. The bioinformatic relevance of the data is drawn from the inter-laboratory comparisons as well as from the recalculation of all data sets submitted by the different groups. In the following, results of the single groups as well as the centralised reprocessing effort are summarised, demonstrating the added-value of this concerted work.

Influence of Diet on the Proteome of Drosophila Melanogaster as Assessed by Two-Dimensional Gel Electrophoresis and Capillary Liquid Chromatography–Mass Spectrometry: The Hamburger Effect Revisited

PubMed Central

Culwell, Thomas F.; Thulin, Craig D.; Merrell, Karen J.; Graves, Steven W.

2008-01-01

Proteomic biomarker discovery has been called into question. Diamandis hypothesized that seemingly trivial factors, such as eating a hamburger, may cause sufficient proteomic change as to confound proteomic differences. This has been termed the hamburger effect. Little is known about the variability of complex proteomes in response to the environment. Two methods—two-dimensional gel electrophoresis (2DGE) and capillary liquid chromatography–electrospray ionization time-of-flight mass spectrometry (LCMS)—were used to study the hamburger effect in two cross-sections of the soluble fruit fly proteome. 2DGE measured abundant proteins, whereas LCMS measured small proteins and peptides. Proteomic differences between males and females were first evaluated to assess the discriminatory capability of the methods. Likewise, wild-type and white-eyed flies were analyzed as a further demonstration that genetically based proteomic differences could be observed above the background analytical variation. Then dietary interventions were imposed. Ethanol was added to the diet of some populations without significant proteomic effect. However, after a 24-h fast, proteomic differences were found using LCMS but not 2DGE. Even so, only three of ~1000 molecular species were altered significantly, suggesting that the influence of even an extreme diet change produced only modest proteomic variability, and that much of the fruit fly proteome remains relatively constant in response to diet. These experiments suggest that proteomics can be a viable approach to biomarker discovery. PMID:19137114

The quantitative and condition-dependent Escherichia coli proteome

PubMed Central

Schmidt, Alexander; Kochanowski, Karl; Vedelaar, Silke; Ahrné, Erik; Volkmer, Benjamin; Callipo, Luciano; Knoops, Kèvin; Bauer, Manuel; Aebersold, Ruedi; Heinemann, Matthias

2016-01-01

Measuring precise concentrations of proteins can provide insights into biological processes. Here, we use efficient protein extraction and sample fractionation and state-of-the-art quantitative mass spectrometry techniques to generate a comprehensive, condition-dependent protein abundance map of Escherichia coli. We measure cellular protein concentrations for 55% of predicted E. coli genes (>2300 proteins) under 22 different experimental conditions and identify methylation and N-terminal protein acetylations previously not known to be prevalent in bacteria. We uncover system-wide proteome allocation, expression regulation, and post-translational adaptations. These data provide a valuable resource for the systems biology and broader E. coli research communities. PMID:26641532

Proteome Studies of Filamentous Fungi

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baker, Scott E.; Panisko, Ellen A.

2011-04-20

The continued fast pace of fungal genome sequence generation has enabled proteomic analysis of a wide breadth of organisms that span the breadth of the Kingdom Fungi. There is some phylogenetic bias to the current catalog of fungi with reasonable DNA sequence databases (genomic or EST) that could be analyzed at a global proteomic level. However, the rapid development of next generation sequencing platforms has lowered the cost of genome sequencing such that in the near future, having a genome sequence will no longer be a time or cost bottleneck for downstream proteomic (and transcriptomic) analyses. High throughput, non-gel basedmore » proteomics offers a snapshot of proteins present in a given sample at a single point in time. There are a number of different variations on the general method and technologies for identifying peptides in a given sample. We present a method that can serve as a “baseline” for proteomic studies of fungi.« less

Recent 5-year Findings and Technological Advances in the Proteomic Study of HIV-associated Disorders.

PubMed

Zhang, Lijun; Jia, Xiaofang; Jin, Jun-O; Lu, Hongzhou; Tan, Zhimi

2017-04-01

Human immunodeficiency virus-1 (HIV-1) mainly relies on host factors to complete its life cycle. Hence, it is very important to identify HIV-regulated host proteins. Proteomics is an excellent technique for this purpose because of its high throughput and sensitivity. In this review, we summarized current technological advances in proteomics, including general isobaric tags for relative and absolute quantitation (iTRAQ) and stable isotope labeling by amino acids in cell culture (SILAC), as well as subcellular proteomics and investigation of posttranslational modifications. Furthermore, we reviewed the applications of proteomics in the discovery of HIV-related diseases and HIV infection mechanisms. Proteins identified by proteomic studies might offer new avenues for the diagnosis and treatment of HIV infection and the related diseases. Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

Proteome studies of filamentous fungi.

PubMed

Baker, Scott E; Panisko, Ellen A

2011-01-01

The continued fast pace of fungal genome sequence generation has enabled proteomic analysis of a wide variety of organisms that span the breadth of the Kingdom Fungi. There is some phylogenetic bias to the current catalog of fungi with reasonable DNA sequence databases (genomic or EST) that could be analyzed at a global proteomic level. However, the rapid development of next generation sequencing platforms has lowered the cost of genome sequencing such that in the near future, having a genome sequence will no longer be a time or cost bottleneck for downstream proteomic (and transcriptomic) analyses. High throughput, nongel-based proteomics offers a snapshot of proteins present in a given sample at a single point in time. There are a number of variations on the general methods and technologies for identifying peptides in a given sample. We present a method that can serve as a "baseline" for proteomic studies of fungi.

The wheat chloroplastic proteome.

PubMed

Kamal, Abu Hena Mostafa; Cho, Kun; Choi, Jong-Soon; Bae, Kwang-Hee; Komatsu, Setsuko; Uozumi, Nobuyuki; Woo, Sun Hee

2013-11-20

With the availability of plant genome sequencing, analysis of plant proteins with mass spectrometry has become promising and admired. Determining the proteome of a cell is still a challenging assignment, which is convoluted by proteome dynamics and convolution. Chloroplast is fastidious curiosity for plant biologists due to their intricate biochemical pathways for indispensable metabolite functions. In this review, an overview on proteomic studies conducted in wheat with a special focus on subcellular proteomics of chloroplast, salt and water stress. In recent years, we and other groups have attempted to understand the photosynthesis in wheat and abiotic stress under salt imposed and water deficit during vegetative stage. Those studies provide interesting results leading to better understanding of the photosynthesis and identifying the stress-responsive proteins. Indeed, recent studies aimed at resolving the photosynthesis pathway in wheat. Proteomic analysis combining two complementary approaches such as 2-DE and shotgun methods couple to high through put mass spectrometry (LTQ-FTICR and MALDI-TOF/TOF) in order to better understand the responsible proteins in photosynthesis and abiotic stress (salt and water) in wheat chloroplast will be focused. In this review we discussed the identification of the most abundant protein in wheat chloroplast and stress-responsive under salt and water stress in chloroplast of wheat seedlings, thus providing the proteomic view of the events during the development of this seedling under stress conditions. Chloroplast is fastidious curiosity for plant biologists due to their intricate biochemical pathways for indispensable metabolite functions. An overview on proteomic studies conducted in wheat with a special focus on subcellular proteomics of chloroplast, salt and water stress. We have attempted to understand the photosynthesis in wheat and abiotic stress under salt imposed and water deficit during seedling stage. Those studies provide interesting results leading to a better understanding of the photosynthesis and identifying the stress-responsive proteins. In reality, our studies aspired at resolving the photosynthesis pathway in wheat. Proteomic analysis united two complementary approaches such as Tricine SDS-PAGE and 2-DE methods couple to high through put mass spectrometry (LTQ-FTICR and MALDI-TOF/TOF) in order to better understand the responsible proteins in photosynthesis and abiotic stress (salt and water) in wheat chloroplast will be highlighted. This article is part of a Special Issue entitled: Translational Plant Proteomics. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

Quantifying Integrated Proteomic Responses to Iron Stress in the Globally Important Marine Diazotroph Trichodesmium

PubMed Central

Snow, Joseph T.; Polyviou, Despo; Skipp, Paul; Chrismas, Nathan A. M.; Hitchcock, Andrew; Geider, Richard; Moore, C. Mark; Bibby, Thomas S.

2015-01-01

Trichodesmium is a biogeochemically important marine cyanobacterium, responsible for a significant proportion of the annual ‘new’ nitrogen introduced into the global ocean. These non-heterocystous filamentous diazotrophs employ a potentially unique strategy of near-concurrent nitrogen fixation and oxygenic photosynthesis, potentially burdening Trichodesmium with a particularly high iron requirement due to the iron-binding proteins involved in these processes. Iron availability may therefore have a significant influence on the biogeography of Trichodesmium. Previous investigations of molecular responses to iron stress in this keystone marine microbe have largely been targeted. Here a holistic approach was taken using a label-free quantitative proteomics technique (MSE) to reveal a sophisticated multi-faceted proteomic response of Trichodesmium erythraeum IMS101 to iron stress. Increased abundances of proteins known to be involved in acclimation to iron stress and proteins known or predicted to be involved in iron uptake were observed, alongside decreases in the abundances of iron-binding proteins involved in photosynthesis and nitrogen fixation. Preferential loss of proteins with a high iron content contributed to overall reductions of 55–60% in estimated proteomic iron requirements. Changes in the abundances of iron-binding proteins also suggested the potential importance of alternate photosynthetic pathways as Trichodesmium reallocates the limiting resource under iron stress. Trichodesmium therefore displays a significant and integrated proteomic response to iron availability that likely contributes to the ecological success of this species in the ocean. PMID:26562022

The state of proteome profiling in the fungal genus Aspergillus.

PubMed

Kim, Yonghyun; Nandakumar, M P; Marten, Mark R

2008-03-01

Aspergilli are an important genus of filamentous fungi that contribute to a multibillion dollar industry. Since many fungal genome sequencing were recently completed, it would be advantageous to profile their proteome to better understand the fungal cell factory. Here, we review proteomic data generated for the Aspergilli in recent years. Thus far, a combined total of 28 cell surface, 102 secreted and 139 intracellular proteins have been identified based on 10 different studies on Aspergillus proteomics. A summary proteome map highlighting identified proteins in major metabolic pathway is presented.

Functional proteomic analysis reveals the involvement of KIAA1199 in breast cancer growth, motility and invasiveness

PubMed Central

2014-01-01

Background KIAA1199 is a recently identified novel gene that is up-regulated in human cancer with poor survival. Our proteomic study on signaling polarity in chemotactic cells revealed KIAA1199 as a novel protein target that may be involved in cellular chemotaxis and motility. In the present study, we examined the functional significance of KIAA1199 expression in breast cancer growth, motility and invasiveness. Methods We validated the previous microarray observation by tissue microarray immunohistochemistry using a TMA slide containing 12 breast tumor tissue cores and 12 corresponding normal tissues. We performed the shRNA-mediated knockdown of KIAA1199 in MDA-MB-231 and HS578T cells to study the role of this protein in cell proliferation, migration and apoptosis in vitro. We studied the effects of KIAA1199 knockdown in vivo in two groups of mice (n = 5). We carried out the SILAC LC-MS/MS based proteomic studies on the involvement of KIAA1199 in breast cancer. Results KIAA1199 mRNA and protein was significantly overexpressed in breast tumor specimens and cell lines as compared with non-neoplastic breast tissues from large-scale microarray and studies of breast cancer cell lines and tumors. To gain deeper insights into the novel role of KIAA1199 in breast cancer, we modulated KIAA1199 expression using shRNA-mediated knockdown in two breast cancer cell lines (MDA-MB-231 and HS578T), expressing higher levels of KIAA1199. The KIAA1199 knockdown cells showed reduced motility and cell proliferation in vitro. Moreover, when the knockdown cells were injected into the mammary fat pads of female athymic nude mice, there was a significant decrease in tumor incidence and growth. In addition, quantitative proteomic analysis revealed that knockdown of KIAA1199 in breast cancer (MDA-MB-231) cells affected a broad range of cellular functions including apoptosis, metabolism and cell motility. Conclusions Our findings indicate that KIAA1199 may play an important role in breast tumor growth and invasiveness, and that it may represent a novel target for biomarker development and a novel therapeutic target for breast cancer. PMID:24628760

Proteomic analysis of specific brain proteins in aged SAMP8 mice treated with alpha-lipoic acid: implications for aging and age-related neurodegenerative disorders.

PubMed

Poon, H Fai; Farr, Susan A; Thongboonkerd, Visith; Lynn, Bert C; Banks, William A; Morley, John E; Klein, Jon B; Butterfield, D Allan

2005-01-01

Free radical-mediated damage to neuronal membrane components has been implicated in the etiology of Alzheimer's disease (AD) and aging. The senescence accelerated prone mouse strain 8 (SAMP8) exhibits age-related deterioration in memory and learning along with increased oxidative markers. Therefore, SAMP8 is a suitable model to study brain aging and, since aging is the major risk factor for AD and SAMP8 exhibits many of the biochemical findings of AD, perhaps as a model for and the early phase of AD. Our previous studies reported higher oxidative stress markers in brains of 12-month-old SAMP8 mice when compared to that of 4-month-old SAMP8 mice. Further, we have previously shown that injecting the mice with alpha-lipoic acid (LA) reversed brain lipid peroxidation, protein oxidation, as well as the learning and memory impairments in SAMP8 mice. Recently, we reported the use of proteomics to identify proteins that are expressed differently and/or modified oxidatively in aged SAMP8 brains. In order to understand how LA reverses the learning and memory deficits of aged SAMP8 mice, in the current study, we used proteomics to compare the expression levels and specific carbonyl levels of proteins in brains from 12-month-old SAMP8 mice treated or not treated with LA. We found that the expressions of the three brain proteins (neurofilament triplet L protein, alpha-enolase, and ubiquitous mitochondrial creatine kinase) were increased significantly and that the specific carbonyl levels of the three brain proteins (lactate dehydrogenase B, dihydropyrimidinase-like protein 2, and alpha-enolase) were significantly decreased in the aged SAMP8 mice treated with LA. These findings suggest that the improved learning and memory observed in LA-injected SAMP8 mice may be related to the restoration of the normal condition of specific proteins in aged SAMP8 mouse brain. Moreover, our current study implicates neurofilament triplet L protein, alpha-enolase, ubiquitous mitochondrial creatine kinase, lactate dehydrogenase B, and dihydropyrimidinase-like protein 2 in process associated with learning and memory of SAMP8 mice.

Applications of Proteomic Technologies to Toxicology

EPA Science Inventory

Proteomics is the large-scale study of gene expression at the protein level. This cutting edge technology has been extensively applied to toxicology research recently. The up-to-date development of proteomics has presented the toxicology community with an unprecedented opportunit...

Interlaboratory studies and initiatives developing standards for proteomics

PubMed Central

Ivanov, Alexander R.; Colangelo, Christopher M.; Dufresne, Craig P.; Friedman, David B.; Lilley, Kathryn S.; Mechtler, Karl; Phinney, Brett S.; Rose, Kristie L.; Rudnick, Paul A.; Searle, Brian C.; Shaffer, Scott A.; Weintraub, Susan T.

2013-01-01

Proteomics is a rapidly transforming interdisciplinary field of research that embraces a diverse set of analytical approaches to tackle problems in fundamental and applied biology. This view-point article highlights the benefits of interlaboratory studies and standardization initiatives to enable investigators to address many of the challenges found in proteomics research. Among these initiatives, we discuss our efforts on a comprehensive performance standard for characterizing PTMs by MS that was recently developed by the Association of Biomolecular Resource Facilities (ABRF) Proteomics Standards Research Group (sPRG). PMID:23319436

Sys-BodyFluid: a systematical database for human body fluid proteome research

PubMed Central

Li, Su-Jun; Peng, Mao; Li, Hong; Liu, Bo-Shu; Wang, Chuan; Wu, Jia-Rui; Li, Yi-Xue; Zeng, Rong

2009-01-01

Recently, body fluids have widely become an important target for proteomic research and proteomic study has produced more and more body fluid related protein data. A database is needed to collect and analyze these proteome data. Thus, we developed this web-based body fluid proteome database Sys-BodyFluid. It contains eleven kinds of body fluid proteomes, including plasma/serum, urine, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, synovial fluid, nipple aspirate fluid, tear fluid, seminal fluid, human milk and amniotic fluid. Over 10 000 proteins are presented in the Sys-BodyFluid. Sys-BodyFluid provides the detailed protein annotations, including protein description, Gene Ontology, domain information, protein sequence and involved pathways. These proteome data can be retrieved by using protein name, protein accession number and sequence similarity. In addition, users can query between these different body fluids to get the different proteins identification information. Sys-BodyFluid database can facilitate the body fluid proteomics and disease proteomics research as a reference database. It is available at http://www.biosino.org/bodyfluid/. PMID:18978022

Sys-BodyFluid: a systematical database for human body fluid proteome research.

PubMed

Li, Su-Jun; Peng, Mao; Li, Hong; Liu, Bo-Shu; Wang, Chuan; Wu, Jia-Rui; Li, Yi-Xue; Zeng, Rong

2009-01-01

Recently, body fluids have widely become an important target for proteomic research and proteomic study has produced more and more body fluid related protein data. A database is needed to collect and analyze these proteome data. Thus, we developed this web-based body fluid proteome database Sys-BodyFluid. It contains eleven kinds of body fluid proteomes, including plasma/serum, urine, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, synovial fluid, nipple aspirate fluid, tear fluid, seminal fluid, human milk and amniotic fluid. Over 10,000 proteins are presented in the Sys-BodyFluid. Sys-BodyFluid provides the detailed protein annotations, including protein description, Gene Ontology, domain information, protein sequence and involved pathways. These proteome data can be retrieved by using protein name, protein accession number and sequence similarity. In addition, users can query between these different body fluids to get the different proteins identification information. Sys-BodyFluid database can facilitate the body fluid proteomics and disease proteomics research as a reference database. It is available at http://www.biosino.org/bodyfluid/.

Major depressive disorder: insight into candidate cerebrospinal fluid protein biomarkers from proteomics studies.

PubMed

Al Shweiki, Mhd Rami; Oeckl, Patrick; Steinacker, Petra; Hengerer, Bastian; Schönfeldt-Lecuona, Carlos; Otto, Markus

2017-06-01

Major Depressive Disorder (MDD) is the leading cause of global disability, and an increasing body of literature suggests different cerebrospinal fluid (CSF) proteins as biomarkers of MDD. The aim of this review is to summarize the suggested CSF biomarkers and to analyze the MDD proteomics studies of CSF and brain tissues for promising biomarker candidates. Areas covered: The review includes the human studies found by a PubMed search using the following terms: 'depression cerebrospinal fluid biomarker', 'major depression biomarker CSF', 'depression CSF biomarker', 'proteomics depression', 'proteomics biomarkers in depression', 'proteomics CSF biomarker in depression', and 'major depressive disorder CSF'. The literature analysis highlights promising biomarker candidates and demonstrates conflicting results on others. It reveals 42 differentially regulated proteins in MDD that were identified in more than one proteomics study. It discusses the diagnostic potential of the biomarker candidates and their association with the suggested pathologies. Expert commentary: One ultimate goal of finding biomarkers for MDD is to improve the diagnostic accuracy to achieve better treatment outcomes; due to the heterogeneous nature of MDD, using bio-signatures could be a good strategy to differentiate MDD from other neuropsychiatric disorders. Notably, further validation studies of the suggested biomarkers are still needed.

In Silico Prediction and Validation of Novel RNA Binding Proteins and Residues in the Human Proteome.

PubMed

Chowdhury, Shomeek; Zhang, Jian; Kurgan, Lukasz

2018-05-28

Deciphering a complete landscape of protein-RNA interactions in the human proteome remains an elusive challenge. We computationally elucidate RNA binding proteins (RBPs) using an approach that complements previous efforts. We employ two modern complementary sequence-based methods that provide accurate predictions from the structured and the intrinsically disordered sequences, even in the absence of sequence similarity to the known RBPs. We generate and analyze putative RNA binding residues on the whole proteome scale. Using a conservative setting that ensures low, 5% false positive rate, we identify 1511 putative RBPs that include 281 known RBPs and 166 RBPs that were previously predicted. We empirically demonstrate that these overlaps are statistically significant. We also validate the putative RBPs based on two major hallmarks of their RNA binding residues: high levels of evolutionary conservation and enrichment in charged amino acids. Moreover, we show that the novel RBPs are significantly under-annotated functionally which coincides with the fact that they were not yet found to interact with RNAs. We provide two examples of our novel putative RBPs for which there is recent evidence of their interactions with RNAs. The dataset of novel putative RBPs and RNA binding residues for the future hypothesis generation is provided in the Supporting Information. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantitation of peptides from non-invasive skin tapings using isotope dilution and tandem mass spectrometry.

PubMed

Reisdorph, Nichole; Armstrong, Michael; Powell, Roger; Quinn, Kevin; Legg, Kevin; Leung, Donald; Reisdorph, Rick

2018-05-01

Previous work from our laboratories utilized a novel skin taping method and mass spectrometry-based proteomics to discover clinical biomarkers of skin conditions; these included atopic dermatitis, Staphylococcus aureus colonization, and eczema herpeticum. While suitable for discovery purposes, semi-quantitative proteomics is generally time-consuming and expensive. Furthermore, depending on the method used, discovery-based proteomics can result in high variation and inadequate sensitivity to detect low abundant peptides. Therefore, we strove to develop a rapid, sensitive, and reproducible method to quantitate disease-related proteins from skin tapings. We utilized isotopically-labeled peptides and tandem mass spectrometry to obtain absolute quantitation values on 14 peptides from 7 proteins; these proteins had shown previous importance in skin disease. The method demonstrated good reproducibility, dynamic range, and linearity (R 2  > 0.993) when n = 3 standards were analyzed across 0.05-2.5 pmol. The method was used to determine if differences exist between skin proteins in a small group of atopic versus non-atopic individuals (n = 12). While only minimal differences were found, peptides were detected in all samples and exhibited good correlation between peptides for 5 of the 7 proteins (R 2  = 0.71-0.98). This method can be applied to larger cohorts to further establish the relationships of these proteins to skin disease. Copyright © 2017. Published by Elsevier B.V.

Elucidation of cross-species proteomic effects in human and hominin bone proteome identification through a bioinformatics experiment.

PubMed

Welker, F

2018-02-20

The study of ancient protein sequences is increasingly focused on the analysis of older samples, including those of ancient hominins. The analysis of such ancient proteomes thereby potentially suffers from "cross-species proteomic effects": the loss of peptide and protein identifications at increased evolutionary distances due to a larger number of protein sequence differences between the database sequence and the analyzed organism. Error-tolerant proteomic search algorithms should theoretically overcome this problem at both the peptide and protein level; however, this has not been demonstrated. If error-tolerant searches do not overcome the cross-species proteomic issue then there might be inherent biases in the identified proteomes. Here, a bioinformatics experiment is performed to test this using a set of modern human bone proteomes and three independent searches against sequence databases at increasing evolutionary distances: the human (0 Ma), chimpanzee (6-8 Ma) and orangutan (16-17 Ma) reference proteomes, respectively. Incorrectly suggested amino acid substitutions are absent when employing adequate filtering criteria for mutable Peptide Spectrum Matches (PSMs), but roughly half of the mutable PSMs were not recovered. As a result, peptide and protein identification rates are higher in error-tolerant mode compared to non-error-tolerant searches but did not recover protein identifications completely. Data indicates that peptide length and the number of mutations between the target and database sequences are the main factors influencing mutable PSM identification. The error-tolerant results suggest that the cross-species proteomics problem is not overcome at increasing evolutionary distances, even at the protein level. Peptide and protein loss has the potential to significantly impact divergence dating and proteome comparisons when using ancient samples as there is a bias towards the identification of conserved sequences and proteins. Effects are minimized between moderately divergent proteomes, as indicated by almost complete recovery of informative positions in the search against the chimpanzee proteome (≈90%, 6-8 Ma). This provides a bioinformatic background to future phylogenetic and proteomic analysis of ancient hominin proteomes, including the future description of novel hominin amino acid sequences, but also has negative implications for the study of fast-evolving proteins in hominins, non-hominin animals, and ancient bacterial proteins in evolutionary contexts.

Use of proteomic methods in the analysis of human body fluids in Alzheimer research.

PubMed

Zürbig, Petra; Jahn, Holger

2012-12-01

Proteomics is the study of the entire population of proteins and peptides in an organism or a part of it, such as a cell, tissue, or fluids like cerebrospinal fluid, plasma, serum, urine, or saliva. It is widely assumed that changes in the composition of the proteome may reflect disease states and provide clues to its origin, eventually leading to targets for new treatments. The ability to perform large-scale proteomic studies now is based jointly on recent advances in our analytical methods. Separation techniques like CE and 2DE have developed and matured. Detection methods like MS have also improved greatly in the last 5 years. These developments have also driven the fields of bioinformatics, needed to deal with the increased data production and systems biology. All these developing methods offer specific advantages but also come with certain limitations. This review describes the different proteomic methods used in the field, their limitations, and their possible pitfalls. Based on a literature search in PubMed, we identified 112 studies that applied proteomic techniques to identify biomarkers for Alzheimer disease. This review describes the results of these studies on proteome changes in human body fluids of Alzheimer patients reviewing the most important studies. We extracted a list of 366 proteins and peptides that were identified by these studies as potential targets in Alzheimer research. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Computer applications making rapid advances in high throughput microbial proteomics (HTMP).

PubMed

Anandkumar, Balakrishna; Haga, Steve W; Wu, Hui-Fen

2014-02-01

The last few decades have seen the rise of widely-available proteomics tools. From new data acquisition devices, such as MALDI-MS and 2DE to new database searching softwares, these new products have paved the way for high throughput microbial proteomics (HTMP). These tools are enabling researchers to gain new insights into microbial metabolism, and are opening up new areas of study, such as protein-protein interactions (interactomics) discovery. Computer software is a key part of these emerging fields. This current review considers: 1) software tools for identifying the proteome, such as MASCOT or PDQuest, 2) online databases of proteomes, such as SWISS-PROT, Proteome Web, or the Proteomics Facility of the Pathogen Functional Genomics Resource Center, and 3) software tools for applying proteomic data, such as PSI-BLAST or VESPA. These tools allow for research in network biology, protein identification, functional annotation, target identification/validation, protein expression, protein structural analysis, metabolic pathway engineering and drug discovery.

Novel Phage Group Infecting Lactobacillus delbrueckii subsp. lactis, as Revealed by Genomic and Proteomic Analysis of Bacteriophage Ldl1

PubMed Central

Casey, Eoghan; Mahony, Jennifer; Neve, Horst; Noben, Jean-Paul; Dal Bello, Fabio

2014-01-01

Ldl1 is a virulent phage infecting the dairy starter Lactobacillus delbrueckii subsp. lactis LdlS. Electron microscopy analysis revealed that this phage exhibits a large head and a long tail and bears little resemblance to other characterized phages infecting Lactobacillus delbrueckii. In vitro propagation of this phage revealed a latent period of 30 to 40 min and a burst size of 59.9 ± 1.9 phage particles. Comparative genomic and proteomic analyses showed remarkable similarity between the genome of Ldl1 and that of Lactobacillus plantarum phage ATCC 8014-B2. The genomic and proteomic characteristics of Ldl1 demonstrate that this phage does not belong to any of the four previously recognized L. delbrueckii phage groups, necessitating the creation of a new group, called group e, thus adding to the knowledge on the diversity of phages targeting strains of this industrially important lactic acid bacterial species. PMID:25501478

Proteome Speciation by Mass Spectrometry: Characterization of Composite Protein Mixtures in Milk Replacers.

PubMed

Gaspari, Marco; Chiesa, Luca; Nicastri, Annalisa; Gabriele, Caterina; Harper, Valeria; Britti, Domenico; Cuda, Giovanni; Procopio, Antonio

2016-12-06

The ability of tandem mass spectrometry to determine the primary structure of proteolytic peptides can be exploited to trace back the organisms from which the corresponding proteins were extracted. This information can be important when food products, such as protein powders, can be supplemented with lower-quality starting materials. In order to dissect the origin of proteinaceous material composing a given unknown mixture, a two-step database search strategy for bottom-up nanoscale liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) data was implemented. A single nanoLC-MS/MS analysis was sufficient not only to determine the qualitative composition of the mixtures under examination, but also to assess the relative percent composition of the various proteomes, if dedicated calibration curves were previously generated. The approach of two-step database search for qualitative analysis and proteome total ion current (pTIC) calculation for quantitative analysis was applied to several binary and ternary mixtures which mimic the composition of milk replacers typically used in calf feeding.

Targeted proteomic assays for quantitation of proteins identified by proteogenomic analysis of ovarian cancer

DOE PAGES

Song, Ehwang; Gao, Yuqian; Wu, Chaochao; ...

2017-07-19

Here, mass spectrometry (MS) based targeted proteomic methods such as selected reaction monitoring (SRM) are becoming the method of choice for preclinical verification of candidate protein biomarkers. The Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute has investigated the standardization and analytical validation of the SRM assays and demonstrated robust analytical performance on different instruments across different laboratories. An Assay Portal has also been established by CPTAC to provide the research community a resource consisting of large set of targeted MS-based assays, and a depository to share assays publicly, providing that assays meet the guidelines proposed bymore » CPTAC. Herein, we report 98 SRM assays covering 70 candidate protein biomarkers previously reported as associated with ovarian cancer that have been thoroughly characterized according to the CPTAC Assay Characterization Guidance Document. The experiments, methods and results for characterizing these SRM assays for their MS response, repeatability, selectivity, stability, and reproducible detection of endogenous analytes are described in detail.« less

In-depth analysis of protein inference algorithms using multiple search engines and well-defined metrics.

PubMed

Audain, Enrique; Uszkoreit, Julian; Sachsenberg, Timo; Pfeuffer, Julianus; Liang, Xiao; Hermjakob, Henning; Sanchez, Aniel; Eisenacher, Martin; Reinert, Knut; Tabb, David L; Kohlbacher, Oliver; Perez-Riverol, Yasset

2017-01-06

In mass spectrometry-based shotgun proteomics, protein identifications are usually the desired result. However, most of the analytical methods are based on the identification of reliable peptides and not the direct identification of intact proteins. Thus, assembling peptides identified from tandem mass spectra into a list of proteins, referred to as protein inference, is a critical step in proteomics research. Currently, different protein inference algorithms and tools are available for the proteomics community. Here, we evaluated five software tools for protein inference (PIA, ProteinProphet, Fido, ProteinLP, MSBayesPro) using three popular database search engines: Mascot, X!Tandem, and MS-GF+. All the algorithms were evaluated using a highly customizable KNIME workflow using four different public datasets with varying complexities (different sample preparation, species and analytical instruments). We defined a set of quality control metrics to evaluate the performance of each combination of search engines, protein inference algorithm, and parameters on each dataset. We show that the results for complex samples vary not only regarding the actual numbers of reported protein groups but also concerning the actual composition of groups. Furthermore, the robustness of reported proteins when using databases of differing complexities is strongly dependant on the applied inference algorithm. Finally, merging the identifications of multiple search engines does not necessarily increase the number of reported proteins, but does increase the number of peptides per protein and thus can generally be recommended. Protein inference is one of the major challenges in MS-based proteomics nowadays. Currently, there are a vast number of protein inference algorithms and implementations available for the proteomics community. Protein assembly impacts in the final results of the research, the quantitation values and the final claims in the research manuscript. Even though protein inference is a crucial step in proteomics data analysis, a comprehensive evaluation of the many different inference methods has never been performed. Previously Journal of proteomics has published multiple studies about other benchmark of bioinformatics algorithms (PMID: 26585461; PMID: 22728601) in proteomics studies making clear the importance of those studies for the proteomics community and the journal audience. This manuscript presents a new bioinformatics solution based on the KNIME/OpenMS platform that aims at providing a fair comparison of protein inference algorithms (https://github.com/KNIME-OMICS). Six different algorithms - ProteinProphet, MSBayesPro, ProteinLP, Fido and PIA- were evaluated using the highly customizable workflow on four public datasets with varying complexities. Five popular database search engines Mascot, X!Tandem, MS-GF+ and combinations thereof were evaluated for every protein inference tool. In total >186 proteins lists were analyzed and carefully compare using three metrics for quality assessments of the protein inference results: 1) the numbers of reported proteins, 2) peptides per protein, and the 3) number of uniquely reported proteins per inference method, to address the quality of each inference method. We also examined how many proteins were reported by choosing each combination of search engines, protein inference algorithms and parameters on each dataset. The results show that using 1) PIA or Fido seems to be a good choice when studying the results of the analyzed workflow, regarding not only the reported proteins and the high-quality identifications, but also the required runtime. 2) Merging the identifications of multiple search engines gives almost always more confident results and increases the number of peptides per protein group. 3) The usage of databases containing not only the canonical, but also known isoforms of proteins has a small impact on the number of reported proteins. The detection of specific isoforms could, concerning the question behind the study, compensate for slightly shorter reports using the parsimonious reports. 4) The current workflow can be easily extended to support new algorithms and search engine combinations. Copyright © 2016. Published by Elsevier B.V.

Protein profile of mouse ovarian follicles grown in vitro.

PubMed

Anastácio, Amandine; Rodriguez-Wallberg, Kenny A; Chardonnet, Solenne; Pionneau, Cédric; Fédérici, Christian; Almeida Santos, Teresa; Poirot, Catherine

2017-12-01

Could the follicle proteome be mapped by identifying specific proteins that are common or differ between three developmental stages from the secondary follicle (SF) to the antrum-like stage? From a total of 1401 proteins identified in the follicles, 609 were common to the three developmental stages investigated and 444 were found uniquely at one of the stages. The importance of the follicle as a functional structure has been recognized; however, up-to-date the proteome of the whole follicle has not been described. A few studies using proteomics have previously reported on either isolated fully-grown oocytes before or after meiosis resumption or cumulus cells. The experimental design included a validated mice model for isolation and individual culture of SFs. The system was chosen as it allows continuous evaluation of follicle growth and selection of follicles for analysis at pre-determined developmental stages: SF, complete Slavjanski membrane rupture (SMR) and antrum-like cavity (AF). The experiments were repeated 13 times independently to acquire the material that was analyzed by proteomics. SFs (n = 2166) were isolated from B6CBA/F1 female mice (n = 42), 12 days old, from 15 l. About half of the follicles isolated as SF were analyzed as such (n = 1143) and pooled to obtain 139 μg of extracted protein. Both SMR (n = 359) and AF (n = 124) were obtained after individual culture of 1023 follicles in a microdrop system under oil, selected for analysis and pooled, to obtain 339 μg and 170 μg of protein, respectively. The follicle proteome was analyzed combining isoelectric focusing (IEF) fractionation with 1D and 2D LC-MS/MS analysis to enhance protein identification. The three protein lists were submitted to the 'Compare gene list' tool in the PANTHER website to gain insights on the Gene Ontology Biological processes present and to Ingenuity Pathway Analysis to highlight protein networks. A label-free quantification was performed with 1D LC-MS/MS analyses to emphasize proteins with different expression profiles between the three follicular stages. Supplementary western blot analysis (using new biological replicates) was performed to confirm the expression variations of three proteins during follicle development in vitro. It was found that 609 out of 1401 identified proteins were common to the three follicle developmental stages investigated. Some proteins were identified uniquely at one stage: 71 of the 775 identified proteins in SF, 181 of 1092 in SMR and 192 of 1100 in AF. Additional qualitative and quantitative analysis highlighted 44 biological processes over-represented in our samples compared to the Mus musculus gene database. In particular, it was possible to identify proteins implicated in the cell cycle, calcium ion binding and glycolysis, with specific expressions and abundance, throughout in vitro follicle development. Data are available via ProteomeXchange with identifier PXD006227. The proteome analyses described in this study were performed after in vitro development. Despite fractionation of the samples before LC-MS/MS, proteomic approaches are not exhaustive, thus proteins that are not identified in a group are not necessarily absent from that group, although they are likely to be less abundant. This study allowed a general view of proteins implicated in follicle development in vitro and it represents the most complete catalog of the whole follicle proteome available so far. Not only were well known proteins of the oocyte identified but also proteins that are probably expressed only in granulosa cells. This study was supported by the Portuguese Foundation for Science and Technology, FCT (PhD fellowship SFRH/BD/65299/2009 to A.A.), the Swedish Childhood Cancer Foundation (PR 2014-0144 to K.A.R-.W.) and Stockholm County Council to K.A.R-.W. The authors of the study have no conflict of interest to report. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.

New Strategies and Challenges in Lung Proteomics and Metabolomics. An Official American Thoracic Society Workshop Report.

PubMed

Bowler, Russell P; Wendt, Chris H; Fessler, Michael B; Foster, Matthew W; Kelly, Rachel S; Lasky-Su, Jessica; Rogers, Angela J; Stringer, Kathleen A; Winston, Brent W

2017-12-01

This document presents the proceedings from the workshop entitled, "New Strategies and Challenges in Lung Proteomics and Metabolomics" held February 4th-5th, 2016, in Denver, Colorado. It was sponsored by the National Heart Lung Blood Institute, the American Thoracic Society, the Colorado Biological Mass Spectrometry Society, and National Jewish Health. The goal of this workshop was to convene, for the first time, relevant experts in lung proteomics and metabolomics to discuss and overcome specific challenges in these fields that are unique to the lung. The main objectives of this workshop were to identify, review, and/or understand: (1) emerging technologies in metabolomics and proteomics as applied to the study of the lung; (2) the unique composition and challenges of lung-specific biological specimens for metabolomic and proteomic analysis; (3) the diverse informatics approaches and databases unique to metabolomics and proteomics, with special emphasis on the lung; (4) integrative platforms across genetic and genomic databases that can be applied to lung-related metabolomic and proteomic studies; and (5) the clinical applications of proteomics and metabolomics. The major findings and conclusions of this workshop are summarized at the end of the report, and outline the progress and challenges that face these rapidly advancing fields.

Proteomic Analysis of the Neurotrophic Effect of Gelidium amansii in Primary Cultured Neurons.

PubMed

Hannan, Md Abdul; Mohibbullah, Md; Hong, Yong-Ki; Moon, Il Soo

2017-03-01

Gelidium amansii is an edible and economically important red alga consumed in South Eastern Asia. In previous studies, we reported that the ethanol extracts of G. amansii (GAE) has promising modulatory activity with respect to the morphological and functional maturation of hippocampal neurons in culture. In this study, we show that the chloroform (CHCl 3 ) subfraction of GAE and the ethyl acetate (EtOAc) fraction dose-dependently promoted neurite outgrowth, and their effects were comparable with that of GAE. We further assessed in cultured cortical neurons, proteins differentially expressed in the presence/absence of the GAE, CHCl 3 subfraction, and the EtOAc fraction by 2D-PAGE and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Proteomic data revealed that a number of proteins responsible for multiple cellular and biochemical functions vital for neuronal development and maturation were significantly upregulated in neurons treated with the GAE, CHCl 3 subfraction, and the EtOAc fraction. Of the identified proteins, profilin 2a, septin 7, cdc42, protein phosphatase 2A, DA11, eukaryotic translation initiation factor 5A-1, and γ-enolase are known to play important roles in neuritogenesis and dendritic arborization. Immunofluorescence data demonstrate that GAE-treated hippocampal neurons showed greater intensity ratios in the expressions of the septin 7 and cdc42 compared to vehicle control, validating their proteomic profiles. Together these results suggest that the GAE/CHCl 3 subfraction and EtOAc fraction promote neurite development by up or downregulating several key proteins.

Proteomic identification of Profilin1 as a corepressor of estrogen receptor alpha in MCF7 breast cancer cells.

PubMed

Kanaujiya, Jitendra Kumar; Lochab, Savita; Kapoor, Isha; Pal, Pooja; Datta, Dipak; Bhatt, Madan L B; Sanyal, Sabyasachi; Behre, Gerhard; Trivedi, Arun Kumar

2013-07-01

Nuclear receptor coregulators play an important role in the transcriptional regulation of nuclear receptors. In the present study, we aimed to identify estrogen receptor α (ERα) interacting proteins in Tamoxifen treated MCF7 cells. Using in vitro GST-pull down assay with ERα ligand-binding domain (ERα-LBD) and MS-based proteomics approach we identified Profilin1 as a novel ERα interacting protein. Profilin1 contains I/LXX/L/H/I amino acid signature motif required for corepressor interaction with ERα. We show that these two proteins physically interact with each other both in vitro as well as in vivo by GST-pull down and coimmunoprecipitation, respectively. We further show that these two proteins also colocalize together in the nucleus. Previous studies have reported reduced expression of Profilin1 in breast cancer; and here we found that Tamoxifen increases Profilin1 expression in MCF7 cells. Our data demonstrate that over expression of Profilin1 inhibits ERα-mediated transcriptional activation as well as its downstream target genes in ERα positive breast cancer cells MCF7. In addition, Profilin1 overexpression in MCF7 cells leads to inhibition of cell proliferation that apparently is due to enhanced apoptosis. In nutshell, these data indicate that MS-based proteomics approach identifies a novel ERα interacting protein Profilin1 that serves as a putative corepressor of ERα functions. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Highly abundant defense proteins in human sweat as revealed by targeted proteomics and label-free quantification mass spectrometry.

PubMed

Csősz, É; Emri, G; Kalló, G; Tsaprailis, G; Tőzsér, J

2015-10-01

The healthy human skin with its effective antimicrobial defense system forms an efficient barrier against invading pathogens. There is evidence suggesting that the composition of this chemical barrier varies between diseases, making the easily collected sweat an ideal candidate for biomarker discoveries. Our aim was to provide information about the normal composition of the sweat, and to study the chemical barrier found at the surface of skin. Sweat samples from healthy individuals were collected during sauna bathing, and the global protein panel was analysed by label-free mass spectrometry. SRM-based targeted proteomic methods were designed and stable isotope labelled reference peptides were used for method validation. Ninety-five sweat proteins were identified, 20 of them were novel proteins. It was shown that dermcidin is the most abundant sweat protein, and along with apolipoprotein D, clusterin, prolactin-inducible protein and serum albumin, they make up 91% of secreted sweat proteins. The roles of these highly abundant proteins were reviewed; all of which have protective functions, highlighting the importance of sweat glands in composing the first line of innate immune defense system, and maintaining the epidermal barrier integrity. Our findings with regard to the proteins forming the chemical barrier of the skin as determined by label-free quantification and targeted proteomics methods are in accordance with previous studies, and can be further used as a starting point for non-invasive sweat biomarker research. © 2015 European Academy of Dermatology and Venereology.

Mass spectrometry-based identification of allergens from Curvularia pallescens, a prevalent aerospore in India.

PubMed

Dey, Debarati; Saha, Bodhisattwa; Sircar, Gaurab; Ghosal, Kavita; Bhattacharya, Swati Gupta

2016-07-01

The worldwide prevalence of fungal allergy in recent years has augmented mining allergens from yet unexplored ones. Curvularia pallescens (CP) being a dominant aerospore in India and a major sensitiser on a wide range of allergic population, pose a serious threat to human health. Therefore, we aimed to identify novel allergens from CP in our present study. A cohort of 22 CP-sensitised patients was selected by positive Skin prick grade. Individual sera exhibited elevated specific IgE level and significant histamine release on a challenge with antigenic extract of CP. First gel-based profiling of CP proteome was done by 1- and 2-dimensional gel. Parallel 1- and 2-dimensional immunoblot were performed applying individual as well as pooled patient sera. Identification of the sero-reactive spots from the 2-dimensional gel was found to be challenging as CP was not previously sequenced. Hence, mass spectrometry-based proteomic workflow consisting of conventional database search was not alone sufficient. Therefore, de novo sequencing preceded homology search was implemented for further identification. Altogether 11 allergenic proteins including Brn-1, vacuolar protease, and fructose-bis-phosphate aldolase were identified with high statistical confidence (p<0.05). This is the first study to report on any allergens from CP. This kind of proteome-based analysis provided a catalogue of CP allergens that would lead an improved way of diagnosis and therapy of CP-related allergy. Copyright © 2016 Elsevier B.V. All rights reserved.

Validation of a robust proteomic analysis carried out on formalin-fixed paraffin-embedded tissues of the pancreas obtained from mouse and human.

PubMed

Kojima, Kyoko; Bowersock, Gregory J; Kojima, Chinatsu; Klug, Christopher A; Grizzle, William E; Mobley, James A

2012-11-01

A number of reports have recently emerged with focus on extraction of proteins from formalin-fixed paraffin-embedded (FFPE) tissues for MS analysis; however, reproducibility and robustness as compared to flash frozen controls is generally overlooked. The goal of this study was to identify and validate a practical and highly robust approach for the proteomics analysis of FFPE tissues. FFPE and matched frozen pancreatic tissues obtained from mice (n = 8) were analyzed using 1D-nanoLC-MS(MS)(2) following work up with commercially available kits. The chosen approach for FFPE tissues was found to be highly comparable to that of frozen. In addition, the total number of unique peptides identified between the two groups was highly similar, with 958 identified for FFPE and 1070 identified for frozen, with protein identifications that corresponded by approximately 80%. This approach was then applied to archived human FFPE pancreatic cancer specimens (n = 11) as compared to uninvolved tissues (n = 8), where 47 potential pancreatic ductal adenocarcinoma markers were identified as significantly increased, of which 28 were previously reported. Further, these proteins share strongly overlapping pathway associations to pancreatic cancer that include estrogen receptor α. Together, these data support the validation of an approach for the proteomic analysis of FFPE tissues that is straightforward and highly robust, which can also be effectively applied toward translational studies of disease. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

2D-DIGE-based proteome expression changes in leaves of rice seedlings exposed to low-level gamma radiation at Iitate village, Fukushima

PubMed Central

Hayashi, Gohei; Moro, Carlo F; Rohila, Jai Singh; Shibato, Junko; Kubo, Akihiro; Imanaka, Tetsuji; Kimura, Shinzo; Ozawa, Shoji; Fukutani, Satoshi; Endo, Satoru; Ichikawa, Katsuki; Agrawal, Ganesh Kumar; Shioda, Seiji; Hori, Motohide; Fukumoto, Manabu; Rakwal, Randeep

2015-01-01

The present study continues our previous research on investigating the biological effects of low-level gamma radiation in rice at the heavily contaminated Iitate village in Fukushima, by extending the experiments to unraveling the leaf proteome. 14-days-old plants of Japonica rice (Oryza sativa L. cv. Nipponbare) were subjected to gamma radiation level of upto 4 µSv/h, for 72 h. Following exposure, leaf samples were taken from the around 190 µSv/3 d exposed seedling and total proteins were extracted. The gamma irradiated leaf and control leaf (harvested at the start of the experiment) protein lysates were used in a 2-D differential gel electrophoresis (2D-DIGE) experiment using CyDye labeling in order to asses which spots were differentially represented, a novelty of the study. 2D-DIGE analysis revealed 91 spots with significantly different expression between samples (60 positive, 31 negative). MALDI-TOF and TOF/TOF mass spectrometry analyses revealed those as comprising of 59 different proteins (50 up-accumulated, 9 down-accumulated). The identified proteins were subdivided into 10 categories, according to their biological function, which indicated that the majority of the differentially expressed proteins consisted of the general (non-energy) metabolism and stress response categories. Proteome-wide data point to some effects of low-level gamma radiation exposure on the metabolism of rice leaves. PMID:26451896

Genome, Proteome and Structure of a T7-Like Bacteriophage of the Kiwifruit Canker Phytopathogen Pseudomonas syringae pv. actinidiae.

PubMed

Frampton, Rebekah A; Acedo, Elena Lopez; Young, Vivienne L; Chen, Danni; Tong, Brian; Taylor, Corinda; Easingwood, Richard A; Pitman, Andrew R; Kleffmann, Torsten; Bostina, Mihnea; Fineran, Peter C

2015-06-24

Pseudomonas syringae pv. actinidiae is an economically significant pathogen responsible for severe bacterial canker of kiwifruit (Actinidia sp.). Bacteriophages infecting this phytopathogen have potential as biocontrol agents as part of an integrated approach to the management of bacterial canker, and for use as molecular tools to study this bacterium. A variety of bacteriophages were previously isolated that infect P. syringae pv. actinidiae, and their basic properties were characterized to provide a framework for formulation of these phages as biocontrol agents. Here, we have examined in more detail φPsa17, a phage with the capacity to infect a broad range of P. syringae pv. actinidiae strains and the only member of the Podoviridae in this collection. Particle morphology was visualized using cryo-electron microscopy, the genome was sequenced, and its structural proteins were analysed using shotgun proteomics. These studies demonstrated that φPsa17 has a 40,525 bp genome, is a member of the T7likevirus genus and is closely related to the pseudomonad phages φPSA2 and gh-1. Eleven structural proteins (one scaffolding) were detected by proteomics and φPsa17 has a capsid of approximately 60 nm in diameter. No genes indicative of a lysogenic lifecycle were identified, suggesting the phage is obligately lytic. These features indicate that φPsa17 may be suitable for formulation as a biocontrol agent of P. syringae pv. actinidiae.

Organ tropic metastatic secretomes and exosomes in breast cancer

DTIC Science & Technology

2017-10-01

integrins, functionally relevant in organ- 11 tropic, specifically lung-tropic breast cancer metastasis, and tested these samples by ELISA (protein...guided by the results of our exosomal proteomics analysis, we had previously performed ELISA assays for plasma-derived exosomal integrins in breast

Elucidation of salt stress defense and tolerance mechanisms of crop plants using proteomics--current achievements and perspectives.

PubMed

Barkla, Bronwyn J; Castellanos-Cervantes, Thelma; de León, José L Diaz; Matros, Andrea; Mock, Hans-Peter; Perez-Alfocea, Francisco; Salekdeh, Ghasem H; Witzel, Katja; Zörb, Christian

2013-06-01

Salinity is a major threat limiting the productivity of crop plants. A clear demand for improving the salinity tolerance of the major crop plants is imposed by the rapidly growing world population. This review summarizes the achievements of proteomic studies to elucidate the response mechanisms of selected model and crop plants to cope with salinity stress. We also aim at identifying research areas, which deserve increased attention in future proteome studies, as a prerequisite to identify novel targets for breeding strategies. Such areas include the impact of plant-microbial communities on the salinity tolerance of crops under field conditions, the importance of hormone signaling in abiotic stress tolerance, and the significance of control mechanisms underlying the observed changes in the proteome patterns. We briefly highlight the impact of novel tools for future proteome studies and argue for the use of integrated approaches. The evaluation of genetic resources by means of novel automated phenotyping facilities will have a large impact on the application of proteomics especially in combination with metabolomics or transcriptomics. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantitative proteomic analysis reveals a simple strategy of global resource allocation in bacteria

PubMed Central

Hui, Sheng; Silverman, Josh M; Chen, Stephen S; Erickson, David W; Basan, Markus; Wang, Jilong; Hwa, Terence; Williamson, James R

2015-01-01

A central aim of cell biology was to understand the strategy of gene expression in response to the environment. Here, we study gene expression response to metabolic challenges in exponentially growing Escherichia coli using mass spectrometry. Despite enormous complexity in the details of the underlying regulatory network, we find that the proteome partitions into several coarse-grained sectors, with each sector's total mass abundance exhibiting positive or negative linear relations with the growth rate. The growth rate-dependent components of the proteome fractions comprise about half of the proteome by mass, and their mutual dependencies can be characterized by a simple flux model involving only two effective parameters. The success and apparent generality of this model arises from tight coordination between proteome partition and metabolism, suggesting a principle for resource allocation in proteome economy of the cell. This strategy of global gene regulation should serve as a basis for future studies on gene expression and constructing synthetic biological circuits. Coarse graining may be an effective approach to derive predictive phenomenological models for other ‘omics’ studies. PMID:25678603

Environmental Microbial Community Proteomics: Status, Challenges and Perspectives.

PubMed

Wang, Da-Zhi; Kong, Ling-Fen; Li, Yuan-Yuan; Xie, Zhang-Xian

2016-08-05

Microbial community proteomics, also termed metaproteomics, is an emerging field within the area of microbiology, which studies the entire protein complement recovered directly from a complex environmental microbial community at a given point in time. Although it is still in its infancy, microbial community proteomics has shown its powerful potential in exploring microbial diversity, metabolic potential, ecological function and microbe-environment interactions. In this paper, we review recent advances achieved in microbial community proteomics conducted in diverse environments, such as marine and freshwater, sediment and soil, activated sludge, acid mine drainage biofilms and symbiotic communities. The challenges facing microbial community proteomics are also discussed, and we believe that microbial community proteomics will greatly enhance our understanding of the microbial world and its interactions with the environment.

Proteomics of filamentous fungi.

PubMed

Kim, Yonghyun; Nandakumar, M P; Marten, Mark R

2007-09-01

Proteomic analysis, defined here as the global assessment of cellular proteins expressed in a particular biological state, is a powerful tool that can provide a systematic understanding of events at the molecular level. Proteomic studies of filamentous fungi have only recently begun to appear in the literature, despite the prevalence of these organisms in the biotechnology industry, and their importance as both human and plant pathogens. Here, we review recent publications that have used a proteomic approach to develop a better understanding of filamentous fungi, highlighting sample preparation methods and whole-cell cytoplasmic proteomics, as well as subproteomics of cell envelope, mitochondrial and secreted proteins.

Structural bioinformatics of the human spliceosomal proteome

PubMed Central

Korneta, Iga; Magnus, Marcin; Bujnicki, Janusz M.

2012-01-01

In this work, we describe the results of a comprehensive structural bioinformatics analysis of the spliceosomal proteome. We used fold recognition analysis to complement prior data on the ordered domains of 252 human splicing proteins. Examples of newly identified domains include a PWI domain in the U5 snRNP protein 200K (hBrr2, residues 258–338), while examples of previously known domains with a newly determined fold include the DUF1115 domain of the U4/U6 di-snRNP protein 90K (hPrp3, residues 540–683). We also established a non-redundant set of experimental models of spliceosomal proteins, as well as constructed in silico models for regions without an experimental structure. The combined set of structural models is available for download. Altogether, over 90% of the ordered regions of the spliceosomal proteome can be represented structurally with a high degree of confidence. We analyzed the reduced spliceosomal proteome of the intron-poor organism Giardia lamblia, and as a result, we proposed a candidate set of ordered structural regions necessary for a functional spliceosome. The results of this work will aid experimental and structural analyses of the spliceosomal proteins and complexes, and can serve as a starting point for multiscale modeling of the structure of the entire spliceosome. PMID:22573172

Missing Value Monitoring Enhances the Robustness in Proteomics Quantitation.

PubMed

Matafora, Vittoria; Corno, Andrea; Ciliberto, Andrea; Bachi, Angela

2017-04-07

In global proteomic analysis, it is estimated that proteins span from millions to less than 100 copies per cell. The challenge of protein quantitation by classic shotgun proteomic techniques relies on the presence of missing values in peptides belonging to low-abundance proteins that lowers intraruns reproducibility affecting postdata statistical analysis. Here, we present a new analytical workflow MvM (missing value monitoring) able to recover quantitation of missing values generated by shotgun analysis. In particular, we used confident data-dependent acquisition (DDA) quantitation only for proteins measured in all the runs, while we filled the missing values with data-independent acquisition analysis using the library previously generated in DDA. We analyzed cell cycle regulated proteins, as they are low abundance proteins with highly dynamic expression levels. Indeed, we found that cell cycle related proteins are the major components of the missing values-rich proteome. Using the MvM workflow, we doubled the number of robustly quantified cell cycle related proteins, and we reduced the number of missing values achieving robust quantitation for proteins over ∼50 molecules per cell. MvM allows lower quantification variance among replicates for low abundance proteins with respect to DDA analysis, which demonstrates the potential of this novel workflow to measure low abundance, dynamically regulated proteins.

Metabolic switches and adaptations deduced from the proteomes of Streptomyces coelicolor wild type and phoP mutant grown in batch culture.

PubMed

Thomas, Louise; Hodgson, David A; Wentzel, Alexander; Nieselt, Kay; Ellingsen, Trond E; Moore, Jonathan; Morrissey, Edward R; Legaie, Roxane; Wohlleben, Wolfgang; Rodríguez-García, Antonio; Martín, Juan F; Burroughs, Nigel J; Wellington, Elizabeth M H; Smith, Margaret C M

2012-02-01

Bacteria in the genus Streptomyces are soil-dwelling oligotrophs and important producers of secondary metabolites. Previously, we showed that global messenger RNA expression was subject to a series of metabolic and regulatory switches during the lifetime of a fermentor batch culture of Streptomyces coelicolor M145. Here we analyze the proteome from eight time points from the same fermentor culture and, because phosphate availability is an important regulator of secondary metabolite production, compare this to the proteome of a similar time course from an S. coelicolor mutant, INB201 (ΔphoP), defective in the control of phosphate utilization. The proteomes provide a detailed view of enzymes involved in central carbon and nitrogen metabolism. Trends in protein expression over the time courses were deduced from a protein abundance index, which also revealed the importance of stress pathway proteins in both cultures. As expected, the ΔphoP mutant was deficient in expression of PhoP-dependent genes, and several putatively compensatory metabolic and regulatory pathways for phosphate scavenging were detected. Notably there is a succession of switches that coordinately induce the production of enzymes for five different secondary metabolite biosynthesis pathways over the course of the batch cultures.

A proteomic approach to identify endosomal cargoes controlling cancer invasiveness

PubMed Central

Diaz-Vera, Jesica; Palmer, Sarah; Hernandez-Fernaud, Juan Ramon; Dornier, Emmanuel; Mitchell, Louise E.; Macpherson, Iain; Edwards, Joanne; Zanivan, Sara

2017-01-01

ABSTRACT We have previously shown that Rab17, a small GTPase associated with epithelial polarity, is specifically suppressed by ERK2 (also known as MAPK1) signalling to promote an invasive phenotype. However, the mechanisms through which Rab17 loss permits invasiveness, and the endosomal cargoes that are responsible for mediating this, are unknown. Using quantitative mass spectrometry-based proteomics, we have found that knockdown of Rab17 leads to a highly selective reduction in the cellular levels of a v-SNARE (Vamp8). Moreover, proteomics and immunofluorescence indicate that Vamp8 is associated with Rab17 at late endosomes. Reduced levels of Vamp8 promote transition between ductal carcinoma in situ (DCIS) and a more invasive phenotype. We developed an unbiased proteomic approach to elucidate the complement of receptors that redistributes between endosomes and the plasma membrane, and have pin-pointed neuropilin-2 (NRP2) as a key pro-invasive cargo of Rab17- and Vamp8-regulated trafficking. Indeed, reduced Rab17 or Vamp8 levels lead to increased mobilisation of NRP2-containing late endosomes and upregulated cell surface expression of NRP2. Finally, we show that NRP2 is required for the basement membrane disruption that accompanies the transition between DCIS and a more invasive phenotype. PMID:28062852

The proteome of Hypobaric Induced Hypoxic Lung: Insights from Temporal Proteomic Profiling for Biomarker Discovery

PubMed Central

Ahmad, Yasmin; Sharma, Narendra K.; Ahmad, Mohammad Faiz; Sharma, Manish; Garg, Iti; Srivastava, Mousami; Bhargava, Kalpana

2015-01-01

Exposure to high altitude induces physiological responses due to hypoxia. Lungs being at the first level to face the alterations in oxygen levels are critical to counter and balance these changes. Studies have been done analysing pulmonary proteome alterations in response to exposure to hypobaric hypoxia. However, such studies have reported the alterations at specific time points and do not reflect the gradual proteomic changes. These studies also identify the various biochemical pathways and responses induced after immediate exposure and the resolution of these effects in challenge to hypobaric hypoxia. In the present study, using 2-DE/MS approach, we attempt to resolve these shortcomings by analysing the proteome alterations in lungs in response to different durations of exposure to hypobaric hypoxia. Our study thus highlights the gradual and dynamic changes in pulmonary proteome following hypobaric hypoxia. For the first time, we also report the possible consideration of SULT1A1, as a biomarker for the diagnosis of high altitude pulmonary edema (HAPE). Higher SULT1A1 levels were observed in rats as well as in humans exposed to high altitude, when compared to sea-level controls. This study can thus form the basis for identifying biomarkers for diagnostic and prognostic purposes in responses to hypobaric hypoxia. PMID:26022216

The quest of the human proteome and the missing proteins: digging deeper.

PubMed

Reddy, Panga Jaipal; Ray, Sandipan; Srivastava, Sanjeeva

2015-05-01

Given the diverse range of transcriptional and post-transcriptional mechanisms of gene regulation, the estimates of the human proteome is likely subject to scientific surprises as the field of proteomics has gained momentum worldwide. In this regard, the establishment of the "Human Proteome Draft" using high-resolution mass spectrometry (MS), tissue microarrays, and immunohistochemistry by three independent research groups (laboratories of Pandey, Kuster, and Uhlen) accelerated the pace of proteomics research. The Chromosome Centric Human Proteome Project (C-HPP) has taken initiative towards the completion of the Human Proteome Project (HPP) so as to understand the proteomics correlates of common complex human diseases and biological diversity, not to mention person-to-person and population differences in response to drugs, nutrition, vaccines, and other health interventions and host-environment interactions. Although high-resolution MS-based and antibody microarray approaches have shown enormous promises, we are still unable to map the whole human proteome due to the presence of numerous "missing proteins." In December 2014, at the Indian Institute of Technology Bombay, Mumbai the 6(th) Annual Meeting of the Proteomics Society, India (PSI) and the International Proteomics Conference was held. As part of this interdisciplinary summit, a panel discussion session on "The Quest of the Human Proteome and Missing Proteins" was organized. Eminent scientists in the field of proteomics and systems biology, including Akhilesh Pandey, Gilbert S. Omenn, Mark S. Baker, and Robert L. Mortiz, shed light on different aspects of the human proteome drafts and missing proteins. Importantly, the possible reasons for the "missing proteins" in shotgun MS workflow were identified and debated by experts as low tissue expression, lack of enzymatic digestion site, or protein lost during extraction, among other contributing factors. To capture the missing proteins, the experts' collective view was to study the wider tissue range with multiple digesting enzymes and follow targeted proteomics workflow in particular. On the innovation trajectory from the proteomics laboratory to novel proteomics diagnostics and therapeutics in society, we will also need new conceptual frames for translation science and innovation strategy in proteomics. These will embody both technical as well as rigorous social science and humanities considerations to understand the correlates of the proteome from cell to society.

Platelet proteome reveals novel pathways of platelet activation and platelet-mediated immunoregulation in dengue.

PubMed

Trugilho, Monique Ramos de Oliveira; Hottz, Eugenio Damaceno; Brunoro, Giselle Villa Flor; Teixeira-Ferreira, André; Carvalho, Paulo Costa; Salazar, Gustavo Adolfo; Zimmerman, Guy A; Bozza, Fernando A; Bozza, Patrícia T; Perales, Jonas

2017-05-01

Dengue is the most prevalent human arbovirus disease worldwide. Dengue virus (DENV) infection causes syndromes varying from self-limiting febrile illness to severe dengue. Although dengue pathophysiology is not completely understood, it is widely accepted that increased inflammation plays important roles in dengue pathogenesis. Platelets are blood cells classically known as effectors of hemostasis which have been increasingly recognized to have major immune and inflammatory activities. Nevertheless, the phenotype and effector functions of platelets in dengue pathogenesis are not completely understood. Here we used quantitative proteomics to investigate the protein content of platelets in clinical samples from patients with dengue compared to platelets from healthy donors. Our assays revealed a set of 252 differentially abundant proteins. In silico analyses associated these proteins with key molecular events including platelet activation and inflammatory responses, and with events not previously attributed to platelets during dengue infection including antigen processing and presentation, proteasome activity, and expression of histones. From these results, we conducted functional assays using samples from a larger cohort of patients and demonstrated evidence for platelet activation indicated by P-selectin (CD62P) translocation and secretion of granule-stored chemokines by platelets. In addition, we found evidence that DENV infection triggers HLA class I synthesis and surface expression by a mechanism depending on functional proteasome activity. Furthermore, we demonstrate that cell-free histone H2A released during dengue infection binds to platelets, increasing platelet activation. These findings are consistent with functional importance of HLA class I, proteasome subunits, and histones that we found exclusively in proteome analysis of platelets in samples from dengue patients. Our study provides the first in-depth characterization of the platelet proteome in dengue, and sheds light on new mechanisms of platelet activation and platelet-mediated immune and inflammatory responses.

Architecture of a Host-Parasite Interface: Complex Targeting Mechanisms Revealed Through Proteomics.

PubMed

Gadelha, Catarina; Zhang, Wenzhu; Chamberlain, James W; Chait, Brian T; Wickstead, Bill; Field, Mark C

2015-07-01

Surface membrane organization and composition is key to cellular function, and membrane proteins serve many essential roles in endocytosis, secretion, and cell recognition. The surface of parasitic organisms, however, is a double-edged sword; this is the primary interface between parasites and their hosts, and those crucial cellular processes must be carried out while avoiding elimination by the host immune defenses. For extracellular African trypanosomes, the surface is partitioned such that all endo- and exocytosis is directed through a specific membrane region, the flagellar pocket, in which it is thought the majority of invariant surface proteins reside. However, very few of these proteins have been identified, severely limiting functional studies, and hampering the development of potential treatments. Here we used an integrated biochemical, proteomic and bioinformatic strategy to identify surface components of the human parasite Trypanosoma brucei. This surface proteome contains previously known flagellar pocket proteins as well as multiple novel components, and is significantly enriched in proteins that are essential for parasite survival. Molecules with receptor-like properties are almost exclusively parasite-specific, whereas transporter-like proteins are conserved in model organisms. Validation shows that the majority of surface proteome constituents are bona fide surface-associated proteins and, as expected, most present at the flagellar pocket. Moreover, the largest systematic analysis of trypanosome surface molecules to date provides evidence that the cell surface is compartmentalized into three distinct domains with free diffusion of molecules in each, but selective, asymmetric traffic between. This work provides a paradigm for the compartmentalization of a cell surface and a resource for its analysis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Mastitomics, the integrated omics of bovine milk in an experimental model of Streptococcus uberis mastitis: 2. Label-free relative quantitative proteomics† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6mb00290k Click here for additional data file.

PubMed Central

Mudaliar, Manikhandan; Tassi, Riccardo; Thomas, Funmilola C.; McNeilly, Tom N.; Weidt, Stefan K.; McLaughlin, Mark; Wilson, David; Burchmore, Richard; Herzyk, Pawel; Eckersall, P. David

2016-01-01

Mastitis, inflammation of the mammary gland, is the most common and costly disease of dairy cattle in the western world. It is primarily caused by bacteria, with Streptococcus uberis as one of the most prevalent causative agents. To characterize the proteome during Streptococcus uberis mastitis, an experimentally induced model of intramammary infection was used. Milk whey samples obtained from 6 cows at 6 time points were processed using label-free relative quantitative proteomics. This proteomic analysis complements clinical, bacteriological and immunological studies as well as peptidomic and metabolomic analysis of the same challenge model. A total of 2552 non-redundant bovine peptides were identified, and from these, 570 bovine proteins were quantified. Hierarchical cluster analysis and principal component analysis showed clear clustering of results by stage of infection, with similarities between pre-infection and resolution stages (0 and 312 h post challenge), early infection stages (36 and 42 h post challenge) and late infection stages (57 and 81 h post challenge). Ingenuity pathway analysis identified upregulation of acute phase protein pathways over the course of infection, with dominance of different acute phase proteins at different time points based on differential expression analysis. Antimicrobial peptides, notably cathelicidins and peptidoglycan recognition protein, were upregulated at all time points post challenge and peaked at 57 h, which coincided with 10 000-fold decrease in average bacterial counts. The integration of clinical, bacteriological, immunological and quantitative proteomics and other-omic data provides a more detailed systems level view of the host response to mastitis than has been achieved previously. PMID:27412694

Platelet proteome reveals novel pathways of platelet activation and platelet-mediated immunoregulation in dengue

PubMed Central

Teixeira-Ferreira, André; Carvalho, Paulo Costa; Salazar, Gustavo Adolfo; Zimmerman, Guy A.; Perales, Jonas

2017-01-01

Dengue is the most prevalent human arbovirus disease worldwide. Dengue virus (DENV) infection causes syndromes varying from self-limiting febrile illness to severe dengue. Although dengue pathophysiology is not completely understood, it is widely accepted that increased inflammation plays important roles in dengue pathogenesis. Platelets are blood cells classically known as effectors of hemostasis which have been increasingly recognized to have major immune and inflammatory activities. Nevertheless, the phenotype and effector functions of platelets in dengue pathogenesis are not completely understood. Here we used quantitative proteomics to investigate the protein content of platelets in clinical samples from patients with dengue compared to platelets from healthy donors. Our assays revealed a set of 252 differentially abundant proteins. In silico analyses associated these proteins with key molecular events including platelet activation and inflammatory responses, and with events not previously attributed to platelets during dengue infection including antigen processing and presentation, proteasome activity, and expression of histones. From these results, we conducted functional assays using samples from a larger cohort of patients and demonstrated evidence for platelet activation indicated by P-selectin (CD62P) translocation and secretion of granule-stored chemokines by platelets. In addition, we found evidence that DENV infection triggers HLA class I synthesis and surface expression by a mechanism depending on functional proteasome activity. Furthermore, we demonstrate that cell-free histone H2A released during dengue infection binds to platelets, increasing platelet activation. These findings are consistent with functional importance of HLA class I, proteasome subunits, and histones that we found exclusively in proteome analysis of platelets in samples from dengue patients. Our study provides the first in-depth characterization of the platelet proteome in dengue, and sheds light on new mechanisms of platelet activation and platelet-mediated immune and inflammatory responses. PMID:28542641

A comparative proteomics method for multiple samples based on a 18O-reference strategy and a quantitation and identification-decoupled strategy.

PubMed

Wang, Hongbin; Zhang, Yongqian; Gui, Shuqi; Zhang, Yong; Lu, Fuping; Deng, Yulin

2017-08-15

Comparisons across large numbers of samples are frequently necessary in quantitative proteomics. Many quantitative methods used in proteomics are based on stable isotope labeling, but most of these are only useful for comparing two samples. For up to eight samples, the iTRAQ labeling technique can be used. For greater numbers of samples, the label-free method has been used, but this method was criticized for low reproducibility and accuracy. An ingenious strategy has been introduced, comparing each sample against a 18 O-labeled reference sample that was created by pooling equal amounts of all samples. However, it is necessary to use proportion-known protein mixtures to investigate and evaluate this new strategy. Another problem for comparative proteomics of multiple samples is the poor coincidence and reproducibility in protein identification results across samples. In present study, a method combining 18 O-reference strategy and a quantitation and identification-decoupled strategy was investigated with proportion-known protein mixtures. The results obviously demonstrated that the 18 O-reference strategy had greater accuracy and reliability than other previously used comparison methods based on transferring comparison or label-free strategies. By the decoupling strategy, the quantification data acquired by LC-MS and the identification data acquired by LC-MS/MS are matched and correlated to identify differential expressed proteins, according to retention time and accurate mass. This strategy made protein identification possible for all samples using a single pooled sample, and therefore gave a good reproducibility in protein identification across multiple samples, and allowed for optimizing peptide identification separately so as to identify more proteins. Copyright © 2017 Elsevier B.V. All rights reserved.

Comparative Monomethylarginine Proteomics Suggests that Protein Arginine Methyltransferase 1 (PRMT1) is a Significant Contributor to Arginine Monomethylation in Toxoplasma gondii

PubMed Central

Yakubu, Rama R.; Silmon de Monerri, Natalie C.; Nieves, Edward; Kim, Kami; Weiss, Louis M.

2017-01-01

Arginine methylation is a common posttranslational modification found on nuclear and cytoplasmic proteins that has roles in transcriptional regulation, RNA metabolism and DNA repair. The protozoan parasite Toxoplasma gondii has a complex life cycle requiring transcriptional plasticity and has unique transcriptional regulatory pathways. Arginine methylation may play an important part in transcriptional regulation and splicing biology in this organism. The T. gondii genome contains five putative protein arginine methyltransferases (PRMTs), of which PRMT1 is important for cell division and growth. In order to better understand the function(s) of the posttranslational modification monomethyl arginine (MMA) in T. gondii, we performed a proteomic analysis of MMA proteins using affinity purification employing anti-MMA specific antibodies followed by mass spectrometry. The arginine monomethylome of T. gondii contains a large number of RNA binding proteins and multiple ApiAP2 transcription factors, suggesting a role for arginine methylation in RNA biology and transcriptional regulation. Surprisingly, 90% of proteins that are arginine monomethylated were detected as being phosphorylated in a previous phosphoproteomics study which raises the possibility of interplay between MMA and phosphorylation in this organism. Supporting this, a number of kinases are also arginine methylated. Because PRMT1 is thought to be a major PRMT in T. gondii, an organism which lacks a MMA-specific PRMT, we applied comparative proteomics to understand how PRMT1 might contribute to the MMA proteome in T. gondii. We identified numerous putative PRMT1 substrates, which include RNA binding proteins, transcriptional regulators (e.g. AP2 transcription factors), and kinases. Together, these data highlight the importance of MMA and PRMT1 in arginine methylation in T. gondii, as a potential regulator of a large number of processes including RNA biology and transcription. PMID:28143887

Detailed analysis of the plasma extracellular vesicle proteome after separation from lipoproteins.

PubMed

Karimi, Nasibeh; Cvjetkovic, Aleksander; Jang, Su Chul; Crescitelli, Rossella; Hosseinpour Feizi, Mohammad Ali; Nieuwland, Rienk; Lötvall, Jan; Lässer, Cecilia

2018-02-13

The isolation of extracellular vesicles (EVs) from blood is of great importance to understand the biological role of circulating EVs and to develop EVs as biomarkers of disease. Due to the concurrent presence of lipoprotein particles, however, blood is one of the most difficult body fluids to isolate EVs from. The aim of this study was to develop a robust method to isolate and characterise EVs from blood with minimal contamination by plasma proteins and lipoprotein particles. Plasma and serum were collected from healthy subjects, and EVs were isolated by size-exclusion chromatography (SEC), with most particles being present in fractions 8-12, while the bulk of the plasma proteins was present in fractions 11-28. Vesicle markers peaked in fractions 7-11; however, the same fractions also contained lipoprotein particles. The purity of EVs was improved by combining a density cushion with SEC to further separate lipoprotein particles from the vesicles, which reduced the contamination of lipoprotein particles by 100-fold. Using this novel isolation procedure, a total of 1187 proteins were identified in plasma EVs by mass spectrometry, of which several proteins are known as EV-associated proteins but have hitherto not been identified in the previous proteomic studies of plasma EVs. This study shows that SEC alone is unable to completely separate plasma EVs from lipoprotein particles. However, combining SEC with a density cushion significantly improved the separation of EVs from lipoproteins and allowed for a detailed analysis of the proteome of plasma EVs, thus making blood a viable source for EV biomarker discovery.

Proteomic study of periovarian adipose tissue in 17β-estradiol-treated and untreated ovariectomized rats.

PubMed

Amengual-Cladera, Emilia; Capllonch-Amer, Gabriela; Lladó, Isabel; Gianotti, Magdalena; Proenza, Ana M

2016-04-01

Taking into account the sexual dimorphism previously found in white adipose tissue (WAT) regarding mitochondrial function and biogenesis, as well as insulin sensitivity, the aim of this study was to go further into the role of sex hormones in this dimorphism. To achieve this objective, we used ovariectomized rats and performed a screening by means of proteomic analyses of the periovarian WAT, combined with a study of the protein levels of specific factors involved in mitochondrial function. Rats were ovariectomized at 5 weeks of age and subcutaneously injected every 48 h with corn-oil (OVX group) or with 17β-estradiol (E2, 10 μg/kg body mass; OVX + E2 group) for 4 weeks prior to sacrifice. Beside proteomic analysis, protein levels of Transcription Factor A, Mitochondrial (TFAM), cytochrome oxidase (COX)II, and COXIV were determined by Western blot, and mRNA levels of peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α, ERα, ERβ, lipoprotein lipase (LPL), peroxisome proliferator-activated receptor-γ (PPARγ), and adiponectin were quantified by real-time PCR. Our results show that ovariectomy leads to an increase in anabolic processes and inflammatory protein levels as well as to a decrease in some of the markers of mitochondrial function, which are restored, at least in part, by E2 supplementation. Indeed, this E2 supplementation seems to be counteracted by a decline in ERα and in the ERα to ERβ ratio values that could be directed to avoid an over-stimulation of the E2 signaling pathway, given the possibility of an activation of extra-gonadal steroid biosynthetic pathways.

Thalamic Proteome Changes and Behavioral Impairments in Thiamine-deficient Rats.

PubMed

Nunes, Polliana Toledo; Gómez-Mendoza, Diana Paola; Rezende, Cristiana Perdigão; Figueiredo, Henrique César Pereira; Ribeiro, Angela Maria

2018-06-09

Thiamine deficiency (TD) has been used as an experimental model in rodents to study the molecular mechanisms of neurodegeneration and its association with behavioral changes. The aims of the present study were to investigate the spatial cognitive performance of pyrithiamine-induced thiamine deficiency (PTD) in adult male rats and disclose the thalamic proteome alterations caused by a severe TD episode. After the onset of the neurological signs, such as seizure and/or loss of righting reflex, the TD treatment was interrupted. Following 15 days of recovery, all rats were submitted to the spatial cognitive tasks in the Morris Water Maze (MWM). The results show that the PTD rats exhibited deficits during the learning process, which was reverted by repeated training. However, despite the spatial cognitive recovery, some protein changes were not reversible. The proteomic analysis, using label-free quantification, revealed deregulation of 183 thalamic proteins. Using bioinformatic tools, these proteins were categorized according to Gene Ontology functional annotation and metabolic pathways. We show that a severe TD affects proteins involved in different biological processes, such as, oxidative stress, neurotransmitter synthesis and synaptic vesicle cycle. These could explain the outcome in neurotransmitter release changes caused by TD, previously observed by our group and by other authors. These findings disclose the role of key proteins and metabolic pathways probably involved in the neurodegeneration process induced by TD. These proteins represent relevant molecular targets for future studies focusing also on the molecular basis of selective vulnerability of some brain areas to TD insult. Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.

High Concentrations of Atmospheric Ammonia Induce Alterations in the Hepatic Proteome of Broilers (Gallus gallus): An iTRAQ-Based Quantitative Proteomic Analysis

PubMed Central

Zhang, Jize; Li, Cong; Tang, Xiangfang; Lu, Qingping; Sa, Renna; Zhang, Hongfu

2015-01-01

With the development of the poultry industry, ammonia, as a main contaminant in the air, is causing increasing problems with broiler health. To date, most studies of ammonia toxicity have focused on the nervous system and the gastrointestinal tract in mammals. However, few detailed studies have been conducted on the hepatic response to ammonia toxicity in poultry. The molecular mechanisms that underlie these effects remain unclear. In the present study, our group applied isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomic analysis to investigate changes in the protein profile change in hepatic tissue of broilers exposed to high concentrations of atmospheric ammonia, with the goal of characterizing the molecular mechanisms of chronic liver injury from exposure to high ambient levels of ammonia. Overall, 30 differentially expressed proteins that are involved in nutrient metabolism (energy, lipid, and amino acid), immune response, transcriptional and translational regulation, stress response, and detoxification were identified. In particular, two of these proteins, beta-1 galactosidase (GLB1) and a kinase (PRKA) anchor protein 8-like (AKAP8 L), were previously suggested to be potential biomarkers of chronic liver injury. In addition to the changes in the protein profile, serum parameters and histochemical analyses of hepatic tissue also showed extensive hepatic damage in ammonia-exposed broilers. Altogether, these findings suggest that longtime exposure to high concentrations of atmospheric ammonia can trigger chronic hepatic injury in broilers via different mechanisms, providing new information that can be used for intervention using nutritional strategies in the future. PMID:25901992

Proteomic analysis of trans-hemispheric motor cortex reorganization following contralateral C7 nerve transfer

PubMed Central

Yuan, Yin; Xu, Xiu-yue; Lao, Jie; Zhao, Xin

2018-01-01

Nerve transfer is the most common treatment for total brachial plexus avulsion injury. After nerve transfer, the movement of the injured limb may be activated by certain movements of the healthy limb at the early stage of recovery, i.e., trans-hemispheric reorganization. Previous studies have focused on functional magnetic resonance imaging and changes in brain-derived neurotrophic factor and growth associated protein 43, but there have been no proteomics studies. In this study, we designed a rat model of total brachial plexus avulsion injury involving contralateral C7 nerve transfer. Isobaric tags for relative and absolute quantitation and western blot assay were then used to screen differentially expressed proteins in bilateral motor cortices. We found that most differentially expressed proteins in both cortices of upper limb were associated with nervous system development and function (including neuron differentiation and development, axonogenesis, and guidance), microtubule and cytoskeleton organization, synapse plasticity, and transmission of nerve impulses. Two key differentially expressed proteins, neurofilament light (NFL) and Thy-1, were identified. In contralateral cortex, the NFL level was upregulated 2 weeks after transfer and downregulated at 1 and 5 months. The Thy-1 level was upregulated from 1 to 5 months. In the affected cortex, the NFL level increased gradually from 1 to 5 months. Western blot results of key differentially expressed proteins were consistent with the proteomic findings. These results indicate that NFL and Thy-1 play an important role in trans-hemispheric organization following total brachial plexus root avulsion and contralateral C7 nerve transfer. PMID:29557385

Evolution of complexity in the zebrafish synapse proteome

PubMed Central

Bayés, Àlex; Collins, Mark O.; Reig-Viader, Rita; Gou, Gemma; Goulding, David; Izquierdo, Abril; Choudhary, Jyoti S.; Emes, Richard D.; Grant, Seth G. N.

2017-01-01

The proteome of human brain synapses is highly complex and is mutated in over 130 diseases. This complexity arose from two whole-genome duplications early in the vertebrate lineage. Zebrafish are used in modelling human diseases; however, its synapse proteome is uncharacterized, and whether the teleost-specific genome duplication (TSGD) influenced complexity is unknown. We report the characterization of the proteomes and ultrastructure of central synapses in zebrafish and analyse the importance of the TSGD. While the TSGD increases overall synapse proteome complexity, the postsynaptic density (PSD) proteome of zebrafish has lower complexity than mammals. A highly conserved set of ∼1,000 proteins is shared across vertebrates. PSD ultrastructural features are also conserved. Lineage-specific proteome differences indicate that vertebrate species evolved distinct synapse types and functions. The data sets are a resource for a wide range of studies and have important implications for the use of zebrafish in modelling human synaptic diseases. PMID:28252024

A New Mass Spectrometry-compatible Degradable Surfactant for Tissue Proteomics

PubMed Central

Chang, Ying-Hua; Gregorich, Zachery R.; Chen, Albert J.; Hwang, Leekyoung; Guner, Huseyin; Yu, Deyang; Zhang, Jianyi; Ge, Ying

2015-01-01

Tissue proteomics is increasingly recognized for its role in biomarker discovery and disease mechanism investigation. However, protein solubility remains a significant challenge in mass spectrometry (MS)-based tissue proteomics. Conventional surfactants such as sodium dodecyl sulfate (SDS), the preferred surfactant for protein solubilization, are not compatible with MS. Herein, we have screened a library of surfactant-like compounds and discovered an MS-compatible degradable surfactant (MaSDeS) for tissue proteomics that solubilizes all categories of proteins with performance comparable to SDS. The use of MaSDeS in the tissue extraction significantly improves the total number of protein identifications from commonly used tissues, including tissue from the heart, liver, and lung. Notably, MaSDeS significantly enriches membrane proteins, which are often under-represented in proteomics studies. The acid degradable nature of MaSDeS makes it amenable for high-throughput mass spectrometry-based proteomics. In addition, the thermostability of MaSDeS allows for its use in experiments requiring high temperature to facilitate protein extraction and solubilization. Furthermore, we have shown that MaSDeS outperforms the other MS-compatible surfactants in terms of overall protein solubility and the total number of identified proteins in tissue proteomics. Thus, the use of MaSDeS will greatly advance tissue proteomics and realize its potential in basic biomedical and clinical research. MaSDeS could be utilized in a variety of proteomics studies as well as general biochemical and biological experiments that employ surfactants for protein solubilization. PMID:25589168

Interlaboratory Study Characterizing a Yeast Performance Standard for Benchmarking LC-MS Platform Performance*

PubMed Central

Paulovich, Amanda G.; Billheimer, Dean; Ham, Amy-Joan L.; Vega-Montoto, Lorenzo; Rudnick, Paul A.; Tabb, David L.; Wang, Pei; Blackman, Ronald K.; Bunk, David M.; Cardasis, Helene L.; Clauser, Karl R.; Kinsinger, Christopher R.; Schilling, Birgit; Tegeler, Tony J.; Variyath, Asokan Mulayath; Wang, Mu; Whiteaker, Jeffrey R.; Zimmerman, Lisa J.; Fenyo, David; Carr, Steven A.; Fisher, Susan J.; Gibson, Bradford W.; Mesri, Mehdi; Neubert, Thomas A.; Regnier, Fred E.; Rodriguez, Henry; Spiegelman, Cliff; Stein, Stephen E.; Tempst, Paul; Liebler, Daniel C.

2010-01-01

Optimal performance of LC-MS/MS platforms is critical to generating high quality proteomics data. Although individual laboratories have developed quality control samples, there is no widely available performance standard of biological complexity (and associated reference data sets) for benchmarking of platform performance for analysis of complex biological proteomes across different laboratories in the community. Individual preparations of the yeast Saccharomyces cerevisiae proteome have been used extensively by laboratories in the proteomics community to characterize LC-MS platform performance. The yeast proteome is uniquely attractive as a performance standard because it is the most extensively characterized complex biological proteome and the only one associated with several large scale studies estimating the abundance of all detectable proteins. In this study, we describe a standard operating protocol for large scale production of the yeast performance standard and offer aliquots to the community through the National Institute of Standards and Technology where the yeast proteome is under development as a certified reference material to meet the long term needs of the community. Using a series of metrics that characterize LC-MS performance, we provide a reference data set demonstrating typical performance of commonly used ion trap instrument platforms in expert laboratories; the results provide a basis for laboratories to benchmark their own performance, to improve upon current methods, and to evaluate new technologies. Additionally, we demonstrate how the yeast reference, spiked with human proteins, can be used to benchmark the power of proteomics platforms for detection of differentially expressed proteins at different levels of concentration in a complex matrix, thereby providing a metric to evaluate and minimize preanalytical and analytical variation in comparative proteomics experiments. PMID:19858499

Systems Biology Analysis of Zymomonas mobilis ZM4 Ethanol Stress Responses

PubMed Central

Yang, Shihui; Pan, Chongle; Tschaplinski, Timothy J.; Hurst, Gregory B.; Engle, Nancy L.; Zhou, Wen; Dam, PhuongAn; Xu, Ying; Rodriguez, Miguel; Dice, Lezlee; Johnson, Courtney M.; Davison, Brian H.; Brown, Steven D.

2013-01-01

Background Zymomonas mobilis ZM4 is a capable ethanologenic bacterium with high ethanol productivity and ethanol tolerance. Previous studies indicated that several stress-related proteins and changes in the ZM4 membrane lipid composition may contribute to ethanol tolerance. However, the molecular mechanisms of its ethanol stress response have not been elucidated fully. Methodology/Principal Findings In this study, ethanol stress responses were investigated using systems biology approaches. Medium supplementation with an initial 47 g/L (6% v/v) ethanol reduced Z. mobilis ZM4 glucose consumption, growth rate and ethanol productivity compared to that of untreated controls. A proteomic analysis of early exponential growth identified about one thousand proteins, or approximately 55% of the predicted ZM4 proteome. Proteins related to metabolism and stress response such as chaperones and key regulators were more abundant in the early ethanol stress condition. Transcriptomic studies indicated that the response of ZM4 to ethanol is dynamic, complex and involves many genes from all the different functional categories. Most down-regulated genes were related to translation and ribosome biogenesis, while the ethanol-upregulated genes were mostly related to cellular processes and metabolism. Transcriptomic data were used to update Z. mobilis ZM4 operon models. Furthermore, correlations among the transcriptomic, proteomic and metabolic data were examined. Among significantly expressed genes or proteins, we observe higher correlation coefficients when fold-change values are higher. Conclusions Our study has provided insights into the responses of Z. mobilis to ethanol stress through an integrated “omics” approach for the first time. This systems biology study elucidated key Z. mobilis ZM4 metabolites, genes and proteins that form the foundation of its distinctive physiology and its multifaceted response to ethanol stress. PMID:23874800

Marine proteomics: a critical assessment of an emerging technology.

PubMed

Slattery, Marc; Ankisetty, Sridevi; Corrales, Jone; Marsh-Hunkin, K Erica; Gochfeld, Deborah J; Willett, Kristine L; Rimoldi, John M

2012-10-26

The application of proteomics to marine sciences has increased in recent years because the proteome represents the interface between genotypic and phenotypic variability and, thus, corresponds to the broadest possible biomarker for eco-physiological responses and adaptations. Likewise, proteomics can provide important functional information regarding biosynthetic pathways, as well as insights into mechanism of action, of novel marine natural products. The goal of this review is to (1) explore the application of proteomics methodologies to marine systems, (2) assess the technical approaches that have been used, and (3) evaluate the pros and cons of this proteomic research, with the intent of providing a critical analysis of its future roles in marine sciences. To date, proteomics techniques have been utilized to investigate marine microbe, plant, invertebrate, and vertebrate physiology, developmental biology, seafood safety, susceptibility to disease, and responses to environmental change. However, marine proteomics studies often suffer from poor experimental design, sample processing/optimization difficulties, and data analysis/interpretation issues. Moreover, a major limitation is the lack of available annotated genomes and proteomes for most marine organisms, including several "model species". Even with these challenges in mind, there is no doubt that marine proteomics is a rapidly expanding and powerful integrative molecular research tool from which our knowledge of the marine environment, and the natural products from this resource, will be significantly expanded.

Proteomics Insights into Autophagy.

PubMed

Cudjoe, Emmanuel K; Saleh, Tareq; Hawkridge, Adam M; Gewirtz, David A

2017-10-01

Autophagy, a conserved cellular process by which cells recycle their contents either to maintain basal homeostasis or in response to external stimuli, has for the past two decades become one of the most studied physiological processes in cell biology. The 2016 Nobel Prize in Medicine and Biology awarded to Dr. Ohsumi Yoshinori, one of the first scientists to characterize this cellular mechanism, attests to its importance. The induction and consequent completion of the process of autophagy results in wide ranging changes to the cellular proteome as well as the secretome. MS-based proteomics affords the ability to measure, in an unbiased manner, the ubiquitous changes that occur when autophagy is initiated and progresses in the cell. The continuous improvements and advances in mass spectrometers, especially relating to ionization sources and detectors, coupled with advances in proteomics experimental design, has made it possible to study autophagy, among other process, in great detail. Innovative labeling strategies and protein separation techniques as well as complementary methods including immuno-capture/blotting/staining have been used in proteomics studies to provide more specific protein identification. In this review, we will discuss recent advances in proteomics studies focused on autophagy. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Proteomic Analysis of the Human Skin Proteome after In Vivo Treatment with Sodium Dodecyl Sulphate

PubMed Central

Parkinson, Erika; Skipp, Paul; Aleksic, Maja; Garrow, Andrew; Dadd, Tony; Hughes, Michael; Clough, Geraldine; O′Connor, C. David

2014-01-01

Background Skin has a variety of functions that are incompletely understood at the molecular level. As the most accessible tissue in the body it often reveals the first signs of inflammation or infection and also represents a potentially valuable source of biomarkers for several diseases. In this study we surveyed the skin proteome qualitatively using gel electrophoresis, liquid chromatography tandem mass spectrometry (GeLC-MS/MS) and quantitatively using an isobaric tagging strategy (iTRAQ) to characterise the response of human skin following exposure to sodium dodecyl sulphate (SDS). Results A total of 653 skin proteins were assigned, 159 of which were identified using GeLC-MS/MS and 616 using iTRAQ, representing the most comprehensive proteomic study in human skin tissue. Statistical analysis of the available iTRAQ data did not reveal any significant differences in the measured skin proteome after 4 hours exposure to the model irritant SDS. Conclusions This study represents the first step in defining the critical response to an irritant at the level of the proteome and provides a valuable resource for further studies at the later stages of irritant exposure. PMID:24849295

Quantitative proteomics to study carbapenem resistance in Acinetobacter baumannii

PubMed Central

Tiwari, Vishvanath; Tiwari, Monalisa

2014-01-01

Acinetobacter baumannii is an opportunistic pathogen causing pneumonia, respiratory infections and urinary tract infections. The prevalence of this lethal pathogen increases gradually in the clinical setup where it can grow on artificial surfaces, utilize ethanol as a carbon source. Moreover it resists desiccation. Carbapenems, a β-lactam, are the most commonly prescribed drugs against A. baumannii. Resistance against carbapenem has emerged in Acinetobacter baumannii which can create significant health problems and is responsible for high morbidity and mortality. With the development of quantitative proteomics, a considerable progress has been made in the study of carbapenem resistance of Acinetobacter baumannii. Recent updates showed that quantitative proteomics has now emerged as an important tool to understand the carbapenem resistance mechanism in Acinetobacter baumannii. Present review also highlights the complementary nature of different quantitative proteomic methods used to study carbapenem resistance and suggests to combine multiple proteomic methods for understanding the response to antibiotics by Acinetobacter baumannii. PMID:25309531

Design and analysis issues in quantitative proteomics studies.

PubMed

Karp, Natasha A; Lilley, Kathryn S

2007-09-01

Quantitative proteomics is the comparison of distinct proteomes which enables the identification of protein species which exhibit changes in expression or post-translational state in response to a given stimulus. Many different quantitative techniques are being utilized and generate large datasets. Independent of the technique used, these large datasets need robust data analysis to ensure valid conclusions are drawn from such studies. Approaches to address the problems that arise with large datasets are discussed to give insight into the types of statistical analyses of data appropriate for the various experimental strategies that can be employed by quantitative proteomic studies. This review also highlights the importance of employing a robust experimental design and highlights various issues surrounding the design of experiments. The concepts and examples discussed within will show how robust design and analysis will lead to confident results that will ensure quantitative proteomics delivers.

A Proteomic Study of Clavibacter Michiganensis Subsp. Michiganensis Culture Supernatants

PubMed Central

Hiery, Eva; Poetsch, Ansgar; Moosbauer, Tanja; Amin, Bushra; Hofmann, Jörg; Burkovski, Andreas

2015-01-01

Clavibacter michiganensis, subsp. michiganensis is a Gram-positive plant pathogen infecting tomato (Solanum lycopersicum). Despite a considerable economic importance due to significant losses of infected plants and fruits, knowledge about virulence factors of C. michiganensis subsp. michiganensis and host-pathogen interactions on a molecular level are rather limited. In the study presented here, the proteome of culture supernatants from C. michiganensis subsp. michiganensis NCPPB382 was analyzed. In total, 1872 proteins were identified in M9 and 1766 proteins in xylem mimicking medium. Filtration of supernatants before protein precipitation reduced these to 1276 proteins in M9 and 976 proteins in the xylem mimicking medium culture filtrate. The results obtained indicate that C. michiganensis subsp. michiganensis reacts to a sucrose- and glucose-depleted medium similar to the xylem sap by utilizing amino acids and host cell polymers as well as their degradation products, mainly peptides, amino acids and various C5 and C6 sugars. Interestingly, the bacterium expresses the previously described virulence factors Pat-1 and CelA not exclusively after host cell contact in planta but already in M9 minimal and xylem mimicking medium. PMID:28248277

Label-free quantitative proteomic analysis of human plasma-derived microvesicles to find protein signatures of abdominal aortic aneurysms.

PubMed

Martinez-Pinna, Roxana; Gonzalez de Peredo, Anne; Monsarrat, Bernard; Burlet-Schiltz, Odile; Martin-Ventura, Jose Luis

2014-08-01

To find potential biomarkers of abdominal aortic aneurysms (AAA), we performed a differential proteomic study based on human plasma-derived microvesicles. Exosomes and microparticles isolated from plasma of AAA patients and control subjects (n = 10 each group) were analyzed by a label-free quantitative MS-based strategy. Homemade and publicly available software packages have been used for MS data analysis. The application of two kinds of bioinformatic tools allowed us to find differential protein profiles from AAA patients. Some of these proteins found by the two analysis methods belong to main pathological mechanisms of AAA such as oxidative stress, immune-inflammation, and thrombosis. Data analysis from label-free MS-based experiments requires the use of sophisticated bioinformatic approaches to perform quantitative studies from complex protein mixtures. The application of two of these bioinformatic tools provided us a preliminary list of differential proteins found in plasma-derived microvesicles not previously associated to AAA, which could help us to understand the pathological mechanisms related to this disease. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Statistical Methods for Proteomic Biomarker Discovery based on Feature Extraction or Functional Modeling Approaches.

PubMed

Morris, Jeffrey S

2012-01-01

In recent years, developments in molecular biotechnology have led to the increased promise of detecting and validating biomarkers, or molecular markers that relate to various biological or medical outcomes. Proteomics, the direct study of proteins in biological samples, plays an important role in the biomarker discovery process. These technologies produce complex, high dimensional functional and image data that present many analytical challenges that must be addressed properly for effective comparative proteomics studies that can yield potential biomarkers. Specific challenges include experimental design, preprocessing, feature extraction, and statistical analysis accounting for the inherent multiple testing issues. This paper reviews various computational aspects of comparative proteomic studies, and summarizes contributions I along with numerous collaborators have made. First, there is an overview of comparative proteomics technologies, followed by a discussion of important experimental design and preprocessing issues that must be considered before statistical analysis can be done. Next, the two key approaches to analyzing proteomics data, feature extraction and functional modeling, are described. Feature extraction involves detection and quantification of discrete features like peaks or spots that theoretically correspond to different proteins in the sample. After an overview of the feature extraction approach, specific methods for mass spectrometry ( Cromwell ) and 2D gel electrophoresis ( Pinnacle ) are described. The functional modeling approach involves modeling the proteomic data in their entirety as functions or images. A general discussion of the approach is followed by the presentation of a specific method that can be applied, wavelet-based functional mixed models, and its extensions. All methods are illustrated by application to two example proteomic data sets, one from mass spectrometry and one from 2D gel electrophoresis. While the specific methods presented are applied to two specific proteomic technologies, MALDI-TOF and 2D gel electrophoresis, these methods and the other principles discussed in the paper apply much more broadly to other expression proteomics technologies.

Proteomic changes in chicken plasma induced by Salmonella typhimurium lipopolysaccharides

USDA-ARS?s Scientific Manuscript database

Lipopolysaccharides (LPS) are cell wall components of gram-negative bacteria that cause inflammation and sickness through genetic and proteomic activation. The objective of our study was to identify the proteomic changes in plasma associated with inflammation induced by LPS treatment. Five-week-old ...

Wheat proteomics: proteome modulation and abiotic stress acclimation

PubMed Central

Komatsu, Setsuko; Kamal, Abu H. M.; Hossain, Zahed

2014-01-01

Cellular mechanisms of stress sensing and signaling represent the initial plant responses to adverse conditions. The development of high-throughput “Omics” techniques has initiated a new era of the study of plant molecular strategies for adapting to environmental changes. However, the elucidation of stress adaptation mechanisms in plants requires the accurate isolation and characterization of stress-responsive proteins. Because the functional part of the genome, namely the proteins and their post-translational modifications, are critical for plant stress responses, proteomic studies provide comprehensive information about the fine-tuning of cellular pathways that primarily involved in stress mitigation. This review summarizes the major proteomic findings related to alterations in the wheat proteomic profile in response to abiotic stresses. Moreover, the strengths and weaknesses of different sample preparation techniques, including subcellular protein extraction protocols, are discussed in detail. The continued development of proteomic approaches in combination with rapidly evolving bioinformatics tools and interactive databases will facilitate understanding of the plant mechanisms underlying stress tolerance. PMID:25538718

Systematic Comparison of Label-Free, Metabolic Labeling, and Isobaric Chemical Labeling for Quantitative Proteomics on LTQ Orbitrap Velos

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Zhou; Adams, Rachel M; Chourey, Karuna

2012-01-01

A variety of quantitative proteomics methods have been developed, including label-free, metabolic labeling, and isobaric chemical labeling using iTRAQ or TMT. Here, these methods were compared in terms of the depth of proteome coverage, quantification accuracy, precision, and reproducibility using a high-performance hybrid mass spectrometer, LTQ Orbitrap Velos. Our results show that (1) the spectral counting method provides the deepest proteome coverage for identification, but its quantification performance is worse than labeling-based approaches, especially the quantification reproducibility; (2) metabolic labeling and isobaric chemical labeling are capable of accurate, precise, and reproducible quantification and provide deep proteome coverage for quantification. Isobaricmore » chemical labeling surpasses metabolic labeling in terms of quantification precision and reproducibility; (3) iTRAQ and TMT perform similarly in all aspects compared in the current study using a CID-HCD dual scan configuration. Based on the unique advantages of each method, we provide guidance for selection of the appropriate method for a quantitative proteomics study.« less

Current advances in esophageal cancer proteomics.

PubMed

Uemura, Norihisa; Kondo, Tadashi

2015-06-01

We review the current status of proteomics for esophageal cancer (EC) from a clinician's viewpoint. The ultimate goal of cancer proteomics is the improvement of clinical outcome. The proteome as a functional translation of the genome is a straightforward representation of genomic mechanisms that trigger carcinogenesis. Cancer proteomics has identified the mechanisms of carcinogenesis and tumor progression, detected biomarker candidates for early diagnosis, and provided novel therapeutic targets for personalized treatments. Our review focuses on three major topics in EC proteomics: diagnostics, treatment, and molecular mechanisms. We discuss the major histological differences between EC types, i.e., esophageal squamous cell carcinoma and adenocarcinoma, and evaluate the clinical significance of published proteomics studies, including promising diagnostic biomarkers and novel therapeutic targets, which should be further validated prior to launching clinical trials. Multi-disciplinary collaborations between basic scientists, clinicians, and pathologists should be established for inter-institutional validation. In conclusion, EC proteomics has provided significant results, which after thorough validation, should lead to the development of novel clinical tools and improvement of the clinical outcome for esophageal cancer patients. This article is part of a Special Issue entitled: Medical Proteomics. Copyright © 2014 Elsevier B.V. All rights reserved.

Proteomics reveals the effects of sustained weight loss on the human plasma proteome.

PubMed

Geyer, Philipp E; Wewer Albrechtsen, Nicolai J; Tyanova, Stefka; Grassl, Niklas; Iepsen, Eva W; Lundgren, Julie; Madsbad, Sten; Holst, Jens J; Torekov, Signe S; Mann, Matthias

2016-12-22

Sustained weight loss is a preferred intervention in a wide range of metabolic conditions, but the effects on an individual's health state remain ill-defined. Here, we investigate the plasma proteomes of a cohort of 43 obese individuals that had undergone 8 weeks of 12% body weight loss followed by a year of weight maintenance. Using mass spectrometry-based plasma proteome profiling, we measured 1,294 plasma proteomes. Longitudinal monitoring of the cohort revealed individual-specific protein levels with wide-ranging effects of losing weight on the plasma proteome reflected in 93 significantly affected proteins. The adipocyte-secreted SERPINF1 and apolipoprotein APOF1 were most significantly regulated with fold changes of -16% and +37%, respectively (P < 10 -13 ), and the entire apolipoprotein family showed characteristic differential regulation. Clinical laboratory parameters are reflected in the plasma proteome, and eight plasma proteins correlated better with insulin resistance than the known marker adiponectin. Nearly all study participants benefited from weight loss regarding a ten-protein inflammation panel defined from the proteomics data. We conclude that plasma proteome profiling broadly evaluates and monitors intervention in metabolic diseases. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

Crystal Structure and Proteomics Analysis of Empty Virus-like Particles of Cowpea Mosaic Virus

PubMed Central

Huynh, Nhung T.; Hesketh, Emma L.; Saxena, Pooja; Meshcheriakova, Yulia; Ku, You-Chan; Hoang, Linh T.; Johnson, John E.; Ranson, Neil A.; Lomonossoff, George P.; Reddy, Vijay S.

2016-01-01

SUMMARY Empty virus-like particles (eVLPs) of Cowpea mosaic virus (CPMV) are currently being utilized as reagents in various biomedical and nanotechnology applications. Here, we report the crystal structure of CPMV eVLPs determined using X-ray crystallography at 2.3 Å resolution and compare it with previously reported cryo-electron microscopy (cryo-EM) of eVLPs and virion crystal structures. Although the X-ray and cryo-EM structures of eVLPs are mostly similar, there exist significant differences at the C terminus of the small (S) subunit. The intact C terminus of the S subunit plays a critical role in enabling the efficient assembly of CPMV virions and eVLPs, but undergoes proteolysis after particle formation. In addition, we report the results of mass spectrometry-based proteomics analysis of coat protein subunits from CPMV eVLPs and virions that identify the C termini of S subunits undergo proteolytic cleavages at multiple sites instead of a single cleavage site as previously observed. PMID:27021160

The Use of Functional Genomics in Conjunction with Metabolomics for Mycobacterium tuberculosis Research

PubMed Central

Swanepoel, Conrad C.

2014-01-01

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a fatal infectious disease, resulting in 1.4 million deaths globally per annum. Over the past three decades, genomic studies have been conducted in an attempt to elucidate the functionality of the genome of the pathogen. However, many aspects of this complex genome remain largely unexplored, as approaches like genomics, proteomics, and transcriptomics have failed to characterize them successfully. In turn, metabolomics, which is relatively new to the “omics” revolution, has shown great potential for investigating biological systems or their modifications. Furthermore, when these data are interpreted in combination with previously acquired genomics, proteomics and transcriptomics data, using what is termed a systems biology approach, a more holistic understanding of these systems can be achieved. In this review we discuss how metabolomics has contributed so far to characterizing TB, with emphasis on the resulting improved elucidation of M. tuberculosis in terms of (1) metabolism, (2) growth and replication, (3) pathogenicity, and (4) drug resistance, from the perspective of systems biology. PMID:24771957

Integration of Proteomic, Transcriptional, and Interactome Data Reveals Hidden Signaling Components

PubMed Central

Huang, Shao-shan Carol; Fraenkel, Ernest

2009-01-01

Cellular signaling and regulatory networks underlie fundamental biological processes such as growth, differentiation, and response to the environment. Although there are now various high-throughput methods for studying these processes, knowledge of them remains fragmentary. Typically, the vast majority of hits identified by transcriptional, proteomic, and genetic assays lie outside of the expected pathways. These unexpected components of the cellular response are often the most interesting, because they can provide new insights into biological processes and potentially reveal new therapeutic approaches. However, they are also the most difficult to interpret. We present a technique, based on the Steiner tree problem, that uses previously reported protein-protein and protein-DNA interactions to determine how these hits are organized into functionally coherent pathways, revealing many components of the cellular response that are not readily apparent in the original data. Applied simultaneously to phosphoproteomic and transcriptional data for the yeast pheromone response, it identifies changes in diverse cellular processes that extend far beyond the expected pathways. PMID:19638617

Global Survey of Protein Expression during Gonadal Sex Determination in Mice*

PubMed Central

Ewen, Katherine; Baker, Mark; Wilhelm, Dagmar; Aitken, R. John; Koopman, Peter

2009-01-01

The development of an embryo as male or female depends on differentiation of the gonads as either testes or ovaries. A number of genes are known to be important for gonadal differentiation, but our understanding of the regulatory networks underpinning sex determination remains fragmentary. To advance our understanding of sexual development beyond the transcriptome level, we performed the first global survey of the mouse gonad proteome at the time of sex determination by using two-dimensional nanoflow LC-MS/MS. The resulting data set contains a total of 1037 gene products (154 non-redundant and 883 redundant proteins) identified from 620 peptides. Functional classification and biological network construction suggested that the identified proteins primarily serve in RNA post-transcriptional modification and trafficking, protein synthesis and folding, and post-translational modification. The data set contains potential novel regulators of gonad development and sex determination not revealed previously by transcriptomics and proteomics studies and more than 60 proteins with potential links to human disorders of sexual development. PMID:19617587

Global Analysis of Palmitoylated Proteins in Toxoplasma gondii.

PubMed

Foe, Ian T; Child, Matthew A; Majmudar, Jaimeen D; Krishnamurthy, Shruthi; van der Linden, Wouter A; Ward, Gary E; Martin, Brent R; Bogyo, Matthew

2015-10-14

Post-translational modifications (PTMs) such as palmitoylation are critical for the lytic cycle of the protozoan parasite Toxoplasma gondii. While palmitoylation is involved in invasion, motility, and cell morphology, the proteins that utilize this PTM remain largely unknown. Using a chemical proteomic approach, we report a comprehensive analysis of palmitoylated proteins in T. gondii, identifying a total of 282 proteins, including cytosolic, membrane-associated, and transmembrane proteins. From this large set of palmitoylated targets, we validate palmitoylation of proteins involved in motility (myosin light chain 1, myosin A), cell morphology (PhIL1), and host cell invasion (apical membrane antigen 1, AMA1). Further studies reveal that blocking AMA1 palmitoylation enhances the release of AMA1 and other invasion-related proteins from apical secretory organelles, suggesting a previously unrecognized role for AMA1. These findings suggest that palmitoylation is ubiquitous throughout the T. gondii proteome and reveal insights into the biology of this important human pathogen. Copyright © 2015 Elsevier Inc. All rights reserved.

Transcriptome and proteome analysis of Salmonella enterica serovar Typhimurium systemic infection of wild type and immune-deficient mice

PubMed Central

Oshota, Olusegun; Fookes, Maria; Schreiber, Fernanda; Chaudhuri, Roy R.; Yu, Lu; Clare, Simon; Choudhary, Jyoti; Thomson, Nicholas R.; Lio, Pietro

2017-01-01

Salmonella enterica are a threat to public health. Current vaccines are not fully effective. The ability to grow in infected tissues within phagocytes is required for S. enterica virulence in systemic disease. As the infection progresses the bacteria are exposed to a complex host immune response. Consequently, in order to continue growing in the tissues, S. enterica requires the coordinated regulation of fitness genes. Bacterial gene regulation has so far been investigated largely using exposure to artificial environmental conditions or to in vitro cultured cells, and little information is available on how S. enterica adapts in vivo to sustain cell division and survival. We have studied the transcriptome, proteome and metabolic flux of Salmonella, and the transcriptome of the host during infection of wild type C57BL/6 and immune-deficient gp91-/-phox mice. Our analyses advance the understanding of how S. enterica and the host behaves during infection to a more sophisticated level than has previously been reported. PMID:28796780

Identification of Salmonella Typhimurium deubiquitinase SseL substrates by immunoaffinity enrichment and quantitative proteomic analysis

DOE PAGES

Nakayasu, Ernesto S.; Sydor, Michael A.; Brown, Roslyn N.; ...

2015-07-06

Ubiquitination is a key protein post-translational modification that regulates many important cellular pathways and whose levels are regulated by equilibrium between the activities of ubiquitin ligases and deubiquitinases. Here we present a method to identify specific deubiquitinase substrates based on treatment of cell lysates with recombinant enzymes, immunoaffinity purification and global quantitative proteomic analysis. As model system to identify substrates, we used a virulence-related deubiquitinase secreted by Salmonella enterica serovar Typhimurium into the host cells, SseL. By using this approach two SseL substrates were identified in RAW 264.7 murine macrophage-like cell line, S100A6 and het-erogeneous nuclear ribonuclear protein K, inmore » addition to the previously reported K63-linked ubiquitin chains. These substrates were further validated by a combination of enzymatic and binding assays. Finally, this method can be used for the systematic identification of substrates of deubiquitinases from other organisms and applied to study their functions in physiology and disease.« less

Bicarbonate Induced Redox Proteome Changes in Arabidopsis Suspension Cells.

PubMed

Yin, Zepeng; Balmant, Kelly; Geng, Sisi; Zhu, Ning; Zhang, Tong; Dufresne, Craig; Dai, Shaojun; Chen, Sixue

2017-01-01

Climate change as a result of increasing atmospheric CO 2 affects plant growth and productivity. CO 2 is not only a carbon donor for photosynthesis but also an environmental signal that can perturb cellular redox homeostasis and lead to modifications of redox-sensitive proteins. Although redox regulation of protein functions has emerged as an important mechanism in several biological processes, protein redox modifications and how they function in plant CO 2 response remain unclear. Here a new iodoTMTRAQ proteomics technology was employed to analyze changes in protein redox modifications in Arabidopsis thaliana suspension cells in response to bicarbonate (mimic of elevated CO 2 ) in a time-course study. A total of 47 potential redox-regulated proteins were identified with functions in carbohydrate and energy metabolism, transport, ROS scavenging, cell structure modulation and protein turnover. This inventory of previously unknown redox responsive proteins in Arabidopsis bicarbonate responses lays a foundation for future research toward understanding the molecular mechanisms underlying plant CO 2 responses.

Bicarbonate Induced Redox Proteome Changes in Arabidopsis Suspension Cells

PubMed Central

Yin, Zepeng; Balmant, Kelly; Geng, Sisi; Zhu, Ning; Zhang, Tong; Dufresne, Craig; Dai, Shaojun; Chen, Sixue

2017-01-01

Climate change as a result of increasing atmospheric CO2 affects plant growth and productivity. CO2 is not only a carbon donor for photosynthesis but also an environmental signal that can perturb cellular redox homeostasis and lead to modifications of redox-sensitive proteins. Although redox regulation of protein functions has emerged as an important mechanism in several biological processes, protein redox modifications and how they function in plant CO2 response remain unclear. Here a new iodoTMTRAQ proteomics technology was employed to analyze changes in protein redox modifications in Arabidopsis thaliana suspension cells in response to bicarbonate (mimic of elevated CO2) in a time-course study. A total of 47 potential redox-regulated proteins were identified with functions in carbohydrate and energy metabolism, transport, ROS scavenging, cell structure modulation and protein turnover. This inventory of previously unknown redox responsive proteins in Arabidopsis bicarbonate responses lays a foundation for future research toward understanding the molecular mechanisms underlying plant CO2 responses. PMID:28184230

Identification of Salmonella Typhimurium deubiquitinase SseL substrates by immunoaffinity enrichment and quantitative proteomic analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakayasu, Ernesto S.; Sydor, Michael A.; Brown, Roslyn N.

Ubiquitination is a key protein post-translational modification that regulates many important cellular pathways and whose levels are regulated by equilibrium between the activities of ubiquitin ligases and deubiquitinases. Here we present a method to identify specific deubiquitinase substrates based on treatment of cell lysates with recombinant enzymes, immunoaffinity purification and global quantitative proteomic analysis. As model system to identify substrates, we used a virulence-related deubiquitinase secreted by Salmonella enterica serovar Typhimurium into the host cells, SseL. By using this approach two SseL substrates were identified in RAW 264.7 murine macrophage-like cell line, S100A6 and het-erogeneous nuclear ribonuclear protein K, inmore » addition to the previously reported K63-linked ubiquitin chains. These substrates were further validated by a combination of enzymatic and binding assays. Finally, this method can be used for the systematic identification of substrates of deubiquitinases from other organisms and applied to study their functions in physiology and disease.« less

Proteome map of Aspergillus nidulans during osmoadaptation.

PubMed

Kim, Yonghyun; Nandakumar, M P; Marten, Mark R

2007-09-01

The model filamentous fungus Aspergillus nidulans, when grown in a moderate level of osmolyte (+0.6M KCl), was previously found to have a significantly reduced cell wall elasticity (Biotech Prog, 21:292, 2005). In this study, comparative proteomic analysis via two-dimensional gel electrophoresis (2de) and matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) mass spectrometry was used to assess molecular level events associated with this phenomenon. Thirty of 90 differentially expressed proteins were identified. Sequence homology and conserved domains were used to assign probable function to twenty-one proteins currently annotated as "hypothetical." In osmoadapted cells, there was an increased expression of glyceraldehyde-3-phosphate dehydrogenase and aldehyde dehydrogenase, as well as a decreased expression of enolase, suggesting an increased glycerol biosynthesis and decreased use of the TCA cycle. There also was an increased expression of heat shock proteins and Shp1-like protein degradation protein, implicating increased protein turnover. Five novel osmoadaptation proteins of unknown functions were also identified.

Proteomic and metabolomic analysis of the carotenogenic yeast Xanthophyllomyces dendrorhous using different carbon sources.

PubMed

Martinez-Moya, Pilar; Niehaus, Karsten; Alcaíno, Jennifer; Baeza, Marcelo; Cifuentes, Víctor

2015-04-12

Astaxanthin is a potent antioxidant with increasing biotechnological interest. In Xanthophyllomyces dendrorhous, a natural source of this pigment, carotenogenesis is a complex process regulated through several mechanisms, including the carbon source. X. dendrorhous produces more astaxanthin when grown on a non-fermentable carbon source, while decreased astaxanthin production is observed in the presence of high glucose concentrations. In the present study, we used a comparative proteomic and metabolomic analysis to characterize the yeast response when cultured in minimal medium supplemented with glucose (fermentable) or succinate (non-fermentable). A total of 329 proteins were identified from the proteomic profiles, and most of these proteins were associated with carotenogenesis, lipid and carbohydrate metabolism, and redox and stress responses. The metabolite profiles revealed 92 metabolites primarily associated with glycolysis, the tricarboxylic acid cycle, amino acids, organic acids, sugars and phosphates. We determined the abundance of proteins and metabolites of the central pathways of yeast metabolism and examined the influence of these molecules on carotenogenesis. Similar to previous proteomic-stress response studies, we observed modulation of abundance from several redox, stress response, carbohydrate and lipid enzymes. Additionally, the accumulation of trehalose, absence of key ROS response enzymes, an increased abundance of the metabolites of the pentose phosphate pathway and tricarboxylic acid cycle suggested an association between the accumulation of astaxanthin and oxidative stress in the yeast. Moreover, we observed the increased abundance of late carotenogenesis enzymes during astaxanthin accumulation under succinate growth conditions. The use of succinate as a carbon source in X. dendrorhous cultures increases the availability of acetyl-CoA for the astaxanthin production compared with glucose, likely reflecting the positive regulation of metabolic enzymes of the tricarboxylic acid and glyoxylate cycles. The high metabolite level generated in this pathway could increase the cellular respiration rate, producing reactive oxygen species, which induces carotenogenesis.

Maternal serum proteome changes between the first and third trimester of pregnancy in rural southern Nepal.

PubMed

Scholl, P F; Cole, R N; Ruczinski, I; Gucek, M; Diez, R; Rennie, A; Nathasingh, C; Schulze, K; Christian, P; Yager, J D; Groopman, J D; West, K P

2012-05-01

Characterization of normal changes in the serum proteome during pregnancy may enhance understanding of maternal physiology and lead to the development of new gestational biomarkers. In 23 Nepalese pregnant women who delivered at term, two-dimensional difference in-gel electrophoresis (DIGE) was used to assess changes in relative protein abundance between paired serum samples collected in the first and third trimesters. One-hundred and forty-five of over 700 protein spots in DIGE gels (pI 4.2-6.8) exhibited nominally significant (p < 0.05) differences in abundance across trimesters. Additional filtering using a Bonferroni correction reduced the number of significant (p < 0.00019) spots to 61. Mass spectrometric analysis detected 38 proteins associated with gestational age, cytoskeletal remodeling, blood pressure regulation, lipid and nutrient transport, and inflammation. One new protein, pregnancy-specific β-glycoprotein 4 was detected. A follow-up isotope tagging for relative and absolute quantitation (iTRAQ) experiment of six mothers from the DIGE study revealed 111 proteins, of which 11 exhibited significant (p < 0.05) differences between trimesters. Four of these proteins: gelsolin, complement C1r subcomponent, α-1-acid glycoprotein, and α-1B-glycoprotein also changed in the DIGE analysis. Although not previously associated with normal pregnancy, gelsolin decreased in abundance by the third trimester (p < 0.01) in DIGE, iTRAQ and Western analyses. Changes in abundance of proteins in serum that are associated with syncytiotrophoblasts (gelsolin, pregnancy-specific β-1 glycoprotein 1 and β-2-glycoprotein I) probably reflect dynamics of a placental proteome shed into maternal circulation during pregnancy. Measurement of changes in the maternal serum proteome, when linked with birth outcomes, may yield biomarkers for tracking reproductive health in resource poor settings in future studies. Published by Elsevier Ltd.

Proteomic analysis of ligamentum flavum from patients with lumbar spinal stenosis.

PubMed

Kamita, Masahiro; Mori, Taiki; Sakai, Yoshihito; Ito, Sadayuki; Gomi, Masahiro; Miyamoto, Yuko; Harada, Atsushi; Niida, Shumpei; Yamada, Tesshi; Watanabe, Ken; Ono, Masaya

2015-05-01

Lumbar spinal stenosis (LSS) is a syndromic degenerative spinal disease and is characterized by spinal canal narrowing with subsequent neural compression causing gait disturbances. Although LSS is a major age-related musculoskeletal disease that causes large decreases in the daily living activities of the elderly, its molecular pathology has not been investigated using proteomics. Thus, we used several proteomic technologies to analyze the ligamentum flavum (LF) of individuals with LSS. Using comprehensive proteomics with strong cation exchange fractionation, we detected 1288 proteins in these LF samples. A GO analysis of the comprehensive proteome revealed that more than 30% of the identified proteins were extracellular. Next, we used 2D image converted analysis of LC/MS to compare LF obtained from individuals with LSS to that obtained from individuals with disc herniation (nondegenerative control). We detected 64 781 MS peaks and identified 1675 differentially expressed peptides derived from 286 proteins. We verified four differentially expressed proteins (fibronectin, serine protease HTRA1, tenascin, and asporin) by quantitative proteomics using SRM/MRM. The present proteomic study is the first to identify proteins from degenerated and hypertrophied LF in LSS, which will help in studying LSS. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biomarker Discovery and Mechanistic Studies of Prostate Cancer Using Targeted Proteomic Approaches

DTIC Science & Technology

2010-07-01

1-0431 TITLE: Biomarker Discovery and Mechanistic Studies of Prostate Cancer Using Targeted Proteomic Approaches PRINCIPAL INVESTIGATOR...June 2010 4. TITLE AND SUBTITLE Biomarker Discovery and Mechanistic Studies of Prostate Cancer Using Targeted Proteomic 5a. CONTRACT NUMBER...1-0430; W81XWH-08-1-0431; Grant sponsor: NIH/NCRR COBRE Grant; Grant number: 1P20RR020171; Grant sponsor: NIH/NIDDK Grant; Grant number: R01DK053525

Proteomic study of acute respiratory distress syndrome: current knowledge and implications for drug development

PubMed Central

Levitt, Joseph E.; Rogers, Angela J.

2017-01-01

The acute respiratory distress syndrome (ARDS) is a common cause of acute respiratory failure, and is associated with substantial mortality and morbidity. Dozens of clinical trials targeting ARDS have failed, with no drug specifically targeting lung injury in widespread clinical use. Thus, the need for drug development in ARDS is great. Targeted proteomic studies in ARDS have identified many key pathways in the disease, including inflammation, epithelial injury, endothelial injury or activation, and disordered coagulation and repair. Recent studies reveal the potential for proteomic changes to identify novel subphenotypes of ARDS patients who may be most likely to respond to therapy and could thus be targeted for enrollment in clinical trials. Nontargeted studies of proteomics in ARDS are just beginning and have the potential to identify novel drug targets and key pathways in the disease. Proteomics will play an important role in phenotyping of patients and developing novel therapies for ARDS in the future. PMID:27031735

Application of targeted quantitative proteomics analysis in human cerebrospinal fluid using a liquid chromatography matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometer (LC MALDI TOF/TOF) platform.

PubMed

Pan, Sheng; Rush, John; Peskind, Elaine R; Galasko, Douglas; Chung, Kathryn; Quinn, Joseph; Jankovic, Joseph; Leverenz, James B; Zabetian, Cyrus; Pan, Catherine; Wang, Yan; Oh, Jung Hun; Gao, Jean; Zhang, Jianpeng; Montine, Thomas; Zhang, Jing

2008-02-01

Targeted quantitative proteomics by mass spectrometry aims to selectively detect one or a panel of peptides/proteins in a complex sample and is particularly appealing for novel biomarker verification/validation because it does not require specific antibodies. Here, we demonstrated the application of targeted quantitative proteomics in searching, identifying, and quantifying selected peptides in human cerebrospinal spinal fluid (CSF) using a matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometer (MALDI TOF/TOF)-based platform. The approach involved two major components: the use of isotopic-labeled synthetic peptides as references for targeted identification and quantification and a highly selective mass spectrometric analysis based on the unique characteristics of the MALDI instrument. The platform provides high confidence for targeted peptide detection in a complex system and can potentially be developed into a high-throughput system. Using the liquid chromatography (LC) MALDI TOF/TOF platform and the complementary identification strategy, we were able to selectively identify and quantify a panel of targeted peptides in the whole proteome of CSF without prior depletion of abundant proteins. The effectiveness and robustness of the approach associated with different sample complexity, sample preparation strategies, as well as mass spectrometric quantification were evaluated. Other issues related to chromatography separation and the feasibility for high-throughput analysis were also discussed. Finally, we applied targeted quantitative proteomics to analyze a subset of previously identified candidate markers in CSF samples of patients with Parkinson's disease (PD) at different stages and Alzheimer's disease (AD) along with normal controls.

Quantitative and temporal proteome analysis of butyrate-treated colorectal cancer cells.

PubMed

Tan, Hwee Tong; Tan, Sandra; Lin, Qingsong; Lim, Teck Kwang; Hew, Choy Leong; Chung, Maxey C M

2008-06-01

Colorectal cancer is one of the most common cancers in developed countries, and its incidence is negatively associated with high dietary fiber intake. Butyrate, a short-chain fatty acid fermentation by-product of fiber induces cell maturation with the promotion of growth arrest, differentiation, and/or apoptosis of cancer cells. The stimulation of cell maturation by butyrate in colonic cancer cells follows a temporal progression from the early phase of growth arrest to the activation of apoptotic cascades. Previously we performed two-dimensional DIGE to identify differentially expressed proteins induced by 24-h butyrate treatment of HCT-116 colorectal cancer cells. Herein we used quantitative proteomics approaches using iTRAQ (isobaric tags for relative and absolute quantitation), a stable isotope labeling methodology that enables multiplexing of four samples, for a temporal study of HCT-116 cells treated with butyrate. In addition, cleavable ICAT, which selectively tags cysteine-containing proteins, was also used, and the results complemented those obtained from the iTRAQ strategy. Selected protein targets were validated by real time PCR and Western blotting. A model is proposed to illustrate our findings from this temporal analysis of the butyrate-responsive proteome that uncovered several integrated cellular processes and pathways involved in growth arrest, apoptosis, and metastasis. These signature clusters of butyrate-regulated pathways are potential targets for novel chemopreventive and therapeutic drugs for treatment of colorectal cancer.

Multiomics reveal non-alcoholic fatty liver disease in rats following chronic exposure to an ultra-low dose of Roundup herbicide

PubMed Central

Mesnage, Robin; Renney, George; Séralini, Gilles-Eric; Ward, Malcolm; Antoniou, Michael N.

2017-01-01

The impairment of liver function by low environmentally relevant doses of glyphosate-based herbicides (GBH) is still a debatable and unresolved matter. Previously we have shown that rats administered for 2 years with 0.1 ppb (50 ng/L glyphosate equivalent dilution; 4 ng/kg body weight/day daily intake) of a Roundup GBH formulation showed signs of enhanced liver injury as indicated by anatomorphological, blood/urine biochemical changes and transcriptome profiling. Here we present a multiomic study combining metabolome and proteome liver analyses to obtain further insight into the Roundup-induced pathology. Proteins significantly disturbed (214 out of 1906 detected, q < 0.05) were involved in organonitrogen metabolism and fatty acid β-oxidation. Proteome disturbances reflected peroxisomal proliferation, steatosis and necrosis. The metabolome analysis (55 metabolites altered out of 673 detected, p < 0.05) confirmed lipotoxic conditions and oxidative stress by showing an activation of glutathione and ascorbate free radical scavenger systems. Additionally, we found metabolite alterations associated with hallmarks of hepatotoxicity such as γ-glutamyl dipeptides, acylcarnitines, and proline derivatives. Overall, metabolome and proteome disturbances showed a substantial overlap with biomarkers of non-alcoholic fatty liver disease and its progression to steatohepatosis and thus confirm liver functional dysfunction resulting from chronic ultra-low dose GBH exposure. PMID:28067231

Proteomic Analysis of Mouse Oocytes Identifies PRMT7 as a Reprogramming Factor that Replaces SOX2 in the Induction of Pluripotent Stem Cells.

PubMed

Wang, Bingyuan; Pfeiffer, Martin J; Drexler, Hannes C A; Fuellen, Georg; Boiani, Michele

2016-08-05

The reprogramming process that leads to induced pluripotent stem cells (iPSCs) may benefit from adding oocyte factors to Yamanaka's reprogramming cocktail (OCT4, SOX2, KLF4, with or without MYC; OSK(M)). We previously searched for such facilitators of reprogramming (the reprogrammome) by applying label-free LC-MS/MS analysis to mouse oocytes, producing a catalog of 28 candidates that are (i) able to robustly access the cell nucleus and (ii) shared between mature mouse oocytes and pluripotent embryonic stem cells. In the present study, we hypothesized that our 28 reprogrammome candidates would also be (iii) abundant in mature oocytes, (iv) depleted after the oocyte-to-embryo transition, and (v) able to potentiate or replace the OSKM factors. Using LC-MS/MS and isotopic labeling methods, we found that the abundance profiles of the 28 proteins were below those of known oocyte-specific and housekeeping proteins. Of the 28 proteins, only arginine methyltransferase 7 (PRMT7) changed substantially during mouse embryogenesis and promoted the conversion of mouse fibroblasts into iPSCs. Specifically, PRMT7 replaced SOX2 in a factor-substitution assay, yielding iPSCs. These findings exemplify how proteomics can be used to prioritize the functional analysis of reprogrammome candidates. The LC-MS/MS data are available via ProteomeXchange with identifier PXD003093.

Proteomic approaches in research of cyanobacterial photosynthesis.

PubMed

Battchikova, Natalia; Angeleri, Martina; Aro, Eva-Mari

2015-10-01

Oxygenic photosynthesis in cyanobacteria, algae, and plants is carried out by a fabulous pigment-protein machinery that is amazingly complicated in structure and function. Many different approaches have been undertaken to characterize the most important aspects of photosynthesis, and proteomics has become the essential component in this research. Here we describe various methods which have been used in proteomic research of cyanobacteria, and demonstrate how proteomics is implemented into on-going studies of photosynthesis in cyanobacterial cells.

Proteomics analysis of malignant and benign prostate tissue by 2D DIGE/MS reveals new insights into proteins involved in prostate cancer.

PubMed

Davalieva, Katarina; Kostovska, Ivana Maleva; Kiprijanovska, Sanja; Markoska, Katerina; Kubelka-Sabit, Katerina; Filipovski, Vanja; Stavridis, Sotir; Stankov, Oliver; Komina, Selim; Petrusevska, Gordana; Polenakovic, Momir

2015-10-01

The key to a more effective diagnosis, prognosis, and therapeutic management of prostate cancer (PCa) could lie in the direct analysis of cancer tissue. In this study, by comparative proteomics analysis of PCa and benign prostate hyperplasia (BPH) tissues we attempted to elucidate the proteins and regulatory pathways involved in this disease. The samples used in this study were fresh surgical tissues with clinically and histologically confirmed PCa (n = 19) and BPH (n = 33). We used two dimensional difference in gel electrophoresis (2D DIGE) coupled with mass spectrometry (MS) and bioinformatics analysis. Thirty-nine spots with statistically significant 1.8-fold variation or more in abundance, corresponding to 28 proteins were identified. The IPA analysis pointed out to 3 possible networks regulated within MAPK, ERK, TGFB1, and ubiquitin pathways. Thirteen of the identified proteins, namely, constituents of the intermediate filaments (KRT8, KRT18, DES), potential tumor suppressors (ARHGAP1, AZGP1, GSTM2, and MFAP4), transport and membrane organization proteins (FABP5, GC, and EHD2), chaperons (FKBP4 and HSPD1) and known cancer marker (NME1) have been associated with prostate and other cancers by numerous proteomics, genomics or functional studies. We evidenced for the first time the dysregulation of 9 proteins (CSNK1A1, ARID5B, LYPLA1, PSMB6, RABEP1, TALDO1, UBE2N, PPP1CB, and SERPINB1) that may have role in PCa. The UBE2N, PSMB6, and PPP1CB, involved in cell cycle regulation and progression were evaluated by Western blot analysis which confirmed significantly higher abundances of UBE2N and PSMB6 and significantly lower abundance of PPP1CB in PCa. In addition to the identification of substantial number of proteins with known association with PCa, the proteomic approach in this study revealed proteins not previously clearly related to PCa, providing a starting point for further elucidation of their function in disease initiation and progression. © 2015 Wiley Periodicals, Inc.

Proteomics in the investigation of HIV-1 interactions with host proteins.

PubMed

Li, Ming

2015-02-01

Productive HIV-1 infection depends on host machinery, including a broad array of cellular proteins. Proteomics has played a significant role in the discovery of HIV-1 host proteins. In this review, after a brief survey of the HIV-1 host proteins that were discovered by proteomic analyses, I focus on analyzing the interactions between the virion and host proteins, as well as the technologies and strategies used in those proteomic studies. With the help of proteomics, the identification and characterization of HIV-1 host proteins can be translated into novel antiretroviral therapeutics. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

"Does understanding the brain need proteomics and does understanding proteomics need brains?"--Second HUPO HBPP Workshop hosted in Paris.

PubMed

Hamacher, Michael; Klose, Joachim; Rossier, Jean; Marcus, Katrin; Meyer, Helmut E

2004-07-01

The second Human Brain Proteome Project (HBPP) Workshop of the Human Proteome Organisation (HUPO) took place at the Ecole Supérieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI) from April 23-24, 2004. During two days, more than 70 attendees from Europe, Asia and the US came together to decide basic strategic approaches, standards and the beginning of a pilot phase prior to further studies of the human brain proteome. The international consortium presented the technological and scientific portfolio and scheduled the time table for the next year.

Proteomic dissection of plant responses to various pathogens.

PubMed

Fang, Xianping; Chen, Jianping; Dai, Liangying; Ma, Huasheng; Zhang, Hengmu; Yang, Jian; Wang, Fang; Yan, Chengqi

2015-05-01

During their growth and development, plants are vulnerable to the effects of a variety of pathogens. Proteomics technology plays an important role in research studies of plant defense mechanisms by mining the expression changes of proteins in response to various biotic stresses. This review article provides a comprehensive overview of the latest developments in international proteomic research on plant biotic stress. It summarizes the methods commonly used in plant proteomic research to investigate biotic stress, analyze the protein responses of plants in adverse conditions, and reviews the applications of proteomics combined with transgenic technology in plant protection. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Compositional complexity of the mitochondrial proteome of a unicellular eukaryote (Acanthamoeba castellanii, supergroup Amoebozoa) rivals that of animals, fungi, and plants.

PubMed

Gawryluk, Ryan M R; Chisholm, Kenneth A; Pinto, Devanand M; Gray, Michael W

2014-09-23

We present a combined proteomic and bioinformatic investigation of mitochondrial proteins from the amoeboid protist Acanthamoeba castellanii, the first such comprehensive investigation in a free-living member of the supergroup Amoebozoa. This protist was chosen both for its phylogenetic position (as a sister to animals and fungi) and its ecological ubiquity and physiological flexibility. We report 1033 A. castellanii mitochondrial protein sequences, 709 supported by mass spectrometry data (676 nucleus-encoded and 33 mitochondrion-encoded), including two previously unannotated mtDNA-encoded proteins, which we identify as highly divergent mitochondrial ribosomal proteins. Other notable findings include duplicate proteins for all of the enzymes of the tricarboxylic acid (TCA) cycle-which, along with the identification of a mitochondrial malate synthase-isocitrate lyase fusion protein, suggests the interesting possibility that the glyoxylate cycle operates in A. castellanii mitochondria. Additionally, the A. castellanii genome encodes an unusually high number (at least 29) of mitochondrion-targeted pentatricopeptide repeat (PPR) proteins, organellar RNA metabolism factors in other organisms. We discuss several key mitochondrial pathways, including DNA replication, transcription and translation, protein degradation, protein import and Fe-S cluster biosynthesis, highlighting similarities and differences in these pathways in other eukaryotes. In compositional and functional complexity, the mitochondrial proteome of A. castellanii rivals that of multicellular eukaryotes. Comprehensive proteomic surveys of mitochondria have been undertaken in a limited number of predominantly multicellular eukaryotes. This phylogenetically narrow perspective constrains and biases our insights into mitochondrial function and evolution, as it neglects protists, which account for most of the evolutionary and functional diversity within eukaryotes. We report here the first comprehensive investigation of the mitochondrial proteome in a member (A. castellanii) of the eukaryotic supergroup Amoebozoa. Through a combination of tandem mass spectrometry (MS/MS) and in silico data mining, we have retrieved 1033 candidate mitochondrial protein sequences, 709 having MS support. These data were used to reconstruct the metabolic pathways and protein complexes of A. castellanii mitochondria, and were integrated with data from other characterized mitochondrial proteomes to augment our understanding of mitochondrial proteome evolution. Our results demonstrate the power of combining direct proteomic and bioinformatic approaches in the discovery of novel mitochondrial proteins, both nucleus-encoded and mitochondrion-encoded, and highlight the compositional complexity of the A. castellanii mitochondrial proteome, which rivals that of animals, fungi and plants. Copyright © 2014 Elsevier B.V. All rights reserved.

Proteomics: Protein Identification Using Online Databases

ERIC Educational Resources Information Center

Eurich, Chris; Fields, Peter A.; Rice, Elizabeth

2012-01-01

Proteomics is an emerging area of systems biology that allows simultaneous study of thousands of proteins expressed in cells, tissues, or whole organisms. We have developed this activity to enable high school or college students to explore proteomic databases using mass spectrometry data files generated from yeast proteins in a college laboratory…

Unraveling snake venom complexity with 'omics' approaches: challenges and perspectives.

PubMed

Zelanis, André; Tashima, Alexandre Keiji

2014-09-01

The study of snake venom proteomes (venomics) has been experiencing a burst of reports, however the comprehensive knowledge of the dynamic range of proteins present within a single venom, the set of post-translational modifications (PTMs) as well as the lack of a comprehensive database related to venom proteins are among the main challenges in venomics research. The phenotypic plasticity in snake venom proteomes together with their inherent toxin proteoform diversity, points out to the use of integrative analysis in order to better understand their actual complexity. In this regard, such a systems venomics task should encompass the integration of data from transcriptomic and proteomic studies (specially the venom gland proteome), the identification of biological PTMs, and the estimation of artifactual proteomes and peptidomes generated by sample handling procedures. Copyright © 2014 Elsevier Ltd. All rights reserved.

Characterization of the human aqueous humour proteome: A comparison of the genders.

PubMed

Perumal, Natarajan; Manicam, Caroline; Steinicke, Matthias; Funke, Sebastian; Pfeiffer, Norbert; Grus, Franz H

2017-01-01

Aqueous humour (AH) is an important biologic fluid that maintains normal intraocular pressure and contains proteins that regulate the homeostasis of ocular tissues. Any alterations in the protein compositions are correlated to the pathogenesis of various ocular disorders. In recent years, gender-based medicine has emerged as an important research focus considering the prevalence of certain diseases, which are higher in a particular sex. Nevertheless, the inter-gender variations in the AH proteome are unknown. Therefore, this study endeavoured to characterize the AH proteome to assess the differences between genders. Thirty AH samples of patients who underwent cataract surgery were categorized according to their gender. Label-free quantitative discovery mass spectrometry-based proteomics strategy was employed to characterize the AH proteome. A total of 147 proteins were identified with a false discovery rate of less than 1% and only the top 10 major AH proteins make up almost 90% of the total identified proteins. A large number of proteins identified were correlated to defence, immune and inflammatory mechanisms, and response to wounding. Four proteins were found to be differentially abundant between the genders, comprising SERPINF1, SERPINA3, SERPING1 and PTGDS. The findings emerging from our study provide the first insight into the gender-based proteome differences in the AH and also highlight the importance in considering potential sex-dependent changes in the proteome of ocular pathologies in future studies employing the AH.

Proteomics of survival structures of fungal pathogens.

PubMed

Loginov, Dmitry; Šebela, Marek

2016-09-25

Fungal pathogens are causal agents of numerous human, animal, and plant diseases. They employ various infection modes to overcome host defense systems. Infection mechanisms of different fungi have been subjected to many comprehensive studies. These investigations have been facilitated by the development of various '-omics' techniques, and proteomics has one of the leading roles in this regard. Fungal conidia and sclerotia could be considered the most important structures for pathogenesis as their germination is one of the first steps towards a host infection. They represent interesting objects for proteomic studies because of the presence of unique proteins with unexplored biotechnological potential required for pathogen viability, development and the subsequent host infection. Proteomic peculiarities of survival structures of different fungi, including those of biotechnological significance (e.g., Asperillus fumigatus, A. nidulans, Metarhizium anisopliae), in a dormant state, as well as changes in the protein production during early stages of fungal development are the subjects of the present review. We focused on biological aspects of proteomic studies of fungal survival structures rather than on an evaluation of proteomic approaches. For that reason, proteins that have been identified in this context are discussed from the point of view of their involvement in different biological processes and possible functions assigned to them. This is the first review paper summarizing recent advances in proteomics of fungal survival structures. Copyright © 2016 Elsevier B.V. All rights reserved.

The Human Plasma Proteome Draft of 2017: Building on the Human Plasma PeptideAtlas from Mass Spectrometry and Complementary Assays.

PubMed

Schwenk, Jochen M; Omenn, Gilbert S; Sun, Zhi; Campbell, David S; Baker, Mark S; Overall, Christopher M; Aebersold, Ruedi; Moritz, Robert L; Deutsch, Eric W

2017-12-01

Human blood plasma provides a highly accessible window to the proteome of any individual in health and disease. Since its inception in 2002, the Human Proteome Organization's Human Plasma Proteome Project (HPPP) has been promoting advances in the study and understanding of the full protein complement of human plasma and on determining the abundance and modifications of its components. In 2017, we review the history of the HPPP and the advances of human plasma proteomics in general, including several recent achievements. We then present the latest 2017-04 build of Human Plasma PeptideAtlas, which yields ∼43 million peptide-spectrum matches and 122,730 distinct peptide sequences from 178 individual experiments at a 1% protein-level FDR globally across all experiments. Applying the latest Human Proteome Project Data Interpretation Guidelines, we catalog 3509 proteins that have at least two non-nested uniquely mapping peptides of nine amino acids or more and >1300 additional proteins with ambiguous evidence. We apply the same two-peptide guideline to historical PeptideAtlas builds going back to 2006 and examine the progress made in the past ten years in plasma proteome coverage. We also compare the distribution of proteins in historical PeptideAtlas builds in various RNA abundance and cellular localization categories. We then discuss advances in plasma proteomics based on targeted mass spectrometry as well as affinity assays, which during early 2017 target ∼2000 proteins. Finally, we describe considerations about sample handling and study design, concluding with an outlook for future advances in deciphering the human plasma proteome.

Virtual Labs in proteomics: new E-learning tools.

PubMed

Ray, Sandipan; Koshy, Nicole Rachel; Reddy, Panga Jaipal; Srivastava, Sanjeeva

2012-05-17

Web-based educational resources have gained enormous popularity recently and are increasingly becoming a part of modern educational systems. Virtual Labs are E-learning platforms where learners can gain the experience of practical experimentation without any direct physical involvement on real bench work. They use computerized simulations, models, videos, animations and other instructional technologies to create interactive content. Proteomics being one of the most rapidly growing fields of the biological sciences is now an important part of college and university curriculums. Consequently, many E-learning programs have started incorporating the theoretical and practical aspects of different proteomic techniques as an element of their course work in the form of Video Lectures and Virtual Labs. To this end, recently we have developed a Virtual Proteomics Lab at the Indian Institute of Technology Bombay, which demonstrates different proteomics techniques, including basic and advanced gel and MS-based protein separation and identification techniques, bioinformatics tools and molecular docking methods, and their applications in different biological samples. This Tutorial will discuss the prominent Virtual Labs featuring proteomics content, including the Virtual Proteomics Lab of IIT-Bombay, and E-resources available for proteomics study that are striving to make proteomic techniques and concepts available and accessible to the student and research community. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP 14). Details can be found at: http://www.proteomicstutorials.org/. Copyright © 2012 Elsevier B.V. All rights reserved.

Advances in proteomics research for peanut genetics and breeding

USDA-ARS?s Scientific Manuscript database

Crop trait improvement aimed at increased yield and quality relies on an understanding of the biology of the plant, particular protein-protein interactions. In this regard, the application of “-omics” techniques combined with field-level agronomy is poised to deliver novel insight into previously u...

Plasma-based proteomics reveals immune response, complement and coagulation cascades pathway shifts in heat-stressed lactating dairy cows.

PubMed

Min, Li; Cheng, Jianbo; Zhao, Shengguo; Tian, He; Zhang, Yangdong; Li, Songli; Yang, Hongjian; Zheng, Nan; Wang, Jiaqi

2016-09-02

Heat stress (HS) has an enormous economic impact on the dairy industry. In recent years, many researchers have investigated changes in the gene expression and metabolomics profiles in dairy cows caused by HS. However, the proteomics profiles of heat-stressed dairy cows have not yet been completely elucidated. We compared plasma proteomics from HS-free and heat-stressed dairy cows using an iTRAQ labeling approach. After the depletion of high abundant proteins in the plasma, 1472 proteins were identified. Of these, 85 proteins were differentially abundant in cows exposed to HS relative to HS-free. Database searches combined with GO and KEGG pathway enrichment analyses revealed that many components of the complement and coagulation cascades were altered in heat-stressed cows compared with HS-free cows. Of these, many factors in the complement system (including complement components C1, C3, C5, C6, C7, C8, and C9, complement factor B, and factor H) were down-regulated by HS, while components of the coagulation system (including coagulation factors, vitamin K-dependent proteins, and fibrinogens) were up-regulated by HS. In conclusion, our results indicate that HS decreases plasma levels of complement system proteins, suggesting that immune function is impaired in dairy cows exposed to HS. Though many aspects of heat stress (HS) have been extensively researched, relatively little is known about the proteomics profile changes that occur during heat exposure. In this work, we employed a proteomics approach to investigate differential abundance of plasma proteins in HS-free and heat-stressed dairy cows. Database searches combined with GO and KEGG pathway enrichment analyses revealed that HS resulted in a decrease in complement components, suggesting that heat-stressed dairy cows have impaired immune function. In addition, through integrative analyses of proteomics and previous metabolomics, we showed enhanced glycolysis, lipid metabolic pathway shifts, and nitrogen repartitioning in dairy cows exposed to HS. Our findings expand our current knowledge on the effects of HS on plasma proteomics in dairy cows and offer a new perspective for future research. Copyright © 2016 Elsevier B.V. All rights reserved.

Proteomic profiling reveals dopaminergic regulation of progenitor cell functions of goldfish radial glial cells in vitro.

PubMed

Xing, Lei; Martyniuk, Christopher J; Esau, Crystal; Da Fonte, Dillon F; Trudeau, Vance L

2016-07-20

Radial glial cells (RGCs) are stem-like cells found in the developing and adult central nervous system. They function as both a scaffold to guide neuron migration and as progenitor cells that support neurogenesis. Our previous study revealed a close anatomical relationship between dopamine neurons and RGCs in the telencephalon of female goldfish. In this study, label-free proteomics was used to identify the proteins in a primary RGC culture and to determine the proteome response to the selective dopamine D1 receptor agonist SKF 38393 (10μM), in order to better understand dopaminergic regulation of RGCs. A total of 689 unique proteins were identified in the RGCs and these were classified into biological and pathological pathways. Proteins such as nucleolin (6.9-fold) and ependymin related protein 1 (4.9-fold) were increased in abundance while proteins triosephosphate isomerase (10-fold) and phosphoglycerate dehydrogenase (5-fold) were decreased in abundance. Pathway analysis revealed that proteins that consistently changed in abundance across biological replicates were related to small molecules such as ATP, lipids and steroids, hormones, glucose, cyclic AMP and Ca(2+). Sub-network enrichment analysis suggested that estrogen receptor signaling, among other transcription factors, is regulated by D1 receptor activation. This suggests that these signaling pathways are correlated to dopaminergic regulation of radial glial cell functions. Most proteins down-regulated by SKF 38393 were involved in cell cycle/proliferation, growth, death, and survival, which suggests that dopamine inhibits the progenitor-related processes of radial glial cells. Examples of differently expressed proteins including triosephosphate isomerase, nucleolin, phosphoglycerate dehydrogenase and capping protein (actin filament) muscle Z-line beta were validated by qPCR and western blot, which were consistent with MS/MS data in the direction of change. This is the first study to characterize the RGC proteome on a large scale in a vertebrate species. These data provide novel insight into glial protein networks that are associated with neuroendocrine function and neurogenesis in the teleost brain. While the role of radial glial cells in organizing brain structure and neurogenesis has been well studied, protein profiling experiments in this unique cell type has not been conducted. This study is the first to profile the proteome of goldfish radial glial cells in culture and to study the regulation of progenitor functions of radial glial cells by the neurotransmitter dopamine. This study provides the foundation for molecular network analysis in fish radial glial cells, and identifies cellular processes and signaling pathways in these cells with roles in neurogenesis and neuroendocrine function. Lastly, this study begins to characterize signatures and biomarkers for specific neuroendocrine and neurogenesis disruptors. Copyright © 2016 Elsevier B.V. All rights reserved.

Label-free proteome of water buffalo (Bubalus bubalis) seminal plasma.

PubMed

Brito, Mayara F; Auler, Patrícia A; Tavares, Guilherme C; Rezende, Cristiana P; Almeida, Gabriel M F; Pereira, Felipe L; Leal, Carlos A G; Moura, Arlindo de Alencar; Figueiredo, Henrique C P; Henry, Marc

2018-06-11

The study aimed to describe the Bubalus bubalis seminal plasma proteome using a label-free shotgun UDMS E approach. A total of 859 nonredundant proteins were identified across five biological replicates with stringent identification. Proteins specifically related to sperm maturation and protection, capacitation, fertilization and metabolic activity were detected in the buffalo seminal fluid. In conclusion, we provide a comprehensive proteomic profile of buffalo seminal plasma, which establishes a foundation for further studies designed to understand regulation of sperm function and discovery of novel biomarkers for fertility. MS data are available in the ProteomeXchange with identifier PXD003728. © 2018 Blackwell Verlag GmbH.

Mitochondrial Proteome Studies in Seeds during Germination

PubMed Central

Czarna, Malgorzata; Kolodziejczak, Marta; Janska, Hanna

2016-01-01

Seed germination is considered to be one of the most critical phases in the plant life cycle, establishing the next generation of a plant species. It is an energy-demanding process that requires functioning mitochondria. One of the earliest events of seed germination is progressive development of structurally simple and metabolically quiescent promitochondria into fully active and cristae-containing mitochondria, known as mitochondrial biogenesis. This is a complex and tightly regulated process, which is accompanied by sequential and dynamic gene expression, protein synthesis, and post-translational modifications. The aim of this review is to give a comprehensive summary of seed mitochondrial proteome studies during germination of various plant model organisms. We describe different gel-based and gel-free proteomic approaches used to characterize mitochondrial proteomes of germinating seeds as well as challenges and limitations of these proteomic studies. Furthermore, the dynamic changes in the abundance of the mitochondrial proteomes of germinating seeds are illustrated, highlighting numerous mitochondrial proteins involved in respiration, tricarboxycylic acid (TCA) cycle, metabolism, import, and stress response as potentially important for seed germination. We then review seed mitochondrial protein carbonylation, phosphorylation, and S-nitrosylation as well as discuss the possible link between these post-translational modifications (PTMs) and the regulation of seed germination. PMID:28248229

Application of an Improved Proteomics Method for Abundant Protein Cleanup: Molecular and Genomic Mechanisms Study in Plant Defense*

PubMed Central

Zhang, Yixiang; Gao, Peng; Xing, Zhuo; Jin, Shumei; Chen, Zhide; Liu, Lantao; Constantino, Nasie; Wang, Xinwang; Shi, Weibing; Yuan, Joshua S.; Dai, Susie Y.

2013-01-01

High abundance proteins like ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) impose a consistent challenge for the whole proteome characterization using shot-gun proteomics. To address this challenge, we developed and evaluated Polyethyleneimine Assisted Rubisco Cleanup (PARC) as a new method by combining both abundant protein removal and fractionation. The new approach was applied to a plant insect interaction study to validate the platform and investigate mechanisms for plant defense against herbivorous insects. Our results indicated that PARC can effectively remove Rubisco, improve the protein identification, and discover almost three times more differentially regulated proteins. The significantly enhanced shot-gun proteomics performance was translated into in-depth proteomic and molecular mechanisms for plant insect interaction, where carbon re-distribution was used to play an essential role. Moreover, the transcriptomic validation also confirmed the reliability of PARC analysis. Finally, functional studies were carried out for two differentially regulated genes as revealed by PARC analysis. Insect resistance was induced by over-expressing either jacalin-like or cupin-like genes in rice. The results further highlighted that PARC can serve as an effective strategy for proteomics analysis and gene discovery. PMID:23943779

The role of proteomics in studies of protein moonlighting.

PubMed

Beynon, Robert J; Hammond, Dean; Harman, Victoria; Woolerton, Yvonne

2014-12-01

The increasing acceptance that proteins may exert multiple functions in the cell brings with it new analytical challenges that will have an impact on the field of proteomics. Many proteomics workflows begin by destroying information about the interactions between different proteins, and the reduction of a complex protein mixture to constituent peptides also scrambles information about the combinatorial potential of post-translational modifications. To bring the focus of proteomics on to the domain of protein moonlighting will require novel analytical and quantitative approaches.

Changes in mouse whole saliva soluble proteome induced by tannin-enriched diet

PubMed Central

2010-01-01

Background Previous studies suggested that dietary tannin ingestion may induce changes in mouse salivary proteins in addition to the primarily studied proline-rich proteins (PRPs). The aim of the present study was to determine the protein expression changes induced by condensed tannin intake on the fraction of mouse whole salivary proteins that are unable to form insoluble tannin-protein complexes. Two-dimensional polyacrylamide gel electrophoresis protein separation was used, followed by protein identification by mass spectrometry. Results Fifty-seven protein spots were excised from control group gels, and 21 different proteins were identified. With tannin consumption, the expression levels of one α-amylase isoform and one unidentified protein increased, whereas acidic mammalian chitinase and Muc10 decreased. Additionally, two basic spots that stained pink with Coomassie Brilliant Blue R-250 were newly observed, suggesting that some induced PRPs may remain uncomplexed or form soluble complexes with tannins. Conclusion This proteomic analysis provides evidence that other salivary proteins, in addition to tannin-precipitating proteins, are affected by tannin ingestion. Changes in the expression levels of the acidic mammalian chitinase precursor and in one of the 14 salivary α-amylase isoforms underscores the need to further investigate their role in tannin ingestion. PMID:21159160

Microbial Interactions in Plants: Perspectives and Applications of Proteomics.

PubMed

Imam, Jahangir; Shukla, Pratyoosh; Mandal, Nimai Prasad; Variar, Mukund

2017-01-01

The structure and function of proteins involved in plant-microbe interactions is investigated through large-scale proteomics technology in a complex biological sample. Since the whole genome sequences are now available for several plant species and microbes, proteomics study has become easier, accurate and huge amount of data can be generated and analyzed during plant-microbe interactions. Proteomics approaches are highly important and relevant in many studies and showed that only genomics approaches are not sufficient enough as much significant information are lost as the proteins and not the genes coding them are final product that is responsible for the observed phenotype. Novel approaches in proteomics are developing continuously enabling the study of the various aspects in arrangements and configuration of proteins and its functions. Its application is becoming more common and frequently used in plant-microbe interactions with the advancement in new technologies. They are more used for the portrayal of cell and extracellular destructiveness and pathogenicity variables delivered by pathogens. This distinguishes the protein level adjustments in host plants when infected with pathogens and advantageous partners. This review provides a brief overview of different proteomics technology which is currently available followed by their exploitation to study the plant-microbe interaction. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Dentistry proteomics: from laboratory development to clinical practice.

PubMed

Rezende, Taia M B; Lima, Stella M F; Petriz, Bernardo A; Silva, Osmar N; Freire, Mirna S; Franco, Octávio L

2013-12-01

Despite all the dental information acquired over centuries and the importance of proteome research, the cross-link between these two areas only emerged around mid-nineties. Proteomic tools can help dentistry in the identification of risk factors, early diagnosis, prevention, and systematic control that will promote the evolution of treatment in all dentistry specialties. This review mainly focuses on the evolution of dentistry in different specialties based on proteomic research and how these tools can improve knowledge in dentistry. The subjects covered are an overview of proteomics in dentistry, specific information on different fields in dentistry (dental structure, restorative dentistry, endodontics, periodontics, oral pathology, oral surgery, and orthodontics) and future directions. There are many new proteomic technologies that have never been used in dentistry studies and some dentistry areas that have never been explored by proteomic tools. It is expected that a greater integration of these areas will help to understand what is still unknown in oral health and disease. Copyright © 2013 Wiley Periodicals, Inc.

Proteomics in Argentina - limitations and future perspectives: A special emphasis on meat proteomics.

PubMed

Fadda, Silvina; Almeida, André M

2015-11-01

Argentina is one of the most relevant countries in Latin America, playing a major role in regional economics, culture and science. Over the last 80 years, Argentinean history has been characterized by several upward and downward phases that had major consequences on the development of science in the country and most recently on proteomics. In this article, we characterize the evolution of Proteomics sciences in Argentina over the last decade and a half. We describe the proteomics publication output of the country in the framework of the regional and international contexts, demonstrating that Argentina is solidly anchored in a regional context, showing results similar to other emergent and Latin American countries, albeit still far from the European, American or Australian realities. We also provide a case-study on the importance of Proteomics to a specific sector in the area of food science: the use of bacteria of technological interest, highlighting major achievements obtained by Argentinean proteomics scientists. Finally, we provide a general picture of the endeavors being undertaken by Argentinean Proteomics scientists and their international collaborators to promote the Proteomics-based research with the new generation of scientists and PhD students in both Argentina and other countries in the Southern cone. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Perturbations in the Urinary Exosome in Transplant Rejection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sigdel, Tara K.; NG, Yolanda; Lee, Sangho

Background: Urine exosomes, vesicles exocytosed into urine by all renal epithelial cell types, occur under normal physiologic and disease states. Exosome contents may mirror disease-specific proteome perturbations in kidney injury. Analysis methodologies for the exosomal fraction of the urinary proteome were developed and for comparing the urinary exosomal fraction versus unfractionated proteome for biomarker discovery. Methods: Urine exosomes were isolated by centrifugal filtration from mid-stream, second morning void, urine samples collected from kidney transplant recipients with and without biopsy matched acute rejection. The proteomes of unfractionated whole urine (Uw) and urine exosomes (Uexo) underwent mass spectrometry-based quantitative proteomics analysis. Themore » proteome data were analyzed for significant differential protein abundances in acute rejection (AR). Results: Identifications of 1018 and 349 proteins, Uw and Uexo fractions, respectively, demonstrated a 279 protein overlap between the two urinary compartments with 25%(70) of overlapping proteins unique to Uexoand represented membrane bound proteins (p=9.31e-7). Of 349 urine exosomal proteins identified in transplant patients 220 were not previously identified in the normal urine exosomal fraction. Uexo proteins (11), functioning in the inflammatory / stress response, were more abundant in patients with biopsy-confirmed acute rejection, 3 of which were exclusive to Uexo. Uexo AR-specific biomarkers (8) were also detected in Uw, but since they were observed at significantly lower abundances in Uw, they were not significant for AR in Uw. Conclusions: A rapid urinary exosome isolation method and quantitative measurement of enriched Uexo proteins was applied. Urine proteins specific to the exosomal fraction were detected either in unfractionated urine (at low abundances) or by Uexo fraction analysis. Perturbed proteins in the exosomal compartment of urine collected from kidney transplant patients were specific to inflammatory responses, and were not observed in the Uexo fraction from normal healthy subjects. Uexo specific protein alterations in renal disease provide potential mechanistic insights and offer a unique panel of sensitive biomarkers for monitoring for acute transplant rejection.« less

AT_CHLORO, a comprehensive chloroplast proteome database with subplastidial localization and curated information on envelope proteins.

PubMed

Ferro, Myriam; Brugière, Sabine; Salvi, Daniel; Seigneurin-Berny, Daphné; Court, Magali; Moyet, Lucas; Ramus, Claire; Miras, Stéphane; Mellal, Mourad; Le Gall, Sophie; Kieffer-Jaquinod, Sylvie; Bruley, Christophe; Garin, Jérôme; Joyard, Jacques; Masselon, Christophe; Rolland, Norbert

2010-06-01

Recent advances in the proteomics field have allowed a series of high throughput experiments to be conducted on chloroplast samples, and the data are available in several public databases. However, the accurate localization of many chloroplast proteins often remains hypothetical. This is especially true for envelope proteins. We went a step further into the knowledge of the chloroplast proteome by focusing, in the same set of experiments, on the localization of proteins in the stroma, the thylakoids, and envelope membranes. LC-MS/MS-based analyses first allowed building the AT_CHLORO database (http://www.grenoble.prabi.fr/protehome/grenoble-plant-proteomics/), a comprehensive repertoire of the 1323 proteins, identified by 10,654 unique peptide sequences, present in highly purified chloroplasts and their subfractions prepared from Arabidopsis thaliana leaves. This database also provides extensive proteomics information (peptide sequences and molecular weight, chromatographic retention times, MS/MS spectra, and spectral count) for a unique chloroplast protein accurate mass and time tag database gathering identified peptides with their respective and precise analytical coordinates, molecular weight, and retention time. We assessed the partitioning of each protein in the three chloroplast compartments by using a semiquantitative proteomics approach (spectral count). These data together with an in-depth investigation of the literature were compiled to provide accurate subplastidial localization of previously known and newly identified proteins. A unique knowledge base containing extensive information on the proteins identified in envelope fractions was thus obtained, allowing new insights into this membrane system to be revealed. Altogether, the data we obtained provide unexpected information about plastidial or subplastidial localization of some proteins that were not suspected to be associated to this membrane system. The spectral counting-based strategy was further validated as the compartmentation of well known pathways (for instance, photosynthesis and amino acid, fatty acid, or glycerolipid biosynthesis) within chloroplasts could be dissected. It also allowed revisiting the compartmentation of the chloroplast metabolism and functions.

Changes over lactation in breast milk serum proteins involved in the maturation of immune and digestive system of the infant.

PubMed

Zhang, Lina; de Waard, Marita; Verheijen, Hester; Boeren, Sjef; Hageman, Jos A; van Hooijdonk, Toon; Vervoort, Jacques; van Goudoever, Johannes B; Hettinga, Kasper

2016-09-16

To objective of this study was to better understand the biological functions of breast milk proteins in relation to the growth and development of infants over the first six months of life. Breast milk samples from four individual women collected at seven time points in the first six months after delivery were analyzed by filter aided sample preparation and dimethyl labeling combined with liquid chromatography tandem mass spectrometry. A total of 247 and 200 milk serum proteins were identified and quantified, respectively. The milk serum proteome showed a high similarity (80% overlap) on the qualitative level between women and over lactation. The quantitative changes in milk serum proteins were mainly caused by three groups of proteins, enzymes, and transport and immunity proteins. Of these 21 significantly changed proteins, 30% were transport proteins, such as serum albumin and fatty acid binding protein, which are both involved in transporting nutrients to the infant. The decrease of the enzyme bile salt-activated lipase as well as the immunity proteins immunoglobulins and lactoferrin coincide with the gradual maturation of the digestive and immune system of infants. The human milk serum proteome didn't differ qualitatively but it did quantitatively, both between mothers and as lactation advanced. The changes of the breast milk serum proteome over lactation corresponded with the development of the digestive and immune system of infants. Breast milk proteins provide nutrition, but also contribute to healthy development of infants. Despite the previously reported large number of identified breast milk proteins and their changes over lactation, less is known on the changes of these proteins in individual mothers. This study is the first to determine the qualitative and quantitative changes of milk proteome over lactation between individual mothers. The results indicate that the differences in the milk proteome between individual mothers are more related to the quantitative level than qualitative level. The correlation between the changes of milk proteins and the gradual maturation of the gastrointestinal tract and immune system in infants, contributes to a better understanding of the biological functions of human milk proteins for the growth and development of infants. Copyright © 2016 Elsevier B.V. All rights reserved.

Proteome dynamics of cold-acclimating Rhododendron species contrasting in their freezing tolerance and thermonasty response

USDA-ARS?s Scientific Manuscript database

In the present study we used 2D-DIGE technique to document the Rhododendron proteome during the seasonal development of cold hardiness. We selected two genotypes with different cold hardiness levels. This enabled us to perform comparative analysis of their proteome profiles and screen differentially...

Schizophrenia proteomics: biomarkers on the path to laboratory medicine?

PubMed Central

Lakhan, Shaheen Emmanuel

2006-01-01

Over two million Americans are afflicted with schizophrenia, a debilitating mental health disorder with a unique symptomatic and epidemiological profile. Genomics studies have hinted towards candidate schizophrenia susceptibility chromosomal loci and genes. Modern proteomic tools, particularly mass spectrometry and expression scanning, aim to identify both pathogenic-revealing and diagnostically significant biomarkers. Only a few studies on basic proteomics have been conducted for psychiatric disorders relative to the plethora of cancer specific experiments. One such proteomic utility enables the discovery of proteins and biological marker fingerprinting profiling techniques (SELDI-TOF-MS), and then subjects them to tandem mass spectrometric fragmentation and de novo protein sequencing (MALDI-TOF/TOF-MS) for the accurate identification and characterization of the proteins. Such utilities can explain the pathogenesis of neuro-psychiatric disease, provide more objective testing methods, and further demonstrate a biological basis to mental illness. Although clinical proteomics in schizophrenia have yet to reveal a biomarker with diagnostic specificity, methods that better characterize the disorder using endophenotypes can advance findings. Schizophrenia biomarkers could potentially revolutionize its psychopharmacology, changing it into a more hypothesis and genomic/proteomic-driven science. PMID:16846510

Top-down Proteomics: Technology Advancements and Applications to Heart Diseases

PubMed Central

Cai, Wenxuan; Tucholski, Trisha M.; Gregorich, Zachery R.; Ge, Ying

2016-01-01

Introduction Diseases of the heart are a leading cause of morbidity and mortality for both men and women worldwide, and impose significant economic burdens on the healthcare systems. Despite substantial effort over the last several decades, the molecular mechanisms underlying diseases of the heart remain poorly understood. Areas covered Altered protein post-translational modifications (PTMs) and protein isoform switching are increasingly recognized as important disease mechanisms. Top-down high-resolution mass spectrometry (MS)-based proteomics has emerged as the most powerful method for the comprehensive analysis of PTMs and protein isoforms. Here, we will review recent technology developments in the field of top-down proteomics, as well as highlight recent studies utilizing top-down proteomics to decipher the cardiac proteome for the understanding of the molecular mechanisms underlying diseases of the heart. Expert commentary Top-down proteomics is a premier method for the global and comprehensive study of protein isoforms and their PTMs, enabling the identification of novel protein isoforms and PTMs, characterization of sequence variations, and quantification of disease-associated alterations. Despite significant challenges, continuous development of top-down proteomics technology will greatly aid the dissection of the molecular mechanisms underlying diseases of the hearts for the identification of novel biomarkers and therapeutic targets. PMID:27448560

MitoMiner: a data warehouse for mitochondrial proteomics data

PubMed Central

Smith, Anthony C.; Blackshaw, James A.; Robinson, Alan J.

2012-01-01

MitoMiner (http://mitominer.mrc-mbu.cam.ac.uk/) is a data warehouse for the storage and analysis of mitochondrial proteomics data gathered from publications of mass spectrometry and green fluorescent protein tagging studies. In MitoMiner, these data are integrated with data from UniProt, Gene Ontology, Online Mendelian Inheritance in Man, HomoloGene, Kyoto Encyclopaedia of Genes and Genomes and PubMed. The latest release of MitoMiner stores proteomics data sets from 46 studies covering 11 different species from eumetazoa, viridiplantae, fungi and protista. MitoMiner is implemented by using the open source InterMine data warehouse system, which provides a user interface allowing users to upload data for analysis, personal accounts to store queries and results and enables queries of any data in the data model. MitoMiner also provides lists of proteins for use in analyses, including the new MitoMiner mitochondrial proteome reference sets that specify proteins with substantial experimental evidence for mitochondrial localization. As further mitochondrial proteomics data sets from normal and diseased tissue are published, MitoMiner can be used to characterize the variability of the mitochondrial proteome between tissues and investigate how changes in the proteome may contribute to mitochondrial dysfunction and mitochondrial-associated diseases such as cancer, neurodegenerative diseases, obesity, diabetes, heart failure and the ageing process. PMID:22121219

Proteome-wide analysis and diel proteomic profiling of the cyanobacterium Arthrospira platensis PCC 8005.

PubMed

Matallana-Surget, Sabine; Derock, Jérémy; Leroy, Baptiste; Badri, Hanène; Deschoenmaeker, Frédéric; Wattiez, Ruddy

2014-01-01

The filamentous cyanobacterium Arthrospira platensis has a long history of use as a food supply and it has been used by the European Space Agency in the MELiSSA project, an artificial microecosystem which supports life during long-term manned space missions. This study assesses progress in the field of cyanobacterial shotgun proteomics and light/dark diurnal cycles by focusing on Arthrospira platensis. Several fractionation workflows including gel-free and gel-based protein/peptide fractionation procedures were used and combined with LC-MS/MS analysis, enabling the overall identification of 1306 proteins, which represents 21% coverage of the theoretical proteome. A total of 30 proteins were found to be significantly differentially regulated under light/dark growth transition. Interestingly, most of the proteins showing differential abundance were related to photosynthesis, the Calvin cycle and translation processes. A novel aspect and major achievement of this work is the successful improvement of the cyanobacterial proteome coverage using a 3D LC-MS/MS approach, based on an immobilized metal affinity chromatography, a suitable tool that enabled us to eliminate the most abundant protein, the allophycocyanin. We also demonstrated that cell growth follows a light/dark cycle in A. platensis. This preliminary proteomic study has highlighted new characteristics of the Arthrospira platensis proteome in terms of diurnal regulation.

Proteome-Wide Analysis and Diel Proteomic Profiling of the Cyanobacterium Arthrospira platensis PCC 8005

PubMed Central

Matallana-Surget, Sabine; Derock, Jérémy; Leroy, Baptiste; Badri, Hanène; Deschoenmaeker, Frédéric; Wattiez, Ruddy

2014-01-01

The filamentous cyanobacterium Arthrospira platensis has a long history of use as a food supply and it has been used by the European Space Agency in the MELiSSA project, an artificial microecosystem which supports life during long-term manned space missions. This study assesses progress in the field of cyanobacterial shotgun proteomics and light/dark diurnal cycles by focusing on Arthrospira platensis. Several fractionation workflows including gel-free and gel-based protein/peptide fractionation procedures were used and combined with LC-MS/MS analysis, enabling the overall identification of 1306 proteins, which represents 21% coverage of the theoretical proteome. A total of 30 proteins were found to be significantly differentially regulated under light/dark growth transition. Interestingly, most of the proteins showing differential abundance were related to photosynthesis, the Calvin cycle and translation processes. A novel aspect and major achievement of this work is the successful improvement of the cyanobacterial proteome coverage using a 3D LC-MS/MS approach, based on an immobilized metal affinity chromatography, a suitable tool that enabled us to eliminate the most abundant protein, the allophycocyanin. We also demonstrated that cell growth follows a light/dark cycle in A. platensis. This preliminary proteomic study has highlighted new characteristics of the Arthrospira platensis proteome in terms of diurnal regulation. PMID:24914774

Proteomic approaches to understanding the role of the cytoskeleton in host-defense mechanisms

PubMed Central

Radulovic, Marko; Godovac-Zimmermann, Jasminka

2014-01-01

The cytoskeleton is a cellular scaffolding system whose functions include maintenance of cellular shape, enabling cellular migration, division, intracellular transport, signaling and membrane organization. In addition, in immune cells, the cytoskeleton is essential for phagocytosis. Following the advances in proteomics technology over the past two decades, cytoskeleton proteome analysis in resting and activated immune cells has emerged as a possible powerful approach to expand our understanding of cytoskeletal composition and function. However, so far there have only been a handful of studies of the cytoskeleton proteome in immune cells. This article considers promising proteomics strategies that could augment our understanding of the role of the cytoskeleton in host-defense mechanisms. PMID:21329431

Biomarkers of systemic lupus erythematosus identified using mass spectrometry-based proteomics: a systematic review.

PubMed

Nicolaou, Orthodoxia; Kousios, Andreas; Hadjisavvas, Andreas; Lauwerys, Bernard; Sokratous, Kleitos; Kyriacou, Kyriacos

2017-05-01

Advances in mass spectrometry technologies have created new opportunities for discovering novel protein biomarkers in systemic lupus erythematosus (SLE). We performed a systematic review of published reports on proteomic biomarkers identified in SLE patients using mass spectrometry-based proteomics and highlight their potential disease association and clinical utility. Two electronic databases, MEDLINE and EMBASE, were systematically searched up to July 2015. The methodological quality of studies included in the review was performed according to Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines. Twenty-five studies were included in the review, identifying 241 SLE candidate proteomic biomarkers related to various aspects of the disease including disease diagnosis and activity or pinpointing specific organ involvement. Furthermore, 13 of the 25 studies validated their results for a selected number of biomarkers in an independent cohort, resulting in the validation of 28 candidate biomarkers. It is noteworthy that 11 candidate biomarkers were identified in more than one study. A significant number of potential proteomic biomarkers that are related to a number of aspects of SLE have been identified using mass spectrometry proteomic approaches. However, further studies are required to assess the utility of these biomarkers in routine clinical practice. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Proteome Characterization of Leaves in Common Bean

PubMed Central

Robison, Faith M.; Heuberger, Adam L.; Brick, Mark A.; Prenni, Jessica E.

2015-01-01

Dry edible bean (Phaseolus vulgaris L.) is a globally relevant food crop. The bean genome was recently sequenced and annotated allowing for proteomics investigations aimed at characterization of leaf phenotypes important to agriculture. The objective of this study was to utilize a shotgun proteomics approach to characterize the leaf proteome and to identify protein abundance differences between two bean lines with known variation in their physiological resistance to biotic stresses. Overall, 640 proteins were confidently identified. Among these are proteins known to be involved in a variety of molecular functions including oxidoreductase activity, binding peroxidase activity, and hydrolase activity. Twenty nine proteins were found to significantly vary in abundance (p-value < 0.05) between the two bean lines, including proteins associated with biotic stress. To our knowledge, this work represents the first large scale shotgun proteomic analysis of beans and our results lay the groundwork for future studies designed to investigate the molecular mechanisms involved in pathogen resistance. PMID:28248269

Drought-Responsive Mechanisms in Plant Leaves Revealed by Proteomics.

PubMed

Wang, Xiaoli; Cai, Xiaofeng; Xu, Chenxi; Wang, Quanhua; Dai, Shaojun

2016-10-18

Plant drought tolerance is a complex trait that requires a global view to understand its underlying mechanism. The proteomic aspects of plant drought response have been extensively investigated in model plants, crops and wood plants. In this review, we summarize recent proteomic studies on drought response in leaves to reveal the common and specialized drought-responsive mechanisms in different plants. Although drought-responsive proteins exhibit various patterns depending on plant species, genotypes and stress intensity, proteomic analyses show that dominant changes occurred in sensing and signal transduction, reactive oxygen species scavenging, osmotic regulation, gene expression, protein synthesis/turnover, cell structure modulation, as well as carbohydrate and energy metabolism. In combination with physiological and molecular results, proteomic studies in leaves have helped to discover some potential proteins and/or metabolic pathways for drought tolerance. These findings provide new clues for understanding the molecular basis of plant drought tolerance.

Drought-Responsive Mechanisms in Plant Leaves Revealed by Proteomics

PubMed Central

Wang, Xiaoli; Cai, Xiaofeng; Xu, Chenxi; Wang, Quanhua; Dai, Shaojun

2016-01-01

Plant drought tolerance is a complex trait that requires a global view to understand its underlying mechanism. The proteomic aspects of plant drought response have been extensively investigated in model plants, crops and wood plants. In this review, we summarize recent proteomic studies on drought response in leaves to reveal the common and specialized drought-responsive mechanisms in different plants. Although drought-responsive proteins exhibit various patterns depending on plant species, genotypes and stress intensity, proteomic analyses show that dominant changes occurred in sensing and signal transduction, reactive oxygen species scavenging, osmotic regulation, gene expression, protein synthesis/turnover, cell structure modulation, as well as carbohydrate and energy metabolism. In combination with physiological and molecular results, proteomic studies in leaves have helped to discover some potential proteins and/or metabolic pathways for drought tolerance. These findings provide new clues for understanding the molecular basis of plant drought tolerance. PMID:27763546

Proteomic platform for the identification of proteins in olive (Olea europaea) pulp.

PubMed

Capriotti, Anna Laura; Cavaliere, Chiara; Foglia, Patrizia; Piovesana, Susy; Samperi, Roberto; Stampachiacchiere, Serena; Laganà, Aldo

2013-10-24

The nutritional and cancer-protective properties of the oil extracted mechanically from the ripe fruits of Olea europaea trees are attracting constantly more attention worldwide. The preparation of high-quality protein samples from plant tissues for proteomic analysis poses many challenging problems. In this study we employed a proteomic platform based on two different extraction methods, SDS and CHAPS based protocols, followed by two precipitation protocols, TCA/acetone and MeOH precipitation, in order to increase the final number of identified proteins. The use of advanced MS techniques in combination with the Swissprot and NCBI Viridiplantae databases and TAIR10 Arabidopsis database allowed us to identify 1265 proteins, of which 22 belong to O. europaea. The application of this proteomic platform for protein extraction and identification will be useful also for other proteomic studies on recalcitrant plant/fruit tissues. Copyright © 2013. Published by Elsevier B.V.

Proteomics of Plant Pathogenic Fungi

PubMed Central

González-Fernández, Raquel; Prats, Elena; Jorrín-Novo, Jesús V.

2010-01-01

Plant pathogenic fungi cause important yield losses in crops. In order to develop efficient and environmental friendly crop protection strategies, molecular studies of the fungal biological cycle, virulence factors, and interaction with its host are necessary. For that reason, several approaches have been performed using both classical genetic, cell biology, and biochemistry and the modern, holistic, and high-throughput, omic techniques. This work briefly overviews the tools available for studying Plant Pathogenic Fungi and is amply focused on MS-based Proteomics analysis, based on original papers published up to December 2009. At a methodological level, different steps in a proteomic workflow experiment are discussed. Separate sections are devoted to fungal descriptive (intracellular, subcellular, extracellular) and differential expression proteomics and interactomics. From the work published we can conclude that Proteomics, in combination with other techniques, constitutes a powerful tool for providing important information about pathogenicity and virulence factors, thus opening up new possibilities for crop disease diagnosis and crop protection. PMID:20589070

Proteomics of plant pathogenic fungi.

PubMed

González-Fernández, Raquel; Prats, Elena; Jorrín-Novo, Jesús V

2010-01-01

Plant pathogenic fungi cause important yield losses in crops. In order to develop efficient and environmental friendly crop protection strategies, molecular studies of the fungal biological cycle, virulence factors, and interaction with its host are necessary. For that reason, several approaches have been performed using both classical genetic, cell biology, and biochemistry and the modern, holistic, and high-throughput, omic techniques. This work briefly overviews the tools available for studying Plant Pathogenic Fungi and is amply focused on MS-based Proteomics analysis, based on original papers published up to December 2009. At a methodological level, different steps in a proteomic workflow experiment are discussed. Separate sections are devoted to fungal descriptive (intracellular, subcellular, extracellular) and differential expression proteomics and interactomics. From the work published we can conclude that Proteomics, in combination with other techniques, constitutes a powerful tool for providing important information about pathogenicity and virulence factors, thus opening up new possibilities for crop disease diagnosis and crop protection.

Functional and morphological analysis of putative chemoreception-related proteins in the lone star tick, Amblyomma americanum

USDA-ARS?s Scientific Manuscript database

Ehrlichiosis, a potentially fatal infection, is caused by rickettsial bacteria transmitted by the lone star tick, Amblyomma americanum. We previously analyzed the chemosensory appendage proteome of A. americanum as part of a project to develop new chemosensory-based vector control methods. Among the...

Improved Recovery and Identification of Membrane Proteins from Rat Hepatic Cells using a Centrifugal Proteomic Reactor*

PubMed Central

Zhou, Hu; Wang, Fangjun; Wang, Yuwei; Ning, Zhibin; Hou, Weimin; Wright, Theodore G.; Sundaram, Meenakshi; Zhong, Shumei; Yao, Zemin; Figeys, Daniel

2011-01-01

Despite their importance in many biological processes, membrane proteins are underrepresented in proteomic analysis because of their poor solubility (hydrophobicity) and often low abundance. We describe a novel approach for the identification of plasma membrane proteins and intracellular microsomal proteins that combines membrane fractionation, a centrifugal proteomic reactor for streamlined protein extraction, protein digestion and fractionation by centrifugation, and high performance liquid chromatography-electrospray ionization-tandem MS. The performance of this approach was illustrated for the study of the proteome of ER and Golgi microsomal membranes in rat hepatic cells. The centrifugal proteomic reactor identified 945 plasma membrane proteins and 955 microsomal membrane proteins, of which 63 and 47% were predicted as bona fide membrane proteins, respectively. Among these proteins, >800 proteins were undetectable by the conventional in-gel digestion approach. The majority of the membrane proteins only identified by the centrifugal proteomic reactor were proteins with ≥2 transmembrane segments or proteins with high molecular mass (e.g. >150 kDa) and hydrophobicity. The improved proteomic reactor allowed the detection of a group of endocytic and/or signaling receptor proteins on the plasma membrane, as well as apolipoproteins and glycerolipid synthesis enzymes that play a role in the assembly and secretion of apolipoprotein B100-containing very low density lipoproteins. Thus, the centrifugal proteomic reactor offers a new analytical tool for structure and function studies of membrane proteins involved in lipid and lipoprotein metabolism. PMID:21749988

Comprehensive Proteomic Analysis of Human Milk-derived Extracellular Vesicles Unveils a Novel Functional Proteome Distinct from Other Milk Components*

PubMed Central

van Herwijnen, Martijn J.C.; Zonneveld, Marijke I.; Goerdayal, Soenita; Nolte – 't Hoen, Esther N.M.; Garssen, Johan; Stahl, Bernd; Maarten Altelaar, A.F.; Redegeld, Frank A.; Wauben, Marca H.M.

2016-01-01

Breast milk contains several macromolecular components with distinctive functions, whereby milk fat globules and casein micelles mainly provide nutrition to the newborn, and whey contains molecules that can stimulate the newborn's developing immune system and gastrointestinal tract. Although extracellular vesicles (EV) have been identified in breast milk, their physiological function and composition has not been addressed in detail. EV are submicron sized vehicles released by cells for intercellular communication via selectively incorporated lipids, nucleic acids, and proteins. Because of the difficulty in separating EV from other milk components, an in-depth analysis of the proteome of human milk-derived EV is lacking. In this study, an extensive LC-MS/MS proteomic analysis was performed of EV that had been purified from breast milk of seven individual donors using a recently established, optimized density-gradient-based EV isolation protocol. A total of 1963 proteins were identified in milk-derived EV, including EV-associated proteins like CD9, Annexin A5, and Flotillin-1, with a remarkable overlap between the different donors. Interestingly, 198 of the identified proteins are not present in the human EV database Vesiclepedia, indicating that milk-derived EV harbor proteins not yet identified in EV of different origin. Similarly, the proteome of milk-derived EV was compared with that of other milk components. For this, data from 38 published milk proteomic studies were combined in order to construct the total milk proteome, which consists of 2698 unique proteins. Remarkably, 633 proteins identified in milk-derived EV have not yet been identified in human milk to date. Interestingly, these novel proteins include proteins involved in regulation of cell growth and controlling inflammatory signaling pathways, suggesting that milk-derived EVs could support the newborn's developing gastrointestinal tract and immune system. Overall, this study provides an expansion of the whole milk proteome and illustrates that milk-derived EV are macromolecular components with a unique functional proteome. PMID:27601599

Proteomic Profiling of β-hCG-Induced Spheres in BRCA1 Defective Triple Negative Breast Cancer Cells.

PubMed

Sengodan, Satheesh Kumar; Rajan, Arathi; Hemalatha, Sreelatha Krishnakumar; Nadhan, Revathy; Jaleel, Abdul; Srinivas, Priya

2018-01-05

Previously, we identified that β-hCG is expressed by BRCA1 mutated but not wild type breast cancers in vitro/in vivo and exhibited a novel event in β-hCG overexpressing BRCA1 mutated HCC1937 cells where the cells were able to form spheres (HCC1937 β spheres) in adherent cell culture plates even in the absence of any growth factors. These spheres express stem cell and EMT markers. In the present study, we carried out the total proteomic profiling of these HCC1937 β spheres obtained from BRCA1 defective β-hCG expressing stable breast cancer cells to analyze the cell signaling pathways that are active in these cells. Functional annotation revealed proteins (164 cellular and 97 secretory) predominantly involved in oxygen binding, nucleosome assembly, cytoskeleton organization, protein folding, etc. Many of the proteins identified from HCC1937 β spheres in this study are also up regulated in breast cancers, which are directly linked with poor prognosis in human cancer samples as analyzed using TCGA data set. Survival analysis shows that β-hCG expressing cancer patients are linked with poor survival rate. Interestingly, hemoglobins were identified at both cellular and secretory level in HCC1937 β spheres and experiments after treating with ROS inducers revealed that β-hCG induces hemoglobin and protects the cancer cells during oxidative stress. Our proteomic data strongly propose β-hCG as an oncogenic molecule associated with BRCA1 mutation, and hence, targeting β-hCG could be a strategy to treat BRCA1 defective breast cancers.

Quantitative proteomics analysis reveals perturbation of lipid metabolic pathways in the liver of Atlantic cod (Gadus morhua) treated with PCB 153.

PubMed

Yadetie, Fekadu; Oveland, Eystein; Døskeland, Anne; Berven, Frode; Goksøyr, Anders; Karlsen, Odd André

2017-04-01

PCB 153 is one of the most abundant PCB congeners detected in biological samples. It is a persistent compound that is still present in the environment despite the ban on production and use of PCBs in the late 1970s. It has strong tendencies to bioaccumulate and biomagnify in biota, and studies have suggested that it is an endocrine and metabolic disruptor. In order to study mechanisms of toxicity, we exposed Atlantic cod (Gadus morhua) to various doses of PCB 153 (0, 0.5, 2 and 8mg/kg body weight) for two weeks and examined the effects on expression of liver proteins using label-free quantitative proteomics. Label-free liquid chromatography-mass spectrometry analysis of the liver proteome resulted in the quantification of 1272 proteins, of which 78 proteins were differentially regulated in the PCB 153-treated dose groups compared to the control group. Functional enrichment analysis showed that pathways significantly affected are related to lipid metabolism, cytoskeletal remodeling, cell cycle and cell adhesion. Importantly, the main effects appear to be on lipid metabolism, with up-regulation of enzymes in the de novo fatty acid synthesis pathway, consistent with previous transcriptomics results. Increased plasma triglyceride levels were also observed in the PCB 153 treated fish, in agreement with the induction of the lipogenic genes and proteins. The results suggest that PCB 153 perturbs lipid metabolism in the Atlantic cod liver. Elevated levels of lipogenic enzymes and plasma triglycerides further suggest increased synthesis of fatty acids and triglycerides. Copyright © 2017 Elsevier B.V. All rights reserved.

Chromosomal abnormalities and molecular landscape of metastasizing mucinous salivary adenocarcinoma

PubMed Central

Panaccione, Alex; Zhang, Yi; Mi, Yanfang; Mitani, Yoshitsugu; Yan, Guo; Prasad, Manju L.; McDonald, W. Hayes; El-Naggar, Adel K.; Yarbrough, Wendell G.; Ivanov, Sergey V.

2017-01-01

Background Mucinous adenocarcinoma of the salivary gland (MAC) is a lethal cancer with unknown molecular etiology and a high propensity to lymph node metastasis. Mostly due to its orphan status, MAC remains one of the least explored cancers that lacks cell lines and mouse models that could help translational and pre-clinical studies. Surgery with or without radiation remains the only treatment modality but poor overall survival (10-year, 44%) underscores the urgent need for mechanism-based therapies. Methods We developed the first patient-derived xenograft (PDX) model for pre-clinical MAC studies and a cell line that produces aggressively growing tumors after subcutaneous injection into nude mice. We performed cytogenetic, exome, and proteomic profiling of MAC to identify driving mutations, therapeutic targets, and pathways involved in aggressive cancers based on TCGA database mining and GEO analysis. Results: We identified in MAC KRAS (G13D) and TP53 (R213X) mutations that have been previously reported as drivers in a variety of highly aggressive cancers. Somatic mutations were also found in KDM6A, KMT2D, and other genes frequently mutated in colorectal and other cancers: FAT1, NBEA, RELN, RLP1B, and ZFHX3. Proteomic analysis of MAC implied epigenetic up-regulation of a genetic program involved in proliferation and cancer stem cell maintenance. Conclusion Genomic and proteomic analyses provided the first insight into potential molecular drivers of MAC metastases pointing at common mechanisms of CSC propagation in aggressive cancers. The in vitro/in vivo models that we created should aid in the development and validation of new treatment strategies against MAC. PMID:28249646

Global Analysis of Mannitol 2-Dehydrogenase in Lactobacillus reuteri CRL 1101 during Mannitol Production through Enzymatic, Genetic and Proteomic Approaches.

PubMed

Ortiz, Maria Eugenia; Bleckwedel, Juliana; Fadda, Silvina; Picariello, Gianluca; Hebert, Elvira M; Raya, Raúl R; Mozzi, Fernanda

2017-01-01

Several plants, fungi, algae, and certain bacteria produce mannitol, a polyol derived from fructose. Mannitol has multiple industrial applications in the food, pharmaceutical, and medical industries, being mainly used as a non-metabolizable sweetener in foods. Many heterofermentative lactic acid bacteria synthesize mannitol when an alternative electron acceptor such as fructose is present in the medium. In previous work, we reported the ability of Lactobacillus reuteri CRL 1101 to efficiently produce mannitol from sugarcane molasses as carbon source at constant pH of 5.0; the activity of the enzyme mannitol 2-dehydrogenase (MDH) responsible for the fructose conversion into mannitol being highest during the log cell growth phase. Here, a detailed assessment of the MDH activity and relative expression of the mdh gene during the growth of L. reuteri CRL 1101 in the presence of fructose is presented. It was observed that MDH was markedly induced by the presence of fructose. A direct correlation between the maximum MDH enzyme activity and a high level of mdh transcript expression during the log-phase of cells grown in a fructose-containing chemically defined medium was detected. Furthermore, two proteomic approaches (2DE and shotgun proteomics) applied in this study confirmed the inducible expression of MDH in L. reuteri. A global study of the effect of fructose on activity, mdh gene, and protein expressions of MDH in L. reuteri is thus for the first time presented. This work represents a deep insight into the polyol formation by a Lactobacillus strain with biotechnological potential in the nutraceutics and pharmaceutical areas.

Global Analysis of Mannitol 2-Dehydrogenase in Lactobacillus reuteri CRL 1101 during Mannitol Production through Enzymatic, Genetic and Proteomic Approaches

PubMed Central

Ortiz, Maria Eugenia; Bleckwedel, Juliana; Fadda, Silvina; Picariello, Gianluca; Hebert, Elvira M.; Raya, Raúl R.

2017-01-01

Several plants, fungi, algae, and certain bacteria produce mannitol, a polyol derived from fructose. Mannitol has multiple industrial applications in the food, pharmaceutical, and medical industries, being mainly used as a non-metabolizable sweetener in foods. Many heterofermentative lactic acid bacteria synthesize mannitol when an alternative electron acceptor such as fructose is present in the medium. In previous work, we reported the ability of Lactobacillus reuteri CRL 1101 to efficiently produce mannitol from sugarcane molasses as carbon source at constant pH of 5.0; the activity of the enzyme mannitol 2-dehydrogenase (MDH) responsible for the fructose conversion into mannitol being highest during the log cell growth phase. Here, a detailed assessment of the MDH activity and relative expression of the mdh gene during the growth of L. reuteri CRL 1101 in the presence of fructose is presented. It was observed that MDH was markedly induced by the presence of fructose. A direct correlation between the maximum MDH enzyme activity and a high level of mdh transcript expression during the log-phase of cells grown in a fructose-containing chemically defined medium was detected. Furthermore, two proteomic approaches (2DE and shotgun proteomics) applied in this study confirmed the inducible expression of MDH in L. reuteri. A global study of the effect of fructose on activity, mdh gene, and protein expressions of MDH in L. reuteri is thus for the first time presented. This work represents a deep insight into the polyol formation by a Lactobacillus strain with biotechnological potential in the nutraceutics and pharmaceutical areas. PMID:28060932

Proteomic Characterization of Yersinia pestis Virulence

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chromy, B; Murphy, G; Gonzales, A

2005-01-05

Yersinia pestis, the etiological agent of plague, functions via the Type III secretion mechanism whereby virulence factors are induced upon interactions with a mammalian host. Here, the Y. pestis proteome was studied by two-dimensional differential gel electrophoresis (2-D DIGE) under physiologically relevant growth conditions mimicking the calcium concentrations and temperatures that the pathogen would encounter in the flea vector and upon interaction with the mammalian host. Over 4100 individual protein spots were detected of which hundreds were differentially expressed in the entire comparative experiment. A total of 43 proteins that were differentially expressed between the vector and host growth conditionsmore » were identified by mass spectrometry. Expected differences in expression were observed for several known virulence factors including catalase-peroxidase (KatY), murine toxin (Ymt), plasminogen activator (Pla), and F1 capsule antigen (Caf1), as well as putative virulence factors. Chaperone proteins and signaling molecules hypothesized to be involved in virulence due to their role in Type III secretion were also identified. Other differentially expressed proteins not previously reported to contribute to virulence are candidates for more detailed mechanistic studies, representing potential new virulence determinants. For example, several sugar metabolism proteins were differentially regulated in response to lower calcium and higher temperature, suggesting these proteins, while not directly connected to virulence, either represent a metabolic switch for survival in the host environment or may facilitate production of virulence factors. Results presented here contribute to a more thorough understanding of the virulence mechanism of Y. pestis through proteomic characterization of the pathogen under induced virulence.« less

Effects of Hypertension and Exercise on Cardiac Proteome Remodelling

PubMed Central

Petriz, Bernardo A.; Franco, Octavio L.

2014-01-01

Left ventricle hypertrophy is a common outcome of pressure overload stimulus closely associated with hypertension. This process is triggered by adverse molecular signalling, gene expression, and proteome alteration. Proteomic research has revealed that several molecular targets are associated with pathologic cardiac hypertrophy, including angiotensin II, endothelin-1 and isoproterenol. Several metabolic, contractile, and stress-related proteins are shown to be altered in cardiac hypertrophy derived by hypertension. On the other hand, exercise is a nonpharmacologic agent used for hypertension treatment, where cardiac hypertrophy induced by exercise training is characterized by improvement in cardiac function and resistance against ischemic insult. Despite the scarcity of proteomic research performed with exercise, healthy and pathologic heart proteomes are shown to be modulated in a completely different way. Hence, the altered proteome induced by exercise is mostly associated with cardioprotective aspects such as contractile and metabolic improvement and physiologic cardiac hypertrophy. The present review, therefore, describes relevant studies involving the molecular characteristics and alterations from hypertensive-induced and exercise-induced hypertrophy, as well as the main proteomic research performed in this field. Furthermore, proteomic research into the effect of hypertension on other target-demerged organs is examined. PMID:24877123

Comparative and Quantitative Global Proteomics Approaches: An Overview

PubMed Central

Deracinois, Barbara; Flahaut, Christophe; Duban-Deweer, Sophie; Karamanos, Yannis

2013-01-01

Proteomics became a key tool for the study of biological systems. The comparison between two different physiological states allows unravelling the cellular and molecular mechanisms involved in a biological process. Proteomics can confirm the presence of proteins suggested by their mRNA content and provides a direct measure of the quantity present in a cell. Global and targeted proteomics strategies can be applied. Targeted proteomics strategies limit the number of features that will be monitored and then optimise the methods to obtain the highest sensitivity and throughput for a huge amount of samples. The advantage of global proteomics strategies is that no hypothesis is required, other than a measurable difference in one or more protein species between the samples. Global proteomics methods attempt to separate quantify and identify all the proteins from a given sample. This review highlights only the different techniques of separation and quantification of proteins and peptides, in view of a comparative and quantitative global proteomics analysis. The in-gel and off-gel quantification of proteins will be discussed as well as the corresponding mass spectrometry technology. The overview is focused on the widespread techniques while keeping in mind that each approach is modular and often recovers the other. PMID:28250403

Top-down Proteomics in Health and Disease: Challenges and Opportunities

PubMed Central

Gregorich, Zachery R.; Ge, Ying

2014-01-01

Proteomics is essential for deciphering how molecules interact as a system and for understanding the functions of cellular systems in human disease; however, the unique characteristics of the human proteome, which include a high dynamic range of protein expression and extreme complexity due to a plethora of post-translational modifications (PTMs) and sequence variations, make such analyses challenging. An emerging “top-down” mass spectrometry (MS)-based proteomics approach, which provides a “bird’s eye” view of all proteoforms, has unique advantages for the assessment of PTMs and sequence variations. Recently, a number of studies have showcased the potential of top-down proteomics for unraveling of disease mechanisms and discovery of new biomarkers. Nevertheless, the top-down approach still faces significant challenges in terms of protein solubility, separation, and the detection of large intact proteins, as well as the under-developed data analysis tools. Consequently, new technological developments are urgently needed to advance the field of top-down proteomics. Herein, we intend to provide an overview of the recent applications of top-down proteomics in biomedical research. Moreover, we will outline the challenges and opportunities facing top-down proteomics strategies aimed at understanding and diagnosing human diseases. PMID:24723472