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

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.

PatternLab for proteomics 4.0: A one-stop shop for analyzing shotgun proteomic data

PubMed Central

Carvalho, Paulo C; Lima, Diogo B; Leprevost, Felipe V; Santos, Marlon D M; Fischer, Juliana S G; Aquino, Priscila F; Moresco, James J; Yates, John R; Barbosa, Valmir C

2017-01-01

PatternLab for proteomics is an integrated computational environment that unifies several previously published modules for analyzing shotgun proteomic data. PatternLab contains modules for formatting sequence databases, performing peptide spectrum matching, statistically filtering and organizing shotgun proteomic data, extracting quantitative information from label-free and chemically labeled data, performing statistics for differential proteomics, displaying results in a variety of graphical formats, performing similarity-driven studies with de novo sequencing data, analyzing time-course experiments, and helping with the understanding of the biological significance of data in the light of the Gene Ontology. Here we describe PatternLab for proteomics 4.0, which closely knits together all of these modules in a self-contained environment, covering the principal aspects of proteomic data analysis as a freely available and easily installable software package. All updates to PatternLab, as well as all new features added to it, have been tested over the years on millions of mass spectra. PMID:26658470

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.

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.

Quantitative Analysis of Global Proteome and Lysine Acetylome Reveal the Differential Impacts of VPA and SAHA on HL60 Cells.

PubMed

Zhu, Xiaoyu; Liu, Xin; Cheng, Zhongyi; Zhu, Jun; Xu, Lei; Wang, Fengsong; Qi, Wulin; Yan, Jiawei; Liu, Ning; Sun, Zimin; Liu, Huilan; Peng, Xiaojun; Hao, Yingchan; Zheng, Nan; Wu, Quan

2016-01-29

Valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA) are both HDAC inhibitors (HDACi). Previous studies indicated that both inhibitors show therapeutic effects on acute myeloid leukaemia (AML), while the differential impacts of the two different HDACi on AML treatment still remains elusive. In this study, using 3-plex SILAC based quantitative proteomics technique, anti-acetyllysine antibody based affinity enrichment, high resolution LC-MS/MS and intensive bioinformatic analysis, the quantitative proteome and acetylome in SAHA and VPA treated AML HL60 cells were extensively studied. In total, 5,775 proteins and 1,124 lysine acetylation sites were successfully obtained in response to VAP and SAHA treatment. It is found that VPA and SAHA treatment differently induced proteome and acetylome profiling in AML HL60 cells. This study revealed the differential impacts of VPA and SAHA on proteome/acetylome in AML cells, deepening our understanding of HDAC inhibitor mediated AML therapeutics.

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.

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

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.

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.

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.

Implementation of proteomics for cancer research: past, present, and future.

PubMed

Karimi, Parisa; Shahrokni, Armin; Ranjbar, Mohammad R Nezami

2014-01-01

Cancer is the leading cause of the death, accounts for about 13% of all annual deaths worldwide. Many different fields of science are collaborating together studying cancer to improve our knowledge of this lethal disease, and find better solutions for diagnosis and treatment. Proteomics is one of the most recent and rapidly growing areas in molecular biology that helps understanding cancer from an omics data analysis point of view. The human proteome project was officially initiated in 2008. Proteomics enables the scientists to interrogate a variety of biospecimens for their protein contents and measure the concentrations of these proteins. Current necessary equipment and technologies for cancer proteomics are mass spectrometry, protein microarrays, nanotechnology and bioinformatics. In this paper, we provide a brief review on proteomics and its application in cancer research. After a brief introduction including its definition, we summarize the history of major previous work conducted by researchers, followed by an overview on the role of proteomics in cancer studies. We also provide a list of different utilities in cancer proteomics and investigate their advantages and shortcomings from theoretical and practical angles. Finally, we explore some of the main challenges and conclude the paper with future directions in this field.

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.

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.

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.

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.

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.

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

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.

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.

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

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.

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.

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.

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

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

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.

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

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.

[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.

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.

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

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

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

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

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.

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.

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.

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.

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.

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.

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

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).

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.

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

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.

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.

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.

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.

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

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

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.

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

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

Protein complexes, big data, machine learning and integrative proteomics: lessons learned over a decade of systematic analysis of protein interaction networks.

PubMed

Havugimana, Pierre C; Hu, Pingzhao; Emili, Andrew

2017-10-01

Elucidation of the networks of physical (functional) interactions present in cells and tissues is fundamental for understanding the molecular organization of biological systems, the mechanistic basis of essential and disease-related processes, and for functional annotation of previously uncharacterized proteins (via guilt-by-association or -correlation). After a decade in the field, we felt it timely to document our own experiences in the systematic analysis of protein interaction networks. Areas covered: Researchers worldwide have contributed innovative experimental and computational approaches that have driven the rapidly evolving field of 'functional proteomics'. These include mass spectrometry-based methods to characterize macromolecular complexes on a global-scale and sophisticated data analysis tools - most notably machine learning - that allow for the generation of high-quality protein association maps. Expert commentary: Here, we recount some key lessons learned, with an emphasis on successful workflows, and challenges, arising from our own and other groups' ongoing efforts to generate, interpret and report proteome-scale interaction networks in increasingly diverse biological contexts.

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

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.

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

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 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.

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

"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.

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.

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.

Proteome analysis of Arabidopsis seedlings exposed to bacterial volatiles.

PubMed

Kwon, Young Sang; Ryu, Choong-Min; Lee, Soohyun; Park, Hyo Bee; Han, Ki Soo; Lee, Jung Han; Lee, Kyunghee; Chung, Woo Sik; Jeong, Mi-Jeong; Kim, Hee Kyu; Bae, Dong-Won

2010-11-01

Plant root-associated bacteria (rhizobacteria) elicit plant basal immunity referred to as induced systemic resistance (ISR) against multiple pathogens. Among multi-bacterial determinants involving such ISR, the induction of ISR and promotion of growth by bacterial volatile compounds was previously reported. To exploit global de novo expression of plant proteins by bacterial volatiles, proteomic analysis was performed after exposure of Arabidopsis plants to the rhizobacterium Bacillus subtilis GB03. Ethylene biosynthesis enzymes were significantly up-regulated. Analysis by quantitative reverse transcriptase polymerase chain reaction confirmed that ethylene biosynthesis-related genes SAM-2, ACS4, ACS12, and ACO2 as well as ethylene response genes, ERF1, GST2, and CHIB were up-regulated by the exposure to bacterial volatiles. More interestingly, the emission of bacterial volatiles significantly up-regulated both key defense mechanisms mediated by jasmonic acid and salicylic acid signaling pathways. In addition, high accumulation of antioxidant proteins also provided evidence of decreased sensitivity to reactive oxygen species during the elicitation of ISR by bacterial volatiles. The present results suggest that the proteomic analysis of plant defense responses in bacterial volatile-mediated ISR can reveal the mechanisms of plant basal defenses orchestrated by endogenous ethylene production pathways and the generation of reactive oxygen species.

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

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.

Skyline: an open source document editor for creating and analyzing targeted proteomics experiments.

PubMed

MacLean, Brendan; Tomazela, Daniela M; Shulman, Nicholas; Chambers, Matthew; Finney, Gregory L; Frewen, Barbara; Kern, Randall; Tabb, David L; Liebler, Daniel C; MacCoss, Michael J

2010-04-01

Skyline is a Windows client application for targeted proteomics method creation and quantitative data analysis. It is open source and freely available for academic and commercial use. The Skyline user interface simplifies the development of mass spectrometer methods and the analysis of data from targeted proteomics experiments performed using selected reaction monitoring (SRM). Skyline supports using and creating MS/MS spectral libraries from a wide variety of sources to choose SRM filters and verify results based on previously observed ion trap data. Skyline exports transition lists to and imports the native output files from Agilent, Applied Biosystems, Thermo Fisher Scientific and Waters triple quadrupole instruments, seamlessly connecting mass spectrometer output back to the experimental design document. The fast and compact Skyline file format is easily shared, even for experiments requiring many sample injections. A rich array of graphs displays results and provides powerful tools for inspecting data integrity as data are acquired, helping instrument operators to identify problems early. The Skyline dynamic report designer exports tabular data from the Skyline document model for in-depth analysis with common statistical tools. Single-click, self-updating web installation is available at http://proteome.gs.washington.edu/software/skyline. This web site also provides access to instructional videos, a support board, an issues list and a link to the source code project.

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

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.

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.

CAPER 3.0: A Scalable Cloud-Based System for Data-Intensive Analysis of Chromosome-Centric Human Proteome Project Data Sets.

PubMed

Yang, Shuai; Zhang, Xinlei; Diao, Lihong; Guo, Feifei; Wang, Dan; Liu, Zhongyang; Li, Honglei; Zheng, Junjie; Pan, Jingshan; Nice, Edouard C; Li, Dong; He, Fuchu

2015-09-04

The Chromosome-centric Human Proteome Project (C-HPP) aims to catalog genome-encoded proteins using a chromosome-by-chromosome strategy. As the C-HPP proceeds, the increasing requirement for data-intensive analysis of the MS/MS data poses a challenge to the proteomic community, especially small laboratories lacking computational infrastructure. To address this challenge, we have updated the previous CAPER browser into a higher version, CAPER 3.0, which is a scalable cloud-based system for data-intensive analysis of C-HPP data sets. CAPER 3.0 uses cloud computing technology to facilitate MS/MS-based peptide identification. In particular, it can use both public and private cloud, facilitating the analysis of C-HPP data sets. CAPER 3.0 provides a graphical user interface (GUI) to help users transfer data, configure jobs, track progress, and visualize the results comprehensively. These features enable users without programming expertise to easily conduct data-intensive analysis using CAPER 3.0. Here, we illustrate the usage of CAPER 3.0 with four specific mass spectral data-intensive problems: detecting novel peptides, identifying single amino acid variants (SAVs) derived from known missense mutations, identifying sample-specific SAVs, and identifying exon-skipping events. CAPER 3.0 is available at http://prodigy.bprc.ac.cn/caper3.

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.

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.

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

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

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...

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

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.

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

Software Analysis of Uncorrelated MS1 Peaks for Discovery of Post-Translational Modifications.

PubMed

Pascal, Bruce D; West, Graham M; Scharager-Tapia, Catherina; Flefil, Ricardo; Moroni, Tina; Martinez-Acedo, Pablo; Griffin, Patrick R; Carvalloza, Anthony C

2015-12-01

The goal in proteomics to identify all peptides in a complex mixture has been largely addressed using various LC MS/MS approaches, such as data dependent acquisition, SRM/MRM, and data independent acquisition instrumentation. Despite these developments, many peptides remain unsequenced, often due to low abundance, poor fragmentation patterns, or data analysis difficulties. Many of the unidentified peptides exhibit strong evidence in high resolution MS(1) data and are frequently post-translationally modified, playing a significant role in biological processes. Proteomics Workbench (PWB) software was developed to automate the detection and visualization of all possible peptides in MS(1) data, reveal candidate peptides not initially identified, and build inclusion lists for subsequent MS(2) analysis to uncover new identifications. We used this software on existing data on the autophagy regulating kinase Ulk1 as a proof of concept for this method, as we had already manually identified a number of phosphorylation sites Dorsey, F. C. et al (J. Proteome. Res. 8(11), 5253-5263 (2009)). PWB found all previously identified sites of phosphorylation. The software has been made freely available at http://www.proteomicsworkbench.com . Graphical Abstract ᅟ.

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

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.

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.

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.

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 (

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

The Escherichia coli O157:H7 cattle immuno-proteome includes outer membrane protein A (OmpA), a modulator of adherence to bovine recto-anal junction squamous epithelial (RSE) cells

PubMed Central

Kudva, Indira T.; Krastins, Bryan; Torres, Alfredo G.; Griffin, Robert W.; Sheng, Haiqing; Sarracino, David A.; Hovde, Carolyn J.; Calderwood, Stephen B.; John, Manohar

2015-01-01

SUMMARY Building on previous studies, we defined the repertoire of proteins comprising the immuno-proteome of E. coli O157:H7 (O157) cultured in DMEM supplemented with norepinephrine (NE; O157 immuno-proteome), a β-adrenergic hormone that regulates E. coli O157 gene expression in the gastrointestinal tract, using a variation of a novel proteomics-based platform proteome mining tool for antigen discovery, called Proteomics-based Expression Library Screening (PELS; Kudva et al., 2006). The E. coli O157 immuno-proteome (O157-IP) comprised 91 proteins, and included those identified previously using PELS, and also proteins comprising DMEM- and bovine rumen fluid- proteomes. Outer membrane protein A (OmpA), a common component of the above proteomes, and reportedly a contributor to E. coli O157 adherence to cultured Hep-2 epithelial cells, was interestingly found to be a modulator rather than a contributor to E. coli O157 adherence to bovine recto-anal junction squamous epithelial (RSE) cells. Our results point to a role for yet to be identified members of the O157-IP in E. coli O157 adherence to RSE-cells, and additionally implicate a possible role for the OmpA regulator, TdcA, in the expression of such adhesins. Our observations have implications for development of efficacious vaccines for preventing E. coli O157 colonization of the bovine gastrointestinal tract. PMID:25643951

BioInfra.Prot: A comprehensive proteomics workflow including data standardization, protein inference, expression analysis and data publication.

PubMed

Turewicz, Michael; Kohl, Michael; Ahrens, Maike; Mayer, Gerhard; Uszkoreit, Julian; Naboulsi, Wael; Bracht, Thilo; Megger, Dominik A; Sitek, Barbara; Marcus, Katrin; Eisenacher, Martin

2017-11-10

The analysis of high-throughput mass spectrometry-based proteomics data must address the specific challenges of this technology. To this end, the comprehensive proteomics workflow offered by the de.NBI service center BioInfra.Prot provides indispensable components for the computational and statistical analysis of this kind of data. These components include tools and methods for spectrum identification and protein inference, protein quantification, expression analysis as well as data standardization and data publication. All particular methods of the workflow which address these tasks are state-of-the-art or cutting edge. As has been shown in previous publications, each of these methods is adequate to solve its specific task and gives competitive results. However, the methods included in the workflow are continuously reviewed, updated and improved to adapt to new scientific developments. All of these particular components and methods are available as stand-alone BioInfra.Prot services or as a complete workflow. Since BioInfra.Prot provides manifold fast communication channels to get access to all components of the workflow (e.g., via the BioInfra.Prot ticket system: bioinfraprot@rub.de) users can easily benefit from this service and get support by experts. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Identification of BAG3 target proteins in anaplastic thyroid cancer cells by proteomic analysis.

PubMed

Galdiero, Francesca; Bello, Anna Maria; Spina, Anna; Capiluongo, Anna; Liuu, Sophie; De Marco, Margot; Rosati, Alessandra; Capunzo, Mario; Napolitano, Maria; Vuttariello, Emilia; Monaco, Mario; Califano, Daniela; Turco, Maria Caterina; Chiappetta, Gennaro; Vinh, Joëlle; Chiappetta, Giovanni

2018-01-30

BAG3 protein is an apoptosis inhibitor and is highly expressed in Anaplastic Thyroid Cancer. We investigated the entire set of proteins modulated by BAG3 silencing in the human anaplastic thyroid 8505C cancer cells by using the Stable-Isotope Labeling by Amino acids in Cell culture strategy combined with mass spectrometry analysis. By this approach we identified 37 up-regulated and 54 down-regulated proteins in BAG3-silenced cells. Many of these proteins are reportedly involved in tumor progression, invasiveness and resistance to therapies. We focused our attention on an oncogenic protein, CAV1, and a tumor suppressor protein, SERPINB2, that had not previously been reported to be modulated by BAG3. Their expression levels in BAG3-silenced cells were confirmed by qRT-PCR and western blot analyses, disclosing two novel targets of BAG3 pro-tumor activity. We also examined the dataset of proteins obtained by the quantitative proteomics analysis using two tools, Downstream Effect Analysis and Upstream Regulator Analysis of the Ingenuity Pathways Analysis software. Our analyses confirm the association of the proteome profile observed in BAG3-silenced cells with an increase in cell survival and a decrease in cell proliferation and invasion, and highlight the possible involvement of four tumor suppressor miRNAs and TP53/63 proteins in BAG3 activity.

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.

Comprehensive genome-wide proteomic analysis of human placental tissue for the Chromosome-Centric Human Proteome Project.

PubMed

Lee, Hyoung-Joo; Jeong, Seul-Ki; Na, Keun; Lee, Min Jung; Lee, Sun Hee; Lim, Jong-Sun; Cha, Hyun-Jeong; Cho, Jin-Young; Kwon, Ja-Young; Kim, Hoguen; Song, Si Young; Yoo, Jong Shin; Park, Young Mok; Kim, Hail; Hancock, William S; Paik, Young-Ki

2013-06-07

As a starting point of the Chromosome-Centric Human Proteome Project (C-HPP), we established strategies of genome-wide proteomic analysis, including protein identification, quantitation of disease-specific proteins, and assessment of post-translational modifications, using paired human placental tissues from healthy and preeclampsia patients. This analysis resulted in identification of 4239 unique proteins with high confidence (two or more unique peptides with a false discovery rate less than 1%), covering 21% of approximately 20, 059 (Ensembl v69, Oct 2012) human proteins, among which 28 proteins exhibited differentially expressed preeclampsia-specific proteins. When these proteins are assigned to all human chromosomes, the pattern of the newly identified placental protein population is proportional to that of the gene count distribution of each chromosome. We also identified 219 unique N-linked glycopeptides, 592 unique phosphopeptides, and 66 chromosome 13-specific proteins. In particular, protein evidence of 14 genes previously known to be specifically up-regulated in human placenta was verified by mass spectrometry. With respect to the functional implication of these proteins, 38 proteins were found to be involved in regulatory factor biosynthesis or the immune system in the placenta, but the molecular mechanism of these proteins during pregnancy warrants further investigation. As far as we know, this work produced the highest number of proteins identified in the placenta and will be useful for annotating and mapping all proteins encoded in the human genome.

Skyline: an open source document editor for creating and analyzing targeted proteomics experiments

PubMed Central

MacLean, Brendan; Tomazela, Daniela M.; Shulman, Nicholas; Chambers, Matthew; Finney, Gregory L.; Frewen, Barbara; Kern, Randall; Tabb, David L.; Liebler, Daniel C.; MacCoss, Michael J.

2010-01-01

Summary: Skyline is a Windows client application for targeted proteomics method creation and quantitative data analysis. It is open source and freely available for academic and commercial use. The Skyline user interface simplifies the development of mass spectrometer methods and the analysis of data from targeted proteomics experiments performed using selected reaction monitoring (SRM). Skyline supports using and creating MS/MS spectral libraries from a wide variety of sources to choose SRM filters and verify results based on previously observed ion trap data. Skyline exports transition lists to and imports the native output files from Agilent, Applied Biosystems, Thermo Fisher Scientific and Waters triple quadrupole instruments, seamlessly connecting mass spectrometer output back to the experimental design document. The fast and compact Skyline file format is easily shared, even for experiments requiring many sample injections. A rich array of graphs displays results and provides powerful tools for inspecting data integrity as data are acquired, helping instrument operators to identify problems early. The Skyline dynamic report designer exports tabular data from the Skyline document model for in-depth analysis with common statistical tools. Availability: Single-click, self-updating web installation is available at http://proteome.gs.washington.edu/software/skyline. This web site also provides access to instructional videos, a support board, an issues list and a link to the source code project. Contact: brendanx@u.washington.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:20147306

Characterization of the human submandibular/sublingual saliva glycoproteome using lectin affinity chromatography coupled to Multidimensional Protein Identification Technology

PubMed Central

Gonzalez-Begne, Mireya; Lu, Bingwen; Liao, Lujian; Xu, Tao; Bedi, Gurrinder; Melvin, James E.; Yates, John R.

2011-01-01

In-depth analysis of the salivary proteome is fundamental to understanding the functions of salivary proteins in the oral cavity and to reveal disease biomarkers involved in different pathophysiological conditions, with the ultimate goal of improving patient diagnosis and prognosis. Submandibular and sublingual glands contribute saliva rich in glycoproteins to the total saliva output, making them valuable sources for glycoproteomic analysis. Lectin-affinity chromatography coupled to mass spectrometry-based shotgun proteomics was used to explore the submandibular/sublingual (SM/SL) saliva glycoproteome. A total of 262 N- and O-linked glycoproteins were identified by multidimensional protein identification technology (MudPIT). Only 38 were previously described in SM and SL salivas from the human salivary N-linked glycoproteome, while 224 were unique. Further comparison analysis with SM/SL saliva of the human saliva proteome, revealed 125 glycoproteins not formerly reported in this secretion. KEGG pathway analyses demonstrated that many of these glycoproteins are involved in processes such as complement and coagulation cascades, cell communication, glycosphingolipid biosynthesis neo-lactoseries, O-glycan biosynthesis, glycan structures-biosynthesis 2, starch and sucrose metabolism, peptidoglycan biosynthesis or others pathways. In summary, lectin-affinity chromatography coupled to MudPIT mass spectrometry identified many novel glycoproteins in SM/SL saliva. These new additions to the salivary proteome may prove to be a critical step for providing reliable biomarkers in the diagnosis of a myriad of oral and systemic diseases. PMID:21936497

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

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

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.

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.

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.

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

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.

GProX, a user-friendly platform for bioinformatics analysis and visualization of quantitative proteomics data.

PubMed

Rigbolt, Kristoffer T G; Vanselow, Jens T; Blagoev, Blagoy

2011-08-01

Recent technological advances have made it possible to identify and quantify thousands of proteins in a single proteomics experiment. As a result of these developments, the analysis of data has become the bottleneck of proteomics experiment. To provide the proteomics community with a user-friendly platform for comprehensive analysis, inspection and visualization of quantitative proteomics data we developed the Graphical Proteomics Data Explorer (GProX)(1). The program requires no special bioinformatics training, as all functions of GProX are accessible within its graphical user-friendly interface which will be intuitive to most users. Basic features facilitate the uncomplicated management and organization of large data sets and complex experimental setups as well as the inspection and graphical plotting of quantitative data. These are complemented by readily available high-level analysis options such as database querying, clustering based on abundance ratios, feature enrichment tests for e.g. GO terms and pathway analysis tools. A number of plotting options for visualization of quantitative proteomics data is available and most analysis functions in GProX create customizable high quality graphical displays in both vector and bitmap formats. The generic import requirements allow data originating from essentially all mass spectrometry platforms, quantitation strategies and software to be analyzed in the program. GProX represents a powerful approach to proteomics data analysis providing proteomics experimenters with a toolbox for bioinformatics analysis of quantitative proteomics data. The program is released as open-source and can be freely downloaded from the project webpage at http://gprox.sourceforge.net.

GProX, a User-Friendly Platform for Bioinformatics Analysis and Visualization of Quantitative Proteomics Data*

PubMed Central

Rigbolt, Kristoffer T. G.; Vanselow, Jens T.; Blagoev, Blagoy

2011-01-01

Recent technological advances have made it possible to identify and quantify thousands of proteins in a single proteomics experiment. As a result of these developments, the analysis of data has become the bottleneck of proteomics experiment. To provide the proteomics community with a user-friendly platform for comprehensive analysis, inspection and visualization of quantitative proteomics data we developed the Graphical Proteomics Data Explorer (GProX)1. The program requires no special bioinformatics training, as all functions of GProX are accessible within its graphical user-friendly interface which will be intuitive to most users. Basic features facilitate the uncomplicated management and organization of large data sets and complex experimental setups as well as the inspection and graphical plotting of quantitative data. These are complemented by readily available high-level analysis options such as database querying, clustering based on abundance ratios, feature enrichment tests for e.g. GO terms and pathway analysis tools. A number of plotting options for visualization of quantitative proteomics data is available and most analysis functions in GProX create customizable high quality graphical displays in both vector and bitmap formats. The generic import requirements allow data originating from essentially all mass spectrometry platforms, quantitation strategies and software to be analyzed in the program. GProX represents a powerful approach to proteomics data analysis providing proteomics experimenters with a toolbox for bioinformatics analysis of quantitative proteomics data. The program is released as open-source and can be freely downloaded from the project webpage at http://gprox.sourceforge.net. PMID:21602510

Crystal Structure and Proteomics Analysis of Empty Virus-like Particles of Cowpea Mosaic Virus.

PubMed

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

2016-04-05

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. Copyright © 2016 Elsevier Ltd. All rights reserved.

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.

Notice of Pre-Application Webinar (RFA-CA-15-021, RFA-CA-15-022, RFA-CA-15-023) | Office of Cancer Clinical Proteomics Research

Cancer.gov

The National Cancer Institute will hold a public pre-application webinar on Friday, December 11 at 12:00 p.m. (EST) for the Funding Opportunity Announcements (FOAs) RFA-CA-15-021 entitled “Proteome Characterization Centers for Clinical Proteomic Tumor Analysis Consortium (U24), RFA-CA-15-022 entitled “Proteogenomic Translational Research Centers for Clinical Proteomic Tumor Analysis Consortium (U01)”, and RFA-CA-15-023 entitled “Proteogenomic Data Analysis Centers for Clinical Proteomic Tumor Analysis Consortium (U24)”.

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.

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.

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.

Adaptation of Decoy Fusion Strategy for Existing Multi-Stage Search Workflows

NASA Astrophysics Data System (ADS)

Ivanov, Mark V.; Levitsky, Lev I.; Gorshkov, Mikhail V.

2016-09-01

A number of proteomic database search engines implement multi-stage strategies aiming at increasing the sensitivity of proteome analysis. These approaches often employ a subset of the original database for the secondary stage of analysis. However, if target-decoy approach (TDA) is used for false discovery rate (FDR) estimation, the multi-stage strategies may violate the underlying assumption of TDA that false matches are distributed uniformly across the target and decoy databases. This violation occurs if the numbers of target and decoy proteins selected for the second search are not equal. Here, we propose a method of decoy database generation based on the previously reported decoy fusion strategy. This method allows unbiased TDA-based FDR estimation in multi-stage searches and can be easily integrated into existing workflows utilizing popular search engines and post-search algorithms.

PeptideDepot: flexible relational database for visual analysis of quantitative proteomic data and integration of existing protein information.

PubMed

Yu, Kebing; Salomon, Arthur R

2009-12-01

Recently, dramatic progress has been achieved in expanding the sensitivity, resolution, mass accuracy, and scan rate of mass spectrometers able to fragment and identify peptides through MS/MS. Unfortunately, this enhanced ability to acquire proteomic data has not been accompanied by a concomitant increase in the availability of flexible tools allowing users to rapidly assimilate, explore, and analyze this data and adapt to various experimental workflows with minimal user intervention. Here we fill this critical gap by providing a flexible relational database called PeptideDepot for organization of expansive proteomic data sets, collation of proteomic data with available protein information resources, and visual comparison of multiple quantitative proteomic experiments. Our software design, built upon the synergistic combination of a MySQL database for safe warehousing of proteomic data with a FileMaker-driven graphical user interface for flexible adaptation to diverse workflows, enables proteomic end-users to directly tailor the presentation of proteomic data to the unique analysis requirements of the individual proteomics lab. PeptideDepot may be deployed as an independent software tool or integrated directly with our high throughput autonomous proteomic pipeline used in the automated acquisition and post-acquisition analysis of proteomic data.

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.

Software Analysis of Uncorrelated MS1 Peaks for Discovery of Post-Translational Modifications

NASA Astrophysics Data System (ADS)

Pascal, Bruce D.; West, Graham M.; Scharager-Tapia, Catherina; Flefil, Ricardo; Moroni, Tina; Martinez-Acedo, Pablo; Griffin, Patrick R.; Carvalloza, Anthony C.

2015-12-01

The goal in proteomics to identify all peptides in a complex mixture has been largely addressed using various LC MS/MS approaches, such as data dependent acquisition, SRM/MRM, and data independent acquisition instrumentation. Despite these developments, many peptides remain unsequenced, often due to low abundance, poor fragmentation patterns, or data analysis difficulties. Many of the unidentified peptides exhibit strong evidence in high resolution MS1 data and are frequently post-translationally modified, playing a significant role in biological processes. Proteomics Workbench (PWB) software was developed to automate the detection and visualization of all possible peptides in MS1 data, reveal candidate peptides not initially identified, and build inclusion lists for subsequent MS2 analysis to uncover new identifications. We used this software on existing data on the autophagy regulating kinase Ulk1 as a proof of concept for this method, as we had already manually identified a number of phosphorylation sites Dorsey, F. C. et al (J. Proteome. Res. 8(11), 5253-5263 (2009)). PWB found all previously identified sites of phosphorylation. The software has been made freely available at http://www.proteomicsworkbench.com .

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).

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.

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

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.

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

Grade-Dependent Metabolic Reprogramming in Kidney Cancer Revealed by Combined Proteomics and Metabolomics Analysis [Combined Proteomics and Metabolomics Analysis Reveals Grade-Dependent Metabolism Pathways in Kidney Cancer

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

Wettersten, Hiromi I.; Hakimi, A. Ari; Morin, Dexter

Kidney cancer [or renal cell carcinoma (RCC)] is known as “the internist's tumor” because it has protean systemic manifestations, suggesting that it utilizes complex, nonphysiologic metabolic pathways. Given the increasing incidence of this cancer and its lack of effective therapeutic targets, we undertook an extensive analysis of human RCC tissue employing combined grade-dependent proteomics and metabolomics analysis to determine how metabolic reprogramming occurring in this disease allows it to escape available therapeutic approaches. After validation experiments in RCC cell lines that were wild-type or mutant for the Von Hippel–Lindau tumor suppressor, in characterizing higher-grade tumors, we found that the Warburgmore » effect is relatively more prominent at the expense of the tricarboxylic acid cycle and oxidative metabolism in general. Further, we found that the glutamine metabolism pathway acts to inhibit reactive oxygen species, as evidenced by an upregulated glutathione pathway, whereas the β-oxidation pathway is inhibited, leading to increased fatty acylcarnitines. In support of findings from previous urine metabolomics analyses, we also documented tryptophan catabolism associated with immune suppression, which was highly represented in RCC compared with other metabolic pathways. Altogether, our results offer a rationale to evaluate novel antimetabolic treatment strategies being developed in other disease settings as therapeutic strategies in RCC« less

Grade-Dependent Metabolic Reprogramming in Kidney Cancer Revealed by Combined Proteomics and Metabolomics Analysis [Combined Proteomics and Metabolomics Analysis Reveals Grade-Dependent Metabolism Pathways in Kidney Cancer

DOE PAGES

Wettersten, Hiromi I.; Hakimi, A. Ari; Morin, Dexter; ...

2015-05-07

Kidney cancer [or renal cell carcinoma (RCC)] is known as “the internist's tumor” because it has protean systemic manifestations, suggesting that it utilizes complex, nonphysiologic metabolic pathways. Given the increasing incidence of this cancer and its lack of effective therapeutic targets, we undertook an extensive analysis of human RCC tissue employing combined grade-dependent proteomics and metabolomics analysis to determine how metabolic reprogramming occurring in this disease allows it to escape available therapeutic approaches. After validation experiments in RCC cell lines that were wild-type or mutant for the Von Hippel–Lindau tumor suppressor, in characterizing higher-grade tumors, we found that the Warburgmore » effect is relatively more prominent at the expense of the tricarboxylic acid cycle and oxidative metabolism in general. Further, we found that the glutamine metabolism pathway acts to inhibit reactive oxygen species, as evidenced by an upregulated glutathione pathway, whereas the β-oxidation pathway is inhibited, leading to increased fatty acylcarnitines. In support of findings from previous urine metabolomics analyses, we also documented tryptophan catabolism associated with immune suppression, which was highly represented in RCC compared with other metabolic pathways. Altogether, our results offer a rationale to evaluate novel antimetabolic treatment strategies being developed in other disease settings as therapeutic strategies in RCC« less

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

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

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

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.

Organellar proteome analyses of ricin toxin-treated HeLa cells.

PubMed

Liao, Peng; Li, Yunhu; Li, Hongyang; Liu, Wensen

2016-07-01

Apoptosis triggered by ricin toxin (RT) has previously been associated with certain cellular organellar compartments, but the diversity in the composition of the organellar proteins remains unclear. Here, we applied a shotgun proteomics strategy to examine the differential expression of proteins in the mitochondria, nuclei, and cytoplasm of HeLa cells treated and not treated with RT. Data were combined with a global bioinformatics analysis and experimental confirmations. A total of 3107 proteins were identified. Bioinformatics predictors (Proteome Analyst, WoLF PSORT, TargetP, MitoPred, Nucleo, MultiLoc, and k-nearest neighbor) and a Bayesian model that integrated these predictors were used to predict the locations of 1349 distinct organellar proteins. Our data indicate that the Bayesian model was more efficient than the individual implementation of these predictors. Additionally, a Biomolecular Interaction Network (BIN) analysis was used to identify 149 BIN subnetworks. Our experimental confirmations indicate that certain apoptosis-related proteins (e.g. cytochrome c, enolase, lamin B, Bax, and Drp1) were found to be translocated and had variable expression levels. These results provide new insights for the systematic understanding of RT-induced apoptosis responses. © The Author(s) 2014.

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

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

Low Cost, Scalable Proteomics Data Analysis Using Amazon's Cloud Computing Services and Open Source Search Algorithms

PubMed Central

Halligan, Brian D.; Geiger, Joey F.; Vallejos, Andrew K.; Greene, Andrew S.; Twigger, Simon N.

2009-01-01

One of the major difficulties for many laboratories setting up proteomics programs has been obtaining and maintaining the computational infrastructure required for the analysis of the large flow of proteomics data. We describe a system that combines distributed cloud computing and open source software to allow laboratories to set up scalable virtual proteomics analysis clusters without the investment in computational hardware or software licensing fees. Additionally, the pricing structure of distributed computing providers, such as Amazon Web Services, allows laboratories or even individuals to have large-scale computational resources at their disposal at a very low cost per run. We provide detailed step by step instructions on how to implement the virtual proteomics analysis clusters as well as a list of current available preconfigured Amazon machine images containing the OMSSA and X!Tandem search algorithms and sequence databases on the Medical College of Wisconsin Proteomics Center website (http://proteomics.mcw.edu/vipdac). PMID:19358578

Low cost, scalable proteomics data analysis using Amazon's cloud computing services and open source search algorithms.

PubMed

Halligan, Brian D; Geiger, Joey F; Vallejos, Andrew K; Greene, Andrew S; Twigger, Simon N

2009-06-01

One of the major difficulties for many laboratories setting up proteomics programs has been obtaining and maintaining the computational infrastructure required for the analysis of the large flow of proteomics data. We describe a system that combines distributed cloud computing and open source software to allow laboratories to set up scalable virtual proteomics analysis clusters without the investment in computational hardware or software licensing fees. Additionally, the pricing structure of distributed computing providers, such as Amazon Web Services, allows laboratories or even individuals to have large-scale computational resources at their disposal at a very low cost per run. We provide detailed step-by-step instructions on how to implement the virtual proteomics analysis clusters as well as a list of current available preconfigured Amazon machine images containing the OMSSA and X!Tandem search algorithms and sequence databases on the Medical College of Wisconsin Proteomics Center Web site ( http://proteomics.mcw.edu/vipdac ).

PeptideDepot: Flexible Relational Database for Visual Analysis of Quantitative Proteomic Data and Integration of Existing Protein Information

PubMed Central

Yu, Kebing; Salomon, Arthur R.

2010-01-01

Recently, dramatic progress has been achieved in expanding the sensitivity, resolution, mass accuracy, and scan rate of mass spectrometers able to fragment and identify peptides through tandem mass spectrometry (MS/MS). Unfortunately, this enhanced ability to acquire proteomic data has not been accompanied by a concomitant increase in the availability of flexible tools allowing users to rapidly assimilate, explore, and analyze this data and adapt to a variety of experimental workflows with minimal user intervention. Here we fill this critical gap by providing a flexible relational database called PeptideDepot for organization of expansive proteomic data sets, collation of proteomic data with available protein information resources, and visual comparison of multiple quantitative proteomic experiments. Our software design, built upon the synergistic combination of a MySQL database for safe warehousing of proteomic data with a FileMaker-driven graphical user interface for flexible adaptation to diverse workflows, enables proteomic end-users to directly tailor the presentation of proteomic data to the unique analysis requirements of the individual proteomics lab. PeptideDepot may be deployed as an independent software tool or integrated directly with our High Throughput Autonomous Proteomic Pipeline (HTAPP) used in the automated acquisition and post-acquisition analysis of proteomic data. PMID:19834895

Proteomes and Phosphoproteomes of Anther and Pollen: Availability and Progress.

PubMed

Zhang, Zaibao; Hu, Menghui; Feng, Xiaobing; Gong, Andong; Cheng, Lin; Yuan, Hongyu

2017-10-01

In flowering plants, anther development plays crucial role in sexual reproduction. Within the anther, microspore mother cells meiosis produces microspores, which further develop into pollen grains that play decisive role in plant reproduction. Previous studies on anther biology mainly focused on single gene functions relying on genetic and molecular methods. Recently, anther development has been expanded from multiple OMICS approaches like transcriptomics, proteomics/phosphoproteomics, and metabolomics. The development of proteomics techniques allowing increased proteome coverage and quantitative measurements of proteins which can characterize proteomes and their modulation during normal development, biotic and abiotic stresses in anther development. In this review, we summarize the achievements of proteomics and phosphoproteomics with anther and pollen organs from model plant and crop species (i.e. Arabidopsis, rice, tobacco). The increased proteomic information facilitated translation of information from the models to crops and thus aid in agricultural improvement. © 2017 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 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

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 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

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

New Funding Opportunity Announcements (FOAs): Reissuance of Clinical Proteomic Tumor Analysis Consortium (CPTAC) | Office of Cancer Clinical Proteomics Research

Cancer.gov

The National Cancer Institute is soliciting applications for the reissuance of its Clinical Proteomic Tumor Analysis Consortium (CPTAC) program.   CPTAC will support broad efforts focused on several cancer types to explore further the complexities of cancer proteomes and their connections to abnormalities in cancer genomes.

ProteomeVis: a web app for exploration of protein properties from structure to sequence evolution across organisms' proteomes.

PubMed

Razban, Rostam M; Gilson, Amy I; Durfee, Niamh; Strobelt, Hendrik; Dinkla, Kasper; Choi, Jeong-Mo; Pfister, Hanspeter; Shakhnovich, Eugene I

2018-05-08

Protein evolution spans time scales and its effects span the length of an organism. A web app named ProteomeVis is developed to provide a comprehensive view of protein evolution in the S. cerevisiae and E. coli proteomes. ProteomeVis interactively creates protein chain graphs, where edges between nodes represent structure and sequence similarities within user-defined ranges, to study the long time scale effects of protein structure evolution. The short time scale effects of protein sequence evolution are studied by sequence evolutionary rate (ER) correlation analyses with protein properties that span from the molecular to the organismal level. We demonstrate the utility and versatility of ProteomeVis by investigating the distribution of edges per node in organismal protein chain universe graphs (oPCUGs) and putative ER determinants. S. cerevisiae and E. coli oPCUGs are scale-free with scaling constants of 1.79 and 1.56, respectively. Both scaling constants can be explained by a previously reported theoretical model describing protein structure evolution (Dokholyan et al., 2002). Protein abundance most strongly correlates with ER among properties in ProteomeVis, with Spearman correlations of -0.49 (p-value<10-10) and -0.46 (p-value<10-10) for S. cerevisiae and E. coli, respectively. This result is consistent with previous reports that found protein expression to be the most important ER determinant (Zhang and Yang, 2015). ProteomeVis is freely accessible at http://proteomevis.chem.harvard.edu. Supplementary data are available at Bioinformatics. shakhnovich@chemistry.harvard.edu.

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

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.

CPTAC | Office of Cancer Clinical Proteomics Research

Cancer.gov

The National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium (CPTAC) is a national effort to accelerate the understanding of the molecular basis of cancer through the application of large-scale proteome and genome analysis, or proteogenomics.

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.

Characterization of the Mouse Pancreatic Islet Proteome and Comparative Analysis with Other Mouse Tissues

PubMed Central

Petyuk, Vladislav A.; Qian, Wei-Jun; Hinault, Charlotte; Gritsenko, Marina A.; Singhal, Mudita; Monroe, Matthew E.; Camp, David G.; Kulkarni, Rohit N.; Smith, Richard D.

2009-01-01

The pancreatic islets of Langerhans, and especially the insulin-producing beta cells, play a central role in the maintenance of glucose homeostasis. Alterations in the expression of multiple proteins in the islets that contribute to the maintenance of islet function are likely to underlie the pathogenesis of type 2 diabetes. To identify proteins that constitute the islet proteome, we provide the first comprehensive proteomic characterization of pancreatic islets for mouse, the most commonly used animal model in diabetes research. Using strong cation exchange fractionation coupled with reversed phase LC-MS/MS we report the confident identification of 17,350 different tryptic peptides covering 2,612 proteins having at least two unique peptides per protein. The dataset also identified ~60 post-translationally modified peptides including oxidative modifications and phosphorylation. While many of the identified phosphorylation sites corroborate those previously known, the oxidative modifications observed on cysteinyl residues reveal potentially novel information suggesting a role for oxidative stress in islet function. Comparative analysis with 15 available proteomic datasets from other mouse tissues and cells revealed a set of 133 proteins predominantly expressed in pancreatic islets. This unique set of proteins, in addition to those with known functions such as peptide hormones secreted from the islets, contains several proteins with as yet unknown functions. The mouse islet protein and peptide database accessible at http://ncrr.pnl.gov, provides an important reference resource for the research community to facilitate research in the diabetes and metabolism fields. PMID:18570455

Proteomic analysis of grapevine resistance induced by Trichoderma harzianum T39 reveals specific defence pathways activated against downy mildew

PubMed Central

Perazzolli, Michele

2012-01-01

Downy mildew is caused by the oomycete Plasmopara viticola and is one of the most serious diseases of grapevine. The beneficial microorganism Trichoderma harzianum T39 (T39) has previously been shown to induce plant-mediated resistance and to reduce the severity of downy mildew in susceptible grapevines. In order to better understand the cellular processes associated with T39-induced resistance, the proteomic and histochemical changes activated by T39 in grapevine were investigated before and 1 day after P. viticola inoculation. A comprehensive proteomic analysis of T39-induced resistance in grapevine was performed using an eight-plex iTRAQ protocol, resulting in the identification and quantification of a total of 800 proteins. Most of the proteins directly affected by T39 were found to be involved in signal transduction, indicating activation of a complete microbial recognition machinery. Moreover, T39-induced resistance was associated with rapid accumulation of reactive oxygen species and callose at infection sites, as well as changes in abundance of proteins involved in response to stress and redox balance, indicating an active defence response to downy mildew. On the other hand, proteins affected by P. viticola in control plants mainly decreased in abundance, possibly reflecting the establishment of a compatible interaction. Finally, the high-throughput iTRAQ protocol allowed de novo peptide sequencing, which will be used to improve annotation of the Vitis vinifera cv. Pinot Noir proteome. PMID:23105132

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

Large-Scale and Deep Quantitative Proteome Profiling Using Isobaric Labeling Coupled with Two-Dimensional LC-MS/MS

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

Gritsenko, Marina A.; Xu, Zhe; Liu, Tao

Comprehensive, quantitative information on abundances of proteins and their post-translational modifications (PTMs) can potentially provide novel biological insights into diseases pathogenesis and therapeutic intervention. Herein, we introduce a quantitative strategy utilizing isobaric stable isotope-labelling techniques combined with two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) for large-scale, deep quantitative proteome profiling of biological samples or clinical specimens such as tumor tissues. The workflow includes isobaric labeling of tryptic peptides for multiplexed and accurate quantitative analysis, basic reversed-phase LC fractionation and concatenation for reduced sample complexity, and nano-LC coupled to high resolution and high mass accuracy MS analysis for high confidence identification andmore » quantification of proteins. This proteomic analysis strategy has been successfully applied for in-depth quantitative proteomic analysis of tumor samples, and can also be used for integrated proteome and PTM characterization, as well as comprehensive quantitative proteomic analysis across samples from large clinical cohorts.« less

Large-Scale and Deep Quantitative Proteome Profiling Using Isobaric Labeling Coupled with Two-Dimensional LC-MS/MS.

PubMed

Gritsenko, Marina A; Xu, Zhe; Liu, Tao; Smith, Richard D

2016-01-01

Comprehensive, quantitative information on abundances of proteins and their posttranslational modifications (PTMs) can potentially provide novel biological insights into diseases pathogenesis and therapeutic intervention. Herein, we introduce a quantitative strategy utilizing isobaric stable isotope-labeling techniques combined with two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) for large-scale, deep quantitative proteome profiling of biological samples or clinical specimens such as tumor tissues. The workflow includes isobaric labeling of tryptic peptides for multiplexed and accurate quantitative analysis, basic reversed-phase LC fractionation and concatenation for reduced sample complexity, and nano-LC coupled to high resolution and high mass accuracy MS analysis for high confidence identification and quantification of proteins. This proteomic analysis strategy has been successfully applied for in-depth quantitative proteomic analysis of tumor samples and can also be used for integrated proteome and PTM characterization, as well as comprehensive quantitative proteomic analysis across samples from large clinical cohorts.

Human body fluid proteome analysis

PubMed Central

Hu, Shen; Loo, Joseph A.; Wong, David T.

2010-01-01

The focus of this article is to review the recent advances in proteome analysis of human body fluids, including plasma/serum, urine, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, synovial fluid, nipple aspirate fluid, tear fluid, and amniotic fluid, as well as its applications to human disease biomarker discovery. We aim to summarize the proteomics technologies currently used for global identification and quantification of body fluid proteins, and elaborate the putative biomarkers discovered for a variety of human diseases through human body fluid proteome (HBFP) analysis. Some critical concerns and perspectives in this emerging field are also discussed. With the advances made in proteomics technologies, the impact of HBFP analysis in the search for clinically relevant disease biomarkers would be realized in the future. PMID:17083142

Human body fluid proteome analysis.

PubMed

Hu, Shen; Loo, Joseph A; Wong, David T

2006-12-01

The focus of this article is to review the recent advances in proteome analysis of human body fluids, including plasma/serum, urine, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, synovial fluid, nipple aspirate fluid, tear fluid, and amniotic fluid, as well as its applications to human disease biomarker discovery. We aim to summarize the proteomics technologies currently used for global identification and quantification of body fluid proteins, and elaborate the putative biomarkers discovered for a variety of human diseases through human body fluid proteome (HBFP) analysis. Some critical concerns and perspectives in this emerging field are also discussed. With the advances made in proteomics technologies, the impact of HBFP analysis in the search for clinically relevant disease biomarkers would be realized in the future.

Comparative genomics analysis of field isolates of Aspergillus flavus and A. parasiticus to explain phenotypic variation in oxidative stress tolerance and host preference

USDA-ARS?s Scientific Manuscript database

Aflatoxin contamination of peanut and other crops is a major concern for producers globally, and has been shown to be exacerbated by drought stress. Previous transcriptomic and proteomic examination of the responses of isolates of Aspergillus flavus to drought-related oxidative stress in vitro have ...

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.

MASPECTRAS: a platform for management and analysis of proteomics LC-MS/MS data

PubMed Central

Hartler, Jürgen; Thallinger, Gerhard G; Stocker, Gernot; Sturn, Alexander; Burkard, Thomas R; Körner, Erik; Rader, Robert; Schmidt, Andreas; Mechtler, Karl; Trajanoski, Zlatko

2007-01-01

Background The advancements of proteomics technologies have led to a rapid increase in the number, size and rate at which datasets are generated. Managing and extracting valuable information from such datasets requires the use of data management platforms and computational approaches. Results We have developed the MAss SPECTRometry Analysis System (MASPECTRAS), a platform for management and analysis of proteomics LC-MS/MS data. MASPECTRAS is based on the Proteome Experimental Data Repository (PEDRo) relational database schema and follows the guidelines of the Proteomics Standards Initiative (PSI). Analysis modules include: 1) import and parsing of the results from the search engines SEQUEST, Mascot, Spectrum Mill, X! Tandem, and OMSSA; 2) peptide validation, 3) clustering of proteins based on Markov Clustering and multiple alignments; and 4) quantification using the Automated Statistical Analysis of Protein Abundance Ratios algorithm (ASAPRatio). The system provides customizable data retrieval and visualization tools, as well as export to PRoteomics IDEntifications public repository (PRIDE). MASPECTRAS is freely available at Conclusion Given the unique features and the flexibility due to the use of standard software technology, our platform represents significant advance and could be of great interest to the proteomics community. PMID:17567892

Integrated Proteomic Approaches for Understanding Toxicity of Environmental Chemicals

EPA Science Inventory

To apply quantitative proteomic analysis to the evaluation of toxicity of environmental chemicals, we have developed an integrated proteomic technology platform. This platform has been applied to the analysis of the toxic effects and pathways of many important environmental chemi...

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

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.

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.

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

CPTAC Proteomics Data on UCSC Genome Browser | Office of Cancer Clinical Proteomics Research

Cancer.gov

The National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium scientists are working together with the University of California, Santa Cruz (UCSC) Genomics Institute to provide public access to cancer proteomics data via the UCSC Genome Browser. This effort extends accessibility of the CPTAC data to more researchers and provides an additional level of analysis to assist the cancer biology community.

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

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.

Quantitative proteome analysis of barley seeds using ruthenium(II)-tris-(bathophenanthroline-disulphonate) staining.

PubMed

Witzel, Katja; Surabhi, Giridara-Kumar; Jyothsnakumari, Gottimukkala; Sudhakar, Chinta; Matros, Andrea; Mock, Hans-Peter

2007-04-01

This paper describes the application of the recently introduced fluorescence stain Ruthenium(II)-tris-(bathophenanthroline-disulphonate) (RuBP) on a comparative proteome analysis of two phenotypically different barley lines. We carried out an analysis of protein patterns from 2-D gels of the parental lines of the Oregon Wolfe Barley mapping population DOM and REC and stained with either the conventional colloidal Coomassie Brilliant Blue (cCBB) or with the novel RuBP solution. We wished to experimentally verify the usefulness of such a stain in evaluating the complex pattern of a seed proteome, in comparison to the previously used cCBB staining technique. To validate the efficiency of visualization by both stains, we first compared the overall number of detected protein spots. On average, 790 spots were visible by cCBB staining and 1200 spots by RuBP staining. Then, the intensity of a set of spots was assessed, and changes in relative abundance were determined using image analysis software. As expected, staining with RuBP performed better in quantitation in terms of sensitivity and dynamic range. Furthermore, spots from a cultivar-specific region in the protein map were chosen for identification to asses the gain of biological information due to the staining procedure. From this particular region, eight spots were visualized exclusively by RuBP and identification was successful for all spots, proving the ability to identify even very low abundant proteins. Performance in MS analysis was comparable for both protein stains. Proteins were identified by MALDI-TOF MS peptide mass fingerprinting. This approach was not successful for all spots, due to the restricted entry number for barley in the database. Therefore, we subsequently used LC-ESI-Q-TOF MS/MS and de novo sequencing for identification. Because only an insufficient number of proteins from barley is annotated, an EST-based identification strategy was chosen for our experiment. We wished to test whether under these limitations the application of a more sensitive stain would lead to a more advanced proteome approach. In summary, we demonstrate here that the application of RuBP as an economical but reliable and sensitive fluorescence stain is highly suitable for quantitative proteome analysis of plant seeds.

Deterministic protein inference for shotgun proteomics data provides new insights into Arabidopsis pollen development and function

PubMed Central

Grobei, Monica A.; Qeli, Ermir; Brunner, Erich; Rehrauer, Hubert; Zhang, Runxuan; Roschitzki, Bernd; Basler, Konrad; Ahrens, Christian H.; Grossniklaus, Ueli

2009-01-01

Pollen, the male gametophyte of flowering plants, represents an ideal biological system to study developmental processes, such as cell polarity, tip growth, and morphogenesis. Upon hydration, the metabolically quiescent pollen rapidly switches to an active state, exhibiting extremely fast growth. This rapid switch requires relevant proteins to be stored in the mature pollen, where they have to retain functionality in a desiccated environment. Using a shotgun proteomics approach, we unambiguously identified ∼3500 proteins in Arabidopsis pollen, including 537 proteins that were not identified in genetic or transcriptomic studies. To generate this comprehensive reference data set, which extends the previously reported pollen proteome by a factor of 13, we developed a novel deterministic peptide classification scheme for protein inference. This generally applicable approach considers the gene model–protein sequence–protein accession relationships. It allowed us to classify and eliminate ambiguities inherently associated with any shotgun proteomics data set, to report a conservative list of protein identifications, and to seamlessly integrate data from previous transcriptomics studies. Manual validation of proteins unambiguously identified by a single, information-rich peptide enabled us to significantly reduce the false discovery rate, while keeping valuable identifications of shorter and lower abundant proteins. Bioinformatic analyses revealed a higher stability of pollen proteins compared to those of other tissues and implied a protein family of previously unknown function in vesicle trafficking. Interestingly, the pollen proteome is most similar to that of seeds, indicating physiological similarities between these developmentally distinct tissues. PMID:19546170

Reevaluation of the Coding Potential and Proteomic Analysis of the BAC Derived Rhesus Cytomegalovirus Strain 68-1

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

Malouli, Daniel; Nakayasu, Ernesto S.; Viswanathan, Kasinath

2012-09-01

Cytomegaloviruses are highly host restricted resulting in co-speciation with their hosts. As a natural pathogen of rhesus macaques (RM), Rhesus Cytomegalovirus (RhCMV) has therefore emerged as a highly relevant experimental model for pathogenesis and vaccine development due to its close evolutionary relationship to human CMV (HCMV). To date, most in vivo experiments performed with RhCMV employed strain 68-1 cloned as bacterial artificial chromosome (BAC). However, the complete genome sequence of the 68-1 BAC has not been determined. Furthermore, the gene content of the RhCMV genome is unknown and previous open reading frame (ORF) predictions relied solely on uninterrupted ORFs withmore » an arbitrary cutoff of 300bp. To obtain a more precise picture of the actual proteins encoded by the most commonly used molecular clone of RhCMV we re-evaluated the RhCMV 68-1 BAC-genome by whole genome shotgun sequencing and determined the protein content of the resulting RhCMV virions by proteomics. By additionally comparing the RhCMV genome to that of several closely related Old World Monkey (OWM) CMVs we were able to filter out many unlikely ORFs and obtain a simplified map of the RhCMV genome. This comparative genomics analysis eliminated many genes previously characterized as RhCMV-specific while consolidating a high conservation of ORFs among OWM-CMVs and between RhCMV and HCMV. Moreover, virion proteomics independently validated the revised ORF predictions since only proteins encoded by predicted ORFs could be detected. Taken together these data suggest a much higher conservation of genome and virion structure between CMVs of humans, apes and OWMs than previously assumed. Remarkably, BAC-derived RhCMV is able to establish and maintain persistent infection despite the lack of multiple genes homologous to HCMV genes involved in tissue tropism.« less

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.

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

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

Growth in spaceflight hardware results in alterations to the transcriptome and proteome

NASA Astrophysics Data System (ADS)

Basu, Proma; Kruse, Colin P. S.; Luesse, Darron R.; Wyatt, Sarah E.

2017-11-01

The Biological Research in Canisters (BRIC) hardware has been used to house many biology experiments on both the Space Transport System (STS, commonly known as the space shuttle) and the International Space Station (ISS). However, microscopic examination of Arabidopsis seedlings by Johnson et al. (2015) indicated the hardware itself may affect cell morphology. The experiment herein was designed to assess the effects of the BRIC-Petri Dish Fixation Units (BRIC-PDFU) hardware on the transcriptome and proteome of Arabidopsis seedlings. To our knowledge, this is the first transcriptomic and proteomic comparison of Arabidopsis seedlings grown with and without hardware. Arabidopsis thaliana wild-type Columbia (Col-0) seeds were sterilized and bulk plated on forty-four 60 mm Petri plates, of which 22 were integrated into the BRIC-PDFU hardware and 22 were maintained in closed containers at Ohio University. Seedlings were grown for approximately 3 days, fixed with RNAlater® and stored at -80 °C prior to RNA and protein extraction, with proteins separated into membrane and soluble fractions prior to analysis. The RNAseq analysis identified 1651 differentially expressed genes; MS/MS analysis identified 598 soluble and 589 membrane proteins differentially abundant both at p < .05. Fold enrichment analysis of gene ontology terms related to differentially expressed transcripts and proteins highlighted a variety of stress responses. Some of these genes and proteins have been previously identified in spaceflight experiments, indicating that these genes and proteins may be perturbed by both conditions.

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.

Ecto-Fc MS identifies ligand-receptor interactions through extracellular domain Fc fusion protein baits and shotgun proteomic analysis

PubMed Central

Savas, Jeffrey N.; De Wit, Joris; Comoletti, Davide; Zemla, Roland; Ghosh, Anirvan

2015-01-01

Ligand-receptor interactions represent essential biological triggers which regulate many diverse and important cellular processes. We have developed a discovery-based proteomic biochemical protocol which couples affinity purification with multidimensional liquid chromatographic tandem mass spectrometry (LCLC-MS/MS) and bioinformatic analysis. Compared to previous approaches, our analysis increases sensitivity, shortens analysis duration, and boosts comprehensiveness. In this protocol, receptor extracellular domains are fused with the Fc region of IgG to generate fusion proteins that are purified from transfected HEK293T cells. These “ecto-Fcs” are coupled to protein A beads and serve as baits for binding assays with prey proteins extracted from rodent brain. After capture, the affinity purified proteins are digested into peptides and comprehensively analyzed by LCLC-MS/MS with ion trap mass spectrometers. In four working days, this protocol can generate shortlists of candidate ligand-receptor protein-protein interactions. Our “Ecto-Fc MS” approach outperforms antibody-based approaches and provides a reproducible and robust framework to identify extracellular ligand – receptor interactions. PMID:25101821

Proteomic analysis of a rare urinary stone composed of calcium carbonate and calcium oxalate dihydrate: a case report.

PubMed

Kaneko, Kiyoko; Matsuta, Yosuke; Moriyama, Manabu; Yasuda, Makoto; Chishima, Noriharu; Yamaoka, Noriko; Fukuuchi, Tomoko; Miyazawa, Katsuhito; Suzuki, Koji

2014-03-01

The objective of the present study was to investigate the matrix protein of a rare urinary stone that contained calcium carbonate. A urinary stone was extracted from a 34-year-old male patient with metabolic alkalosis. After X-ray diffractometry and infrared analysis of the stone, proteomic analysis was carried out. The resulting mass spectra were evaluated with protein search software, and matrix proteins were identified. X-ray diffraction and infrared analysis confirmed that the stone contained calcium carbonate and calcium oxalate dihydrate. Of the identified 53 proteins, 24 have not been previously reported from calcium oxalate- or calcium phosphate-containing stones. The protease inhibitors and several proteins related to cell adhesion or the cytoskeleton were identified for the first time. We analyzed in detail a rare urinary stone composed of calcium carbonate and calcium oxalate dihydrate. Considering the formation of a calcium carbonate stone, the new identified proteins should play an important role on the urolithiasis process in alkaline condition. © 2013 The Japanese Urological Association.

Proteomic analysis of laser capture microscopy purified myotendinous junction regions from muscle sections

PubMed Central

2014-01-01

The myotendinous junction is a specialized structure of the muscle fibre enriched in mechanosensing complexes, including costameric proteins and core elements of the z-disc. Here, laser capture microdissection was applied to purify membrane regions from the myotendinous junctions of mouse skeletal muscles, which were then processed for proteomic analysis. Sarcolemma sections from the longitudinal axis of the muscle fibre were used as control for the specificity of the junctional preparation. Gene ontology term analysis of the combined lists indicated a statistically significant enrichment in membrane-associated proteins. The myotendinous junction preparation contained previously uncharacterized proteins, a number of z-disc costameric ligands (e.g., actinins, capZ, αB cristallin, filamin C, cypher, calsarcin, desmin, FHL1, telethonin, nebulin, titin and an enigma-like protein) and other proposed players of sarcomeric stretch sensing and signalling, such as myotilin and the three myomesin homologs. A subset were confirmed by immunofluorescence analysis as enriched at the myotendinous junction, suggesting that laser capture microdissection from muscle sections is a valid approach to identify novel myotendinous junction players potentially involved in mechanotransduction pathways. PMID:25071420

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.

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

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

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

FunRich proteomics software analysis, let the fun begin!

PubMed

Benito-Martin, Alberto; Peinado, Héctor

2015-08-01

Protein MS analysis is the preferred method for unbiased protein identification. It is normally applied to a large number of both small-scale and high-throughput studies. However, user-friendly computational tools for protein analysis are still needed. In this issue, Mathivanan and colleagues (Proteomics 2015, 15, 2597-2601) report the development of FunRich software, an open-access software that facilitates the analysis of proteomics data, providing tools for functional enrichment and interaction network analysis of genes and proteins. FunRich is a reinterpretation of proteomic software, a standalone tool combining ease of use with customizable databases, free access, and graphical representations. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Top-down proteomics for the analysis of proteolytic events - Methods, applications and perspectives.

PubMed

Tholey, Andreas; Becker, Alexander

2017-11-01

Mass spectrometry based proteomics is an indispensable tool for almost all research areas relevant for the understanding of proteolytic processing, ranging from the identification of substrates, products and cleavage sites up to the analysis of structural features influencing protease activity. The majority of methods for these studies are based on bottom-up proteomics performing analysis at peptide level. As this approach is characterized by a number of pitfalls, e.g. loss of molecular information, there is an ongoing effort to establish top-down proteomics, performing separation and MS analysis both at intact protein level. We briefly introduce major approaches of bottom-up proteomics used in the field of protease research and highlight the shortcomings of these methods. We then discuss the present state-of-the-art of top-down proteomics. Together with the discussion of known challenges we show the potential of this approach and present a number of successful applications of top-down proteomics in protease research. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John. Copyright © 2017 Elsevier B.V. All rights reserved.

Improved False Discovery Rate Estimation Procedure for Shotgun Proteomics.

PubMed

Keich, Uri; Kertesz-Farkas, Attila; Noble, William Stafford

2015-08-07

Interpreting the potentially vast number of hypotheses generated by a shotgun proteomics experiment requires a valid and accurate procedure for assigning statistical confidence estimates to identified tandem mass spectra. Despite the crucial role such procedures play in most high-throughput proteomics experiments, the scientific literature has not reached a consensus about the best confidence estimation methodology. In this work, we evaluate, using theoretical and empirical analysis, four previously proposed protocols for estimating the false discovery rate (FDR) associated with a set of identified tandem mass spectra: two variants of the target-decoy competition protocol (TDC) of Elias and Gygi and two variants of the separate target-decoy search protocol of Käll et al. Our analysis reveals significant biases in the two separate target-decoy search protocols. Moreover, the one TDC protocol that provides an unbiased FDR estimate among the target PSMs does so at the cost of forfeiting a random subset of high-scoring spectrum identifications. We therefore propose the mix-max procedure to provide unbiased, accurate FDR estimates in the presence of well-calibrated scores. The method avoids biases associated with the two separate target-decoy search protocols and also avoids the propensity for target-decoy competition to discard a random subset of high-scoring target identifications.

In Planta Proteomics and Proteogenomics of the Biotrophic Barley Fungal Pathogen Blumeria graminis f. sp. hordei*

PubMed Central

Bindschedler, Laurence V.; Burgis, Timothy A.; Mills, Davinia J. S.; Ho, Jenny T. C.; Cramer, Rainer; Spanu, Pietro D.

2009-01-01

To further our understanding of powdery mildew biology during infection, we undertook a systematic shotgun proteomics analysis of the obligate biotroph Blumeria graminis f. sp. hordei at different stages of development in the host. Moreover we used a proteogenomics approach to feed information into the annotation of the newly sequenced genome. We analyzed and compared the proteomes from three stages of development representing different functions during the plant-dependent vegetative life cycle of this fungus. We identified 441 proteins in ungerminated spores, 775 proteins in epiphytic sporulating hyphae, and 47 proteins from haustoria inside barley leaf epidermal cells and used the data to aid annotation of the B. graminis f. sp. hordei genome. We also compared the differences in the protein complement of these key stages. Although confirming some of the previously reported findings and models derived from the analysis of transcriptome dynamics, our results also suggest that the intracellular haustoria are subject to stress possibly as a result of the plant defense strategy, including the production of reactive oxygen species. In addition, a number of small haustorial proteins with a predicted N-terminal signal peptide for secretion were identified in infected tissues: these represent candidate effector proteins that may play a role in controlling host metabolism and immunity. PMID:19602707

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.

Improved False Discovery Rate Estimation Procedure for Shotgun Proteomics

PubMed Central

2016-01-01

Interpreting the potentially vast number of hypotheses generated by a shotgun proteomics experiment requires a valid and accurate procedure for assigning statistical confidence estimates to identified tandem mass spectra. Despite the crucial role such procedures play in most high-throughput proteomics experiments, the scientific literature has not reached a consensus about the best confidence estimation methodology. In this work, we evaluate, using theoretical and empirical analysis, four previously proposed protocols for estimating the false discovery rate (FDR) associated with a set of identified tandem mass spectra: two variants of the target-decoy competition protocol (TDC) of Elias and Gygi and two variants of the separate target-decoy search protocol of Käll et al. Our analysis reveals significant biases in the two separate target-decoy search protocols. Moreover, the one TDC protocol that provides an unbiased FDR estimate among the target PSMs does so at the cost of forfeiting a random subset of high-scoring spectrum identifications. We therefore propose the mix-max procedure to provide unbiased, accurate FDR estimates in the presence of well-calibrated scores. The method avoids biases associated with the two separate target-decoy search protocols and also avoids the propensity for target-decoy competition to discard a random subset of high-scoring target identifications. PMID:26152888

iTRAQ-Based Proteomics Analysis and Network Integration for Kernel Tissue Development in Maize

PubMed Central

Dong, Yongbin; Wang, Qilei; Du, Chunguang; Xiong, Wenwei; Li, Xinyu; Zhu, Sailan; Li, Yuling

2017-01-01

Grain weight is one of the most important yield components and a developmentally complex structure comprised of two major compartments (endosperm and pericarp) in maize (Zea mays L.), however, very little is known concerning the coordinated accumulation of the numerous proteins involved. Herein, we used isobaric tags for relative and absolute quantitation (iTRAQ)-based comparative proteomic method to analyze the characteristics of dynamic proteomics for endosperm and pericarp during grain development. Totally, 9539 proteins were identified for both components at four development stages, among which 1401 proteins were non-redundant, 232 proteins were specific in pericarp and 153 proteins were specific in endosperm. A functional annotation of the identified proteins revealed the importance of metabolic and cellular processes, and binding and catalytic activities for the tissue development. Three and 76 proteins involved in 49 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were integrated for the specific endosperm and pericarp proteins, respectively, reflecting their complex metabolic interactions. In addition, four proteins with important functions and different expression levels were chosen for gene cloning and expression analysis. Different concordance between mRNA level and the protein abundance was observed across different proteins, stages, and tissues as in previous research. These results could provide useful message for understanding the developmental mechanisms in grain development in maize. PMID:28837076

Effect of hypoxia on lung gene expression and proteomic profile: insights into the pulmonary surfactant response

PubMed Central

Olmeda, Bárbara; Umstead, Todd M.; Silveyra, Patricia; Pascual, Alberto; López-Barneo, José; Phelps, David S.; Floros, Joanna; Pérez-Gil, Jesús

2014-01-01

Exposure of lung to hypoxia has been previously reported to be associated with significant alterations in the protein content of bronchoalveolar lavage (BAL) and lung tissue. In the present work we have used a proteomic approach to describe the changes in protein complement induced by moderate long-term hypoxia (rats exposed to 10% O2 for 72 hours) in BAL and lung tissue, with a special focus on the proteins associated with pulmonary surfactant, which could indicate adaptation of this system to limited oxygen availability. The analysis of the general proteomic profile indicates a hypoxia-induced increase in proteins associated with inflammation both in lavage and lung tissue. Analysis at mRNA and protein levels revealed no significant changes induced by hypoxia on the content in surfactant proteins or their apparent oligomeric state. In contrast, we detected a hypoxia-induced significant increase in the expression and accumulation of hemoglobin in lung tissue, at both mRNA and protein levels, as well as an accumulation of hemoglobin both in BAL and associated with surface-active membranes of the pulmonary surfactant complex. Evaluation of pulmonary surfactant surface activity from hypoxic rats showed no alterations in its spreading ability, ruling out inhibition by increased levels of serum or inflammatory proteins. PMID:24576641

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

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

Proteomic Identification of Heat Shock-Induced Danger Signals in a Melanoma Cell Lysate Used in Dendritic Cell-Based Cancer Immunotherapy.

PubMed

González, Fermín E; Chernobrovkin, Alexey; Pereda, Cristián; García-Salum, Tamara; Tittarelli, Andrés; López, Mercedes N; Salazar-Onfray, Flavio; Zubarev, Roman A

2018-01-01

Autologous dendritic cells (DCs) loaded with cancer cell-derived lysates have become a promising tool in cancer immunotherapy. During the last decade, we demonstrated that vaccination of advanced melanoma patients with autologous tumor antigen presenting cells (TAPCells) loaded with an allogeneic heat shock- (HS-) conditioned melanoma cell-derived lysate (called TRIMEL) is able to induce an antitumor immune response associated with a prolonged patient survival. TRIMEL provides not only a broad spectrum of potential melanoma-associated antigens but also danger signals that are crucial in the induction of a committed mature DC phenotype. However, potential changes induced by heat conditioning on the proteome of TRIMEL are still unknown. The identification of newly or differentially expressed proteins under defined stress conditions is relevant for understanding the lysate immunogenicity. Here, we characterized the proteomic profile of TRIMEL in response to HS treatment. A quantitative label-free proteome analysis of over 2800 proteins was performed, with 91 proteins that were found to be regulated by HS treatment: 18 proteins were overexpressed and 73 underexpressed. Additionally, 32 proteins were only identified in the HS-treated TRIMEL and 26 in non HS-conditioned samples. One protein from the overexpressed group and two proteins from the HS-exclusive group were previously described as potential damage-associated molecular patterns (DAMPs). Some of the HS-induced proteins, such as haptoglobin, could be also considered as DAMPs and candidates for further immunological analysis in the establishment of new putative danger signals with immunostimulatory functions.

Proteomic Identification of Heat Shock-Induced Danger Signals in a Melanoma Cell Lysate Used in Dendritic Cell-Based Cancer Immunotherapy

PubMed Central

Chernobrovkin, Alexey; Pereda, Cristián; García-Salum, Tamara; Tittarelli, Andrés; López, Mercedes N.; Salazar-Onfray, Flavio

2018-01-01

Autologous dendritic cells (DCs) loaded with cancer cell-derived lysates have become a promising tool in cancer immunotherapy. During the last decade, we demonstrated that vaccination of advanced melanoma patients with autologous tumor antigen presenting cells (TAPCells) loaded with an allogeneic heat shock- (HS-) conditioned melanoma cell-derived lysate (called TRIMEL) is able to induce an antitumor immune response associated with a prolonged patient survival. TRIMEL provides not only a broad spectrum of potential melanoma-associated antigens but also danger signals that are crucial in the induction of a committed mature DC phenotype. However, potential changes induced by heat conditioning on the proteome of TRIMEL are still unknown. The identification of newly or differentially expressed proteins under defined stress conditions is relevant for understanding the lysate immunogenicity. Here, we characterized the proteomic profile of TRIMEL in response to HS treatment. A quantitative label-free proteome analysis of over 2800 proteins was performed, with 91 proteins that were found to be regulated by HS treatment: 18 proteins were overexpressed and 73 underexpressed. Additionally, 32 proteins were only identified in the HS-treated TRIMEL and 26 in non HS-conditioned samples. One protein from the overexpressed group and two proteins from the HS-exclusive group were previously described as potential damage-associated molecular patterns (DAMPs). Some of the HS-induced proteins, such as haptoglobin, could be also considered as DAMPs and candidates for further immunological analysis in the establishment of new putative danger signals with immunostimulatory functions. PMID:29744371

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.

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.

Experimental design and data-analysis in label-free quantitative LC/MS proteomics: A tutorial with MSqRob.

PubMed

Goeminne, Ludger J E; Gevaert, Kris; Clement, Lieven

2018-01-16

Label-free shotgun proteomics is routinely used to assess proteomes. However, extracting relevant information from the massive amounts of generated data remains difficult. This tutorial provides a strong foundation on analysis of quantitative proteomics data. We provide key statistical concepts that help researchers to design proteomics experiments and we showcase how to analyze quantitative proteomics data using our recent free and open-source R package MSqRob, which was developed to implement the peptide-level robust ridge regression method for relative protein quantification described by Goeminne et al. MSqRob can handle virtually any experimental proteomics design and outputs proteins ordered by statistical significance. Moreover, its graphical user interface and interactive diagnostic plots provide easy inspection and also detection of anomalies in the data and flaws in the data analysis, allowing deeper assessment of the validity of results and a critical review of the experimental design. Our tutorial discusses interactive preprocessing, data analysis and visualization of label-free MS-based quantitative proteomics experiments with simple and more complex designs. We provide well-documented scripts to run analyses in bash mode on GitHub, enabling the integration of MSqRob in automated pipelines on cluster environments (https://github.com/statOmics/MSqRob). The concepts outlined in this tutorial aid in designing better experiments and analyzing the resulting data more appropriately. The two case studies using the MSqRob graphical user interface will contribute to a wider adaptation of advanced peptide-based models, resulting in higher quality data analysis workflows and more reproducible results in the proteomics community. We also provide well-documented scripts for experienced users that aim at automating MSqRob on cluster environments. Copyright © 2017 Elsevier B.V. All rights reserved.

Functional Module Search in Protein Networks based on Semantic Similarity Improves the Analysis of Proteomics Data*

PubMed Central

Boyanova, Desislava; Nilla, Santosh; Klau, Gunnar W.; Dandekar, Thomas; Müller, Tobias; Dittrich, Marcus

2014-01-01

The continuously evolving field of proteomics produces increasing amounts of data while improving the quality of protein identifications. Albeit quantitative measurements are becoming more popular, many proteomic studies are still based on non-quantitative methods for protein identification. These studies result in potentially large sets of identified proteins, where the biological interpretation of proteins can be challenging. Systems biology develops innovative network-based methods, which allow an integrated analysis of these data. Here we present a novel approach, which combines prior knowledge of protein-protein interactions (PPI) with proteomics data using functional similarity measurements of interacting proteins. This integrated network analysis exactly identifies network modules with a maximal consistent functional similarity reflecting biological processes of the investigated cells. We validated our approach on small (H9N2 virus-infected gastric cells) and large (blood constituents) proteomic data sets. Using this novel algorithm, we identified characteristic functional modules in virus-infected cells, comprising key signaling proteins (e.g. the stress-related kinase RAF1) and demonstrate that this method allows a module-based functional characterization of cell types. Analysis of a large proteome data set of blood constituents resulted in clear separation of blood cells according to their developmental origin. A detailed investigation of the T-cell proteome further illustrates how the algorithm partitions large networks into functional subnetworks each representing specific cellular functions. These results demonstrate that the integrated network approach not only allows a detailed analysis of proteome networks but also yields a functional decomposition of complex proteomic data sets and thereby provides deeper insights into the underlying cellular processes of the investigated system. PMID:24807868

ProteoSign: an end-user online differential proteomics statistical analysis platform.

PubMed

Efstathiou, Georgios; Antonakis, Andreas N; Pavlopoulos, Georgios A; Theodosiou, Theodosios; Divanach, Peter; Trudgian, David C; Thomas, Benjamin; Papanikolaou, Nikolas; Aivaliotis, Michalis; Acuto, Oreste; Iliopoulos, Ioannis

2017-07-03

Profiling of proteome dynamics is crucial for understanding cellular behavior in response to intrinsic and extrinsic stimuli and maintenance of homeostasis. Over the last 20 years, mass spectrometry (MS) has emerged as the most powerful tool for large-scale identification and characterization of proteins. Bottom-up proteomics, the most common MS-based proteomics approach, has always been challenging in terms of data management, processing, analysis and visualization, with modern instruments capable of producing several gigabytes of data out of a single experiment. Here, we present ProteoSign, a freely available web application, dedicated in allowing users to perform proteomics differential expression/abundance analysis in a user-friendly and self-explanatory way. Although several non-commercial standalone tools have been developed for post-quantification statistical analysis of proteomics data, most of them are not end-user appealing as they often require very stringent installation of programming environments, third-party software packages and sometimes further scripting or computer programming. To avoid this bottleneck, we have developed a user-friendly software platform accessible via a web interface in order to enable proteomics laboratories and core facilities to statistically analyse quantitative proteomics data sets in a resource-efficient manner. ProteoSign is available at http://bioinformatics.med.uoc.gr/ProteoSign and the source code at https://github.com/yorgodillo/ProteoSign. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

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.

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

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

Derivative component analysis for mass spectral serum proteomic profiles.

PubMed

Han, Henry

2014-01-01

As a promising way to transform medicine, mass spectrometry based proteomics technologies have seen a great progress in identifying disease biomarkers for clinical diagnosis and prognosis. However, there is a lack of effective feature selection methods that are able to capture essential data behaviors to achieve clinical level disease diagnosis. Moreover, it faces a challenge from data reproducibility, which means that no two independent studies have been found to produce same proteomic patterns. Such reproducibility issue causes the identified biomarker patterns to lose repeatability and prevents it from real clinical usage. In this work, we propose a novel machine-learning algorithm: derivative component analysis (DCA) for high-dimensional mass spectral proteomic profiles. As an implicit feature selection algorithm, derivative component analysis examines input proteomics data in a multi-resolution approach by seeking its derivatives to capture latent data characteristics and conduct de-noising. We further demonstrate DCA's advantages in disease diagnosis by viewing input proteomics data as a profile biomarker via integrating it with support vector machines to tackle the reproducibility issue, besides comparing it with state-of-the-art peers. Our results show that high-dimensional proteomics data are actually linearly separable under proposed derivative component analysis (DCA). As a novel multi-resolution feature selection algorithm, DCA not only overcomes the weakness of the traditional methods in subtle data behavior discovery, but also suggests an effective resolution to overcoming proteomics data's reproducibility problem and provides new techniques and insights in translational bioinformatics and machine learning. The DCA-based profile biomarker diagnosis makes clinical level diagnostic performances reproducible across different proteomic data, which is more robust and systematic than the existing biomarker discovery based diagnosis. Our findings demonstrate the feasibility and power of the proposed DCA-based profile biomarker diagnosis in achieving high sensitivity and conquering the data reproducibility issue in serum proteomics. Furthermore, our proposed derivative component analysis suggests the subtle data characteristics gleaning and de-noising are essential in separating true signals from red herrings for high-dimensional proteomic profiles, which can be more important than the conventional feature selection or dimension reduction. In particular, our profile biomarker diagnosis can be generalized to other omics data for derivative component analysis (DCA)'s nature of generic data analysis.

Tumor Cold Ischemia | Office of Cancer Clinical Proteomics Research

Cancer.gov

In a recently published manuscript in the journal of Molecular and Cellular Proteomics, researchers from the National Cancer Institutes (NCI) Clinical Proteomic Tumor Analysis Consortium (CPTAC) investigated the effect of cold ischemia on the proteome of fresh frozen tumors.

Proteomics wants cRacker: automated standardized data analysis of LC-MS derived proteomic data.

PubMed

Zauber, Henrik; Schulze, Waltraud X

2012-11-02

The large-scale analysis of thousands of proteins under various experimental conditions or in mutant lines has gained more and more importance in hypothesis-driven scientific research and systems biology in the past years. Quantitative analysis by large scale proteomics using modern mass spectrometry usually results in long lists of peptide ion intensities. The main interest for most researchers, however, is to draw conclusions on the protein level. Postprocessing and combining peptide intensities of a proteomic data set requires expert knowledge, and the often repetitive and standardized manual calculations can be time-consuming. The analysis of complex samples can result in very large data sets (lists with several 1000s to 100,000 entries of different peptides) that cannot easily be analyzed using standard spreadsheet programs. To improve speed and consistency of the data analysis of LC-MS derived proteomic data, we developed cRacker. cRacker is an R-based program for automated downstream proteomic data analysis including data normalization strategies for metabolic labeling and label free quantitation. In addition, cRacker includes basic statistical analysis, such as clustering of data, or ANOVA and t tests for comparison between treatments. Results are presented in editable graphic formats and in list files.

Building ProteomeTools based on a complete synthetic human proteome

PubMed Central

Zolg, Daniel P.; Wilhelm, Mathias; Schnatbaum, Karsten; Zerweck, Johannes; Knaute, Tobias; Delanghe, Bernard; Bailey, Derek J.; Gessulat, Siegfried; Ehrlich, Hans-Christian; Weininger, Maximilian; Yu, Peng; Schlegl, Judith; Kramer, Karl; Schmidt, Tobias; Kusebauch, Ulrike; Deutsch, Eric W.; Aebersold, Ruedi; Moritz, Robert L.; Wenschuh, Holger; Moehring, Thomas; Aiche, Stephan; Huhmer, Andreas; Reimer, Ulf; Kuster, Bernhard

2018-01-01

The ProteomeTools project builds molecular and digital tools from the human proteome to facilitate biomedical and life science research. Here, we report the generation and multimodal LC-MS/MS analysis of >330,000 synthetic tryptic peptides representing essentially all canonical human gene products and exemplify the utility of this data. The resource will be extended to >1 million peptides and all data will be shared with the community via ProteomicsDB and proteomeXchange. PMID:28135259

Comparative shotgun proteomics using spectral count data and quasi-likelihood modeling.

PubMed

Li, Ming; Gray, William; Zhang, Haixia; Chung, Christine H; Billheimer, Dean; Yarbrough, Wendell G; Liebler, Daniel C; Shyr, Yu; Slebos, Robbert J C

2010-08-06

Shotgun proteomics provides the most powerful analytical platform for global inventory of complex proteomes using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and allows a global analysis of protein changes. Nevertheless, sampling of complex proteomes by current shotgun proteomics platforms is incomplete, and this contributes to variability in assessment of peptide and protein inventories by spectral counting approaches. Thus, shotgun proteomics data pose challenges in comparing proteomes from different biological states. We developed an analysis strategy using quasi-likelihood Generalized Linear Modeling (GLM), included in a graphical interface software package (QuasiTel) that reads standard output from protein assemblies created by IDPicker, an HTML-based user interface to query shotgun proteomic data sets. This approach was compared to four other statistical analysis strategies: Student t test, Wilcoxon rank test, Fisher's Exact test, and Poisson-based GLM. We analyzed the performance of these tests to identify differences in protein levels based on spectral counts in a shotgun data set in which equimolar amounts of 48 human proteins were spiked at different levels into whole yeast lysates. Both GLM approaches and the Fisher Exact test performed adequately, each with their unique limitations. We subsequently compared the proteomes of normal tonsil epithelium and HNSCC using this approach and identified 86 proteins with differential spectral counts between normal tonsil epithelium and HNSCC. We selected 18 proteins from this comparison for verification of protein levels between the individual normal and tumor tissues using liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM-MS). This analysis confirmed the magnitude and direction of the protein expression differences in all 6 proteins for which reliable data could be obtained. Our analysis demonstrates that shotgun proteomic data sets from different tissue phenotypes are sufficiently rich in quantitative information and that statistically significant differences in proteins spectral counts reflect the underlying biology of the samples.

Comparative Shotgun Proteomics Using Spectral Count Data and Quasi-Likelihood Modeling

PubMed Central

2010-01-01

Shotgun proteomics provides the most powerful analytical platform for global inventory of complex proteomes using liquid chromatography−tandem mass spectrometry (LC−MS/MS) and allows a global analysis of protein changes. Nevertheless, sampling of complex proteomes by current shotgun proteomics platforms is incomplete, and this contributes to variability in assessment of peptide and protein inventories by spectral counting approaches. Thus, shotgun proteomics data pose challenges in comparing proteomes from different biological states. We developed an analysis strategy using quasi-likelihood Generalized Linear Modeling (GLM), included in a graphical interface software package (QuasiTel) that reads standard output from protein assemblies created by IDPicker, an HTML-based user interface to query shotgun proteomic data sets. This approach was compared to four other statistical analysis strategies: Student t test, Wilcoxon rank test, Fisher’s Exact test, and Poisson-based GLM. We analyzed the performance of these tests to identify differences in protein levels based on spectral counts in a shotgun data set in which equimolar amounts of 48 human proteins were spiked at different levels into whole yeast lysates. Both GLM approaches and the Fisher Exact test performed adequately, each with their unique limitations. We subsequently compared the proteomes of normal tonsil epithelium and HNSCC using this approach and identified 86 proteins with differential spectral counts between normal tonsil epithelium and HNSCC. We selected 18 proteins from this comparison for verification of protein levels between the individual normal and tumor tissues using liquid chromatography−multiple reaction monitoring mass spectrometry (LC−MRM-MS). This analysis confirmed the magnitude and direction of the protein expression differences in all 6 proteins for which reliable data could be obtained. Our analysis demonstrates that shotgun proteomic data sets from different tissue phenotypes are sufficiently rich in quantitative information and that statistically significant differences in proteins spectral counts reflect the underlying biology of the samples. PMID:20586475

Analysis of high accuracy, quantitative proteomics data in the MaxQB database.

PubMed

Schaab, Christoph; Geiger, Tamar; Stoehr, Gabriele; Cox, Juergen; Mann, Matthias

2012-03-01

MS-based proteomics generates rapidly increasing amounts of precise and quantitative information. Analysis of individual proteomic experiments has made great strides, but the crucial ability to compare and store information across different proteome measurements still presents many challenges. For example, it has been difficult to avoid contamination of databases with low quality peptide identifications, to control for the inflation in false positive identifications when combining data sets, and to integrate quantitative data. Although, for example, the contamination with low quality identifications has been addressed by joint analysis of deposited raw data in some public repositories, we reasoned that there should be a role for a database specifically designed for high resolution and quantitative data. Here we describe a novel database termed MaxQB that stores and displays collections of large proteomics projects and allows joint analysis and comparison. We demonstrate the analysis tools of MaxQB using proteome data of 11 different human cell lines and 28 mouse tissues. The database-wide false discovery rate is controlled by adjusting the project specific cutoff scores for the combined data sets. The 11 cell line proteomes together identify proteins expressed from more than half of all human genes. For each protein of interest, expression levels estimated by label-free quantification can be visualized across the cell lines. Similarly, the expression rank order and estimated amount of each protein within each proteome are plotted. We used MaxQB to calculate the signal reproducibility of the detected peptides for the same proteins across different proteomes. Spearman rank correlation between peptide intensity and detection probability of identified proteins was greater than 0.8 for 64% of the proteome, whereas a minority of proteins have negative correlation. This information can be used to pinpoint false protein identifications, independently of peptide database scores. The information contained in MaxQB, including high resolution fragment spectra, is accessible to the community via a user-friendly web interface at http://www.biochem.mpg.de/maxqb.

[Methods of quantitative proteomics].

PubMed

Kopylov, A T; Zgoda, V G

2007-01-01

In modern science proteomic analysis is inseparable from other fields of systemic biology. Possessing huge resources quantitative proteomics operates colossal information on molecular mechanisms of life. Advances in proteomics help researchers to solve complex problems of cell signaling, posttranslational modification, structure and functional homology of proteins, molecular diagnostics etc. More than 40 various methods have been developed in proteomics for quantitative analysis of proteins. Although each method is unique and has certain advantages and disadvantages all these use various isotope labels (tags). In this review we will consider the most popular and effective methods employing both chemical modifications of proteins and also metabolic and enzymatic methods of isotope labeling.

Comprehensive Analysis of Temporal Alterations in Cellular Proteome of Bacillus subtilis under Curcumin Treatment

PubMed Central

Reddy, Panga Jaipal; Sinha, Sneha; Ray, Sandipan; Sathe, Gajanan J.; Chatterjee, Aditi; Prasad, T. S. Keshava; Dhali, Snigdha; Srikanth, Rapole; Panda, Dulal; Srivastava, Sanjeeva

2015-01-01

Curcumin is a natural dietary compound with antimicrobial activity against various gram positive and negative bacteria. This study aims to investigate the proteome level alterations in Bacillus subtilis due to curcumin treatment and identification of its molecular/cellular targets to understand the mechanism of action. We have performed a comprehensive proteomic analysis of B. subtilis AH75 strain at different time intervals of curcumin treatment (20, 60 and 120 min after the drug exposure, three replicates) to compare the protein expression profiles using two complementary quantitative proteomic techniques, 2D-DIGE and iTRAQ. To the best of our knowledge, this is the first comprehensive longitudinal investigation describing the effect of curcumin treatment on B. subtilis proteome. The proteomics analysis revealed several interesting targets such UDP-N-acetylglucosamine 1-carboxyvinyltransferase 1, putative septation protein SpoVG and ATP-dependent Clp protease proteolytic subunit. Further, in silico pathway analysis using DAVID and KOBAS has revealed modulation of pathways related to the fatty acid metabolism and cell wall synthesis, which are crucial for cell viability. Our findings revealed that curcumin treatment lead to inhibition of the cell wall and fatty acid synthesis in addition to differential expression of many crucial proteins involved in modulation of bacterial metabolism. Findings obtained from proteomics analysis were further validated using 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) assay for respiratory activity, resazurin assay for metabolic activity and membrane integrity assay by potassium and inorganic phosphate leakage measurement. The gene expression analysis of selected cell wall biosynthesis enzymes has strengthened the proteomics findings and indicated the major effect of curcumin on cell division. PMID:25874956

Comprehensive analysis of temporal alterations in cellular proteome of Bacillus subtilis under curcumin treatment.

PubMed

Reddy, Panga Jaipal; Sinha, Sneha; Ray, Sandipan; Sathe, Gajanan J; Chatterjee, Aditi; Prasad, T S Keshava; Dhali, Snigdha; Srikanth, Rapole; Panda, Dulal; Srivastava, Sanjeeva

2015-01-01

Curcumin is a natural dietary compound with antimicrobial activity against various gram positive and negative bacteria. This study aims to investigate the proteome level alterations in Bacillus subtilis due to curcumin treatment and identification of its molecular/cellular targets to understand the mechanism of action. We have performed a comprehensive proteomic analysis of B. subtilis AH75 strain at different time intervals of curcumin treatment (20, 60 and 120 min after the drug exposure, three replicates) to compare the protein expression profiles using two complementary quantitative proteomic techniques, 2D-DIGE and iTRAQ. To the best of our knowledge, this is the first comprehensive longitudinal investigation describing the effect of curcumin treatment on B. subtilis proteome. The proteomics analysis revealed several interesting targets such UDP-N-acetylglucosamine 1-carboxyvinyltransferase 1, putative septation protein SpoVG and ATP-dependent Clp protease proteolytic subunit. Further, in silico pathway analysis using DAVID and KOBAS has revealed modulation of pathways related to the fatty acid metabolism and cell wall synthesis, which are crucial for cell viability. Our findings revealed that curcumin treatment lead to inhibition of the cell wall and fatty acid synthesis in addition to differential expression of many crucial proteins involved in modulation of bacterial metabolism. Findings obtained from proteomics analysis were further validated using 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) assay for respiratory activity, resazurin assay for metabolic activity and membrane integrity assay by potassium and inorganic phosphate leakage measurement. The gene expression analysis of selected cell wall biosynthesis enzymes has strengthened the proteomics findings and indicated the major effect of curcumin on cell division.

Improved chip design for integrated solid-phase microextraction in on-line proteomic sample preparation.

PubMed

Bergkvist, Jonas; Ekström, Simon; Wallman, Lars; Löfgren, Mikael; Marko-Varga, György; Nilsson, Johan; Laurell, Thomas

2002-04-01

A recently introduced silicon microextraction chip (SMEC), used for on-line proteomic sample preparation, has proved to facilitate the process of protein identification by sample clean up and enrichment of peptides. It is demonstrated that a novel grid-SMEC design improves the operating characteristics for solid-phase microextraction, by reducing dispersion effects and thereby improving the sample preparation conditions. The structures investigated in this paper are treated both numerically and experimentally. The numerical approach is based on finite element analysis of the microfluidic flow in the microchip. The analysis is accomplished by use of the computational fluid dynamics-module FLOTRAN in the ANSYS software package. The modeling and analysis of the previously reported weir-SMEC design indicates some severe drawbacks, that can be reduced by changing the microextraction chip geometry to the grid-SMEC design. The overall analytical performance was thereby improved and also verified by experimental work. Matrix-assisted laser desorption/ionization mass spectra of model peptides extracted from both the weir-SMEC and the new grid-SMEC support the numerical analysis results. Further use of numerical modeling and analysis of the SMEC structures is also discussed and suggested in this work.

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.

Analyzing large-scale proteomics projects with latent semantic indexing.

PubMed

Klie, Sebastian; Martens, Lennart; Vizcaíno, Juan Antonio; Côté, Richard; Jones, Phil; Apweiler, Rolf; Hinneburg, Alexander; Hermjakob, Henning

2008-01-01

Since the advent of public data repositories for proteomics data, readily accessible results from high-throughput experiments have been accumulating steadily. Several large-scale projects in particular have contributed substantially to the amount of identifications available to the community. Despite the considerable body of information amassed, very few successful analyses have been performed and published on this data, leveling off the ultimate value of these projects far below their potential. A prominent reason published proteomics data is seldom reanalyzed lies in the heterogeneous nature of the original sample collection and the subsequent data recording and processing. To illustrate that at least part of this heterogeneity can be compensated for, we here apply a latent semantic analysis to the data contributed by the Human Proteome Organization's Plasma Proteome Project (HUPO PPP). Interestingly, despite the broad spectrum of instruments and methodologies applied in the HUPO PPP, our analysis reveals several obvious patterns that can be used to formulate concrete recommendations for optimizing proteomics project planning as well as the choice of technologies used in future experiments. It is clear from these results that the analysis of large bodies of publicly available proteomics data by noise-tolerant algorithms such as the latent semantic analysis holds great promise and is currently underexploited.

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.

Evolution of Clinical Proteomics and its Role in Medicine | Office of Cancer Clinical Proteomics Research

Cancer.gov

NCI's Office of Cancer Clinical Proteomics Research authored a review of the current state of clinical proteomics in the peer-reviewed Journal of Proteome Research. The review highlights outcomes from the CPTC program and also provides a thorough overview of the different technologies that have pushed the field forward. Additionally, the review provides a vision for moving the field forward through linking advances in genomic and proteomic analysis to develop new, molecularly targeted interventions.

ApoptoProteomics, an integrated database for analysis of proteomics data obtained from apoptotic cells.

PubMed

Arntzen, Magnus Ø; Thiede, Bernd

2012-02-01

Apoptosis is the most commonly described form of programmed cell death, and dysfunction is implicated in a large number of human diseases. Many quantitative proteome analyses of apoptosis have been performed to gain insight in proteins involved in the process. This resulted in large and complex data sets that are difficult to evaluate. Therefore, we developed the ApoptoProteomics database for storage, browsing, and analysis of the outcome of large scale proteome analyses of apoptosis derived from human, mouse, and rat. The proteomics data of 52 publications were integrated and unified with protein annotations from UniProt-KB, the caspase substrate database homepage (CASBAH), and gene ontology. Currently, more than 2300 records of more than 1500 unique proteins were included, covering a large proportion of the core signaling pathways of apoptosis. Analysis of the data set revealed a high level of agreement between the reported changes in directionality reported in proteomics studies and expected apoptosis-related function and may disclose proteins without a current recognized involvement in apoptosis based on gene ontology. Comparison between induction of apoptosis by the intrinsic and the extrinsic apoptotic signaling pathway revealed slight differences. Furthermore, proteomics has significantly contributed to the field of apoptosis in identifying hundreds of caspase substrates. The database is available at http://apoptoproteomics.uio.no.

ApoptoProteomics, an Integrated Database for Analysis of Proteomics Data Obtained from Apoptotic Cells*

PubMed Central

Arntzen, Magnus Ø.; Thiede, Bernd

2012-01-01

Apoptosis is the most commonly described form of programmed cell death, and dysfunction is implicated in a large number of human diseases. Many quantitative proteome analyses of apoptosis have been performed to gain insight in proteins involved in the process. This resulted in large and complex data sets that are difficult to evaluate. Therefore, we developed the ApoptoProteomics database for storage, browsing, and analysis of the outcome of large scale proteome analyses of apoptosis derived from human, mouse, and rat. The proteomics data of 52 publications were integrated and unified with protein annotations from UniProt-KB, the caspase substrate database homepage (CASBAH), and gene ontology. Currently, more than 2300 records of more than 1500 unique proteins were included, covering a large proportion of the core signaling pathways of apoptosis. Analysis of the data set revealed a high level of agreement between the reported changes in directionality reported in proteomics studies and expected apoptosis-related function and may disclose proteins without a current recognized involvement in apoptosis based on gene ontology. Comparison between induction of apoptosis by the intrinsic and the extrinsic apoptotic signaling pathway revealed slight differences. Furthermore, proteomics has significantly contributed to the field of apoptosis in identifying hundreds of caspase substrates. The database is available at http://apoptoproteomics.uio.no. PMID:22067098

Clinical proteomic analysis of scrub typhus infection.

PubMed

Park, Edmond Changkyun; Lee, Sang-Yeop; Yun, Sung Ho; Choi, Chi-Won; Lee, Hayoung; Song, Hyun Seok; Jun, Sangmi; Kim, Gun-Hwa; Lee, Chang-Seop; Kim, Seung Il

2018-01-01

Scrub typhus is an acute and febrile infectious disease caused by the Gram-negative α-proteobacterium Orientia tsutsugamushi from the family Rickettsiaceae that is widely distributed in Northern, Southern and Eastern Asia. In the present study, we analysed the serum proteome of scrub typhus patients to investigate specific clinical protein patterns in an attempt to explain pathophysiology and discover potential biomarkers of infection. Serum samples were collected from three patients (before and after treatment with antibiotics) and three healthy subjects. One-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by liquid chromatography-tandem mass spectrometry was performed to identify differentially abundant proteins using quantitative proteomic approaches. Bioinformatic analysis was then performed using Ingenuity Pathway Analysis. Proteomic analysis identified 236 serum proteins, of which 32 were differentially expressed in normal subjects, naive scrub typhus patients and patients treated with antibiotics. Comparative bioinformatic analysis of the identified proteins revealed up-regulation of proteins involved in immune responses, especially complement system, following infection with O. tsutsugamushi , and normal expression was largely rescued by antibiotic treatment. This is the first proteomic study of clinical serum samples from scrub typhus patients. Proteomic analysis identified changes in protein expression upon infection with O. tsutsugamushi and following antibiotic treatment. Our results provide valuable information for further investigation of scrub typhus therapy and diagnosis.

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.

PeptidePicker: a scientific workflow with web interface for selecting appropriate peptides for targeted proteomics experiments.

PubMed

Mohammed, Yassene; Domański, Dominik; Jackson, Angela M; Smith, Derek S; Deelder, André M; Palmblad, Magnus; Borchers, Christoph H

2014-06-25

One challenge in Multiple Reaction Monitoring (MRM)-based proteomics is to select the most appropriate surrogate peptides to represent a target protein. We present here a software package to automatically generate these most appropriate surrogate peptides for an LC/MRM-MS analysis. Our method integrates information about the proteins, their tryptic peptides, and the suitability of these peptides for MRM which is available online in UniProtKB, NCBI's dbSNP, ExPASy, PeptideAtlas, PRIDE, and GPMDB. The scoring algorithm reflects our knowledge in choosing the best candidate peptides for MRM, based on the uniqueness of the peptide in the targeted proteome, its physiochemical properties, and whether it previously has been observed. The modularity of the workflow allows further extension and additional selection criteria to be incorporated. We have developed a simple Web interface where the researcher provides the protein accession number, the subject organism, and peptide-specific options. Currently, the software is designed for human and mouse proteomes, but additional species can be easily be added. Our software improved the peptide selection by eliminating human error, considering multiple data sources and all of the isoforms of the protein, and resulted in faster peptide selection - approximately 50 proteins per hour compared to 8 per day. Compiling a list of optimal surrogate peptides for target proteins to be analyzed by LC/MRM-MS has been a cumbersome process, in which expert researchers retrieved information from different online repositories and used their own reasoning to find the most appropriate peptides. Our scientific workflow automates this process by integrating information from different data sources including UniProt, Global Proteome Machine, NCBI's dbSNP, and PeptideAtlas, simulating the researchers' reasoning, and incorporating their knowledge of how to select the best proteotypic peptides for an MRM analysis. The developed software can help to standardize the selection of peptides, eliminate human error, and increase productivity. Copyright © 2014 Elsevier B.V. All rights reserved.

Proteomics analysis in frozen horse mackerel previously high-pressure processed.

PubMed

Pazos, Manuel; Méndez, Lucía; Vázquez, Manuel; Aubourg, Santiago P

2015-10-15

The effect of high-pressure processing (HPP) (150, 300 and 450 MPa for 0, 2.5 and 5 min) on total sodium dodecyl sulphate (SDS)-soluble and sarcoplasmic proteins in frozen (-10 °C for 3 months) horse mackerel (Trachurus trachurus) was evaluated. Proteomics tools based on image analysis of SDS-PAGE protein gels and protein identification by tandem mass spectrometry (MS/MS) were applied. Although total SDS-soluble fraction indicated no important changes induced by HPP, this processing modified the 1-D SDS-PAGE sarcoplasmic patterns in a direct-dependent manner and exerted a selective effect on particular proteins depending on processing conditions. Thus, application of the highest pressure (450 MPa) provoked a significant degradation of phosphoglycerate mutase 2, glycogen phosphorylase muscle form, pyruvate kinase muscle isozyme, beta-enolase and triosephosphate isomerase and phosphoglucomutase-1. Conversely, protein bands assigned to tropomyosin alpha-1 chain, fast myotomal muscle troponin T and parvalbumin beta 2 increased their intensity after applying a 450-MPa processing. Copyright © 2015 Elsevier Ltd. All rights reserved.

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.

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

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

TRIC: an automated alignment strategy for reproducible protein quantification in targeted proteomics.

PubMed

Röst, Hannes L; Liu, Yansheng; D'Agostino, Giuseppe; Zanella, Matteo; Navarro, Pedro; Rosenberger, George; Collins, Ben C; Gillet, Ludovic; Testa, Giuseppe; Malmström, Lars; Aebersold, Ruedi

2016-09-01

Next-generation mass spectrometric (MS) techniques such as SWATH-MS have substantially increased the throughput and reproducibility of proteomic analysis, but ensuring consistent quantification of thousands of peptide analytes across multiple liquid chromatography-tandem MS (LC-MS/MS) runs remains a challenging and laborious manual process. To produce highly consistent and quantitatively accurate proteomics data matrices in an automated fashion, we developed TRIC (http://proteomics.ethz.ch/tric/), a software tool that utilizes fragment-ion data to perform cross-run alignment, consistent peak-picking and quantification for high-throughput targeted proteomics. TRIC reduced the identification error compared to a state-of-the-art SWATH-MS analysis without alignment by more than threefold at constant recall while correcting for highly nonlinear chromatographic effects. On a pulsed-SILAC experiment performed on human induced pluripotent stem cells, TRIC was able to automatically align and quantify thousands of light and heavy isotopic peak groups. Thus, TRIC fills a gap in the pipeline for automated analysis of massively parallel targeted proteomics data sets.

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...

Establishing a leaf proteome reference map for Ginkgo biloba provides insight into potential ethnobotanical uses

USDA-ARS?s Scientific Manuscript database

Although ginkgo (Maidenhair tree, Ginkgo biloba L.) is an ancient medicinal and ornamental tree, there has not previously been any systematic proteomic study of the leaves. Herein we describe results from the initial study identifying abundant ginkgo leaf proteins and present a gel reference map. Pr...

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.

Quantitative phosphoproteomics reveals new roles for the protein phosphatase PP6 in mitotic cells.

PubMed

Rusin, Scott F; Schlosser, Kate A; Adamo, Mark E; Kettenbach, Arminja N

2015-10-13

Protein phosphorylation is an important regulatory mechanism controlling mitotic progression. Protein phosphatase 6 (PP6) is an essential enzyme with conserved roles in chromosome segregation and spindle assembly from yeast to humans. We applied a baculovirus-mediated gene silencing approach to deplete HeLa cells of the catalytic subunit of PP6 (PP6c) and analyzed changes in the phosphoproteome and proteome in mitotic cells by quantitative mass spectrometry-based proteomics. We identified 408 phosphopeptides on 272 proteins that increased and 298 phosphopeptides on 220 proteins that decreased in phosphorylation upon PP6c depletion in mitotic cells. Motif analysis of the phosphorylated sites combined with bioinformatics pathway analysis revealed previously unknown PP6c-dependent regulatory pathways. Biochemical assays demonstrated that PP6c opposed casein kinase 2-dependent phosphorylation of the condensin I subunit NCAP-G, and cellular analysis showed that depletion of PP6c resulted in defects in chromosome condensation and segregation in anaphase, consistent with dysregulation of condensin I function in the absence of PP6 activity. Copyright © 2015, American Association for the Advancement of Science.

Quantitative phosphoproteomics reveals new roles for the protein phosphatase PP6 in mitotic cells

PubMed Central

Rusin, Scott F.; Schlosser, Kate A.; Adamo, Mark E.; Kettenbach, Arminja N.

2017-01-01

Protein phosphorylation is an important regulatory mechanism controlling mitotic progression. Protein phosphatase 6 (PP6) is an essential enzyme with conserved roles in chromosome segregation and spindle assembly from yeast to humans. We applied a baculovirus-mediated gene silencing approach to deplete HeLa cells of the catalytic subunit of PP6 (PP6c) and analyzed changes in the phosphoproteome and proteome in mitotic cells by quantitative mass spectrometry–based proteomics. We identified 408 phosphopeptides on 272 proteins that increased and 298 phosphopeptides on 220 proteins that decreased in phosphorylation upon PP6c depletion in mitotic cells. Motif analysis of the phosphorylated sites combined with bioinformatics pathway analysis revealed previously unknown PP6c–dependent regulatory pathways. Biochemical assays demonstrated that PP6c opposed casein kinase 2–dependent phosphorylation of the condensin I subunit NCAP-G, and cellular analysis showed that depletion of PP6c resulted in defects in chromosome condensation and segregation in anaphase, consistent with dysregulation of condensin I function in the absence of PP6 activity. PMID:26462736

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.

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.

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

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

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.

Preprocessing and Analysis of LC-MS-Based Proteomic Data

PubMed Central

Tsai, Tsung-Heng; Wang, Minkun; Ressom, Habtom W.

2016-01-01

Liquid chromatography coupled with mass spectrometry (LC-MS) has been widely used for profiling protein expression levels. This chapter is focused on LC-MS data preprocessing, which is a crucial step in the analysis of LC-MS based proteomics. We provide a high-level overview, highlight associated challenges, and present a step-by-step example for analysis of data from LC-MS based untargeted proteomic study. Furthermore, key procedures and relevant issues with the subsequent analysis by multiple reaction monitoring (MRM) are discussed. PMID:26519169

Growth in spaceflight hardware results in alterations to the transcriptome and proteome.

PubMed

Basu, Proma; Kruse, Colin P S; Luesse, Darron R; Wyatt, Sarah E

2017-11-01

The Biological Research in Canisters (BRIC) hardware has been used to house many biology experiments on both the Space Transport System (STS, commonly known as the space shuttle) and the International Space Station (ISS). However, microscopic examination of Arabidopsis seedlings by Johnson et al. (2015) indicated the hardware itself may affect cell morphology. The experiment herein was designed to assess the effects of the BRIC-Petri Dish Fixation Units (BRIC-PDFU) hardware on the transcriptome and proteome of Arabidopsis seedlings. To our knowledge, this is the first transcriptomic and proteomic comparison of Arabidopsis seedlings grown with and without hardware. Arabidopsis thaliana wild-type Columbia (Col-0) seeds were sterilized and bulk plated on forty-four 60 mm Petri plates, of which 22 were integrated into the BRIC-PDFU hardware and 22 were maintained in closed containers at Ohio University. Seedlings were grown for approximately 3 days, fixed with RNAlater ® and stored at -80 °C prior to RNA and protein extraction, with proteins separated into membrane and soluble fractions prior to analysis. The RNAseq analysis identified 1651 differentially expressed genes; MS/MS analysis identified 598 soluble and 589 membrane proteins differentially abundant both at p < .05. Fold enrichment analysis of gene ontology terms related to differentially expressed transcripts and proteins highlighted a variety of stress responses. Some of these genes and proteins have been previously identified in spaceflight experiments, indicating that these genes and proteins may be perturbed by both conditions. Copyright © 2017 The Committee on Space Research (COSPAR). Published by Elsevier Ltd. All rights reserved.

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.

NCI's Proteome Characterization Centers Announced | Office of Cancer Clinical Proteomics Research

Cancer.gov

The National Cancer Institute (NCI), part of the National Institutes of Health, announces the launch of a Clinical Proteomic Tumor Analysis Consortium (CPTAC). CPTAC is a comprehensive, coordinated team effort to accelerate the understanding of the molecular basis of cancer through the application of robust, quantitative, proteomic technologies and workflows.

Proteomics in medical microbiology.

PubMed

Cash, P

2000-04-01

The techniques of proteomics (high resolution two-dimensional electrophoresis and protein characterisation) are widely used for microbiological research to analyse global protein synthesis as an indicator of gene expression. The rapid progress in microbial proteomics has been achieved through the wide availability of whole genome sequences for a number of bacterial groups. Beyond providing a basic understanding of microbial gene expression, proteomics has also played a role in medical areas of microbiology. Progress has been made in the use of the techniques for investigating the epidemiology and taxonomy of human microbial pathogens, the identification of novel pathogenic mechanisms and the analysis of drug resistance. In each of these areas, proteomics has provided new insights that complement genomic-based investigations. This review describes the current progress in these research fields and highlights some of the technical challenges existing for the application of proteomics in medical microbiology. The latter concern the analysis of genetically heterogeneous bacterial populations and the integration of the proteomic and genomic data for these bacteria. The characterisation of the proteomes of bacterial pathogens growing in their natural hosts remains a future challenge.

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.

CPTAC Prospective Biospecimen Collection Solicitation | Office of Cancer Clinical Proteomics Research

Cancer.gov

A new funding opportunity in support of the National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium (CPTAC) seeks to prospectively procure tumor samples, collected for proteomics investigation.

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.

A complete mass spectrometric map for the analysis of the yeast proteome and its application to quantitative trait analysis

PubMed Central

Picotti, Paola; Clement-Ziza, Mathieu; Lam, Henry; Campbell, David S.; Schmidt, Alexander; Deutsch, Eric W.; Röst, Hannes; Sun, Zhi; Rinner, Oliver; Reiter, Lukas; Shen, Qin; Michaelson, Jacob J.; Frei, Andreas; Alberti, Simon; Kusebauch, Ulrike; Wollscheid, Bernd; Moritz, Robert; Beyer, Andreas; Aebersold, Ruedi

2013-01-01

Complete reference maps or datasets, like the genomic map of an organism, are highly beneficial tools for biological and biomedical research. Attempts to generate such reference datasets for a proteome so far failed to reach complete proteome coverage, with saturation apparent at approximately two thirds of the proteomes tested, even for the most thoroughly characterized proteomes. Here, we used a strategy based on high-throughput peptide synthesis and mass spectrometry to generate a close to complete reference map (97% of the genome-predicted proteins) of the S. cerevisiae proteome. We generated two versions of this mass spectrometric map one supporting discovery- (shotgun) and the other hypothesis-driven (targeted) proteomic measurements. The two versions of the map, therefore, constitute a complete set of proteomic assays to support most studies performed with contemporary proteomic technologies. The reference libraries can be browsed via a web-based repository and associated navigation tools. To demonstrate the utility of the reference libraries we applied them to a protein quantitative trait locus (pQTL) analysis, which requires measurement of the same peptides over a large number of samples with high precision. Protein measurements over a set of 78 S. cerevisiae strains revealed a complex relationship between independent genetic loci, impacting on the levels of related proteins. Our results suggest that selective pressure favors the acquisition of sets of polymorphisms that maintain the stoichiometry of protein complexes and pathways. PMID:23334424

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.

Top-down venomics of the East African green mamba, Dendroaspis angusticeps, and the black mamba, Dendroaspis polylepis, highlight the complexity of their toxin arsenals.

PubMed

Petras, Daniel; Heiss, Paul; Harrison, Robert A; Süssmuth, Roderich D; Calvete, Juan J

2016-09-02

We report the characterization, by combination of high-resolution on-line molecular mass and disulfide bond profiling and top-down MS/MS analysis, of the venom proteomes of two congeneric African snake species of medical importance, Dendroaspis angusticeps (green mamba) and D. polylepis (black mamba). Each of these mamba venoms comprised more than two-hundred polypeptides belonging to just a few toxin families. Both venom proteomes are overwhelmingly composed of post-synaptically-acting short- and long-chain neurotoxins that potently inhibit muscle- and neuronal-type nicotinic acetylcholine receptors; muscarinic cardiotoxins; and dendrotoxins, that block some of the Kv1, n-class of K+ channels. However, the identity of the major proteins and their relative abundances exhibit marked interspecific variation. In addition, the greater resolution of the top-down venomic analytical approach revealed previously undetected protein species, isoforms and proteoforms, including the identification and precise location of modified lysine residues in a number of proteins in both venoms, but particularly in green mamba toxins. This comparative top-down venomic analysis unveiled the untapped complexity of Dendroaspis venoms and lays the foundations for rationalizing the notably different potency of green and black mamba lethal arsenals at locus resolution. We report the characterization, by combination of high-resolution on-line molecular mass and disulfide bond profiling and top-down MS/MS analysis, of the venom proteomes of two congeneric African snake species of medical importance, Dendroaspis angusticeps (green mamba) and D. polylepis (black mamba). Each of these mamba venoms comprised more than two-hundred polypeptides belonging to just a few toxin families. Both venom proteomes are overwhelmingly composed of post-synaptically-acting short- and long-chain neurotoxins that potently inhibit muscle- and neuronal-type nicotinic acetylcholine receptors; muscarinic cardiotoxins; and dendrotoxins, that block some of the Kv1, n-class of K+ channels. However, the identity of the major proteins and their relative abundances exhibit marked interspecific variation. In addition, the greater resolution of the top-down venomic analytical approach revealed previously undetected protein species, isoforms and proteoforms, including the identification and precise location of modified lysine residues in a number of proteins in both venoms, but particularly in green mamba toxins. This comparative top-down venomic analysis unveiled the untapped complexity of Dendroaspis venoms and lays the foundations for rationalizing the notably different potency of green and black mamba lethal arsenals at locus resolution.

Proteomics approaches advance our understanding of plant self-incompatibility response.

PubMed

Sankaranarayanan, Subramanian; Jamshed, Muhammad; Samuel, Marcus A

2013-11-01

Self-incompatibility (SI) in plants is a genetic mechanism that prevents self-fertilization and promotes out-crossing needed to maintain genetic diversity. SI has been classified into two broad categories: the gametophytic self-incompatibility (GSI) and the sporophytic self-incompatibility (SSI) based on the genetic mechanisms involved in 'self' pollen rejection. Recent proteomic approaches to identify potential candidates involved in SI have shed light onto a number of previously unidentified mechanisms required for SI response. SI proteome research has progressed from the use of isoelectric focusing in early days to the latest third-generation technique of comparative isobaric tag for relative and absolute quantitation (iTRAQ) used in recent times. We will focus on the proteome-based approaches used to study self-incompatibility (GSI and SSI), recent developments in the field of incompatibility research with emphasis on SSI and future prospects of using proteomic approaches to study self-incompatibility.

NCI Launches Proteomics Assay Portal | Office of Cancer Clinical Proteomics Research

Cancer.gov

In a paper recently published by the journal Nature Methods, Investigators from the National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium (NCI-CPTAC) announced the launch of a proteomics Assay Portal for multiple reaction monitoring-mass spectrometry (MRM-MS) assays.  This community web-based repository for well-characterized quantitative proteomic assays currently consists of 456 unique peptide assays to 282 unique proteins and ser

Molecular phenotype of zebrafish ovarian follicle by serial analysis of gene expression and proteomic profiling, and comparison with the transcriptomes of other animals

PubMed Central

Knoll-Gellida, Anja; André, Michèle; Gattegno, Tamar; Forgue, Jean; Admon, Arie; Babin, Patrick J

2006-01-01

Background The ability of an oocyte to develop into a viable embryo depends on the accumulation of specific maternal information and molecules, such as RNAs and proteins. A serial analysis of gene expression (SAGE) was carried out in parallel with proteomic analysis on fully-grown ovarian follicles from zebrafish (Danio rerio). The data obtained were compared with ovary/follicle/egg molecular phenotypes of other animals, published or available in public sequence databases. Results Sequencing of 27,486 SAGE tags identified 11,399 different ones, including 3,329 tags with an occurrence superior to one. Fifty-eight genes were expressed at over 0.15% of the total population and represented 17.34% of the mRNA population identified. The three most expressed transcripts were a rhamnose-binding lectin, beta-actin 2, and a transcribed locus similar to the H2B histone family. Comparison with the large-scale expressed sequence tags sequencing approach revealed highly expressed transcripts that were not previously known to be expressed at high levels in fish ovaries, like the short-sized polarized metallothionein 2 transcript. A higher sensitivity for the detection of transcripts with a characterized maternal genetic contribution was also demonstrated compared to large-scale sequencing of cDNA libraries. Ferritin heavy polypeptide 1, heat shock protein 90-beta, lactate dehydrogenase B4, beta-actin isoforms, tubulin beta 2, ATP synthase subunit 9, together with 40 S ribosomal protein S27a, were common highly-expressed transcripts of vertebrate ovary/unfertilized egg. Comparison of transcriptome and proteome data revealed that transcript levels provide little predictive value with respect to the extent of protein abundance. All the proteins identified by proteomic analysis of fully-grown zebrafish follicles had at least one transcript counterpart, with two exceptions: eosinophil chemotactic cytokine and nothepsin. Conclusion This study provides a complete sequence data set of maternal mRNA stored in zebrafish germ cells at the end of oogenesis. This catalogue contains highly-expressed transcripts that are part of a vertebrate ovarian expressed gene signature. Comparison of transcriptome and proteome data identified downregulated transcripts or proteins potentially incorporated in the oocyte by endocytosis. The molecular phenotype described provides groundwork for future experimental approaches aimed at identifying functionally important stored maternal transcripts and proteins involved in oogenesis and early stages of embryo development. PMID:16526958

A Simple Method for Rapid Depletion of Rubisco from Soybean (Glycine max) Leaf for Proteomic Analysis of Lower Abundance Proteins

USDA-ARS?s Scientific Manuscript database

2-DE analysis of complex plant proteomes has limited dynamic resolution because only abundant proteins can be detected. Proteomic assessment of the low abundance proteins within leaf tissue is difficult when it is comprised of 30 – 50% of the CO2 fixation enzyme Rubisco. Resolution can be improved t...

Xylem sap proteomics.

PubMed

de Bernonville, Thomas Dugé; Albenne, Cécile; Arlat, Matthieu; Hoffmann, Laurent; Lauber, Emmanuelle; Jamet, Elisabeth

2014-01-01

Proteomic analysis of xylem sap has recently become a major field of interest to understand several biological questions related to plant development and responses to environmental clues. The xylem sap appears as a dynamic fluid undergoing changes in its proteome upon abiotic and biotic stresses. Unlike cell compartments which are amenable to purification in sufficient amount prior to proteomic analysis, the xylem sap has to be collected in particular conditions to avoid contamination by intracellular proteins and to obtain enough material. A model plant like Arabidopsis thaliana is not suitable for such an analysis because efficient harvesting of xylem sap is difficult. The analysis of the xylem sap proteome also requires specific procedures to concentrate proteins and to focus on proteins predicted to be secreted. Indeed, xylem sap proteins appear to be synthesized and secreted in the root stele or to originate from dying differentiated xylem cells. This chapter describes protocols to collect xylem sap from Brassica species and to prepare total and N-glycoprotein extracts for identification of proteins by mass spectrometry analyses and bioinformatics.

Highly Reproducible Label Free Quantitative Proteomic Analysis of RNA Polymerase Complexes*

PubMed Central

Mosley, Amber L.; Sardiu, Mihaela E.; Pattenden, Samantha G.; Workman, Jerry L.; Florens, Laurence; Washburn, Michael P.

2011-01-01

The use of quantitative proteomics methods to study protein complexes has the potential to provide in-depth information on the abundance of different protein components as well as their modification state in various cellular conditions. To interrogate protein complex quantitation using shotgun proteomic methods, we have focused on the analysis of protein complexes using label-free multidimensional protein identification technology and studied the reproducibility of biological replicates. For these studies, we focused on three highly related and essential multi-protein enzymes, RNA polymerase I, II, and III from Saccharomyces cerevisiae. We found that label-free quantitation using spectral counting is highly reproducible at the protein and peptide level when analyzing RNA polymerase I, II, and III. In addition, we show that peptide sampling does not follow a random sampling model, and we show the need for advanced computational models to predict peptide detection probabilities. In order to address these issues, we used the APEX protocol to model the expected peptide detectability based on whole cell lysate acquired using the same multidimensional protein identification technology analysis used for the protein complexes. Neither method was able to predict the peptide sampling levels that we observed using replicate multidimensional protein identification technology analyses. In addition to the analysis of the RNA polymerase complexes, our analysis provides quantitative information about several RNAP associated proteins including the RNAPII elongation factor complexes DSIF and TFIIF. Our data shows that DSIF and TFIIF are the most highly enriched RNAP accessory factors in Rpb3-TAP purifications and demonstrate our ability to measure low level associated protein abundance across biological replicates. In addition, our quantitative data supports a model in which DSIF and TFIIF interact with RNAPII in a dynamic fashion in agreement with previously published reports. PMID:21048197

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.

An orthology-based analysis of pathogenic protozoa impacting global health: an improved comparative genomics approach with prokaryotes and model eukaryote orthologs.

PubMed

Cuadrat, Rafael R C; da Serra Cruz, Sérgio Manuel; Tschoeke, Diogo Antônio; Silva, Edno; Tosta, Frederico; Jucá, Henrique; Jardim, Rodrigo; Campos, Maria Luiza M; Mattoso, Marta; Dávila, Alberto M R

2014-08-01

A key focus in 21(st) century integrative biology and drug discovery for neglected tropical and other diseases has been the use of BLAST-based computational methods for identification of orthologous groups in pathogenic organisms to discern orthologs, with a view to evaluate similarities and differences among species, and thus allow the transfer of annotation from known/curated proteins to new/non-annotated ones. We used here a profile-based sensitive methodology to identify distant homologs, coupled to the NCBI's COG (Unicellular orthologs) and KOG (Eukaryote orthologs), permitting us to perform comparative genomics analyses on five protozoan genomes. OrthoSearch was used in five protozoan proteomes showing that 3901 and 7473 orthologs can be identified by comparison with COG and KOG proteomes, respectively. The core protozoa proteome inferred was 418 Protozoa-COG orthologous groups and 704 Protozoa-KOG orthologous groups: (i) 31.58% (132/418) belongs to the category J (translation, ribosomal structure, and biogenesis), and 9.81% (41/418) to the category O (post-translational modification, protein turnover, chaperones) using COG; (ii) 21.45% (151/704) belongs to the categories J, and 13.92% (98/704) to the O using KOG. The phylogenomic analysis showed four well-supported clades for Eukarya, discriminating Multicellular [(i) human, fly, plant and worm] and Unicellular [(ii) yeast, (iii) fungi, and (iv) protozoa] species. These encouraging results attest to the usefulness of the profile-based methodology for comparative genomics to accelerate semi-automatic re-annotation, especially of the protozoan proteomes. This approach may also lend itself for applications in global health, for example, in the case of novel drug target discovery against pathogenic organisms previously considered difficult to research with traditional drug discovery tools.

An Orthology-Based Analysis of Pathogenic Protozoa Impacting Global Health: An Improved Comparative Genomics Approach with Prokaryotes and Model Eukaryote Orthologs

PubMed Central

Cuadrat, Rafael R. C.; da Serra Cruz, Sérgio Manuel; Tschoeke, Diogo Antônio; Silva, Edno; Tosta, Frederico; Jucá, Henrique; Jardim, Rodrigo; Campos, Maria Luiza M.; Mattoso, Marta

2014-01-01

Abstract A key focus in 21st century integrative biology and drug discovery for neglected tropical and other diseases has been the use of BLAST-based computational methods for identification of orthologous groups in pathogenic organisms to discern orthologs, with a view to evaluate similarities and differences among species, and thus allow the transfer of annotation from known/curated proteins to new/non-annotated ones. We used here a profile-based sensitive methodology to identify distant homologs, coupled to the NCBI's COG (Unicellular orthologs) and KOG (Eukaryote orthologs), permitting us to perform comparative genomics analyses on five protozoan genomes. OrthoSearch was used in five protozoan proteomes showing that 3901 and 7473 orthologs can be identified by comparison with COG and KOG proteomes, respectively. The core protozoa proteome inferred was 418 Protozoa-COG orthologous groups and 704 Protozoa-KOG orthologous groups: (i) 31.58% (132/418) belongs to the category J (translation, ribosomal structure, and biogenesis), and 9.81% (41/418) to the category O (post-translational modification, protein turnover, chaperones) using COG; (ii) 21.45% (151/704) belongs to the categories J, and 13.92% (98/704) to the O using KOG. The phylogenomic analysis showed four well-supported clades for Eukarya, discriminating Multicellular [(i) human, fly, plant and worm] and Unicellular [(ii) yeast, (iii) fungi, and (iv) protozoa] species. These encouraging results attest to the usefulness of the profile-based methodology for comparative genomics to accelerate semi-automatic re-annotation, especially of the protozoan proteomes. This approach may also lend itself for applications in global health, for example, in the case of novel drug target discovery against pathogenic organisms previously considered difficult to research with traditional drug discovery tools. PMID:24960463

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

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.

Proteomics-based identification of differentially abundant proteins reveals adaptation mechanisms of Xanthomonas citri subsp. citri during Citrus sinensis infection.

PubMed

Moreira, Leandro M; Soares, Márcia R; Facincani, Agda P; Ferreira, Cristiano B; Ferreira, Rafael M; Ferro, Maria I T; Gozzo, Fábio C; Felestrino, Érica B; Assis, Renata A B; Garcia, Camila Carrião M; Setubal, João C; Ferro, Jesus A; de Oliveira, Julio C F

2017-07-11

Xanthomonas citri subsp. citri (Xac) is the causal agent of citrus canker. A proteomic analysis under in planta infectious and non-infectious conditions was conducted in order to increase our knowledge about the adaptive process of Xac during infection. For that, a 2D-based proteomic analysis of Xac at 1, 3 and 5 days after inoculation, in comparison to Xac growth in NB media was carried out and followed by MALDI-TOF-TOF identification of 124 unique differentially abundant proteins. Among them, 79 correspond to up-regulated proteins in at least one of the three stages of infection. Our results indicate an important role of proteins related to biofilm synthesis, lipopolysaccharides biosynthesis, and iron uptake and metabolism as possible modulators of plant innate immunity, and revealed an intricate network of proteins involved in reactive oxygen species adaptation during Plants` Oxidative Burst response. We also identified proteins previously unknown to be involved in Xac-Citrus interaction, including the hypothetical protein XAC3981. A mutant strain for this gene has proved to be non-pathogenic in respect to classical symptoms of citrus canker induced in compatible plants. This is the first time that a protein repertoire is shown to be active and working in an integrated manner during the infection process in a compatible host, pointing to an elaborate mechanism for adaptation of Xac once inside the plant.

Comparative genomic, proteomic and exoproteomic analyses of three Pseudomonas strains reveals novel insights into the phosphorus scavenging capabilities of soil bacteria

PubMed Central

Murphy, Andrew R. J.; Scanlan, David J.; Bending, Gary D.; Jones, Alexandra M. E.; Moore, Jonathan D.; Goodall, Andrew; Hammond, John P.; Wellington, Elizabeth M. H.

2016-01-01

Summary Bacteria that inhabit the rhizosphere of agricultural crops can have a beneficial effect on crop growth. One such mechanism is the microbial‐driven solubilization and remineralization of complex forms of phosphorus (P). It is known that bacteria secrete various phosphatases in response to low P conditions. However, our understanding of their global proteomic response to P stress is limited. Here, exoproteomic analysis of Pseudomonas putida BIRD‐1 (BIRD‐1), Pseudomonas fluorescens SBW25 and Pseudomonas stutzeri DSM4166 was performed in unison with whole‐cell proteomic analysis of BIRD‐1 grown under phosphate (Pi) replete and Pi deplete conditions. Comparative exoproteomics revealed marked heterogeneity in the exoproteomes of each Pseudomonas strain in response to Pi depletion. In addition to well‐characterized members of the PHO regulon such as alkaline phosphatases, several proteins, previously not associated with the response to Pi depletion, were also identified. These included putative nucleases, phosphotriesterases, putative phosphonate transporters and outer membrane proteins. Moreover, in BIRD‐1, mutagenesis of the master regulator, phoBR, led us to confirm the addition of several novel PHO‐dependent proteins. Our data expands knowledge of the Pseudomonas PHO regulon, including species that are frequently used as bioinoculants, opening up the potential for more efficient and complete use of soil complexed P. PMID:27233093

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.

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.

Regulation of Growth Response to Water Stress in the Soybean Primary Root. I. Proteomic Analysis Reveals Region-Specific Regulation of Phenylpropanoid Metabolism and Control of Free Iron in the Elongation Zone.

USDA-ARS?s Scientific Manuscript database

In water-stressed soybean primary roots, elongation was maintained at well-watered rates in the apical 4 mm (region 1) but was progressively inhibited in the 4-8 mm region (region 2), which exhibits maximum elongation in well-watered roots. These responses are similar to previous results for the mai...

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.

Establishing Substantial Equivalence: Proteomics

NASA Astrophysics Data System (ADS)

Lovegrove, Alison; Salt, Louise; Shewry, Peter R.

Wheat is a major crop in world agriculture and is consumed after processing into a range of food products. It is therefore of great importance to determine the consequences (intended and unintended) of transgenesis in wheat and whether genetically modified lines are substantially equivalent to those produced by conventional plant breeding. Proteomic analysis is one of several approaches which can be used to address these questions. Two-dimensional PAGE (2D PAGE) remains the most widely available method for proteomic analysis, but is notoriously difficult to reproduce between laboratories. We therefore describe methods which have been developed as standard operating procedures in our laboratory to ensure the reproducibility of proteomic analyses of wheat using 2D PAGE analysis of grain proteins.

Integrated Analysis of Transcriptomic and Proteomic Data

PubMed Central

Haider, Saad; Pal, Ranadip

2013-01-01

Until recently, understanding the regulatory behavior of cells has been pursued through independent analysis of the transcriptome or the proteome. Based on the central dogma, it was generally assumed that there exist a direct correspondence between mRNA transcripts and generated protein expressions. However, recent studies have shown that the correlation between mRNA and Protein expressions can be low due to various factors such as different half lives and post transcription machinery. Thus, a joint analysis of the transcriptomic and proteomic data can provide useful insights that may not be deciphered from individual analysis of mRNA or protein expressions. This article reviews the existing major approaches for joint analysis of transcriptomic and proteomic data. We categorize the different approaches into eight main categories based on the initial algorithm and final analysis goal. We further present analogies with other domains and discuss the existing research problems in this area. PMID:24082820

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.

freeQuant: A Mass Spectrometry Label-Free Quantification Software Tool for Complex Proteome Analysis.

PubMed

Deng, Ning; Li, Zhenye; Pan, Chao; Duan, Huilong

2015-01-01

Study of complex proteome brings forward higher request for the quantification method using mass spectrometry technology. In this paper, we present a mass spectrometry label-free quantification tool for complex proteomes, called freeQuant, which integrated quantification with functional analysis effectively. freeQuant consists of two well-integrated modules: label-free quantification and functional analysis with biomedical knowledge. freeQuant supports label-free quantitative analysis which makes full use of tandem mass spectrometry (MS/MS) spectral count, protein sequence length, shared peptides, and ion intensity. It adopts spectral count for quantitative analysis and builds a new method for shared peptides to accurately evaluate abundance of isoforms. For proteins with low abundance, MS/MS total ion count coupled with spectral count is included to ensure accurate protein quantification. Furthermore, freeQuant supports the large-scale functional annotations for complex proteomes. Mitochondrial proteomes from the mouse heart, the mouse liver, and the human heart were used to evaluate the usability and performance of freeQuant. The evaluation showed that the quantitative algorithms implemented in freeQuant can improve accuracy of quantification with better dynamic range.

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.

HTAPP: High-Throughput Autonomous Proteomic Pipeline

PubMed Central

Yu, Kebing; Salomon, Arthur R.

2011-01-01

Recent advances in the speed and sensitivity of mass spectrometers and in analytical methods, the exponential acceleration of computer processing speeds, and the availability of genomic databases from an array of species and protein information databases have led to a deluge of proteomic data. The development of a lab-based automated proteomic software platform for the automated collection, processing, storage, and visualization of expansive proteomic datasets is critically important. The high-throughput autonomous proteomic pipeline (HTAPP) described here is designed from the ground up to provide critically important flexibility for diverse proteomic workflows and to streamline the total analysis of a complex proteomic sample. This tool is comprised of software that controls the acquisition of mass spectral data along with automation of post-acquisition tasks such as peptide quantification, clustered MS/MS spectral database searching, statistical validation, and data exploration within a user-configurable lab-based relational database. The software design of HTAPP focuses on accommodating diverse workflows and providing missing software functionality to a wide range of proteomic researchers to accelerate the extraction of biological meaning from immense proteomic data sets. Although individual software modules in our integrated technology platform may have some similarities to existing tools, the true novelty of the approach described here is in the synergistic and flexible combination of these tools to provide an integrated and efficient analysis of proteomic samples. PMID:20336676

Growth and Proteome Response of Temperate and Polar Diatoms Thalassiosira pseudonana and Chaetoceros sp. to variations in Cobalt and Zinc: Identification of the High-Affinity Zinc Transporter and Potential for Use as a Biomarker

NASA Astrophysics Data System (ADS)

Kellogg, M. M.; Moran, D. M.; McIlvin, M. R.; Allen, A. E.; Saito, M. A.

2016-02-01

Marine diatoms such as the temperate Thalassiosira pseudonana (Tp) and the polar Chaetoceros sp (Ch) are known to be important contributors to marine primary productivity and the global carbon cycle. The nutritional use of zinc (Zn) in diatoms and the ability to substitute cobalt (Co) for Zn has been previously demonstrated to be of importance in their growth and biochemistry. We conducted physiological experiments with Zn and Co on these diatoms and analyzed their proteomic response. Growth studies involving Tp confirmed previous studies' findings showing Zn/Co substitution, while studies on Ch showed a toxic response to high Zn abundances. Proteome responses of Tp to Zn limitation identified a putative and previously unidentified transporter that was undetectable at high Zn concentration and became highly abundant at two lower Zn concentrations. The distribution of this protein in nature and its potential use as a Zn stress biomarker in diatoms will be discussed.

Achievements and perspectives of top-down proteomics.

PubMed

Armirotti, Andrea; Damonte, Gianluca

2010-10-01

Over the last years, top-down (TD) MS has gained a remarkable space in proteomics, rapidly trespassing the limit between a promising approach and a solid, established technique. Several research groups worldwide have implemented TD analysis in their routine work on proteomics, deriving structural information on proteins with the level of accuracy that is impossible to achieve with classical bottom-up approaches. Complete maps of PTMs and assessment of single aminoacid polymorphisms are only a few of the results that can be obtained with this technique. Despite some existing technical and economical limitations, TD analysis is at present the most powerful instrument for MS-based proteomics and its implementation in routine workflow is a rapidly approaching turning point in proteomics. In this review article, the state-of-the-art of TD approach is described along with its major advantages and drawbacks and the most recent trends in TD analysis are discussed. References for all the covered topics are reported in the text, with the aim to support both newcomers and mass spectrometrists already introduced to TD proteomics.

Toward the Standardization of Mitochondrial Proteomics: The Italian Mitochondrial Human Proteome Project Initiative.

PubMed

Alberio, Tiziana; Pieroni, Luisa; Ronci, Maurizio; Banfi, Cristina; Bongarzone, Italia; Bottoni, Patrizia; Brioschi, Maura; Caterino, Marianna; Chinello, Clizia; Cormio, Antonella; Cozzolino, Flora; Cunsolo, Vincenzo; Fontana, Simona; Garavaglia, Barbara; Giusti, Laura; Greco, Viviana; Lucacchini, Antonio; Maffioli, Elisa; Magni, Fulvio; Monteleone, Francesca; Monti, Maria; Monti, Valentina; Musicco, Clara; Petrosillo, Giuseppe; Porcelli, Vito; Saletti, Rosaria; Scatena, Roberto; Soggiu, Alessio; Tedeschi, Gabriella; Zilocchi, Mara; Roncada, Paola; Urbani, Andrea; Fasano, Mauro

2017-12-01

The Mitochondrial Human Proteome Project aims at understanding the function of the mitochondrial proteome and its crosstalk with the proteome of other organelles. Being able to choose a suitable and validated enrichment protocol of functional mitochondria, based on the specific needs of the downstream proteomics analysis, would greatly help the researchers in the field. Mitochondrial fractions from ten model cell lines were prepared using three enrichment protocols and analyzed on seven different LC-MS/MS platforms. All data were processed using neXtProt as reference database. The data are available for the Human Proteome Project purposes through the ProteomeXchange Consortium with the identifier PXD007053. The processed data sets were analyzed using a suite of R routines to perform a statistical analysis and to retrieve subcellular and submitochondrial localizations. Although the overall number of identified total and mitochondrial proteins was not significantly dependent on the enrichment protocol, specific line to line differences were observed. Moreover, the protein lists were mapped to a network representing the functional mitochondrial proteome, encompassing mitochondrial proteins and their first interactors. More than 80% of the identified proteins resulted in nodes of this network but with a different ability in coisolating mitochondria-associated structures for each enrichment protocol/cell line pair.

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.

Multidimensional proteomics for cell biology.

PubMed

Larance, Mark; Lamond, Angus I

2015-05-01

The proteome is a dynamic system in which each protein has interconnected properties - dimensions - that together contribute to the phenotype of a cell. Measuring these properties has proved challenging owing to their diversity and dynamic nature. Advances in mass spectrometry-based proteomics now enable the measurement of multiple properties for thousands of proteins, including their abundance, isoform expression, turnover rate, subcellular localization, post-translational modifications and interactions. Complementing these experimental developments are new data analysis, integration and visualization tools as well as data-sharing resources. Together, these advances in the multidimensional analysis of the proteome are transforming our understanding of various cellular and physiological processes.

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.

Proteome Profiles of Outer Membrane Vesicles and Extracellular Matrix of Pseudomonas aeruginosa Biofilms.

PubMed

Couto, Narciso; Schooling, Sarah R; Dutcher, John R; Barber, Jill

2015-10-02

In the present work, two different proteomic platforms, gel-based and gel-free, were used to map the matrix and outer membrane vesicle exoproteomes of Pseudomonas aeruginosa PAO1 biofilms. These two proteomic strategies allowed us a confident identification of 207 and 327 proteins from enriched outer membrane vesicles and whole matrix isolated from biofilms. Because of the physicochemical characteristics of these subproteomes, the two strategies showed complementarity, and thus, the most comprehensive analysis of P. aeruginosa exoproteome to date was achieved. Under our conditions, outer membrane vesicles contribute approximately 20% of the whole matrix proteome, demonstrating that membrane vesicles are an important component of the matrix. The proteomic profiles were analyzed in terms of their biological context, namely, a biofilm. Accordingly relevant metabolic processes involved in cellular adaptation to the biofilm lifestyle as well as those related to P. aeruginosa virulence capabilities were a key feature of the analyses. The diversity of the matrix proteome corroborates the idea of high heterogeneity within the biofilm; cells can display different levels of metabolism and can adapt to local microenvironments making this proteomic analysis challenging. In addition to analyzing our own primary data, we extend the analysis to published data by other groups in order to deepen our understanding of the complexity inherent within biofilm populations.

Advances of Proteomic Sciences in Dentistry.

PubMed

Khurshid, Zohaib; Zohaib, Sana; Najeeb, Shariq; Zafar, Muhammad Sohail; Rehman, Rabia; Rehman, Ihtesham Ur

2016-05-13

Applications of proteomics tools revolutionized various biomedical disciplines such as genetics, molecular biology, medicine, and dentistry. The aim of this review is to highlight the major milestones in proteomics in dentistry during the last fifteen years. Human oral cavity contains hard and soft tissues and various biofluids including saliva and crevicular fluid. Proteomics has brought revolution in dentistry by helping in the early diagnosis of various diseases identified by the detection of numerous biomarkers present in the oral fluids. This paper covers the role of proteomics tools for the analysis of oral tissues. In addition, dental materials proteomics and their future directions are discussed.

Halobacterium salinarum NRC-1 PeptideAtlas: toward strategies for targeted proteomics and improved proteome coverage.

PubMed

Van, Phu T; Schmid, Amy K; King, Nichole L; Kaur, Amardeep; Pan, Min; Whitehead, Kenia; Koide, Tie; Facciotti, Marc T; Goo, Young Ah; Deutsch, Eric W; Reiss, David J; Mallick, Parag; Baliga, Nitin S

2008-09-01

The relatively small numbers of proteins and fewer possible post-translational modifications in microbes provide a unique opportunity to comprehensively characterize their dynamic proteomes. We have constructed a PeptideAtlas (PA) covering 62.7% of the predicted proteome of the extremely halophilic archaeon Halobacterium salinarum NRC-1 by compiling approximately 636 000 tandem mass spectra from 497 mass spectrometry runs in 88 experiments. Analysis of the PA with respect to biophysical properties of constituent peptides, functional properties of parent proteins of detected peptides, and performance of different mass spectrometry approaches has highlighted plausible strategies for improving proteome coverage and selecting signature peptides for targeted proteomics. Notably, discovery of a significant correlation between absolute abundances of mRNAs and proteins has helped identify low abundance of proteins as the major limitation in peptide detection. Furthermore, we have discovered that iTRAQ labeling for quantitative proteomic analysis introduces a significant bias in peptide detection by mass spectrometry. Therefore, despite identifying at least one proteotypic peptide for almost all proteins in the PA, a context-dependent selection of proteotypic peptides appears to be the most effective approach for targeted proteomics.

Stable isotope labelling methods in mass spectrometry-based quantitative proteomics.

PubMed

Chahrour, Osama; Cobice, Diego; Malone, John

2015-09-10

Mass-spectrometry based proteomics has evolved as a promising technology over the last decade and is undergoing a dramatic development in a number of different areas, such as; mass spectrometric instrumentation, peptide identification algorithms and bioinformatic computational data analysis. The improved methodology allows quantitative measurement of relative or absolute protein amounts, which is essential for gaining insights into their functions and dynamics in biological systems. Several different strategies involving stable isotopes label (ICAT, ICPL, IDBEST, iTRAQ, TMT, IPTL, SILAC), label-free statistical assessment approaches (MRM, SWATH) and absolute quantification methods (AQUA) are possible, each having specific strengths and weaknesses. Inductively coupled plasma mass spectrometry (ICP-MS), which is still widely recognised as elemental detector, has recently emerged as a complementary technique to the previous methods. The new application area for ICP-MS is targeting the fast growing field of proteomics related research, allowing absolute protein quantification using suitable elemental based tags. This document describes the different stable isotope labelling methods which incorporate metabolic labelling in live cells, ICP-MS based detection and post-harvest chemical label tagging for protein quantification, in addition to summarising their pros and cons. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

Synaptic proteomics as a means to identify the molecular basis of mental illness: Are we getting there?

PubMed

Reig-Viader, Rita; Sindreu, Carlos; Bayés, Àlex

2018-06-08

Synapses are centrally involved in many brain disorders, particularly in psychiatric and neurodevelopmental ones. However, our current understanding of the proteomic alterations affecting synaptic performance in the majority of mental illnesses is limited. As a result, novel pharmacotherapies with improved neurological efficacy have been scarce over the past decades. The main goal of synaptic proteomics in the context of mental illnesses is to identify dysregulated molecular mechanisms underlying these conditions. Here we reviewed and performed a meta-analysis of previous neuroproteomic research to identify proteins that may be consistently dysregulated in one or several mental disorders. Notably, we found very few proteins reproducibly altered among independent experiments for any given condition or between conditions, indicating that we are still far from identifying key pathophysiological mechanisms of mental illness. We suggest that future research in the field will require higher levels of standardization and larger-scale experiments to address the challenge posed by biological and methodological variability. We strongly believe that more resources should be placed in this field as the need to identify the molecular roots of mental illnesses is highly pressing. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

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.

CPTAC Collaborates with Molecular & Cellular Proteomics to Address Reproducibility in Targeted Assay Development | Office of Cancer Clinical Proteomics Research

Cancer.gov

The journal Molecular & Cellular Proteomics (MCP), in collaboration with the Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI), part of the National Institutes of Health, announce new guidelines and requirements for papers describing the development and application of targeted mass spectrometry measurements of peptides, modified peptides and proteins (Mol Cell Proteomics 2017; PMID: 28183812).  NCI’s participation is part of NIH’s overall effort to address the r

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.

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

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

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

YPED: An Integrated Bioinformatics Suite and Database for Mass Spectrometry-based Proteomics Research

PubMed Central

Colangelo, Christopher M.; Shifman, Mark; Cheung, Kei-Hoi; Stone, Kathryn L.; Carriero, Nicholas J.; Gulcicek, Erol E.; Lam, TuKiet T.; Wu, Terence; Bjornson, Robert D.; Bruce, Can; Nairn, Angus C.; Rinehart, Jesse; Miller, Perry L.; Williams, Kenneth R.

2015-01-01

We report a significantly-enhanced bioinformatics suite and database for proteomics research called Yale Protein Expression Database (YPED) that is used by investigators at more than 300 institutions worldwide. YPED meets the data management, archival, and analysis needs of a high-throughput mass spectrometry-based proteomics research ranging from a single laboratory, group of laboratories within and beyond an institution, to the entire proteomics community. The current version is a significant improvement over the first version in that it contains new modules for liquid chromatography–tandem mass spectrometry (LC–MS/MS) database search results, label and label-free quantitative proteomic analysis, and several scoring outputs for phosphopeptide site localization. In addition, we have added both peptide and protein comparative analysis tools to enable pairwise analysis of distinct peptides/proteins in each sample and of overlapping peptides/proteins between all samples in multiple datasets. We have also implemented a targeted proteomics module for automated multiple reaction monitoring (MRM)/selective reaction monitoring (SRM) assay development. We have linked YPED’s database search results and both label-based and label-free fold-change analysis to the Skyline Panorama repository for online spectra visualization. In addition, we have built enhanced functionality to curate peptide identifications into an MS/MS peptide spectral library for all of our protein database search identification results. PMID:25712262

QPROT: Statistical method for testing differential expression using protein-level intensity data in label-free quantitative proteomics.

PubMed

Choi, Hyungwon; Kim, Sinae; Fermin, Damian; Tsou, Chih-Chiang; Nesvizhskii, Alexey I

2015-11-03

We introduce QPROT, a statistical framework and computational tool for differential protein expression analysis using protein intensity data. QPROT is an extension of the QSPEC suite, originally developed for spectral count data, adapted for the analysis using continuously measured protein-level intensity data. QPROT offers a new intensity normalization procedure and model-based differential expression analysis, both of which account for missing data. Determination of differential expression of each protein is based on the standardized Z-statistic based on the posterior distribution of the log fold change parameter, guided by the false discovery rate estimated by a well-known Empirical Bayes method. We evaluated the classification performance of QPROT using the quantification calibration data from the clinical proteomic technology assessment for cancer (CPTAC) study and a recently published Escherichia coli benchmark dataset, with evaluation of FDR accuracy in the latter. QPROT is a statistical framework with computational software tool for comparative quantitative proteomics analysis. It features various extensions of QSPEC method originally built for spectral count data analysis, including probabilistic treatment of missing values in protein intensity data. With the increasing popularity of label-free quantitative proteomics data, the proposed method and accompanying software suite will be immediately useful for many proteomics laboratories. This article is part of a Special Issue entitled: Computational Proteomics. Copyright © 2015 Elsevier B.V. All rights reserved.

YPED: an integrated bioinformatics suite and database for mass spectrometry-based proteomics research.

PubMed

Colangelo, Christopher M; Shifman, Mark; Cheung, Kei-Hoi; Stone, Kathryn L; Carriero, Nicholas J; Gulcicek, Erol E; Lam, TuKiet T; Wu, Terence; Bjornson, Robert D; Bruce, Can; Nairn, Angus C; Rinehart, Jesse; Miller, Perry L; Williams, Kenneth R

2015-02-01

We report a significantly-enhanced bioinformatics suite and database for proteomics research called Yale Protein Expression Database (YPED) that is used by investigators at more than 300 institutions worldwide. YPED meets the data management, archival, and analysis needs of a high-throughput mass spectrometry-based proteomics research ranging from a single laboratory, group of laboratories within and beyond an institution, to the entire proteomics community. The current version is a significant improvement over the first version in that it contains new modules for liquid chromatography-tandem mass spectrometry (LC-MS/MS) database search results, label and label-free quantitative proteomic analysis, and several scoring outputs for phosphopeptide site localization. In addition, we have added both peptide and protein comparative analysis tools to enable pairwise analysis of distinct peptides/proteins in each sample and of overlapping peptides/proteins between all samples in multiple datasets. We have also implemented a targeted proteomics module for automated multiple reaction monitoring (MRM)/selective reaction monitoring (SRM) assay development. We have linked YPED's database search results and both label-based and label-free fold-change analysis to the Skyline Panorama repository for online spectra visualization. In addition, we have built enhanced functionality to curate peptide identifications into an MS/MS peptide spectral library for all of our protein database search identification results. Copyright © 2015 The Authors. Production and hosting by Elsevier Ltd.. All rights reserved.

Halobacterium salinarum NRC-1 PeptideAtlas: strategies for targeted proteomics

PubMed Central

Van, Phu T.; Schmid, Amy K.; King, Nichole L.; Kaur, Amardeep; Pan, Min; Whitehead, Kenia; Koide, Tie; Facciotti, Marc T.; Goo, Young-Ah; Deutsch, Eric W.; Reiss, David J.; Mallick, Parag; Baliga, Nitin S.

2009-01-01

The relatively small numbers of proteins and fewer possible posttranslational modifications in microbes provides a unique opportunity to comprehensively characterize their dynamic proteomes. We have constructed a Peptide Atlas (PA) for 62.7% of the predicted proteome of the extremely halophilic archaeon Halobacterium salinarum NRC-1 by compiling approximately 636,000 tandem mass spectra from 497 mass spectrometry runs in 88 experiments. Analysis of the PA with respect to biophysical properties of constituent peptides, functional properties of parent proteins of detected peptides, and performance of different mass spectrometry approaches has helped highlight plausible strategies for improving proteome coverage and selecting signature peptides for targeted proteomics. Notably, discovery of a significant correlation between absolute abundances of mRNAs and proteins has helped identify low abundance of proteins as the major limitation in peptide detection. Furthermore we have discovered that iTRAQ labeling for quantitative proteomic analysis introduces a significant bias in peptide detection by mass spectrometry. Therefore, despite identifying at least one proteotypic peptide for almost all proteins in the PA, a context-dependent selection of proteotypic peptides appears to be the most effective approach for targeted proteomics. PMID:18652504

[Techniques for rapid production of monoclonal antibodies for use with antibody technology].

PubMed

Kamada, Haruhiko

2012-01-01

A monoclonal antibody (Mab), due to its specific binding ability to a target protein, can potentially be one of the most useful tools for the functional analysis of proteins in recent proteomics-based research. However, the production of Mab is a very time-consuming and laborious process (i.e., preparation of recombinant antigens, immunization of animals, preparation of hybridomas), making it the rate-limiting step in using Mabs in high-throughput proteomics research, which heavily relies on comprehensive and rapid methods. Therefore, there is a great demand for new methods to efficiently generate Mabs against a group of proteins identified by proteome analysis. Here, we describe a useful method called "Antibody proteomic technique" for the rapid generations of Mabs to pharmaceutical target, which were identified by proteomic analyses of disease samples (ex. tumor tissue, etc.). We also introduce another method to find profitable targets on vasculature, which is called "Vascular proteomic technique". Our results suggest that this method for the rapid generation of Mabs to proteins may be very useful in proteomics-based research as well as in clinical applications.

Prestroke Proteomic Changes in Cerebral Microvessels in Stroke-Prone, Transgenic[hCETP]-Hyperlipidemic, Dahl Salt-Sensitive Hypertensive Rats

PubMed Central

Bergerat, Agnes; Decano, Julius; Wu, Chang-Jiun; Choi, Hyungwon; Nesvizhskii, Alexey I; Moran, Ann Marie; Ruiz-Opazo, Nelson; Steffen, Martin; Herrera, Victoria LM

2011-01-01

Stroke is the third leading cause of death in the United States with high rates of morbidity among survivors. The search to fill the unequivocal need for new therapeutic approaches would benefit from unbiased proteomic analyses of animal models of spontaneous stroke in the prestroke stage. Since brain microvessels play key roles in neurovascular coupling, we investigated prestroke microvascular proteome changes. Proteomic analysis of cerebral cortical microvessels (cMVs) was done by tandem mass spectrometry comparing two prestroke time points. Metaprotein-pathway analyses of proteomic spectral count data were done to identify risk factor–induced changes, followed by QSPEC-analyses of individual protein changes associated with increased stroke susceptibility. We report 26 cMV proteome profiles from male and female stroke-prone and non–stroke-prone rats at 2 months and 4.5 months of age prior to overt stroke events. We identified 1,934 proteins by two or more peptides. Metaprotein pathway analysis detected age-associated changes in energy metabolism and cell-to-microenvironment interactions, as well as sex-specific changes in energy metabolism and endothelial leukocyte transmigration pathways. Stroke susceptibility was associated independently with multiple protein changes associated with ischemia, angiogenesis or involved in blood brain barrier (BBB) integrity. Immunohistochemical analysis confirmed aquaporin-4 and laminin-α1 induction in cMVs, representative of proteomic changes with >65 Bayes factor (BF), associated with stroke susceptibility. Altogether, proteomic analysis demonstrates significant molecular changes in ischemic cerebral microvasculature in the prestroke stage, which could contribute to the observed model phenotype of microhemorrhages and postischemic hemorrhagic transformation. These pathways comprise putative targets for translational research of much needed novel diagnostic and therapeutic approaches for stroke. PMID:21519634

Physiological and Proteomics Analyses Reveal Low-Phosphorus Stress Affected the Regulation of Photosynthesis in Soybean.

PubMed

Chu, Shanshan; Li, Hongyan; Zhang, Xiangqian; Yu, Kaiye; Chao, Maoni; Han, Suoyi; Zhang, Dan

2018-06-06

Previous studies have revealed a significant genetic relationship between phosphorus (P)-efficiency and photosynthesis-related traits in soybean. In this study, we used proteome profiling in combination with expression analysis, biochemical investigations, and leaf ultrastructural analysis to identify the underlying physiological and molecular responses. The expression analysis and ultrastructural analysis showed that the photosynthesis key genes were decreased at transcript levels and the leaf mesophyll and chloroplast were severely damaged after low-P stress. Approximately 55 protein spots showed changes under low-P condition by mass spectrometry, of which 17 were involved in various photosynthetic processes. Further analysis revealed the depression of photosynthesis caused by low-P stress mainly involves the regulation of leaf structure, adenosine triphosphate (ATP) synthesis, absorption and transportation of CO₂, photosynthetic electron transport, production of assimilatory power, and levels of enzymes related to the Calvin cycle. In summary, our findings indicated that the existence of a stringent relationship between P supply and the genomic control of photosynthesis in soybean. As an important strategy to protect soybean photosynthesis, P could maintain the stability of cell structure, up-regulate the enzymes’ activities, recover the process of photosystem II (PSII), and induce the expression of low-P responsive genes and proteins.

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

Song, Ehwang; Gao, Yuqian; Wu, Chaochao

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

Current state of the art for enhancing urine biomarker discovery

PubMed Central

Harpole, Michael; Davis, Justin; Espina, Virginia

2016-01-01

Urine is a highly desirable biospecimen for biomarker analysis because it can be collected recurrently by non-invasive techniques, in relatively large volumes. Urine contains cellular elements, biochemicals, and proteins derived from glomerular filtration of plasma, renal tubule excretion, and urogenital tract secretions that reflect, at a given time point, an individual's metabolic and pathophysiologic state. High-resolution mass spectrometry, coupled with state of the art fractionation systems are revealing the plethora of diagnostic/prognostic proteomic information existing within urinary exosomes, glycoproteins, and proteins. Affinity capture pre-processing techniques such as combinatorial peptide ligand libraries and biomarker harvesting hydrogel nanoparticles are enabling measurement/identification of previously undetectable urinary proteins. Future challenges in the urinary proteomics field include a) defining either single or multiple, universally applicable data normalization methods for comparing results within and between individual patients/data sets, and b) defining expected urinary protein levels in healthy individuals. PMID:27232439

CPTAC Accelerates Precision Proteomics Biomedical Research | Office of Cancer Clinical Proteomics Research

Cancer.gov

The accurate quantitation of proteins or peptides using Mass Spectrometry (MS) is gaining prominence in the biomedical research community as an alternative method for analyte measurement. The Clinical Proteomic Tumor Analysis Consortium (CPTAC) investigators have been at the forefront in the promotion of reproducible MS techniques, through the development and application of standardized proteomic methods for protein quantitation on biologically relevant samples.

Combination of Metabolomic and Proteomic Analysis Revealed Different Features among Lactobacillus delbrueckii Subspecies bulgaricus and lactis Strains While In Vivo Testing in the Model Organism Caenorhabditis elegans Highlighted Probiotic Properties

PubMed Central

Zanni, Elena; Schifano, Emily; Motta, Sara; Sciubba, Fabio; Palleschi, Claudio; Mauri, Pierluigi; Perozzi, Giuditta; Uccelletti, Daniela; Devirgiliis, Chiara; Miccheli, Alfredo

2017-01-01

Lactobacillus delbrueckii represents a technologically relevant member of lactic acid bacteria, since the two subspecies bulgaricus and lactis are widely associated with fermented dairy products. In the present work, we report the characterization of two commercial strains belonging to L. delbrueckii subspecies bulgaricus, lactis and a novel strain previously isolated from a traditional fermented fresh cheese. A phenomic approach was performed by combining metabolomic and proteomic analysis of the three strains, which were subsequently supplemented as food source to the model organism Caenorhabditis elegans, with the final aim to evaluate their possible probiotic effects. Restriction analysis of 16S ribosomal DNA revealed that the novel foodborne strain belonged to L. delbrueckii subspecies lactis. Proteomic and metabolomic approaches showed differences in folate, aminoacid and sugar metabolic pathways among the three strains. Moreover, evaluation of C. elegans lifespan, larval development, brood size, and bacterial colonization capacity demonstrated that L. delbrueckii subsp. bulgaricus diet exerted beneficial effects on nematodes. On the other hand, both L. delbrueckii subsp. lactis strains affected lifespan and larval development. We have characterized three strains belonging to L. delbrueckii subspecies bulgaricus and lactis highlighting their divergent origin. In particular, the two closely related isolates L. delbrueckii subspecies lactis display different galactose metabolic capabilities. Moreover, the L. delbrueckii subspecies bulgaricus strain demonstrated potential probiotic features. Combination of omic platforms coupled with in vivo screening in the simple model organism C. elegans is a powerful tool to characterize industrially relevant bacterial isolates. PMID:28702021

Combination of Metabolomic and Proteomic Analysis Revealed Different Features among Lactobacillus delbrueckii Subspecies bulgaricus and lactis Strains While In Vivo Testing in the Model Organism Caenorhabditis elegans Highlighted Probiotic Properties.

PubMed

Zanni, Elena; Schifano, Emily; Motta, Sara; Sciubba, Fabio; Palleschi, Claudio; Mauri, Pierluigi; Perozzi, Giuditta; Uccelletti, Daniela; Devirgiliis, Chiara; Miccheli, Alfredo

2017-01-01

Lactobacillus delbrueckii represents a technologically relevant member of lactic acid bacteria, since the two subspecies bulgaricus and lactis are widely associated with fermented dairy products. In the present work, we report the characterization of two commercial strains belonging to L. delbrueckii subspecies bulgaricus , lactis and a novel strain previously isolated from a traditional fermented fresh cheese. A phenomic approach was performed by combining metabolomic and proteomic analysis of the three strains, which were subsequently supplemented as food source to the model organism Caenorhabditis elegans , with the final aim to evaluate their possible probiotic effects. Restriction analysis of 16S ribosomal DNA revealed that the novel foodborne strain belonged to L. delbrueckii subspecies lactis . Proteomic and metabolomic approaches showed differences in folate, aminoacid and sugar metabolic pathways among the three strains. Moreover, evaluation of C. elegans lifespan, larval development, brood size, and bacterial colonization capacity demonstrated that L. delbrueckii subsp. bulgaricus diet exerted beneficial effects on nematodes. On the other hand, both L. delbrueckii subsp. lactis strains affected lifespan and larval development. We have characterized three strains belonging to L. delbrueckii subspecies bulgaricus and lactis highlighting their divergent origin. In particular, the two closely related isolates L. delbrueckii subspecies lactis display different galactose metabolic capabilities. Moreover, the L. delbrueckii subspecies bulgaricus strain demonstrated potential probiotic features. Combination of omic platforms coupled with in vivo screening in the simple model organism C. elegans is a powerful tool to characterize industrially relevant bacterial isolates.

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.

Michael T. Guarnieri | NREL

Science.gov Websites

accumulation," J. Proteomics (2013) "Comparative Proteomics Lends Insight into Genotype-Specific Pathogenicity," J. Proteomics (2013) "De Novo Transcriptomic Analysis of Hydrogen Production in the amino acid changes in the small envelope protein and rescued by a novel glycosolation site," J

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.

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

An Extended Proteome Map of the Lysosomal Membrane Reveals Novel Potential Transporters*

PubMed Central

Chapel, Agnès; Kieffer-Jaquinod, Sylvie; Sagné, Corinne; Verdon, Quentin; Ivaldi, Corinne; Mellal, Mourad; Thirion, Jaqueline; Jadot, Michel; Bruley, Christophe; Garin, Jérôme; Gasnier, Bruno; Journet, Agnès

2013-01-01

Lysosomes are membrane-bound endocytic organelles that play a major role in degrading cell macromolecules and recycling their building blocks. A comprehensive knowledge of the lysosome function requires an extensive description of its content, an issue partially addressed by previous proteomic analyses. However, the proteins underlying many lysosomal membrane functions, including numerous membrane transporters, remain unidentified. We performed a comparative, semi-quantitative proteomic analysis of rat liver lysosome-enriched and lysosome-nonenriched membranes and used spectral counts to evaluate the relative abundance of proteins. Among a total of 2,385 identified proteins, 734 proteins were significantly enriched in the lysosomal fraction, including 207 proteins already known or predicted as endo-lysosomal and 94 proteins without any known or predicted subcellular localization. The remaining 433 proteins had been previously assigned to other subcellular compartments but may in fact reside on lysosomes either predominantly or as a secondary location. Many membrane-associated complexes implicated in diverse processes such as degradation, membrane trafficking, lysosome biogenesis, lysosome acidification, signaling, and nutrient sensing were enriched in the lysosomal fraction. They were identified to an unprecedented extent as most, if not all, of their subunits were found and retained by our screen. Numerous transporters were also identified, including 46 novel potentially lysosomal proteins. We expressed 12 candidates in HeLa cells and observed that most of them colocalized with the lysosomal marker LAMP1, thus confirming their lysosomal residency. This list of candidate lysosomal proteins substantially increases our knowledge of the lysosomal membrane and provides a basis for further characterization of lysosomal functions. PMID:23436907

Spatially-Resolved Proteomics: Rapid Quantitative Analysis of Laser Capture Microdissected Alveolar Tissue Samples

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

Clair, Geremy; Piehowski, Paul D.; Nicola, Teodora

Global proteomics approaches allow characterization of whole tissue lysates to an impressive depth. However, it is now increasingly recognized that to better understand the complexity of multicellular organisms, global protein profiling of specific spatially defined regions/substructures of tissues (i.e. spatially-resolved proteomics) is essential. Laser capture microdissection (LCM) enables microscopic isolation of defined regions of tissues preserving crucial spatial information. However, current proteomics workflows entail several manual sample preparation steps and are challenged by the microscopic mass-limited samples generated by LCM, and that impact measurement robustness, quantification, and throughput. Here, we coupled LCM with a fully automated sample preparation workflow thatmore » with a single manual step allows: protein extraction, tryptic digestion, peptide cleanup and LC-MS/MS analysis of proteomes from microdissected tissues. Benchmarking against the current state of the art in ultrasensitive global proteomic analysis, our approach demonstrated significant improvements in quantification and throughput. Using our LCM-SNaPP proteomics approach, we characterized to a depth of more than 3,400 proteins, the ontogeny of protein changes during normal lung development in laser capture microdissected alveolar tissue containing ~4,000 cells per sample. Importantly, the data revealed quantitative changes for 350 low abundance transcription factors and signaling molecules, confirming earlier transcript-level observations and defining seven modules of coordinated transcription factor/signaling molecule expression patterns, suggesting that a complex network of temporal regulatory control directs normal lung development with epigenetic regulation fine-tuning pre-natal developmental processes. Our LCM-proteomics approach facilitates efficient, spatially-resolved, ultrasensitive global proteomics analyses in high-throughput that will be enabling for several clinical and biological applications.« less

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

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

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

Sialome of a Generalist Lepidopteran Herbivore: Identification of Transcripts and Proteins from Helicoverpa armigera Labial Salivary Glands

PubMed Central

Celorio-Mancera, Maria de la Paz; Courtiade, Juliette; Muck, Alexander; Heckel, David G.; Musser, Richard O.; Vogel, Heiko

2011-01-01

Although the importance of insect saliva in insect-host plant interactions has been acknowledged, there is very limited information on the nature and complexity of the salivary proteome in lepidopteran herbivores. We inspected the labial salivary transcriptome and proteome of Helicoverpa armigera, an important polyphagous pest species. To identify the majority of the salivary proteins we have randomly sequenced 19,389 expressed sequence tags (ESTs) from a normalized cDNA library of salivary glands. In parallel, a non-cytosolic enriched protein fraction was obtained from labial salivary glands and subjected to two-dimensional gel electrophoresis (2-DE) and de novo peptide sequencing. This procedure allowed comparison of peptides and EST sequences and enabled us to identify 65 protein spots from the secreted labial saliva 2DE proteome. The mass spectrometry analysis revealed ecdysone, glucose oxidase, fructosidase, carboxyl/cholinesterase and an uncharacterized protein previously detected in H. armigera midgut proteome. Consistently, their corresponding transcripts are among the most abundant in our cDNA library. We did find redundancy of sequence identification of saliva-secreted proteins suggesting multiple isoforms. As expected, we found several enzymes responsible for digestion and plant offense. In addition, we identified non-digestive proteins such as an arginine kinase and abundant proteins of unknown function. This identification of secreted salivary gland proteins allows a more comprehensive understanding of insect feeding and poses new challenges for the elucidation of protein function. PMID:22046331

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.

Advances of Proteomic Sciences in Dentistry

PubMed Central

Khurshid, Zohaib; Zohaib, Sana; Najeeb, Shariq; Zafar, Muhammad Sohail; Rehman, Rabia; Rehman, Ihtesham Ur

2016-01-01

Applications of proteomics tools revolutionized various biomedical disciplines such as genetics, molecular biology, medicine, and dentistry. The aim of this review is to highlight the major milestones in proteomics in dentistry during the last fifteen years. Human oral cavity contains hard and soft tissues and various biofluids including saliva and crevicular fluid. Proteomics has brought revolution in dentistry by helping in the early diagnosis of various diseases identified by the detection of numerous biomarkers present in the oral fluids. This paper covers the role of proteomics tools for the analysis of oral tissues. In addition, dental materials proteomics and their future directions are discussed. PMID:27187379

Recent advances in proteomics of cereals.

PubMed

Bansal, Monika; Sharma, Madhu; Kanwar, Priyanka; Goyal, Aakash

Cereals contribute a major part of human nutrition and are considered as an integral source of energy for human diets. With genomic databases already available in cereals such as rice, wheat, barley, and maize, the focus has now moved to proteome analysis. Proteomics studies involve the development of appropriate databases based on developing suitable separation and purification protocols, identification of protein functions, and can confirm their functional networks based on already available data from other sources. Tremendous progress has been made in the past decade in generating huge data-sets for covering interactions among proteins, protein composition of various organs and organelles, quantitative and qualitative analysis of proteins, and to characterize their modulation during plant development, biotic, and abiotic stresses. Proteomics platforms have been used to identify and improve our understanding of various metabolic pathways. This article gives a brief review of efforts made by different research groups on comparative descriptive and functional analysis of proteomics applications achieved in the cereal science so far.

SAFE Software and FED Database to Uncover Protein-Protein Interactions using Gene Fusion Analysis.

PubMed

Tsagrasoulis, Dimosthenis; Danos, Vasilis; Kissa, Maria; Trimpalis, Philip; Koumandou, V Lila; Karagouni, Amalia D; Tsakalidis, Athanasios; Kossida, Sophia

2012-01-01

Domain Fusion Analysis takes advantage of the fact that certain proteins in a given proteome A, are found to have statistically significant similarity with two separate proteins in another proteome B. In other words, the result of a fusion event between two separate proteins in proteome B is a specific full-length protein in proteome A. In such a case, it can be safely concluded that the protein pair has a common biological function or even interacts physically. In this paper, we present the Fusion Events Database (FED), a database for the maintenance and retrieval of fusion data both in prokaryotic and eukaryotic organisms and the Software for the Analysis of Fusion Events (SAFE), a computational platform implemented for the automated detection, filtering and visualization of fusion events (both available at: http://www.bioacademy.gr/bioinformatics/projects/ProteinFusion/index.htm). Finally, we analyze the proteomes of three microorganisms using these tools in order to demonstrate their functionality.

SAFE Software and FED Database to Uncover Protein-Protein Interactions using Gene Fusion Analysis

PubMed Central

Tsagrasoulis, Dimosthenis; Danos, Vasilis; Kissa, Maria; Trimpalis, Philip; Koumandou, V. Lila; Karagouni, Amalia D.; Tsakalidis, Athanasios; Kossida, Sophia

2012-01-01

Domain Fusion Analysis takes advantage of the fact that certain proteins in a given proteome A, are found to have statistically significant similarity with two separate proteins in another proteome B. In other words, the result of a fusion event between two separate proteins in proteome B is a specific full-length protein in proteome A. In such a case, it can be safely concluded that the protein pair has a common biological function or even interacts physically. In this paper, we present the Fusion Events Database (FED), a database for the maintenance and retrieval of fusion data both in prokaryotic and eukaryotic organisms and the Software for the Analysis of Fusion Events (SAFE), a computational platform implemented for the automated detection, filtering and visualization of fusion events (both available at: http://www.bioacademy.gr/bioinformatics/projects/ProteinFusion/index.htm). Finally, we analyze the proteomes of three microorganisms using these tools in order to demonstrate their functionality. PMID:22267904

Connecting genomic alterations to cancer biology with proteomics: the NCI Clinical Proteomic Tumor Analysis Consortium.

PubMed

Ellis, Matthew J; Gillette, Michael; Carr, Steven A; Paulovich, Amanda G; Smith, Richard D; Rodland, Karin K; Townsend, R Reid; Kinsinger, Christopher; Mesri, Mehdi; Rodriguez, Henry; Liebler, Daniel C

2013-10-01

The National Cancer Institute (NCI) Clinical Proteomic Tumor Analysis Consortium is applying the latest generation of proteomic technologies to genomically annotated tumors from The Cancer Genome Atlas (TCGA) program, a joint initiative of the NCI and the National Human Genome Research Institute. By providing a fully integrated accounting of DNA, RNA, and protein abnormalities in individual tumors, these datasets will illuminate the complex relationship between genomic abnormalities and cancer phenotypes, thus producing biologic insights as well as a wave of novel candidate biomarkers and therapeutic targets amenable to verification using targeted mass spectrometry methods. ©2013 AACR.

Advances in Proteomics Data Analysis and Display Using an Accurate Mass and Time Tag Approach

PubMed Central

Zimmer, Jennifer S.D.; Monroe, Matthew E.; Qian, Wei-Jun; Smith, Richard D.

2007-01-01

Proteomics has recently demonstrated utility in understanding cellular processes on the molecular level as a component of systems biology approaches and for identifying potential biomarkers of various disease states. The large amount of data generated by utilizing high efficiency (e.g., chromatographic) separations coupled to high mass accuracy mass spectrometry for high-throughput proteomics analyses presents challenges related to data processing, analysis, and display. This review focuses on recent advances in nanoLC-FTICR-MS-based proteomics approaches and the accompanying data processing tools that have been developed to display and interpret the large volumes of data being produced. PMID:16429408

Rice proteome analysis: a step toward functional analysis of the rice genome.

PubMed

Komatsu, Setsuko; Tanaka, Naoki

2005-03-01

The technique of proteome analysis using 2-DE has the power to monitor global changes that occur in the protein complement of tissues and subcellular compartments. In this review, we describe construction of the rice proteome database, the cataloging of rice proteins, and the functional characterization of some of the proteins identified. Initially, proteins extracted from various tissues and organelles were separated by 2-DE and an image analyzer was used to construct a display or reference map of the proteins. The rice proteome database currently contains 23 reference maps based on 2-DE of proteins from different rice tissues and subcellular compartments. These reference maps comprise 13 129 rice proteins, and the amino acid sequences of 5092 of these proteins are entered in the database. Major proteins involved in growth or stress responses have been identified by using a proteomics approach and some of these proteins have unique functions. Furthermore, initial work has also begun on analyzing the phosphoproteome and protein-protein interactions in rice. The information obtained from the rice proteome database will aid in the molecular cloning of rice genes and in predicting the function of unknown proteins.

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

Quantitative Proteomic Analysis of Wheat Seeds during Artificial Ageing and Priming Using the Isobaric Tandem Mass Tag Labeling

PubMed Central

Lv, Yangyong; Zhang, Shuaibing; Wang, Jinshui; Hu, Yuansen

2016-01-01

Wheat (Triticum aestivum L.) is an important crop worldwide. The physiological deterioration of seeds during storage and seed priming is closely associated with germination, and thus contributes to plant growth and subsequent grain yields. In this study, wheat seeds during different stages of artificial ageing (45°C; 50% relative humidity; 98%, 50%, 20%, and 1% Germination rates) and priming (hydro-priming treatment) were subjected to proteomics analysis through a proteomic approach based on the isobaric tandem mass tag labeling. A total of 162 differentially expressed proteins (DEPs) mainly involved in metabolism, energy supply, and defense/stress responses, were identified during artificial ageing and thus validated previous physiological and biochemical studies. These DEPs indicated that the inability to protect against ageing leads to the incremental decomposition of the stored substance, impairment of metabolism and energy supply, and ultimately resulted in seed deterioration. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the up-regulated proteins involved in seed ageing were mainly enriched in ribosome, whereas the down-regulated proteins were mainly accumulated in energy supply (starch and sucrose metabolism) and stress defense (ascorbate and aldarate metabolism). Proteins, including hemoglobin 1, oleosin, agglutinin, and non-specific lipid-transfer proteins, were first identified in aged seeds and might be regarded as new markers of seed deterioration. Of the identified proteins, 531 DEPs were recognized during seed priming compared with unprimed seeds. In contrast to the up-regulated DEPs in seed ageing, several up-regulated DEPs in priming were involved in energy supply (tricarboxylic acid cycle, glycolysis, and fatty acid oxidation), anabolism (amino acids, and fatty acid synthesis), and cell growth/division. KEGG and protein-protein interaction analysis indicated that the up-regulated proteins in seed priming were mainly enriched in amino acid synthesis, stress defense (plant-pathogen interactions, and ascorbate and aldarate metabolism), and energy supply (oxidative phosphorylation and carbon metabolism). Therefore, DEPs associated with seed ageing and priming can be used to characterize seed vigor and optimize germination enhancement treatments. This work reveals new proteomic insights into protein changes that occur during seed deterioration and priming. PMID:27632285

Differential proteomics analysis to identify proteins and pathways associated with male sterility of soybean using iTRAQ-based strategy.

PubMed

Li, Jiajia; Ding, Xianlong; Han, Shaohuai; He, Tingting; Zhang, Hao; Yang, Longshu; Yang, Shouping; Gai, Junyi

2016-04-14

To further elucidate the molecular mechanism of cytoplasmic male sterility (CMS) in soybean, a differential proteomic analysis was completed between the CMS line NJCMS1A and its maintainer NJCMS1B using iTRAQ-based strategy. As a result, 180 differential abundance proteins (DAPs) were identified, of which, 60 were down-regulated and 120 were up-regulated in NJCMS1A compared with NJCMS1B. Bioinformatic analysis showed that 167 DAPs were annotated in 41 Gene Ontology functional groups, 106 DAPs were classified into 20 clusters of orthologous groups of protein categories, and 128 DAPs were enrichment in 53 KEGG pathways. Fifteen differential level proteins/genes with the same expression pattern were identified in the further conjoint analysis of DAPs and the previously reported differential expression genes. Moreover, multiple reaction monitoring test, qRT-PCR analysis and enzyme activity assay validated that the iTRAQ results were reliable. Based on functional analysis of DAPs, we concluded that male sterility in NJCMS1A might be related to insufficiencies in energy supply, unbalance of protein synthesis and degradation, disruption of flavonoid synthesis, programmed cell death, abnormalities of substance metabolism, etc. These results might facilitate our understanding of the molecular mechanisms behind CMS in soybean. Soybean is an important global crop that provides protein and oil. Heterosis is a significantly potential approach to increase the yield of soybean. Cytoplasmic male sterility (CMS) plays a vital role in the production of hybrid seeds. However, the genetic and molecular mechanisms of male sterility in soybean still need to be further elucidated. In the present paper, a differential proteomic analysis was carried out and the results showed that several key proteins involved in key pathways were associated with male sterility in soybean. This work provides a new insight to understand the genetic and molecular mechanisms underlying CMS in soybean. Copyright © 2016 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

Automated image alignment for 2D gel electrophoresis in a high-throughput proteomics pipeline.

PubMed

Dowsey, Andrew W; Dunn, Michael J; Yang, Guang-Zhong

2008-04-01

The quest for high-throughput proteomics has revealed a number of challenges in recent years. Whilst substantial improvements in automated protein separation with liquid chromatography and mass spectrometry (LC/MS), aka 'shotgun' proteomics, have been achieved, large-scale open initiatives such as the Human Proteome Organization (HUPO) Brain Proteome Project have shown that maximal proteome coverage is only possible when LC/MS is complemented by 2D gel electrophoresis (2-DE) studies. Moreover, both separation methods require automated alignment and differential analysis to relieve the bioinformatics bottleneck and so make high-throughput protein biomarker discovery a reality. The purpose of this article is to describe a fully automatic image alignment framework for the integration of 2-DE into a high-throughput differential expression proteomics pipeline. The proposed method is based on robust automated image normalization (RAIN) to circumvent the drawbacks of traditional approaches. These use symbolic representation at the very early stages of the analysis, which introduces persistent errors due to inaccuracies in modelling and alignment. In RAIN, a third-order volume-invariant B-spline model is incorporated into a multi-resolution schema to correct for geometric and expression inhomogeneity at multiple scales. The normalized images can then be compared directly in the image domain for quantitative differential analysis. Through evaluation against an existing state-of-the-art method on real and synthetically warped 2D gels, the proposed analysis framework demonstrates substantial improvements in matching accuracy and differential sensitivity. High-throughput analysis is established through an accelerated GPGPU (general purpose computation on graphics cards) implementation. Supplementary material, software and images used in the validation are available at http://www.proteomegrid.org/rain/.

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.

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.

Overlap of proteomics biomarkers between women with pre-eclampsia and PCOS: a systematic review and biomarker database integration

PubMed Central

Khan, Gulafshana Hafeez; Galazis, Nicolas; Docheva, Nikolina; Layfield, Robert; Atiomo, William

2015-01-01

STUDY QUESTION Do any proteomic biomarkers previously identified for pre-eclampsia (PE) overlap with those identified in women with polycystic ovary syndrome (PCOS). SUMMARY ANSWER Five previously identified proteomic biomarkers were found to be common in women with PE and PCOS when compared with controls. WHAT IS KNOWN ALREADY Various studies have indicated an association between PCOS and PE; however, the pathophysiological mechanisms supporting this association are not known. STUDY DESIGN, SIZE, DURATION A systematic review and update of our PCOS proteomic biomarker database was performed, along with a parallel review of PE biomarkers. The study included papers from 1980 to December 2013. PARTICIPANTS/MATERIALS, SETTING, METHODS In all the studies analysed, there were a total of 1423 patients and controls. The number of proteomic biomarkers that were catalogued for PE was 192. MAIN RESULTS AND THE ROLE OF CHANCE Five proteomic biomarkers were shown to be differentially expressed in women with PE and PCOS when compared with controls: transferrin, fibrinogen α, β and γ chain variants, kininogen-1, annexin 2 and peroxiredoxin 2. In PE, the biomarkers were identified in serum, plasma and placenta and in PCOS, the biomarkers were identified in serum, follicular fluid, and ovarian and omental biopsies. LIMITATIONS, REASONS FOR CAUTION The techniques employed to detect proteomics have limited ability in identifying proteins that are of low abundance, some of which may have a diagnostic potential. The sample sizes and number of biomarkers identified from these studies do not exclude the risk of false positives, a limitation of all biomarker studies. The biomarkers common to PE and PCOS were identified from proteomic analyses of different tissues. WIDER IMPLICATIONS OF THE FINDINGS This data amalgamation of the proteomic studies in PE and in PCOS, for the first time, discovered a panel of five biomarkers for PE which are common to women with PCOS, including transferrin, fibrinogen α, β and γ chain variants, kininogen-1, annexin 2 and peroxiredoxin 2. If validated, these biomarkers could provide a useful framework for the knowledge infrastructure in this area. To accomplish this goal, a well co-ordinated multidisciplinary collaboration of clinicians, basic scientists and mathematicians is vital. STUDY FUNDING/COMPETING INTEREST(S) No financial support was obtained for this project. There are no conflicts of interest. PMID:25351721

Recent advances on multidimensional liquid chromatography-mass spectrometry for proteomics: from qualitative to quantitative analysis--a review.

PubMed

Wu, Qi; Yuan, Huiming; Zhang, Lihua; Zhang, Yukui

2012-06-20

With the acceleration of proteome research, increasing attention has been paid to multidimensional liquid chromatography-mass spectrometry (MDLC-MS) due to its high peak capacity and separation efficiency. Recently, many efforts have been put to improve MDLC-based strategies including "top-down" and "bottom-up" to enable highly sensitive qualitative and quantitative analysis of proteins, as well as accelerate the whole analytical procedure. Integrated platforms with combination of sample pretreatment, multidimensional separations and identification were also developed to achieve high throughput and sensitive detection of proteomes, facilitating highly accurate and reproducible quantification. This review summarized the recent advances of such techniques and their applications in qualitative and quantitative analysis of proteomes. Copyright © 2012 Elsevier B.V. All rights reserved.

Proteomic analysis of tissue samples in translational breast cancer research.

PubMed

Gromov, Pavel; Moreira, José M A; Gromova, Irina

2014-06-01

In the last decade, many proteomic technologies have been applied, with varying success, to the study of tissue samples of breast carcinoma for protein expression profiling in order to discover protein biomarkers/signatures suitable for: characterization and subtyping of tumors; early diagnosis, and both prognosis and prediction of outcome of chemotherapy. The purpose of this review is to critically appraise what has been achieved to date using proteomic technologies and to bring forward novel strategies - based on the analysis of clinically relevant samples - that promise to accelerate the translation of basic discoveries into the daily breast cancer clinical practice. In particular, we address major issues in experimental design by reviewing the strengths and weaknesses of current proteomic strategies in the context of the analysis of human breast tissue specimens.

From proteomics to systems biology: MAPA, MASS WESTERN, PROMEX, and COVAIN as a user-oriented platform.

PubMed

Weckwerth, Wolfram; Wienkoop, Stefanie; Hoehenwarter, Wolfgang; Egelhofer, Volker; Sun, Xiaoliang

2014-01-01

Genome sequencing and systems biology are revolutionizing life sciences. Proteomics emerged as a fundamental technique of this novel research area as it is the basis for gene function analysis and modeling of dynamic protein networks. Here a complete proteomics platform suited for functional genomics and systems biology is presented. The strategy includes MAPA (mass accuracy precursor alignment; http://www.univie.ac.at/mosys/software.html ) as a rapid exploratory analysis step; MASS WESTERN for targeted proteomics; COVAIN ( http://www.univie.ac.at/mosys/software.html ) for multivariate statistical analysis, data integration, and data mining; and PROMEX ( http://www.univie.ac.at/mosys/databases.html ) as a database module for proteogenomics and proteotypic peptides for targeted analysis. Moreover, the presented platform can also be utilized to integrate metabolomics and transcriptomics data for the analysis of metabolite-protein-transcript correlations and time course analysis using COVAIN. Examples for the integration of MAPA and MASS WESTERN data, proteogenomic and metabolic modeling approaches for functional genomics, phosphoproteomics by integration of MOAC (metal-oxide affinity chromatography) with MAPA, and the integration of metabolomics, transcriptomics, proteomics, and physiological data using this platform are presented. All software and step-by-step tutorials for data processing and data mining can be downloaded from http://www.univie.ac.at/mosys/software.html.

QIL1 is a novel mitochondrial protein required for MICOS complex stability and cristae morphology.

PubMed

Guarani, Virginia; McNeill, Elizabeth M; Paulo, Joao A; Huttlin, Edward L; Fröhlich, Florian; Gygi, Steven P; Van Vactor, David; Harper, J Wade

2015-05-21

The mitochondrial contact site and cristae junction (CJ) organizing system (MICOS) dynamically regulate mitochondrial membrane architecture. Through systematic proteomic analysis of human MICOS, we identified QIL1 (C19orf70) as a novel conserved MICOS subunit. QIL1 depletion disrupted CJ structure in cultured human cells and in Drosophila muscle and neuronal cells in vivo. In human cells, mitochondrial disruption correlated with impaired respiration. Moreover, increased mitochondrial fragmentation was observed upon QIL1 depletion in flies. Using quantitative proteomics, we show that loss of QIL1 resulted in MICOS disassembly with the accumulation of a MIC60-MIC19-MIC25 sub-complex and degradation of MIC10, MIC26, and MIC27. Additionally, we demonstrated that in QIL1-depleted cells, overexpressed MIC10 fails to significantly restore its interaction with other MICOS subunits and SAMM50. Collectively, our work uncovers a previously unrecognized subunit of the MICOS complex, necessary for CJ integrity, cristae morphology, and mitochondrial function and provides a resource for further analysis of MICOS architecture.

QIL1 is a novel mitochondrial protein required for MICOS complex stability and cristae morphology

PubMed Central

Guarani, Virginia; McNeill, Elizabeth M; Paulo, Joao A; Huttlin, Edward L; Fröhlich, Florian; Gygi, Steven P; Van Vactor, David; Harper, J Wade

2015-01-01

The mitochondrial contact site and cristae junction (CJ) organizing system (MICOS) dynamically regulate mitochondrial membrane architecture. Through systematic proteomic analysis of human MICOS, we identified QIL1 (C19orf70) as a novel conserved MICOS subunit. QIL1 depletion disrupted CJ structure in cultured human cells and in Drosophila muscle and neuronal cells in vivo. In human cells, mitochondrial disruption correlated with impaired respiration. Moreover, increased mitochondrial fragmentation was observed upon QIL1 depletion in flies. Using quantitative proteomics, we show that loss of QIL1 resulted in MICOS disassembly with the accumulation of a MIC60-MIC19-MIC25 sub-complex and degradation of MIC10, MIC26, and MIC27. Additionally, we demonstrated that in QIL1-depleted cells, overexpressed MIC10 fails to significantly restore its interaction with other MICOS subunits and SAMM50. Collectively, our work uncovers a previously unrecognized subunit of the MICOS complex, necessary for CJ integrity, cristae morphology, and mitochondrial function and provides a resource for further analysis of MICOS architecture. DOI: http://dx.doi.org/10.7554/eLife.06265.001 PMID:25997101

Identifying technical aliases in SELDI mass spectra of complex mixtures of proteins

PubMed Central

2013-01-01

Background Biomarker discovery datasets created using mass spectrum protein profiling of complex mixtures of proteins contain many peaks that represent the same protein with different charge states. Correlated variables such as these can confound the statistical analyses of proteomic data. Previously we developed an algorithm that clustered mass spectrum peaks that were biologically or technically correlated. Here we demonstrate an algorithm that clusters correlated technical aliases only. Results In this paper, we propose a preprocessing algorithm that can be used for grouping technical aliases in mass spectrometry protein profiling data. The stringency of the variance allowed for clustering is customizable, thereby affecting the number of peaks that are clustered. Subsequent analysis of the clusters, instead of individual peaks, helps reduce difficulties associated with technically-correlated data, and can aid more efficient biomarker identification. Conclusions This software can be used to pre-process and thereby decrease the complexity of protein profiling proteomics data, thus simplifying the subsequent analysis of biomarkers by decreasing the number of tests. The software is also a practical tool for identifying which features to investigate further by purification, identification and confirmation. PMID:24010718

Advancing Cell Biology Through Proteomics in Space and Time (PROSPECTS)*

PubMed Central

Lamond, Angus I.; Uhlen, Mathias; Horning, Stevan; Makarov, Alexander; Robinson, Carol V.; Serrano, Luis; Hartl, F. Ulrich; Baumeister, Wolfgang; Werenskiold, Anne Katrin; Andersen, Jens S.; Vorm, Ole; Linial, Michal; Aebersold, Ruedi; Mann, Matthias

2012-01-01

The term “proteomics” encompasses the large-scale detection and analysis of proteins and their post-translational modifications. Driven by major improvements in mass spectrometric instrumentation, methodology, and data analysis, the proteomics field has burgeoned in recent years. It now provides a range of sensitive and quantitative approaches for measuring protein structures and dynamics that promise to revolutionize our understanding of cell biology and molecular mechanisms in both human cells and model organisms. The Proteomics Specification in Time and Space (PROSPECTS) Network is a unique EU-funded project that brings together leading European research groups, spanning from instrumentation to biomedicine, in a collaborative five year initiative to develop new methods and applications for the functional analysis of cellular proteins. This special issue of Molecular and Cellular Proteomics presents 16 research papers reporting major recent progress by the PROSPECTS groups, including improvements to the resolution and sensitivity of the Orbitrap family of mass spectrometers, systematic detection of proteins using highly characterized antibody collections, and new methods for absolute as well as relative quantification of protein levels. Manuscripts in this issue exemplify approaches for performing quantitative measurements of cell proteomes and for studying their dynamic responses to perturbation, both during normal cellular responses and in disease mechanisms. Here we present a perspective on how the proteomics field is moving beyond simply identifying proteins with high sensitivity toward providing a powerful and versatile set of assay systems for characterizing proteome dynamics and thereby creating a new “third generation” proteomics strategy that offers an indispensible tool for cell biology and molecular medicine. PMID:22311636

Comparative analysis of inflamed and non-inflamed colon biopsies reveals strong proteomic inflammation profile in patients with ulcerative colitis

PubMed Central

2012-01-01

Background Accurate diagnostic and monitoring tools for ulcerative colitis (UC) are missing. Our aim was to describe the proteomic profile of UC and search for markers associated with disease exacerbation. Therefore, we aimed to characterize specific proteins associated with inflamed colon mucosa from patients with acute UC using mass spectrometry-based proteomic analysis. Methods Biopsies were sampled from rectum, sigmoid colon and left colonic flexure from twenty patients with active proctosigmoiditis and from four healthy controls for proteomics and histology. Proteomic profiles of whole colonic biopsies were characterized using 2D-gel electrophoresis, and peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was applied for identification of differently expressed protein spots. Results A total of 597 spots were annotated by image analysis and 222 of these had a statistically different protein level between inflamed and non-inflamed tissue in the patient group. Principal component analysis clearly grouped non-inflamed samples separately from the inflamed samples indicating that the proteomic signature of colon mucosa with acute UC is strong. Totally, 43 individual protein spots were identified, including proteins involved in energy metabolism (triosephosphate isomerase, glycerol-3-phosphate-dehydrogenase, alpha enolase and L-lactate dehydrogenase B-chain) and in oxidative stress (superoxide dismutase, thioredoxins and selenium binding protein). Conclusions A distinct proteomic profile of inflamed tissue in UC patients was found. Specific proteins involved in energy metabolism and oxidative stress were identified as potential candidate markers for UC. PMID:22726388

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.

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.

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.

Low-molecular-weight color pI markers to monitor on-line the peptide focusing process in OFFGEL fractionation.

PubMed

Michelland, Sylvie; Bourgoin-Voillard, Sandrine; Cunin, Valérie; Tollance, Axel; Bertolino, Pascal; Slais, Karel; Seve, Michel

2017-08-01

High-throughput mass spectrometry-based proteomic analysis requires peptide fractionation to simplify complex biological samples and increase proteome coverage. OFFGEL fractionation technology became a common method to separate peptides or proteins using isoelectric focusing in an immobilized pH gradient. However, the OFFGEL focusing process may be further optimized and controlled in terms of separation time and pI resolution. Here we evaluated OFFGEL technology to separate peptides from different samples in the presence of low-molecular-weight (LMW) color pI markers to visualize the focusing process. LMW color pI markers covering a large pH range were added to the peptide mixture before OFFGEL fractionation using a 24-wells device encompassing the pH range 3-10. We also explored the impact of LMW color pI markers on peptide fractionation labeled previously for iTRAQ. Then, fractionated peptides were separated by RP_HPLC prior to MS analysis using MALDI-TOF/TOF mass spectrometry in MS and MS/MS modes. Here we report the performance of the peptide focusing process in the presence of LMW color pI markers as on-line trackers during the OFFGEL process and the possibility to use them as pI controls for peptide focusing. This method improves the workflow for peptide fractionation in a bottom-up proteomic approach with or without iTRAQ labeling. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Investigation of Pokemon-regulated proteins in hepatocellular carcinoma using mass spectrometry-based multiplex quantitative proteomics.

PubMed

Bi, Xin; Jin, Yibao; Gao, Xiang; Liu, Feng; Gao, Dan; Jiang, Yuyang; Liu, Hongxia

2013-01-01

Pokemon is a transcription regulator involved in embryonic development, cellular differentiation and oncogenesis. It is aberrantly overexpressed in multiple human cancers including Hepatocellular carcinoma (HCC) and is considered as a promising biomarker for HCC. In this work, the isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics strategy was used to investigate the proteomic profile associated with Pokemon in human HCC cell line QGY7703 and human hepatocyte line HL7702. Samples were labeled with four-plex iTRAQ reagents followed by two-dimensional liquid chromatography coupled with tandem mass spectrometry analysis. A total of 24 differentially expressed proteins were selected as significant. Nine proteins were potentially up-regulated by Pokemon while 15 proteins were potentially down-regulated and many proteins were previously identified as potential biomarkers for HCC. Gene ontology (GO) term enrichment revealed that the listed proteins were mainly involved in DNA metabolism and biosynthesis process. The changes of glucose-6-phosphate 1-dehydrogenase (G6PD, up-regulated) and ribonucleoside-diphosphate reductase large sub-unit (RIM1, down-regulated) were validated by Western blotting analysis and denoted as Pokemon's function of oncogenesis. We also found that Pokemon potentially repressed the expression of highly clustered proteins (MCM3, MCM5, MCM6, MCM7) which played key roles in promoting DNA replication. Altogether, our results may help better understand the role of Pokemon in HCC and promote the clinical applications.

Physiological and proteomic analyses of Fe(III)-reducing co-cultures of Desulfotomaculum reducens MI-1 and Geobacter sulfurreducens PCA.

PubMed

Otwell, Anne E; Callister, Stephen J; Sherwood, Robert W; Zhang, Sheng; Goldman, Abby R; Smith, Richard D; Richardson, Ruth E

2018-06-15

We established Fe(III)-reducing co-cultures of two species of metal-reducing bacteria, the Gram-positive Desulfotomaculum reducens MI-1 and the Gram-negative Geobacter sulfurreducens PCA. Co-cultures were given pyruvate, a substrate that D. reducens can ferment and use as electron donor for Fe(III) reduction. G. sulfurreducens relied upon products of pyruvate oxidation by D. reducens (acetate, hydrogen) for use as electron donor in the co-culture. Co-cultures reduced Fe(III) to Fe(II) robustly, and Fe(II) was consistently detected earlier in co-cultures than pure cultures. Notably, faster cell growth, and correspondingly faster pyruvate oxidation, was observed by D. reducens in co-cultures. Global comparative proteomic analysis was performed to observe differential protein abundance during co-culture vs. pure culture growth. Proteins previously associated with Fe(III) reduction in G. sulfurreducens, namely c-type cytochromes and type IV pili proteins, were significantly increased in abundance in co-cultures relative to pure cultures. D. reducens ribosomal proteins were significantly increased in co-cultures, likely a reflection of faster growth rates observed for D. reducens cells while in co-culture. Furthermore, we developed multiple reaction monitoring (MRM) assays to quantitate specific biomarker peptides. The assays were validated in pure and co-cultures, and protein abundance ratios from targeted MRM and global proteomic analysis correlate significantly. © 2018 John Wiley & Sons Ltd.

Proteomic profiling and pathway analysis of the response of rat renal proximal convoluted tubules to metabolic acidosis

PubMed Central

Schauer, Kevin L.; Freund, Dana M.; Prenni, Jessica E.

2013-01-01

Metabolic acidosis is a relatively common pathological condition that is defined as a decrease in blood pH and bicarbonate concentration. The renal proximal convoluted tubule responds to this condition by increasing the extraction of plasma glutamine and activating ammoniagenesis and gluconeogenesis. The combined processes increase the excretion of acid and produce bicarbonate ions that are added to the blood to partially restore acid-base homeostasis. Only a few cytosolic proteins, such as phosphoenolpyruvate carboxykinase, have been determined to play a role in the renal response to metabolic acidosis. Therefore, further analysis was performed to better characterize the response of the cytosolic proteome. Proximal convoluted tubule cells were isolated from rat kidney cortex at various times after onset of acidosis and fractionated to separate the soluble cytosolic proteins from the remainder of the cellular components. The cytosolic proteins were analyzed using two-dimensional liquid chromatography and tandem mass spectrometry (MS/MS). Spectral counting along with average MS/MS total ion current were used to quantify temporal changes in relative protein abundance. In all, 461 proteins were confidently identified, of which 24 exhibited statistically significant changes in abundance. To validate these techniques, several of the observed abundance changes were confirmed by Western blotting. Data from the cytosolic fractions were then combined with previous proteomic data, and pathway analyses were performed to identify the primary pathways that are activated or inhibited in the proximal convoluted tubule during the onset of metabolic acidosis. PMID:23804448

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.

Combining proteomics and metabolite analyses to unravel cadmium stress-response in poplar leaves.

PubMed

Kieffer, Pol; Planchon, Sébastien; Oufir, Mouhssin; Ziebel, Johanna; Dommes, Jacques; Hoffmann, Lucien; Hausman, Jean-François; Renaut, Jenny

2009-01-01

A proteomic analysis of poplar leaves exposed to cadmium, combined with biochemical analysis of pigments and carbohydrates revealed changes in primary carbon metabolism. Proteomic results suggested that photosynthesis was slightly affected. Together with a growth inhibition, photoassimilates were less needed for developmental processes and could be stored in the form of hexoses or complex sugars, acting also as osmoprotectants. Simultaneously, mitochondrial respiration was upregulated, providing energy needs of cadmium-exposed plants.

Understanding the Mechanism of Thermotolerance Distinct From Heat Shock Response Through Proteomic Analysis of Industrial Strains of Saccharomyces cerevisiae*

PubMed Central

Shui, Wenqing; Xiong, Yun; Xiao, Weidi; Qi, Xianni; Zhang, Yong; Lin, Yuping; Guo, Yufeng; Zhang, Zhidan; Wang, Qinhong; Ma, Yanhe

2015-01-01

Saccharomyces cerevisiae has been intensively studied in responses to different environmental stresses such as heat shock through global omic analysis. However, the S. cerevisiae industrial strains with superior thermotolerance have not been explored in any proteomic studies for elucidating the tolerance mechanism. Recently a new diploid strain was obtained through evolutionary engineering of a parental industrial strain, and it exhibited even higher resistance to prolonged thermal stress. Herein, we performed iTRAQ-based quantitative proteomic analysis on both the parental and evolved industrial strains to further understand the mechanism of thermotolerant adaptation. Out of ∼2600 quantifiable proteins from biological quadruplicates, 193 and 204 proteins were differentially regulated in the parental and evolved strains respectively during heat-stressed growth. The proteomic response of the industrial strains cultivated under prolonged thermal stress turned out to be substantially different from that of the laboratory strain exposed to sudden heat shock. Further analysis of transcription factors underlying the proteomic perturbation also indicated the distinct regulatory mechanism of thermotolerance. Finally, a cochaperone Mdj1 and a metabolic enzyme Adh1 were selected to investigate their roles in mediating heat-stressed growth and ethanol production of yeasts. Our proteomic characterization of the industrial strain led to comprehensive understanding of the molecular basis of thermotolerance, which would facilitate future improvement in the industrially important trait of S. cerevisiae by rational engineering. PMID:25926660

A Comprehensive Guide for Performing Sample Preparation and Top-Down Protein Analysis

PubMed Central

Padula, Matthew P.; Berry, Iain J.; O′Rourke, Matthew B.; Raymond, Benjamin B.A.; Santos, Jerran; Djordjevic, Steven P.

2017-01-01

Methodologies for the global analysis of proteins in a sample, or proteome analysis, have been available since 1975 when Patrick O′Farrell published the first paper describing two-dimensional gel electrophoresis (2D-PAGE). This technique allowed the resolution of single protein isoforms, or proteoforms, into single ‘spots’ in a polyacrylamide gel, allowing the quantitation of changes in a proteoform′s abundance to ascertain changes in an organism′s phenotype when conditions change. In pursuit of the comprehensive profiling of the proteome, significant advances in technology have made the identification and quantitation of intact proteoforms from complex mixtures of proteins more routine, allowing analysis of the proteome from the ‘Top-Down’. However, the number of proteoforms detected by Top-Down methodologies such as 2D-PAGE or mass spectrometry has not significantly increased since O’Farrell’s paper when compared to Bottom-Up, peptide-centric techniques. This article explores and explains the numerous methodologies and technologies available to analyse the proteome from the Top-Down with a strong emphasis on the necessity to analyse intact proteoforms as a better indicator of changes in biology and phenotype. We arrive at the conclusion that the complete and comprehensive profiling of an organism′s proteome is still, at present, beyond our reach but the continuing evolution of protein fractionation techniques and mass spectrometry brings comprehensive Top-Down proteome profiling closer. PMID:28387712

A Comprehensive Guide for Performing Sample Preparation and Top-Down Protein Analysis.

PubMed

Padula, Matthew P; Berry, Iain J; O Rourke, Matthew B; Raymond, Benjamin B A; Santos, Jerran; Djordjevic, Steven P

2017-04-07

Methodologies for the global analysis of proteins in a sample, or proteome analysis, have been available since 1975 when Patrick O'Farrell published the first paper describing two-dimensional gel electrophoresis (2D-PAGE). This technique allowed the resolution of single protein isoforms, or proteoforms, into single 'spots' in a polyacrylamide gel, allowing the quantitation of changes in a proteoform's abundance to ascertain changes in an organism's phenotype when conditions change. In pursuit of the comprehensive profiling of the proteome, significant advances in technology have made the identification and quantitation of intact proteoforms from complex mixtures of proteins more routine, allowing analysis of the proteome from the 'Top-Down'. However, the number of proteoforms detected by Top-Down methodologies such as 2D-PAGE or mass spectrometry has not significantly increased since O'Farrell's paper when compared to Bottom-Up, peptide-centric techniques. This article explores and explains the numerous methodologies and technologies available to analyse the proteome from the Top-Down with a strong emphasis on the necessity to analyse intact proteoforms as a better indicator of changes in biology and phenotype. We arrive at the conclusion that the complete and comprehensive profiling of an organism's proteome is still, at present, beyond our reach but the continuing evolution of protein fractionation techniques and mass spectrometry brings comprehensive Top-Down proteome profiling closer.

Processing Shotgun Proteomics Data on the Amazon Cloud with the Trans-Proteomic Pipeline*

PubMed Central

Slagel, Joseph; Mendoza, Luis; Shteynberg, David; Deutsch, Eric W.; Moritz, Robert L.

2015-01-01

Cloud computing, where scalable, on-demand compute cycles and storage are available as a service, has the potential to accelerate mass spectrometry-based proteomics research by providing simple, expandable, and affordable large-scale computing to all laboratories regardless of location or information technology expertise. We present new cloud computing functionality for the Trans-Proteomic Pipeline, a free and open-source suite of tools for the processing and analysis of tandem mass spectrometry datasets. Enabled with Amazon Web Services cloud computing, the Trans-Proteomic Pipeline now accesses large scale computing resources, limited only by the available Amazon Web Services infrastructure, for all users. The Trans-Proteomic Pipeline runs in an environment fully hosted on Amazon Web Services, where all software and data reside on cloud resources to tackle large search studies. In addition, it can also be run on a local computer with computationally intensive tasks launched onto the Amazon Elastic Compute Cloud service to greatly decrease analysis times. We describe the new Trans-Proteomic Pipeline cloud service components, compare the relative performance and costs of various Elastic Compute Cloud service instance types, and present on-line tutorials that enable users to learn how to deploy cloud computing technology rapidly with the Trans-Proteomic Pipeline. We provide tools for estimating the necessary computing resources and costs given the scale of a job and demonstrate the use of cloud enabled Trans-Proteomic Pipeline by performing over 1100 tandem mass spectrometry files through four proteomic search engines in 9 h and at a very low cost. PMID:25418363

Processing shotgun proteomics data on the Amazon cloud with the trans-proteomic pipeline.

PubMed

Slagel, Joseph; Mendoza, Luis; Shteynberg, David; Deutsch, Eric W; Moritz, Robert L

2015-02-01

Cloud computing, where scalable, on-demand compute cycles and storage are available as a service, has the potential to accelerate mass spectrometry-based proteomics research by providing simple, expandable, and affordable large-scale computing to all laboratories regardless of location or information technology expertise. We present new cloud computing functionality for the Trans-Proteomic Pipeline, a free and open-source suite of tools for the processing and analysis of tandem mass spectrometry datasets. Enabled with Amazon Web Services cloud computing, the Trans-Proteomic Pipeline now accesses large scale computing resources, limited only by the available Amazon Web Services infrastructure, for all users. The Trans-Proteomic Pipeline runs in an environment fully hosted on Amazon Web Services, where all software and data reside on cloud resources to tackle large search studies. In addition, it can also be run on a local computer with computationally intensive tasks launched onto the Amazon Elastic Compute Cloud service to greatly decrease analysis times. We describe the new Trans-Proteomic Pipeline cloud service components, compare the relative performance and costs of various Elastic Compute Cloud service instance types, and present on-line tutorials that enable users to learn how to deploy cloud computing technology rapidly with the Trans-Proteomic Pipeline. We provide tools for estimating the necessary computing resources and costs given the scale of a job and demonstrate the use of cloud enabled Trans-Proteomic Pipeline by performing over 1100 tandem mass spectrometry files through four proteomic search engines in 9 h and at a very low cost. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Translational plant proteomics: a perspective.

PubMed

Agrawal, Ganesh Kumar; Pedreschi, Romina; Barkla, Bronwyn J; Bindschedler, Laurence Veronique; Cramer, Rainer; Sarkar, Abhijit; Renaut, Jenny; Job, Dominique; Rakwal, Randeep

2012-08-03

Translational proteomics is an emerging sub-discipline of the proteomics field in the biological sciences. Translational plant proteomics aims to integrate knowledge from basic sciences to translate it into field applications to solve issues related but not limited to the recreational and economic values of plants, food security and safety, and energy sustainability. In this review, we highlight the substantial progress reached in plant proteomics during the past decade which has paved the way for translational plant proteomics. Increasing proteomics knowledge in plants is not limited to model and non-model plants, proteogenomics, crop improvement, and food analysis, safety, and nutrition but to many more potential applications. Given the wealth of information generated and to some extent applied, there is the need for more efficient and broader channels to freely disseminate the information to the scientific community. This article is part of a Special Issue entitled: Translational Proteomics. Copyright © 2012 Elsevier B.V. All rights reserved.

Biochemical and genetic analysis of the yeast proteome with a movable ORF collection

PubMed Central

Gelperin, Daniel M.; White, Michael A.; Wilkinson, Martha L.; Kon, Yoshiko; Kung, Li A.; Wise, Kevin J.; Lopez-Hoyo, Nelson; Jiang, Lixia; Piccirillo, Stacy; Yu, Haiyuan; Gerstein, Mark; Dumont, Mark E.; Phizicky, Eric M.; Snyder, Michael; Grayhack, Elizabeth J.

2005-01-01

Functional analysis of the proteome is an essential part of genomic research. To facilitate different proteomic approaches, a MORF (moveable ORF) library of 5854 yeast expression plasmids was constructed, each expressing a sequence-verified ORF as a C-terminal ORF fusion protein, under regulated control. Analysis of 5573 MORFs demonstrates that nearly all verified ORFs are expressed, suggests the authenticity of 48 ORFs characterized as dubious, and implicates specific processes including cytoskeletal organization and transcriptional control in growth inhibition caused by overexpression. Global analysis of glycosylated proteins identifies 109 new confirmed N-linked and 345 candidate glycoproteins, nearly doubling the known yeast glycome. PMID:16322557

Mass spectrometry based proteomics profiling as diagnostic tool in oncology: current status and future perspective.

PubMed

Findeisen, Peter; Neumaier, Michael

2009-01-01

Proteomics analysis has been heralded as a novel tool for identifying new and specific biomarkers that may improve diagnosis and monitoring of various disease states. Recent years have brought a number of proteomics profiling technologies. Although proteomics profiling has resulted in the detection of disease-associated differences and modification of proteins, current proteomics technologies display certain limitations that are hampering the introduction of these new technologies into clinical laboratory diagnostics and routine applications. In this review, we summarize current advances in mass spectrometry based biomarker discovery. The promises and challenges of this new technology are discussed with particular emphasis on diagnostic perspectives of mass-spectrometry based proteomics profiling for malignant diseases.

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.

Nanoliter-Scale Oil-Air-Droplet Chip-Based Single Cell Proteomic Analysis.

PubMed

Li, Zi-Yi; Huang, Min; Wang, Xiu-Kun; Zhu, Ying; Li, Jin-Song; Wong, Catherine C L; Fang, Qun

2018-04-17

Single cell proteomic analysis provides crucial information on cellular heterogeneity in biological systems. Herein, we describe a nanoliter-scale oil-air-droplet (OAD) chip for achieving multistep complex sample pretreatment and injection for single cell proteomic analysis in the shotgun mode. By using miniaturized stationary droplet microreaction and manipulation techniques, our system allows all sample pretreatment and injection procedures to be performed in a nanoliter-scale droplet with minimum sample loss and a high sample injection efficiency (>99%), thus substantially increasing the analytical sensitivity for single cell samples. We applied the present system in the proteomic analysis of 100 ± 10, 50 ± 5, 10, and 1 HeLa cell(s), and protein IDs of 1360, 612, 192, and 51 were identified, respectively. The OAD chip-based system was further applied in single mouse oocyte analysis, with 355 protein IDs identified at the single oocyte level, which demonstrated its special advantages of high enrichment of sequence coverage, hydrophobic proteins, and enzymatic digestion efficiency over the traditional in-tube system.

Data from quantitative label free proteomics analysis of rat spleen.

PubMed

Dudekula, Khadar; Le Bihan, Thierry

2016-09-01

The dataset presented in this work has been obtained using a label-free quantitative proteomic analysis of rat spleen. A robust method for extraction of proteins from rat spleen tissue and LC-MS-MS analysis was developed using a urea and SDS-based buffer. Different fractionation methods were compared. A total of 3484 different proteins were identified from the pool of all experiments run in this study (a total of 2460 proteins with at least two peptides). A total of 1822 proteins were identified from nine non-fractionated pulse gels, 2288 proteins and 2864 proteins were identified by SDS-PAGE fractionation into three and five fractions respectively. The proteomics data are deposited in ProteomeXchange Consortium via PRIDE PXD003520, Progenesis and Maxquant output are presented in the supported information. The generated list of proteins under different regimes of fractionation allow assessing the nature of the identified proteins; variability in the quantitative analysis associated with the different sampling strategy and allow defining a proper number of replicates for future quantitative analysis.

Top-Down Proteomics and Direct Surface Sampling of Neonatal Dried Blood Spots: Diagnosis of Unknown Hemoglobin Variants

NASA Astrophysics Data System (ADS)

Edwards, Rebecca L.; Griffiths, Paul; Bunch, Josephine; Cooper, Helen J.

2012-11-01

We have previously shown that liquid microjunction surface sampling of dried blood spots coupled with high resolution top-down mass spectrometry may be used for screening of common hemoglobin variants HbS, HbC, and HbD. In order to test the robustness of the approach, we have applied the approach to unknown hemoglobin variants. Six neonatal dried blood spot samples that had been identified as variants, but which could not be diagnosed by current screening methods, were analyzed by direct surface sampling top-down mass spectrometry. Both collision-induced dissociation and electron transfer dissociation mass spectrometry were employed. Four of the samples were identified as β-chain variants: two were heterozygous Hb D-Iran, one was heterozygous Hb Headington, and one was heterozygous Hb J-Baltimore. The fifth sample was identified as the α-chain variant heterozygous Hb Phnom Penh. Analysis of the sixth sample suggested that it did not in fact contain a variant. Adoption of the approach in the clinic would require speed in both data collection and interpretation. To address that issue, we have compared manual data analysis with freely available data analysis software (ProsightPTM). The results demonstrate the power of top-down proteomics for hemoglobin variant analysis in newborn samples.

Sherlock Holmes and the proteome--a detective story.

PubMed

Righetti, Pier Giorgio; Boschetti, Egisto

2007-02-01

The performance of a hexapeptide ligand library in capturing the 'hidden proteome' is illustrated and evaluated. This library, insolubilized on an organic polymer and available under the trade name 'Equalizer Bead Technology', acts by capturing all components of a given proteome, by concentrating rare and very rare proteins, and simultaneously diluting the abundant ones. This results in a proteome of 'normalized' relative abundances, amenable to analysis by MS and any other analytical tool. Examples are given of analysis of human urine and serum, as well as cell and tissue lysates, such as Escherichia coli and Saccharomyces cerevisiae extracts. Another important application is impurity tracking and polishing of recombinant DNA products, especially biopharmaceuticals meant for human consumption.

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

Analysis of PNGase F-resistant N-glycopeptides using SugarQb for Proteome Discoverer 2.1 reveals cryptic substrate specificities.

PubMed

Stadlmann, Johannes; Hoi, David M; Taubenschmid, Jasmin; Mechtler, Karl; Penninger, Josef M

2018-05-18

SugarQb (www.imba.oeaw.ac.at/sugarqb) is a freely available collection of computational tools for the automated identification of intact glycopeptides from high-resolution HCD MS/MS data-sets in the Proteome Discoverer environment. We report the migration of SugarQb to the latest and free version of Proteome Discoverer 2.1, and apply it to the analysis of PNGase F-resistant N-glycopeptides from mouse embryonic stem cells. The analysis of intact glycopeptides highlights unexpected technical limitations to PNGase F-dependent glycoproteomic workflows at the proteome level, and warrants a critical re-interpretation of seminal data-sets in the context of N-glycosylation-site prediction. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

MAPU: Max-Planck Unified database of organellar, cellular, tissue and body fluid proteomes

PubMed Central

Zhang, Yanling; Zhang, Yong; Adachi, Jun; Olsen, Jesper V.; Shi, Rong; de Souza, Gustavo; Pasini, Erica; Foster, Leonard J.; Macek, Boris; Zougman, Alexandre; Kumar, Chanchal; Wiśniewski, Jacek R.; Jun, Wang; Mann, Matthias

2007-01-01

Mass spectrometry (MS)-based proteomics has become a powerful technology to map the protein composition of organelles, cell types and tissues. In our department, a large-scale effort to map these proteomes is complemented by the Max-Planck Unified (MAPU) proteome database. MAPU contains several body fluid proteomes; including plasma, urine, and cerebrospinal fluid. Cell lines have been mapped to a depth of several thousand proteins and the red blood cell proteome has also been analyzed in depth. The liver proteome is represented with 3200 proteins. By employing high resolution MS and stringent validation criteria, false positive identification rates in MAPU are lower than 1:1000. Thus MAPU datasets can serve as reference proteomes in biomarker discovery. MAPU contains the peptides identifying each protein, measured masses, scores and intensities and is freely available at using a clickable interface of cell or body parts. Proteome data can be queried across proteomes by protein name, accession number, sequence similarity, peptide sequence and annotation information. More than 4500 mouse and 2500 human proteins have already been identified in at least one proteome. Basic annotation information and links to other public databases are provided in MAPU and we plan to add further analysis tools. PMID:17090601

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.

Comparative Testis Tissue Proteomics Using 2-Dye Versus 3-Dye DIGE Analysis.

PubMed

Holland, Ashling

2018-01-01

Comparative tissue proteomics aims to analyze alterations of the proteome in response to a stimulus. Two-dimensional difference gel electrophoresis (2D-DIGE) is a modified and advanced form of 2D gel electrophoresis. DIGE is a powerful biochemical method that compares two or three protein samples on the same analytical gel, and can be used to establish differentially expressed protein levels between healthy normal and diseased pathological tissue sample groups. Minimal DIGE labeling can be used via a 2-dye system with Cy3 and Cy5 or a 3-dye system with Cy2, Cy3, and Cy5 to fluorescently label samples with CyDye flours pre-electrophoresis. DIGE circumvents gel-to-gel variability by multiplexing samples to a single gel and through the use of a pooled internal standard for normalization. This form of quantitative high-resolution proteomics facilitates the comparative analysis and evaluation of tissue protein compositions. Comparing tissue groups under different conditions is crucially important for advancing the biomedical field by characterization of cellular processes, understanding pathophysiological development and tissue biomarker discovery. This chapter discusses 2D-DIGE as a comparative tissue proteomic technique and describes in detail the experimental steps required for comparative proteomic analysis employing both options of 2-dye and 3-dye DIGE minimal labeling.

Studies of a biochemical factory: tomato trichome deep expressed sequence tag sequencing and proteomics.

PubMed

Schilmiller, Anthony L; Miner, Dennis P; Larson, Matthew; McDowell, Eric; Gang, David R; Wilkerson, Curtis; Last, Robert L

2010-07-01

Shotgun proteomics analysis allows hundreds of proteins to be identified and quantified from a single sample at relatively low cost. Extensive DNA sequence information is a prerequisite for shotgun proteomics, and it is ideal to have sequence for the organism being studied rather than from related species or accessions. While this requirement has limited the set of organisms that are candidates for this approach, next generation sequencing technologies make it feasible to obtain deep DNA sequence coverage from any organism. As part of our studies of specialized (secondary) metabolism in tomato (Solanum lycopersicum) trichomes, 454 sequencing of cDNA was combined with shotgun proteomics analyses to obtain in-depth profiles of genes and proteins expressed in leaf and stem glandular trichomes of 3-week-old plants. The expressed sequence tag and proteomics data sets combined with metabolite analysis led to the discovery and characterization of a sesquiterpene synthase that produces beta-caryophyllene and alpha-humulene from E,E-farnesyl diphosphate in trichomes of leaf but not of stem. This analysis demonstrates the utility of combining high-throughput cDNA sequencing with proteomics experiments in a target tissue. These data can be used for dissection of other biochemical processes in these specialized epidermal cells.

Studies of a Biochemical Factory: Tomato Trichome Deep Expressed Sequence Tag Sequencing and Proteomics1[W][OA

PubMed Central

Schilmiller, Anthony L.; Miner, Dennis P.; Larson, Matthew; McDowell, Eric; Gang, David R.; Wilkerson, Curtis; Last, Robert L.

2010-01-01

Shotgun proteomics analysis allows hundreds of proteins to be identified and quantified from a single sample at relatively low cost. Extensive DNA sequence information is a prerequisite for shotgun proteomics, and it is ideal to have sequence for the organism being studied rather than from related species or accessions. While this requirement has limited the set of organisms that are candidates for this approach, next generation sequencing technologies make it feasible to obtain deep DNA sequence coverage from any organism. As part of our studies of specialized (secondary) metabolism in tomato (Solanum lycopersicum) trichomes, 454 sequencing of cDNA was combined with shotgun proteomics analyses to obtain in-depth profiles of genes and proteins expressed in leaf and stem glandular trichomes of 3-week-old plants. The expressed sequence tag and proteomics data sets combined with metabolite analysis led to the discovery and characterization of a sesquiterpene synthase that produces β-caryophyllene and α-humulene from E,E-farnesyl diphosphate in trichomes of leaf but not of stem. This analysis demonstrates the utility of combining high-throughput cDNA sequencing with proteomics experiments in a target tissue. These data can be used for dissection of other biochemical processes in these specialized epidermal cells. PMID:20431087

Challenges and Strategies for Proteome Analysis of the Interaction of Human Pathogenic Fungi with Host Immune Cells.

PubMed

Krüger, Thomas; Luo, Ting; Schmidt, Hella; Shopova, Iordana; Kniemeyer, Olaf

2015-12-14

Opportunistic human pathogenic fungi including the saprotrophic mold Aspergillus fumigatus and the human commensal Candida albicans can cause severe fungal infections in immunocompromised or critically ill patients. The first line of defense against opportunistic fungal pathogens is the innate immune system. Phagocytes such as macrophages, neutrophils and dendritic cells are an important pillar of the innate immune response and have evolved versatile defense strategies against microbial pathogens. On the other hand, human-pathogenic fungi have sophisticated virulence strategies to counteract the innate immune defense. In this context, proteomic approaches can provide deeper insights into the molecular mechanisms of the interaction of host immune cells with fungal pathogens. This is crucial for the identification of both diagnostic biomarkers for fungal infections and therapeutic targets. Studying host-fungal interactions at the protein level is a challenging endeavor, yet there are few studies that have been undertaken. This review draws attention to proteomic techniques and their application to fungal pathogens and to challenges, difficulties, and limitations that may arise in the course of simultaneous dual proteome analysis of host immune cells interacting with diverse morphotypes of fungal pathogens. On this basis, we discuss strategies to overcome these multifaceted experimental and analytical challenges including the viability of immune cells during co-cultivation, the increased and heterogeneous protein complexity of the host proteome dynamically interacting with the fungal proteome, and the demands on normalization strategies in terms of relative quantitative proteome analysis.

Quantitative proteomic analysis of paired colorectal cancer and non-tumorigenic tissues reveals signature proteins and perturbed pathways involved in CRC progression and metastasis.

PubMed

Sethi, Manveen K; Thaysen-Andersen, Morten; Kim, Hoguen; Park, Cheol Keun; Baker, Mark S; Packer, Nicolle H; Paik, Young-Ki; Hancock, William S; Fanayan, Susan

2015-08-03

Modern proteomics has proven instrumental in our understanding of the molecular deregulations associated with the development and progression of cancer. Herein, we profile membrane-enriched proteome of tumor and adjacent normal tissues from eight CRC patients using label-free nanoLC-MS/MS-based quantitative proteomics and advanced pathway analysis. Of the 948 identified proteins, 184 proteins were differentially expressed (P<0.05, fold change>1.5) between the tumor and non-tumor tissue (69 up-regulated and 115 down-regulated in tumor tissues). The CRC tumor and non-tumor tissues clustered tightly in separate groups using hierarchical cluster analysis of the differentially expressed proteins, indicating a strong CRC-association of this proteome subset. Specifically, cancer associated proteins such as FN1, TNC, DEFA1, ITGB2, MLEC, CDH17, EZR and pathways including actin cytoskeleton and RhoGDI signaling were deregulated. Stage-specific proteome signatures were identified including up-regulated ribosomal proteins and down-regulated annexin proteins in early stage CRC. Finally, EGFR(+) CRC tissues showed an EGFR-dependent down-regulation of cell adhesion molecules, relative to EGFR(-) tissues. Taken together, this study provides a detailed map of the altered proteome and associated protein pathways in CRC, which enhances our mechanistic understanding of CRC biology and opens avenues for a knowledge-driven search for candidate CRC protein markers. Copyright © 2015 Elsevier B.V. All rights reserved.

Analysis of the variability of human normal urine by 2D-GE reveals a "public" and a "private" proteome.

PubMed

Molina, Laurence; Salvetat, Nicolas; Ameur, Randa Ben; Peres, Sabine; Sommerer, Nicolas; Jarraya, Fayçal; Ayadi, Hammadi; Molina, Franck; Granier, Claude

2011-12-10

The characterization of the normal urinary proteome is steadily progressing and represents a major interest in the assessment of clinical urinary biomarkers. To estimate quantitatively the variability of the normal urinary proteome, urines of 20 healthy people were collected. We first evaluated the impact of the sample conservation temperature on urine proteome integrity. Keeping the urine sample at RT or at +4°C until storage at -80°C seems the best way for long-term storage of samples for 2D-GE analysis. The quantitative variability of the normal urinary proteome was estimated on the 20 urines mapped by 2D-GE. The occurrence of the 910 identified spots was analysed throughout the gels and represented in a virtual 2D gel. Sixteen percent of the spots were found to occur in all samples and 23% occurred in at least 90% of urines. About 13% of the protein spots were present only in 10% or less of the samples, thus representing the most variable part of the normal urinary proteome. Twenty proteins corresponding to a fraction of the fully conserved spots were identified by mass spectrometry. In conclusion, a "public" urinary proteome, common to healthy individuals, seems to coexist with a "private" urinary proteome, which is more specific to each individual. Copyright © 2011 Elsevier B.V. All rights reserved.

Transformative Impact of Proteomics on Cardiovascular Health and Disease: A Scientific Statement From the American Heart Association.

PubMed

Lindsey, Merry L; Mayr, Manuel; Gomes, Aldrin V; Delles, Christian; Arrell, D Kent; Murphy, Anne M; Lange, Richard A; Costello, Catherine E; Jin, Yu-Fang; Laskowitz, Daniel T; Sam, Flora; Terzic, Andre; Van Eyk, Jennifer; Srinivas, Pothur R

2015-09-01

The year 2014 marked the 20th anniversary of the coining of the term proteomics. The purpose of this scientific statement is to summarize advances over this period that have catalyzed our capacity to address the experimental, translational, and clinical implications of proteomics as applied to cardiovascular health and disease and to evaluate the current status of the field. Key successes that have energized the field are delineated; opportunities for proteomics to drive basic science research, facilitate clinical translation, and establish diagnostic and therapeutic healthcare algorithms are discussed; and challenges that remain to be solved before proteomic technologies can be readily translated from scientific discoveries to meaningful advances in cardiovascular care are addressed. Proteomics is the result of disruptive technologies, namely, mass spectrometry and database searching, which drove protein analysis from 1 protein at a time to protein mixture analyses that enable large-scale analysis of proteins and facilitate paradigm shifts in biological concepts that address important clinical questions. Over the past 20 years, the field of proteomics has matured, yet it is still developing rapidly. The scope of this statement will extend beyond the reaches of a typical review article and offer guidance on the use of next-generation proteomics for future scientific discovery in the basic research laboratory and clinical settings. © 2015 American Heart Association, Inc.

Proteomic analysis of acquired tamoxifen resistance in MCF-7 cells reveals expression signatures associated with enhanced migration

PubMed Central

2012-01-01

Introduction Acquired tamoxifen resistance involves complex signaling events that are not yet fully understood. Successful therapeutic intervention to delay the onset of hormone resistance depends critically on mechanistic elucidation of viable molecular targets associated with hormone resistance. This study was undertaken to investigate the global proteomic alterations in a tamoxifen resistant MCF-7 breast cancer cell line obtained by long term treatment of the wild type MCF-7 cell line with 4-hydroxytamoxifen (4-OH Tam). Methods We cultured MCF-7 cells with 4-OH Tam over a period of 12 months to obtain the resistant cell line. A gel-free, quantitative proteomic method was used to identify and quantify the proteome of the resistant cell line. Nano-flow high-performance liquid chromatography coupled to high resolution Fourier transform mass spectrometry was used to analyze fractionated peptide mixtures that were isobarically labeled from the resistant and control cell lysates. Real time quantitative PCR and Western blots were used to verify selected proteomic changes. Lentiviral vector transduction was used to generate MCF-7 cells stably expressing S100P. Online pathway analysis was performed to assess proteomic signatures in tamoxifen resistance. Survival analysis was done to evaluate clinical relevance of altered proteomic expressions. Results Quantitative proteomic analysis revealed a wide breadth of signaling events during transition to acquired tamoxifen resistance. A total of 629 proteins were found significantly changed with 364 up-regulated and 265 down-regulated. Collectively, these changes demonstrated the suppressed state of estrogen receptor (ER) and ER-regulated genes, activated survival signaling and increased migratory capacity of the resistant cell line. The protein S100P was found to play a critical role in conferring tamoxifen resistance and enhanced cell motility. Conclusions Our data demonstrate that the adaptive changes in the proteome of tamoxifen resistant breast cancer cells are characterized by down-regulated ER signaling, activation of alternative survival pathways, and enhanced cell motility through regulation of the actin cytoskeleton dynamics. Evidence also emerged that S100P mediates acquired tamoxifen resistance and migration capacity. PMID:22417809

A comparative proteomic strategy for subcellular proteome research: ICAT approach coupled with bioinformatics prediction to ascertain rat liver mitochondrial proteins and indication of mitochondrial localization for catalase.

PubMed

Jiang, Xiao-Sheng; Dai, Jie; Sheng, Quan-Hu; Zhang, Lei; Xia, Qi-Chang; Wu, Jia-Rui; Zeng, Rong

2005-01-01

Subcellular proteomics, as an important step to functional proteomics, has been a focus in proteomic research. However, the co-purification of "contaminating" proteins has been the major problem in all the subcellular proteomic research including all kinds of mitochondrial proteome research. It is often difficult to conclude whether these "contaminants" represent true endogenous partners or artificial associations induced by cell disruption or incomplete purification. To solve such a problem, we applied a high-throughput comparative proteome experimental strategy, ICAT approach performed with two-dimensional LC-MS/MS analysis, coupled with combinational usage of different bioinformatics tools, to study the proteome of rat liver mitochondria prepared with traditional centrifugation (CM) or further purified with a Nycodenz gradient (PM). A total of 169 proteins were identified and quantified convincingly in the ICAT analysis, in which 90 proteins have an ICAT ratio of PM:CM>1.0, while another 79 proteins have an ICAT ratio of PM:CM<1.0. Almost all the proteins annotated as mitochondrial according to Swiss-Prot annotation, bioinformatics prediction, and literature reports have a ratio of PM:CM>1.0, while proteins annotated as extracellular or secreted, cytoplasmic, endoplasmic reticulum, ribosomal, and so on have a ratio of PM:CM<1.0. Catalase and AP endonuclease 1, which have been known as peroxisomal and nuclear, respectively, have shown a ratio of PM:CM>1.0, confirming the reports about their mitochondrial location. Moreover, the 125 proteins with subcellular location annotation have been used as a testing dataset to evaluate the efficiency for ascertaining mitochondrial proteins by ICAT analysis and the bioinformatics tools such as PSORT, TargetP, SubLoc, MitoProt, and Predotar. The results indicated that ICAT analysis coupled with combinational usage of different bioinformatics tools could effectively ascertain mitochondrial proteins and distinguish contaminant proteins and even multilocation proteins. Using such a strategy, many novel proteins, known proteins without subcellular location annotation, and even known proteins that have been annotated as other locations have been strongly indicated for their mitochondrial location.

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...

Complex and extensive post-transcriptional regulation revealed by integrative proteomic and transcriptomic analysis of metabolite stress response in Clostridium acetobutylicum.

PubMed

Venkataramanan, Keerthi P; Min, Lie; Hou, Shuyu; Jones, Shawn W; Ralston, Matthew T; Lee, Kelvin H; Papoutsakis, E Terry

2015-01-01

Clostridium acetobutylicum is a model organism for both clostridial biology and solvent production. The organism is exposed to its own toxic metabolites butyrate and butanol, which trigger an adaptive stress response. Integrative analysis of proteomic and RNAseq data may provide novel insights into post-transcriptional regulation. The identified iTRAQ-based quantitative stress proteome is made up of 616 proteins with a 15 % genome coverage. The differentially expressed proteome correlated poorly with the corresponding differential RNAseq transcriptome. Up to 31 % of the differentially expressed proteins under stress displayed patterns opposite to those of the transcriptome, thus suggesting significant post-transcriptional regulation. The differential proteome of the translation machinery suggests that cells employ a different subset of ribosomal proteins under stress. Several highly upregulated proteins but with low mRNA levels possessed mRNAs with long 5'UTRs and strong RBS scores, thus supporting the argument that regulatory elements on the long 5'UTRs control their translation. For example, the oxidative stress response rubrerythrin was upregulated only at the protein level up to 40-fold without significant mRNA changes. We also identified many leaderless transcripts, several displaying different transcriptional start sites, thus suggesting mRNA-trimming mechanisms under stress. Downregulation of Rho and partner proteins pointed to changes in transcriptional elongation and termination under stress. The integrative proteomic-transcriptomic analysis demonstrated complex expression patterns of a large fraction of the proteome. Such patterns could not have been detected with one or the other omic analyses. Our analysis proposes the involvement of specific molecular mechanisms of post-transcriptional regulation to explain the observed complex stress response.

MASH Suite Pro: A Comprehensive Software Tool for Top-Down Proteomics*

PubMed Central

Cai, Wenxuan; Guner, Huseyin; Gregorich, Zachery R.; Chen, Albert J.; Ayaz-Guner, Serife; Peng, Ying; Valeja, Santosh G.; Liu, Xiaowen; Ge, Ying

2016-01-01

Top-down mass spectrometry (MS)-based proteomics is arguably a disruptive technology for the comprehensive analysis of all proteoforms arising from genetic variation, alternative splicing, and posttranslational modifications (PTMs). However, the complexity of top-down high-resolution mass spectra presents a significant challenge for data analysis. In contrast to the well-developed software packages available for data analysis in bottom-up proteomics, the data analysis tools in top-down proteomics remain underdeveloped. Moreover, despite recent efforts to develop algorithms and tools for the deconvolution of top-down high-resolution mass spectra and the identification of proteins from complex mixtures, a multifunctional software platform, which allows for the identification, quantitation, and characterization of proteoforms with visual validation, is still lacking. Herein, we have developed MASH Suite Pro, a comprehensive software tool for top-down proteomics with multifaceted functionality. MASH Suite Pro is capable of processing high-resolution MS and tandem MS (MS/MS) data using two deconvolution algorithms to optimize protein identification results. In addition, MASH Suite Pro allows for the characterization of PTMs and sequence variations, as well as the relative quantitation of multiple proteoforms in different experimental conditions. The program also provides visualization components for validation and correction of the computational outputs. Furthermore, MASH Suite Pro facilitates data reporting and presentation via direct output of the graphics. Thus, MASH Suite Pro significantly simplifies and speeds up the interpretation of high-resolution top-down proteomics data by integrating tools for protein identification, quantitation, characterization, and visual validation into a customizable and user-friendly interface. We envision that MASH Suite Pro will play an integral role in advancing the burgeoning field of top-down proteomics. PMID:26598644

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.

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.

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

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 analysis of human follicular fluid associated with successful in vitro fertilization.

PubMed

Shen, Xiaofang; Liu, Xin; Zhu, Peng; Zhang, Yuhua; Wang, Jiahui; Wang, Yanwei; Wang, Wenting; Liu, Juan; Li, Ning; Liu, Fujun

2017-07-27

Human follicular fluid (HFF) provides a key environment for follicle development and oocyte maturation, and contributes to oocyte quality and in vitro fertilization (IVF) outcome. To better understand folliculogenesis in the ovary, a proteomic strategy based on dual reverse phase high performance liquid chromatography (RP-HPLC) coupled to matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (LC-MALDI TOF/TOF MS) was used to investigate the protein profile of HFF from women undergoing successful IVF. A total of 219 unique high-confidence (False Discovery Rate (FDR) < 0.01) HFF proteins were identified by searching the reviewed Swiss-Prot human database (20,183 sequences), and MS data were further verified by western blot. PANTHER showed HFF proteins were involved in complement and coagulation cascade, growth factor and hormone, immunity, and transportation, KEGG indicated their pathway, and STRING demonstrated their interaction networks. In comparison, 32% and 50% of proteins have not been reported in previous human follicular fluid and plasma. Our HFF proteome research provided a new complementary high-confidence dataset of folliculogenesis and oocyte maturation environment. Those proteins associated with innate immunity, complement cascade, blood coagulation, and angiogenesis might serve as the biomarkers of female infertility and IVF outcome, and their pathways facilitated a complete exhibition of reproductive process.

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

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

Morphine biosynthesis in opium poppy involves two cell types: sieve elements and laticifers.

PubMed

Onoyovwe, Akpevwe; Hagel, Jillian M; Chen, Xue; Khan, Morgan F; Schriemer, David C; Facchini, Peter J

2013-10-01

Immunofluorescence labeling and shotgun proteomics were used to establish the cell type-specific localization of morphine biosynthesis in opium poppy (Papaver somniferum). Polyclonal antibodies for each of six enzymes involved in converting (R)-reticuline to morphine detected corresponding antigens in sieve elements of the phloem, as described previously for all upstream enzymes transforming (S)-norcoclaurine to (S)-reticuline. Validated shotgun proteomics performed on whole-stem and latex total protein extracts generated 2031 and 830 distinct protein families, respectively. Proteins corresponding to nine morphine biosynthetic enzymes were represented in the whole stem, whereas only four of the final five pathway enzymes were detected in the latex. Salutaridine synthase was detected in the whole stem, but not in the latex subproteome. The final three enzymes converting thebaine to morphine were among the most abundant active latex proteins despite a limited occurrence in laticifers suggested by immunofluorescence labeling. Multiple charge isoforms of two key O-demethylases in the latex were revealed by two-dimensional immunoblot analysis. Salutaridine biosynthesis appears to occur only in sieve elements, whereas conversion of thebaine to morphine is predominant in adjacent laticifers, which contain morphine-rich latex. Complementary use of immunofluorescence labeling and shotgun proteomics has substantially resolved the cellular localization of morphine biosynthesis in opium poppy.

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.

Time-resolved Global and Chromatin Proteomics during Herpes Simplex Virus Type 1 (HSV-1) Infection*

PubMed Central

Kulej, Katarzyna; Avgousti, Daphne C.; Sidoli, Simone; Herrmann, Christin; Della Fera, Ashley N.; Kim, Eui Tae; Garcia, Benjamin A.; Weitzman, Matthew D.

2017-01-01

Herpes simplex virus (HSV-1) lytic infection results in global changes to the host cell proteome and the proteins associated with host chromatin. We present a system level characterization of proteome dynamics during infection by performing a multi-dimensional analysis during HSV-1 lytic infection of human foreskin fibroblast (HFF) cells. Our study includes identification and quantification of the host and viral proteomes, phosphoproteomes, chromatin bound proteomes and post-translational modifications (PTMs) on cellular histones during infection. We analyzed proteomes across six time points of virus infection (0, 3, 6, 9, 12 and 15 h post-infection) and clustered trends in abundance using fuzzy c-means. Globally, we accurately quantified more than 4000 proteins, 200 differently modified histone peptides and 9000 phosphorylation sites on cellular proteins. In addition, we identified 67 viral proteins and quantified 571 phosphorylation events (465 with high confidence site localization) on viral proteins, which is currently the most comprehensive map of HSV-1 phosphoproteome. We investigated chromatin bound proteins by proteomic analysis of the high-salt chromatin fraction and identified 510 proteins that were significantly different in abundance during infection. We found 53 histone marks significantly regulated during virus infection, including a steady increase of histone H3 acetylation (H3K9ac and H3K14ac). Our data provide a resource of unprecedented depth for human and viral proteome dynamics during infection. Collectively, our results indicate that the proteome composition of the chromatin of HFF cells is highly affected during HSV-1 infection, and that phosphorylation events are abundant on viral proteins. We propose that our epi-proteomics approach will prove to be important in the characterization of other model infectious systems that involve changes to chromatin composition. PMID:28179408

Immunogenic membrane-associated proteins of Mycobacterium tuberculosis revealed by proteomics.

PubMed

Sinha, Sudhir; Kosalai, K; Arora, Shalini; Namane, Abdelkader; Sharma, Pawan; Gaikwad, Anil N; Brodin, Priscille; Cole, Stewart T

2005-07-01

Membrane-associated proteins of Mycobacterium tuberculosis offer a challenge, as well as an opportunity, in the quest for better therapeutic and prophylactic interventions against tuberculosis. The authors have previously reported that extraction with the detergent Triton X-114 (TX-114) is a useful step in proteomic analysis of mycobacterial cell membranes, and detergent-soluble membrane proteins of mycobacteria are potent stimulators of human T cells. In this study 1-D and 2-D gel electrophoresis-based protocols were used for the analysis of proteins in the TX-114 extract of M. tuberculosis membranes. Peptide mass mapping (using MALDI-TOF-MS, matrix assisted laser desorption/ionization time of flight mass spectrometry) of 116 samples led to the identification of 105 proteins, 9 of which were new to the M. tuberculosis proteome. Functional orthologues of 73 of these proteins were also present in Mycobacterium leprae, suggesting their relative importance. Bioinformatics predicted that as many as 73% of the proteins had a hydrophobic disposition. 1-D gel electrophoresis revealed more hydrophobic/transmembrane and basic proteins than 2-D gel electrophoresis. Identified proteins fell into the following major categories: protein synthesis, cell wall biogenesis/architecture and conserved hypotheticals/unknowns. To identify immunodominant proteins of the detergent phase (DP), 14 low-molecular-mass fractions prepared by continuous-elution gel electrophoresis were subjected to T cell activation assays using blood samples from BCG-vaccinated healthy donors from a tuberculosis endemic area. Analysis of the responses (cell proliferation and IFN-gamma production) showed that the immunodominance of certain DP fractions was most probably due to ribosomal proteins, which is consistent with both their specificity for mycobacteria and their abundance. Other membrane-associated proteins, including transmembrane proteins/lipoproteins and ESAT-6, did not appear to contribute significantly to the observed T cell responses.

Machine Learning-based Classification of Diffuse Large B-cell Lymphoma Patients by Their Protein Expression Profiles.

PubMed

Deeb, Sally J; Tyanova, Stefka; Hummel, Michael; Schmidt-Supprian, Marc; Cox, Juergen; Mann, Matthias

2015-11-01

Characterization of tumors at the molecular level has improved our knowledge of cancer causation and progression. Proteomic analysis of their signaling pathways promises to enhance our understanding of cancer aberrations at the functional level, but this requires accurate and robust tools. Here, we develop a state of the art quantitative mass spectrometric pipeline to characterize formalin-fixed paraffin-embedded tissues of patients with closely related subtypes of diffuse large B-cell lymphoma. We combined a super-SILAC approach with label-free quantification (hybrid LFQ) to address situations where the protein is absent in the super-SILAC standard but present in the patient samples. Shotgun proteomic analysis on a quadrupole Orbitrap quantified almost 9,000 tumor proteins in 20 patients. The quantitative accuracy of our approach allowed the segregation of diffuse large B-cell lymphoma patients according to their cell of origin using both their global protein expression patterns and the 55-protein signature obtained previously from patient-derived cell lines (Deeb, S. J., D'Souza, R. C., Cox, J., Schmidt-Supprian, M., and Mann, M. (2012) Mol. Cell. Proteomics 11, 77-89). Expression levels of individual segregation-driving proteins as well as categories such as extracellular matrix proteins behaved consistently with known trends between the subtypes. We used machine learning (support vector machines) to extract candidate proteins with the highest segregating power. A panel of four proteins (PALD1, MME, TNFAIP8, and TBC1D4) is predicted to classify patients with low error rates. Highly ranked proteins from the support vector analysis revealed differential expression of core signaling molecules between the subtypes, elucidating aspects of their pathobiology. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Quantitative Proteomics Reveals Temporal Proteomic Changes in Signaling Pathways during BV2 Mouse Microglial Cell Activation.

PubMed

Woo, Jongmin; Han, Dohyun; Wang, Joseph Injae; Park, Joonho; Kim, Hyunsoo; Kim, Youngsoo

2017-09-01

The development of systematic proteomic quantification techniques in systems biology research has enabled one to perform an in-depth analysis of cellular systems. We have developed a systematic proteomic approach that encompasses the spectrum from global to targeted analysis on a single platform. We have applied this technique to an activated microglia cell system to examine changes in the intracellular and extracellular proteomes. Microglia become activated when their homeostatic microenvironment is disrupted. There are varying degrees of microglial activation, and we chose to focus on the proinflammatory reactive state that is induced by exposure to such stimuli as lipopolysaccharide (LPS) and interferon-gamma (IFN-γ). Using an improved shotgun proteomics approach, we identified 5497 proteins in the whole-cell proteome and 4938 proteins in the secretome that were associated with the activation of BV2 mouse microglia by LPS or IFN-γ. Of the differentially expressed proteins in stimulated microglia, we classified pathways that were related to immune-inflammatory responses and metabolism. Our label-free parallel reaction monitoring (PRM) approach made it possible to comprehensively measure the hyper-multiplex quantitative value of each protein by high-resolution mass spectrometry. Over 450 peptides that corresponded to pathway proteins and direct or indirect interactors via the STRING database were quantified by label-free PRM in a single run. Moreover, we performed a longitudinal quantification of secreted proteins during microglial activation, in which neurotoxic molecules that mediate neuronal cell loss in the brain are released. These data suggest that latent pathways that are associated with neurodegenerative diseases can be discovered by constructing and analyzing a pathway network model of proteins. Furthermore, this systematic quantification platform has tremendous potential for applications in large-scale targeted analyses. The proteomics data for discovery and label-free PRM analysis have been deposited to the ProteomeXchange Consortium with identifiers and , respectively.

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

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

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

Proteomic approaches and their application to plant gravitropism.

PubMed

Basu, Proma; Luesse, Darron R; Wyatt, Sarah E

2015-01-01

Proteomics is a powerful technique that allows researchers a window into how an organism responds to a mutation, a specific environment, or at a distinct point during development by quantifying relative protein abundance and posttranslational modifications. Here, we describe methods for the proteomic analysis of Arabidopsis thaliana tissue. Extraction protocols are provided for isolation of soluble, plasma membrane, and tonoplast proteins. In addition, basic analysis and quality metrics for MS/MS data are discussed. The protocols outlined have the potential to unlock new avenues of research that are not possible through basic genetics or transcriptomic approaches. By combining proteomic information with known gene regulatory patterns, researchers can gain a complete picture of how molecular pathways, such as those required for gravitropism, are initiated, regulated, and terminated.

Announcing the Launch of CPTAC’s Proteogenomics DREAM Challenge | Office of Cancer Clinical Proteomics Research

Cancer.gov

This week, we are excited to announce the launch of the National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium (CPTAC) Proteogenomics Computational DREAM Challenge.  The aim of this Challenge is to encourage the generation of computational methods for extracting information from the cancer proteome and for linking those data to genomic and transcriptomic information.  The specific goals are to predict proteomic and phosphoproteomic data from other multiple data types including transcriptomics and genetics.

GENOMIC AND PROTEOMIC ANALYSIS OF SURROGATE TISSUES FOR ASSESSING TOXIC EXPOSURES AND DISEASE STATES

EPA Science Inventory

Genomic and Proteomic Analysis of Surrogate Tissues for Assessing Toxic Exposures and Disease States
David J. Dix and John C. Rockett
Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, USEPA, ...

Proteomic analysis of cell cycle progression in asynchronous cultures, including mitotic subphases, using PRIMMUS

PubMed Central

Whigham, Arlene; Clarke, Rosemary; Brenes-Murillo, Alejandro J; Estes, Brett; Madhessian, Diana; Lundberg, Emma; Wadsworth, Patricia

2017-01-01

The temporal regulation of protein abundance and post-translational modifications is a key feature of cell division. Recently, we analysed gene expression and protein abundance changes during interphase under minimally perturbed conditions (Ly et al., 2014, 2015). Here, we show that by using specific intracellular immunolabelling protocols, FACS separation of interphase and mitotic cells, including mitotic subphases, can be combined with proteomic analysis by mass spectrometry. Using this PRIMMUS (PRoteomic analysis of Intracellular iMMUnolabelled cell Subsets) approach, we now compare protein abundance and phosphorylation changes in interphase and mitotic fractions from asynchronously growing human cells. We identify a set of 115 phosphorylation sites increased during G2, termed ‘early risers’. This set includes phosphorylation of S738 on TPX2, which we show is important for TPX2 function and mitotic progression. Further, we use PRIMMUS to provide the first a proteome-wide analysis of protein abundance remodeling between prophase, prometaphase and anaphase. PMID:29052541

Progress in Top-Down Proteomics and the Analysis of Proteoforms

PubMed Central

Toby, Timothy K.; Fornelli, Luca; Kelleher, Neil L.

2017-01-01

From a molecular perspective, enactors of function in biology are intact proteins that can be variably modified at the genetic, transcriptional, or post-translational level. Over the past 30 years, mass spectrometry (MS) has become a powerful method for the analysis of proteomes. Prevailing bottom-up proteomics operates at the level of the peptide, leading to issues with protein inference, connectivity, and incomplete sequence/modification information. Top-down proteomics (TDP), alternatively, applies MS at the proteoform level to analyze intact proteins with diverse sources of intramolecular complexity preserved during analysis. Fortunately, advances in prefractionation workflows, MS instrumentation, and dissociation methods for whole-protein ions have helped TDP emerge as an accessible and potentially disruptive modality with increasingly translational value. In this review, we discuss technical and conceptual advances in TDP, along with the growing power of proteoform-resolved measurements in clinical and translational research. PMID:27306313

Sex-Specific Biology of the Human Malaria Parasite Revealed from the Proteomes of Mature Male and Female Gametocytes.

PubMed

Miao, Jun; Chen, Zhao; Wang, Zenglei; Shrestha, Sony; Li, Xiaolian; Li, Runze; Cui, Liwang

2017-04-01

The gametocytes of the malaria parasites are obligate for perpetuating the parasite's life cycle through mosquitoes, but the sex-specific biology of gametocytes is poorly understood. We generated a transgenic line in the human malaria parasite Plasmodium falciparum , which allowed us to accurately separate male and female gametocytes by flow cytometry. In-depth analysis of the proteomes by liquid chromatography-tandem mass spectrometry identified 1244 and 1387 proteins in mature male and female gametocytes, respectively. GFP-tagging of nine selected proteins confirmed their sex-partitions to be agreeable with the results from the proteomic analysis. The sex-specific proteomes showed significant differences that are consistent with the divergent functions of the two sexes. Although the male-specific proteome (119 proteins) is enriched in proteins associated with the flagella and genome replication, the female-specific proteome (262 proteins) is more abundant in proteins involved in metabolism, translation and organellar functions. Compared with the Plasmodium berghei sex-specific proteomes, this study revealed both extensive conservation and considerable divergence between these two species, which reflect the disparities between the two species in proteins involved in cytoskeleton, lipid metabolism and protein degradation. Comparison with three sex-specific proteomes allowed us to obtain high-confidence lists of 73 and 89 core male- and female-specific/biased proteins conserved in Plasmodium The identification of sex-specific/biased proteomes in Plasmodium lays a solid foundation for understanding the molecular mechanisms underlying the unique sex-specific biology in this early-branching eukaryote. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Sex-Specific Biology of the Human Malaria Parasite Revealed from the Proteomes of Mature Male and Female Gametocytes *

PubMed Central

Miao, Jun; Chen, Zhao; Wang, Zenglei; Shrestha, Sony; Li, Xiaolian; Li, Runze; Cui, Liwang

2017-01-01

The gametocytes of the malaria parasites are obligate for perpetuating the parasite's life cycle through mosquitoes, but the sex-specific biology of gametocytes is poorly understood. We generated a transgenic line in the human malaria parasite Plasmodium falciparum, which allowed us to accurately separate male and female gametocytes by flow cytometry. In-depth analysis of the proteomes by liquid chromatography-tandem mass spectrometry identified 1244 and 1387 proteins in mature male and female gametocytes, respectively. GFP-tagging of nine selected proteins confirmed their sex-partitions to be agreeable with the results from the proteomic analysis. The sex-specific proteomes showed significant differences that are consistent with the divergent functions of the two sexes. Although the male-specific proteome (119 proteins) is enriched in proteins associated with the flagella and genome replication, the female-specific proteome (262 proteins) is more abundant in proteins involved in metabolism, translation and organellar functions. Compared with the Plasmodium berghei sex-specific proteomes, this study revealed both extensive conservation and considerable divergence between these two species, which reflect the disparities between the two species in proteins involved in cytoskeleton, lipid metabolism and protein degradation. Comparison with three sex-specific proteomes allowed us to obtain high-confidence lists of 73 and 89 core male- and female-specific/biased proteins conserved in Plasmodium. The identification of sex-specific/biased proteomes in Plasmodium lays a solid foundation for understanding the molecular mechanisms underlying the unique sex-specific biology in this early-branching eukaryote. PMID:28126901

Discovery of potential protein biomarkers of lung adenocarcinoma in bronchoalveolar lavage fluid by SWATH MS data-independent acquisition and targeted data extraction.

PubMed

Ortea, I; Rodríguez-Ariza, A; Chicano-Gálvez, E; Arenas Vacas, M S; Jurado Gámez, B

2016-04-14

Lung cancer currently ranks as the neoplasia with the highest global mortality rate. Although some improvements have been introduced in recent years, new advances in diagnosis are required in order to increase survival rates. New mildly invasive endoscopy-based diagnostic techniques include the collection of bronchoalveolar lavage fluid (BALF), which is discarded after using a portion of the fluid for standard pathological procedures. BALF proteomic analysis can contribute to clinical practice with more sensitive biomarkers, and can complement cytohistological studies by aiding in the diagnosis, prognosis, and subtyping of lung cancer, as well as the monitoring of treatment response. The range of quantitative proteomics methodologies used for biomarker discovery is currently being broadened with the introduction of data-independent acquisition (DIA) analysis-related approaches that address the massive quantitation of the components of a proteome. Here we report for the first time a DIA-based quantitative proteomics study using BALF as the source for the discovery of potential lung cancer biomarkers. The results have been encouraging in terms of the number of identified and quantified proteins. A panel of candidate protein biomarkers for adenocarcinoma in BALF is reported; this points to the activation of the complement network as being strongly over-represented and suggests this pathway as a potential target for lung cancer research. In addition, the results reported for haptoglobin, complement C4-A, and glutathione S-transferase pi are consistent with previous studies, which indicates that these proteins deserve further consideration as potential lung cancer biomarkers in BALF. Our study demonstrates that the analysis of BALF proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS), combining a simple sample pre-treatment and SWATH DIA MS, is a useful method for the discovery of potential lung cancer biomarkers. Bronchoalveolar lavage fluid (BALF) analysis can contribute to clinical practice with more sensitive biomarkers, thus complementing cytohistological studies in order to aid in the diagnosis, prognosis, and subtyping of lung cancer, as well as the monitoring of treatment response. Here we report a panel of candidate protein biomarkers for adenocarcinoma in BALF. Forty-four proteins showed a fold-change higher than 3.75 among adenocarcinoma patients compared with controls. This report is the first DIA-based quantitative proteomics study to use bronchoalveolar lavage fluid (BALF) as a matrix for discovering potential biomarkers. The results are encouraging in terms of the number of identified and quantified proteins, demonstrating that the analysis of BALF proteins by a SWATH approach is a useful method for the discovery of potential biomarkers of pulmonary diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

Bacterial membrane proteomics.

PubMed

Poetsch, Ansgar; Wolters, Dirk

2008-10-01

About one quarter to one third of all bacterial genes encode proteins of the inner or outer bacterial membrane. These proteins perform essential physiological functions, such as the import or export of metabolites, the homeostasis of metal ions, the extrusion of toxic substances or antibiotics, and the generation or conversion of energy. The last years have witnessed completion of a plethora of whole-genome sequences of bacteria important for biotechnology or medicine, which is the foundation for proteome and other functional genome analyses. In this review, we discuss the challenges in membrane proteome analysis, starting from sample preparation and leading to MS-data analysis and quantification. The current state of available proteomics technologies as well as their advantages and disadvantages will be described with a focus on shotgun proteomics. Then, we will briefly introduce the most abundant proteins and protein families present in bacterial membranes before bacterial membrane proteomics studies of the last years will be presented. It will be shown how these works enlarged our knowledge about the physiological adaptations that take place in bacteria during fine chemical production, bioremediation, protein overexpression, and during infections. Furthermore, several examples from literature demonstrate the suitability of membrane proteomics for the identification of antigens and different pathogenic strains, as well as the elucidation of membrane protein structure and function.

Cell death proteomics database: consolidating proteomics data on cell death.

PubMed

Arntzen, Magnus Ø; Bull, Vibeke H; Thiede, Bernd

2013-05-03

Programmed cell death is a ubiquitous process of utmost importance for the development and maintenance of multicellular organisms. More than 10 different types of programmed cell death forms have been discovered. Several proteomics analyses have been performed to gain insight in proteins involved in the different forms of programmed cell death. To consolidate these studies, we have developed the cell death proteomics (CDP) database, which comprehends data from apoptosis, autophagy, cytotoxic granule-mediated cell death, excitotoxicity, mitotic catastrophe, paraptosis, pyroptosis, and Wallerian degeneration. The CDP database is available as a web-based database to compare protein identifications and quantitative information across different experimental setups. The proteomics data of 73 publications were integrated and unified with protein annotations from UniProt-KB and gene ontology (GO). Currently, more than 6,500 records of more than 3,700 proteins are included in the CDP. Comparing apoptosis and autophagy using overrepresentation analysis of GO terms, the majority of enriched processes were found in both, but also some clear differences were perceived. Furthermore, the analysis revealed differences and similarities of the proteome between autophagosomal and overall autophagy. The CDP database represents a useful tool to consolidate data from proteome analyses of programmed cell death and is available at http://celldeathproteomics.uio.no.

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 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.

The alternative Medicago truncatula defense proteome of ROS—defective transgenic roots during early microbial infection

PubMed Central

Kiirika, Leonard M.; Schmitz, Udo; Colditz, Frank

2014-01-01

ROP-type GTPases of plants function as molecular switches within elementary signal transduction pathways such as the regulation of ROS synthesis via activation of NADPH oxidases (RBOH-respiratory burst oxidase homolog in plants). Previously, we reported that silencing of the Medicago truncatula GTPase MtROP9 led to reduced ROS production and suppressed induction of ROS-related enzymes in transgenic roots (MtROP9i) infected with pathogenic (Aphanomyces euteiches) and symbiotic microorganisms (Glomus intraradices, Sinorhizobium meliloti). While fungal infections were enhanced, S. meliloti infection was drastically impaired. In this study, we investigate the temporal proteome response of M. truncatula MtROP9i transgenic roots during the same microbial interactions under conditions of deprived potential to synthesize ROS. In comparison with control roots (Mtvector), we present a comprehensive proteomic analysis using sensitive MS protein identification. For four early infection time-points (1, 3, 5, 24 hpi), 733 spots were found to be different in abundance: 213 spots comprising 984 proteins (607 unique) were identified after S. meliloti infection, 230 spots comprising 796 proteins (580 unique) after G. intraradices infection, and 290 spots comprising 1240 proteins (828 unique) after A. euteiches infection. Data evaluation by GelMap in combination with a heatmap tool allowed recognition of key proteome changes during microbial interactions under conditions of hampered ROS synthesis. Overall, the number of induced proteins in MtROP9i was low as compared with controls, indicating a dual function of ROS in defense signaling as well as alternative response patterns activated during microbial infection. Qualitative analysis of induced proteins showed that enzymes linked to ROS production and scavenging were highly induced in control roots, while in MtROP9i the majority of proteins were involved in alternative defense pathways such as cell wall and protein degradation. PMID:25101099

Universal Solid-phase Reversible Sample-Prep for Concurrent Proteome and N-glycome Characterization

PubMed Central

Zhou, Hui; Morley, Samantha; Kostel, Stephen; Freeman, Michael R.; Joshi, Vivek; Brewster, David; Lee, Richard S.

2017-01-01

SUMMARY We describe a novel Solid-phase Reversible Sample-Prep (SRS) platform, which enables rapid sample preparation for concurrent proteome and N-glycome characterization by mass spectrometry. SRS utilizes a uniquely functionalized, silica-based bead that has strong affinity toward proteins with minimal-to-no affinity for peptides and other small molecules. By leveraging the inherent size difference between, SRS permits high-capacity binding of proteins, rapid removal of small molecules (detergents, metabolites, salts, etc.), extensive manipulation including enzymatic and chemical treatments on beads-bound proteins, and easy recovery of N-glycans and peptides. The efficacy of SRS was evaluated in a wide range of biological samples including single glycoprotein, whole cell lysate, murine tissues, and human urine. To further demonstrate the SRS platform, we coupled a quantitative strategy to SRS to investigate the differences between DU145 prostate cancer cells and its DIAPH3-silenced counterpart. Our previous studies suggested that DIAPH3 silencing in DU145 prostate cancer cells induced transition to an amoeboid phenotype that correlated with tumor progression and metastasis. In this analysis we identified distinct proteomic and N-glycomic alterations between the two cells. Intriguingly, a metastasis-associated tyrosine kinase receptor ephrin-type-A receptor (EPHA2) was highly upregulated in DIAPH3-silenced cells, indicating underling connection between EPHA2 and DIAPH3. Moreover, distinct alterations in the N-glycome were identified, suggesting a cross-link between DIAPH3 and glycosyltransferase networks. Overall, SRS is an enabling universal sample preparation strategy that is not size limited and has the capability to efficiently prepare and clean peptides and N-glycans concurrently from nearly all sample types. Conceptually, SRS can be utilized for the analysis of other posttranslational modifications, and the unique surface chemistry can be further transformed for high-throughput automation. The technical simplicity, robustness, and modularity of SRS make it a highly promising technology with great potential in proteomic-based research. PMID:26791391

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.

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

Urinary proteomics for prediction of mortality in patients with type 2 diabetes and microalbuminuria.

PubMed

Currie, Gemma E; von Scholten, Bernt Johan; Mary, Sheon; Flores Guerrero, Jose-Luis; Lindhardt, Morten; Reinhard, Henrik; Jacobsen, Peter K; Mullen, William; Parving, Hans-Henrik; Mischak, Harald; Rossing, Peter; Delles, Christian

2018-04-06

The urinary proteomic classifier CKD273 has shown promise for prediction of progressive diabetic nephropathy (DN). Whether it is also a determinant of mortality and cardiovascular disease in patients with microalbuminuria (MA) is unknown. Urine samples were obtained from 155 patients with type 2 diabetes and confirmed microalbuminuria. Proteomic analysis was undertaken using capillary electrophoresis coupled to mass spectrometry to determine the CKD273 classifier score. A previously defined CKD273 threshold of 0.343 for identification of DN was used to categorise the cohort in Kaplan-Meier and Cox regression models with all-cause mortality as the primary endpoint. Outcomes were traced through national health registers after 6 years. CKD273 correlated with urine albumin excretion rate (UAER) (r = 0.481, p = <0.001), age (r = 0.238, p = 0.003), coronary artery calcium (CAC) score (r = 0.236, p = 0.003), N-terminal pro-brain natriuretic peptide (NT-proBNP) (r = 0.190, p = 0.018) and estimated glomerular filtration rate (eGFR) (r = 0.265, p = 0.001). On multivariate analysis only UAER (β = 0.402, p < 0.001) and eGFR (β = - 0.184, p = 0.039) were statistically significant determinants of CKD273. Twenty participants died during follow-up. CKD273 was a determinant of mortality (log rank [Mantel-Cox] p = 0.004), and retained significance (p = 0.048) after adjustment for age, sex, blood pressure, NT-proBNP and CAC score in a Cox regression model. A multidimensional biomarker can provide information on outcomes associated with its primary diagnostic purpose. Here we demonstrate that the urinary proteomic classifier CKD273 is associated with mortality in individuals with type 2 diabetes and MA even when adjusted for other established cardiovascular and renal biomarkers.

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.

A proteomics performance standard to support measurement quality in proteomics.

PubMed

Beasley-Green, Ashley; Bunk, David; Rudnick, Paul; Kilpatrick, Lisa; Phinney, Karen

2012-04-01

The emergence of MS-based proteomic platforms as a prominent technology utilized in biochemical and biomedical research has increased the need for high-quality MS measurements. To address this need, National Institute of Standards and Technology (NIST) reference material (RM) 8323 yeast protein extract is introduced as a proteomics quality control material for benchmarking the preanalytical and analytical performance of proteomics-based experimental workflows. RM 8323 yeast protein extract is based upon the well-characterized eukaryote Saccharomyces cerevisiae and can be utilized in the design and optimization of proteomics-based methodologies from sample preparation to data analysis. To demonstrate its utility as a proteomics quality control material, we coupled LC-MS/MS measurements of RM 8323 with the NIST MS Quality Control (MSQC) performance metrics to quantitatively assess the LC-MS/MS instrumentation parameters that influence measurement accuracy, repeatability, and reproducibility. Due to the complexity of the yeast proteome, we also demonstrate how NIST RM 8323, along with the NIST MSQC performance metrics, can be used in the evaluation and optimization of proteomics-based sample preparation methods. © 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.

Expanding the bovine milk proteome through extensive fractionation.

PubMed

Nissen, Asger; Bendixen, Emøke; Ingvartsen, Klaus Lønne; Røntved, Christine Maria

2013-01-01

Bovine milk is an agricultural product of tremendous value worldwide. It contains proteins, fat, lactose, vitamins, and minerals. It provides nutrition and immunological protection (e.g., in the gastrointestinal tract) to the newborn and young calf. It also forms an important part of human nutrition. The repertoire of proteins in milk (i.e., its proteome) is vast and complex. The milk proteome can be described in detail by mass spectrometry-based proteomics. However, the high concentration of dominating proteins in milk reduces mass spectrometry detection sensitivity and limits detection of low abundant proteins. Further, the general health and udder health of the dairy cows delivering the milk may influence the composition of the milk proteome. To gain a more exhaustive and true picture of the milk proteome, we performed an extensive preanalysis fractionation of raw composite milk collected from documented healthy cows in early lactation. Four simple and industrially applicable techniques exploring the physical and chemical properties of milk, including acidification, filtration, and centrifugation, were used for separation of the proteins. This resulted in 5 different fractions, whose content of proteins were compared with the proteins of nonfractionated milk using 2-dimensional liquid chromatography tandem mass spectrometry analysis. To validate the proteome analysis, spectral counts and ELISA were performed on 7 proteins using the ELISA for estimation of the detection sensitivity limit of the 2-dimensional liquid chromatography tandem mass spectrometry analysis. Each fractionation technique resulted in identification of a unique subset of proteins. However, high-speed centrifugation of milk to whey was by far the best method to achieve high and repeatable proteome coverage. The total number of milk proteins initially detected in nonfractionated milk and the fractions were 635 in 2 replicates. Removal of dominant proteins and filtering for redundancy across the different fractions reduced the number to 376 unique proteins in 2 replicates. In addition, 366 proteins were detected by this process in 1 replicate. Hence, by applying different fractionation techniques to milk, we expanded the milk proteome. The milk proteome map may serve as a reference for scientists working in the dairy sector. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Quantitative label-free proteomic analysis of human urine to identify novel candidate protein biomarkers for schistosomiasis.

PubMed

Onile, Olugbenga Samson; Calder, Bridget; Soares, Nelson C; Anumudu, Chiaka I; Blackburn, Jonathan M

2017-11-01

Schistosomiasis is a chronic neglected tropical disease that is characterized by continued inflammatory challenges to the exposed population and it has been established as a possible risk factor in the aetiology of bladder cancer. Improved diagnosis of schistosomiasis and its associated pathology is possible through mass spectrometry to identify biomarkers among the infected population, which will influence early detection of the disease and its subtle morbidity. A high-throughput proteomic approach was used to analyse human urine samples for 49 volunteers from Eggua, a schistosomiasis endemic community in South-West, Nigeria. The individuals were previously screened for Schistosoma haematobium and structural bladder pathologies via microscopy and ultrasonography respectively. Samples were categorised into schistosomiasis, schistosomiasis with bladder pathology, bladder pathology, and a normal healthy control group. These samples were analysed to identify potential protein biomarkers. A total of 1306 proteins and 9701 unique peptides were observed in this study (FDR = 0.01). Fifty-four human proteins were found to be potential biomarkers for schistosomiasis and bladder pathologies due to schistosomiasis by label-free quantitative comparison between groups. Thirty-six (36) parasite-derived potential biomarkers were also identified, which include some existing putative schistosomiasis biomarkers that have been previously reported. Some of these proteins include Elongation factor 1 alpha, phosphopyruvate hydratase, histone H4 and heat shock proteins (HSP 60, HSP 70). These findings provide an in-depth analysis of potential schistosoma and human host protein biomarkers for diagnosis of chronic schistosomiasis caused by Schistosoma haematobium and its pathogenesis.

Serum proteome profiling in canine idiopathic dilated cardiomyopathy using TMT-based quantitative proteomics approach.

PubMed

Bilić, Petra; Guillemin, Nicolas; Kovačević, Alan; Beer Ljubić, Blanka; Jović, Ines; Galan, Asier; Eckersall, Peter David; Burchmore, Richard; Mrljak, Vladimir

2018-05-15

Idiopathic dilated cardiomyopathy (iDCM) is a primary myocardial disorder with an unknown aetiology, characterized by reduced contractility and ventricular dilation of the left or both ventricles. Naturally occurring canine iDCM was used herein to identify serum proteomic signature of the disease compared to the healthy state, providing an insight into underlying mechanisms and revealing proteins with biomarker potential. To achieve this, we used high-throughput label-based quantitative LC-MS/MS proteomics approach and bioinformatics analysis of the in silico inferred interactome protein network created from the initial list of differential proteins. To complement the proteomic analysis, serum biochemical parameters and levels of know biomarkers of cardiac function were measured. Several proteins with biomarker potential were identified, such as inter-alpha-trypsin inhibitor heavy chain H4, microfibril-associated glycoprotein 4 and apolipoprotein A-IV, which were validated using an independent method (Western blotting) and showed high specificity and sensitivity according to the receiver operating characteristic curve analysis. Bioinformatics analysis revealed involvement of different pathways in iDCM, such as complement cascade activation, lipoprotein particles dynamics, elastic fibre formation, GPCR signalling and respiratory electron transport chain. Idiopathic dilated cardiomyopathy is a severe primary myocardial disease of unknown cause, affecting both humans and dogs. This study is a contribution to the canine heart disease research by means of proteomic and bioinformatic state of the art analyses, following similar approach in human iDCM research. Importantly, we used serum as non-invasive and easily accessible biological source of information and contributed to the scarce data on biofluid proteome research on this topic. Bioinformatics analysis revealed biological pathways modulated in canine iDCM with potential of further targeted research. Also, several proteins with biomarker potential have been identified and successfully validated. Copyright © 2018 Elsevier B.V. All rights reserved.

Optimizing of MALDI-ToF-based low-molecular-weight serum proteome pattern analysis in detection of breast cancer patients; the effect of albumin removal on classification performance.

PubMed

Pietrowska, M; Marczak, L; Polanska, J; Nowicka, E; Behrent, K; Tarnawski, R; Stobiecki, M; Polanski, A; Widlak, P

2010-01-01

Mass spectrometry-based analysis of the serum proteome allows identifying multi-peptide patterns/signatures specific for blood of cancer patients, thus having high potential value for cancer diagnostics. However, because of problems with optimization and standardization of experimental and computational design, none of identified proteome patterns/signatures was approved for diagnostics in clinical practice as yet. Here we compared two methods of serum sample preparation for mass spectrometry-based proteome pattern analysis aimed to identify biomarkers that could be used in early detection of breast cancer patients. Blood samples were collected in a group of 92 patients diagnosed at early (I and II) stages of the disease before the start of therapy, and in a group of age-matched healthy controls (104 women). Serum specimens were purified and analyzed using MALDI-ToF spectrometry, either directly or after membrane filtration (50 kDa cut-off) to remove albumin and other large serum proteins. Mass spectra of the low-molecular-weight fraction (2-10 kDa) of the serum proteome were resolved using the Gaussian mixture decomposition, and identified spectral components were used to build classifiers that differentiated samples from breast cancer patients and healthy persons. Mass spectra of complete serum and membrane-filtered albumin-depleted samples have apparently different structure and peaks specific for both types of samples could be identified. The optimal classifier built for the complete serum specimens consisted of 8 spectral components, and had 81% specificity and 72% sensitivity, while that built for the membrane-filtered samples consisted of 4 components, and had 80% specificity and 81% sensitivity. We concluded that pre-processing of samples to remove albumin might be recommended before MALDI-ToF mass spectrometric analysis of the low-molecular-weight components of human serum Keywords: albumin removal; breast cancer; clinical proteomics; mass spectrometry; pattern analysis; serum proteome.

Comparative analysis of genomics and proteomics in Bacillus thuringiensis 4.0718.

PubMed

Rang, Jie; He, Hao; Wang, Ting; Ding, Xuezhi; Zuo, Mingxing; Quan, Meifang; Sun, Yunjun; Yu, Ziquan; Hu, Shengbiao; Xia, Liqiu

2015-01-01

Bacillus thuringiensis is a widely used biopesticide that produced various insecticidal active substances during its life cycle. Separation and purification of numerous insecticide active substances have been difficult because of the relatively short half-life of such substances. On the other hand, substances can be synthetized at different times during development, so samples at different stages have to be studied, further complicating the analysis. A dual genomic and proteomic approach would enhance our ability to identify such substances, and particularily using mass spectrometry-based proteomic methods. The comparative analysis for genomic and proteomic data have showed that not all of the products deduced from the annotated genome could be identified among the proteomic data. For instance, genome annotation results showed that 39 coding sequences in the whole genome were related to insect pathogenicity, including five cry genes. However, Cry2Ab, Cry1Ia, Cytotoxin K, Bacteriocin, Exoenzyme C3 and Alveolysin could not be detected in the proteomic data obtained. The sporulation-related proteins were also compared analysis, results showed that the great majority sporulation-related proteins can be detected by mass spectrometry. This analysis revealed Spo0A~P, SigF, SigE(+), SigK(+) and SigG(+), all known to play an important role in the process of spore formation regulatory network, also were displayed in the proteomic data. Through the comparison of the two data sets, it was possible to infer that some genes were silenced or were expressed at very low levels. For instance, found that cry2Ab seems to lack a functional promoter while cry1Ia may not be expressed due to the presence of transposons. With this comparative study a relatively complete database can be constructed and used to transform hereditary material, thereby prompting the high expression of toxic proteins. A theoretical basis is provided for constructing highly virulent engineered bacteria and for promoting the application of proteogenomics in the life sciences.

MAPU: Max-Planck Unified database of organellar, cellular, tissue and body fluid proteomes.

PubMed

Zhang, Yanling; Zhang, Yong; Adachi, Jun; Olsen, Jesper V; Shi, Rong; de Souza, Gustavo; Pasini, Erica; Foster, Leonard J; Macek, Boris; Zougman, Alexandre; Kumar, Chanchal; Wisniewski, Jacek R; Jun, Wang; Mann, Matthias

2007-01-01

Mass spectrometry (MS)-based proteomics has become a powerful technology to map the protein composition of organelles, cell types and tissues. In our department, a large-scale effort to map these proteomes is complemented by the Max-Planck Unified (MAPU) proteome database. MAPU contains several body fluid proteomes; including plasma, urine, and cerebrospinal fluid. Cell lines have been mapped to a depth of several thousand proteins and the red blood cell proteome has also been analyzed in depth. The liver proteome is represented with 3200 proteins. By employing high resolution MS and stringent validation criteria, false positive identification rates in MAPU are lower than 1:1000. Thus MAPU datasets can serve as reference proteomes in biomarker discovery. MAPU contains the peptides identifying each protein, measured masses, scores and intensities and is freely available at http://www.mapuproteome.com using a clickable interface of cell or body parts. Proteome data can be queried across proteomes by protein name, accession number, sequence similarity, peptide sequence and annotation information. More than 4500 mouse and 2500 human proteins have already been identified in at least one proteome. Basic annotation information and links to other public databases are provided in MAPU and we plan to add further analysis tools.

PACOM: A Versatile Tool for Integrating, Filtering, Visualizing, and Comparing Multiple Large Mass Spectrometry Proteomics Data Sets.

PubMed

Martínez-Bartolomé, Salvador; Medina-Aunon, J Alberto; López-García, Miguel Ángel; González-Tejedo, Carmen; Prieto, Gorka; Navajas, Rosana; Salazar-Donate, Emilio; Fernández-Costa, Carolina; Yates, John R; Albar, Juan Pablo

2018-04-06

Mass-spectrometry-based proteomics has evolved into a high-throughput technology in which numerous large-scale data sets are generated from diverse analytical platforms. Furthermore, several scientific journals and funding agencies have emphasized the storage of proteomics data in public repositories to facilitate its evaluation, inspection, and reanalysis. (1) As a consequence, public proteomics data repositories are growing rapidly. However, tools are needed to integrate multiple proteomics data sets to compare different experimental features or to perform quality control analysis. Here, we present a new Java stand-alone tool, Proteomics Assay COMparator (PACOM), that is able to import, combine, and simultaneously compare numerous proteomics experiments to check the integrity of the proteomic data as well as verify data quality. With PACOM, the user can detect source of errors that may have been introduced in any step of a proteomics workflow and that influence the final results. Data sets can be easily compared and integrated, and data quality and reproducibility can be visually assessed through a rich set of graphical representations of proteomics data features as well as a wide variety of data filters. Its flexibility and easy-to-use interface make PACOM a unique tool for daily use in a proteomics laboratory. PACOM is available at https://github.com/smdb21/pacom .

Proteomic analysis on mangrove plant Avicennia marina leaves reveals nitric oxide enhances the salt tolerance by up-regulating photosynthetic and energy metabolic protein expression.

PubMed

Shen, Zhi-Jun; Chen, Juan; Ghoto, Kabir; Hu, Wen-Jun; Gao, Gui-Feng; Luo, Mei-Rong; Li, Zan; Simon, Martin; Zhu, Xue-Yi; Zheng, Hai-Lei

2018-06-15

Avicennia marina (Forsk.) Vierh is one of the most salt-tolerant mangrove species. Our previous study revealed that nitric oxide (NO) enhanced the salt tolerance of A. marina by promoting salt secretion and Na+ sequestration under salt stress. However, little is known about the regulation of NO on proteomic profiling for this mangrove species. In this study, we used sodium nitroprusside (SNP), an NO donor, to investigate the regulatory mechanism of NO on salt tolerance of A. marina according to physiological and proteomic aspects. Photosynthesis data showed that the reduction in photosynthesis caused by high salinity treatment (400 mM NaCl) could be partially recovered by addition of SNP (100 μM). Further analysis revealed that the high salinity treatment could induce not only the stomatal limitation but also non-stomatal limitation on photosynthetic reduction, while SNP addition could restore the non-stomatal limitation, implying that the application of SNP was beneficial to the metabolic process in leaves. Proteomic analysis identified 49 differentially expressed proteins involved in various biological processes such as photosynthesis, energy metabolism, primary metabolism, RNA transcription, protein translation and stress response proteins. Under high salinity treatment, the abundances of proteins related to photosynthesis, such as ribulose-phosphate 3-epimerase (RPE, spot 3), RuBisCO large subunit (RBCL, spot 4, 5, 24), RuBisCO activase A (RCA, spot 17, 18) and quinine oxidoreductase-like protein isoform 1 (QOR1, spot 23), were significantly decreased. However, the abundance of proteins such as RBCL (spot 5, 9) and QOR1 (spot 23) were increased by SNP addition. In addition, exogenous NO supply alleviated salt tolerance by increasing the accumulation of some proteins involved in energy metabolism (spot 15), primary metabolism (spot 25, 45, 46), RNA transcription (spot 36) and stress response proteins (spot 12, 21, 26, 37, 43). The transcriptional levels of nine selected proteins were mostly consistent with their protein abundance except spot 46. Overall, the presented data demonstrated that NO has a positive effect on improving salt tolerance in A. marina by regulating the protein abundance involved in photosynthesis, energy metabolism, primary metabolism and stress response.

Advanced proteomic liquid chromatography

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

Xie, Fang; Smith, Richard D.; Shen, Yufeng

2012-10-26

Liquid chromatography coupled with mass spectrometry is the predominant platform used to analyze proteomics samples consisting of large numbers of proteins and their proteolytic products (e.g., truncated polypeptides) and spanning a wide range of relative concentrations. This review provides an overview of advanced capillary liquid chromatography techniques and methodologies that greatly improve separation resolving power and proteomics analysis coverage, sensitivity, and throughput.

CPTAC Contributes to Healthdata.gov | Office of Cancer Clinical Proteomics Research

Cancer.gov

Recently, proteomic data generated by the Clinical Proteomic Tumor Analysis Consortium (CPTAC) funded by National Cancer Institute (NCI) was highlighted to the wider research community at Healthdata.gov. Healthdata.gov aims to make health data more accessible to entrepreneurs, researchers, and policy makers in the hopes of improving health outcomes f

Teaching Expression Proteomics: From the Wet-Lab to the Laptop

ERIC Educational Resources Information Center

Teixeira, Miguel C.; Santos, Pedro M.; Rodrigues, Catarina; Sa-Correia, Isabel

2009-01-01

Expression proteomics has become, in recent years, a key genome-wide expression approach in fundamental and applied life sciences. This postgenomic technology aims the quantitative analysis of all the proteins or protein forms (the so-called proteome) of a given organism in a given environmental and genetic context. It is a challenge to provide…

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.

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.

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.

Proteomic analysis of chromoplasts from six crop species reveals insights into chromoplast function and development

USDA-ARS?s Scientific Manuscript database

Chromoplasts are unique plastids that accumulate massive amounts of carotenoids. To gain a general and comparative characterization of chromoplast proteins, we performed proteomic analysis of chromoplasts from six carotenoid-rich crops: watermelon, tomato, carrot, orange cauliflower, red papaya, and...

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.

Recent advances in mass spectrometry-based proteomics of gastric cancer.

PubMed

Kang, Changwon; Lee, Yejin; Lee, J Eugene

2016-10-07

The last decade has witnessed remarkable technological advances in mass spectrometry-based proteomics. The development of proteomics techniques has enabled the reliable analysis of complex proteomes, leading to the identification and quantification of thousands of proteins in gastric cancer cells, tissues, and sera. This quantitative information has been used to profile the anomalies in gastric cancer and provide insights into the pathogenic mechanism of the disease. In this review, we mainly focus on the advances in mass spectrometry and quantitative proteomics that were achieved in the last five years and how these up-and-coming technologies are employed to track biochemical changes in gastric cancer cells. We conclude by presenting a perspective on quantitative proteomics and its future applications in the clinic and translational gastric cancer research.

Proteomics identifies the composition and manufacturing recipe of the 2500-year old sourdough bread from Subeixi cemetery in China.

PubMed

Shevchenko, Anna; Yang, Yimin; Knaust, Andrea; Thomas, Henrik; Jiang, Hongen; Lu, Enguo; Wang, Changsui; Shevchenko, Andrej

2014-06-13

We report on the geLC-MS/MS proteomics analysis of cereals and cereal food excavated in Subeixi cemetery (500-300BC) in Xinjiang, China. Proteomics provided direct evidence that at the Subexi sourdough bread was made from barley and broomcorn millet by leavening with a renewable starter comprising baker's yeast and lactic acid bacteria. The baking recipe and flour composition indicated that barley and millet bread belonged to the staple food already in the first millennium BC and suggested the role of Turpan basin as a major route for cultural communication between Western and Eastern Eurasia in antiquity. This article is part of a Special Issue entitled: Proteomics of non-model organisms. We demonstrate that organic residues of thousand year old foods unearthed by archeological excavations can be analyzed by geLC-MS/MS proteomics with good representation of protein source organisms and coverage of sequences of identified proteins. In-depth look into the foods proteome identifies the food type and its individual ingredients, reveals ancient food processing technologies, projects their social and economic impact and provides evidence of intercultural communication between ancient populations. Proteomics analysis of ancient organic residues is direct, quantitative and informative and therefore has the potential to develop into a valuable, generally applicable tool in archaeometry. This article is part of a Special Issue entitled: Proteomics of non-model organisms. Copyright © 2013. Published by Elsevier B.V.

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.

The iron-responsive microsomal proteome of Aspergillus fumigatus.

PubMed

Moloney, Nicola M; Owens, Rebecca A; Meleady, Paula; Henry, Michael; Dolan, Stephen K; Mulvihill, Eoin; Clynes, Martin; Doyle, Sean

2016-03-16

Aspergillus fumigatus is an opportunistic fungal pathogen. Siderophore biosynthesis and iron acquisition are essential for virulence. Yet, limited data exist with respect to the adaptive nature of the fungal microsomal proteome under iron-limiting growth conditions, as encountered during host infection. Here, we demonstrate that under siderophore biosynthetic conditions--significantly elevated fusarinine C (FSC) and triacetylfusarinine C (TAFC) production (p<0.0001), extensive microsomal proteome remodelling occurs. Specifically, a four-fold enrichment of transmembrane-containing proteins was observed with respect to whole cell lysates following ultracentrifugation-based microsomal extraction. Comparative label-free proteomic analysis of microsomal extracts, isolated following iron-replete and -deplete growth, identified 710 unique proteins. Scatterplot analysis (MaxQuant) demonstrated high correlation amongst biological replicates from each growth condition (Pearson correlation >0.96 within groups; biological replicates (n=4)). Quantitative and qualitative comparison revealed 231 proteins with a significant change in abundance between the iron-replete and iron-deplete conditions (p<0.05, fold change ≥ 2), with 96 proteins showing increased abundance and 135 with decreased abundance following iron limitation, including predicted siderophore transporters. Fluorescently labelled FSC was only sequestered following A. fumigatus growth under iron-limiting conditions. Interestingly, human sera exhibited significantly increased reactivity (p<0.0001) against microsomal protein extracts obtained following iron-deplete growth. The opportunistic fungal pathogen Aspergillus fumigatus must acquire iron to facilitate growth and pathogenicity. Iron-chelating non-ribosomal peptides, termed siderophores, mediate iron uptake via membrane-localised transporter proteins. Here we demonstrate for the first time that growth of A. fumigatus under iron-deplete conditions, concomitant with siderophore biosynthesis, leads to an extensive remodelling of the microsomal proteome which includes significantly altered levels of 231 constituent proteins (96 increased and 135 decreased in abundance), many of which have not previously been localised to the microsome. We also demonstrate the first synthesis of a fluorescent version of fusarinine C, an extracellular A. fumigatus siderophore, and its uptake and localization under iron-restricted conditions. This infers the use of an A. fumigatus siderophore as a 'Trojan horse' to potentiate the efficacy of anti-fungal drugs. Finally, in addition to revealing the Aspergillus-specific IgG reactivity in normal human sera against microsomal proteins, there appears to be a significantly increased reactivity against microsomal proteins obtained following iron-restricted growth. We hypothesise that iron-limiting environment in humans, which has evolved to nutritionally limit pathogen growth in vivo, may also alter the fungal microsomal proteome. Copyright © 2016 Elsevier B.V. All rights reserved.

Proteomic analysis of cow, yak, buffalo, goat and camel milk whey proteins: quantitative differential expression patterns.

PubMed

Yang, Yongxin; Bu, Dengpan; Zhao, Xiaowei; Sun, Peng; Wang, Jiaqi; Zhou, Lingyun

2013-04-05

To aid in unraveling diverse genetic and biological unknowns, a proteomic approach was used to analyze the whey proteome in cow, yak, buffalo, goat, and camel milk based on the isobaric tag for relative and absolute quantification (iTRAQ) techniques. This analysis is the first to produce proteomic data for the milk from the above-mentioned animal species: 211 proteins have been identified and 113 proteins have been categorized according to molecular function, cellular components, and biological processes based on gene ontology annotation. The results of principal component analysis showed significant differences in proteomic patterns among goat, camel, cow, buffalo, and yak milk. Furthermore, 177 differentially expressed proteins were submitted to advanced hierarchical clustering. The resulting clustering pattern included three major sample clusters: (1) cow, buffalo, and yak milk; (2) goat, cow, buffalo, and yak milk; and (3) camel milk. Certain proteins were chosen as characterization traits for a given species: whey acidic protein and quinone oxidoreductase for camel milk, biglycan for goat milk, uncharacterized protein (Accession Number: F1MK50 ) for yak milk, clusterin for buffalo milk, and primary amine oxidase for cow milk. These results help reveal the quantitative milk whey proteome pattern for analyzed species. This provides information for evaluating adulteration of specific specie milk and may provide potential directions for application of specific milk protein production based on physiological differences among animal species.

Quantitative Analysis of the Human Milk Whey Proteome Reveals Developing Milk and Mammary-Gland Functions across the First Year of Lactation

PubMed Central

Zhang, Qiang; Cundiff, Judy K.; Maria, Sarah D.; McMahon, Robert J.; Woo, Jessica G.; Davidson, Barbara S.; Morrow, Ardythe L.

2013-01-01

In-depth understanding of the changing functions of human milk (HM) proteins and the corresponding physiological adaptions of the lactating mammary gland has been inhibited by incomplete knowledge of the HM proteome. We analyzed the HM whey proteome (n = 10 women with samples at 1 week and 1, 3, 6, 9 and 12 months) using a quantitative proteomic approach. One thousand three hundred and thirty three proteins were identified with 615 being quantified. Principal component analysis revealed a transition in the HM whey proteome-throughout the first year of lactation. Abundance changes in IgG, sIgA and sIgM display distinct features during the first year. Complement components and other acute-phase proteins are generally at higher levels in early lactation. Proteomic analysis further suggests that the sources of milk fatty acids (FA) shift from more direct blood influx to more de novo mammary synthesis over lactation. The abundances of the majority of glycoproteins decline over lactation, which is consistent with increased enzyme expression in glycoprotein degradation and decreased enzyme expression in glycoprotein synthesis. Cellular detoxification machinery may be transformed as well, thereby accommodating increased metabolic activities in late lactation. The multiple developing functions of HM proteins and the corresponding mammary adaption become more apparent from this study. PMID:28250401

Molecular Diagnosis and Biomarker Identification on SELDI proteomics data by ADTBoost method.

PubMed

Wang, Lu-Yong; Chakraborty, Amit; Comaniciu, Dorin

2005-01-01

Clinical proteomics is an emerging field that will have great impact on molecular diagnosis, identification of disease biomarkers, drug discovery and clinical trials in the post-genomic era. Protein profiling in tissues and fluids in disease and pathological control and other proteomics techniques will play an important role in molecular diagnosis with therapeutics and personalized healthcare. We introduced a new robust diagnostic method based on ADTboost algorithm, a novel algorithm in proteomics data analysis to improve classification accuracy. It generates classification rules, which are often smaller and easier to interpret. This method often gives most discriminative features, which can be utilized as biomarkers for diagnostic purpose. Also, it has a nice feature of providing a measure of prediction confidence. We carried out this method in amyotrophic lateral sclerosis (ALS) disease data acquired by surface enhanced laser-desorption/ionization-time-of-flight mass spectrometry (SELDI-TOF MS) experiments. Our method is shown to have outstanding prediction capacity through the cross-validation, ROC analysis results and comparative study. Our molecular diagnosis method provides an efficient way to distinguish ALS disease from neurological controls. The results are expressed in a simple and straightforward alternating decision tree format or conditional format. We identified most discriminative peaks in proteomic data, which can be utilized as biomarkers for diagnosis. It will have broad application in molecular diagnosis through proteomics data analysis and personalized medicine in this post-genomic era.

Proteome Characterization Centers - TCGA

Cancer.gov

The centers, a component of NCI’s Clinical Proteomic Tumor Analysis Consortium, will analyze a subset of TCGA samples to define proteins translated from cancer genomes and their related biological processes.

Matrilin-1 expression is increased in the vertebral column of Atlantic salmon (Salmo salar L.) individuals displaying spinal fusions.

PubMed

Pedersen, Mona E; Takle, Harald; Ytteborg, Elisabeth; Veiseth-Kent, Eva; Enersen, Grethe; Færgestad, Ellen; Baeverfjord, Grete; Hannesson, Kirsten O

2011-12-01

We have previously characterized the development of vertebral fusions induced by elevated water temperature in Atlantic salmon. Molecular markers of bone and cartilage development together with histology were used to understand the complex pathology and mechanism in the development of this spinal malformation. In this study, we wanted to use proteomics, a non-hypothetical approach to screen for possible new markers involved in the fusion process. Proteins extracted from non-deformed and fused vertebrae of Atlantic salmon were therefore compared by two-dimensional electrophoresis (2DE) and MALDI-TOF analysis. Data analysis of protein spots in the 2DE gels demonstrated matrilin-1, also named cartilage matrix protein, to be the most highly up-regulated protein in fused compared with non-deformed vertebrae. Furthermore, real-time PCR analysis showed strong up-regulation of matrilin-1 mRNA in fused vertebrae. Immunohistochemistry demonstrated induced matrilin-1 expression in trans-differentiating cells undergoing a metaplastic shift toward chondrocytes in fusing vertebrae, whereas abundant expression was demonstrated in cartilaginous tissue and chordocytes of both non-deformed and fused vertebrae. These results identifies matrilin-1 as a new interesting candidate in the fusion process, and ratify the use of proteomic as a valuable technique to screen for markers involved in vertebral pathogenesis.

"Omics" of Food-Borne Gastroenteritis: Global Proteomic and Mutagenic Analysis of Salmonella enterica Serovar Enteritidis.

PubMed

Arunima, Aryashree; Yelamanchi, Soujanya D; Padhi, Chandrashekhar; Jaiswal, Sangeeta; Ryan, Daniel; Gupta, Bhawna; Sathe, Gajanan; Advani, Jayshree; Gowda, Harsha; Prasad, T S Keshava; Suar, Mrutyunjay

2017-10-01

Salmonella Enteritidis causes food-borne gastroenteritis by the two type three secretion systems (TTSS). TTSS-1 mediates invasion through intestinal lining, and TTSS-2 facilitates phagocytic survival. The pathogens' ability to infect effectively under TTSS-1-deficient background in host's phagocytes is poorly understood. Therefore, pathobiological understanding of TTSS-1-defective nontyphoidal Salmonellosis is highly important. We performed a comparative global proteomic analysis of the isogenic TTSS-1 mutant of Salmonella Enteritidis (M1511) and its wild-type isolate P125109. Our results showed 43 proteins were differentially expressed. Functional annotation further revealed that differentially expressed proteins belong to pathogenesis, tRNA and ncRNA metabolic processes. Three proteins, tryptophan subunit alpha chain, citrate lyase subunit alpha, and hypothetical protein 3202, were selected for in vitro analysis based on their functional annotations. Deletion mutants generated for the above proteins in the M1511 strain showed reduced intracellular survival inside macrophages in vitro. In sum, this study provides mass spectrometry-based evidence for seven hypothetical proteins, which will be subject of future investigations. Our study identifies proteins influencing virulence of Salmonella in the host. The study complements and further strengthens previously published research on proteins involved in enteropathogenesis of Salmonella and extends their role in noninvasive Salmonellosis.

Grade-dependent metabolic reprogramming in kidney cancer revealed by combined proteomics and metabolomics analysis

PubMed Central

Wettersten, Hiromi I.; Hakimi, A. Ari; Morin, Dexter; Bianchi, Cristina; Johnstone, Megan E.; Donohoe, Dallas R.; Trott, Josephine F.; Aboud, Omran Abu; Stirdivant, Steven; Neri, Bruce; Wolfert, Robert; Stewart, Benjamin; Perego, Roberto; Hsieh, James J.; Weiss, Robert H.

2015-01-01

Kidney cancer (or renal cell carcinoma [RCC]) is known as “the internist’s tumor” because it has protean systemic manifestations suggesting it utilizes complex, non-physiologic metabolic pathways. Given the increasing incidence of this cancer and its lack of effective therapeutic targets, we undertook an extensive analysis of human RCC tissue employing combined grade-dependent proteomics and metabolomics analysis to determine how metabolic reprogramming occurring in this disease allows it to escape available therapeutic approaches. After validation experiments in RCC cell lines that were wild-type or mutant for the VHL tumor suppressor, in characterizing higher grade tumors we found that the Warburg effect is relatively more prominent at the expense of the tricarboxylic acid cycle and oxidative metabolism in general. Further, we found that the glutamine metabolism pathway acts to inhibit reactive oxygen species, as evidenced by an upregulated glutathione pathway, while the β-oxidation pathway is inhibited leading to increased fatty acyl-carnitines. In support of findings from previous urine metabolomics analyses, we also documented tryptophan catabolism associated with immune suppression, which was highly represented in RCC compared to other metabolic pathways. Together, our results offer a rationale to evaluate novel anti-metabolic treatment strategies being developed in other disease settings as therapeutic strategies in RCC. PMID:25952651

Optimization of quantitative proteomic analysis of clots generated from plasma of patients with venous thromboembolism.

PubMed

Stachowicz, Aneta; Siudut, Jakub; Suski, Maciej; Olszanecki, Rafał; Korbut, Ryszard; Undas, Anetta; Wiśniewski, Jacek R

2017-01-01

It is well known that fibrin network binds a large variety of proteins, including inhibitors and activators of fibrinolysis, which may affect clot properties, such as stability and susceptibility to fibrinolysis. Specific plasma clot composition differs between individuals and may change in disease states. However, the plasma clot proteome has not yet been in-depth analyzed, mainly due to technical difficulty related to the presence of a highly abundant protein-fibrinogen and fibrin that forms a plasma clot. The aim of our study was to optimize quantitative proteomic analysis of fibrin clots prepared ex vivo from citrated plasma of the peripheral blood drawn from patients with prior venous thromboembolism (VTE). We used a multiple enzyme digestion filter aided sample preparation, a multienzyme digestion (MED) FASP method combined with LC-MS/MS analysis performed on a Proxeon Easy-nLC System coupled to the Q Exactive HF mass spectrometer. We also evaluated the impact of peptide fractionation with pipet-tip strong anion exchange (SAX) method on the obtained results. Our proteomic approach revealed 476 proteins repeatedly identified in the plasma fibrin clots from patients with VTE including extracellular vesicle-derived proteins, lipoproteins, fibrinolysis inhibitors, and proteins involved in immune responses. The MED FASP method using three different enzymes: LysC, trypsin and chymotrypsin increased the number of identified peptides and proteins and their sequence coverage as compared to a single step digestion. Peptide fractionation with a pipet-tip strong anion exchange (SAX) protocol increased the depth of proteomic analyses, but also extended the time needed for sample analysis with LC-MS/MS. The MED FASP method combined with a label-free quantification is an excellent proteomic approach for the analysis of fibrin clots prepared ex vivo from citrated plasma of patients with prior VTE.

Exploring the Human Plasma Proteome for Humoral Mediators of Remote Ischemic Preconditioning - A Word of Caution

PubMed Central

Helgeland, Erik; Breivik, Lars Ertesvåg; Vaudel, Marc; Svendsen, Øyvind Sverre; Garberg, Hilde; Nordrehaug, Jan Erik; Berven, Frode Steingrimsen; Jonassen, Anne Kristine

2014-01-01

Despite major advances in early revascularization techniques, cardiovascular diseases are still the leading cause of death worldwide, and myocardial infarctions contribute heavily to this. Over the past decades, it has become apparent that reperfusion of blood to a previously ischemic area of the heart causes damage in and of itself, and that this ischemia reperfusion induced injury can be reduced by up to 50% by mechanical manipulation of the blood flow to the heart. The recent discovery of remote ischemic preconditioning (RIPC) provides a non-invasive approach of inducing this cardioprotection at a distance. Finding its endogenous mediators and their operative mode is an important step toward increasing the ischemic tolerance. The release of humoral factor(s) upon RIPC was recently demonstrated and several candidate proteins were published as possible mediators of the cardioprotection. Before clinical applicability, these potential biomarkers and their efficiency must be validated, a task made challenging by the large heterogeneity in reported data and results. Here, in an attempt to reproduce and provide more experimental data on these mediators, we conducted an unbiased in-depth analysis of the human plasma proteome before and after RIPC. From the 68 protein markers reported in the literature, only 28 could be mapped to manually reviewed (Swiss-Prot) protein sequences. 23 of them were monitored in our untargeted experiment. However, their significant regulation could not be reproducibly estimated. In fact, among the 394 plasma proteins we accurately quantified, no significant regulation could be confidently and reproducibly assessed. This indicates that it is difficult to both monitor and reproduce published data from experiments exploring for RIPC induced plasma proteomic regulations, and suggests that further work should be directed towards small humoral factors. To simplify this task, we made our proteomic dataset available via ProteomeXchange, where scientists can mine for novel potential targets. PMID:25333471

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.

Method and platform standardization in MRM-based quantitative plasma proteomics.

PubMed

Percy, Andrew J; Chambers, Andrew G; Yang, Juncong; Jackson, Angela M; Domanski, Dominik; Burkhart, Julia; Sickmann, Albert; Borchers, Christoph H

2013-12-16

There exists a growing demand in the proteomics community to standardize experimental methods and liquid chromatography-mass spectrometry (LC/MS) platforms in order to enable the acquisition of more precise and accurate quantitative data. This necessity is heightened by the evolving trend of verifying and validating candidate disease biomarkers in complex biofluids, such as blood plasma, through targeted multiple reaction monitoring (MRM)-based approaches with stable isotope-labeled standards (SIS). Considering the lack of performance standards for quantitative plasma proteomics, we previously developed two reference kits to evaluate the MRM with SIS peptide approach using undepleted and non-enriched human plasma. The first kit tests the effectiveness of the LC/MRM-MS platform (kit #1), while the second evaluates the performance of an entire analytical workflow (kit #2). Here, these kits have been refined for practical use and then evaluated through intra- and inter-laboratory testing on 6 common LC/MS platforms. For an identical panel of 22 plasma proteins, similar concentrations were determined, regardless of the kit, instrument platform, and laboratory of analysis. These results demonstrate the value of the kit and reinforce the utility of standardized methods and protocols. The proteomics community needs standardized experimental protocols and quality control methods in order to improve the reproducibility of MS-based quantitative data. This need is heightened by the evolving trend for MRM-based validation of proposed disease biomarkers in complex biofluids such as blood plasma. We have developed two kits to assist in the inter- and intra-laboratory quality control of MRM experiments: the first kit tests the effectiveness of the LC/MRM-MS platform (kit #1), while the second evaluates the performance of an entire analytical workflow (kit #2). In this paper, we report the use of these kits in intra- and inter-laboratory testing on 6 common LC/MS platforms. This article is part of a Special Issue entitled: Standardization and Quality Control in Proteomics. © 2013.

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

Tissue proteomics of the low-molecular weight proteome using an integrated cLC-ESI-QTOFMS approach.

PubMed

Alvarez, MeiHwa Tanielle Bench; Shah, Dipti Jigar; Thulin, Craig D; Graves, Steven W

2013-05-01

Analysis of the protein/peptide composition of tissue has provided meaningful insights into tissue biology and even disease mechanisms. However, little has been published regarding top down methods to investigate lower molecular weight (MW) (500-5000 Da) species in tissue. Here, we evaluate a tissue proteomics approach involving tissue homogenization followed by depletion of large proteins and then cLC-MS (where c stands for capillary) analysis to interrogate the low MW/low abundance tissue proteome. In the development of this method, sheep heart, lung, liver, kidney, and spleen were surveyed to test our ability to observe tissue differences. After categorical tissue differences were demonstrated, a detailed study of this method's reproducibility was undertaken to determine whether or not it is suitable for analyzing more subtle differences in the abundance of small proteins and peptides. Our results suggest that this method should be useful in exploring the low MW proteome of tissues. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Noninvasive diagnosis of intraamniotic infection: proteomic biomarkers in vaginal fluid.

PubMed

Hitti, Jane; Lapidus, Jodi A; Lu, Xinfang; Reddy, Ashok P; Jacob, Thomas; Dasari, Surendra; Eschenbach, David A; Gravett, Michael G; Nagalla, Srinivasa R

2010-07-01

We analyzed the vaginal fluid proteome to identify biomarkers of intraamniotic infection among women in preterm labor. Proteome analysis was performed on vaginal fluid specimens from women with preterm labor, using multidimensional liquid chromatography, tandem mass spectrometry, and label-free quantification. Enzyme immunoassays were used to quantify candidate proteins. Classification accuracy for intraamniotic infection (positive amniotic fluid bacterial culture and/or interleukin-6 >2 ng/mL) was evaluated using receiver-operator characteristic curves obtained by logistic regression. Of 170 subjects, 30 (18%) had intraamniotic infection. Vaginal fluid proteome analysis revealed 338 unique proteins. Label-free quantification identified 15 proteins differentially expressed in intraamniotic infection, including acute-phase reactants, immune modulators, high-abundance amniotic fluid proteins and extracellular matrix-signaling factors; these findings were confirmed by enzyme immunoassay. A multi-analyte algorithm showed accurate classification of intraamniotic infection. Vaginal fluid proteome analyses identified proteins capable of discriminating between patients with and without intraamniotic infection. Copyright (c) 2010 Mosby, Inc. All rights reserved.

Hepatic Proteomic Analysis Revealed Altered Metabolic Pathways in Insulin Resistant Akt1+/-/Akt2-/-Mice

PubMed Central

Pedersen, Brian A; Wang, Weiwen; Taylor, Jared F; Khattab, Omar S; Chen, Yu-Han; Edwards, Robert A; Yazdi, Puya G; Wang, Ping H

2015-01-01

Objective The aim of this study was to identify liver proteome changes in a mouse model of severe insulin resistance and markedly decreased leptin levels. Methods Two-dimensional differential gel electrophoresis was utilized to identify liver proteome changes in AKT1+/-/AKT2-/- mice. Proteins with altered levels were identified with tandem mass spectrometry. Ingenuity Pathway analysis was performed for the interpretation of the biological significance of the observed proteomic changes. Results 11 proteins were identified from 2 biological replicates to be differentially expressed by a ratio of at least 1.3 between age-matched insulin resistant (Akt1+/-/Akt2-/-) and wild type mice. Albumin and mitochondrial ornithine aminotransferase were detected from multiple spots, which suggest post-translational modifications. Enzymes of the urea cycle were common members of top regulated pathways. Conclusion Our results help to unveil the regulation of the liver proteome underlying altered metabolism in an animal model of severe insulin resistance. PMID:26455965

Proteomic Analysis of Mitotic RNA Polymerase II Reveals Novel Interactors and Association With Proteins Dysfunctional in Disease*

PubMed Central

Möller, André; Xie, Sheila Q.; Hosp, Fabian; Lang, Benjamin; Phatnani, Hemali P.; James, Sonya; Ramirez, Francisco; Collin, Gayle B.; Naggert, Jürgen K.; Babu, M. Madan; Greenleaf, Arno L.; Selbach, Matthias; Pombo, Ana

2012-01-01

RNA polymerase II (RNAPII) transcribes protein-coding genes in eukaryotes and interacts with factors involved in chromatin remodeling, transcriptional activation, elongation, and RNA processing. Here, we present the isolation of native RNAPII complexes using mild extraction conditions and immunoaffinity purification. RNAPII complexes were extracted from mitotic cells, where they exist dissociated from chromatin. The proteomic content of native complexes in total and size-fractionated extracts was determined using highly sensitive LC-MS/MS. Protein associations with RNAPII were validated by high-resolution immunolocalization experiments in both mitotic cells and in interphase nuclei. Functional assays of transcriptional activity were performed after siRNA-mediated knockdown. We identify >400 RNAPII associated proteins in mitosis, among these previously uncharacterized proteins for which we show roles in transcriptional elongation. We also identify, as novel functional RNAPII interactors, two proteins involved in human disease, ALMS1 and TFG, emphasizing the importance of gene regulation for normal development and physiology. PMID:22199231

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

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

Global changes of the RNA-bound proteome during the maternal-to-zygotic transition in Drosophila

PubMed Central

Sysoev, Vasiliy O.; Fischer, Bernd; Frese, Christian K.; Gupta, Ishaan; Krijgsveld, Jeroen; Hentze, Matthias W.; Castello, Alfredo; Ephrussi, Anne

2016-01-01

The maternal-to-zygotic transition (MZT) is a process that occurs in animal embryos at the earliest developmental stages, during which maternally deposited mRNAs and other molecules are degraded and replaced by products of the zygotic genome. The zygotic genome is not activated immediately upon fertilization, and in the pre-MZT embryo post-transcriptional control by RNA-binding proteins (RBPs) orchestrates the first steps of development. To identify relevant Drosophila RBPs organism-wide, we refined the RNA interactome capture method for comparative analysis of the pre- and post-MZT embryos. We determine 523 proteins as high-confidence RBPs, half of which were not previously reported to bind RNA. Comparison of the RNA interactomes of pre- and post-MZT embryos reveals high dynamicity of the RNA-bound proteome during early development, and suggests active regulation of RNA binding of some RBPs. This resource provides unprecedented insight into the system of RBPs that govern the earliest steps of Drosophila development. PMID:27378189

Short term changes in the proteome of human cerebral organoids induced by 5-MeO-DMT.

PubMed

Dakic, Vanja; Minardi Nascimento, Juliana; Costa Sartore, Rafaela; Maciel, Renata de Moraes; de Araujo, Draulio B; Ribeiro, Sidarta; Martins-de-Souza, Daniel; Rehen, Stevens K

2017-10-09

Dimethyltryptamines are entheogenic serotonin-like molecules present in traditional Amerindian medicine recently associated with cognitive gains, antidepressant effects, and changes in brain areas related to attention. Legal restrictions and the lack of adequate experimental models have limited the understanding of how such substances impact human brain metabolism. Here we used shotgun mass spectrometry to explore proteomic differences induced by 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) on human cerebral organoids. Out of the 6,728 identified proteins, 934 were found differentially expressed in 5-MeO-DMT-treated cerebral organoids. In silico analysis reinforced previously reported anti-inflammatory actions of 5-MeO-DMT and revealed modulatory effects on proteins associated with long-term potentiation, the formation of dendritic spines, including those involved in cellular protrusion formation, microtubule dynamics, and cytoskeletal reorganization. Our data offer the first insight about molecular alterations caused by 5-MeO-DMT in human cerebral organoids.

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.

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 analysis of pollination-induced corolla senescence in petunia.

PubMed

Bai, Shuangyi; Willard, Belinda; Chapin, Laura J; Kinter, Michael T; Francis, David M; Stead, Anthony D; Jones, Michelle L

2010-02-01

Senescence represents the last phase of petal development during which macromolecules and organelles are degraded and nutrients are recycled to developing tissues. To understand better the post-transcriptional changes regulating petal senescence, a proteomic approach was used to profile protein changes during the senescence of Petuniaxhybrida 'Mitchell Diploid' corollas. Total soluble proteins were extracted from unpollinated petunia corollas at 0, 24, 48, and 72 h after flower opening and at 24, 48, and 72 h after pollination. Two-dimensional gel electrophoresis (2-DE) was used to identify proteins that were differentially expressed in non-senescing (unpollinated) and senescing (pollinated) corollas, and image analysis was used to determine which proteins were up- or down-regulated by the experimentally determined cut-off of 2.1-fold for P <0.05. One hundred and thirty-three differentially expressed protein spots were selected for sequencing. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine the identity of these proteins. Searching translated EST databases and the NCBI non-redundant protein database, it was possible to assign a putative identification to greater than 90% of these proteins. Many of the senescence up-regulated proteins were putatively involved in defence and stress responses or macromolecule catabolism. Some proteins, not previously characterized during flower senescence, were identified, including an orthologue of the tomato abscisic acid stress ripening protein 4 (ASR4). Gene expression patterns did not always correlate with protein expression, confirming that both proteomic and genomic approaches will be required to obtain a detailed understanding of the regulation of petal senescence.

Proteomic insights into the protective mechanisms of an in vitro oxidative stress model of early stage Parkinson's disease.

PubMed

Bauereis, Brian; Haskins, William E; Lebaron, Richard G; Renthal, Robert

2011-01-13

Previous studies in Parkinson's disease (PD) models suggest that early events along the path to neurodegeneration involve activation of the ubiquitin-proteasome system (UPS), endoplasmic reticulum-associated degradation (ERAD), and the unfolded protein response (UPR) pathways, in both the sporadic and familial forms of the disease, and thus ER stress may be a common feature. Furthermore, impairments in protein degradation have been linked to oxidative stress as well as pathways associated with ER stress. We hypothesize that oxidative stress is a primary initiator in a multi-factorial cascade driving dopaminergic (DA) neurons towards death in the early stages of the disease. We now report results from proteomic analysis of a rotenone-induced oxidative stress model of PD in the human neuroblastoma cell line, SH-SY5Y. Cells were exposed to sub-micromolar concentrations of rotenone for 48h prior to whole cell protein extraction and shotgun proteomic analysis. Evidence for activation of the UPR comes from our observation of up-regulated binding immunoglobulin protein (BiP), heat shock proteins, and foldases. We also observed up-regulation of proteins that contribute to the degradation of misfolded or unfolded proteins controlled by the UPS and ERAD pathways. Activation of the UPR may allow neurons to maintain protein homeostasis in the cytosol and ER despite an increase in reactive oxygen species due to oxidative stress, and activation of the UPS and ERAD may further augment clean-up and quality control in the cell. Copyright © 2010 Elsevier Ireland 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

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

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

Enhanced Missing Proteins Detection in NCI60 Cell Lines Using an Integrative Search Engine Approach.

PubMed

Guruceaga, Elizabeth; Garin-Muga, Alba; Prieto, Gorka; Bejarano, Bartolomé; Marcilla, Miguel; Marín-Vicente, Consuelo; Perez-Riverol, Yasset; Casal, J Ignacio; Vizcaíno, Juan Antonio; Corrales, Fernando J; Segura, Victor

2017-12-01

The Human Proteome Project (HPP) aims deciphering the complete map of the human proteome. In the past few years, significant efforts of the HPP teams have been dedicated to the experimental detection of the missing proteins, which lack reliable mass spectrometry evidence of their existence. In this endeavor, an in depth analysis of shotgun experiments might represent a valuable resource to select a biological matrix in design validation experiments. In this work, we used all the proteomic experiments from the NCI60 cell lines and applied an integrative approach based on the results obtained from Comet, Mascot, OMSSA, and X!Tandem. This workflow benefits from the complementarity of these search engines to increase the proteome coverage. Five missing proteins C-HPP guidelines compliant were identified, although further validation is needed. Moreover, 165 missing proteins were detected with only one unique peptide, and their functional analysis supported their participation in cellular pathways as was also proposed in other studies. Finally, we performed a combined analysis of the gene expression levels and the proteomic identifications from the common cell lines between the NCI60 and the CCLE project to suggest alternatives for further validation of missing protein observations.

Enhanced Missing Proteins Detection in NCI60 Cell Lines Using an Integrative Search Engine Approach

PubMed Central

2017-01-01

The Human Proteome Project (HPP) aims deciphering the complete map of the human proteome. In the past few years, significant efforts of the HPP teams have been dedicated to the experimental detection of the missing proteins, which lack reliable mass spectrometry evidence of their existence. In this endeavor, an in depth analysis of shotgun experiments might represent a valuable resource to select a biological matrix in design validation experiments. In this work, we used all the proteomic experiments from the NCI60 cell lines and applied an integrative approach based on the results obtained from Comet, Mascot, OMSSA, and X!Tandem. This workflow benefits from the complementarity of these search engines to increase the proteome coverage. Five missing proteins C-HPP guidelines compliant were identified, although further validation is needed. Moreover, 165 missing proteins were detected with only one unique peptide, and their functional analysis supported their participation in cellular pathways as was also proposed in other studies. Finally, we performed a combined analysis of the gene expression levels and the proteomic identifications from the common cell lines between the NCI60 and the CCLE project to suggest alternatives for further validation of missing protein observations. PMID:28960077

Hepatic SILAC proteomic data from PANDER transgenic model.

PubMed

Athanason, Mark G; Stevens, Stanley M; Burkhardt, Brant R

2016-12-01

This article contains raw and processed data related to research published in "Quantitative Proteomic Profiling Reveals Hepatic Lipogenesis and Liver X Receptor Activation in the PANDER Transgenic Model" (M.G. Athanason, W.A. Ratliff, D. Chaput, C.B. MarElia, M.N. Kuehl, S.M., Jr. Stevens, B.R. Burkhardt (2016)) [1], and was generated by "spike-in" SILAC-based proteomic analysis of livers obtained from the PANcreatic-Derived factor (PANDER) transgenic mouse (PANTG) under various metabolic conditions [1]. The mass spectrometry output of the PANTG and wild-type B6SJLF mice liver tissue and resulting proteome search from MaxQuant 1.2.2.5 employing the Andromeda search algorithm against the UniprotKB reference database for Mus musculus has been deposited to the ProteomeXchange Consortium (http://www.proteomexchange.org) via the PRIDE partner repository with dataset identifiers PRIDE: PXD004171 and doi:10.6019/PXD004171. Protein ratio values representing PANTG/wild-type obtained by MaxQuant analysis were input into the Perseus processing suite to determine statistical significance using the Significance A outlier test (p<0.05). Differentially expressed proteins using this approach were input into Ingenuity Pathway Analysis to determined altered pathways and upstream regulators that were altered in PANTG mice.

Evaluation of different multidimensional LC-MS/MS pipelines for iTRAQ-based proteomic analysis of potato tubers in response to cold storage

USDA-ARS?s Scientific Manuscript database

Cold-induced sweetening in potato tubers is a costly problem for food industry. To systematically identify the proteins associated with this process, we employed a comparative proteomics approach using isobaric, stable isotope coded labels to compare the proteomes of potato tubers after 0 and 5 mont...