Comparative Proteomic Analysis of Whole-Gut Lavage Fluid and Pancreatic Juice Reveals a Less Invasive Method of Sampling Pancreatic Secretions

PubMed Central

Rocker, Jana M; Tan, Marcus C; Thompson, Lee W; Contreras, Carlo M; DiPalma, Jack A; Pannell, Lewis K

2016-01-01

OBJECTIVES: There are currently no reliable, non-invasive screening tests for pancreatic ductal adenocarcinoma. The fluid secreted from the pancreatic ductal system (“pancreatic juice”) has been well-studied as a potential source of cancer biomarkers. However, it is invasive to collect. We recently observed that the proteomic profile of intestinal effluent from the bowel in response to administration of an oral bowel preparation solution (also known as whole-gut lavage fluid, WGLF) contains large amounts of pancreas-derived proteins. We therefore hypothesized that the proteomic profile is similar to that of pancreatic juice. In this study, we compared the proteomic profiles of 77 patients undergoing routine colonoscopy with the profiles of 19 samples of pure pancreatic juice collected during surgery. METHODS: WGLF was collected from patients undergoing routine colonoscopy, and pancreatic juice was collected from patients undergoing pancreatic surgery. Protein was isolated from both samples using an optimized method and analyzed by LC-MS/MS. Identified proteins were compared between samples and groups to determine similarity of the two fluids. We then compared our results with literature reports of pancreatic juice-based studies to determine similarity. RESULTS: We found 104 proteins in our pancreatic juice samples, of which 90% were also found in our WGLF samples. The majority (67%) of the total proteins found in the WGLF were common to pancreatic juice, with intestine-specific proteins making up a smaller proportion. CONCLUSIONS: WGLF and pancreatic juice appear to have similar proteomic profiles. This supports the notion that WGLF is a non-invasive, surrogate bio-fluid for pancreatic juice. Further studies are required to further elucidate its role in the diagnosis of pancreatic cancer. PMID:27228405

Proteomics reveals the effects of sustained weight loss on the human plasma proteome.

PubMed

Geyer, Philipp E; Wewer Albrechtsen, Nicolai J; Tyanova, Stefka; Grassl, Niklas; Iepsen, Eva W; Lundgren, Julie; Madsbad, Sten; Holst, Jens J; Torekov, Signe S; Mann, Matthias

2016-12-22

Sustained weight loss is a preferred intervention in a wide range of metabolic conditions, but the effects on an individual's health state remain ill-defined. Here, we investigate the plasma proteomes of a cohort of 43 obese individuals that had undergone 8 weeks of 12% body weight loss followed by a year of weight maintenance. Using mass spectrometry-based plasma proteome profiling, we measured 1,294 plasma proteomes. Longitudinal monitoring of the cohort revealed individual-specific protein levels with wide-ranging effects of losing weight on the plasma proteome reflected in 93 significantly affected proteins. The adipocyte-secreted SERPINF1 and apolipoprotein APOF1 were most significantly regulated with fold changes of -16% and +37%, respectively (P < 10 -13 ), and the entire apolipoprotein family showed characteristic differential regulation. Clinical laboratory parameters are reflected in the plasma proteome, and eight plasma proteins correlated better with insulin resistance than the known marker adiponectin. Nearly all study participants benefited from weight loss regarding a ten-protein inflammation panel defined from the proteomics data. We conclude that plasma proteome profiling broadly evaluates and monitors intervention in metabolic diseases. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

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

PubMed Central

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

2015-01-01

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

Proteome dynamics of cold-acclimating Rhododendron species contrasting in their freezing tolerance and thermonasty response

USDA-ARS?s Scientific Manuscript database

In the present study we used 2D-DIGE technique to document the Rhododendron proteome during the seasonal development of cold hardiness. We selected two genotypes with different cold hardiness levels. This enabled us to perform comparative analysis of their proteome profiles and screen differentially...

Schizophrenia proteomics: biomarkers on the path to laboratory medicine?

PubMed Central

Lakhan, Shaheen Emmanuel

2006-01-01

Over two million Americans are afflicted with schizophrenia, a debilitating mental health disorder with a unique symptomatic and epidemiological profile. Genomics studies have hinted towards candidate schizophrenia susceptibility chromosomal loci and genes. Modern proteomic tools, particularly mass spectrometry and expression scanning, aim to identify both pathogenic-revealing and diagnostically significant biomarkers. Only a few studies on basic proteomics have been conducted for psychiatric disorders relative to the plethora of cancer specific experiments. One such proteomic utility enables the discovery of proteins and biological marker fingerprinting profiling techniques (SELDI-TOF-MS), and then subjects them to tandem mass spectrometric fragmentation and de novo protein sequencing (MALDI-TOF/TOF-MS) for the accurate identification and characterization of the proteins. Such utilities can explain the pathogenesis of neuro-psychiatric disease, provide more objective testing methods, and further demonstrate a biological basis to mental illness. Although clinical proteomics in schizophrenia have yet to reveal a biomarker with diagnostic specificity, methods that better characterize the disorder using endophenotypes can advance findings. Schizophrenia biomarkers could potentially revolutionize its psychopharmacology, changing it into a more hypothesis and genomic/proteomic-driven science. PMID:16846510

High-throughput and targeted in-depth mass spectrometry-based approaches for biofluid profiling and biomarker discovery.

PubMed

Jimenez, Connie R; Piersma, Sander; Pham, Thang V

2007-12-01

Proteomics aims to create a link between genomic information, biological function and disease through global studies of protein expression, modification and protein-protein interactions. Recent advances in key proteomics tools, such as mass spectrometry (MS) and (bio)informatics, provide tremendous opportunities for biomarker-related clinical applications. In this review, we focus on two complementary MS-based approaches with high potential for the discovery of biomarker patterns and low-abundant candidate biomarkers in biofluids: high-throughput matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy-based methods for peptidome profiling and label-free liquid chromatography-based methods coupled to MS for in-depth profiling of biofluids with a focus on subproteomes, including the low-molecular-weight proteome, carrier-bound proteome and N-linked glycoproteome. The two approaches differ in their aims, throughput and sensitivity. We discuss recent progress and challenges in the analysis of plasma/serum and proximal fluids using these strategies and highlight the potential of liquid chromatography-MS-based proteomics of cancer cell and tumor secretomes for the discovery of candidate blood-based biomarkers. Strategies for candidate validation are also described.

Analysis of proteome profile in germinating soybean seed, and its comparison with rice showing the styles of reserves mobilization in different crops.

PubMed

Han, Chao; Yin, Xiaojian; He, Dongli; Yang, Pingfang

2013-01-01

Seed germination is a complex physiological process during which mobilization of nutrient reserves happens. In different crops, this event might be mediated by different regulatory and metabolic pathways. Proteome profiling has been proved to be an efficient way that can help us to construct these pathways. However, no such studies have been performed in soybean germinating seeds up to date. Proteome profiling was conducted through one-dimensional gel electrophoresis followed by liquid chromatography and tandem mass spectrometry strategy in the germinating seeds of soybean (glycine max). Comprehensive comparisons were also carried out between rice and soybean germinating seeds. 764 proteins belonging to 14 functional groups were identified and metabolism related proteins were the largest group. Deep analyses of the proteins and pathways showed that lipids were degraded through lipoxygenase dependent pathway and proteins were degraded through both protease and 26S proteosome system, and the lipoxygenase could also help to remove the reactive oxygen species during the rapid mobilization of reserves of soybean germinating seeds. The differences between rice and soybean germinating seeds proteome profiles indicate that each crop species has distinct mechanism for reserves mobilization during germination. Different reserves could be converted into starches before they are totally utilized during the germination in different crops seeds. This study is the first comprehensive analysis of proteome profile in germinating soybean seeds to date. The data presented in this paper will improve our understanding of the physiological and biochemical status in the imbibed soybean seeds just prior to germination. Comparison of the protein profile with that of germinating rice seeds gives us new insights on mobilization of nutrient reserves during the germination of crops seeds.

Differential proteomic profiling of primary and recurrent chordomas.

PubMed

Chen, Su; Xu, Wei; Jiao, Jian; Jiang, Dongjie; Liu, Jian; Chen, Tenghui; Wan, Zongmiao; Xu, Leqin; Zhou, Zhenhua; Xiao, Jianru

2015-05-01

Chordomas are locally destructive tumors with high rates of recurrence and a poor prognosis. The mechanisms involved in chordoma recurrence remain largely unknown. In the present study, we examined the proteomic profile of a chordoma primary tumor (CSO) and a recurrent tumor (CSR) through mass spectrum in a chordoma patient who underwent surgery. Bioinformatic analysis of the profile showed that 359 proteins had a significant expression difference and 21 pathways had a striking alteration between the CSO and the CSR. The CSR showed a significant increase in carbohydrate metabolism. Immunohistochemistry (IHC) confirmed that the cancer stem cell marker activated leukocyte cell adhesion molecule (ALCAM or CD166) expression level was higher in the recurrent than that in the primary tumor. The present study analyzed the proteomic profile change between CSO and CSR and identified a new biomarker ALCAM in recurrent chordomas. This finding sheds light on unraveling the pathophysiology of chordoma recurrence and on exploring more effective prognostic biomarkers and targeted therapies against this devastating disease.

Differential expression profiling of serum proteins and metabolites for biomarker discovery

NASA Astrophysics Data System (ADS)

Roy, Sushmita Mimi; Anderle, Markus; Lin, Hua; Becker, Christopher H.

2004-11-01

A liquid chromatography-mass spectrometry (LC-MS) proteomics and metabolomics platform is presented for quantitative differential expression analysis. Proteome profiles obtained from 1.5 [mu]L of human serum show ~5000 de-isotoped and quantifiable molecular ions. Approximately 1500 metabolites are observed from 100 [mu]L of serum. Quantification is based on reproducible sample preparation and linear signal intensity as a function of concentration. The platform is validated using human serum, but is generally applicable to all biological fluids and tissues. The median coefficient of variation (CV) for ~5000 proteomic and ~1500 metabolomic molecular ions is approximately 25%. For the case of C-reactive protein, results agree with quantification by immunoassay. The independent contributions of two sources of variance, namely sample preparation and LC-MS analysis, are respectively quantified as 20.4 and 15.1% for the proteome, and 19.5 and 13.5% for the metabolome, for median CV values. Furthermore, biological diversity for ~20 healthy individuals is estimated by measuring the variance of ~6500 proteomic and metabolomic molecular ions in sera for each sample; the median CV is 22.3% for the proteome and 16.7% for the metabolome. Finally, quantitative differential expression profiling is applied to a clinical study comparing healthy individuals and rheumatoid arthritis (RA) patients.

Proteomic Profiling of Rat Thyroarytenoid Muscle

ERIC Educational Resources Information Center

Welham, Nathan V.; Marriott, Gerard; Bless, Diane M.

2006-01-01

Purpose: Proteomic methodologies offer promise in elucidating the systemwide cellular and molecular processes that characterize normal and diseased thyroarytenoid (TA) muscle. This study examined methodological issues central to the application of 2-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D SDS-PAGE) to the study of…

Metabolome and proteome profiling of complex I deficiency induced by rotenone.

PubMed

Gielisch, Ina; Meierhofer, David

2015-01-02

Complex I (CI; NADH dehydrogenase) deficiency causes mitochondrial diseases, including Leigh syndrome. A variety of clinical symptoms of CI deficiency are known, including neurodegeneration. Here, we report an integrative study combining liquid chromatography-mass spectrometry (LC-MS)-based metabolome and proteome profiling in CI deficient HeLa cells. We report a rapid LC-MS-based method for the relative quantification of targeted metabolome profiling with an additional layer of confidence by applying multiple reaction monitoring (MRM) ion ratios for further identity confirmation and robustness. The proteome was analyzed by label-free quantification (LFQ). More than 6000 protein groups were identified. Pathway and network analyses revealed that the respiratory chain was highly deregulated, with metabolites such as FMN, FAD, NAD(+), and ADP, direct players of the OXPHOS system, and metabolites of the TCA cycle decreased up to 100-fold. Synthesis of functional iron-sulfur clusters, which are of central importance for the electron transfer chain, and degradation products like bilirubin were also significantly reduced. Glutathione metabolism on the pathway level, as well as individual metabolite components such as NADPH, glutathione (GSH), and oxidized glutathione (GSSG), was downregulated. Overall, metabolome and proteome profiles in CI deficient cells correlated well, supporting our integrated approach.

Genetic differences in the serum proteome of horses, donkeys and mules are detectable by protein profiling.

PubMed

Henze, Andrea; Aumer, Franziska; Grabner, Arthur; Raila, Jens; Schweigert, Florian J

2011-10-01

Although horses and donkeys belong to the same genus, their genetic characteristics probably result in specific proteomes and post-translational modifications (PTM) of proteins. Since PTM can alter protein properties, specific PTM may contribute to species-specific characteristics. Therefore, the aim of the present study was to analyse differences in serum protein profiles of horses and donkeys as well as mules, which combine the genetic backgrounds of both species. Additionally, changes in PTM of the protein transthyretin (TTR) were analysed. Serum protein profiles of each species (five animals per species) were determined using strong anion exchanger ProteinChips® (Bio-Rad, Munich, Germany) in combination with surface-enhanced laser desorption ionisation-time of flight MS. The PTM of TTR were analysed subsequently by immunoprecipitation in combination with matrix-assisted laser desorption ionisation-time of flight MS. Protein profiling revealed species-specific differences in the proteome, with some protein peaks present in all three species as well as protein peaks that were unique for donkeys and mules, horses and mules or for horses alone. The molecular weight of TTR of horses and donkeys differed by 30 Da, and both species revealed several modified forms of TTR besides the native form. The mass spectra of mules represented a merging of TTR spectra of horses and donkeys. In summary, the present study indicated that there are substantial differences in the proteome of horses and donkeys. Additionally, the results probably indicate that the proteome of mules reveal a higher similarity to donkeys than to horses.

Comparative proteomics and protein profile related to phenolic compounds and antioxidant activity in germinated Oryza sativa 'KDML105' and Thai brown rice 'Mali Daeng' for better nutritional value.

PubMed

Maksup, Sarunyaporn; Pongpakpian, Sarintip; Roytrakul, Sittiruk; Cha-Um, Suriyan; Supaibulwatana, Kanyaratt

2018-01-01

Brown rice (BR) and germinated brown rice (GBR) are considered as prime sources of carbohydrate and bioactive compounds for more than half of the populations worldwide. Several studies have reported on the proteomics of BR and GBR; however, the proteomic profiles related to the synthesis of bioactive compounds are less well documented. In the present study, BR and GBR were used in a comparative analysis of the proteomic and bioactive compound profiles for two famous Thai rice varieties: Khao Dawk Mali 105 (KDML) and Mali Daeng (MD). The proteomes of KDML and MD revealed differences in the expression patterns of proteins after germination. Total phenolic compound content, anthocyanin contents and antioxidant activity of red rice MD was approximately 2.6-, 2.2- and 9.2-fold higher, respectively, compared to that of the white rice KDML. Moreover, GBR of MD showed higher total anthocyanin content and greater antioxidant activity, which is consistent with the increase expression of several proteins involved in the biosynthesis of phenolic compounds and protection against oxidative stress. Red rice MD exhibits higher nutrient values compared to white rice KDML and the appropriate germination of brown rice could represent a method for improving health-related benefits. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Activity-based proteomics of enzyme superfamilies: serine hydrolases as a case study.

PubMed

Simon, Gabriel M; Cravatt, Benjamin F

2010-04-09

Genome sequencing projects have uncovered thousands of uncharacterized enzymes in eukaryotic and prokaryotic organisms. Deciphering the physiological functions of enzymes requires tools to profile and perturb their activities in native biological systems. Activity-based protein profiling has emerged as a powerful chemoproteomic strategy to achieve these objectives through the use of chemical probes that target large swaths of enzymes that share active-site features. Here, we review activity-based protein profiling and its implementation to annotate the enzymatic proteome, with particular attention given to probes that target serine hydrolases, a diverse superfamily of enzymes replete with many uncharacterized members.

Label-Free LC-MS/MS Proteomic Analysis of Cerebrospinal Fluid Identifies Protein/Pathway Alterations and Candidate Biomarkers for Amyotrophic Lateral Sclerosis.

PubMed

Collins, Mahlon A; An, Jiyan; Hood, Brian L; Conrads, Thomas P; Bowser, Robert P

2015-11-06

Analysis of the cerebrospinal fluid (CSF) proteome has proven valuable to the study of neurodegenerative disorders. To identify new protein/pathway alterations and candidate biomarkers for amyotrophic lateral sclerosis (ALS), we performed comparative proteomic profiling of CSF from sporadic ALS (sALS), healthy control (HC), and other neurological disease (OND) subjects using label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 1712 CSF proteins were detected and relatively quantified by spectral counting. Levels of several proteins with diverse biological functions were significantly altered in sALS samples. Enrichment analysis was used to link these alterations to biological pathways, which were predominantly related to inflammation, neuronal activity, and extracellular matrix regulation. We then used our CSF proteomic profiles to create a support vector machines classifier capable of discriminating training set ALS from non-ALS (HC and OND) samples. Four classifier proteins, WD repeat-containing protein 63, amyloid-like protein 1, SPARC-like protein 1, and cell adhesion molecule 3, were identified by feature selection and externally validated. The resultant classifier distinguished ALS from non-ALS samples with 83% sensitivity and 100% specificity in an independent test set. Collectively, our results illustrate the utility of CSF proteomic profiling for identifying ALS protein/pathway alterations and candidate disease biomarkers.

Technological advances for deciphering the complexity of psychiatric disorders: merging proteomics with cell biology.

PubMed

Wesseling, Hendrik; Guest, Paul C; Lago, Santiago G; Bahn, Sabine

2014-08-01

Proteomic studies have increased our understanding of the molecular pathways affected in psychiatric disorders. Mass spectrometry and two-dimensional gel electrophoresis analyses of post-mortem brain samples from psychiatric patients have revealed effects on synaptic, cytoskeletal, antioxidant and mitochondrial protein networks. Multiplex immunoassay profiling studies have found alterations in hormones, growth factors, transport and inflammation-related proteins in serum and plasma from living first-onset patients. Despite these advances, there are still difficulties in translating these findings into platforms for improved treatment of patients and for discovery of new drugs with better efficacy and side effect profiles. This review describes how the next phase of proteomic investigations in psychiatry should include stringent replication studies for validation of biomarker candidates and functional follow-up studies which can be used to test the impact on physiological function. All biomarker candidates should now be tested in series with traditional and emerging cell biological approaches. This should include investigations of the effects of post-translational modifications, protein dynamics and network analyses using targeted proteomic approaches. Most importantly, there is still an urgent need for development of disease-relevant cellular models for improved translation of proteomic findings into a means of developing novel drug treatments for patients with these life-altering disorders.

Saliva analysis by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) in orthodontic treatment: first pilot study.

PubMed

Ciavarella, Domenico; Mastrovincenzo, Mario; D'Onofrio, Valentina; Chimenti, Claudio; Parziale, Vincenzo; Barbato, Ersilia; Lo Muzio, Lorenzo

2011-11-01

SELDI-TOF-MS (Surface-Enhanced Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry) allows the generation of an accurate protein profile from minimal amounts of biological samples and may executes proteomic profile of saliva. The aim of this work is to compare the proteomic profile of saliva of patients in orthodontic treatment to the beginning of treatment and after three months by using the surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) technology. Saliva was collected from 14 patients, between the 11 and 17 years, to the beginning of the orthodontic treatment and after three months. Specimens were centrifuged (10 min, 13000 x g); the Q10 ProteinChips were prepared according to the manufacturer's instructions and were loaded with the supernatants. A saturated solution of sinapinic acid was used as energy-absorbing matrix. The analysis was performed in a m/z range from 2500 to 25000 Da, and the proteomic profiles were compared by a specific data analysis software. Saliva (5 mL) was collected by spitting directly into a clean 15 mL conical tube. The samples were then aliquotted and stored at -80°C until use. Profile of saliva of patients before orthodontic treatment present a number of peaks different respect profile of saliva after three months of treatment. The average intensities of peaks at m/z 3372, 5232, 4045 and 10128 were significantly higher after three months then at beginning of treatment in the same patients and among these one. The Roc Plot has demonstrated high sensitivity and specificity. Many differences were noted in salivary proteomic profile obtained using the SELDI-TOF-MS technology in patients in orthodontic treatment to beginning and after three months. These data suggest that the proteomic analysis of saliva is a promising new tool for a non-invasive study of oral mucosa and bone changes. Copyright © 2011 Società Italiana di Ortodonzia SIDO. Published by Elsevier Srl. All rights reserved.

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.

Human borna disease virus infection impacts host proteome and histone lysine acetylation in human oligodendroglia cells

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

Liu, Xia; Department of Neurology, The Fifth People's Hospital of Shanghai, School of Medicine, Fudan University, Shanghai, 200240; Zhao, Libo

2014-09-15

Background: Borna disease virus (BDV) replicates in the nucleus and establishes persistent infections in mammalian hosts. A human BDV strain was used to address the first time, how BDV infection impacts the proteome and histone lysine acetylation (Kac) of human oligodendroglial (OL) cells, thus allowing a better understanding of infection-driven pathophysiology in vitro. Methods: Proteome and histone lysine acetylation were profiled through stable isotope labeling for cell culture (SILAC)-based quantitative proteomics. The quantifiable proteome was annotated using bioinformatics. Histone acetylation changes were validated by biochemistry assays. Results: Post BDV infection, 4383 quantifiable differential proteins were identified and functionally annotated tomore » metabolism pathways, immune response, DNA replication, DNA repair, and transcriptional regulation. Sixteen of the thirty identified Kac sites in core histones presented altered acetylation levels post infection. Conclusions: BDV infection using a human strain impacted the whole proteome and histone lysine acetylation in OL cells. - Highlights: • A human strain of BDV (BDV Hu-H1) was used to infect human oligodendroglial cells (OL cells). • This study is the first to reveal the host proteomic and histone Kac profiles in BDV-infected OL cells. • BDV infection affected the expression of many transcription factors and several HATs and HDACs.« less

Label-free proteome of water buffalo (Bubalus bubalis) seminal plasma.

PubMed

Brito, Mayara F; Auler, Patrícia A; Tavares, Guilherme C; Rezende, Cristiana P; Almeida, Gabriel M F; Pereira, Felipe L; Leal, Carlos A G; Moura, Arlindo de Alencar; Figueiredo, Henrique C P; Henry, Marc

2018-06-11

The study aimed to describe the Bubalus bubalis seminal plasma proteome using a label-free shotgun UDMS E approach. A total of 859 nonredundant proteins were identified across five biological replicates with stringent identification. Proteins specifically related to sperm maturation and protection, capacitation, fertilization and metabolic activity were detected in the buffalo seminal fluid. In conclusion, we provide a comprehensive proteomic profile of buffalo seminal plasma, which establishes a foundation for further studies designed to understand regulation of sperm function and discovery of novel biomarkers for fertility. MS data are available in the ProteomeXchange with identifier PXD003728. © 2018 Blackwell Verlag GmbH.

Hospitalized Premature Infants Are Colonized by Related Bacterial Strains with Distinct Proteomic Profiles

PubMed Central

Xiong, Weili; Olm, Matthew R.; Thomas, Brian C.; Baker, Robyn; Firek, Brian; Morowitz, Michael J.; Hettich, Robert L.

2018-01-01

ABSTRACT During the first weeks of life, microbial colonization of the gut impacts human immune system maturation and other developmental processes. In premature infants, aberrant colonization has been implicated in the onset of necrotizing enterocolitis (NEC), a life-threatening intestinal disease. To study the premature infant gut colonization process, genome-resolved metagenomics was conducted on 343 fecal samples collected during the first 3 months of life from 35 premature infants housed in a neonatal intensive care unit, 14 of whom developed NEC, and metaproteomic measurements were made on 87 samples. Microbial community composition and proteomic profiles remained relatively stable on the time scale of a week, but the proteome was more variable. Although genetically similar organisms colonized many infants, most infants were colonized by distinct strains with metabolic profiles that could be distinguished using metaproteomics. Microbiome composition correlated with infant, antibiotics administration, and NEC diagnosis. Communities were found to cluster into seven primary types, and community type switched within infants, sometimes multiple times. Interestingly, some communities sampled from the same infant at subsequent time points clustered with those of other infants. In some cases, switches preceded onset of NEC; however, no species or community type could account for NEC across the majority of infants. In addition to a correlation of protein abundances with organism replication rates, we found that organism proteomes correlated with overall community composition. Thus, this genome-resolved proteomics study demonstrated that the contributions of individual organisms to microbiome development depend on microbial community context. PMID:29636439

Multilineage potential and proteomic profiling of human dental stem cells derived from a single donor

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

Patil, Rajreddy; Kumar, B. Mohana; Lee, Won-Jae

Dental tissues provide an alternative autologous source of mesenchymal stem cells (MSCs) for regenerative medicine. In this study, we isolated human dental MSCs of follicle, pulp and papilla tissue from a single donor tooth after impacted third molar extraction by excluding the individual differences. We then compared the morphology, proliferation rate, expression of MSC-specific and pluripotency markers, and in vitro differentiation ability into osteoblasts, adipocytes, chondrocytes and functional hepatocyte-like cells (HLCs). Finally, we analyzed the protein expression profiles of undifferentiated dental MSCs using 2DE coupled with MALDI-TOF-MS. Three types of dental MSCs largely shared similar morphology, proliferation potential, expression ofmore » surface markers and pluripotent transcription factors, and differentiation ability into osteoblasts, adipocytes, and chondrocytes. Upon hepatogenic induction, all MSCs were transdifferentiated into functional HLCs, and acquired hepatocyte functions by showing their ability for glycogen storage and urea production. Based on the proteome profiling results, we identified nineteen proteins either found commonly or differentially expressed among the three types of dental MSCs. In conclusion, three kinds of dental MSCs from a single donor tooth possessed largely similar cellular properties and multilineage potential. Further, these dental MSCs had similar proteomic profiles, suggesting their interchangeable applications for basic research and call therapy. - Highlights: • Isolated and characterized three types of human dental MSCs from a single donor. • MSCs of dental follicle, pulp and papilla had largely similar biological properties. • All MSCs were capable of transdifferentiating into functional hepatocyte-like cells. • 2DE proteomics with MALDI-TOF/MS identified 19 proteins in three types of MSCs. • Similar proteomic profiles suggest interchangeable applications of dental MSCs.« less

Proteomic Profiling of Bladders from Mice Exposed with Sodium Arsenite

EPA Science Inventory

Arsenic, an environmental contaminant, has been linked with cancer of the bladder in humans. To study the mode of action of arsenic, female CH3 mice were exposed to 85 ppm sodium arsenite in their drinking water for 30 days. Following the exposure a comparative proteomic analysis...

Trauma-associated Human Neutrophil Alterations Revealed by Comparative Proteomics Profiling

PubMed Central

Zhou, Jian-Ying; Krovvidi, Ravi K.; Gao, Yuqian; Gao, Hong; Petritis, Brianne O.; De, Asit; Miller-Graziano, Carol; Bankey, Paul E.; Petyuk, Vladislav A.; Nicora, Carrie D.; Clauss, Therese R; Moore, Ronald J.; Shi, Tujin; Brown, Joseph N.; Kaushal, Amit; Xiao, Wenzhong; Davis, Ronald W.; Maier, Ronald V.; Tompkins, Ronald G.; Qian, Wei-Jun; Camp, David G.; Smith, Richard D.

2013-01-01

PURPOSE Polymorphonuclear neutrophils (PMNs) play an important role in mediating the innate immune response after severe traumatic injury; however, the cellular proteome response to traumatic condition is still largely unknown. EXPERIMENTAL DESIGN We applied 2D-LC-MS/MS based shotgun proteomics to perform comparative proteome profiling of human PMNs from severe trauma patients and healthy controls. RESULTS A total of 197 out of ~2500 proteins (being identified with at least two peptides) were observed with significant abundance changes following the injury. The proteomics data were further compared with transcriptomics data for the same genes obtained from an independent patient cohort. The comparison showed that the protein abundance changes for the majority of proteins were consistent with the mRNA abundance changes in terms of directions of changes. Moreover, increased protein secretion was suggested as one of the mechanisms contributing to the observed discrepancy between protein and mRNA abundance changes. Functional analyses of the altered proteins showed that many of these proteins were involved in immune response, protein biosynthesis, protein transport, NRF2-mediated oxidative stress response, the ubiquitin-proteasome system, and apoptosis pathways. CONCLUSIONS AND CLINICAL RELEVANCE Our data suggest increased neutrophil activation and inhibited neutrophil apoptosis in response to trauma. The study not only reveals an overall picture of functional neutrophil response to trauma at the proteome level, but also provides a rich proteomics data resource of trauma-associated changes in the neutrophil that will be valuable for further studies of the functions of individual proteins in PMNs. PMID:23589343

A human protein atlas for normal and cancer tissues based on antibody proteomics.

PubMed

Uhlén, Mathias; Björling, Erik; Agaton, Charlotta; Szigyarto, Cristina Al-Khalili; Amini, Bahram; Andersen, Elisabet; Andersson, Ann-Catrin; Angelidou, Pia; Asplund, Anna; Asplund, Caroline; Berglund, Lisa; Bergström, Kristina; Brumer, Harry; Cerjan, Dijana; Ekström, Marica; Elobeid, Adila; Eriksson, Cecilia; Fagerberg, Linn; Falk, Ronny; Fall, Jenny; Forsberg, Mattias; Björklund, Marcus Gry; Gumbel, Kristoffer; Halimi, Asif; Hallin, Inga; Hamsten, Carl; Hansson, Marianne; Hedhammar, My; Hercules, Görel; Kampf, Caroline; Larsson, Karin; Lindskog, Mats; Lodewyckx, Wald; Lund, Jan; Lundeberg, Joakim; Magnusson, Kristina; Malm, Erik; Nilsson, Peter; Odling, Jenny; Oksvold, Per; Olsson, Ingmarie; Oster, Emma; Ottosson, Jenny; Paavilainen, Linda; Persson, Anja; Rimini, Rebecca; Rockberg, Johan; Runeson, Marcus; Sivertsson, Asa; Sköllermo, Anna; Steen, Johanna; Stenvall, Maria; Sterky, Fredrik; Strömberg, Sara; Sundberg, Mårten; Tegel, Hanna; Tourle, Samuel; Wahlund, Eva; Waldén, Annelie; Wan, Jinghong; Wernérus, Henrik; Westberg, Joakim; Wester, Kenneth; Wrethagen, Ulla; Xu, Lan Lan; Hober, Sophia; Pontén, Fredrik

2005-12-01

Antibody-based proteomics provides a powerful approach for the functional study of the human proteome involving the systematic generation of protein-specific affinity reagents. We used this strategy to construct a comprehensive, antibody-based protein atlas for expression and localization profiles in 48 normal human tissues and 20 different cancers. Here we report a new publicly available database containing, in the first version, approximately 400,000 high resolution images corresponding to more than 700 antibodies toward human proteins. Each image has been annotated by a certified pathologist to provide a knowledge base for functional studies and to allow queries about protein profiles in normal and disease tissues. Our results suggest it should be possible to extend this analysis to the majority of all human proteins thus providing a valuable tool for medical and biological research.

Proteomic Analysis Reveals Age-related Changes in Tendon Matrix Composition, with Age- and Injury-specific Matrix Fragmentation*

PubMed Central

Peffers, Mandy J.; Thorpe, Chavaunne T.; Collins, John A.; Eong, Robin; Wei, Timothy K. J.; Screen, Hazel R. C.; Clegg, Peter D.

2014-01-01

Energy storing tendons, such as the human Achilles and equine superficial digital flexor tendon (SDFT), are highly prone to injury, the incidence of which increases with aging. The cellular and molecular mechanisms that result in increased injury in aged tendons are not well established but are thought to result in altered matrix turnover. However, little attempt has been made to fully characterize the tendon proteome nor determine how the abundance of specific tendon proteins changes with aging and/or injury. The aim of this study was, therefore, to assess the protein profile of normal SDFTs from young and old horses using label-free relative quantification to identify differentially abundant proteins and peptide fragments between age groups. The protein profile of injured SDFTs from young and old horses was also assessed. The results demonstrate distinct proteomic profiles in young and old tendon, with alterations in the levels of proteins involved in matrix organization and regulation of cell tension. Furthermore, we identified several new peptide fragments (neopeptides) present in aged tendons, suggesting that there are age-specific cleavage patterns within the SDFT. Proteomic profile also differed between young and old injured tendon, with a greater number of neopeptides identified in young injured tendon. This study has increased the knowledge of molecular events associated with tendon aging and injury, suggesting that maintenance and repair of tendon tissue may be reduced in aged individuals and may help to explain why the risk of injury increases with aging. PMID:25077967

Lipid remodeling and an altered membrane-associated proteome may drive the differential effects of EPA and DHA treatment on skeletal muscle glucose uptake and protein accretion.

PubMed

Jeromson, Stewart; Mackenzie, Ivor; Doherty, Mary K; Whitfield, Phillip D; Bell, Gordon; Dick, James; Shaw, Andy; Rao, Francesco V; Ashcroft, Stephen P; Philp, Andrew; Galloway, Stuart D R; Gallagher, Iain; Hamilton, D Lee

2018-06-01

In striated muscle, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have differential effects on the metabolism of glucose and differential effects on the metabolism of protein. We have shown that, despite similar incorporation, treatment of C 2 C 12 myotubes (CM) with EPA but not DHA improves glucose uptake and protein accretion. We hypothesized that these differential effects of EPA and DHA may be due to divergent shifts in lipidomic profiles leading to altered proteomic profiles. We therefore carried out an assessment of the impact of treating CM with EPA and DHA on lipidomic and proteomic profiles. Fatty acid methyl esters (FAME) analysis revealed that both EPA and DHA led to similar but substantials changes in fatty acid profiles with the exception of arachidonic acid, which was decreased only by DHA, and docosapentanoic acid (DPA), which was increased only by EPA treatment. Global lipidomic analysis showed that EPA and DHA induced large alterations in the cellular lipid profiles and in particular, the phospholipid classes. Subsequent targeted analysis confirmed that the most differentially regulated species were phosphatidylcholines and phosphatidylethanolamines containing long-chain fatty acids with five (EPA treatment) or six (DHA treatment) double bonds. As these are typically membrane-associated lipid species we hypothesized that these treatments differentially altered the membrane-associated proteome. Stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics of the membrane fraction revealed significant divergence in the effects of EPA and DHA on the membrane-associated proteome. We conclude that the EPA-specific increase in polyunsaturated long-chain fatty acids in the phospholipid fraction is associated with an altered membrane-associated proteome and these may be critical events in the metabolic remodeling induced by EPA treatment.

Key players in neurodegenerative disorders in focus-New insights into the proteomic profile of Alzheimer's disease, schizophrenia, ALS, and multiple sclerosis-24th HUPO BPP Workshop: September 29, 2015, Vancouver, Canada.

PubMed

Schrötter, Andreas; Park, Young Mok; Marcus, Katrin; Martins-de-Souza, Daniel; Nilsson, Peter; Magraoui, Fouzi El; Meyer, Helmut E; Grinberg, Lea T

2016-04-01

The HUPO Brain Proteome Project (HUPO BPP) held its 24th workshop in Vancouver, Canada, September 29, 2015. The focus of the autumn workshop was on new insights into the proteomic profile of Alzheimer's disease, schizophrenia, ALS and multiple sclerosis. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Early Prediction of Lupus Nephritis Using Advanced Proteomics

DTIC Science & Technology

2011-06-01

spectroscopy-based metabolomic profiling , and apolipoprotein D, lipocalin-like prostaglandin D synthetase, hemopexin, ceruloplasmin, -1-B glycoprotein and...will be confirmed and enhanced using NMR- and MS-based metabonomics , by Dr. Michael Kennedy, Miami University. Changes in proteomic profiles will be...based metabolomic profiling , and apolipoprotein D, lipocalin-like prostaglandin D synthetase, hemopexin, ceruloplasmin, -1-B glycoprotein and

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.

Hypothalamus proteomics from mouse models with obesity and anorexia reveals therapeutic targets of appetite regulation

PubMed Central

Manousopoulou, A; Koutmani, Y; Karaliota, S; Woelk, C H; Manolakos, E S; Karalis, K; Garbis, S D

2016-01-01

Objective: This study examined the proteomic profile of the hypothalamus in mice exposed to a high-fat diet (HFD) or with the anorexia of acute illness. This comparison could provide insight on the effects of these two opposite states of energy balance on appetite regulation. Methods: Four to six-week-old male C56BL/6J mice were fed a normal (control 1 group; n=7) or a HFD (HFD group; n=10) for 8 weeks. The control 2 (n=7) and lipopolysaccharide (LPS) groups (n=10) were fed a normal diet for 8 weeks before receiving an injection of saline and LPS, respectively. Hypothalamic regions were analysed using a quantitative proteomics method based on a combination of techniques including iTRAQ stable isotope labeling, orthogonal two-dimensional liquid chromatography hyphenated with nanospray ionization and high-resolution mass spectrometry. Key proteins were validated with quantitative PCR. Results: Quantitative proteomics of the hypothalamous regions profiled a total of 9249 protein groups (q<0.05). Of these, 7718 protein groups were profiled with a minimum of two unique peptides for each. Hierachical clustering of the differentiated proteome revealed distinct proteomic signatures for the hypothalamus under the HFD and LPS nutritional conditions. Literature research with in silico bioinformatics interpretation of the differentiated proteome identified key biological relevant proteins and implicated pathways. Furthermore, the study identified potential pharmacologic targets. In the LPS groups, the anorexigen pro-opiomelanocortin was downregulated. In mice with obesity, nuclear factor-κB, glycine receptor subunit alpha-4 (GlyR) and neuropeptide Y levels were elevated, whereas serotonin receptor 1B levels decreased. Conclusions: High-precision quantitative proteomics revealed that under acute systemic inflammation in the hypothalamus as a response to LPS, homeostatic mechanisms mediating loss of appetite take effect. Conversely, under chronic inflammation in the hypothalamus as a response to HFD, mechanisms mediating a sustained ‘perpetual cycle' of appetite enhancement were observed. The GlyR protein may constitute a novel treatment target for the reduction of central orexigenic signals in obesity. PMID:27110685

Hypothalamus proteomics from mouse models with obesity and anorexia reveals therapeutic targets of appetite regulation.

PubMed

Manousopoulou, A; Koutmani, Y; Karaliota, S; Woelk, C H; Manolakos, E S; Karalis, K; Garbis, S D

2016-04-25

This study examined the proteomic profile of the hypothalamus in mice exposed to a high-fat diet (HFD) or with the anorexia of acute illness. This comparison could provide insight on the effects of these two opposite states of energy balance on appetite regulation. Four to six-week-old male C56BL/6J mice were fed a normal (control 1 group; n=7) or a HFD (HFD group; n=10) for 8 weeks. The control 2 (n=7) and lipopolysaccharide (LPS) groups (n=10) were fed a normal diet for 8 weeks before receiving an injection of saline and LPS, respectively. Hypothalamic regions were analysed using a quantitative proteomics method based on a combination of techniques including iTRAQ stable isotope labeling, orthogonal two-dimensional liquid chromatography hyphenated with nanospray ionization and high-resolution mass spectrometry. Key proteins were validated with quantitative PCR. Quantitative proteomics of the hypothalamous regions profiled a total of 9249 protein groups (q<0.05). Of these, 7718 protein groups were profiled with a minimum of two unique peptides for each. Hierachical clustering of the differentiated proteome revealed distinct proteomic signatures for the hypothalamus under the HFD and LPS nutritional conditions. Literature research with in silico bioinformatics interpretation of the differentiated proteome identified key biological relevant proteins and implicated pathways. Furthermore, the study identified potential pharmacologic targets. In the LPS groups, the anorexigen pro-opiomelanocortin was downregulated. In mice with obesity, nuclear factor-κB, glycine receptor subunit alpha-4 (GlyR) and neuropeptide Y levels were elevated, whereas serotonin receptor 1B levels decreased. High-precision quantitative proteomics revealed that under acute systemic inflammation in the hypothalamus as a response to LPS, homeostatic mechanisms mediating loss of appetite take effect. Conversely, under chronic inflammation in the hypothalamus as a response to HFD, mechanisms mediating a sustained 'perpetual cycle' of appetite enhancement were observed. The GlyR protein may constitute a novel treatment target for the reduction of central orexigenic signals in obesity.

“Exosomics”—A Review of Biophysics, Biology and Biochemistry of Exosomes With a Focus on Human Breast Milk

PubMed Central

de la Torre Gomez, Carolina; Goreham, Renee V.; Bech Serra, Joan J.; Nann, Thomas; Kussmann, Martin

2018-01-01

Exosomes are biomolecular nanostructures released from cells. They carry specific biomolecular information and are mainly researched for their exquisite properties as a biomarker source and delivery system. We introduce exosomes in the context of other extracellular vesicles, describe their biophysical isolation and characterisation and discuss their biochemical profiling. Motivated by our interest in early-life nutrition and health, and corresponding studies enrolling lactating mothers and their infants, we zoom into exosomes derived from human breast milk. We argue that these should be more extensively studied at proteomic and micronutrient profiling level, because breast milk exosomes provide a more specific window into breast milk quality from an immunological (proteomics) and nutritional (micronutrient) perspective. Such enhanced breast milk exosome profiling would thereby complement and enrich the more classical whole breast milk analysis and is expected to deliver more functional insights than the rather descriptive analysis of human milk, or larger fractions thereof, such as milk fat globule membrane. We substantiate our arguments by a bioinformatic analysis of two published proteomic data sets of human breast milk exosomes. PMID:29636770

"Exosomics"-A Review of Biophysics, Biology and Biochemistry of Exosomes With a Focus on Human Breast Milk.

PubMed

de la Torre Gomez, Carolina; Goreham, Renee V; Bech Serra, Joan J; Nann, Thomas; Kussmann, Martin

2018-01-01

Exosomes are biomolecular nanostructures released from cells. They carry specific biomolecular information and are mainly researched for their exquisite properties as a biomarker source and delivery system. We introduce exosomes in the context of other extracellular vesicles, describe their biophysical isolation and characterisation and discuss their biochemical profiling. Motivated by our interest in early-life nutrition and health, and corresponding studies enrolling lactating mothers and their infants, we zoom into exosomes derived from human breast milk. We argue that these should be more extensively studied at proteomic and micronutrient profiling level, because breast milk exosomes provide a more specific window into breast milk quality from an immunological (proteomics) and nutritional (micronutrient) perspective. Such enhanced breast milk exosome profiling would thereby complement and enrich the more classical whole breast milk analysis and is expected to deliver more functional insights than the rather descriptive analysis of human milk, or larger fractions thereof, such as milk fat globule membrane. We substantiate our arguments by a bioinformatic analysis of two published proteomic data sets of human breast milk exosomes.

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

Proteomic profiling of human plasma for cancer biomarker discovery.

PubMed

Huang, Zhao; Ma, Linguang; Huang, Canhua; Li, Qifu; Nice, Edouard C

2017-03-01

Over the past decades, substantial advances have been made in both the early diagnosis and accurate prognosis of many cancers because of the impressive development of novel proteomic strategies. However, it remains difficult to standardize proteomic approaches. In addition, the heterogeneity of proteins in distinct tissues results in incomplete population of the whole proteome, which inevitably limits its clinical practice. As one of the most complex proteomes in the human body, the plasma proteome contains secreted proteins originating from multiple organs and tissues, making it a favorable matrix for comprehensive biomarker discovery. Here, we will discuss the roles of plasma proteome profiling in cancer biomarker discovery and validation, and highlight both the inherent advantages and disadvantages. Although several hurdles lay ahead, further advances in this technology will greatly increase our understanding of cancer biology, reveal new biomarkers and biomarker panels, and open a new avenue for more efficient early diagnosis and surveillance of cancer, leading toward personalized medicine. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization of the Low-Molecular-Weight Human Plasma Peptidome.

PubMed

Greening, David W; Simpson, Richard J

2017-01-01

The human plasma proteome represents an important secreted sub-proteome. Proteomic analysis of blood plasma with mass spectrometry is a challenging task. The high complexity and wide dynamic range of proteins as well as the presence of several proteins at very high concentrations complicate the profiling of the human plasma proteome. The peptidome (or low-molecular-weight fraction, LMF) of the human plasma proteome is an invaluable source of biological information, especially in the context of identifying plasma-based markers of disease. Peptides are generated by active synthesis and proteolytic processing, often yielding proteolytic fragments that mediate a variety of physiological and pathological functions. As such, degradomic studies, investigating cleavage products via peptidomics and top-down proteomics in particular, have warranted significant research interest. However, due to their molecular weight, abundance, and solubility, issues with identifying specific cleavage sites and coverage of peptide fragments remain challenging. Peptidomics is currently focused toward comprehensively studying peptides cleaved from precursor proteins by endogenous proteases. This protocol outlines a standardized rapid and reproducible procedure for peptidomic profiling of human plasma using centrifugal ultrafiltration and mass spectrometry. Ultrafiltration is a convective process that uses anisotropic semipermeable membranes to separate macromolecular species on the basis of size. We have optimized centrifugal ultrafiltration (cellulose triacetate membrane) for plasma fractionation with respect to buffer and solvent composition, centrifugal force, duration, and temperature to facilitate recovery >95% and enrichment of the human plasma peptidome. This method serves as a comprehensive and facile process to enrich and identify a key, underrepresented sub-proteome of human blood plasma.

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

Vascular biology: cellular and molecular profiling.

PubMed

Baird, Alison E; Wright, Violet L

2006-02-01

Our understanding of the mechanisms underlying cerebrovascular atherosclerosis has improved in recent years, but significant gaps remain. New insights into the vascular biological processes that result in ischemic stroke may come from cellular and molecular profiling studies of the peripheral blood. In recent cellular profiling studies, increased levels of a proinflammatory T-cell subset (CD4 (+)CD28 (-)) have been associated with stroke recurrence and death. Expansion of this T-cell subset may occur after ischemic stroke and be a pathogenic mechanism leading to recurrent stroke and death. Increases in certain phenotypes of endothelial cell microparticles have been found in stroke patients relative to controls, possibly indicating a state of increased vascular risk. Molecular profiling approaches include gene expression profiling and proteomic methods that permit large-scale analyses of the transcriptome and the proteome, respectively. Ultimately panels of genes and proteins may be identified that are predictive of stroke risk. Cellular and molecular profiling studies of the peripheral blood and of atherosclerotic plaques may also pave the way for the development of therapeutic agents for primary and secondary stroke prevention.

Proteomic analysis of propiconazole responses in mouse liver: comparison of genomic and proteomic profiles

EPA Science Inventory

We have performed for the first time a comprehensive profiling of changes in protein expression of soluble proteins in livers from mice treated with the mouse liver tumorigen, propiconazole, to uncover the pathways and networks altered by this fungicide. Utilizing twodimensional...

Proteomics of contrasting rice genotypes: Identification of potential targets for raising crops for saline environment.

PubMed

Lakra, Nita; Kaur, Charanpreet; Anwar, Khalid; Singla-Pareek, Sneh Lata; Pareek, Ashwani

2018-05-01

High salinity is one of the major problems in crop productivity, affecting seed germination as well as yield. In order to enhance tolerance of crops towards salinity, it is essential to understand the underlying physiological and molecular mechanisms. In this endeavor, study of contrasting genotypes of the same species differing in their response towards salinity stress can be very useful. In the present study, we have investigated temporal differences in morphological, physiological and proteome profiles of two contrasting genotypes of rice to understand the basis of salt tolerance. When compared to IR64 rice, Pokkali, the salt-tolerant wild genotype, has enhanced capacity to cope with stress, better growth rate and possesses efficient antioxidant system, as well as better photosynthetic machinery. Our proteome studies revealed a higher and an early abundance of proteins involved in stress tolerance and photosynthesis in Pokkali in comparison with IR64, which, in contrast, showed greater changes in metabolic machinery even during early duration of stress. Our findings suggest important differences in physicochemical and proteome profiles of the two genotypes, which may be the basis of observed stress tolerance in the salt-tolerant Pokkali. © 2017 John Wiley & Sons Ltd.

An integrated metabonomic and proteomic study on Kidney-Yin Deficiency Syndrome patients with diabetes mellitus in China.

PubMed

Jiang, Ning; Liu, Hong-fang; Li, Si-di; Zhou, Wen-xia; Zhang, Yong-xiang; Zhang, Qi; Yan, Xian-zhong

2015-06-01

To investigate specific changes in metabolites and proteins of Kidney-Yin Deficiency Syndrome (KYDS) patients with diabetes mellitus (DM) in China. KYDS (n=29) and non-KYDS (n=23) patients with DM were recruited for this study. The KYDS was diagnosed by two senior TCM clinicians separately. The metabonomic and proteomic profiles of the patients were assessed using a metabonomic strategy based on NMR with multivariate analysis and a proteomic strategy based on MALDI-TOF-MS, respectively. Eighteen upregulated peptides and thirty downregulated peptides were observed in the plasma of the KYDS patients. Comparing the proteomic profiles of the KYDS and non-KYDS groups, however, no significantly differentially expressed peptides were found. At the same time, major metabolic alterations were found to distinguish the two groups, including eight significantly changed metabolites (creatinine, citrate, TMAO, phenylalanine, tyrosine, alanine, glycine and taurine). The levels of creatinine, citrate, TMAO, phenylalanine and tyrosine were decreased, whereas the levels of alanine, glycine and taurine were increased in the KYDS patients. These biochemical changes were found to be associated with alterations in amino acid metabolism, energy metabolism and gut microflora. The identification of distinct expression profiles of metabolites and signaling pathways in KYDS patients with DM suggests that there are indeed molecular signatures underlying the principles of 'Syndrome Differentiation' in traditional Chinese medicine.

Proteomic analysis reveals age-related changes in tendon matrix composition, with age- and injury-specific matrix fragmentation.

PubMed

Peffers, Mandy J; Thorpe, Chavaunne T; Collins, John A; Eong, Robin; Wei, Timothy K J; Screen, Hazel R C; Clegg, Peter D

2014-09-12

Energy storing tendons, such as the human Achilles and equine superficial digital flexor tendon (SDFT), are highly prone to injury, the incidence of which increases with aging. The cellular and molecular mechanisms that result in increased injury in aged tendons are not well established but are thought to result in altered matrix turnover. However, little attempt has been made to fully characterize the tendon proteome nor determine how the abundance of specific tendon proteins changes with aging and/or injury. The aim of this study was, therefore, to assess the protein profile of normal SDFTs from young and old horses using label-free relative quantification to identify differentially abundant proteins and peptide fragments between age groups. The protein profile of injured SDFTs from young and old horses was also assessed. The results demonstrate distinct proteomic profiles in young and old tendon, with alterations in the levels of proteins involved in matrix organization and regulation of cell tension. Furthermore, we identified several new peptide fragments (neopeptides) present in aged tendons, suggesting that there are age-specific cleavage patterns within the SDFT. Proteomic profile also differed between young and old injured tendon, with a greater number of neopeptides identified in young injured tendon. This study has increased the knowledge of molecular events associated with tendon aging and injury, suggesting that maintenance and repair of tendon tissue may be reduced in aged individuals and may help to explain why the risk of injury increases with aging. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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

PubMed

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

2013-12-01

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

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

PubMed

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

2017-12-01

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

Proteomic Analysis of Propiconazole Responses in Mouse Liver-Comparison of Genomic and Proteomic Profiles

EPA Science Inventory

We have performed for the first time a comprehensive profiling of changes in protein expression of soluble proteins in livers from mice treated with the mouse liver tumorigen, propiconazole, to uncover the pathways and networks altered by this commonly used fungicide. Utilizing t...

Shotgun proteomic monitoring of Clostridium acetobutylicum during stationary phase of butanol fermentation using xylose and comparison with the exponential phase

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

Sivagnanam, Kumaran; Raghavan, Vijaya G. S.; Shah, Manesh B

2012-01-01

Economically viable production of solvents through acetone butanol ethanol (ABE) fermentation requires a detailed understanding of Clostridium acetobutylicum. This study focuses on the proteomic profiling of C. acetobutylicum ATCC 824 from the stationary phase of ABE fermentation using xylose and compares with the exponential growth by shotgun proteomics approach. Comparative proteomic analysis revealed 22.9% of the C. acetobutylicum genome and 18.6% was found to be common in both exponential and stationary phases. The proteomic profile of C. acetobutylicum changed during the ABE fermentation such that 17 proteins were significantly differentially expressed between the two phases. Specifically, the expression of fivemore » proteins namely, CAC2873, CAP0164, CAP0165, CAC3298, and CAC1742 involved in the solvent production pathway were found to be significantly lower in the stationary phase compared to the exponential growth. Similarly, the expression of fucose isomerase (CAC2610), xylulose kinase (CAC2612), and a putative uncharacterized protein (CAC2611) involved in the xylose utilization pathway were also significantly lower in the stationary phase. These findings provide an insight into the metabolic behavior of C. acetobutylicum between different phases of ABE fermentation using xylose.« less

Proteomic and Bioinformatic Profile of Primary Human Oral Epithelial Cells

PubMed Central

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

2012-01-01

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

Venomous snakes of Costa Rica: biological and medical implications of their venom proteomic profiles analyzed through the strategy of snake venomics.

PubMed

Lomonte, Bruno; Fernández, Julián; Sanz, Libia; Angulo, Yamileth; Sasa, Mahmood; Gutiérrez, José María; Calvete, Juan J

2014-06-13

In spite of its small territory of ~50,000km(2), Costa Rica harbors a remarkably rich biodiversity. Its herpetofauna includes 138 species of snakes, of which sixteen pit vipers (family Viperidae, subfamily Crotalinae), five coral snakes (family Elapidae, subfamily Elapinae), and one sea snake (Family Elapidae, subfamily Hydrophiinae) pose potential hazards to human and animal health. In recent years, knowledge on the composition of snake venoms has expanded dramatically thanks to the development of increasingly fast and sensitive analytical techniques in mass spectrometry and separation science applied to protein characterization. Among several analytical strategies to determine the overall protein/peptide composition of snake venoms, the methodology known as 'snake venomics' has proven particularly well suited and informative, by providing not only a catalog of protein types/families present in a venom, but also a semi-quantitative estimation of their relative abundances. Through a collaborative research initiative between Instituto de Biomedicina de Valencia (IBV) and Instituto Clodomiro Picado (ICP), this strategy has been applied to the study of venoms of Costa Rican snakes, aiming to obtain a deeper knowledge on their composition, geographic and ontogenic variations, relationships to taxonomy, correlation with toxic activities, and discovery of novel components. The proteomic profiles of venoms from sixteen out of the 22 species within the Viperidae and Elapidae families found in Costa Rica have been reported so far, and an integrative view of these studies is hereby presented. In line with other venomic projects by research groups focusing on a wide variety of snakes around the world, these studies contribute to a deeper understanding of the biochemical basis for the diverse toxic profiles evolved by venomous snakes. In addition, these studies provide opportunities to identify novel molecules of potential pharmacological interest. Furthermore, the establishment of venom proteomic profiles offers a fundamental platform to assess the detailed immunorecognition of individual proteins/peptides by therapeutic or experimental antivenoms, an evolving methodology for which the term 'antivenomics' was coined (as described in an accompanying paper in this special issue). Venoms represent an adaptive trait and an example of both divergent and convergent evolution. A deep understanding of the composition of venoms and of the principles governing the evolution of venomous systems is of applied importance for exploring the enormous potential of venoms as sources of chemical and pharmacological novelty but also to fight the consequences of snakebite envenomings. Key to this is the identification of evolutionary and ecological trends at different taxonomical levels. However, the evolution of venomous species and their venoms do not always follow the same course, and the identification of structural and functional convergences and divergences among venoms is often unpredictable by a phylogenetic hypothesis. Snake venomics is a proteomic-centered strategy to deconstruct the complex molecular phenotypes the venom proteomes. The proteomic profiles of venoms from sixteen out of the 22 venomous species within the Viperidae and Elapidae families found in Costa Rica have been completed so far. An integrative view of their venom composition, including the identification of geographic and ontogenic variations, is hereby presented. Venom proteomic profiles offer a fundamental platform to assess the detailed immunorecognition of individual venom components by therapeutic or experimental antivenoms. This aspect is reviewed in the companion paper. This article is part of a Special Issue entitled: Proteomics of non-model organisms. Copyright © 2014 Elsevier B.V. All rights reserved.

Wheat proteomics: proteome modulation and abiotic stress acclimation

PubMed Central

Komatsu, Setsuko; Kamal, Abu H. M.; Hossain, Zahed

2014-01-01

Cellular mechanisms of stress sensing and signaling represent the initial plant responses to adverse conditions. The development of high-throughput “Omics” techniques has initiated a new era of the study of plant molecular strategies for adapting to environmental changes. However, the elucidation of stress adaptation mechanisms in plants requires the accurate isolation and characterization of stress-responsive proteins. Because the functional part of the genome, namely the proteins and their post-translational modifications, are critical for plant stress responses, proteomic studies provide comprehensive information about the fine-tuning of cellular pathways that primarily involved in stress mitigation. This review summarizes the major proteomic findings related to alterations in the wheat proteomic profile in response to abiotic stresses. Moreover, the strengths and weaknesses of different sample preparation techniques, including subcellular protein extraction protocols, are discussed in detail. The continued development of proteomic approaches in combination with rapidly evolving bioinformatics tools and interactive databases will facilitate understanding of the plant mechanisms underlying stress tolerance. PMID:25538718

Extending the Limits of Quantitative Proteome Profiling with Data-Independent Acquisition and Application to Acetaminophen-Treated Three-Dimensional Liver Microtissues*

PubMed Central

Bruderer, Roland; Bernhardt, Oliver M.; Gandhi, Tejas; Miladinović, Saša M.; Cheng, Lin-Yang; Messner, Simon; Ehrenberger, Tobias; Zanotelli, Vito; Butscheid, Yulia; Escher, Claudia; Vitek, Olga; Rinner, Oliver; Reiter, Lukas

2015-01-01

The data-independent acquisition (DIA) approach has recently been introduced as a novel mass spectrometric method that promises to combine the high content aspect of shotgun proteomics with the reproducibility and precision of selected reaction monitoring. Here, we evaluate, whether SWATH-MS type DIA effectively translates into a better protein profiling as compared with the established shotgun proteomics. We implemented a novel DIA method on the widely used Orbitrap platform and used retention-time-normalized (iRT) spectral libraries for targeted data extraction using Spectronaut. We call this combination hyper reaction monitoring (HRM). Using a controlled sample set, we show that HRM outperformed shotgun proteomics both in the number of consistently identified peptides across multiple measurements and quantification of differentially abundant proteins. The reproducibility of HRM in peptide detection was above 98%, resulting in quasi complete data sets compared with 49% of shotgun proteomics. Utilizing HRM, we profiled acetaminophen (APAP)1-treated three-dimensional human liver microtissues. An early onset of relevant proteome changes was revealed at subtoxic doses of APAP. Further, we detected and quantified for the first time human NAPQI-protein adducts that might be relevant for the toxicity of APAP. The adducts were identified on four mitochondrial oxidative stress related proteins (GATM, PARK7, PRDX6, and VDAC2) and two other proteins (ANXA2 and FTCD). Our findings imply that DIA should be the preferred method for quantitative protein profiling. PMID:25724911

Proteome-level interplay between folding and aggregation propensities of proteins.

PubMed

Tartaglia, Gian Gaetano; Vendruscolo, Michele

2010-10-08

With the advent of proteomics, there is an increasing need of tools for predicting the properties of large numbers of proteins by using the information provided by their amino acid sequences, even in the absence of the knowledge of their structures. One of the most important types of predictions concerns whether proteins will fold or aggregate. Here, we study the competition between these two processes by analyzing the relationship between the folding and aggregation propensity profiles for the human and Escherichia coli proteomes. These profiles are calculated, respectively, using the CamFold method, which we introduce in this work, and the Zyggregator method. Our results indicate that the kinetic behavior of proteins is, to a large extent, determined by the interplay between regions of low folding and high aggregation propensities. Copyright © 2010. Published by Elsevier Ltd.

Proteomic characterization and comparison of venoms from two elapid snakes (Bungarus multicinctus and Naja atra) from China.

PubMed

Shan, Lin-Lin; Gao, Jian-Fang; Zhang, Yan-Xia; Shen, Shan-Shan; He, Ying; Wang, Jin; Ma, Xiao-Mei; Ji, Xiang

2016-04-14

Bungarus multicinctus (many-banded krait) and Naja atra (Chinese cobra) are widely distributed and medically important venomous snakes in China; however, their venom proteomic profiles have not been fully compared. Here, we fractionated crude venoms and analyzed them using a combination of proteomic techniques. Three-finger toxins (3-FTx) and phospholipase A2 (PLA2) were most abundant in both species, respectively accounting for 32.6% and 66.4% of total B. multicinctus venom, and 84.3% and 12.2% of total N. atra venom. Venoms from these two species contained one common protein family and six less abundant species-specific protein families. The proteomic profiles of B. multicinctus and N. atra venoms and analysis of toxicological activity in mice suggested that 3-FTx and PLA2 are the major contributors to clinical symptoms caused by envenomation. The venoms differed in enzymatic activity, likely the result of inter-specific variation in the amount of related venom components. Antivenomics assessment revealed that a small number of venom components (3-FTxs and PLA2s in B. multicinctus, and 3-FTxs in N. atra) could not be immunocaptured completely, suggesting that we should pay attention to enhancing the immune response of these components in designing commercial antivenoms for B. multicinctus and N. atra. The proteomic profiles of venoms from two medically important snake species - B. multicinctus and N. atra - have been explored. Quantitative and qualitative differences are evident in both venoms when proteomic profiles and transcriptomic results are compared; this is a reminder that combined approaches are needed to explore the precise composition of snake venom. Two protein families (3-FTx and PLA2) of high abundance in these snake venoms are major players in the biochemical and pharmacological effects of envenomation. Elucidation of the proteomic profiles of these snake venoms is helpful in understanding composition-function relationships and will facilitate the clinical application of antivenoms. Copyright © 2016 Elsevier B.V. All rights reserved.

Proteome comparison for discrimination between honeydew and floral honeys from botanical species Mimosa scabrella Bentham by principal component analysis.

PubMed

Azevedo, Mônia Stremel; Valentim-Neto, Pedro Alexandre; Seraglio, Siluana Katia Tischer; da Luz, Cynthia Fernandes Pinto; Arisi, Ana Carolina Maisonnave; Costa, Ana Carolina Oliveira

2017-10-01

Due to the increasing valuation and appreciation of honeydew honey in many European countries and also to existing contamination among different types of honeys, authentication is an important aspect of quality control with regard to guaranteeing the origin in terms of source (honeydew or floral) and needs to be determined. Furthermore, proteins are minor components of the honey, despite the importance of their physiological effects, and can differ according to the source of the honey. In this context, the aims of this study were to carry out protein extraction from honeydew and floral honeys and to discriminate these honeys from the same botanical species, Mimosa scabrella Bentham, through proteome comparison using two-dimensional gel electrophoresis and principal component analysis. The results showed that the proteome profile and principal component analysis can be a useful tool for discrimination between these types of honey using matched proteins (45 matched spots). Also, the proteome profile showed 160 protein spots in honeydew honey and 84 spots in the floral honey. The protein profile can be a differential characteristic of this type of honey, in view of the importance of proteins as bioactive compounds in honey. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Label-free Quantitative Protein Profiling of vastus lateralis Muscle During Human Aging*

PubMed Central

Théron, Laëtitia; Gueugneau, Marine; Coudy, Cécile; Viala, Didier; Bijlsma, Astrid; Butler-Browne, Gillian; Maier, Andrea; Béchet, Daniel; Chambon, Christophe

2014-01-01

Sarcopenia corresponds to the loss of muscle mass occurring during aging, and is associated with a loss of muscle functionality. Proteomic links the muscle functional changes with protein expression pattern. To better understand the mechanisms involved in muscle aging, we performed a proteomic analysis of Vastus lateralis muscle in mature and older women. For this, a shotgun proteomic method was applied to identify soluble proteins in muscle, using a combination of high performance liquid chromatography and mass spectrometry. A label-free protein profiling was then conducted to quantify proteins and compare profiles from mature and older women. This analysis showed that 35 of the 366 identified proteins were linked to aging in muscle. Most of the proteins were under-represented in older compared with mature women. We built a functional interaction network linking the proteins differentially expressed between mature and older women. The results revealed that the main differences between mature and older women were defined by proteins involved in energy metabolism and proteins from the myofilament and cytoskeleton. This is the first time that label-free quantitative proteomics has been applied to study of aging mechanisms in human skeletal muscle. This approach highlights new elements for elucidating the alterations observed during aging and may lead to novel sarcopenia biomarkers. PMID:24217021

Label-free quantitative protein profiling of vastus lateralis muscle during human aging.

PubMed

Théron, Laëtitia; Gueugneau, Marine; Coudy, Cécile; Viala, Didier; Bijlsma, Astrid; Butler-Browne, Gillian; Maier, Andrea; Béchet, Daniel; Chambon, Christophe

2014-01-01

Sarcopenia corresponds to the loss of muscle mass occurring during aging, and is associated with a loss of muscle functionality. Proteomic links the muscle functional changes with protein expression pattern. To better understand the mechanisms involved in muscle aging, we performed a proteomic analysis of Vastus lateralis muscle in mature and older women. For this, a shotgun proteomic method was applied to identify soluble proteins in muscle, using a combination of high performance liquid chromatography and mass spectrometry. A label-free protein profiling was then conducted to quantify proteins and compare profiles from mature and older women. This analysis showed that 35 of the 366 identified proteins were linked to aging in muscle. Most of the proteins were under-represented in older compared with mature women. We built a functional interaction network linking the proteins differentially expressed between mature and older women. The results revealed that the main differences between mature and older women were defined by proteins involved in energy metabolism and proteins from the myofilament and cytoskeleton. This is the first time that label-free quantitative proteomics has been applied to study of aging mechanisms in human skeletal muscle. This approach highlights new elements for elucidating the alterations observed during aging and may lead to novel sarcopenia biomarkers.

An Anatomically Resolved Mouse Brain Proteome Reveals Parkinson Disease-relevant Pathways *

PubMed Central

Choi, Jong Min; Rousseaux, Maxime W. C.; Malovannaya, Anna; Kim, Jean J.; Kutzera, Joachim; Wang, Yi; Huang, Yin; Zhu, Weimin; Maity, Suman; Zoghbi, Huda Yahya; Qin, Jun

2017-01-01

Here, we present a mouse brain protein atlas that covers 17 surgically distinct neuroanatomical regions of the adult mouse brain, each less than 1 mm3 in size. The protein expression levels are determined for 6,500 to 7,500 gene protein products from each region and over 12,000 gene protein products for the entire brain, documenting the physiological repertoire of mouse brain proteins in an anatomically resolved and comprehensive manner. We explored the utility of our spatially defined protein profiling methods in a mouse model of Parkinson's disease. We compared the proteome from a vulnerable region (substantia nigra pars compacta) of wild type and parkinsonian mice with that of an adjacent, less vulnerable, region (ventral tegmental area) and identified several proteins that exhibited both spatiotemporal- and genotype-restricted changes. We validated the most robustly altered proteins using an alternative profiling method and found that these modifications may highlight potential new pathways for future studies. This proteomic atlas is a valuable resource that offers a practical framework for investigating the molecular intricacies of normal brain function as well as regional vulnerability in neurological diseases. All of the mouse regional proteome profiling data are published on line at http://mbpa.bprc.ac.cn/. PMID:28153913

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.

Functional protease profiling for diagnosis of malignant disease.

PubMed

Findeisen, Peter; Neumaier, Michael

2012-01-01

Clinical proteomic profiling by mass spectrometry (MS) aims at uncovering specific alterations within mass profiles of clinical specimens that are of diagnostic value for the detection and classification of various diseases including cancer. However, despite substantial progress in the field, the clinical proteomic profiling approaches have not matured into routine diagnostic applications so far. Their limitations are mainly related to high-abundance proteins and their complex processing by a multitude of endogenous proteases thus making rigorous standardization difficult. MS is biased towards the detection of low-molecular-weight peptides. Specifically, in serum specimens, the particular fragments of proteolytically degraded proteins are amenable to MS analysis. Proteases are known to be involved in tumour progression and tumour-specific proteases are released into the blood stream presumably as a result of invasive progression and metastasis. Thus, the determination of protease activity in clinical specimens from patients with malignant disease can offer diagnostic and also therapeutic options. The identification of specific substrates for tumour proteases in complex biological samples is challenging, but proteomic screens for proteases/substrate interactions are currently experiencing impressive progress. Such proteomic screens include peptide-based libraries, differential isotope labelling in combination with MS, quantitative degradomic analysis of proteolytically generated neo-N-termini, monitoring the degradation of exogenous reporter peptides with MS, and activity-based protein profiling. In the present article, we summarize and discuss the current status of proteomic techniques to identify tumour-specific protease-substrate interactions for functional protease profiling. Thereby, we focus on the potential diagnostic use of the respective approaches. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Head and neck cancer: proteomic advances and biomarker achievements.

PubMed

Rezende, Taia Maria Berto; de Souza Freire, Mirna; Franco, Octávio Luiz

2010-11-01

Tumors of the head and neck comprise an important neoplasia group, the incidence of which is increasing in many parts of the world. Recent advances in diagnostic and therapeutic techniques for these lesions have yielded novel molecular targets, uncovered signal pathway dominance, and advanced early cancer detection. Proteomics is a powerful tool for investigating the distribution of proteins and small molecules within biological systems through the analysis of different types of samples. The proteomic profiles of different types of cancer have been studied, and this has provided remarkable advances in cancer understanding. This review covers recent advances for head and neck cancer; it encompasses the risk factors, pathogenesis, proteomic tools that can help in understanding cancer, and new proteomic findings in this type of cancer. Copyright © 2010 American Cancer Society.

Improvement of Soybean Products Through the Response Mechanism Analysis Using Proteomic Technique.

PubMed

Wang, Xin; Komatsu, Setsuko

Soybean is rich in protein/vegetable oil and contains several phytochemicals such as isoflavones and phenolic compounds. Because of the predominated nutritional values, soybean is considered as traditional health benefit food. Soybean is a widely cultivated crop; however, its growth and yield are markedly affected by adverse environmental conditions. Proteomic techniques make it feasible to map protein profiles both during soybean growth and under unfavorable conditions. The stress-responsive mechanisms during soybean growth have been uncovered with the help of proteomic studies. In this review, the history of soybean as food and the morphology/physiology of soybean are described. The utilization of proteomics during soybean germination and development is summarized. In addition, the stress-responsive mechanisms explored using proteomic techniques are reviewed in soybean. © 2017 Elsevier Inc. All rights reserved.

Proteomic Profiling of Serial Prediagnostic Serum Samples for Early Detection of Colon Cancer in the U.S. Military.

PubMed

Shao, Stephanie; Neely, Benjamin A; Kao, Tzu-Cheg; Eckhaus, Janet; Bourgeois, Jolie; Brooks, Jasmin; Jones, Elizabeth E; Drake, Richard R; Zhu, Kangmin

2017-05-01

Background: Serum proteomic biomarkers offer a promising approach for early detection of cancer. In this study, we aimed to identify proteomic profiles that could distinguish colon cancer cases from controls using serial prediagnostic serum samples. Methods: This was a nested case-control study of active duty military members. Cases consisted of 264 patients diagnosed with colon cancer between 2001 and 2009. Controls were matched to cases on age, gender, race, serum sample count, and collection date. We identified peaks that discriminated cases from controls using random forest data analysis with a 2/3 training and 1/3 validation dataset. We then included epidemiologic data to see whether further improvement of model performance was obtainable. Proteins that corresponded to discriminatory peaks were identified. Results: Peaks with m/z values of 3,119.32, 2,886.67, 2,939.23, and 5,078.81 were found to discriminate cases from controls with a sensitivity of 69% and a specificity of 67% in the year before diagnosis. When smoking status was included, sensitivity increased to 76% while histories of other cancer and tonsillectomy raised specificity to 76%. Peaks at 2,886.67 and 3,119.32 m/z were identified as histone acetyltransferases while 2,939.24 m/z was a transporting ATPase subunit. Conclusions: Proteomic profiles in the year before cancer diagnosis have the potential to discriminate colon cancer patients from controls, and the addition of epidemiologic information may increase the sensitivity and specificity of discrimination. Impact: Our findings indicate the potential value of using serum prediagnostic proteomic biomarkers in combination with epidemiologic data for early detection of colon cancer. Cancer Epidemiol Biomarkers Prev; 26(5); 711-8. ©2016 AACR . ©2016 American Association for Cancer Research.

Effects of Fe and Mn deficiencies on the protein profiles of tomato (Solanum lycopersicum) xylem sap as revealed by shotgun analyses

USDA-ARS?s Scientific Manuscript database

The aim of this work was to study the effects of Fe and Mn deficiencies on the xylem sap proteome of tomato using a shotgun proteomic approach, with the final goal of elucidating plant response mechanisms to these stresses. This approach yielded 643 proteins reliably identified and quantified with 7...

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.

Activity-based protein profiling: from enzyme chemistry to proteomic chemistry.

PubMed

Cravatt, Benjamin F; Wright, Aaron T; Kozarich, John W

2008-01-01

Genome sequencing projects have provided researchers with a complete inventory of the predicted proteins produced by eukaryotic and prokaryotic organisms. Assignment of functions to these proteins represents one of the principal challenges for the field of proteomics. Activity-based protein profiling (ABPP) has emerged as a powerful chemical proteomic strategy to characterize enzyme function directly in native biological systems on a global scale. Here, we review the basic technology of ABPP, the enzyme classes addressable by this method, and the biological discoveries attributable to its application.

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.

Combining genomic and proteomic approaches for epigenetics research

PubMed Central

Han, Yumiao; Garcia, Benjamin A

2014-01-01

Epigenetics is the study of changes in gene expression or cellular phenotype that do not change the DNA sequence. In this review, current methods, both genomic and proteomic, associated with epigenetics research are discussed. Among them, chromatin immunoprecipitation (ChIP) followed by sequencing and other ChIP-based techniques are powerful techniques for genome-wide profiling of DNA-binding proteins, histone post-translational modifications or nucleosome positions. However, mass spectrometry-based proteomics is increasingly being used in functional biological studies and has proved to be an indispensable tool to characterize histone modifications, as well as DNA–protein and protein–protein interactions. With the development of genomic and proteomic approaches, combination of ChIP and mass spectrometry has the potential to expand our knowledge of epigenetics research to a higher level. PMID:23895656

Magnesium supplementation, metabolic and inflammatory markers, and global genomic and proteomic profiling: a randomized, double-blind, controlled, crossover trial in overweight individuals.

PubMed

Chacko, Sara A; Sul, James; Song, Yiqing; Li, Xinmin; LeBlanc, James; You, Yuko; Butch, Anthony; Liu, Simin

2011-02-01

Dietary magnesium intake has been favorably associated with reduced risk of metabolic outcomes in observational studies; however, few randomized trials have introduced a systems-biology approach to explore molecular mechanisms of pleiotropic metabolic actions of magnesium supplementation. We examined the effects of oral magnesium supplementation on metabolic biomarkers and global genomic and proteomic profiling in overweight individuals. We undertook this randomized, crossover, pilot trial in 14 healthy, overweight volunteers [body mass index (in kg/m(2)) ≥25] who were randomly assigned to receive magnesium citrate (500 mg elemental Mg/d) or a placebo for 4 wk with a 1-mo washout period. Fasting blood and urine specimens were collected according to standardized protocols. Biochemical assays were conducted on blood specimens. RNA was extracted and subsequently hybridized with the Human Gene ST 1.0 array (Affymetrix, Santa Clara, CA). Urine proteomic profiling was analyzed with the CM10 ProteinChip array (Bio-Rad Laboratories, Hercules, CA). We observed that magnesium treatment significantly decreased fasting C-peptide concentrations (change: -0.4 ng/mL after magnesium treatment compared with +0.05 ng/mL after placebo treatment; P = 0.004) and appeared to decrease fasting insulin concentrations (change: -2.2 μU/mL after magnesium treatment compared with 0.0 μU/mL after placebo treatment; P = 0.25). No consistent patterns were observed across inflammatory biomarkers. Gene expression profiling revealed up-regulation of 24 genes and down-regulation of 36 genes including genes related to metabolic and inflammatory pathways such as C1q and tumor necrosis factor-related protein 9 (C1QTNF9) and pro-platelet basic protein (PPBP). Urine proteomic profiling showed significant differences in the expression amounts of several peptides and proteins after treatment. Magnesium supplementation for 4 wk in overweight individuals led to distinct changes in gene expression and proteomic profiling consistent with favorable effects on several metabolic pathways. This trial was registered at clinicaltrials.gov as NCT00737815.

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.

Developmental cigarette smoke exposure II: Hepatic proteome profiles in 6 month old adult offspring.

PubMed

Neal, Rachel E; Chen, Jing; Webb, Cindy; Stocke, Kendall; Gambrell, Caitlin; Greene, Robert M; Pisano, M Michele

2016-10-01

Utilizing a mouse model of 'active' developmental cigarette smoke exposure (CSE) [gestational day (GD) 1 through postnatal day (PD) 21] characterized by offspring low birth weight, the impact of developmental CSE on liver proteome profiles of adult offspring at 6 months of age was determined. Liver tissue was collected from Sham- and CSE-offspring for 2D-SDS-PAGE based proteome analysis with Partial Least Squares-Discriminant Analysis (PLS-DA). A similar study conducted at the cessation of exposure to cigarette smoke documented decreased gluconeogenesis coupled to oxidative stress in weanling offspring. In the current study, exposure throughout development to cigarette smoke resulted in impaired hepatic carbohydrate metabolism, decreased serum glucose levels, and increased gluconeogenic regulatory enzyme abundances during the fed-state coupled to decreased expression of SIRT1 as well as increased PEPCK and PGC1α expression. Together these findings indicate inappropriately timed gluconeogenesis that may reflect impaired insulin signaling in mature offspring exposed to 'active' developmental CSE. Copyright © 2016 Elsevier Inc. All rights reserved.

Proteomic profiling of human pleural effusion using two-dimensional nano liquid chromatography tandem mass spectrometry.

PubMed

Tyan, Yu-Chang; Wu, Hsin-Yi; Lai, Wu-Wei; Su, Wu-Chou; Liao, Pao-Chi

2005-01-01

Pleural effusion, an accumulation of pleural fluid, contains proteins originated from plasma filtrate and, especially when tissues are damaged, parenchyma interstitial spaces of lungs and/or other organs. This study details protein profiles in human pleural effusion from 43 lung adenocarcinoma patients by a two-dimensional nano-high performance liquid chromatography electrospray ionization tandem mass spectrometry (2D nano-HPLC-ESI-MS/MS) system. The experimental results revealed the identification of 1415 unique proteins from human pleural effusion. Among these 124 proteins identified with higher confidence levels, some proteins have not been reported in plasma and may represent proteins specifically present in pleural effusion. These proteins are valuable for mass identification of differentially expressed proteins involved in proteomics database and screening biomarker to further study in human lung adenocarcinoma. The significance of the use of proteomics analysis of human pleural fluid for the search of new lung cancer marker proteins, and for their simultaneous display and analysis in patients suffering from lung disorders has been examined.

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

PubMed Central

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

2013-01-01

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

Benzoate-mediated changes on expression profile of soluble proteins in Serratia sp. DS001.

PubMed

Pandeeti, E V P; Chinnaboina, M R; Siddavattam, D

2009-05-01

To assess differences in protein expression profile associated with shift in carbon source from succinate to benzoate in Serratia sp. DS001 using a proteomics approach. A basic proteome map was generated for the soluble proteins extracted from Serratia sp. DS001 grown in succinate and benzoate. The differently and differentially expressed proteins were identified using ImageMaster 2D Platinum software (GE Healthcare). The identity of the proteins was determined by employing MS or MS/MS. Important enzymes such as Catechol 1,2 dioxygenase and transcriptional regulators that belong to the LysR superfamily were identified. Nearly 70 proteins were found to be differentially expressed when benzoate was used as carbon source. Based on the protein identity and degradation products generated from benzoate it is found that ortho pathway is operational in Serratia sp. DS001. Expression profile of the soluble proteins associated with shift in carbon source was mapped. The study also elucidates degradation pathway of benzoate in Serratia sp. DS001 by correlating the proteomics data with the catabolites of benzoate.

Identification of an Effective Early Signaling Signature during Neo-Vasculogenesis In Vivo by Ex Vivo Proteomic Profiling

PubMed Central

Rohban, Rokhsareh; Reinisch, Andreas; Etchart, Nathalie; Schallmoser, Katharina; Hofmann, Nicole A.; Szoke, Krisztina; Brinchmann, Jan E.; Rad, Ehsan Bonyadi; Rohde, Eva; Strunk, Dirk

2013-01-01

Therapeutic neo-vasculogenesis in vivo can be achieved by the co-transplantation of human endothelial colony-forming progenitor cells (ECFCs) with mesenchymal stem/progenitor cells (MSPCs). The underlying mechanism is not completely understood thus hampering the development of novel stem cell therapies. We hypothesized that proteomic profiling could be used to retrieve the in vivo signaling signature during the initial phase of human neo-vasculogenesis. ECFCs and MSPCs were therefore either transplanted alone or co-transplanted subcutaneously into immune deficient mice. Early cell signaling, occurring within the first 24 hours in vivo, was analyzed using antibody microarray proteomic profiling. Vessel formation and persistence were verified in parallel transplants for up to 24 weeks. Proteomic analysis revealed significant alteration of regulatory components including caspases, calcium/calmodulin-dependent protein kinase, DNA protein kinase, human ErbB2 receptor-tyrosine kinase as well as mitogen-activated protein kinases. Caspase-4 was selected from array results as one therapeutic candidate for targeting vascular network formation in vitro as well as modulating therapeutic vasculogenesis in vivo. As a proof-of-principle, caspase-4 and general caspase-blocking led to diminished endothelial network formation in vitro and significantly decreased vasculogenesis in vivo. Proteomic profiling ex vivo thus unraveled a signaling signature which can be used for target selection to modulate neo-vasculogenesis in vivo. PMID:23826172

Label-free quantitative proteomics to investigate strawberry fruit proteome changes under controlled atmosphere and low temperature storage.

PubMed

Li, Li; Luo, Zisheng; Huang, Xinhong; Zhang, Lu; Zhao, Pengyu; Ma, Hongyuan; Li, Xihong; Ban, Zhaojun; Liu, Xia

2015-04-29

To elucidate the mechanisms contributing to fruit responses to senescence and stressful environmental stimuli under low temperature (LT) and controlled atmosphere (CA) storage, a label-free quantitative proteomic investigation was conducted in strawberry (Fragaria ananassa, Duch. cv. 'Akihime'). Postharvest physiological quality traits including firmness, total soluble solids, total acidity, ascorbic acid and volatile production were characterized following storage under different conditions. The observed post-storage protein expression profiles may be associated with delayed senescence features in strawberry. A total of 454 proteins were identified in differentially treated strawberry fruits. Quantitative analysis, using normalized spectral counts, revealed 73 proteins common to all treatments, which formed three clusters in a hierarchical clustering analysis. The proteins spanned a range of functions in various metabolic pathways and networks involved in carbohydrate and energy metabolism, volatile biosynthesis, phenylpropanoid activity, stress response and protein synthesis, degradation and folding. After CA and LT storage, 16 (13) and 11 (17) proteins, respectively, were significantly increased (decreased) in abundance, while expression profile of 12 proteins was significantly changed by both CA and LT. To summarize, the differential variability of abundance in strawberry proteome, working in a cooperative manner, provided an overview of the biological processes that occurred during CA and LT storage. Controlled atmosphere storage at an optimal temperature is regarded to be an effective postharvest technology to delay fruit senescence and maintain fruit quality during shelf life. Nonetheless, little information on fruit proteomic changes under controlled atmosphere and/or low temperature storage is available. The significance of this paper is that it is the first study employing a label-free approach in the investigation of strawberry fruit response to controlled atmosphere and cold storage. Changes in postharvest physiological quality traits including volatile production, firmness, ascorbic acid, soluble solids and total acidity were also characterized. Significant biological changes associated with senescence were revealed and differentially abundant proteins under various storage conditions were identified. Proteomic profiles were linked to physiological aspects of strawberry fruit senescence in order to provide new insights into possible regulation mechanisms. Findings from this study not only provide proteomic information on fruit regulation, but also pave the way for further quantitative studies at the transcriptomic and metabolomic levels. Copyright © 2015 Elsevier B.V. All rights reserved.

A Combined Metabonomic and Proteomic Approach Identifies Frontal Cortex Changes in a Chronic Phencyclidine Rat Model in Relation to Human Schizophrenia Brain Pathology

PubMed Central

Wesseling, Hendrik; Chan, Man K; Tsang, T M; Ernst, Agnes; Peters, Fabian; Guest, Paul C; Holmes, Elaine; Bahn, Sabine

2013-01-01

Current schizophrenia (SCZ) treatments fail to treat the broad range of manifestations associated with this devastating disorder. Thus, new translational models that reproduce the core pathological features are urgently needed to facilitate novel drug discovery efforts. Here, we report findings from the first comprehensive label-free liquid-mass spectrometry proteomic- and proton nuclear magnetic resonance-based metabonomic profiling of the rat frontal cortex after chronic phencyclidine (PCP) intervention, which induces SCZ-like symptoms. The findings were compared with results from a proteomic profiling of post-mortem prefrontal cortex from SCZ patients and with relevant findings in the literature. Through this approach, we identified proteomic alterations in glutamate-mediated Ca2+ signaling (Ca2+/calmodulin-dependent protein kinase II, PPP3CA, and VISL1), mitochondrial function (GOT2 and PKLR), and cytoskeletal remodeling (ARP3). Metabonomic profiling revealed changes in the levels of glutamate, glutamine, glycine, pyruvate, and the Ca2+ regulator taurine. Effects on similar pathways were also identified in the prefrontal cortex tissue from human SCZ subjects. The discovery of similar but not identical proteomic and metabonomic alterations in the chronic PCP rat model and human brain indicates that this model recapitulates only some of the molecular alterations of the disease. This knowledge may be helpful in understanding mechanisms underlying psychosis, which, in turn, can facilitate improved therapy and drug discovery for SCZ and other psychiatric diseases. Most importantly, these molecular findings suggest that the combined use of multiple models may be required for more effective translation to studies of human SCZ. PMID:23942359

The Response of the Root Proteome to the Synthetic Strigolactone GR24 in Arabidopsis*

PubMed Central

Walton, Alan; Stes, Elisabeth; Goeminne, Geert; Braem, Lukas; Vuylsteke, Marnik; Matthys, Cedrick; De Cuyper, Carolien; Staes, An; Vandenbussche, Jonathan; Boyer, François-Didier; Vanholme, Ruben; Fromentin, Justine; Boerjan, Wout; Gevaert, Kris; Goormachtig, Sofie

2016-01-01

Strigolactones are plant metabolites that act as phytohormones and rhizosphere signals. Whereas most research on unraveling the action mechanisms of strigolactones is focused on plant shoots, we investigated proteome adaptation during strigolactone signaling in the roots of Arabidopsis thaliana. Through large-scale, time-resolved, and quantitative proteomics, the impact of the strigolactone analog rac-GR24 was elucidated on the root proteome of the wild type and the signaling mutant more axillary growth 2 (max2). Our study revealed a clear MAX2-dependent rac-GR24 response: an increase in abundance of enzymes involved in flavonol biosynthesis, which was reduced in the max2–1 mutant. Mass spectrometry-driven metabolite profiling and thin-layer chromatography experiments demonstrated that these changes in protein expression lead to the accumulation of specific flavonols. Moreover, quantitative RT-PCR revealed that the flavonol-related protein expression profile was caused by rac-GR24-induced changes in transcript levels of the corresponding genes. This induction of flavonol production was shown to be activated by the two pure enantiomers that together make up rac-GR24. Finally, our data provide much needed clues concerning the multiple roles played by MAX2 in the roots and a comprehensive view of the rac-GR24-induced response in the root proteome. PMID:27317401

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.

Characterization of the liver tissue interstitial fluid (TIF) proteome indicates potential for application in liver disease biomarker discovery.

PubMed

Sun, Wei; Ma, Jie; Wu, Songfeng; Yang, Dong; Yan, Yujuan; Liu, Kehui; Wang, Jinglan; Sun, Longqin; Chen, Ning; Wei, Handong; Zhu, Yunping; Xing, Baocai; Zhao, Xiaohang; Qian, Xiaohong; Jiang, Ying; He, Fuchu

2010-02-05

Tissue interstitial fluid (TIF) forms the interface between circulating body fluids and intracellular fluid. Pathological alterations of liver cells could be reflected in TIF, making it a promising source of liver disease biomarkers. Mouse liver TIF was extracted, separated by SDS-PAGE, analyzed by linear ion trap mass spectrometer, and 1450 proteins were identified. These proteins may be secreted, shed from membrane vesicles, or represent cellular breakdown products. They show different profiling patterns, quantities, and possibly modification/cleavage of intracellular proteins. The high solubility and even distribution of liver TIF supports its suitability for proteome analysis. Comparison of mouse liver TIF data with liver tissue and plasma proteome data identified major proteins that might be released from liver to plasma and serve as blood biomarkers of liver origin. This result was partially supported by comparison of human liver TIF data with human liver and plasma proteome data. Paired TIFs from tumor and nontumor liver tissues of a hepatocellular carcinoma patient were analyzed and the profile of subtracted differential proteins supports the potential for biomarker discovery in TIF. This study is the first analysis of the liver TIF proteome and provides a foundation for further application of TIF in liver disease biomarker discovery.

Integrative Analysis of Longitudinal Metabolomics Data from a Personal Multi-Omics Profile

PubMed Central

Stanberry, Larissa; Mias, George I.; Haynes, Winston; Higdon, Roger; Snyder, Michael; Kolker, Eugene

2013-01-01

The integrative personal omics profile (iPOP) is a pioneering study that combines genomics, transcriptomics, proteomics, metabolomics and autoantibody profiles from a single individual over a 14-month period. The observation period includes two episodes of viral infection: a human rhinovirus and a respiratory syncytial virus. The profile studies give an informative snapshot into the biological functioning of an organism. We hypothesize that pathway expression levels are associated with disease status. To test this hypothesis, we use biological pathways to integrate metabolomics and proteomics iPOP data. The approach computes the pathways’ differential expression levels at each time point, while taking into account the pathway structure and the longitudinal design. The resulting pathway levels show strong association with the disease status. Further, we identify temporal patterns in metabolite expression levels. The changes in metabolite expression levels also appear to be consistent with the disease status. The results of the integrative analysis suggest that changes in biological pathways may be used to predict and monitor the disease. The iPOP experimental design, data acquisition and analysis issues are discussed within the broader context of personal profiling. PMID:24958148

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.

CSF proteomic fingerprints for HIV-associated cognitive impairment.

PubMed

Laspiur, Juliana Pérez; Anderson, Eric R; Ciborowski, Pawel; Wojna, Valerie; Rozek, Wojciech; Duan, Fenghai; Mayo, Raul; Rodríguez, Elaine; Plaud-Valentín, Marinés; Rodríguez-Orengo, José; Gendelman, Howard E; Meléndez, Loyda M

2007-12-01

Cognitive impairment remains a major complication of advanced human immunodeficiency virus (HIV) infection despite the widespread use of anti-retroviral therapy. Diagnosis is made by exclusion making biomarkers of great potential use. Thus, we used an integrated proteomics platform to assess cerebrospinal fluid protein profiles from 50 HIV-1 seropositive Hispanic women. Nine of 38 proteins identified were unique in those patients with cognitive impairment (CI). These proteins were linked to cell signaling, structural function, and antioxidant activities. This work highlights, in a preliminary manner, the utility of proteomic profiling for biomarker discovery for HIV-1 associated cognitive dysfunction.

CSF proteomic fingerprints for HIV- associated cognitive impairment

PubMed Central

Laspiur, Juliana Pérez; Anderson, Eric R.; Ciborowski, Pawel; Wojna, Valerie; Rozek, Wojciech; Duan, Fenghai; Mayo, Raul; Rodríguez, Elaine; Plaud-Valentín, Marinés; Rodríguez-Orengo, José; Gendelman, Howard E.; Meléndez, Loyda M.

2008-01-01

Cognitive impairment remains a major complication of advanced human immunodeficiency virus (HIV) infection despite the wide spread use of anti-retroviral therapy. Diagnosis is made by exclusion making biomarkers of great potential use. Thus, we used an integrated proteomics platform to assess cerebrospinal fluid protein profiles from 50 HIV-1 seropositive Hispanic women. Nine of 38 proteins identified were unique in those patients with cognitive impairment. These proteins were linked to cell signaling, structural function, and antioxidant activities. This work highlights, in a preliminary manner, the utility of proteomic profiling for biomarker discovery for HIV-1 associated cognitive dysfunction. PMID:17950469

Application of activity-based protein profiling to study enzyme function in adipocytes.

PubMed

Galmozzi, Andrea; Dominguez, Eduardo; Cravatt, Benjamin F; Saez, Enrique

2014-01-01

Activity-based protein profiling (ABPP) is a chemical proteomics approach that utilizes small-molecule probes to determine the functional state of enzymes directly in native systems. ABPP probes selectively label active enzymes, but not their inactive forms, facilitating the characterization of changes in enzyme activity that occur without alterations in protein levels. ABPP can be a tool superior to conventional gene expression and proteomic profiling methods to discover new enzymes active in adipocytes and to detect differences in the activity of characterized enzymes that may be associated with disorders of adipose tissue function. ABPP probes have been developed that react selectively with most members of specific enzyme classes. Here, using as an example the serine hydrolase family that includes many enzymes with critical roles in adipocyte physiology, we describe methods to apply ABPP analysis to the study of adipocyte enzymatic pathways. © 2014 Elsevier Inc. All rights reserved.

Proteomics assisted profiling of antimicrobial peptide signatures from black pepper (Piper nigrum L.).

PubMed

Umadevi, P; Soumya, M; George, Johnson K; Anandaraj, M

2018-05-01

Plant antimicrobial peptides are the interesting source of studies in defense response as they are essential components of innate immunity which exert rapid defense response. In spite of abundant reports on the isolation of antimicrobial peptides (AMPs) from many sources, the profile of AMPs expressed/identified from single crop species under certain stress/physiological condition is still unknown. This work describes the AMP signature profile of black pepper and their expression upon Phytophthora infection using label-free quantitative proteomics strategy. The differential expression of 24 AMPs suggests that a combinatorial strategy is working in the defense network. The 24 AMP signatures belonged to the cationic, anionic, cysteine-rich and cysteine-free group. As the first report on the possible involvement of AMP signature in Phytophthora infection, our results offer a platform for further study on regulation, evolutionary importance and exploitation of theses AMPs as next generation molecules against pathogens.

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.

Alkylation Damage by Lipid Electrophiles Targets Functional Protein Systems*

PubMed Central

Codreanu, Simona G.; Ullery, Jody C.; Zhu, Jing; Tallman, Keri A.; Beavers, William N.; Porter, Ned A.; Marnett, Lawrence J.; Zhang, Bing; Liebler, Daniel C.

2014-01-01

Protein alkylation by reactive electrophiles contributes to chemical toxicities and oxidative stress, but the functional impact of alkylation damage across proteomes is poorly understood. We used Click chemistry and shotgun proteomics to profile the accumulation of proteome damage in human cells treated with lipid electrophile probes. Protein target profiles revealed three damage susceptibility classes, as well as proteins that were highly resistant to alkylation. Damage occurred selectively across functional protein interaction networks, with the most highly alkylation-susceptible proteins mapping to networks involved in cytoskeletal regulation. Proteins with lower damage susceptibility mapped to networks involved in protein synthesis and turnover and were alkylated only at electrophile concentrations that caused significant toxicity. Hierarchical susceptibility of proteome systems to alkylation may allow cells to survive sublethal damage while protecting critical cell functions. PMID:24429493

The proteome of Hypobaric Induced Hypoxic Lung: Insights from Temporal Proteomic Profiling for Biomarker Discovery

PubMed Central

Ahmad, Yasmin; Sharma, Narendra K.; Ahmad, Mohammad Faiz; Sharma, Manish; Garg, Iti; Srivastava, Mousami; Bhargava, Kalpana

2015-01-01

Exposure to high altitude induces physiological responses due to hypoxia. Lungs being at the first level to face the alterations in oxygen levels are critical to counter and balance these changes. Studies have been done analysing pulmonary proteome alterations in response to exposure to hypobaric hypoxia. However, such studies have reported the alterations at specific time points and do not reflect the gradual proteomic changes. These studies also identify the various biochemical pathways and responses induced after immediate exposure and the resolution of these effects in challenge to hypobaric hypoxia. In the present study, using 2-DE/MS approach, we attempt to resolve these shortcomings by analysing the proteome alterations in lungs in response to different durations of exposure to hypobaric hypoxia. Our study thus highlights the gradual and dynamic changes in pulmonary proteome following hypobaric hypoxia. For the first time, we also report the possible consideration of SULT1A1, as a biomarker for the diagnosis of high altitude pulmonary edema (HAPE). Higher SULT1A1 levels were observed in rats as well as in humans exposed to high altitude, when compared to sea-level controls. This study can thus form the basis for identifying biomarkers for diagnostic and prognostic purposes in responses to hypobaric hypoxia. PMID:26022216

Proteomic profile of serum of pregnant women carring a fetus with Down syndrome using nano uplc Q-tof ms/ms technology.

PubMed

López Uriarte, Graciela Arelí; Burciaga Flores, Carlos Horacio; Torres de la Cruz, Víctor Manuel; Medina Aguado, María Magdalena; Gómez Puente, Viviana Maricela; Romero Gutiérrez, Liliana Nayeli; Martínez de Villarreal, Laura Elia

2018-06-01

Prenatal diagnosis of Down syndrome (DS) is based on the calculated risk of maternal age, biochemical and ultrasonographic markers and recently by cfDNA. Differences in proteomic profiles may give an opportunity to find new biomarkers. Characterize proteome of serum of mothers carrying DS fetus. Blood serum samples of three groups of women were obtained, (a) 10 non-pregnant, (b) 10 pregnant with healthy fetus by ultrasound evaluation, (c) nine pregnant with DS fetus. Sample preparation was as follows: Albumin/IgG depletion, desalting, and trypsin digestion; the process was performed in nanoUPLC MS/MS. Data analysis was made with Mass Lynx 4.1 and ProteinLynx Global Server 3.0, peptide and protein recognition by MASCOT algorithm and UNIPROT-Swissprot database. Each group showed different protein profiles. Some proteins were shared between groups. Only sera from pregnant women showed proteins related to immune and clot pathways. Mothers with DS fetus had 42 specific proteins. We found a different serum protein profile in mothers carrying DS fetuses that do not reflect expression of genes in the extra chromosome. Further studies will be necessary to establish the role of these proteins in aneuploid fetus and analyze their possible use as potential biomarkers.

Secretome profiles of immortalized dental follicle cells using iTRAQ-based proteomic analysis.

PubMed

Dou, Lei; Wu, Yan; Yan, Qifang; Wang, Jinhua; Zhang, Yan; Ji, Ping

2017-08-04

Secretomes produced by mesenchymal stromal cells (MSCs) were considered to be therapeutic potential. However, harvesting enough primary MSCs from tissue was time-consuming and costly, which impeded the application of MSCs secretomes. This study was to immortalize MSCs and compare the secretomes profile of immortalized and original MSCs. Human dental follicle cells (DFCs) were isolated and immortalized using pMPH86. The secretome profile of immortalized DFCs (iDFCs) was investigated and compared using iTRAQ labeling combined with mass spectrometry (MS) quantitative proteomics. The MS data was analyzed using ProteinPilotTM software, and then bioinformatic analysis of identified proteins was done. A total of 2092 secreted proteins were detected in conditioned media of iDFCs. Compared with primary DFCs, 253 differently expressed proteins were found in iDFCs secretome (142 up-regulated and 111 down-regulated). Intensive bioinformatic analysis revealed that the majority of secreted proteins were involved in cellular process, metabolic process, biological regulation, cellular component organization or biogenesis, immune system process, developmental process, response to stimulus and signaling. Proteomic profile of cell secretome wasn't largely affected after immortalization converted by this piggyBac immortalization system. The secretome of iDFCs may be a good candidate of primary DFCs for regenerative medicine.

Proteomic analysis of the seed development in Jatropha curcas: from carbon flux to the lipid accumulation.

PubMed

Liu, Hui; Wang, Cuiping; Komatsu, Setsuko; He, Mingxia; Liu, Gongshe; Shen, Shihua

2013-10-08

To characterize the metabolic signatures of lipid accumulation in Jatropha curcas seeds, comparative proteomic technique was employed to profile protein changes during the seed development. Temporal changes in comparative proteome were examined using gels-based proteomic technique at six developmental stages for lipid accumulation. And 104 differentially expressed proteins were identified by MALDI-TOF/TOF tandem mass spectrometry. These protein species were classified into 10 functional categories, and the results demonstrated that protein species related to energy and metabolism were notably accumulated and involved in the carbon flux to lipid accumulation that occurs primarily from early to late stage in seed development. Glycolysis and oxidative pentose phosphate pathways were the major pathways of producing carbon flux, and the glucose-6-phosphate and triose-phosphate are the major carbon source for fatty acid synthesis. Lipid analysis revealed that fatty acid accumulation initiated 25days after flowering at the late stage of seed development of J. curcas. Furthermore, C16:0 was initially synthesized as the precursor for the elongation to C18:1 and C18:2 in the developing seeds of J. curcas. Together, the metabolic signatures on protein changes in seed development provide profound knowledge and perspective insights into understanding lipid network in J. curcas. Due to the abundant oil content in seeds, Jatropha curcas seeds are being considered as the ideal materials for biodiesel. Although several studies had carried out the transcriptomic project to study the genes expression profiles in seed development of J. curcas, these ESTs hadn't been confirmed by qRT-PCR. Yet, the seed development of J. curcas had been described for a pool of developing seeds instead of being characterized systematically. Moreover, cellular metabolic events are also controlled by protein-protein interactions, posttranslational protein modifications, and enzymatic activities which cannot be described by transcriptional profiling approaches alone. In this study, within the overall objective of profiling differential protein abundance in developing J. curcas seeds, we provide a setting of physiological data with dynamic proteomic and qRT-PCR analysis to characterize the metabolic pathways and the relationship between mRNA and protein patterns from early stage to seed filling during the seed development of J. curcas. The construction of J. curcas seed development proteome profiles will significantly increase our understanding of the process of seed development and provide a foundation to examine the dynamic changes of the metabolic network during seed development process and certainly suggest some clues to improve the lipid content of J. curcas seeds. © 2013. Published by Elsevier B.V. All rights reserved.

Proteomic Profiling of the Pituitary Gland in Studies of Psychiatric Disorders.

PubMed

Krishnamurthy, Divya; Rahmoune, Hassan; Guest, Paul C

2017-01-01

Psychiatric disorders have been associated with perturbations of the hypothalamic-pituitary-adrenal axis. Therefore, proteomic studies of the pituitary gland have the potential to provide new insights into the underlying pathways affected in these conditions as well as identify new biomarkers or targets for use in developing improved medications. This chapter describes a protocol for preparation of pituitary protein extracts followed by characterization of the pituitary proteome by label-free liquid chromatography-tandem mass spectrometry in expression mode (LC-MS E ). The main focus was on establishing a method for identifying the major pituitary hormones and accessory proteins as many of these have already been implicated in psychiatric diseases.

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

PubMed

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

2018-06-21

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

A Combined Metabolomic and Proteomic Analysis of Gestational Diabetes Mellitus

PubMed Central

Hajduk, Joanna; Klupczynska, Agnieszka; Dereziński, Paweł; Matysiak, Jan; Kokot, Piotr; Nowak, Dorota M.; Gajęcka, Marzena; Nowak-Markwitz, Ewa; Kokot, Zenon J.

2015-01-01

The aim of this pilot study was to apply a novel combined metabolomic and proteomic approach in analysis of gestational diabetes mellitus. The investigation was performed with plasma samples derived from pregnant women with diagnosed gestational diabetes mellitus (n = 18) and a matched control group (n = 13). The mass spectrometry-based analyses allowed to determine 42 free amino acids and low molecular-weight peptide profiles. Different expressions of several peptides and altered amino acid profiles were observed in the analyzed groups. The combination of proteomic and metabolomic data allowed obtaining the model with a high discriminatory power, where amino acids ethanolamine, l-citrulline, l-asparagine, and peptide ions with m/z 1488.59; 4111.89 and 2913.15 had the highest contribution to the model. The sensitivity (94.44%) and specificity (84.62%), as well as the total group membership classification value (90.32%) calculated from the post hoc classification matrix of a joint model were the highest when compared with a single analysis of either amino acid levels or peptide ion intensities. The obtained results indicated a high potential of integration of proteomic and metabolomics analysis regardless the sample size. This promising approach together with clinical evaluation of the subjects can also be used in the study of other diseases. PMID:26694367

Global proteomics profiling improves drug sensitivity prediction: results from a multi-omics, pan-cancer modeling approach.

PubMed

Ali, Mehreen; Khan, Suleiman A; Wennerberg, Krister; Aittokallio, Tero

2018-04-15

Proteomics profiling is increasingly being used for molecular stratification of cancer patients and cell-line panels. However, systematic assessment of the predictive power of large-scale proteomic technologies across various drug classes and cancer types is currently lacking. To that end, we carried out the first pan-cancer, multi-omics comparative analysis of the relative performance of two proteomic technologies, targeted reverse phase protein array (RPPA) and global mass spectrometry (MS), in terms of their accuracy for predicting the sensitivity of cancer cells to both cytotoxic chemotherapeutics and molecularly targeted anticancer compounds. Our results in two cell-line panels demonstrate how MS profiling improves drug response predictions beyond that of the RPPA or the other omics profiles when used alone. However, frequent missing MS data values complicate its use in predictive modeling and required additional filtering, such as focusing on completely measured or known oncoproteins, to obtain maximal predictive performance. Rather strikingly, the two proteomics profiles provided complementary predictive signal both for the cytotoxic and targeted compounds. Further, information about the cellular-abundance of primary target proteins was found critical for predicting the response of targeted compounds, although the non-target features also contributed significantly to the predictive power. The clinical relevance of the selected protein markers was confirmed in cancer patient data. These results provide novel insights into the relative performance and optimal use of the widely applied proteomic technologies, MS and RPPA, which should prove useful in translational applications, such as defining the best combination of omics technologies and marker panels for understanding and predicting drug sensitivities in cancer patients. Processed datasets, R as well as Matlab implementations of the methods are available at https://github.com/mehr-een/bemkl-rbps. mehreen.ali@helsinki.fi or tero.aittokallio@fimm.fi. Supplementary data are available at Bioinformatics online.

A Method for Label-Free, Differential Top-Down Proteomics.

PubMed

Ntai, Ioanna; Toby, Timothy K; LeDuc, Richard D; Kelleher, Neil L

2016-01-01

Biomarker discovery in the translational research has heavily relied on labeled and label-free quantitative bottom-up proteomics. Here, we describe a new approach to biomarker studies that utilizes high-throughput top-down proteomics and is the first to offer whole protein characterization and relative quantitation within the same experiment. Using yeast as a model, we report procedures for a label-free approach to quantify the relative abundance of intact proteins ranging from 0 to 30 kDa in two different states. In this chapter, we describe the integrated methodology for the large-scale profiling and quantitation of the intact proteome by liquid chromatography-mass spectrometry (LC-MS) without the need for metabolic or chemical labeling. This recent advance for quantitative top-down proteomics is best implemented with a robust and highly controlled sample preparation workflow before data acquisition on a high-resolution mass spectrometer, and the application of a hierarchical linear statistical model to account for the multiple levels of variance contained in quantitative proteomic comparisons of samples for basic and clinical research.

Impact of pyrrolidine-bispyrrole DNA minor groove binding agents and chirality on global proteomic profile in Escherichia Coli.

PubMed

Yang, Ya-Ting; Lin, Chun-Yu; Jeng, Jingyueh; Ong, Chi-Wi

2013-05-23

There is great interest in the design of small molecules that selectively target minor grooves of duplex DNA for controlling specific gene expression implicated in a disease. The design of chiral small molecules for rational drug design has attracted increasing attention due to the chirality of DNA. Yet, there is limited research on the chirality effect of minor groove binders on DNA interaction, especially at the protein expression level. This paper is an attempt to illustrate that DNA binding affinity might not provide a full picture on the biological activities. Drug interacting at the genomic level can be translated to the proteomic level. Here we have illustrated that although the chiral bispyrrole-pyrrolidine-oligoamides, PySSPy and PyRSPy, showed low binding affinity to DNA, their influence at the proteomic level is significant. More importantly, the chirality also plays a role. Two-dimensional proteomic profile to identify the differentially expressed protein in Escherichia coli DH5α (E coli DH5α) were investigated. E coli DH5α incubated with the chiral PySSPy and PyRSPy, diastereomeric at the pyrrolidine ring, showed differential expression of eighteen proteins as observed through two dimensional proteomic profiling. These eighteen proteins identified by MALDI_TOF/TOF MS include antioxidant defense, DNA protection, protein synthesis, chaperone, and stress response proteins. No statistically significant toxicity was observed at the tested drug concentrations as measured via MTT assay. The current results showed that the chiral PySSPy and PyRSPy impact on the proteomic profiling of E coli DH5α, implicating the importance of drug chirality on biological activities at the molecular level.

Profiling modifications for glioblastoma proteome using ultra-tolerant database search: Are the peptide mass shifts biologically relevant or chemically induced?

PubMed

Tarasova, Irina A; Chumakov, Peter M; Moshkovskii, Sergei A; Gorshkov, Mikhail V

2018-05-17

Peptide mass shifts were profiled using ultra-tolerant database search strategy for shotgun proteomics data sets of human glioblastoma cell lines demonstrating strong response to the type I interferon (IFNα-2b) treatment. The main objective of this profiling was revealing the cell response to IFN treatment at the level of protein modifications. To achieve this objective, statistically significant changes in peptide mass shift profiles between IFN treated and untreated glioblastoma samples were analyzed. Detailed analysis of MS/MS spectra allowed further interpretation of the observed mass shifts and differentiation between post-translational and artifact modifications. Malignant cells typically acquire increased sensitivity to viruses due to the deregulated antiviral mechanisms. Therefore, a viral therapy is considered as one of the promising approaches to treat cancer. However, recent studies have demonstrated that malignant cells can preserve intact antiviral mechanisms, e.g. interferon signaling, and develop resistance to virus infection in response to interferon treatment. Post translational modifications, e.g. tyrosine phosphorylation, are the interferon signaling drivers. Thus, comprehensive characterization of modifications is crucially important, yet, most challenging problem in cancer proteomics. Here, we report on the application of the recently introduced ultra-tolerant search strategy for profiling peptide modifications in the human glioblastoma cell lines demonstrating strong response to the type I interferon (IFNα-2b) treatment. The specific aim of the study was identification of statistically significant changes in peptide mass shift profiles between IFN treated and untreated glioblastoma samples, as well as determination of whether these shifts represent the biologically relevant modification. Copyright © 2018 Elsevier B.V. All rights reserved.

Comparative proteomic analysis of rd29A:RdreB1BI transgenic and non-transgenic strawberries exposed to low temperature.

PubMed

Gu, Xianbin; Gao, Zhihong; Zhuang, Weibing; Qiao, Yushan; Wang, Xiuyun; Mi, Lin; Zhang, Zhen; Lin, Zhilin

2013-05-01

Low-temperature stress is one of the major abiotic stresses in plants worldwide, and the dehydration responsive element binding protein (DREB) transcription factor induces expression of genes involved in environmental stress tolerance in plants. A proteomic approach based on two-dimensional gel electrophoresis (2-DE) and subsequent mass spectrometric identification was used to study the changes in the leaf proteome profiles of rd29A:RdreB1BI transgenic and non-transgenic strawberries exposed to low-temperature conditions. By comparing the proteomic profiles, we located 21 protein spots that were reproducibly up- or down-regulated by more than twofold between transgenic and non-transgenic strawberries. Eight identified proteins function in energy and metabolism, four in biosynthetic processes, four were stress and defense related, three spots were identified as cold-stress related expressed sequence tags (ESTs), and two were unknown proteins. The change patterns of low-temperature tolerance proteins, including photosynthetic proteins (RuBisCO large subunit and RuBisCO activase), cytoplasmic Cu/Zn-superoxide dismutase (Cu/Zn-SOD), late embryogenesis abundant protein 14-A (Lea14-A), eukaryotic translation initiation factor 5A (eIF5A), and cold-stress related ESTs, were differentially regulated between non-transgenic and rd29A:RdreB1BI transgenic strawberries. They are likely important gene products in the regulatory network of the RdreB1BI gene. Consequently, this study provides the first characterization of the transgenic strawberry proteome and the predicted target proteins of the RdreB1BI gene by using proteomic approaches. Copyright © 2013 Elsevier GmbH. All rights reserved.

Urinary proteomic profiling in severe obesity and obstructive sleep apnoea with CPAP treatment

PubMed Central

Seetho, Ian W; Ramírez-Torres, Adela; Albalat, Amaya; Mullen, William; Mischak, Harald; Parker, Robert J; Craig, Sonya; Duffy, Nick; Hardy, Kevin J; Burniston, Jatin G; Wilding, John PH

2015-01-01

Introduction Obstructive sleep apnoea (OSA) is common in obesity and is associated with cardiovascular and metabolic complications. Continuous positive airway pressure (CPAP) in OSA may lead to physiological changes reflected in the urinary proteome. The aim of this study was to characterise the urinary proteome in severely obese adult subjects with OSA who were receiving CPAP compared with severely obese subjects without OSA. Methods Severely obese subjects with and without OSA were recruited. Subjects with OSA were receiving CPAP. Body composition and blood pressure measurements were recorded. Urinary samples were analysed by Capillary Electrophoresis–Mass Spectrometry (CE–MS). Results Twenty-seven subjects with OSA-on-CPAP (age 49±7years, BMI 43±7 kg/m2) and 25 controls without OSA (age 52±9years, BMI 39±4 kg/m2) were studied. Age and BMI were not significantly different between groups. Mean CPAP use for OSA patients was 14.5±1.0 months. Metabolic syndrome was present in 14(52%) of those with OSA compared with 6(24%) of controls (p=0.039). A urinary proteome comprising 15 peptides was identified showing differential expression between the groups (p<0.01). Although correction for multiple testing did not reach significance, sequences were determined for 8 peptides demonstrating origins from collagens, fibrinogen beta chain and T-cadherin that may be associated with underlying cardiovascular disease mechanisms in OSA. Conclusions The urinary proteome is compared in OSA with CPAP and without OSA in severe obesity. The effects of CPAP on OSA may lead to changes in the urinary peptides but further research work is needed to investigate the potential role for urinary proteomics in characterising urinary peptide profiles in OSA. PMID:26483946

A cell-based systems biology assessment of human blood to monitor immune responses after influenza vaccination.

PubMed

Hoek, Kristen L; Samir, Parimal; Howard, Leigh M; Niu, Xinnan; Prasad, Nripesh; Galassie, Allison; Liu, Qi; Allos, Tara M; Floyd, Kyle A; Guo, Yan; Shyr, Yu; Levy, Shawn E; Joyce, Sebastian; Edwards, Kathryn M; Link, Andrew J

2015-01-01

Systems biology is an approach to comprehensively study complex interactions within a biological system. Most published systems vaccinology studies have utilized whole blood or peripheral blood mononuclear cells (PBMC) to monitor the immune response after vaccination. Because human blood is comprised of multiple hematopoietic cell types, the potential for masking responses of under-represented cell populations is increased when analyzing whole blood or PBMC. To investigate the contribution of individual cell types to the immune response after vaccination, we established a rapid and efficient method to purify human T and B cells, natural killer (NK) cells, myeloid dendritic cells (mDC), monocytes, and neutrophils from fresh venous blood. Purified cells were fractionated and processed in a single day. RNA-Seq and quantitative shotgun proteomics were performed to determine expression profiles for each cell type prior to and after inactivated seasonal influenza vaccination. Our results show that transcriptomic and proteomic profiles generated from purified immune cells differ significantly from PBMC. Differential expression analysis for each immune cell type also shows unique transcriptomic and proteomic expression profiles as well as changing biological networks at early time points after vaccination. This cell type-specific information provides a more comprehensive approach to monitor vaccine responses.

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

Yeast Interspecies Comparative Proteomics Reveals Divergence in Expression Profiles and Provides Insights into Proteome Resource Allocation and Evolutionary Roles of Gene Duplication*

PubMed Central

Kito, Keiji; Ito, Haruka; Nohara, Takehiro; Ohnishi, Mihoko; Ishibashi, Yuko; Takeda, Daisuke

2016-01-01

Omics analysis is a versatile approach for understanding the conservation and diversity of molecular systems across multiple taxa. In this study, we compared the proteome expression profiles of four yeast species (Saccharomyces cerevisiae, Saccharomyces mikatae, Kluyveromyces waltii, and Kluyveromyces lactis) grown on glucose- or glycerol-containing media. Conserved expression changes across all species were observed only for a small proportion of all proteins differentially expressed between the two growth conditions. Two Kluyveromyces species, both of which exhibited a high growth rate on glycerol, a nonfermentative carbon source, showed distinct species-specific expression profiles. In K. waltii grown on glycerol, proteins involved in the glyoxylate cycle and gluconeogenesis were expressed in high abundance. In K. lactis grown on glycerol, the expression of glycolytic and ethanol metabolic enzymes was unexpectedly low, whereas proteins involved in cytoplasmic translation, including ribosomal proteins and elongation factors, were highly expressed. These marked differences in the types of predominantly expressed proteins suggest that K. lactis optimizes the balance of proteome resource allocation between metabolism and protein synthesis giving priority to cellular growth. In S. cerevisiae, about 450 duplicate gene pairs were retained after whole-genome duplication. Intriguingly, we found that in the case of duplicates with conserved sequences, the total abundance of proteins encoded by a duplicate pair in S. cerevisiae was similar to that of protein encoded by nonduplicated ortholog in Kluyveromyces yeast. Given the frequency of haploinsufficiency, this observation suggests that conserved duplicate genes, even though minor cases of retained duplicates, do not exhibit a dosage effect in yeast, except for ribosomal proteins. Thus, comparative proteomic analyses across multiple species may reveal not only species-specific characteristics of metabolic processes under nonoptimal culture conditions but also provide valuable insights into intriguing biological principles, including the balance of proteome resource allocation and the role of gene duplication in evolutionary history. PMID:26560065

Yeast Interspecies Comparative Proteomics Reveals Divergence in Expression Profiles and Provides Insights into Proteome Resource Allocation and Evolutionary Roles of Gene Duplication.

PubMed

Kito, Keiji; Ito, Haruka; Nohara, Takehiro; Ohnishi, Mihoko; Ishibashi, Yuko; Takeda, Daisuke

2016-01-01

Omics analysis is a versatile approach for understanding the conservation and diversity of molecular systems across multiple taxa. In this study, we compared the proteome expression profiles of four yeast species (Saccharomyces cerevisiae, Saccharomyces mikatae, Kluyveromyces waltii, and Kluyveromyces lactis) grown on glucose- or glycerol-containing media. Conserved expression changes across all species were observed only for a small proportion of all proteins differentially expressed between the two growth conditions. Two Kluyveromyces species, both of which exhibited a high growth rate on glycerol, a nonfermentative carbon source, showed distinct species-specific expression profiles. In K. waltii grown on glycerol, proteins involved in the glyoxylate cycle and gluconeogenesis were expressed in high abundance. In K. lactis grown on glycerol, the expression of glycolytic and ethanol metabolic enzymes was unexpectedly low, whereas proteins involved in cytoplasmic translation, including ribosomal proteins and elongation factors, were highly expressed. These marked differences in the types of predominantly expressed proteins suggest that K. lactis optimizes the balance of proteome resource allocation between metabolism and protein synthesis giving priority to cellular growth. In S. cerevisiae, about 450 duplicate gene pairs were retained after whole-genome duplication. Intriguingly, we found that in the case of duplicates with conserved sequences, the total abundance of proteins encoded by a duplicate pair in S. cerevisiae was similar to that of protein encoded by nonduplicated ortholog in Kluyveromyces yeast. Given the frequency of haploinsufficiency, this observation suggests that conserved duplicate genes, even though minor cases of retained duplicates, do not exhibit a dosage effect in yeast, except for ribosomal proteins. Thus, comparative proteomic analyses across multiple species may reveal not only species-specific characteristics of metabolic processes under nonoptimal culture conditions but also provide valuable insights into intriguing biological principles, including the balance of proteome resource allocation and the role of gene duplication in evolutionary history. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Proteomic Profiling of Paraffin-Embedded Samples Identifies Metaplasia-Specific and Early-Stage Gastric Cancer Biomarkers

PubMed Central

Sousa, Josane F.; Ham, Amy-Joan L.; Whitwell, Corbin; Nam, Ki Taek; Lee, Hyuk-Joon; Yang, Han-Kwang; Kim, Woo Ho; Zhang, Bing; Li, Ming; LaFleur, Bonnie; Liebler, Daniel C.; Goldenring, James R.

2013-01-01

Early diagnosis and curative resection are the predominant factors associated with increased survival in patients with gastric cancer. However, most gastric cancer cases are still diagnosed at later stages. Since most pathologic specimens are archived as FFPE samples, the ability to use them to generate expression profiles can greatly improve cancer biomarker discovery. We sought to uncover new biomarkers for stomach preneoplastic metaplasias and neoplastic lesions by generating proteome profiles using FFPE samples. We combined peptide isoelectric focusing and liquid chromatography–tandem mass spectrometry analysis to generate proteomic profiles from FFPE samples of intestinal-type gastric cancer, metaplasia, and normal mucosa. The expression patterns of selected proteins were analyzed by immunostaining first in single tissue sections from normal stomach, metaplasia, and gastric cancer and later in larger tissue array cohorts. We detected 60 proteins up-regulated and 87 proteins down-regulated during the progression from normal mucosa to metaplasia to gastric cancer. Two of the up-regulated proteins, LTF and DMBT1, were validated as specific markers for spasmolytic polypeptide–expressing metaplasia and intestinal metaplasia, respectively. In cancers, significantly lower levels of DMBT1 or LTF correlated with more advanced disease and worse prognosis. Thus, proteomic profiling using FFPE samples has led to the identification of two novel markers for stomach metaplasias and gastric cancer prognosis. PMID:22944598

Blood Sampling and Preparation Procedures for Proteomic Biomarker Studies of Psychiatric Disorders.

PubMed

Guest, Paul C; Rahmoune, Hassan

2017-01-01

A major challenge in proteomic biomarker discovery and validation for psychiatric diseases is the inherent biological complexity underlying these conditions. There are also many technical issues which hinder this process such as the lack of standardization in sampling, processing and storage of bio-samples in preclinical and clinical settings. This chapter describes a reproducible procedure for sampling blood serum and plasma that is specifically designed for maximizing data quality output in two-dimensional gel electrophoresis, multiplex immunoassay and mass spectrometry profiling studies.

Quantitative Proteomic Profiling of Prostate Cancer Reveals a Role for miR-128 in Prostate Cancer*

PubMed Central

Khan, Amjad P.; Poisson, Laila M.; Bhat, Vadiraja B.; Fermin, Damian; Zhao, Rong; Kalyana-Sundaram, Shanker; Michailidis, George; Nesvizhskii, Alexey I.; Omenn, Gilbert S.; Chinnaiyan, Arul M.; Sreekumar, Arun

2010-01-01

Multiple, complex molecular events characterize cancer development and progression. Deciphering the molecular networks that distinguish organ-confined disease from metastatic disease may lead to the identification of biomarkers of cancer invasion and disease aggressiveness. Although alterations in gene expression have been extensively quantified during neoplastic progression, complementary analyses of proteomic changes have been limited. Here we interrogate the proteomic alterations in a cohort of 15 prostate-derived tissues that included five each from adjacent benign prostate, clinically localized prostate cancer, and metastatic disease from distant sites. The experimental strategy couples isobaric tags for relative and absolute quantitation with multidimensional liquid phase peptide fractionation followed by tandem mass spectrometry. Over 1000 proteins were quantified across the specimens and delineated into clinically localized and metastatic prostate cancer-specific signatures. Included in these class-specific profiles were both proteins that were known to be dysregulated during prostate cancer progression and new ones defined by this study. Enrichment analysis of the prostate cancer-specific proteomic signature, to gain insight into the functional consequences of these alterations, revealed involvement of miR-128-a/b regulation during prostate cancer progression. This finding was validated using real time PCR analysis for microRNA transcript levels in an independent set of 15 clinical specimens. miR-128 levels were elevated in benign prostate epithelial cell lines compared with invasive prostate cancer cells. Knockdown of miR-128 induced invasion in benign prostate epithelial cells, whereas its overexpression attenuated invasion in prostate cancer cells. Taken together, our profiles of the proteomic alterations of prostate cancer progression revealed miR-128 as a potentially important negative regulator of prostate cancer cell invasion. PMID:19955085

Comparative proteomic profiling of soleus, extensor digitorum longus, flexor digitorum brevis and interosseus muscles from the mdx mouse model of Duchenne muscular dystrophy.

PubMed

Carberry, Steven; Brinkmeier, Heinrich; Zhang, Yaxin; Winkler, Claudia K; Ohlendieck, Kay

2013-09-01

Duchenne muscular dystrophy is due to genetic abnormalities in the dystrophin gene and represents one of the most frequent genetic childhood diseases. In the X-linked muscular dystrophy (mdx) mouse model of dystrophinopathy, different subtypes of skeletal muscles are affected to a varying degree albeit the same single base substitution within exon 23 of the dystrophin gene. Thus, to determine potential muscle subtype-specific differences in secondary alterations due to a deficiency in dystrophin, in this study, we carried out a comparative histological and proteomic survey of mdx muscles. We intentionally included the skeletal muscles that are often used for studying the pathomechanism of muscular dystrophy. Histological examinations revealed a significantly higher degree of central nucleation in the soleus and extensor digitorum longus muscles compared with the flexor digitorum brevis and interosseus muscles. Muscular hypertrophy of 20-25% was likewise only observed in the soleus and extensor digitorum longus muscles from mdx mice, but not in the flexor digitorum brevis and interosseus muscles. For proteomic analysis, muscle protein extracts were separated by fluorescence two-dimensional (2D) gel electrophoresis. Proteins with a significant change in their expression were identified by mass spectrometry. Proteomic profiling established an altered abundance of 24, 17, 19 and 5 protein species in the dystrophin-deficient soleus, extensor digitorum longus, flexor digitorum brevis and interosseus muscle, respectively. The key proteomic findings were verified by immunoblot analysis. The identified proteins are involved in the contraction-relaxation cycle, metabolite transport, muscle metabolism and the cellular stress response. Thus, histological and proteomic profiling of muscle subtypes from mdx mice indicated that distinct skeletal muscles are differentially affected by the loss of the membrane cytoskeletal protein, dystrophin. Varying degrees of perturbed protein expression patterns in the muscle subtypes from mdx mice may be due to dissimilar downstream events, including differences in muscle structure or compensatory mechanisms that counteract pathophysiological processes. The interosseus muscle from mdx mice possibly represents a naturally protected phenotype.

Comparative proteomic profiling of soleus, extensor digitorum longus, flexor digitorum brevis and interosseus muscles from the mdx mouse model of Duchenne muscular dystrophy

PubMed Central

CARBERRY, STEVEN; BRINKMEIER, HEINRICH; ZHANG, YAXIN; WINKLER, CLAUDIA K.; OHLENDIECK, KAY

2013-01-01

Duchenne muscular dystrophy is due to genetic abnormalities in the dystrophin gene and represents one of the most frequent genetic childhood diseases. In the X-linked muscular dystrophy (mdx) mouse model of dystrophinopathy, different subtypes of skeletal muscles are affected to a varying degree albeit the same single base substitution within exon 23 of the dystrophin gene. Thus, to determine potential muscle subtype-specific differences in secondary alterations due to a deficiency in dystrophin, in this study, we carried out a comparative histological and proteomic survey of mdx muscles. We intentionally included the skeletal muscles that are often used for studying the pathomechanism of muscular dystrophy. Histological examinations revealed a significantly higher degree of central nucleation in the soleus and extensor digitorum longus muscles compared with the flexor digitorum brevis and interosseus muscles. Muscular hypertrophy of 20–25% was likewise only observed in the soleus and extensor digitorum longus muscles from mdx mice, but not in the flexor digitorum brevis and interosseus muscles. For proteomic analysis, muscle protein extracts were separated by fluorescence two-dimensional (2D) gel electrophoresis. Proteins with a significant change in their expression were identified by mass spectrometry. Proteomic profiling established an altered abundance of 24, 17, 19 and 5 protein species in the dystrophin-deficient soleus, extensor digitorum longus, flexor digitorum brevis and interosseus muscle, respectively. The key proteomic findings were verified by immunoblot analysis. The identified proteins are involved in the contraction-relaxation cycle, metabolite transport, muscle metabolism and the cellular stress response. Thus, histological and proteomic profiling of muscle subtypes from mdx mice indicated that distinct skeletal muscles are differentially affected by the loss of the membrane cytoskeletal protein, dystrophin. Varying degrees of perturbed protein expression patterns in the muscle subtypes from mdx mice may be due to dissimilar downstream events, including differences in muscle structure or compensatory mechanisms that counteract pathophysiological processes. The interosseus muscle from mdx mice possibly represents a naturally protected phenotype. PMID:23828267

In Silico Functional Networks Identified in Fish Nucleated Red Blood Cells by Means of Transcriptomic and Proteomic Profiling.

PubMed

Puente-Marin, Sara; Nombela, Iván; Ciordia, Sergio; Mena, María Carmen; Chico, Verónica; Coll, Julio; Ortega-Villaizan, María Del Mar

2018-04-09

Nucleated red blood cells (RBCs) of fish have, in the last decade, been implicated in several immune-related functions, such as antiviral response, phagocytosis or cytokine-mediated signaling. RNA-sequencing (RNA-seq) and label-free shotgun proteomic analyses were carried out for in silico functional pathway profiling of rainbow trout RBCs. For RNA-seq, a de novo assembly was conducted, in order to create a transcriptome database for RBCs. For proteome profiling, we developed a proteomic method that combined: (a) fractionation into cytosolic and membrane fractions, (b) hemoglobin removal of the cytosolic fraction, (c) protein digestion, and (d) a novel step with pH reversed-phase peptide fractionation and final Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric (LC ESI-MS/MS) analysis of each fraction. Combined transcriptome- and proteome- sequencing data identified, in silico, novel and striking immune functional networks for rainbow trout nucleated RBCs, which are mainly linked to innate and adaptive immunity. Functional pathways related to regulation of hematopoietic cell differentiation, antigen presentation via major histocompatibility complex class II (MHCII), leukocyte differentiation and regulation of leukocyte activation were identified. These preliminary findings further implicate nucleated RBCs in immune function, such as antigen presentation and leukocyte activation.

In Silico Functional Networks Identified in Fish Nucleated Red Blood Cells by Means of Transcriptomic and Proteomic Profiling

PubMed Central

Puente-Marin, Sara; Ciordia, Sergio; Mena, María Carmen; Chico, Verónica; Coll, Julio

2018-01-01

Nucleated red blood cells (RBCs) of fish have, in the last decade, been implicated in several immune-related functions, such as antiviral response, phagocytosis or cytokine-mediated signaling. RNA-sequencing (RNA-seq) and label-free shotgun proteomic analyses were carried out for in silico functional pathway profiling of rainbow trout RBCs. For RNA-seq, a de novo assembly was conducted, in order to create a transcriptome database for RBCs. For proteome profiling, we developed a proteomic method that combined: (a) fractionation into cytosolic and membrane fractions, (b) hemoglobin removal of the cytosolic fraction, (c) protein digestion, and (d) a novel step with pH reversed-phase peptide fractionation and final Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric (LC ESI-MS/MS) analysis of each fraction. Combined transcriptome- and proteome- sequencing data identified, in silico, novel and striking immune functional networks for rainbow trout nucleated RBCs, which are mainly linked to innate and adaptive immunity. Functional pathways related to regulation of hematopoietic cell differentiation, antigen presentation via major histocompatibility complex class II (MHCII), leukocyte differentiation and regulation of leukocyte activation were identified. These preliminary findings further implicate nucleated RBCs in immune function, such as antigen presentation and leukocyte activation. PMID:29642539

Proteomic profiling of eccrine sweat reveals its potential as a diagnostic biofluid for active tuberculosis.

PubMed

Adewole, Olanisun Olufemi; Erhabor, Greg Efosa; Adewole, Temitayo Oluwatoyin; Ojo, Abiodun Oluwasesan; Oshokoya, Harriet; Wolfe, Lisa M; Prenni, Jessica E

2016-05-01

Excessive sweating is a common symptom of the disease and an unexplored biofluid for TB diagnosis; we conducted a proof-of-concept study to identify potential diagnostic biomarkers of active TB in eccrine sweat. We performed a global proteomic profile of eccrine sweat sampled from patients with active pulmonary TB, other lung diseases (non-TB disease), and healthy controls. A comparison of proteomics between Active-TB, Non-TB, and Healthy Controls was done in search for potential biomarkers of active TB. Sweat specimens were pooled from 32 active TB patients, 27 patients with non-TB diseases, and 24 apparently healthy controls, all were negative for HIV. Over 100 unique proteins were identified in the eccrine sweat of all three groups. Twenty-six proteins were exclusively detected in the sweat of patients with active TB while the remaining detected proteins overlapped between three groups. Gene ontology evaluation indicated that the proteins detected uniquely in sweat of active TB patients were involved in immune response and auxiliary protein transport. Gene products for cellular components (e.g. ribosomes) were detected only in active TB patients. Data are available via ProteomeXchange with identifier PXD003224. Proteomics of sweat from active TB patients is a viable approach for biomarker identification, which could be used to develop a nonsputum-based test for detection of active TB. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Salt stress-induced changes in antioxidative defense system and proteome profiles of salt-tolerant and sensitive Frankia strains.

PubMed

Srivastava, Amrita; Singh, Anumeha; Singh, Satya S; Mishra, Arun K

2017-04-16

An appreciation of comparative microbial survival is most easily done while evaluating their adaptive strategies during stress. In the present experiment, antioxidative and whole cell proteome variations based on spectrophotometric analysis and SDS-PAGE and 2-dimensional gel electrophoresis have been analysed among salt-tolerant and salt-sensitive Frankia strains. This is the first report of proteomic basis underlying salt tolerance in these newly isolated Frankia strains from Hippophae salicifolia D. Don. Salt-tolerant strain HsIi10 shows higher increment in the contents of superoxide dismutase, catalase and ascorbate peroxidase as compared to salt-sensitive strain HsIi8. Differential 2-DGE profile has revealed differential profiles for salt-tolerant and salt-sensitive strains. Proteomic confirmation of salt tolerance in the strains with inbuilt efficiency of thriving in nitrogen-deficient locales is a definite advantage for these microbes. This would be equally beneficial for improvement of soil nitrogen status. Efficient protein regulation in HsIi10 suggests further exploration for its potential use as biofertilizer in saline soils.

Quantitative proteomic analysis of whey proteins in the colostrum and mature milk of yak (Bos grunniens).

PubMed

Yang, Yongxin; Zhao, Xiaowei; Yu, Shumin; Cao, Suizhong

2015-02-01

Yak (Bos grunniens) is an important natural resource in mountainous regions. To date, few studies have addressed the differences in the protein profiles of yak colostrum and milk. We used quantitative proteomics to compare the protein profiles of whey from yak colostrum and milk. Milk samples were collected from 21 yaks after calving (1 and 28 d). Whey protein profiles were generated through isobaric tag for relative and absolute quantification (iTRAQ)-labelled proteomics. We identified 183 proteins in milk whey; of these, the expression levels of 86 proteins differed significantly between the whey from colostrum and milk. Haemoglobin expression showed the greatest change; its levels were significantly higher in the whey from colostrum than in mature milk whey. Functional analysis revealed that many of the differentially expressed proteins were associated with biological regulation and response to stimuli. Further, eight differentially expressed proteins involved in the complement and coagulation cascade pathway were enriched in milk whey. These findings add to the general understanding of the protein composition of yak milk, suggest potential functions of the differentially expressed proteins, and provide novel information on the role of colostral components in calf survival. © 2014 Society of Chemical Industry.

Comparative Proteomic and Nutritional Composition Analysis of Independent Transgenic Pigeon Pea Seeds Harboring cry1AcF and cry2Aa Genes and Their Nontransgenic Counterparts.

PubMed

Mishra, Pragya; Singh, Shweta; Rathinam, Maniraj; Nandiganti, Muralimohan; Ram Kumar, Nikhil; Thangaraj, Arulprakash; Thimmegowda, Vinutha; Krishnan, Veda; Mishra, Vagish; Jain, Neha; Rai, Vandna; Pattanayak, Debasis; Sreevathsa, Rohini

2017-02-22

Safety assessment of genetically modified plants is an important aspect prior to deregulation. Demonstration of substantial equivalence of the transgenics compared to their nontransgenic counterparts can be performed using different techniques at various molecular levels. The present study is a first-ever comprehensive evaluation of pigeon pea transgenics harboring two independent cry genes, cry2Aa and cry1AcF. The absence of unintended effects in the transgenic seed components was demonstrated by proteome and nutritional composition profiling. Analysis revealed that no significant differences were found in the various nutritional compositional analyses performed. Additionally, 2-DGE-based proteome analysis of the transgenic and nontransgenic seed protein revealed that there were no major changes in the protein profile, although a minor fold change in the expression of a few proteins was observed. Furthermore, the study also demonstrated that neither the integration of T-DNA nor the expression of the cry genes resulted in the production of unintended effects in the form of new toxins or allergens.

Integrative genomic and proteomic profiling of human neuroblastoma SH-SY5Y cells reveals signatures of endosulfan exposure.

PubMed

Gandhi, Deepa; Tarale, Prashant; Naoghare, Pravin K; Bafana, Amit; Kannan, Krishnamurthi; Sivanesan, Saravanadevi

2016-01-01

Endosulfan, an organochlorine pesticide, is known to induce multiple disorders/abnormalities including neuro-degenerative disorders in many animal species. However, the molecular mechanism of endosulfan induced neuronal alterations is still not well understood. In the present study, the effect of sub-lethal concentration of endosulfan (3 μM) on human neuroblastoma cells (SH-SY5Y) was investigated using genomic and proteomic approaches. Microarray and 2D-PAGE followed by MALDI-TOF-MS analysis revealed differential expression of 831 transcripts and 16 proteins in exposed cells. A gene ontology enrichment analysis revealed that the differentially expressed genes and proteins were involved in variety of cellular events such as neuronal developmental pathway, immune response, cell differentiation, apoptosis, transmission of nerve impulse, axonogenesis, etc. The present study attempted to explore the possible molecular mechanism of endosulfan induced neuronal alterations in SH-SY5Y cells using an integrated genomic and proteomic approach. Based on the gene and protein profile possible mechanisms underlying endosulfan neurotoxicity were predicted. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

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

Activity-based protein profiling for biochemical pathway discovery in cancer

PubMed Central

Nomura, Daniel K.; Dix, Melissa M.; Cravatt, Benjamin F.

2011-01-01

Large-scale profiling methods have uncovered numerous gene and protein expression changes that correlate with tumorigenesis. However, determining the relevance of these expression changes and which biochemical pathways they affect has been hindered by our incomplete understanding of the proteome and its myriad functions and modes of regulation. Activity-based profiling platforms enable both the discovery of cancer-relevant enzymes and selective pharmacological probes to perturb and characterize these proteins in tumour cells. When integrated with other large-scale profiling methods, activity-based proteomics can provide insight into the metabolic and signalling pathways that support cancer pathogenesis and illuminate new strategies for disease diagnosis and treatment. PMID:20703252

Short infusion of paclitaxel imbalances plasmatic lipid metabolism and correlates with cardiac markers of acute damage in patients with breast cancer.

PubMed

Panis, C; Binato, R; Correa, S; Victorino, V J; Dias-Alves, V; Herrera, A C S A; Cecchini, R; Simão, A N C; Barbosa, D S; Pizzatti, L; Abdelhay, E

2017-09-01

Although paclitaxel-based chemotherapy is widely used for treating breast cancer, paclitaxel therapy has been associated with several adverse effects. Such adverse effects have primarily been associated with long-term regimens, but some acute effects are being increasingly reported in the literature. In this context, the present study analyzed the systemic proteomic profiles of women diagnosed with breast cancer at the first cycle of short paclitaxel infusion (n = 30). Proteomic profiles thus obtained were compared with those of breast cancer patients without chemotherapy (n = 50), as well as with those of healthy controls (n = 40). Plasma samples were evaluated by label-free LC-MS to obtain systemic proteomic profiles. Putative dysregulated pathways were identified and validated by in silico analysis of proteomic profiles. Our results identified 188 proteins that were differentially expressed in patients who received a single short paclitaxel infusion when compared to patients who did not receive the infusion. Gene ontology analysis indicated that the cholesterol pathway may be dysregulated by paclitaxel in these patients. Validation analysis showed that paclitaxel treatment significantly reduced plasma high-density lipoprotein levels and increased plasma hydroperoxide levels when compared to breast cancer patients without chemotherapy. Furthermore, augmented C-reactive protein and creatine kinase fraction MB were found to be significantly higher in paclitaxel-treated patients in comparison with healthy controls. Taken together, these data suggest that a single dose of short paclitaxel infusion is sufficient to trigger significant alterations in lipid metabolism, which puts breast cancer patients at risk for increased incidence of cardiovascular disease.

New serum biomarkers for detection of tuberculosis using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry.

PubMed

Liu, Ji-Yan; Jin, Lei; Zhao, Meng-Yuan; Zhang, Xin; Liu, Chi-Bo; Zhang, Yu-Xiang; Li, Fu-Jian; Zhou, Jian-Min; Wang, Hua-Jun; Li, Ji-Cheng

2011-10-01

New technologies for the early detection of tuberculosis (TB) are urgently needed. Pathological changes within an organ might be reflected in proteomic patterns in serum. The aim of the present study was to screen for the potential protein biomarkers in serum for the diagnosis of TB using proteomic fingerprint technology. Proteomic fingerprint technology combining protein chips with surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) was used to profile the serum proteins from 50 patients with TB, 25 patients with lung disease other than TB, and 25 healthy volunteers. The protein fingerprint expression of all the serum samples and the resulting profiles between TB and control groups were analyzed with the Biomarker Wizard system. A total of 30 discriminating m/z peaks were detected that were related to TB (p<0.01). The model of biomarkers constructed by the Biomarker Patterns Software based on the three biomarkers (2024, 8007, and 8598 Da) generated excellent separation between the TB and control groups. The sensitivity was 84.0% and the specificity was 86.0%. Blind test data indicated a sensitivity of 80.0% and a specificity of 84.2%. The data suggested a potential application of SELDI-TOF MS as an effective technology to profile serum proteome, and with pattern analysis, a diagnostic model comprising three potential biomarkers was indicated to differentiate people with TB and healthy controls rapidly and precisely.

Serum Proteomic Profiles In Subjects with Heavy Alcohol Abuse

PubMed Central

Liangpunsakul, Suthat; Lai, Xianyin; Ringham, Heather N.; Crabb, David W.; Witzmann, Frank A.

2009-01-01

Objectives The abuse of alcohol is a major public health problem, and the diagnosis and care of patients with alcohol abuse and dependence is hindered by the lack of tests that can detect dangerous levels of drinking or relapse during therapy. Gastroenterologists and other healthcare providers find it very challenging to obtain an accurate alcohol drinking history. We hypothesized that the effects of ethanol on numerous systems may well be reflected in changes in quantity or qualities of constituent or novel plasma proteins or protein fragments. Organ/tissue-specific proteins may be released into the blood stream when cells are injured by alcohol, or when systemic changes are induced by alcohol, and such proteins would be detected using a proteomic approach. The objective of this pilot study was to determine if there are plasma proteome profiles that correlate with heavy alcohol use. Methods Paired serum samples, before and after intensive alcohol treatment, were obtained from subjects who attended an outpatient alcohol treatment program. Serum proteomic profiles using MALDI –OTOF Mass Spectrometry were compared between pre- and post treatment samples. Results Of 16 subjects who enrolled in the study, 8 were females. The mean age of the study subjects was 49 yrs. The baseline laboratory data showed elevated AST (54 ± 37 IU/L), ALT (37 ± 19 IU/L), and MCV (99 ± 5 fl). Self-reported pre-treatment drinking levels for these subjects averaged 17 ± 7drinks/day and 103 ± 37 drinks/week. Mass spectrometry analyses showed a novel 5.9 kDa protein, a fragment of alpha fibrinogen, isoform 1, that might be might be a new novel marker for abusive alcohol drinking. Conclusions We have shown in this pilot study that several potential protein markers have appeared in mass spectral profiles and that they may be useful clinically to determine the status of alcohol drinking by MALDI –OTOF mass spectrometry, especially a fragment of alpha fibrinogen, isoform 1. However, a large-scale study is needed to confirm and validate our current results. PMID:19672327

Differential proteomics study of platelets in asymptomatic constitutional macrothrombocytopenia: altered levels of cytoskeletal proteins.

PubMed

Karmakar, Shilpita; Saha, Sutapa; Banerjee, Debasis; Chakrabarti, Abhijit

2015-01-01

Harris platelet syndrome (HPS), also known as asymptomatic constitutional macrothrombocytopenia (ACMT), is an autosomal dominant platelet disorder characterized by mild-to-severe thrombocytopenia and giant platelets with normal platelet aggregation and absence of bleeding symptoms. We have attempted a comparative proteomics study for profiling of platelet proteins in healthy vs. pathological states to discover characteristic protein expression changes in macrothrombocytes and decipher the factors responsible for the functionally active yet morphologically distinct platelets. We have used 2-D gel-based protein separation techniques coupled with MALDI-ToF/ToF-based mass spectrometric identification and characterization of the proteins to investigate the differential proteome profiling of platelet proteins isolated from the peripheral blood samples of patients and normal volunteers. Our study revealed altered levels of actin-binding proteins such as myosin light chain, coactosin-like protein, actin-related protein 2/3 complex, and transgelin2 that hint toward the cytoskeletal changes necessary to maintain the structural and functional integrity of macrothrombocytes. We have also observed over expressed levels of peroxiredoxin2 that signifies the prevailing oxidative stress in these cells. Additionally, altered levels of protein disulfide isomerase and transthyretin provide insights into the measures adapted by the macrothrombocytes to maintain their normal functional activity. This first proteomics study of platelets from ACMT may provide an understanding of the structural stability and normal functioning of these platelets in spite of their large size. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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

PubMed

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

2014-01-01

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

A pursuit of lineage-specific and niche-specific proteome features in the world of archaea

PubMed Central

2012-01-01

Background Archaea evoke interest among researchers for two enigmatic characteristics –a combination of bacterial and eukaryotic components in their molecular architectures and an enormous diversity in their life-style and metabolic capabilities. Despite considerable research efforts, lineage- specific/niche-specific molecular features of the whole archaeal world are yet to be fully unveiled. The study offers the first large-scale in silico proteome analysis of all archaeal species of known genome sequences with a special emphasis on methanogenic and sulphur-metabolising archaea. Results Overall amino acid usage in archaea is dominated by GC-bias. But the environmental factors like oxygen requirement or thermal adaptation seem to play important roles in selection of residues with no GC-bias at the codon level. All methanogens, irrespective of their thermal/salt adaptation, show higher usage of Cys and have relatively acidic proteomes, while the proteomes of sulphur-metabolisers have higher aromaticity and more positive charges. Despite of exhibiting thermophilic life-style, korarchaeota possesses an acidic proteome. Among the distinct trends prevailing in COGs (Cluster of Orthologous Groups of proteins) distribution profiles, crenarchaeal organisms display higher intra-order variations in COGs repertoire, especially in the metabolic ones, as compared to euryarchaea. All methanogens are characterised by a presence of 22 exclusive COGs. Conclusions Divergences in amino acid usage, aromaticity/charge profiles and COG repertoire among methanogens and sulphur-metabolisers, aerobic and anaerobic archaea or korarchaeota and nanoarchaeota, as elucidated in the present study, point towards the presence of distinct molecular strategies for niche specialization in the archaeal world. PMID:22691113

A pursuit of lineage-specific and niche-specific proteome features in the world of archaea.

PubMed

Roy Chowdhury, Anindya; Dutta, Chitra

2012-06-12

Archaea evoke interest among researchers for two enigmatic characteristics -a combination of bacterial and eukaryotic components in their molecular architectures and an enormous diversity in their life-style and metabolic capabilities. Despite considerable research efforts, lineage- specific/niche-specific molecular features of the whole archaeal world are yet to be fully unveiled. The study offers the first large-scale in silico proteome analysis of all archaeal species of known genome sequences with a special emphasis on methanogenic and sulphur-metabolising archaea. Overall amino acid usage in archaea is dominated by GC-bias. But the environmental factors like oxygen requirement or thermal adaptation seem to play important roles in selection of residues with no GC-bias at the codon level. All methanogens, irrespective of their thermal/salt adaptation, show higher usage of Cys and have relatively acidic proteomes, while the proteomes of sulphur-metabolisers have higher aromaticity and more positive charges. Despite of exhibiting thermophilic life-style, korarchaeota possesses an acidic proteome. Among the distinct trends prevailing in COGs (Cluster of Orthologous Groups of proteins) distribution profiles, crenarchaeal organisms display higher intra-order variations in COGs repertoire, especially in the metabolic ones, as compared to euryarchaea. All methanogens are characterised by a presence of 22 exclusive COGs. Divergences in amino acid usage, aromaticity/charge profiles and COG repertoire among methanogens and sulphur-metabolisers, aerobic and anaerobic archaea or korarchaeota and nanoarchaeota, as elucidated in the present study, point towards the presence of distinct molecular strategies for niche specialization in the archaeal world.

Gestational Exposure to Bisphenol A Affects the Function and Proteome Profile of F1 Spermatozoa in Adult Mice.

PubMed

Rahman, Md Saidur; Kwon, Woo-Sung; Karmakar, Polash Chandra; Yoon, Sung-Jae; Ryu, Buom-Yong; Pang, Myung-Geol

2017-02-01

Maternal exposure to the endocrine disruptor bisphenol A (BPA) has been linked to offspring reproductive abnormalities. However, exactly how BPA affects offspring fertility remains poorly understood. The aim of the present study was to evaluate the effects of gestational BPA exposure on sperm function, fertility, and proteome profile of F1 spermatozoa in adult mice. Pregnant CD-1 mice (F0) were gavaged with BPA at three different doses (50 μg/kg bw/day, 5 mg/kg bw/day, and 50 mg/kg bw/day) on embryonic days 7 to 14. We investigated the function, fertility, and related processes of F1 spermatozoa at postnatal day 120. We also evaluated protein profiles of F1 spermatozoa to monitor their functional affiliation to disease. BPA inhibited sperm count, motility parameters, and intracellular ATP levels in a dose-dependent manner. These effects appeared to be caused by reduced numbers of stage VIII seminiferous epithelial cells in testis and decreased protein kinase A (PKA) activity and tyrosine phosphorylation in spermatozoa. We also found that BPA compromised average litter size. Proteins differentially expressed in spermatozoa from BPA treatment groups are known to play a critical role in ATP generation, oxidative stress response, fertility, and in the pathogenesis of several diseases. Our study provides mechanistic support for the hypothesis that gestational exposure to BPA alters sperm function and fertility via down-regulation of tyrosine phosphorylation through a PKA-dependent mechanism. In addition, we anticipate that the BPA-induced changes in the sperm proteome might be partly responsible for the observed effects in spermatozoa. Citation: Rahman MS, Kwon WS, Karmakar PC, Yoon SJ, Ryu BY, Pang MG. 2017. Gestational exposure to bisphenol-A affects the function and proteome profile of F1 spermatozoa in adult mice. Environ Health Perspect 125:238-245; http://dx.doi.org/10.1289/EHP378.

Gestational Exposure to Bisphenol A Affects the Function and Proteome Profile of F1 Spermatozoa in Adult Mice

PubMed Central

Rahman, Md Saidur; Kwon, Woo-Sung; Karmakar, Polash Chandra; Yoon, Sung-Jae; Ryu, Buom-Yong; Pang, Myung-Geol

2016-01-01

Background: Maternal exposure to the endocrine disruptor bisphenol A (BPA) has been linked to offspring reproductive abnormalities. However, exactly how BPA affects offspring fertility remains poorly understood. Objectives: The aim of the present study was to evaluate the effects of gestational BPA exposure on sperm function, fertility, and proteome profile of F1 spermatozoa in adult mice. Methods: Pregnant CD-1 mice (F0) were gavaged with BPA at three different doses (50 μg/kg bw/day, 5 mg/kg bw/day, and 50 mg/kg bw/day) on embryonic days 7 to 14. We investigated the function, fertility, and related processes of F1 spermatozoa at postnatal day 120. We also evaluated protein profiles of F1 spermatozoa to monitor their functional affiliation to disease. Results: BPA inhibited sperm count, motility parameters, and intracellular ATP levels in a dose-dependent manner. These effects appeared to be caused by reduced numbers of stage VIII seminiferous epithelial cells in testis and decreased protein kinase A (PKA) activity and tyrosine phosphorylation in spermatozoa. We also found that BPA compromised average litter size. Proteins differentially expressed in spermatozoa from BPA treatment groups are known to play a critical role in ATP generation, oxidative stress response, fertility, and in the pathogenesis of several diseases. Conclusions: Our study provides mechanistic support for the hypothesis that gestational exposure to BPA alters sperm function and fertility via down-regulation of tyrosine phosphorylation through a PKA-dependent mechanism. In addition, we anticipate that the BPA-induced changes in the sperm proteome might be partly responsible for the observed effects in spermatozoa. Citation: Rahman MS, Kwon WS, Karmakar PC, Yoon SJ, Ryu BY, Pang MG. 2017. Gestational exposure to bisphenol-A affects the function and proteome profile of F1 spermatozoa in adult mice. Environ Health Perspect 125:238–245; http://dx.doi.org/10.1289/EHP378 PMID:27384531

Global Cell Proteome Profiling, Phospho-signaling and Quantitative 
Proteomics for Identification of New Biomarkers in Acute Myeloid 
Leukemia Patients

PubMed Central

Aasebø, Elise; Forthun, Rakel B.; Berven, Frode; Selheim, Frode; Hernandez-Valladares, Maria

2016-01-01

The identification of protein biomarkers for acute myeloid leukemia (AML) that could find applications in AML diagnosis and prognosis, treatment and the selection for bone marrow transplant requires substantial comparative analyses of the proteomes from AML patients. In the past years, several studies have suggested some biomarkers for AML diagnosis or AML classification using methods for sample preparation with low proteome coverage and low resolution mass spectrometers. However, most of the studies did not follow up, confirm or validate their candidates with more patient samples. Current proteomics methods, new high resolution and fast mass spectrometers allow the identification and quantification of several thousands of proteins obtained from few tens of μg of AML cell lysate. Enrichment methods for posttranslational modifications (PTM), such as phosphorylation, can isolate several thousands of site-specific phosphorylated peptides from AML patient samples, which subsequently can be quantified with high confidence in new mass spectrometers. While recent reports aiming to propose proteomic or phosphoproteomic biomarkers on the studied AML patient samples have taken advantage of the technological progress, the access to large cohorts of AML patients to sample from and the availability of appropriate control samples still remain challenging. PMID:26306748

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.

Urinary proteomics in renal pathophysiology: Impact of proteinuria.

PubMed

Sancho-Martínez, Sandra M; Prieto-García, Laura; Blanco-Gozalo, Víctor; Fontecha-Barriuso, Miguel; López-Novoa, José M; López-Hernández, Francisco J

2015-06-01

Urinary differential proteomics is used to study renal pathophysiological mechanisms, find novel markers of biological processes and renal diseases, and stratify patients according to proteomic profiles. The proteomic procedure determines the pathophysiological meaning and clinical relevance of results. Urine samples for differential proteomic studies are usually normalized by protein content, regardless of its pathophysiological characteristics. In the field of nephrology, this approach translates into the comparison of a different fraction of the total daily urine output between proteinuric and nonproteinuric samples. Accordingly, alterations in the level of specific proteins found by this method reflect the relative presence of individual proteins in the urine; but they do not necessarily show alterations in their daily excretion, which is a key parameter for the understanding of the pathophysiological meaning of urinary components. For renal pathophysiology studies and clinical biomarker identification or determination, an alternative proteomic concept providing complementary information is based on sample normalization by daily urine output, which directly informs on changes in the daily excretion of individual proteins. This is clinically important because daily excretion (rather than absolute or relative concentration) is the only self-normalized way to evaluate the real meaning of urinary parameters, which is also independent of urine concentration. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Effect of N′-nitrosodimethylamine on red blood cell rheology and proteomic profiles of brain in male albino rats

PubMed Central

Ahmad, Areeba; Fatima, Ravish; Maheshwari, Veena; Ahmad, Riaz

2011-01-01

We investigated the effects of N'-nitrosodimethylamine (NDMA) induced toxicity on red blood cell rheology in male rats and identified bands in proteomic profiles of brain which can be used as novel markers. Polyacrylamide gel electrophoresis (PAGE) profiles exhibited constitutive as well as induced expression of the polypeptides. Remarkably, the molecular weight range of the polypeptides (8–150 kDa) corresponded to that of the family of heat shock proteins. Our results revealed significant changes in blood parameters and showed the presence of acanthocytes, tear drop cells, spicules and cobot rings in the treated categories. Lactate dehydrogenase and esterase zymograms displayed a shift to anaerobic metabolism generating hypoxia-like conditions. This study strongly suggests that NDMA treatment causes acute toxicity leading to cell membrane destruction and alters protein profiles in rats. It is therefore recommended that caution should be exercised in using NDMA to avoid risks, and if at all necessary strategies should be designed to combat such conditions. PMID:22058653

Mild caloric restriction up-regulates the expression of prohibitin: A proteome study

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

Takahashi, Shoko; Masuda, Junko; Shimagami, Hiroshi

2011-02-18

Research highlights: {yields} Proteomic analysis was performed to elucidate physiological alterations induced by mild CR. {yields} The results suggest good reproducibility and possibility to grasp the important response of CR. {yields} The increase in prohibitin abundance was observed in CR groups by proteomic analysis. {yields} We hypothesize that prohibitin might be involved in the longevity induced by CR. -- Abstract: Caloric restriction (CR) is well known to expand lifespan in a variety of species and to retard many age-related diseases. The effects of relatively mild CR on the proteome profile in relation to lifespan have not yet been reported, despitemore » the more extensive studies of the stricter CR conditions. Thus, the present study was conducted to elucidate the protein profiles in rat livers after mild CR for a relatively short time. Young growing rats were fed CR diets (10% and 30% CR) for 1 month. We performed the differential proteomic analysis of the rat livers using two-dimensional electrophoresis combined with MALDI-TOF mass spectrometry. The most remarkable protein among the differentially expressed proteins was found to be prohibitin, the abundance of which was increased by 30% CR. Prohibitin is a ubiquitously expressed protein shown to suppress cell proliferation and to be related to longevity. The increase in prohibitin was observed both in 10% and 30% CR by Western blot analysis. Furthermore, induction of AMP-activated kinase (AMPK) protein, related to the actions of prohibitin in promoting longevity, was observed. The increased prohibitin level in response to subtle CR suggests that this increase may be one of the early events leading to the expansion of lifespan in response to CR.« less

Proteomic Characterization of Central Pacific Oxygen Minimum Zone Microbial Communities

NASA Astrophysics Data System (ADS)

Saunders, J. K.; McIlvin, M. M.; Moran, D.; Held, N.; Futrelle, J.; Webb, E.; Santoro, A.; Dupont, C.; Saito, M.

2018-05-01

Microbial proteomic profiles are excellent for surveying vast expanses of pelagic ecosystems for links between microbial communities and the biogeochemical cycles they mediate. Data from the ProteOMZ expedition supports the utility of this method.

Technological advances and proteomic applications in drug discovery and target deconvolution: identification of the pleiotropic effects of statins.

PubMed

Banfi, Cristina; Baetta, Roberta; Gianazza, Erica; Tremoli, Elena

2017-06-01

Proteomic-based techniques provide a powerful tool for identifying the full spectrum of protein targets of a drug, elucidating its mechanism(s) of action, and identifying biomarkers of its efficacy and safety. Herein, we outline the technological advancements in the field, and illustrate the contribution of proteomics to the definition of the pharmacological profile of statins, which represent the cornerstone of the prevention and treatment of cardiovascular diseases (CVDs). Statins act by inhibiting 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase, thus reducing cholesterol biosynthesis and consequently enhancing the clearance of low-density lipoproteins from the blood; however, HMG-CoA reductase inhibition can result in a multitude of additional effects beyond lipid lowering, known as 'pleiotropic effects'. The case of statins highlights the unique contribution of proteomics to the target profiling of a drug molecule. Copyright © 2017 Elsevier Ltd. All rights reserved.

Proteome profiling of early seed development in Cunninghamia lanceolata (Lamb.) Hook

PubMed Central

Shi, Jisen; Zhen, Yan; Zheng, Ren-Hua

2010-01-01

Knowledge of the proteome of the early gymnosperm embryo could provide important information for optimizing plant cloning procedures and for establishing platforms for research into plant development/regulation and in vitro transgenic studies. Compared with angiosperms, it is more difficult to induce somatic embryogenesis in gymnosperms; success in this endeavour could be increased, however, if proteomic information was available on the complex, dynamic, and multistage processes of gymnosperm embryogenesis in vivo. A proteomic analysis of Chinese fir seeds in six developmental stages was carried out during early embryogenesis. Proteins were extracted from seeds dissected from immature cones and separated by two-dimensional difference gel electrophoresis. Analysis with DeCyder 6.5 software revealed 136 spots that differed in kinetics of appearance. Analysis by liquid chromatography coupled to tandem mass spectrometry and MALDI-TOF mass spectrometry identified proteins represented by 71 of the spots. Functional annotation of these seed proteins revealed their involvement in programmed cell death and chromatin modification, indicating that the proteins may play a central role in determining the number of zygotic embryos generated and controlling embryo patterning and shape remodelling. The analysis also revealed other proteins involved in carbon metabolism, methionine metabolism, energy production, protein storage, synthesis and stabilization, disease/defence, the cytoskeleton, and embryo development. The comprehensive protein expression profiles generated by our study provide new insights into the complex developmental processes in the seeds of the Chinese fir. PMID:20363864

Characterization of human pineal gland proteome.

PubMed

Yelamanchi, Soujanya D; Kumar, Manish; Madugundu, Anil K; Gopalakrishnan, Lathika; Dey, Gourav; Chavan, Sandip; Sathe, Gajanan; Mathur, Premendu P; Gowda, Harsha; Mahadevan, Anita; Shankar, Susarla K; Prasad, T S Keshava

2016-11-15

The pineal gland is a neuroendocrine gland located at the center of the brain. It is known to regulate various physiological functions in the body through secretion of the neurohormone melatonin. Comprehensive characterization of the human pineal gland proteome has not been undertaken to date. We employed a high-resolution mass spectrometry-based approach to characterize the proteome of the human pineal gland. A total of 5874 proteins were identified from the human pineal gland in this study. Of these, 5820 proteins were identified from the human pineal gland for the first time. Interestingly, 1136 proteins from the human pineal gland were found to contain a signal peptide domain, which indicates the secretory nature of these proteins. An unbiased global proteomic profile of this biomedically important organ should benefit molecular research to unravel the role of the pineal gland in neuropsychiatric and neurodegenerative diseases.

Skin toxicology of lead species evaluated by their permeability and proteomic profiles: a comparison of organic and inorganic lead.

PubMed

Pan, Tai-Long; Wang, Pei-Wen; Al-Suwayeh, Saleh A; Chen, Chih-Chieh; Fang, Jia-You

2010-08-01

Lead compounds are known to cause cytotoxicity and genotoxicity. Lead absorption by the skin is an important route through which this metal enters the body. The purpose of this work was to evaluate the skin permeability and toxicological profiles of two lead species, lead acetate and lead nitrate. This study assessed lead-induced toxicity mechanisms by focusing on the histopathology, proteomics, cell growth, and cellular ATP. In vitro skin permeation assays showed that there was no significant difference of lead accumulation within and across the skin between the two lead species. The presence of simulated sweat reduced the skin uptake of lead. The skin deposition of lead acetate was greater than that of lead nitrate with in vivo topical application. On the other hand, lead nitrate produced greater changes in the skin's histology and proteomic profiles compared to lead acetate. Four protein spots which showed significant changes were identified and are discussed in this study. These included glucose-related protein precursor (GRP) 78, K14, alpha-actin, and Rho GDP-dissociation inhibitor 2 (RhoGDI2). These proteins are respectively associated with oxidative stress, apoptosis, wound healing, and proliferation. Lead presented a biphasic pattern on cell growth and intracellular ATP content, with a stimulating effect at low concentrations and an inhibitory effect on cell proliferation at higher concentrations. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

Biomolecular Profiling of Jet Fuel Toxicity Using Proteomics

DTIC Science & Technology

2006-02-28

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

Proteomics: A valuable approach to elucidate spermatozoa post -testicular maturation in the endangered Acipenseridae family.

PubMed

Boccaletto, Pietro; Siddique, Mohammad Abdul Momin; Cosson, Jacky

2018-05-01

Proteomics techniques, such as two-dimensional polyacrylamide gel electrophoresis, mass spectrometry, and differential gel electrophoresis, have been extensively used to describe the protein composition of male gametes in different animals, mainly mammals. They have also provided a deeper understanding of protein functions involved in sperm processes, as in processes that in humans lead to male infertility. However, few studies focus on fish sperm proteomics and even fewer have tried to explore the proteomic profile of Sturgeon spermatozoa. Sturgeon is an endangered, ancient group of fish species exploited mostly for caviar. In this fish group, a part of the process that leads to final functional maturation of spermatozoa so as to have the capability to activate eggs during the fertilization process. This process has a broad similarity to post-testicular maturation in mammals; where spermatozoa leaving the testes must be mixed with seminal fluid along the transit through the Wolffian ducts to modify its surface membrane protein composition, leading to axonemal and acrosomal competence. The aim of this study was to review the current literature on various proteomic techniques, their usefulness in separating, identifying and studying the proteome composition of the fish spermatozoon, as well as their potential applications in studying the post-testicular maturation process in Sturgeon. Such understanding could lead to development of more sophisticated aquaculture techniques, favorable for sturgeon reproduction. Copyright © 2018 Elsevier B.V. All rights reserved.

The Proteome of Native Adult Müller Glial Cells From Murine Retina*

PubMed Central

Hauser, Alexandra; Lepper, Marlen Franziska; Mayo, Rebecca

2016-01-01

To date, the proteomic profiling of Müller cells, the dominant macroglia of the retina, has been hampered because of the absence of suitable enrichment methods. We established a novel protocol to isolate native, intact Müller cells from adult murine retinae at excellent purity which retain in situ morphology and are well suited for proteomic analyses. Two different strategies of sample preparation - an in StageTips (iST) and a subcellular fractionation approach including cell surface protein profiling were used for quantitative liquid chromatography-mass spectrometry (LC-MSMS) comparing Müller cell-enriched to depleted neuronal fractions. Pathway enrichment analyses on both data sets enabled us to identify Müller cell-specific functions which included focal adhesion kinase signaling, signal transduction mediated by calcium as second messenger, transmembrane neurotransmitter transport and antioxidant activity. Pathways associated with RNA processing, cellular respiration and phototransduction were enriched in the neuronal subpopulation. Proteomic results were validated for selected Müller cell genes by quantitative real time PCR, confirming the high expression levels of numerous members of the angiogenic and anti-inflammatory annexins and antioxidant enzymes (e.g. paraoxonase 2, peroxiredoxin 1, 4 and 6). Finally, the significant enrichment of antioxidant proteins in Müller cells was confirmed by measurements on vital retinal cells using the oxidative stress indicator CM-H2DCFDA. In contrast to photoreceptors or bipolar cells, Müller cells were most efficiently protected against H2O2-induced reactive oxygen species formation, which is in line with the protein repertoire identified in the proteomic profiling. Our novel approach to isolate intact glial cells from adult retina in combination with proteomic profiling enabled the identification of novel Müller glia specific proteins, which were validated as markers and for their functional impact in glial physiology. This provides the basis to allow the discovery of novel glial specializations and will enable us to elucidate the role of Müller cells in retinal pathologies — a topic still controversially discussed. PMID:26324419

Global proteomic analysis of two tick-borne emerging zoonotic agents: Anaplasma phagocytophilum and Ehrlichia chaffeensis

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

Lin, Mingqun ..; Kikuchi, Takane; Brewer, Heather M.

2011-02-17

Anaplasma phagocytophilum and Ehrlichia chaffeensis are obligatory intracellular {alpha}-proteobacteria that infect human leukocytes and cause potentially fatal emerging zoonoses. In the present study, we determined global protein expression profiles of these bacteria cultured in the human promyelocytic leukemia cell line, HL-60. Mass spectrometric (MS) analyses identified a total of 1,212 A. phagocytophilum and 1,021 E. chaffeensis proteins, representing 89.3 and 92.3% of the predicted bacterial proteomes, respectively. Nearly all bacterial proteins ({approx}99%) with known functions were expressed, whereas only approximately 80% of hypothetical proteins were detected in infected human cells. Quantitative MS/MS analyses indicated that highly expressed proteins in bothmore » bacteria included chaperones, enzymes involved in biosynthesis and metabolism, and outer membrane proteins, such as A. phagocytophilum P44 and E. chaffeensis P28/OMP-1. Among 113 A. phagocytophilum p44 paralogous genes, 110 of them were expressed and 88 of them were encoded by pseudogenes. In addition, bacterial infection of HL-60 cells up-regulated the expression of human proteins involved mostly in cytoskeleton components, vesicular trafficking, cell signaling, and energy metabolism, but down regulated some pattern recognition receptors involved in innate immunity. Our proteomics data represent a comprehensive analysis of A. phagocytophilum and E. chaffeensis proteomes, and provide a quantitative view of human host protein expression profiles regulated by bacterial infection. The availability of these proteomic data will provide new insights into biology and pathogenesis of these obligatory intracellular pathogens.« less

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

PubMed

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

2015-04-29

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

Proteomic profiling of developing cotton fibers from wild and domesticated Gossypium barbadense.

PubMed

Hu, Guanjing; Koh, Jin; Yoo, Mi-Jeong; Grupp, Kara; Chen, Sixue; Wendel, Jonathan F

2013-10-01

Pima cotton (Gossypium barbadense) is widely cultivated because of its long, strong seed trichomes ('fibers') used for premium textiles. These agronomically advanced fibers were derived following domestication and thousands of years of human-mediated crop improvement. To gain an insight into fiber development and evolution, we conducted comparative proteomic and transcriptomic profiling of developing fiber from an elite cultivar and a wild accession. Analyses using isobaric tag for relative and absolute quantification (iTRAQ) LC-MS/MS technology identified 1317 proteins in fiber. Of these, 205 were differentially expressed across developmental stages, and 190 showed differential expression between wild and cultivated forms, 14.4% of the proteome sampled. Human selection may have shifted the timing of developmental modules, such that some occur earlier in domesticated than in wild cotton. A novel approach was used to detect possible biased expression of homoeologous copies of proteins. Results indicate a significant partitioning of duplicate gene expression at the protein level, but an approximately equal degree of bias for each of the two constituent genomes of allopolyploid cotton. Our results demonstrate the power of complementary transcriptomic and proteomic approaches for the study of the domestication process. They also provide a rich database for mining for functional analyses of cotton improvement or evolution. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

[Search for potential gastric cancer biomarkers using low molecular weight blood plasma proteome profiling by mass spectrometry].

PubMed

Shevchenko, V E; Arnotskaia, N E; Ogorodnikova, E V; Davydov, M M; Ibraev, M A; Turkin, I N; Davydov, M I

2014-01-01

Gastric cancer, one of the most widespread malignant tumors, still lacks reliable serum/plasma biomarkers of its early detection. In this study we have developed, unified, and tested a new methodology for search of gastric cancer biomarkers based on profiling of low molecular weight proteome (LMWP) (1-17 kDa). This approach included three main components: sample pre-fractionation, matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), data analysis by a bioinformatics software package. Applicability and perspectives of the developed approach for detection of potential gastric cancer markers during LMWP analysis have been demonstrated using 69 plasma samples from patients with gastric cancer (stages I-IV) and 238 control samples. The study revealed peptides/polypeptides, which may be potentially used for detection of this pathology.

Quantitative trait loci mapping of the mouse plasma proteome (pQTL).

PubMed

Holdt, Lesca M; von Delft, Annette; Nicolaou, Alexandros; Baumann, Sven; Kostrzewa, Markus; Thiery, Joachim; Teupser, Daniel

2013-02-01

A current challenge in the era of genome-wide studies is to determine the responsible genes and mechanisms underlying newly identified loci. Screening of the plasma proteome by high-throughput mass spectrometry (MALDI-TOF MS) is considered a promising approach for identification of metabolic and disease processes. Therefore, plasma proteome screening might be particularly useful for identifying responsible genes when combined with analysis of variation in the genome. Here, we describe a proteomic quantitative trait locus (pQTL) study of plasma proteome screens in an F(2) intercross of 455 mice mapped with 177 genetic markers across the genome. A total of 69 of 176 peptides revealed significant LOD scores (≥5.35) demonstrating strong genetic regulation of distinct components of the plasma proteome. Analyses were confirmed by mechanistic studies and MALDI-TOF/TOF, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of the two strongest pQTLs: A pQTL for mass-to-charge ratio (m/z) 3494 (LOD 24.9, D11Mit151) was identified as the N-terminal 35 amino acids of hemoglobin subunit A (Hba) and caused by genetic variation in Hba. Another pQTL for m/z 8713 (LOD 36.4; D1Mit111) was caused by variation in apolipoprotein A2 (Apoa2) and cosegregated with HDL cholesterol. Taken together, we show that genome-wide plasma proteome profiling in combination with genome-wide genetic screening aids in the identification of causal genetic variants affecting abundance of plasma proteins.

Quantitative Trait Loci Mapping of the Mouse Plasma Proteome (pQTL)

PubMed Central

Holdt, Lesca M.; von Delft, Annette; Nicolaou, Alexandros; Baumann, Sven; Kostrzewa, Markus; Thiery, Joachim; Teupser, Daniel

2013-01-01

A current challenge in the era of genome-wide studies is to determine the responsible genes and mechanisms underlying newly identified loci. Screening of the plasma proteome by high-throughput mass spectrometry (MALDI-TOF MS) is considered a promising approach for identification of metabolic and disease processes. Therefore, plasma proteome screening might be particularly useful for identifying responsible genes when combined with analysis of variation in the genome. Here, we describe a proteomic quantitative trait locus (pQTL) study of plasma proteome screens in an F2 intercross of 455 mice mapped with 177 genetic markers across the genome. A total of 69 of 176 peptides revealed significant LOD scores (≥5.35) demonstrating strong genetic regulation of distinct components of the plasma proteome. Analyses were confirmed by mechanistic studies and MALDI-TOF/TOF, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of the two strongest pQTLs: A pQTL for mass-to-charge ratio (m/z) 3494 (LOD 24.9, D11Mit151) was identified as the N-terminal 35 amino acids of hemoglobin subunit A (Hba) and caused by genetic variation in Hba. Another pQTL for m/z 8713 (LOD 36.4; D1Mit111) was caused by variation in apolipoprotein A2 (Apoa2) and cosegregated with HDL cholesterol. Taken together, we show that genome-wide plasma proteome profiling in combination with genome-wide genetic screening aids in the identification of causal genetic variants affecting abundance of plasma proteins. PMID:23172855

A Miniaturized Chemical Proteomic Approach for Target Profiling of Clinical Kinase Inhibitors in Tumor Biopsies

PubMed Central

Chamrád, Ivo; Rix, Uwe; Stukalov, Alexey; Gridling, Manuela; Parapatics, Katja; Müller, André C.; Altiok, Soner; Colinge, Jacques; Superti-Furga, Giulio; Haura, Eric B.; Bennett, Keiryn L.

2014-01-01

While targeted therapy based on the idea of attenuating the activity of a preselected, therapeutically relevant protein has become one of the major trends in modern cancer therapy, no truly specific targeted drug has been developed and most clinical agents have displayed a degree of polypharmacology. Therefore, the specificity of anticancer therapeutics has emerged as a highly important but severely underestimated issue. Chemical proteomics is a powerful technique combining postgenomic drug-affinity chromatography with high-end mass spectrometry analysis and bioinformatic data processing to assemble a target profile of a desired therapeutic molecule. Due to high demands on the starting material, however, chemical proteomic studies have been mostly limited to cancer cell lines. Herein, we report a down-scaling of the technique to enable the analysis of very low abundance samples, as those obtained from needle biopsies. By a systematic investigation of several important parameters in pull-downs with the multikinase inhibitor bosutinib, the standard experimental protocol was optimized to 100 µg protein input. At this level, more than 30 well-known targets were detected per single pull-down replicate with high reproducibility. Moreover, as presented by the comprehensive target profile obtained from miniaturized pull-downs with another clinical drug, dasatinib, the optimized protocol seems to be extendable to other drugs of interest. Sixty distinct human and murine targets were finally identified for bosutinib and dasatinib in chemical proteomic experiments utilizing core needle biopsy samples from xenotransplants derived from patient tumor tissue. Altogether, the developed methodology proves robust and generic and holds many promises for the field of personalized health care. PMID:23901793

MALDI-TOF mass spectrometry proteomic phenotyping of clinically relevant fungi.

PubMed

Putignani, Lorenza; Del Chierico, Federica; Onori, Manuela; Mancinelli, Livia; Argentieri, Marta; Bernaschi, Paola; Coltella, Luana; Lucignano, Barbara; Pansani, Laura; Ranno, Stefania; Russo, Cristina; Urbani, Andrea; Federici, Giorgio; Menichella, Donato

2011-03-01

Proteomics is particularly suitable for characterising human pathogens with high life cycle complexity, such as fungi. Protein content and expression levels may be affected by growth states and life cycle morphs and correlate to species and strain variation. Identification and typing of fungi by conventional methods are often difficult, time-consuming and frequently, for unusual species, inconclusive. Proteomic phenotypes from MALDI-TOF MS were employed as analytical and typing expression profiling of yeast, yeast-like species and strain variants in order to achieve a microbial proteomics population study. Spectra from 303 clinical isolates were generated and processed by standard pattern matching with a MALDI-TOF Biotyper (MT). Identifications (IDs) were compared to a reference biochemical-based system (Vitek-2) and, when discordant, MT IDs were verified with genotyping IDs, obtained by sequencing the 25-28S rRNA hypervariable D2 region. Spectra were converted into virtual gel-like formats, and hierarchical clustering analysis was performed for 274 Candida profiles to investigate species and strain typing correlation. MT provided 257/303 IDs consistent with Vitek-2 ones. However, amongst 26/303 discordant MT IDs, only 5 appeared "true". No MT identification was achieved for 20/303 isolates for incompleteness of database species variants. Candida spectra clustering agreed with identified species and topology of Candida albicans and Candida parapsilosis specific dendrograms. MT IDs show a high analytical performance and profiling heterogeneity which seems to complement or even outclass existing typing tools. This variability reflects the high biological complexity of yeasts and may be properly exploited to provide epidemiological tracing and infection dispersion patterns.

Temporal expression profiling of plasma proteins reveals oxidative stress in early stages of Type 1 Diabetes progression

DOE PAGES

Liu, Chih-Wei; Bramer, Lisa; Webb-Robertson, Bobbie-Jo; ...

2017-10-07

We report that blood markers other than islet autoantibodies are greatly needed to indicate the pancreatic beta cell destruction process as early as possible, and more accurately reflect the progression of Type 1 Diabetes Mellitus (T1D). To this end, a longitudinal proteomic profiling of human plasma using TMT-10plex-based LC-MS/MS analysis was performed to track temporal proteomic changes of T1D patients (n = 11) across 9 serial time points, spanning the period of T1D natural progression, in comparison with those of the matching healthy controls (n = 10). To our knowledge, the current study represents the largest (> 2000 proteins measured)more » longitudinal expression profiles of human plasma proteome in T1D research. By applying statistical trend analysis on the temporal expression patterns between T1D and controls, and Benjamini-Hochberg procedure for multiple-testing correction, 13 protein groups were regarded as having statistically significant differences during the entire follow-up period. Moreover, 16 protein groups, which play pivotal roles in response to oxidative stress, have consistently abnormal expression trend before seroconversion to islet autoimmunity. Importantly, the expression trends of two key reactive oxygen species-decomposing enzymes, Catalase and Superoxide dismutase were verified independently by ELISA.« less

Temporal expression profiling of plasma proteins reveals oxidative stress in early stages of Type 1 Diabetes progression

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

Liu, Chih-Wei; Bramer, Lisa; Webb-Robertson, Bobbie-Jo

We report that blood markers other than islet autoantibodies are greatly needed to indicate the pancreatic beta cell destruction process as early as possible, and more accurately reflect the progression of Type 1 Diabetes Mellitus (T1D). To this end, a longitudinal proteomic profiling of human plasma using TMT-10plex-based LC-MS/MS analysis was performed to track temporal proteomic changes of T1D patients (n = 11) across 9 serial time points, spanning the period of T1D natural progression, in comparison with those of the matching healthy controls (n = 10). To our knowledge, the current study represents the largest (> 2000 proteins measured)more » longitudinal expression profiles of human plasma proteome in T1D research. By applying statistical trend analysis on the temporal expression patterns between T1D and controls, and Benjamini-Hochberg procedure for multiple-testing correction, 13 protein groups were regarded as having statistically significant differences during the entire follow-up period. Moreover, 16 protein groups, which play pivotal roles in response to oxidative stress, have consistently abnormal expression trend before seroconversion to islet autoimmunity. Importantly, the expression trends of two key reactive oxygen species-decomposing enzymes, Catalase and Superoxide dismutase were verified independently by ELISA.« less

Multidimensional protein fractionation using ProteomeLab PF 2D™ for profiling amyotrophic lateral sclerosis immunity: A preliminary report

PubMed Central

Schlautman, Joshua D; Rozek, Wojciech; Stetler, Robert; Mosley, R Lee; Gendelman, Howard E; Ciborowski, Pawel

2008-01-01

Background The ProteomeLab™ PF 2D platform is a relatively new approach to global protein profiling. Herein, it was used for investigation of plasma proteome changes in amyotrophic lateral sclerosis (ALS) patients before and during immunization with glatiramer acetate (GA) in a clinical trial. Results The experimental design included immunoaffinity depletion of 12 most abundant proteins from plasma samples with the ProteomeLab™ IgY-12 LC10 column kit as first dimension separation, also referred to as immuno-partitioning. Second and third dimension separations of the enriched proteome were performed on the PF 2D platform utilizing 2D isoelectric focusing and RP-HPLC with the resulting fractions collected for analysis. 1D gel electrophoresis was added as a fourth dimension when sufficient protein was available. Protein identification from collected fractions was performed using nano-LC-MS/MS approach. Analysis of differences in the resulting two-dimensional maps of fractions obtained from the PF 2D and the ability to identify proteins from these fractions allowed sensitivity threshold measurements. Masked proteins in the PF 2D fractions are discussed. Conclusion We offer some insight into the strengths and limitations of this emerging proteomic platform. PMID:18789151

Systems proteomic analysis reveals that clusterin and tissue inhibitor of metalloproteinases 3 increase in leptomeningeal arteries affected by cerebral amyloid angiopathy.

PubMed

Manousopoulou, A; Gatherer, M; Smith, C; Nicoll, J A R; Woelk, C H; Johnson, M; Kalaria, R; Attems, J; Garbis, S D; Carare, R O

2017-10-01

Amyloid beta (Aβ) accumulation in the walls of leptomeningeal arteries as cerebral amyloid angiopathy (CAA) is a major feature of Alzheimer's disease. In this study, we used global quantitative proteomic analysis to examine the hypothesis that the leptomeningeal arteries derived from patients with CAA have a distinct endophenotypic profile compared to those from young and elderly controls. Freshly dissected leptomeningeal arteries from the Newcastle Brain Tissue Resource and Edinburgh Sudden Death Brain Bank from seven elderly (82.9 ± 7.5 years) females with severe capillary and arterial CAA, as well as seven elderly (88.3 ± 8.6 years) and five young (45.4 ± 3.9 years) females without CAA were used in this study. Arteries from four patients with CAA, two young and two elderly controls were individually analysed using quantitative proteomics. Key proteomic findings were then validated using immunohistochemistry. Bioinformatics interpretation of the results showed a significant enrichment of the immune response/classical complement and extracellular matrix remodelling pathways (P < 0.05) in arteries affected by CAA vs. those from young and elderly controls. Clusterin (apolipoprotein J) and tissue inhibitor of metalloproteinases-3 (TIMP3), validated using immunohistochemistry, were shown to co-localize with Aβ and to be up-regulated in leptomeningeal arteries from CAA patients compared to young and elderly controls. Global proteomic profiling of brain leptomeningeal arteries revealed that clusterin and TIMP3 increase in leptomeningeal arteries affected by CAA. We propose that clusterin and TIMP3 could facilitate perivascular clearance and may serve as novel candidate therapeutic targets for CAA. © 2016 The Authors. Neuropathology and Applied Neurobiology published by John Wiley & Sons Ltd on behalf of British Neuropathological Society.

Curcumin exerts its antitumor effects in a context dependent fashion.

PubMed

Kreutz, Dominique; Sinthuvanich, Chomdao; Bileck, Andrea; Janker, Lukas; Muqaku, Besnik; Slany, Astrid; Gerner, Christopher

2018-06-30

Proteome profiling profoundly contributes to the understanding of cell response mechanisms to drug actions. Such knowledge may become a key to improve personalized medicine. In the present study, the effects of the natural remedy curcumin on breast cancer model systems were investigated. MCF-7, ZR-75-1 and TGF-β1 pretreated fibroblasts, mimicking cancer-associated fibroblasts (CAFs), were treated independently as well as in tumor cell/CAF co-cultures. Remarkably, co-culturing with CAF-like cells (CLCs) induced different proteome alterations in MCF-7 and ZR-75-1 cells, respectively. Curcumin significantly induced HMOX1 in single cell type models and co-cultures. However, other curcumin effects differed. In the MCF-7/CLC co-culture, curcumin significantly down-regulated RC3H1, a repressor of inflammatory signaling. In the ZR-75-1/CLC co-culture, curcumin significantly down-regulated PEG10, an anti-apoptotic protein, and induced RRAGA, a pro-apoptotic protein involved in TNF-alpha signaling. Furthermore, curcumin induced AKR1C2, an important enzyme for progesterone metabolism. None of these specific curcumin effects were observed in single cell type cultures. All high-resolution mass spectrometry data are available via ProteomeXchange with the identifier PXD008719. The present data demonstrate that curcumin induces proteome alterations, potentially accounting for its known antitumor effects, in a strongly context-dependent fashion. Better means to understand and potentially predict individual variations of drug effects are urgently required. The present proteome profiling study of curcumin effects demonstrates the massive impact of the cell microenvironment on cell responses to drug action. Co-culture models apparently provide more biologically relevant information regarding curcumin effects than single cell type cultures. Copyright © 2018. Published by Elsevier B.V.

Label-free proteomic analysis of intestinal mucosa proteins in common carp (Cyprinus carpio) infected with Aeromonas hydrophila.

PubMed

Di, Guilan; Li, Hui; Zhang, Chao; Zhao, Yanjing; Zhou, Chuanjiang; Naeem, Sajid; Li, Li; Kong, Xianghui

2017-07-01

Outbreaks of infectious diseases in common carp Cyprinus carpio, a major cultured fish in northern regions of China, constantly result in significant economic losses. Until now, information proteomic on immune defence remains limited. In the present study, a profile of intestinal mucosa immune response in Cyprinus carpio was investigated after 0, 12, 36 and 84 h after challenging tissues with Aeromonas hydrophila at a concentration of 1.4 × 10 8  CFU/mL. Proteomic profiles in different samples were compared using label-free quantitative proteomic approach. Based on MASCOT database search, 1149 proteins were identified in samples after normalisation of proteins. Treated groups 1 (T1) and 2 (T2) were first clustered together and then clustered with control (C group). The distance between C and treated group 3 (T3) represented the maxima according to hierarchical cluster analysis. Therefore, comparative analysis between C and T3 was selected in the following analysis. A total of 115 proteins with differential abundance were detected to show conspicuous expressing variances. A total of 52 up-regulated proteins and 63 down-regulated proteins were detected in T3. Gene ontology analysis showed that identified up-regulated differentially expressed proteins in T3 were mainly localised in the hemoglobin complex, and down-regulated proteins in T3 were mainly localised in the major histocompatibility complex II protein complex. Forty-six proteins of differential abundance (40% of 115) were involved in immune response, with 17 up-regulated and 29 down-regulated proteins detected in T3. This study is the first to report proteome response of carp intestinal mucosa against A. hydrophila infection; information obtained contribute to understanding defence mechanisms of carp intestinal mucosa. Copyright © 2017 Elsevier Ltd. All rights reserved.

Simplifying the human serum proteome for discriminating patients with bipolar disorder of other psychiatry conditions.

PubMed

de Jesus, Jemmyson Romário; Galazzi, Rodrigo Moretto; de Lima, Tatiani Brenelli; Banzato, Cláudio Eduardo Muller; de Almeida Lima E Silva, Luiz Fernando; de Rosalmeida Dantas, Clarissa; Gozzo, Fábio Cézar; Arruda, Marco Aurélio Zezzi

2017-12-01

An exploratory analysis using proteomic strategies in blood serum of patients with bipolar disorder (BD), and with other psychiatric conditions such as Schizophrenia (SCZ), can provide a better understanding of this disorder, as well as their discrimination based on their proteomic profile. The proteomic profile of blood serum samples obtained from patients with BD using lithium or other drugs (N=14), healthy controls, including non-family (HCNF; N=3) and family (HCF; N=9), patients with schizophrenia (SCZ; N=23), and patients using lithium for other psychiatric conditions (OD; N=4) were compared. Four methods for simplifying the serum samples proteome were evaluated for both removing the most abundant proteins and for enriching those of lower-abundance: protein depletion with acetonitrile (ACN), dithiothreitol (DTT), sequential depletion using DTT and ACN, and protein equalization using commercial ProteoMiner® kit (PM). For proteomic evaluation, 2-D DIGE and nanoLC-MS/MS analysis were employed. PM method was the best strategy for removing proteins of high abundance. Through 2-D DIGE gel image comparison, 37 protein spots were found differentially abundant (p<0.05, Student's t-test), which exhibited ≥2.0-fold change of the average value of normalized spot intensities in the serum of SCZ, BD and OD patients compared to subject controls (HCF and HCNF). From these spots detected, 13 different proteins were identified: ApoA1, ApoE, ApoC3, ApoA4, Samp, SerpinA1, TTR, IgK, Alb, VTN, TR, C4A and C4B. Proteomic analysis allowed the discrimination of patients with BD from patients with other mental disorders, such as SCZ. The findings in this exploratory study may also contribute for better understanding the pathophysiology of these disorders and finding potential serum biomarkers for these conditions. Copyright © 2017 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Proteomics of gliomas: Initial biomarker discovery and evolution of technology

PubMed Central

Kalinina, Juliya; Peng, Junmin; Ritchie, James C.; Van Meir, Erwin G.

2011-01-01

Gliomas are a group of aggressive brain tumors that diffusely infiltrate adjacent brain tissues, rendering them largely incurable, even with multiple treatment modalities and agents. Mostly asymptomatic at early stages, they present in several subtypes with astrocytic or oligodendrocytic features and invariably progress to malignant forms. Gliomas are difficult to classify precisely because of interobserver variability during histopathologic grading. Identifying biological signatures of each glioma subtype through protein biomarker profiling of tumor or tumor-proximal fluids is therefore of high priority. Such profiling not only may provide clues regarding tumor classification but may identify clinical biomarkers and pathologic targets for the development of personalized treatments. In the past decade, differential proteomic profiling techniques have utilized tumor, cerebrospinal fluid, and plasma from glioma patients to identify the first candidate diagnostic, prognostic, predictive, and therapeutic response markers, highlighting the potential for glioma biomarker discovery. The number of markers identified, however, has been limited, their reproducibility between studies is unclear, and none have been validated for clinical use. Recent technological advancements in methodologies for high-throughput profiling, which provide easy access, rapid screening, low sample consumption, and accurate protein identification, are anticipated to accelerate brain tumor biomarker discovery. Reliable tools for biomarker verification forecast translation of the biomarkers into clinical diagnostics in the foreseeable future. Herein we update the reader on the recent trends and directions in glioma proteomics, including key findings and established and emerging technologies for analysis, together with challenges we are still facing in identifying and verifying potential glioma biomarkers. PMID:21852429

Use of focused ultrasonication in activity-based profiling of deubiquitinating enzymes in tissue.

PubMed

Nanduri, Bindu; Shack, Leslie A; Rai, Aswathy N; Epperson, William B; Baumgartner, Wes; Schmidt, Ty B; Edelmann, Mariola J

2016-12-15

To develop a reproducible tissue lysis method that retains enzyme function for activity-based protein profiling, we compared four different methods to obtain protein extracts from bovine lung tissue: focused ultrasonication, standard sonication, mortar & pestle method, and homogenization combined with standard sonication. Focused ultrasonication and mortar & pestle methods were sufficiently effective for activity-based profiling of deubiquitinases in tissue, and focused ultrasonication also had the fastest processing time. We used focused-ultrasonicator for subsequent activity-based proteomic analysis of deubiquitinases to test the compatibility of this method in sample preparation for activity-based chemical proteomics. Copyright © 2016 Elsevier Inc. 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 meets blood banking: identification of protein targets for the improvement of platelet quality.

PubMed

Schubert, Peter; Devine, Dana V

2010-01-03

Proteomics has brought new perspectives to the fields of hematology and transfusion medicine in the last decade. The steady improvement of proteomic technology is propelling novel discoveries of molecular mechanisms by studying protein expression, post-translational modifications and protein interactions. This review article focuses on the application of proteomics to the identification of molecular mechanisms leading to the deterioration of blood platelets during storage - a critical aspect in the provision of platelet transfusion products. Several proteomic approaches have been employed to analyse changes in the platelet protein profile during storage and the obtained data now need to be translated into platelet biochemistry in order to connect the results to platelet function. Targeted biochemical applications then allow the identification of points for intervention in signal transduction pathways. Once validated and placed in a transfusion context, these data will provide further understanding of the underlying molecular mechanisms leading to platelet storage lesion. Future aspects of proteomics in blood banking will aim to make use of protein markers identified for platelet storage lesion development to monitor proteome changes when alterations such as the use of additive solutions or pathogen reduction strategies are put in place in order to improve platelet quality for patients. (c) 2009 Elsevier B.V. All rights reserved.

Proteomics of Skeletal Muscle: Focus on Insulin Resistance and Exercise Biology

PubMed Central

Deshmukh, Atul S.

2016-01-01

Skeletal muscle is the largest tissue in the human body and plays an important role in locomotion and whole body metabolism. It accounts for ~80% of insulin stimulated glucose disposal. Skeletal muscle insulin resistance, a primary feature of Type 2 diabetes, is caused by a decreased ability of muscle to respond to circulating insulin. Physical exercise improves insulin sensitivity and whole body metabolism and remains one of the most promising interventions for the prevention of Type 2 diabetes. Insulin resistance and exercise adaptations in skeletal muscle might be a cause, or consequence, of altered protein expressions profiles and/or their posttranslational modifications (PTMs). Mass spectrometry (MS)-based proteomics offer enormous promise for investigating the molecular mechanisms underlying skeletal muscle insulin resistance and exercise-induced adaptation; however, skeletal muscle proteomics are challenging. This review describes the technical limitations of skeletal muscle proteomics as well as emerging developments in proteomics workflow with respect to samples preparation, liquid chromatography (LC), MS and computational analysis. These technologies have not yet been fully exploited in the field of skeletal muscle proteomics. Future studies that involve state-of-the-art proteomics technology will broaden our understanding of exercise-induced adaptations as well as molecular pathogenesis of insulin resistance. This could lead to the identification of new therapeutic targets. PMID:28248217

Aluminum induced physiological and proteomic responses in tea (Camellia sinensis) roots and leaves.

PubMed

Xu, Qingshan; Wang, Yu; Ding, Zhaotang; Fan, Kai; Ma, Dexin; Zhang, Yongliang; Yin, Qi

2017-06-01

Tea (Camellia sinensis (L.) O. Kuntze), is an aluminum (Al) hyperaccumulator and grows well in acid soils. Although Al-induced growth of tea plant has been studied, the proteomic profiles of tea plants in response to Al are unclear. In the present study, the proteomic profiles in tea roots and leaves under Al stress were investigated using iTRAQ proteomics approach. In total, 755 and 1059 differentially expressed proteins were identified in tea roots and leaves, respectively. KEGG enrichment analysis showed that the differentially expressed proteins in roots were mainly involved in 11 pathways whereas those from leaves were mainly involved in 9 pathways. Abundance of most protein functions in glycolytic metabolism were enhanced in tea roots, and proteins involved in photosynthesis were stimulated in tea leaves. The protein ferulate-5-hydroxylase (F5H) in lignin biosynthetic pathway was down-regulated in both roots and leaves. Furthermore, antioxidant enzymes (ascorbate peroxidase, catalase and glutathione S-transferase) and citrate synthesis were accumulated in tea roots in response to Al. The results indicated that active photosynthesis and glycolysis as well as increased activities of antioxidant enzymes can be considered as a possible reason for the stimulatory effects of Al on the growth of tea plants. Additionally, the down-regulation of F5H and the binding of Al and phenolic acids may reduce the accumulation of lignin. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Proteomic profiling of early degenerative retina of RCS rats.

PubMed

Zhu, Zhi-Hong; Fu, Yan; Weng, Chuan-Huang; Zhao, Cong-Jian; Yin, Zheng-Qin

2017-01-01

To identify the underlying cellular and molecular changes in retinitis pigmentosa (RP). Label-free quantification-based proteomics analysis, with its advantages of being more economic and consisting of simpler procedures, has been used with increasing frequency in modern biological research. Dystrophic RCS rats, the first laboratory animal model for the study of RP, possess a similar pathological course as human beings with the diseases. Thus, we employed a comparative proteomics analysis approach for in-depth proteome profiling of retinas from dystrophic RCS rats and non-dystrophic congenic controls through Linear Trap Quadrupole - orbitrap MS/MS, to identify the significant differentially expressed proteins (DEPs). Bioinformatics analyses, including Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation and upstream regulatory analysis, were then performed on these retina proteins. Finally, a Western blotting experiment was carried out to verify the difference in the abundance of transcript factor E2F1. In this study, we identified a total of 2375 protein groups from the retinal protein samples of RCS rats and non-dystrophic congenic controls. Four hundred thirty-four significantly DEPs were selected by Student's t -test. Based on the results of the bioinformatics analysis, we identified mitochondrial dysfunction and transcription factor E2F1 as the key initiation factors in early retinal degenerative process. We showed that the mitochondrial dysfunction and the transcription factor E2F1 substantially contribute to the disease etiology of RP. The results provide a new potential therapeutic approach for this retinal degenerative disease.

Active site specificity profiling datasets of matrix metalloproteinases (MMPs) 1, 2, 3, 7, 8, 9, 12, 13 and 14.

PubMed

Eckhard, Ulrich; Huesgen, Pitter F; Schilling, Oliver; Bellac, Caroline L; Butler, Georgina S; Cox, Jennifer H; Dufour, Antoine; Goebeler, Verena; Kappelhoff, Reinhild; Auf dem Keller, Ulrich; Klein, Theo; Lange, Philipp F; Marino, Giada; Morrison, Charlotte J; Prudova, Anna; Rodriguez, David; Starr, Amanda E; Wang, Yili; Overall, Christopher M

2016-06-01

The data described provide a comprehensive resource for the family-wide active site specificity portrayal of the human matrix metalloproteinase family. We used the high-throughput proteomic technique PICS (Proteomic Identification of protease Cleavage Sites) to comprehensively assay 9 different MMPs. We identified more than 4300 peptide cleavage sites, spanning both the prime and non-prime sides of the scissile peptide bond allowing detailed subsite cooperativity analysis. The proteomic cleavage data were expanded by kinetic analysis using a set of 6 quenched-fluorescent peptide substrates designed using these results. These datasets represent one of the largest specificity profiling efforts with subsequent structural follow up for any protease family and put the spotlight on the specificity similarities and differences of the MMP family. A detailed analysis of this data may be found in Eckhard et al. (2015) [1]. The raw mass spectrometry data and the corresponding metadata have been deposited in PRIDE/ProteomeXchange with the accession number PXD002265.

Nanodroplet processing platform for deep and quantitative proteome profiling of 10-100 mammalian cells.

PubMed

Zhu, Ying; Piehowski, Paul D; Zhao, Rui; Chen, Jing; Shen, Yufeng; Moore, Ronald J; Shukla, Anil K; Petyuk, Vladislav A; Campbell-Thompson, Martha; Mathews, Clayton E; Smith, Richard D; Qian, Wei-Jun; Kelly, Ryan T

2018-02-28

Nanoscale or single-cell technologies are critical for biomedical applications. However, current mass spectrometry (MS)-based proteomic approaches require samples comprising a minimum of thousands of cells to provide in-depth profiling. Here, we report the development of a nanoPOTS (nanodroplet processing in one pot for trace samples) platform for small cell population proteomics analysis. NanoPOTS enhances the efficiency and recovery of sample processing by downscaling processing volumes to <200 nL to minimize surface losses. When combined with ultrasensitive liquid chromatography-MS, nanoPOTS allows identification of ~1500 to ~3000 proteins from ~10 to ~140 cells, respectively. By incorporating the Match Between Runs algorithm of MaxQuant, >3000 proteins are consistently identified from as few as 10 cells. Furthermore, we demonstrate quantification of ~2400 proteins from single human pancreatic islet thin sections from type 1 diabetic and control donors, illustrating the application of nanoPOTS for spatially resolved proteome measurements from clinical tissues.

Nanodroplet processing platform for deep and quantitative proteome profiling of 10–100 mammalian cells

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

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

Nanoscale or single-cell technologies are critical for biomedical applications. However, current mass spectrometry (MS)-based proteomic approaches require samples comprising a minimum of thousands of cells to provide in-depth profiling. Here in this paper, we report the development of a nanoPOTS (nanodroplet processing in one pot for trace samples) platform for small cell population proteomics analysis. NanoPOTS enhances the efficiency and recovery of sample processing by downscaling processing volumes to <200 nL to minimize surface losses. When combined with ultrasensitive liquid chromatography-MS, nanoPOTS allows identification of ~1500 to ~3000 proteins from ~10 to ~140 cells, respectively. By incorporating the Match Betweenmore » Runs algorithm of MaxQuant, >3000 proteins are consistently identified from as few as 10 cells. Furthermore, we demonstrate quantification of ~2400 proteins from single human pancreatic islet thin sections from type 1 diabetic and control donors, illustrating the application of nanoPOTS for spatially resolved proteome measurements from clinical tissues.« less

Nanodroplet processing platform for deep and quantitative proteome profiling of 10–100 mammalian cells

DOE PAGES

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

2018-02-28

Nanoscale or single-cell technologies are critical for biomedical applications. However, current mass spectrometry (MS)-based proteomic approaches require samples comprising a minimum of thousands of cells to provide in-depth profiling. Here in this paper, we report the development of a nanoPOTS (nanodroplet processing in one pot for trace samples) platform for small cell population proteomics analysis. NanoPOTS enhances the efficiency and recovery of sample processing by downscaling processing volumes to <200 nL to minimize surface losses. When combined with ultrasensitive liquid chromatography-MS, nanoPOTS allows identification of ~1500 to ~3000 proteins from ~10 to ~140 cells, respectively. By incorporating the Match Betweenmore » Runs algorithm of MaxQuant, >3000 proteins are consistently identified from as few as 10 cells. Furthermore, we demonstrate quantification of ~2400 proteins from single human pancreatic islet thin sections from type 1 diabetic and control donors, illustrating the application of nanoPOTS for spatially resolved proteome measurements from clinical tissues.« less

Accounting for isotopic clustering in Fourier transform mass spectrometry data analysis for clinical diagnostic studies.

PubMed

Kakourou, Alexia; Vach, Werner; Nicolardi, Simone; van der Burgt, Yuri; Mertens, Bart

2016-10-01

Mass spectrometry based clinical proteomics has emerged as a powerful tool for high-throughput protein profiling and biomarker discovery. Recent improvements in mass spectrometry technology have boosted the potential of proteomic studies in biomedical research. However, the complexity of the proteomic expression introduces new statistical challenges in summarizing and analyzing the acquired data. Statistical methods for optimally processing proteomic data are currently a growing field of research. In this paper we present simple, yet appropriate methods to preprocess, summarize and analyze high-throughput MALDI-FTICR mass spectrometry data, collected in a case-control fashion, while dealing with the statistical challenges that accompany such data. The known statistical properties of the isotopic distribution of the peptide molecules are used to preprocess the spectra and translate the proteomic expression into a condensed data set. Information on either the intensity level or the shape of the identified isotopic clusters is used to derive summary measures on which diagnostic rules for disease status allocation will be based. Results indicate that both the shape of the identified isotopic clusters and the overall intensity level carry information on the class outcome and can be used to predict the presence or absence of the disease.

Statistical design of quantitative mass spectrometry-based proteomic experiments.

PubMed

Oberg, Ann L; Vitek, Olga

2009-05-01

We review the fundamental principles of statistical experimental design, and their application to quantitative mass spectrometry-based proteomics. We focus on class comparison using Analysis of Variance (ANOVA), and discuss how randomization, replication and blocking help avoid systematic biases due to the experimental procedure, and help optimize our ability to detect true quantitative changes between groups. We also discuss the issues of pooling multiple biological specimens for a single mass analysis, and calculation of the number of replicates in a future study. When applicable, we emphasize the parallels between designing quantitative proteomic experiments and experiments with gene expression microarrays, and give examples from that area of research. We illustrate the discussion using theoretical considerations, and using real-data examples of profiling of disease.

Proteomic profiling in MPTP monkey model for early Parkinson disease biomarker discovery

PubMed Central

Lin, Xiangmin; Shi, Min; Gunasingh Masilamoni, Jeyaraj; Dator, Romel; Movius, James; Aro, Patrick; Smith, Yoland; Zhang, Jing

2015-01-01

Identification of reliable and robust biomarkers is crucial to enable early diagnosis of Parkinson disease (PD) and monitoring disease progression. While imperfect, the slow, chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced non-human primate animal model system of parkinsonism is an abundant source of pre-motor or early stage PD biomarker discovery. Here, we present a study of a MPTP rhesus monkey model of PD that utilizes complementary quantitative iTRAQ-based proteomic, glycoproteomics and phosphoproteomics approaches. We compared the glycoprotein, non-glycoprotein, and phosphoprotein profiles in the putamen of asymptomatic and symptomatic MPTP-treated monkeys as well as saline injected controls. We identified 86 glycoproteins, 163 non-glycoproteins, and 71 phosphoproteins differentially expressed in the MPTP-treated groups. Functional analysis of the data sets inferred the biological processes and pathways that link to neurodegeneration in PD and related disorders. Several potential biomarkers identified in this study have already been translated for their usefulness in PD diagnosis in human subjects and further validation investigations are currently under way. In addition to providing potential early PD biomarkers, this comprehensive quantitative proteomic study may also shed insights regarding the mechanisms underlying early PD development. This article is part of a Special Issue entitled: Neuroproteomics: Applications in neuroscience and neurology. PMID:25617661

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.

An Integrated Platform for Isolation, Processing, and Mass Spectrometry-based Proteomic Profiling of Rare Cells in Whole Blood*

PubMed Central

Li, Siyang; Plouffe, Brian D.; Belov, Arseniy M.; Ray, Somak; Wang, Xianzhe; Murthy, Shashi K.; Karger, Barry L.; Ivanov, Alexander R.

2015-01-01

Isolation and molecular characterization of rare cells (e.g. circulating tumor and stem cells) within biological fluids and tissues has significant potential in clinical diagnostics and personalized medicine. The present work describes an integrated platform of sample procurement, preparation, and analysis for deep proteomic profiling of rare cells in blood. Microfluidic magnetophoretic isolation of target cells spiked into 1 ml of blood at the level of 1000–2000 cells/ml, followed by focused acoustics-assisted sample preparation has been coupled with one-dimensional PLOT-LC-MS methodology. The resulting zeptomole detection sensitivity enabled identification of ∼4000 proteins with injection of the equivalent of only 100–200 cells per analysis. The characterization of rare cells in limited volumes of physiological fluids is shown by the isolation and quantitative proteomic profiling of first MCF-7 cells spiked into whole blood as a model system and then two CD133+ endothelial progenitor and hematopoietic cells in whole blood from volunteers. PMID:25755294

Lipoaspirate fluid proteome: A preliminary investigation by LC-MS top-down/bottom-up integrated platform of a high potential biofluid in regenerative medicine.

PubMed

Inserra, Ilaria; Martelli, Claudia; Cipollina, Mara; Cicione, Claudia; Iavarone, Federica; Taranto, Giuseppe Di; Barba, Marta; Castagnola, Massimo; Desiderio, Claudia; Lattanzi, Wanda

2016-04-01

The lipoaspirate fluid (LAF) is emerging as a potentially valuable source in regenerative medicine. In particular, our group recently demonstrated that it is able to exert osteoinductive properties in vitro. This original observation stimulated the investigation of the proteomic component of LAF, by means of LC-ESI-LTQ-Orbitrap-MS top-down/bottom-up integrated approach, which represents the object of the present study. Top-down analyses required the optimization of sample pretreatment procedures to enable the correct investigation of the intact proteome. Bottom-up analyses have been directly applied to untreated samples after monodimensional SDS-PAGE separation. The analysis of the acid-soluble fraction of LAF by top-down approach allowed demonstrating the presence of albumin and hemoglobin fragments (i.e. VV- and LVV-hemorphin-7), thymosins β4 and β10 peptides, ubiquitin and acyl-CoA binding protein; adipogenesis regulatory factor, perilipin-1 fragments, and S100A6, along with their PTMs. Part of the bottom-up proteomic profile was reproducibly found in both tested samples. The bottom-up approach allowed demonstrating the presence of proteins, listed among the components of adipose tissue and/or comprised within the ASCs intracellular content and secreted proteome. Our data provide a first glance on the LAF molecular profile, which is consistent with its tissue environment. LAF appeared to contain bioactive proteins, peptides and paracrine factors, suggesting its potential translational exploitation. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Comparative and network-based proteomic analysis of low dose ethanol- and lipopolysaccharide-induced macrophages.

PubMed

Kamal, Abu Hena M; Fessler, Michael B; Chowdhury, Saiful M

2018-01-01

Macrophages are specialized phagocytes that play an essential role in inflammation, immunity, and tissue repair. Profiling the global proteomic response of macrophages to microbial molecules such as bacterial lipopolysaccharide is key to understanding fundamental mechanisms of inflammatory disease. Ethanol is a widely abused substance that has complex effects on inflammation. Reports have indicated that ethanol can activate or inhibit the lipopolysaccharide receptor, Toll-like Receptor 4, in different settings, with important consequences for liver and neurologic inflammation, but the underlying mechanisms are poorly understood. To profile the sequential effect of low dose ethanol and lipopolysaccharide on macrophages, a gel-free proteomic technique was applied to RAW 264.7 macrophages. Five hundred four differentially expressed proteins were identified and quantified with high confidence using ≥ 5 peptide spectral matches. Among these, 319 proteins were shared across all treatment conditions, and 69 proteins were exclusively identified in ethanol-treated or lipopolysaccharide-stimulated cells. The interactive impact of ethanol and lipopolysaccharide on the macrophage proteome was evaluated using bioinformatics tools, enabling identification of differentially responsive proteins, protein interaction networks, disease- and function-based networks, canonical pathways, and upstream regulators. Five candidate protein coding genes (PGM2, ISYNA1, PARP1, and PSAP) were further validated by qRT-PCR that mostly related to glucose metabolism and fatty acid synthesis pathways. Taken together, this study describes for the first time at a systems level the interaction between ethanol and lipopolysaccharide in the proteomic programming of macrophages, and offers new mechanistic insights into the biology that may underlie the impact of ethanol on infectious and inflammatory disease in humans.

Assessing the impact of transcriptomics, proteomics and metabolomics on fungal phytopathology.

PubMed

Tan, Kar-Chun; Ipcho, Simon V S; Trengove, Robert D; Oliver, Richard P; Solomon, Peter S

2009-09-01

SUMMARY Peer-reviewed literature is today littered with exciting new tools and techniques that are being used in all areas of biology and medicine. Transcriptomics, proteomics and, more recently, metabolomics are three of these techniques that have impacted on fungal plant pathology. Used individually, each of these techniques can generate a plethora of data that could occupy a laboratory for years. When used in combination, they have the potential to comprehensively dissect a system at the transcriptional and translational level. Transcriptomics, or quantitative gene expression profiling, is arguably the most familiar to researchers in the field of fungal plant pathology. Microarrays have been the primary technique for the last decade, but others are now emerging. Proteomics has also been exploited by the fungal phytopathogen community, but perhaps not to its potential. A lack of genome sequence information has frustrated proteomics researchers and has largely contributed to this technique not fulfilling its potential. The coming of the genome sequencing era has partially alleviated this problem. Metabolomics is the most recent of these techniques to emerge and is concerned with the non-targeted profiling of all metabolites in a given system. Metabolomics studies on fungal plant pathogens are only just beginning to appear, although its potential to dissect many facets of the pathogen and disease will see its popularity increase quickly. This review assesses the impact of transcriptomics, proteomics and metabolomics on fungal plant pathology over the last decade and discusses their futures. Each of the techniques is described briefly with further reading recommended. Key examples highlighting the application of these technologies to fungal plant pathogens are also reviewed.

Ixodes ricinus and Its Endosymbiont Midichloria mitochondrii: A Comparative Proteomic Analysis of Salivary Glands and Ovaries.

PubMed

Di Venere, Monica; Fumagalli, Marco; Cafiso, Alessandra; De Marco, Leone; Epis, Sara; Plantard, Olivier; Bardoni, Anna; Salvini, Roberta; Viglio, Simona; Bazzocchi, Chiara; Iadarola, Paolo; Sassera, Davide

2015-01-01

Hard ticks are hematophagous arthropods that act as vectors of numerous pathogenic microorganisms of high relevance in human and veterinary medicine. Ixodes ricinus is one of the most important tick species in Europe, due to its role of vector of pathogenic bacteria such as Borrelia burgdorferi and Anaplasma phagocytophilum, of viruses such as tick borne encephalitis virus and of protozoans as Babesia spp. In addition to these pathogens, I. ricinus harbors a symbiotic bacterium, Midichloria mitochondrii. This is the dominant bacteria associated to I. ricinus, but its biological role is not yet understood. Most M. mitochondrii symbionts are localized in the tick ovaries, and they are transmitted to the progeny. M. mitochondrii bacteria have however also been detected in the salivary glands and saliva of I. ricinus, as well as in the blood of vertebrate hosts of the tick, prompting the hypothesis of an infectious role of this bacterium. To investigate, from a proteomic point of view, the tick I. ricinus and its symbiont, we generated the protein profile of the ovary tissue (OT) and of salivary glands (SG) of adult females of this tick species. To compare the OT and SG profiles, 2-DE profiling followed by LC-MS/MS protein identification were performed. We detected 21 spots showing significant differences in the relative abundance between the OT and SG, ten of which showed 4- to 18-fold increase/decrease in density. This work allowed to establish a method to characterize the proteome of I. ricinus, and to detect multiple proteins that exhibit a differential expression profile in OT and SG. Additionally, we were able to use an immunoproteomic approach to detect a protein from the symbiont. Finally, the method here developed will pave the way for future studies on the proteomics of I. ricinus, with the goals of better understanding the biology of this vector and of its symbiont M. mitochondrii.

Comparative Proteomic Profiling Reveals Molecular Characteristics Associated with Oogenesis and Oocyte Maturation during Ovarian Development of Bactrocera dorsalis (Hendel)

PubMed Central

Li, Ran; Zhang, Meng-Yi; Liu, Yu-Wei; Zhang, Zheng; Smagghe, Guy; Wang, Jin-Jun

2017-01-01

Time-dependent expression of proteins in ovary is important to understand oogenesis in insects. Here, we profiled the proteomes of developing ovaries from Bactrocera dorsalis (Hendel) to obtain information about ovarian development with particular emphasis on differentially expressed proteins (DEPs) involved in oogenesis. A total of 4838 proteins were identified with an average peptide number of 8.15 and sequence coverage of 20.79%. Quantitative proteomic analysis showed that a total of 612 and 196 proteins were differentially expressed in developing and mature ovaries, respectively. Furthermore, 153, 196 and 59 potential target proteins were highly expressed in early, vitellogenic and mature ovaries and most tested DEPs had the similar trends consistent with the respective transcriptional profiles. These proteins were abundantly expressed in pre-vitellogenic and vitellogenic stages, including tropomyosin, vitellogenin, eukaryotic translation initiation factor, heat shock protein, importin protein, vitelline membrane protein, and chorion protein. Several hormone and signal pathway related proteins were also identified during ovarian development including piRNA, notch, insulin, juvenile, and ecdysone hormone signal pathways. This is the first report of a global ovary proteome of a tephritid fruit fly, and may contribute to understanding the complicate processes of ovarian development and exploring the potentially novel pest control targets. PMID:28665301

Profiling the outer membrane proteome during growth and development of the social bacterium Myxococcus xanthus by selective biotinylation and analyses of outer membrane vesicles.

PubMed

Kahnt, Jörg; Aguiluz, Kryssia; Koch, Jürgen; Treuner-Lange, Anke; Konovalova, Anna; Huntley, Stuart; Hoppert, Michael; Søgaard-Andersen, Lotte; Hedderich, Reiner

2010-10-01

Social behavior in the bacterium Myxococcus xanthus relies on contact-dependent activities involving cell-cell and cell-substratum interactions. To identify outer membrane proteins that have a role in these activities, we profiled the outer membrane proteome of growing and starving cells using two strategies. First, outer membrane proteins were enriched by biotinylation of intact cells using the reagent NHS (N-hydroxysuccinimide)-PEO(12) (polyethylene oxide)-biotin with subsequent membrane solubilization and affinity chromatography. Second, the proteome of outer membrane vesicles (OMV) was determined. Comparisons of detected proteins show that these methods have different detection profiles and together provide a comprehensive view of the outer membrane proteome. From 362 proteins identified, 274 (76%) were cell envelope proteins including 64 integral outer membrane proteins and 85 lipoproteins. The majority of these proteins were of unknown function. Among integral outer membrane proteins with homologues of known function, TonB-dependent transporters comprise the largest group. Our data suggest novel functions for these transporters. Among lipoproteins with homologues of known function, proteins with hydrolytic functions comprise the largest group. The luminal load of OMV was enriched for proteins with hydrolytic functions. Our data suggest that OMV have functions in predation and possibly in transfer of intercellular signaling molecules between cells.

Embryology in the era of proteomics.

PubMed

Katz-Jaffe, Mandy G; McReynolds, Susanna

2013-03-15

Proteomic technologies have begun providing evidence that viable embryos possess unique protein profiles. Some of these potential protein biomarkers have been identified as extracellular and could be used in the development of a noninvasive quantitative method for embryo assessment. The field of assisted reproductive technologies would benefit from defining the human embryonic proteome and secretome, thereby expanding our current knowledge of embryonic cellular processes. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Quantitative high-throughput profiling of snake venom gland transcriptomes and proteomes (Ovophis okinavensis and Protobothrops flavoviridis)

PubMed Central

2013-01-01

Background Advances in DNA sequencing and proteomics have facilitated quantitative comparisons of snake venom composition. Most studies have employed one approach or the other. Here, both Illumina cDNA sequencing and LC/MS were used to compare the transcriptomes and proteomes of two pit vipers, Protobothrops flavoviridis and Ovophis okinavensis, which differ greatly in their biology. Results Sequencing of venom gland cDNA produced 104,830 transcripts. The Protobothrops transcriptome contained transcripts for 103 venom-related proteins, while the Ovophis transcriptome contained 95. In both, transcript abundances spanned six orders of magnitude. Mass spectrometry identified peptides from 100% of transcripts that occurred at higher than contaminant (e.g. human keratin) levels, including a number of proteins never before sequenced from snakes. These transcriptomes reveal fundamentally different envenomation strategies. Adult Protobothrops venom promotes hemorrhage, hypotension, incoagulable blood, and prey digestion, consistent with mammalian predation. Ovophis venom composition is less readily interpreted, owing to insufficient pharmacological data for venom serine and metalloproteases, which comprise more than 97.3% of Ovophis transcripts, but only 38.0% of Protobothrops transcripts. Ovophis venom apparently represents a hybrid strategy optimized for frogs and small mammals. Conclusions This study illustrates the power of cDNA sequencing combined with MS profiling. The former quantifies transcript composition, allowing detection of novel proteins, but cannot indicate which proteins are actually secreted, as does MS. We show, for the first time, that transcript and peptide abundances are correlated. This means that MS can be used for quantitative, non-invasive venom profiling, which will be beneficial for studies of endangered species. PMID:24224955

Protein profiling in potato (Solanum tuberosum L.) leaf tissues by differential centrifugation.

PubMed

Lim, Sanghyun; Chisholm, Kenneth; Coffin, Robert H; Peters, Rick D; Al-Mughrabi, Khalil I; Wang-Pruski, Gefu; Pinto, Devanand M

2012-04-06

Foliar diseases, such as late blight, result in serious threats to potato production. As such, potato leaf tissue becomes an important substrate to study biological processes, such as plant defense responses to infection. Nonetheless, the potato leaf proteome remains poorly characterized. Here, we report protein profiling of potato leaf tissues using a modified differential centrifugation approach to separate the leaf tissues into cell wall and cytoplasmic fractions. This method helps to increase the number of identified proteins, including targeted putative cell wall proteins. The method allowed for the identification of 1484 nonredundant potato leaf proteins, of which 364 and 447 were reproducibly identified proteins in the cell wall and cytoplasmic fractions, respectively. Reproducibly identified proteins corresponded to over 70% of proteins identified in each replicate. A diverse range of proteins was identified based on their theoretical pI values, molecular masses, functional classification, and biological processes. Such a protein extraction method is effective for the establishment of a highly qualified proteome profile.

Comparative Proteome Profiling during Cardiac Hypertrophy and Myocardial Infarction Reveals Altered Glucose Oxidation by Differential Activation of Pyruvate Dehydrogenase E1 Component Subunit β.

PubMed

Mitra, Arkadeep; Basak, Trayambak; Ahmad, Shadab; Datta, Kaberi; Datta, Ritwik; Sengupta, Shantanu; Sarkar, Sagartirtha

2015-06-05

Cardiac hypertrophy and myocardial infarction (MI) are two etiologically different disease forms with varied pathological characteristics. However, the precise molecular mechanisms and specific causal proteins associated with these diseases are obscure to date. In this study, a comparative cardiac proteome profiling was performed in Wistar rat models for diseased and control (sham) groups using two-dimensional difference gel electrophoresis followed by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry. Proteins were identified using Protein Pilot™ software (version 4.0) and were subjected to stringent statistical analysis. Alteration of key proteins was validated by Western blot analysis. The differentially expressed protein sets identified in this study were associated with different functional groups, involving various metabolic pathways, stress responses, cytoskeletal organization, apoptotic signaling and other miscellaneous functions. It was further deciphered that altered energy metabolism during hypertrophy in comparison to MI may be predominantly attributed to induced glucose oxidation level, via reduced phosphorylation of pyruvate dehydrogenase E1 component subunit β (PDHE1-B) protein during hypertrophy. This study reports for the first time the global changes in rat cardiac proteome during two etiologically different cardiac diseases and identifies key signaling regulators modulating ontogeny of these two diseases culminating in heart failure. This study also pointed toward differential activation of PDHE1-B that accounts for upregulation of glucose oxidation during hypertrophy. Downstream analysis of altered proteome and the associated modulators would enhance our present knowledge regarding altered pathophysiology of these two etiologically different cardiac disease forms. Copyright © 2014 Elsevier Ltd. All rights reserved.

Complementary transcriptome and proteome profiling in cabbage buds of a recessive male sterile mutant provides new insights into male reproductive development.

PubMed

Ji, Jialei; Yang, Limei; Fang, Zhiyuan; Zhuang, Mu; Zhang, Yangyong; Lv, Honghao; Liu, Yumei; Li, Zhansheng

2018-05-15

Plant male reproductive development is a very complex biological process that involves multiple metabolic pathways. To reveal novel insights into male reproductive development, we conducted an integrated profiling of gene activity in the developing buds of a cabbage recessive genetic male sterile mutant. Using RNA-Seq and label-free quantitative proteomics, 2881 transcripts and 1245 protein species were identified with significant differential abundance between the male sterile line 83121A and its isogenic maintainer line 83121B. Analyses of function annotations and correlations between transcriptome and proteome and protein interaction networks were also conducted, which suggested that the male sterility involves a complex regulatory pattern. Moreover, several key biological processes, such as fatty acid metabolism, tapetosome biosynthesis, amino acid metabolism and protein synthesis and degradation were identified as being of relevance to male reproductive development. A large number of protein species involved in sporopollenin synthesis, amino acid synthesis, ribosome assembly, protein processing in endoplasmic reticulum and lipid transfer were observed to be significantly down-accumulated in 83121A buds, indicating their potential roles in the regulation of cabbage microspore abortion. In summary, the conjoint analysis of the transcriptome and proteome provided a global picture regarding the molecular dynamics in male sterile buds of 83121A. Male sterile mutants are excellent materials for the study of plant male reproductive development. This study revealed the molecular dynamics of recessive male sterility in cabbage at the transcriptome and proteome levels, which deepens our understanding of the metabolic pathways involved in male development. Moreover, the male sterility-related genes identified in this study could provide a reference for the artificial regulation of cabbage fertility by using genetic engineering technology, which may result in potential applications in agriculture such as production of hybrid seeds using male sterility. Copyright © 2018 Elsevier B.V. All rights reserved.

Proteomics and bioinformatics analysis of altered protein expression in the placental villous tissue from early recurrent miscarriage patients.

PubMed

Pan, Hai-Tao; Ding, Hai-Gang; Fang, Min; Yu, Bin; Cheng, Yi; Tan, Ya-Jing; Fu, Qi-Qin; Lu, Bo; Cai, Hong-Guang; Jin, Xin; Xia, Xian-Qing; Zhang, Tao

2018-01-01

Recurrent miscarriage (RM) affects 5% of women, it has an adverse emotional impact on women. Because of the complexities of early development, the mechanism of recurrent miscarriage is still unclear. We hypothesized that abnormal placenta leads to early recurrent miscarriage (ERM). The aim of this study was to identify ERM associated factors in human placenta villous tissue using proteomics. Investigation of these differences in protein expression in parallel profiling is essential to understand the comprehensive pathophysiological mechanism underlying recurrent miscarriage (RM). To gain more insight into mechanisms of recurrent miscarriage (RM), a comparative proteome profile of the human placenta villous tissue in normal and RM pregnancies was analyzed using iTRAQ technology and bioinformatics analysis used by Ingenuity Pathway Analysis (IPA) software. In this study, we employed an iTRAQ based proteomics analysis of four placental villous tissues from patients with early recurrent miscarriage (ERM) and four from normal pregnant women. Finally, we identified 2805 proteins and 79,998 peptides between patients with RM and normal matched group. Further analysis identified 314 differentially expressed proteins in placental villous tissue (≥1.3-fold, Student's t-test, p < 0.05); 209 proteins showed the increased expression while 105 proteins showed decreased expression. These 314 proteins were analyzed by Ingenuity Pathway Analysis (IPA) and were found to play important roles in the growth of embryo. Furthermore, network analysis show that Angiotensinogen (AGT), MAPK14 and Prothrombin (F2) are core factors in early embryonic development. We used another 8 independent samples (4 cases and 4 controls) to cross validation of the proteomic data. This study has identified several proteins that are associated with early development, these results may supply new insight into mechanisms behind recurrent miscarriage. Copyright © 2017 Elsevier Ltd. All rights reserved.

Integrated Proteomic Pipeline Using Multiple Search Engines for a Proteogenomic Study with a Controlled Protein False Discovery Rate.

PubMed

Park, Gun Wook; Hwang, Heeyoun; Kim, Kwang Hoe; Lee, Ju Yeon; Lee, Hyun Kyoung; Park, Ji Yeong; Ji, Eun Sun; Park, Sung-Kyu Robin; Yates, John R; Kwon, Kyung-Hoon; Park, Young Mok; Lee, Hyoung-Joo; Paik, Young-Ki; Kim, Jin Young; Yoo, Jong Shin

2016-11-04

In the Chromosome-Centric Human Proteome Project (C-HPP), false-positive identification by peptide spectrum matches (PSMs) after database searches is a major issue for proteogenomic studies using liquid-chromatography and mass-spectrometry-based large proteomic profiling. Here we developed a simple strategy for protein identification, with a controlled false discovery rate (FDR) at the protein level, using an integrated proteomic pipeline (IPP) that consists of four engrailed steps as follows. First, using three different search engines, SEQUEST, MASCOT, and MS-GF+, individual proteomic searches were performed against the neXtProt database. Second, the search results from the PSMs were combined using statistical evaluation tools including DTASelect and Percolator. Third, the peptide search scores were converted into E-scores normalized using an in-house program. Last, ProteinInferencer was used to filter the proteins containing two or more peptides with a controlled FDR of 1.0% at the protein level. Finally, we compared the performance of the IPP to a conventional proteomic pipeline (CPP) for protein identification using a controlled FDR of <1% at the protein level. Using the IPP, a total of 5756 proteins (vs 4453 using the CPP) including 477 alternative splicing variants (vs 182 using the CPP) were identified from human hippocampal tissue. In addition, a total of 10 missing proteins (vs 7 using the CPP) were identified with two or more unique peptides, and their tryptic peptides were validated using MS/MS spectral pattern from a repository database or their corresponding synthetic peptides. This study shows that the IPP effectively improved the identification of proteins, including alternative splicing variants and missing proteins, in human hippocampal tissues for the C-HPP. All RAW files used in this study were deposited in ProteomeXchange (PXD000395).

Predictive toxicology using systemic biology and liver microfluidic “on chip” approaches: Application to acetaminophen injury

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

Prot, Jean-Matthieu; Bunescu, Andrei; Elena-Herrmann, Bénédicte

2012-03-15

We have analyzed transcriptomic, proteomic and metabolomic profiles of hepatoma cells cultivated inside a microfluidic biochip with or without acetaminophen (APAP). Without APAP, the results show an adaptive cellular response to the microfluidic environment, leading to the induction of anti-oxidative stress and cytoprotective pathways. In presence of APAP, calcium homeostasis perturbation, lipid peroxidation and cell death are observed. These effects can be attributed to APAP metabolism into its highly reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI). That toxicity pathway was confirmed by the detection of GSH-APAP, the large production of 2-hydroxybutyrate and 3-hydroxybutyrate, and methionine, cystine, and histidine consumption in the treatedmore » biochips. Those metabolites have been reported as specific biomarkers of hepatotoxicity and glutathione depletion in the literature. In addition, the integration of the metabolomic, transcriptomic and proteomic collected profiles allowed a more complete reconstruction of the APAP injury pathways. To our knowledge, this work is the first example of a global integration of microfluidic biochip data in toxicity assessment. Our results demonstrate the potential of that new approach to predictive toxicology. -- Highlights: ► We cultivated liver cells in microfluidic biochips ► We integrated transcriptomic, proteomic and metabolomics profiles ► Pathways reconstructions were proposed in control and acetaminophen treated cultures ► Biomarkers were identified ► Comparisons with in vivo studies were proposed.« less

LC–MS Proteomics Analysis of the Insulin/IGF-1-Deficient Caenorhabditis elegans daf-2(e1370) Mutant Reveals Extensive Restructuring of Intermediary Metabolism

PubMed Central

2015-01-01

The insulin/IGF-1 receptor is a major known determinant of dauer formation, stress resistance, longevity, and metabolism in Caenorhabditis elegans. In the past, whole-genome transcript profiling was used extensively to study differential gene expression in response to reduced insulin/IGF-1 signaling, including the expression levels of metabolism-associated genes. Taking advantage of the recent developments in quantitative liquid chromatography mass spectrometry (LC–MS)-based proteomics, we profiled the proteomic changes that occur in response to activation of the DAF-16 transcription factor in the germline-less glp-4(bn2);daf-2(e1370) receptor mutant. Strikingly, the daf-2 profile suggests extensive reorganization of intermediary metabolism, characterized by the upregulation of many core intermediary metabolic pathways. These include glycolysis/gluconeogenesis, glycogenesis, pentose phosphate cycle, citric acid cycle, glyoxylate shunt, fatty acid β-oxidation, one-carbon metabolism, propionate and tyrosine catabolism, and complexes I, II, III, and V of the electron transport chain. Interestingly, we found simultaneous activation of reciprocally regulated metabolic pathways, which is indicative of spatiotemporal coordination of energy metabolism and/or extensive post-translational regulation of these enzymes. This restructuring of daf-2 metabolism is reminiscent to that of hypometabolic dauers, allowing the efficient and economical utilization of internal nutrient reserves and possibly also shunting metabolites through alternative energy-generating pathways to sustain longevity. PMID:24555535

Proteomic and Bioinformatic Studies for the Characterization of Response to Pemetrexed in Platinum Drug Resistant Ovarian Cancer.

PubMed

Severi, Leda; Losi, Lorena; Fonda, Sergio; Taddia, Laura; Gozzi, Gaia; Marverti, Gaetano; Magni, Fulvio; Chinello, Clizia; Stella, Martina; Sheouli, Jalid; Braicu, Elena I; Genovese, Filippo; Lauriola, Angela; Marraccini, Chiara; Gualandi, Alessandra; D'Arca, Domenico; Ferrari, Stefania; Costi, Maria P

2018-01-01

Proteomics and bioinformatics are a useful combined technology for the characterization of protein expression level and modulation associated with the response to a drug and with its mechanism of action. The folate pathway represents an important target in the anticancer drugs therapy. In the present study, a discovery proteomics approach was applied to tissue samples collected from ovarian cancer patients who relapsed after the first-line carboplatin-based chemotherapy and were treated with pemetrexed (PMX), a known folate pathway targeting drug. The aim of the work is to identify the proteomic profile that can be associated to the response to the PMX treatment in pre-treatement tissue. Statistical metrics of the experimental Mass Spectrometry (MS) data were combined with a knowledge-based approach that included bioinformatics and a literature review through ProteinQuest™ tool, to design a protein set of reference (PSR). The PSR provides feedback for the consistency of MS proteomic data because it includes known validated proteins. A panel of 24 proteins with levels that were significantly different in pre-treatment samples of patients who responded to the therapy vs. the non-responder ones, was identified. The differences of the identified proteins were explained for the patients with different outcomes and the known PMX targets were further validated. The protein panel herein identified is ready for further validation in retrospective clinical trials using a targeted proteomic approach. This study may have a general relevant impact on biomarker application for cancer patients therapy selection.

Search for novel circulating cancer chemopreventive biomarkers of dietary rice bran intervention in Apc(Min) mice model of colorectal carcinogenesis, using proteomic and metabolic profiling strategies.

PubMed

Norris, Leonie; Malkar, Aditya; Horner-Glister, Emma; Hakimi, Amirmansoor; Ng, Leong L; Gescher, Andreas J; Creaser, Colin; Sale, Stewart; Jones, Donald J L

2015-09-01

There is strong epidemiological evidence indicating that consumption by humans of whole-grain foods including rice bran may be associated with a low incidence of cancer, especially in the colorectum. Molecular processes associated with cancer development may be retarded by fiber consumption. Consequently, intervention with dietary fiber might be suitable as a cancer chemoprevention strategy in high-risk populations. Here, we searched for putative molecular mechanism-based efficacy biomarkers of rice fiber consumption in the plasma of mice characterized by a genetic propensity to develop gastrointestinal adenomas. The hypothesis was tested that metabolic and proteomic changes in blood reflect the chemopreventive activity of rice bran. Apc(Min) mice received diet supplemented with rice bran at 5, 15, and 30%. Blood and tissue samples were taken. Plasma was subjected to MS-based proteomic and metabolic profiling analyses as well as assessment of hematocrit values. Gastrointestinal tracts were removed and adenomas were counted and their size was measured so that total tumor burden could be calculated. The hypothesis was tested that metabolic and proteomic changes in blood reflect chemopreventive activity. Rice bran consumption reduced adenoma burden and number in a dose-related fashion when compared to controls. Metabolic profiling data demonstrated strong clustering of the groups indicating that metabolic pathways are perturbed. Proteomic analysis identified adiponectin as a molecule that was significantly altered, which may play a role in tumor suppression. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Sequential Extraction Results in Improved Proteome Profiling of Medicinal Plant Pinellia ternata Tubers, Which Contain Large Amounts of High-Abundance Proteins

PubMed Central

An, SuFang; Gong, FangPing; Wang, Wei

2012-01-01

Pinellia ternata tuber is one of the well-known Chinese traditional medicines. In order to understand the pharmacological properties of tuber proteins, it is necessary to perform proteome analysis of P. ternata tubers. However, a few high-abundance proteins (HAPs), mainly mannose-binding lectin (agglutinin), exist in aggregates of various sizes in the tubers and seriously interfere with proteome profiling by two-dimensional electrophoresis (2-DE). Therefore, selective depletion of these HAPs is a prerequisite for enhanced proteome analysis of P. ternata tubers. Based on differential protein solubility, we developed a novel protocol involving two sequential extractions for depletion of some HAPs and prefractionation of tuber proteins prior to 2-DE. The first extraction using 10% acetic acid selectively extracted acid-soluble HAPs and the second extraction using the SDS-containing buffer extracted remaining acid-insoluble proteins. After application of the protocol, 2-DE profiles of P. ternata tuber proteins were greatly improved and more protein spots were detected, especially low-abundance proteins. Moreover, the subunit composition of P. ternata lectin was analyzed by electrophoresis. Native lectin consists of two hydrogen-bonded subunits (11 kDa and 25 kDa) and the 11 kDa subunit was a glycoprotein. Subsequently, major HAPs in the tubers were analyzed by mass spectrometry, with nine protein spots being identified as lectin isoforms. The methodology was easy to perform and required no specialized apparatus. It would be useful for proteome analysis of other tuber plants of Araceae. PMID:23185632

Sequential extraction results in improved proteome profiling of medicinal plant Pinellia ternata tubers, which contain large amounts of high-abundance proteins.

PubMed

Wu, Xiaolin; Xiong, Erhui; An, Sufang; Gong, Fangping; Wang, Wei

2012-01-01

Pinellia ternata tuber is one of the well-known Chinese traditional medicines. In order to understand the pharmacological properties of tuber proteins, it is necessary to perform proteome analysis of P. ternata tubers. However, a few high-abundance proteins (HAPs), mainly mannose-binding lectin (agglutinin), exist in aggregates of various sizes in the tubers and seriously interfere with proteome profiling by two-dimensional electrophoresis (2-DE). Therefore, selective depletion of these HAPs is a prerequisite for enhanced proteome analysis of P. ternata tubers. Based on differential protein solubility, we developed a novel protocol involving two sequential extractions for depletion of some HAPs and prefractionation of tuber proteins prior to 2-DE. The first extraction using 10% acetic acid selectively extracted acid-soluble HAPs and the second extraction using the SDS-containing buffer extracted remaining acid-insoluble proteins. After application of the protocol, 2-DE profiles of P. ternata tuber proteins were greatly improved and more protein spots were detected, especially low-abundance proteins. Moreover, the subunit composition of P. ternata lectin was analyzed by electrophoresis. Native lectin consists of two hydrogen-bonded subunits (11 kDa and 25 kDa) and the 11 kDa subunit was a glycoprotein. Subsequently, major HAPs in the tubers were analyzed by mass spectrometry, with nine protein spots being identified as lectin isoforms. The methodology was easy to perform and required no specialized apparatus. It would be useful for proteome analysis of other tuber plants of Araceae.

Large-Scale Interaction Profiling of Protein Domains Through Proteomic Peptide-Phage Display Using Custom Peptidomes.

PubMed

Seo, Moon-Hyeong; Nim, Satra; Jeon, Jouhyun; Kim, Philip M

2017-01-01

Protein-protein interactions are essential to cellular functions and signaling pathways. We recently combined bioinformatics and custom oligonucleotide arrays to construct custom-made peptide-phage libraries for screening peptide-protein interactions, an approach we call proteomic peptide-phage display (ProP-PD). In this chapter, we describe protocols for phage display for the identification of natural peptide binders for a given protein. We finally describe deep sequencing for the analysis of the proteomic peptide-phage display.

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

Using Proteomics to Understand How Leishmania Parasites Survive inside the Host and Establish Infection

PubMed Central

Veras, Patrícia Sampaio Tavares; Bezerra de Menezes, Juliana Perrone

2016-01-01

Leishmania is a protozoan parasite that causes a wide range of different clinical manifestations in mammalian hosts. It is a major public health risk on different continents and represents one of the most important neglected diseases. Due to the high toxicity of the drugs currently used, and in the light of increasing drug resistance, there is a critical need to develop new drugs and vaccines to control Leishmania infection. Over the past few years, proteomics has become an important tool to understand the underlying biology of Leishmania parasites and host interaction. The large-scale study of proteins, both in parasites and within the host in response to infection, can accelerate the discovery of new therapeutic targets. By studying the proteomes of host cells and tissues infected with Leishmania, as well as changes in protein profiles among promastigotes and amastigotes, scientists hope to better understand the biology involved in the parasite survival and the host-parasite interaction. This review demonstrates the feasibility of proteomics as an approach to identify new proteins involved in Leishmania differentiation and intracellular survival. PMID:27548150

Using Proteomics to Understand How Leishmania Parasites Survive inside the Host and Establish Infection.

PubMed

Veras, Patrícia Sampaio Tavares; Bezerra de Menezes, Juliana Perrone

2016-08-19

Leishmania is a protozoan parasite that causes a wide range of different clinical manifestations in mammalian hosts. It is a major public health risk on different continents and represents one of the most important neglected diseases. Due to the high toxicity of the drugs currently used, and in the light of increasing drug resistance, there is a critical need to develop new drugs and vaccines to control Leishmania infection. Over the past few years, proteomics has become an important tool to understand the underlying biology of Leishmania parasites and host interaction. The large-scale study of proteins, both in parasites and within the host in response to infection, can accelerate the discovery of new therapeutic targets. By studying the proteomes of host cells and tissues infected with Leishmania, as well as changes in protein profiles among promastigotes and amastigotes, scientists hope to better understand the biology involved in the parasite survival and the host-parasite interaction. This review demonstrates the feasibility of proteomics as an approach to identify new proteins involved in Leishmania differentiation and intracellular survival.

Mass spectrometry-based proteomics: from cancer biology to protein biomarkers, drug targets, and clinical applications.

PubMed

Jimenez, Connie R; Verheul, Henk M W

2014-01-01

Proteomics is optimally suited to bridge the gap between genomic information on the one hand and biologic functions and disease phenotypes at the other, since it studies the expression and/or post-translational modification (especially phosphorylation) of proteins--the major cellular players bringing about cellular functions--at a global level in biologic specimens. Mass spectrometry technology and (bio)informatic tools have matured to the extent that they can provide high-throughput, comprehensive, and quantitative protein inventories of cells, tissues, and biofluids in clinical samples at low level. In this article, we focus on next-generation proteomics employing nanoliquid chromatography coupled to high-resolution tandem mass spectrometry for in-depth (phospho)protein profiling of tumor tissues and (proximal) biofluids, with a focus on studies employing clinical material. In addition, we highlight emerging proteogenomic approaches for the identification of tumor-specific protein variants, and targeted multiplex mass spectrometry strategies for large-scale biomarker validation. Below we provide a discussion of recent progress, some research highlights, and challenges that remain for clinical translation of proteomic discoveries.

Serum proteomic profiling for autism using magnetic bead-assisted matrix-assisted laser desorption ionization time-of-flight mass spectrometry: a pilot study.

PubMed

Chen, Yan-Ni; Du, Hui-Ying; Shi, Zhuo-Yue; He, Li; He, Yu-Ying; Wang, Duan

2018-01-24

The pathogenesis of autism spectrum disorders remains elusive and currently there are no diagnostic or predictive biomarkers in autism available. Proteomic profiling has been used in a wide range of neurodevelopmental disorder studies, which could produce deeper perceptions of the molecular bases behind certain disease and potentially becomes useful in discovering biomarkers in autism spectrum disorders. Serum samples were collected from autistic children about 3 years old in age (n = 32) and healthy controls (n = 20) in similar age and gender. The samples were identified specific proteins that are differentially expressed by magnetic bead-based pre-fractionation and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF-MS). Eight protein peaks were significantly different in autistic children from the healthy controls (P < 0.0001). The two peaks with the most significant differences were 6428 and 7758 Da in size. According to differences in serum protein profiles between the autistic children and healthy controls, this study identified a set of differentially expressed proteins those are significant for further evaluation and might function as biomarkers in autism.

Changes of human serum proteome profile during 7-day “dry” immersion

NASA Astrophysics Data System (ADS)

Pakharukova, N. A.; Pastushkova, L. Kh.; Larina, I. M.; Grigoriev, A. I.

2011-05-01

The aim of this study was to characterize changes of serum proteome profile during 7-day "dry" immersion (DI). The experiment with DI consisted of three series: control group without countermeasures (10 men), with using mechanical stimulation (6 men) and low-frequency myostimulation (5 men) as preventive means. Serum samples were fractionated using ClinProt robot (Bruker Daltonics) on magnetic beads (weak cation exchange magnetic beads—MB WCX) prior to mass-spectral profiling. It was obtained 170 peaks after fractionation of serum samples in each group. On 7th immersion day peak areas of fibrinopeptide A ( m/ z=1206; 1464), angiotensin II ( m/ z=1051), high molecular mass kininogen fragment ( m/ z=2133 Da) and C3-fragment of the complement system ( m/ z=1350 Da) were significantly decreased comparing with pre-experimental values of all experimental series. Peak areas of apolipoprotein C III ( m/ z=9419) and C4a fragment of the complement system ( m/ z=3206 Da) were increased. On 7th day of the recovery peak areas of all changed peaks were not close to pre-experimental values. This fact provided evidence of incomplete recovery of an organism after DI. The depth of the alterations had considerable individual variability. Thereby the detected changes of serum proteome profile in the experiment. They indicated a reorganization of the hormonal, immune systems and lipid metabolism. The use of myostimulation and mechanical stimulation as countermeasures partly compensated adverse effects of 7-day dry immersion on the parameters of coagulation system (fibrinopeptide A) and lipid metabolism (apolipoprotein CIII).

Molecular Profiling of Phagocytic Immune Cells in Anopheles gambiae Reveals Integral Roles for Hemocytes in Mosquito Innate Immunity*

PubMed Central

Smith, Ryan C.; King, Jonas G.; Tao, Dingyin; Zeleznik, Oana A.; Brando, Clara; Thallinger, Gerhard G.; Dinglasan, Rhoel R.

2016-01-01

The innate immune response is highly conserved across all eukaryotes and has been studied in great detail in several model organisms. Hemocytes, the primary immune cell population in mosquitoes, are important components of the mosquito innate immune response, yet critical aspects of their biology have remained uncharacterized. Using a novel method of enrichment, we isolated phagocytic granulocytes and quantified their proteomes by mass spectrometry. The data demonstrate that phagocytosis, blood-feeding, and Plasmodium falciparum infection promote dramatic shifts in the proteomic profiles of An. gambiae granulocyte populations. Of interest, large numbers of immune proteins were induced in response to blood feeding alone, suggesting that granulocytes have an integral role in priming the mosquito immune system for pathogen challenge. In addition, we identify several granulocyte proteins with putative roles as membrane receptors, cell signaling, or immune components that when silenced, have either positive or negative effects on malaria parasite survival. Integrating existing hemocyte transcriptional profiles, we also compare differences in hemocyte transcript and protein expression to provide new insight into hemocyte gene regulation and discuss the potential that post-transcriptional regulation may be an important component of hemocyte gene expression. These data represent a significant advancement in mosquito hemocyte biology, providing the first comprehensive proteomic profiling of mosquito phagocytic granulocytes during homeostasis blood-feeding, and pathogen challenge. Together, these findings extend current knowledge to further illustrate the importance of hemocytes in shaping mosquito innate immunity and their principal role in defining malaria parasite survival in the mosquito host. PMID:27624304

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

Toxicogenomics concepts and applications to study hepatic effects of food additives and chemicals

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

Stierum, Rob; Heijne, Wilbert; Kienhuis, Anne

2005-09-01

Transcriptomics, proteomics and metabolomics are genomics technologies with great potential in toxicological sciences. Toxicogenomics involves the integration of conventional toxicological examinations with gene, protein or metabolite expression profiles. An overview together with selected examples of the possibilities of genomics in toxicology is given. The expectations raised by toxicogenomics are earlier and more sensitive detection of toxicity. Furthermore, toxicogenomics will provide a better understanding of the mechanism of toxicity and may facilitate the prediction of toxicity of unknown compounds. Mechanism-based markers of toxicity can be discovered and improved interspecies and in vitro-in vivo extrapolations will drive model developments in toxicology. Toxicologicalmore » assessment of chemical mixtures will benefit from the new molecular biological tools. In our laboratory, toxicogenomics is predominantly applied for elucidation of mechanisms of action and discovery of novel pathway-supported mechanism-based markers of liver toxicity. In addition, we aim to integrate transcriptome, proteome and metabolome data, supported by bioinformatics to develop a systems biology approach for toxicology. Transcriptomics and proteomics studies on bromobenzene-mediated hepatotoxicity in the rat are discussed. Finally, an example is shown in which gene expression profiling together with conventional biochemistry led to the discovery of novel markers for the hepatic effects of the food additives butylated hydroxytoluene, curcumin, propyl gallate and thiabendazole.« less

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

Proteomic profiling of isogenic primary and metastatic medulloblastoma cell lines reveals differential expression of key metastatic factors.

PubMed

Gu, Shuo; Chen, Kai; Yin, Minzhi; Wu, Zhixiang; Wu, Yeming

2017-05-08

Medulloblastoma is the most common malignant brain tumor in children. Around 30% of medulloblastoma patients are diagnosed with metastasis, which often results in a poor prognosis. Unfortunately, molecular mechanisms of medulloblastoma metastasis remain largely unknown. In this study, we employed the recently developed deep proteome analysis approach to quantitatively profile the expression of >10,000 proteins from CHLA-01-MED and CHLA-01R-MED isogenic cell lines derived from the primary and metastatic tumor of the same patient diagnosed with a group IV medulloblastoma. Using statistical analysis, we identified ~1400 significantly altered proteins between the primary and metastatic cell lines including known factors such as placental growth factor (PLGF), LIM homeobox 1 (LHX1) and prominim 1 (PROM1), as well as the negative regulator secreted protein acidic and cysteine rich (SPARC). Additional transwell experiments and immunohistochemical analysis of clinical medulloblastoma samples implicated yes-associated protein 1 (YAP1) as a potential key factor contributing to metastasis. Taken together, our data broadly defines the metastasis-relevant regulated proteome and provides a precious resource for further investigating potential mechanisms of medulloblastoma metastasis. This study represented the first deep proteome analysis of metastatic medulloblastomas and provided a valuable candidate list of altered proteins in metastatic medulloblastomas. The primary data suggested YAP1 as a potential driver for the metastasis of medulloblastoma. These results open up numerous avenues for further investigating the underlying mechanisms of medulloblastoma metastasis and improving the prognosis of medulloblastoma patients. Copyright © 2017 Elsevier B.V. All rights reserved.

Distinct changes in the proteome profile of endometrial tissues in polycystic ovary syndrome compared with healthy fertile women.

PubMed

Amjadi, Fatemehsadat; Mehdizadeh, Mehdi; Ashrafi, Mahnaz; Nasrabadi, Davood; Taleahmad, Sara; Mirzaei, Mehdi; Gupta, Vivek; Salekdeh, Ghasem Hosseini; Aflatoonian, Reza

2018-04-21

What is the molecular basis of infertility related to uterine dysfunction in women with polycystic ovary syndrome (PCOS)? In this study, differences in protein expression between PCOS and normal endometrium were identified using a proteomic approach based on two-dimensional electrophoresis (2-DE) coupled with mass spectrometry (MS). The proteome of endometrium were analysed during the proliferative (on day 2 or 3 before ovulation, n = 6) and luteal phases (on day 3-5 after ovulation, n = 6) from healthy women and PCOS patients (12-14 days after spontaneous bleeding, n = 12). The differentially expressed proteins were categorized based on the biological process using the DAVID bioinformatics resources. Over 803 reproducible protein spots were detected on gels, and 150 protein spots showed different intensities between PCOS and normal women during the proliferative and luteal phases. MS analysis detected 70 proteins out of 150 spots. For four of the 70 proteins, 14-3-3 protein, annexin A5, SERPINA1 and cathepsin D, 2-DE results were validated and localized by Western blot and immunohistochemistry, respectively, and their gene expression profiles were confirmed by real-time quantitative PCR. The obtained results corresponded to the proteomic analysis. The differentially expressed proteins identified are known to be involved in apoptosis, oxidative stress, inflammation and the cytoskeleton. The processes related to the differentially expressed proteins play important roles in fecundity and fecundability. The present study may reveal the cause of various endometrial aberrations as a limiting factor for achieving pregnancy in PCOS women. Copyright © 2018 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Proteomic profiling of early degenerative retina of RCS rats

PubMed Central

Zhu, Zhi-Hong; Fu, Yan; Weng, Chuan-Huang; Zhao, Cong-Jian; Yin, Zheng-Qin

2017-01-01

AIM To identify the underlying cellular and molecular changes in retinitis pigmentosa (RP). METHODS Label-free quantification-based proteomics analysis, with its advantages of being more economic and consisting of simpler procedures, has been used with increasing frequency in modern biological research. Dystrophic RCS rats, the first laboratory animal model for the study of RP, possess a similar pathological course as human beings with the diseases. Thus, we employed a comparative proteomics analysis approach for in-depth proteome profiling of retinas from dystrophic RCS rats and non-dystrophic congenic controls through Linear Trap Quadrupole - orbitrap MS/MS, to identify the significant differentially expressed proteins (DEPs). Bioinformatics analyses, including Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation and upstream regulatory analysis, were then performed on these retina proteins. Finally, a Western blotting experiment was carried out to verify the difference in the abundance of transcript factor E2F1. RESULTS In this study, we identified a total of 2375 protein groups from the retinal protein samples of RCS rats and non-dystrophic congenic controls. Four hundred thirty-four significantly DEPs were selected by Student's t-test. Based on the results of the bioinformatics analysis, we identified mitochondrial dysfunction and transcription factor E2F1 as the key initiation factors in early retinal degenerative process. CONCLUSION We showed that the mitochondrial dysfunction and the transcription factor E2F1 substantially contribute to the disease etiology of RP. The results provide a new potential therapeutic approach for this retinal degenerative disease. PMID:28730077

Clinical proteomics-driven precision medicine for targeted cancer therapy: current overview and future perspectives.

PubMed

Zhou, Li; Wang, Kui; Li, Qifu; Nice, Edouard C; Zhang, Haiyuan; Huang, Canhua

2016-01-01

Cancer is a common disease that is a leading cause of death worldwide. Currently, early detection and novel therapeutic strategies are urgently needed for more effective management of cancer. Importantly, protein profiling using clinical proteomic strategies, with spectacular sensitivity and precision, offer excellent promise for the identification of potential biomarkers that would direct the development of targeted therapeutic anticancer drugs for precision medicine. In particular, clinical sample sources, including tumor tissues and body fluids (blood, feces, urine and saliva), have been widely investigated using modern high-throughput mass spectrometry-based proteomic approaches combined with bioinformatic analysis, to pursue the possibilities of precision medicine for targeted cancer therapy. Discussed in this review are the current advantages and limitations of clinical proteomics, the available strategies of clinical proteomics for the management of precision medicine, as well as the challenges and future perspectives of clinical proteomics-driven precision medicine for targeted cancer therapy.

Unraveling the proteomic profile of mice testis during the initiation of meiosis.

PubMed

Shao, Binbin; Guo, Yueshuai; Wang, Lei; Zhou, Quan; Gao, Tingting; Zheng, Bo; Zheng, Haoyu; Zhou, Tao; Zhou, Zuomin; Guo, Xuejiang; Huang, Xiaoyan; Sha, Jiahao

2015-04-29

In mice, once primordial germ cells (PGCs) are generated, they continue to proliferate and migrate to eventually reach the future gonads. They initiate sexual differentiation after their colonization of the gonads. During this process, retinoic acid (RA) induces meiosis in the female germ cells, which proceeds to the diplotene stage of meiotic prophase I, whereas the male germ cells initiate growth arrest. After birth, meiosis is initiated in mice spermatogonia by their conversion to preleptotene spermatocytes. There are evidences showing the roles of RA in the regulation of spermatogonial differentiation and meiosis initiation. However, it is still not well known on what responds to RA and how RA signaling engages meiosis. Thus, we constructed a proteomic profile of proteins associated with meiosis onset during testis development in mouse and identified 104 differentially expressed proteins (≥1.5 folds). Bioinformatic analysis showed proteins functioning in specific cell processes. The expression patterns of five selected proteins were verified via Western blot, of which we found that Tfrc gene was RA responsive, with a RA responsive element, and could be up regulated by RA in spermatogonial stem cell (SSC) line. Taken together, the results provide an important reference profile for further functional study of meiosis initiation. Spermatogenesis involves mitosis of spermatogonia, meiosis of spermatocytes and spermiogenesis, in which meiosis is a unique event to germ cells, and not in the somatic cells. Till now, the detailed molecular mechanisms of the transition from mitosis to meiosis are still not elucidated. With high-throughput proteomic technology, it is now possible to systemically identify proteins possibly involved. With TMT-6plex based quantification, we identified 104 proteins differentially between testes without meiosis (day 8.5) and those that were meiosis initiated (day 10.5). And a well-known protein essential for meiosis initiation, stra8, was identified to be differentially expressed in the study. And bioinformatic analysis and functional studies revealed several proteins regulated by retinoic acid, a chemical known to regulate the meiosis initiation. Thus, this quantitative proteomic approach can identify meiosis initiation regulating proteins, and further functional studies of these proteins will help elucidate the mechanisms of meiosis initiation. Copyright © 2015. Published by Elsevier B.V.

AgHalo: A Facile Fluorogenic Sensor to Detect Drug-Induced Proteome Stress.

PubMed

Liu, Yu; Fares, Matthew; Dunham, Noah P; Gao, Zi; Miao, Kun; Jiang, Xueyuan; Bollinger, Samuel S; Boal, Amie K; Zhang, Xin

2017-07-17

Drug-induced proteome stress that involves protein aggregation may cause adverse effects and undermine the safety profile of a drug. Safety of drugs is regularly evaluated using cytotoxicity assays that measure cell death. However, these assays provide limited insights into the presence of proteome stress in live cells. A fluorogenic protein sensor is reported to detect drug-induced proteome stress prior to cell death. An aggregation prone Halo-tag mutant (AgHalo) was evolved to sense proteome stress through its aggregation. Detection of such conformational changes was enabled by a fluorogenic ligand that fluoresces upon AgHalo forming soluble aggregates. Using 5 common anticancer drugs, we exemplified detection of differential proteome stress before any cell death was observed. Thus, this sensor can be used to evaluate drug safety in a regime that the current cytotoxicity assays cannot cover and be generally applied to detect proteome stress induced by other toxins. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Finding Biomass Degrading Enzymes Through an Activity-Correlated Quantitative Proteomics Platform (ACPP).

PubMed

Ma, Hongyan; Delafield, Daniel G; Wang, Zhe; You, Jianlan; Wu, Si

2017-04-01

The microbial secretome, known as a pool of biomass (i.e., plant-based materials) degrading enzymes, can be utilized to discover industrial enzyme candidates for biofuel production. Proteomics approaches have been applied to discover novel enzyme candidates through comparing protein expression profiles with enzyme activity of the whole secretome under different growth conditions. However, the activity measurement of each enzyme candidate is needed for confident "active" enzyme assignments, which remains to be elucidated. To address this challenge, we have developed an Activity-Correlated Quantitative Proteomics Platform (ACPP) that systematically correlates protein-level enzymatic activity patterns and protein elution profiles using a label-free quantitative proteomics approach. The ACPP optimized a high performance anion exchange separation for efficiently fractionating complex protein samples while preserving enzymatic activities. The detected enzymatic activity patterns in sequential fractions using microplate-based assays were cross-correlated with protein elution profiles using a customized pattern-matching algorithm with a correlation R-score. The ACPP has been successfully applied to the identification of two types of "active" biomass-degrading enzymes (i.e., starch hydrolysis enzymes and cellulose hydrolysis enzymes) from Aspergillus niger secretome in a multiplexed fashion. By determining protein elution profiles of 156 proteins in A. niger secretome, we confidently identified the 1,4-α-glucosidase as the major "active" starch hydrolysis enzyme (R = 0.96) and the endoglucanase as the major "active" cellulose hydrolysis enzyme (R = 0.97). The results demonstrated that the ACPP facilitated the discovery of bioactive enzymes from complex protein samples in a high-throughput, multiplexing, and untargeted fashion. Graphical Abstract ᅟ.

Finding Biomass Degrading Enzymes Through an Activity-Correlated Quantitative Proteomics Platform (ACPP)

NASA Astrophysics Data System (ADS)

Ma, Hongyan; Delafield, Daniel G.; Wang, Zhe; You, Jianlan; Wu, Si

2017-04-01

The microbial secretome, known as a pool of biomass (i.e., plant-based materials) degrading enzymes, can be utilized to discover industrial enzyme candidates for biofuel production. Proteomics approaches have been applied to discover novel enzyme candidates through comparing protein expression profiles with enzyme activity of the whole secretome under different growth conditions. However, the activity measurement of each enzyme candidate is needed for confident "active" enzyme assignments, which remains to be elucidated. To address this challenge, we have developed an Activity-Correlated Quantitative Proteomics Platform (ACPP) that systematically correlates protein-level enzymatic activity patterns and protein elution profiles using a label-free quantitative proteomics approach. The ACPP optimized a high performance anion exchange separation for efficiently fractionating complex protein samples while preserving enzymatic activities. The detected enzymatic activity patterns in sequential fractions using microplate-based assays were cross-correlated with protein elution profiles using a customized pattern-matching algorithm with a correlation R-score. The ACPP has been successfully applied to the identification of two types of "active" biomass-degrading enzymes (i.e., starch hydrolysis enzymes and cellulose hydrolysis enzymes) from Aspergillus niger secretome in a multiplexed fashion. By determining protein elution profiles of 156 proteins in A. niger secretome, we confidently identified the 1,4-α-glucosidase as the major "active" starch hydrolysis enzyme (R = 0.96) and the endoglucanase as the major "active" cellulose hydrolysis enzyme (R = 0.97). The results demonstrated that the ACPP facilitated the discovery of bioactive enzymes from complex protein samples in a high-throughput, multiplexing, and untargeted fashion.

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.

Proteomic and metabolomic characterization of streptozotocin-induced diabetic nephropathy in TIMP3-deficient mice.

PubMed

Rossi, Claudia; Marzano, Valeria; Consalvo, Ada; Zucchelli, Mirco; Levi Mortera, Stefano; Casagrande, Viviana; Mavilio, Maria; Sacchetta, Paolo; Federici, Massimo; Menghini, Rossella; Urbani, Andrea; Ciavardelli, Domenico

2018-02-01

The tissue inhibitor of metalloproteinase TIMP3 is a stromal protein that restrains the activity of both protease and receptor in the extracellular matrix and has been found to be down-regulated in diabetic nephropathy (DN), the leading cause of end-stage renal disease in developed countries. In order to gain deeper insights on the association of loss of TIMP3 and DN, we performed differential proteomic analysis of kidney and blood metabolic profiling of wild-type and Timp3-knockout mice before and after streptozotocin (STZ) treatment, widely used to induce insulin deficiency and hyperglycemia. Kidney proteomic data and blood metabolic profiles suggest significant alterations of peroxisomal and mitochondrial fatty acids β-oxidation in Timp3-knockout mice compared to wild-type mice under basal condition. These alterations were exacerbated in response to STZ treatment. Proteomic and metabolomic approaches showed that loss of TIMP3 alone or in combination with STZ treatment results in significant alterations of kidney lipid metabolism and peripheral acylcarnitine levels, supporting the idea that loss of TIMP3 may generate a phenotype more prone to DN.

Evaluating two-dimensional electrophoresis profiles of the protein phaseolin as markers of genetic differentiation and seed protein quality in common bean (Phaseolus vulgaris L.).

PubMed

López-Pedrouso, María; Bernal, Javier; Franco, Daniel; Zapata, Carlos

2014-07-23

High-resolution two-dimensional electrophoresis (2-DE) profiles of the protein phaseolin, the major seed storage protein of common bean, display great number of spots with differentially glycosylated and phosphorylated α- and β-type polypeptides. This work aims to test whether these complex profiles can be useful markers of genetic differentiation and seed protein quality in bean populations. The 2-DE phaseolin profile and the amino acid composition were examined in bean seeds from 18 domesticated and wild accessions belonging to the Mesoamerican and Andean gene pools. We found that proteomic distances based on 2-DE profiles were successful in identifying the accessions belonging to each gene pool and outliers distantly related. In addition, accessions identified as outliers from proteomic distances showed the highest levels of methionine content, an essential amino acid deficient in bean seeds. These findings suggest that 2-DE phaseolin profiles provide valuable information with potential of being used in common bean genetic improvement.

Robust Label-free, Quantitative Profiling of Circulating Plasma Microparticle (MP) Associated Proteins*

PubMed Central

Braga-Lagache, Sophie; Buchs, Natasha; Iacovache, Mircea-Ioan; Zuber, Benoît; Jackson, Christopher Benjamin

2016-01-01

Cells of the vascular system release spherical vesicles, called microparticles, in the size range of 0.1–1 μm induced by a variety of stress factors resulting in variable concentrations between health and disease. Furthermore, microparticles have intercellular communication/signaling properties and interfere with inflammation and coagulation pathways. Today's most used analytical technology for microparticle characterization, flow cytometry, is lacking sensitivity and specificity, which might have led to the publication of contradicting results in the past. We propose the use of nano-liquid chromatography two-stage mass spectrometry as a nonbiased tool for quantitative MP proteome analysis. For this, we developed an improved microparticle isolation protocol and quantified the microparticle protein composition of twelve healthy volunteers with a label-free, data-dependent and independent proteomics approach on a quadrupole orbitrap instrument. Using aliquots of 250 μl platelet-free plasma from one individual donor, we achieved excellent reproducibility with an interassay coefficient of variation of 2.7 ± 1.7% (mean ± 1 standard deviation) on individual peptide intensities across 27 acquisitions performed over a period of 3.5 months. We show that the microparticle proteome between twelve healthy volunteers were remarkably similar, and that it is clearly distinguishable from whole cell and platelet lysates. We propose the use of the proteome profile shown in this work as a quality criterion for microparticle purity in proteomics studies. Furthermore, one freeze thaw cycle damaged the microparticle integrity, articulated by a loss of cytoplasm proteins, encompassing a specific set of proteins involved in regulating dynamic structures of the cytoskeleton, and thrombin activation leading to MP clotting. On the other hand, plasma membrane protein composition was unaffected. Finally, we show that multiplexed data-independent acquisition can be used for relative quantification of target proteins using Skyline software. Mass spectrometry data are available via ProteomeXchange (identifier PXD003935) and panoramaweb.org (https://panoramaweb.org/labkey/N1OHMk.url). PMID:27738094

[The proteomic profiling of blood serum of children with gastroesophageal reflux disease].

PubMed

Korkotashvili, L V; Kolesov, S A; Jukova, E A; Vidmanova, T A; Kankova, N Yu; Bashurova, I A; Sidorova, A M; Kulakova, E V

2015-03-01

The mass-spectra of proteome of blood serum from healthy children and children with gastroesophageal reflux disease were received. The technology platform including direct proteome mass-spectrometer profiling after pre-fractional rectification using magnetic particles MB WCX was applied. The significant differences in mass-spectra were established manifesting in detection of more mass-spectrometer peaks and higher indicators of their intensity and area in group of healthy children. The study detected 39 particular peptides and low-molecular proteins predominantly intrinsic to healthy or ill children. It was established that two peptides with molecular mass 925 and 909 Da. are registered only in healthy patients and have no traces in group ofpatients with gastroesophageal reflux disease. The peptide 1564 Da is detected only in blood of children with gastroesophageal reflux disease and totally is absent in healthy children. The research data permitted to reveal specific patterns (signatures) of low-molecular proteins and peptides specific for blood serum of healthy children and patients with gastroesophageal reflux disease. The results testify the availability of singularities in metabolism of low-molecular proteins and can be used as a basis for development of minimally invasive mass-spectrometer system for its diagnostic.

Proteomic analysis associated with coronary artery dilatation caused by Kawasaki disease using serum exosomes.

PubMed

Zhang, Li; Wang, Wei; Bai, Jun; Xu, Yu-Fen; Li, Lai-Qing; Hua, Liang; Deng, Li; Jia, Hong-Ling

2016-05-01

The aim of this study was to investigate the serum exosome proteome profile of coronary artery dilatation (CAD) caused by Kawasaki disease (KD). Two-dimensional electrophoresis was implemented on proteins of serum exosomes obtained from children with CAD caused by KD and from healthy controls. Differentially expressed proteins were identified by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry analysis. We identified 38 differentially expressed proteins (13 up-regulated and 25 down-regulated) from serum exosomes of patients with CAD caused by KD compared with healthy controls. Expression levels of three differentially expressed proteins (leucine-rich alpha-2-glycoprotein, sex hormone-binding globulin, and serotransferrin) were validated using western blot analysis. Classification and protein-protein network analysis showed that they are associated with multiple functional groups involved in the acute inflammatory response, defense response, complement activation, humoral immune response, and response to wounding. The majority of the proteins are involved in the inflammation and coagulation cascades. These findings establish a comprehensive proteome profile of CAD caused by KD and increase our knowledge of scientific insight into its mechanisms. Copyright © 2016 Sociedade Portuguesa de Cardiologia. Published by Elsevier España. All rights reserved.

Proteomic analysis of liver tissue from rainbow trout (Oncorhynchus mykiss) under high rearing density after administration of dietary vitamin E and selenium nanoparticles.

PubMed

Naderi, Mahdi; Keyvanshokooh, Saeed; Salati, Amir Parviz; Ghaedi, Alireza

2017-06-01

The aim of the present study was to investigate the effects of dietary vitamin E (vit E) and selenium nanoparticles (nanoSe) on liver proteome profile of rainbow trout under high density condition. To correlate the proteome modifications with physiological aspects, growth, serum metabolites (cortisol, glucose, lactate, ALT, AST, and ALP), and liver antioxidant-related parameters (SOD, GPx, CAT, and MDA) were also examined. A total of 1275 fish (average weight of 42.6±2.3g) were stocked into 12 tanks at a density of 80kgm -3 . The fish were divided into four groups according to diet: control (basal diet), vit E (500mgkg - 1 vit E-supplemented diet), nanoSe (1mgkg -1 nanoSe-supplemented diet), and combination (500mgkg -1 vit E and 1mgkg -1 nanoSe-supplemented diet). After 60days, the best performance and health status of fish were observed in vit E and combination groups. Supplementation with nanoSe had no significant effects on growth performance. In addition, we compared liver proteome profiles of fish fed with a basal diet (control) and diets supplemented with vit E or nanoSe. Among the identified proteins, GRP78, ATPsyn-d, and HSP70 had an increased abundance in the vit E group, while HPPD and GAPDH showed a decreased abundance. In response to nanoSe supplementation, the expression of MDH, FAA, FBPA, TPI, GRHPR, GNMT, FDH, and Enol was increased. The proteomic data indicate that vit E or nanoSe supplementation can alter the expression of proteins involved in metabolic status of rainbow trout reared under high rearing density. Copyright © 2017 Elsevier Inc. All rights reserved.

Comparative and network-based proteomic analysis of low dose ethanol- and lipopolysaccharide-induced macrophages

PubMed Central

Kamal, Abu Hena M.; Fessler, Michael B.

2018-01-01

Macrophages are specialized phagocytes that play an essential role in inflammation, immunity, and tissue repair. Profiling the global proteomic response of macrophages to microbial molecules such as bacterial lipopolysaccharide is key to understanding fundamental mechanisms of inflammatory disease. Ethanol is a widely abused substance that has complex effects on inflammation. Reports have indicated that ethanol can activate or inhibit the lipopolysaccharide receptor, Toll-like Receptor 4, in different settings, with important consequences for liver and neurologic inflammation, but the underlying mechanisms are poorly understood. To profile the sequential effect of low dose ethanol and lipopolysaccharide on macrophages, a gel-free proteomic technique was applied to RAW 264.7 macrophages. Five hundred four differentially expressed proteins were identified and quantified with high confidence using ≥ 5 peptide spectral matches. Among these, 319 proteins were shared across all treatment conditions, and 69 proteins were exclusively identified in ethanol-treated or lipopolysaccharide-stimulated cells. The interactive impact of ethanol and lipopolysaccharide on the macrophage proteome was evaluated using bioinformatics tools, enabling identification of differentially responsive proteins, protein interaction networks, disease- and function-based networks, canonical pathways, and upstream regulators. Five candidate protein coding genes (PGM2, ISYNA1, PARP1, and PSAP) were further validated by qRT-PCR that mostly related to glucose metabolism and fatty acid synthesis pathways. Taken together, this study describes for the first time at a systems level the interaction between ethanol and lipopolysaccharide in the proteomic programming of macrophages, and offers new mechanistic insights into the biology that may underlie the impact of ethanol on infectious and inflammatory disease in humans. PMID:29481576

Multivariate proteomic profiling identifies novel accessory proteins of coated vesicles

PubMed Central

Antrobus, Robin; Hirst, Jennifer; Bhumbra, Gary S.; Kozik, Patrycja; Jackson, Lauren P.; Sahlender, Daniela A.

2012-01-01

Despite recent advances in mass spectrometry, proteomic characterization of transport vesicles remains challenging. Here, we describe a multivariate proteomics approach to analyzing clathrin-coated vesicles (CCVs) from HeLa cells. siRNA knockdown of coat components and different fractionation protocols were used to obtain modified coated vesicle-enriched fractions, which were compared by stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative mass spectrometry. 10 datasets were combined through principal component analysis into a “profiling” cluster analysis. Overall, 136 CCV-associated proteins were predicted, including 36 new proteins. The method identified >93% of established CCV coat proteins and assigned >91% correctly to intracellular or endocytic CCVs. Furthermore, the profiling analysis extends to less well characterized types of coated vesicles, and we identify and characterize the first AP-4 accessory protein, which we have named tepsin. Finally, our data explain how sequestration of TACC3 in cytosolic clathrin cages causes the severe mitotic defects observed in auxilin-depleted cells. The profiling approach can be adapted to address related cell and systems biological questions. PMID:22472443

Quantitative proteomic view on secreted, cell surface-associated, and cytoplasmic proteins of the methicillin-resistant human pathogen Staphylococcus aureus under iron-limited conditions.

PubMed

Hempel, Kristina; Herbst, Florian-Alexander; Moche, Martin; Hecker, Michael; Becher, Dörte

2011-04-01

Staphylococcus aureus is capable of colonizing and infecting humans by its arsenal of surface-exposed and secreted proteins. Iron-limited conditions in mammalian body fluids serve as a major environmental signal to bacteria to express virulence determinants. Here we present a comprehensive, gel-free, and GeLC-MS/MS-based quantitative proteome profiling of S. aureus under this infection-relevant situation. (14)N(15)N metabolic labeling and three complementing approaches were combined for relative quantitative analyses of surface-associated proteins. The surface-exposed and secreted proteome profiling approaches comprise trypsin shaving, biotinylation, and precipitation of the supernatant. By analysis of the outer subproteomic and cytoplasmic protein fraction, 1210 proteins could be identified including 221 surface-associated proteins. Thus, access was enabled to 70% of the predicted cell wall-associated proteins, 80% of the predicted sortase substrates, two/thirds of lipoproteins and more than 50% of secreted and cytoplasmic proteins. For iron-deficiency, 158 surface-associated proteins were quantified. Twenty-nine proteins were found in altered amounts showing particularly surface-exposed proteins strongly induced, such as the iron-regulated surface determinant proteins IsdA, IsdB, IsdC and IsdD as well as lipid-anchored iron compound-binding proteins. The work presents a crucial subject for understanding S. aureus pathophysiology by the use of methods that allow quantitative surface proteome profiling.

Plasma-based proteomics reveals immune response, complement and coagulation cascades pathway shifts in heat-stressed lactating dairy cows.

PubMed

Min, Li; Cheng, Jianbo; Zhao, Shengguo; Tian, He; Zhang, Yangdong; Li, Songli; Yang, Hongjian; Zheng, Nan; Wang, Jiaqi

2016-09-02

Heat stress (HS) has an enormous economic impact on the dairy industry. In recent years, many researchers have investigated changes in the gene expression and metabolomics profiles in dairy cows caused by HS. However, the proteomics profiles of heat-stressed dairy cows have not yet been completely elucidated. We compared plasma proteomics from HS-free and heat-stressed dairy cows using an iTRAQ labeling approach. After the depletion of high abundant proteins in the plasma, 1472 proteins were identified. Of these, 85 proteins were differentially abundant in cows exposed to HS relative to HS-free. Database searches combined with GO and KEGG pathway enrichment analyses revealed that many components of the complement and coagulation cascades were altered in heat-stressed cows compared with HS-free cows. Of these, many factors in the complement system (including complement components C1, C3, C5, C6, C7, C8, and C9, complement factor B, and factor H) were down-regulated by HS, while components of the coagulation system (including coagulation factors, vitamin K-dependent proteins, and fibrinogens) were up-regulated by HS. In conclusion, our results indicate that HS decreases plasma levels of complement system proteins, suggesting that immune function is impaired in dairy cows exposed to HS. Though many aspects of heat stress (HS) have been extensively researched, relatively little is known about the proteomics profile changes that occur during heat exposure. In this work, we employed a proteomics approach to investigate differential abundance of plasma proteins in HS-free and heat-stressed dairy cows. Database searches combined with GO and KEGG pathway enrichment analyses revealed that HS resulted in a decrease in complement components, suggesting that heat-stressed dairy cows have impaired immune function. In addition, through integrative analyses of proteomics and previous metabolomics, we showed enhanced glycolysis, lipid metabolic pathway shifts, and nitrogen repartitioning in dairy cows exposed to HS. Our findings expand our current knowledge on the effects of HS on plasma proteomics in dairy cows and offer a new perspective for future research. Copyright © 2016 Elsevier B.V. All rights reserved.

Large Scale Proteomic Data and Network-Based Systems Biology Approaches to Explore the Plant World.

PubMed

Di Silvestre, Dario; Bergamaschi, Andrea; Bellini, Edoardo; Mauri, PierLuigi

2018-06-03

The investigation of plant organisms by means of data-derived systems biology approaches based on network modeling is mainly characterized by genomic data, while the potential of proteomics is largely unexplored. This delay is mainly caused by the paucity of plant genomic/proteomic sequences and annotations which are fundamental to perform mass-spectrometry (MS) data interpretation. However, Next Generation Sequencing (NGS) techniques are contributing to filling this gap and an increasing number of studies are focusing on plant proteome profiling and protein-protein interactions (PPIs) identification. Interesting results were obtained by evaluating the topology of PPI networks in the context of organ-associated biological processes as well as plant-pathogen relationships. These examples foreshadow well the benefits that these approaches may provide to plant research. Thus, in addition to providing an overview of the main-omic technologies recently used on plant organisms, we will focus on studies that rely on concepts of module, hub and shortest path, and how they can contribute to the plant discovery processes. In this scenario, we will also consider gene co-expression networks, and some examples of integration with metabolomic data and genome-wide association studies (GWAS) to select candidate genes will be mentioned.

Daily chronomics of proteomic profile in aging and rotenone-induced Parkinson's disease model in male Wistar rat and its modulation by melatonin.

PubMed

Jagota, Anita; Mattam, Ushodaya

2017-08-01

Aging is associated with changes in several basic parameters of circadian timing system (CTS) in mammals leading to circadian dysfunction. We had reported earlier that upon aging and in rotenone induced Parkinson's disease (RIPD) rat model there were significant alterations in the core clock genes expression levels and daily pulses. To identify biomarkers of aging and PD chronomics of proteomic day-night profiles in suprachiasmatic nucleus (SCN), pineal and substantia nigra (SN) in 3 month (m), 12, 24 m and RIPD rat model were studied at two time points i.e. Zeitgeber Time (ZT)-6 (mid-day) and ZT-18 (mid-night). Proteome analysis was done by using two dimensional (2-D) electrophoresis and the spots showing robust day-night variations were identified by using MALDI TOF/TOF analysis. In 3 m rats the number of proteins showing day-night variations were relatively more than 12, 24 m and RIPD rat model in SCN and SN. But in pineal there was increase in number of protein spots showing day-night variations in 24 m. Mass spectroscopy of the protein spots showing robust day night variation in aging and RIPD rats were identified. As melatonin, a multitasking molecule, an endogenous synchronizer of rhythm, an antioxidant and an antiaging drug, declines with aging, the effects of melatonin administration on differential alterations in chronomics of 2-D protein profiles in aging and RIPD male Wistar rats were studied. We report here that the melatonin could be playing an important role in modulating the chronomics of 2-D protein profiles. Additionally, various proteins were identified for the first time in this study showing significant day night variation in SCN, pineal and SN may prove useful towards targeting novel treatments for circadian dysfunction, good health and longevity.

Urine Sample Preparation in 96-Well Filter Plates for Quantitative Clinical Proteomics

PubMed Central

2015-01-01

Urine is an important, noninvasively collected body fluid source for the diagnosis and prognosis of human diseases. Liquid chromatography mass spectrometry (LC-MS) based shotgun proteomics has evolved as a sensitive and informative technique to discover candidate disease biomarkers from urine specimens. Filter-aided sample preparation (FASP) generates peptide samples from protein mixtures of cell lysate or body fluid origin. Here, we describe a FASP method adapted to 96-well filter plates, named 96FASP. Soluble urine concentrates containing ∼10 μg of total protein were processed by 96FASP and LC-MS resulting in 700–900 protein identifications at a 1% false discovery rate (FDR). The experimental repeatability, as assessed by label-free quantification and Pearson correlation analysis for shared proteins among replicates, was high (R ≥ 0.97). Application to urinary pellet lysates which is of particular interest in the context of urinary tract infection analysis was also demonstrated. On average, 1700 proteins (±398) were identified in five experiments. In a pilot study using 96FASP for analysis of eight soluble urine samples, we demonstrated that protein profiles of technical replicates invariably clustered; the protein profiles for distinct urine donors were very different from each other. Robust, highly parallel methods to generate peptide mixtures from urine and other body fluids are critical to increase cost-effectiveness in clinical proteomics projects. This 96FASP method has potential to become a gold standard for high-throughput quantitative clinical proteomics. PMID:24797144

Ultra-sensitive high performance liquid chromatography-laser-induced fluorescence based proteomics for clinical applications.

PubMed

Patil, Ajeetkumar; Bhat, Sujatha; Pai, Keerthilatha M; Rai, Lavanya; Kartha, V B; Chidangil, Santhosh

2015-09-08

An ultra-sensitive high performance liquid chromatography-laser induced fluorescence (HPLC-LIF) based technique has been developed by our group at Manipal, for screening, early detection, and staging for various cancers, using protein profiling of clinical samples like, body fluids, cellular specimens, and biopsy-tissue. More than 300 protein profiles of different clinical samples (serum, saliva, cellular samples and tissue homogenates) from volunteers (normal, and different pre-malignant/malignant conditions) were recorded using this set-up. The protein profiles were analyzed using principal component analysis (PCA) to achieve objective detection and classification of malignant, premalignant and healthy conditions with high sensitivity and specificity. The HPLC-LIF protein profiling combined with PCA, as a routine method for screening, diagnosis, and staging of cervical cancer and oral cancer, is discussed in this paper. In recent years, proteomics techniques have advanced tremendously in life sciences and medical sciences for the detection and identification of proteins in body fluids, tissue homogenates and cellular samples to understand biochemical mechanisms leading to different diseases. Some of the methods include techniques like high performance liquid chromatography, 2D-gel electrophoresis, MALDI-TOF-MS, SELDI-TOF-MS, CE-MS and LC-MS techniques. We have developed an ultra-sensitive high performance liquid chromatography-laser induced fluorescence (HPLC-LIF) based technique, for screening, early detection, and staging for various cancers, using protein profiling of clinical samples like, body fluids, cellular specimens, and biopsy-tissue. More than 300 protein profiles of different clinical samples (serum, saliva, cellular samples and tissue homogenates) from healthy and volunteers with different malignant conditions were recorded by using this set-up. The protein profile data were analyzed using principal component analysis (PCA) for objective classification and detection of malignant, premalignant and healthy conditions. The method is extremely sensitive to detect proteins with limit of detection of the order of femto-moles. The HPLC-LIF combined with PCA as a potential proteomic method for the diagnosis of oral cancer and cervical cancer has been discussed in this paper. This article is part of a Special Issue entitled: Proteomics in India. Copyright © 2015 Elsevier B.V. All rights reserved.

Proteomic profiling of white muscle from freshwater catfish Rita rita.

PubMed

Mohanty, Bimal Prasanna; Mitra, Tandrima; Banerjee, Sudeshna; Bhattacharjee, Soma; Mahanty, Arabinda; Ganguly, Satabdi; Purohit, Gopal Krishna; Karunakaran, Dhanasekar; Mohanty, Sasmita

2015-06-01

Muscle tissues contribute 34-48 % of the total body mass in fish. Proteomic analysis enables better understanding of the skeletal muscle physiology and metabolism. A proteome map reflects the general fingerprinting of the fish species and has the potential to identify novel proteins which could serve as biomarkers for many aspects of aquaculture including fish physiology and growth, flesh quality, food safety and aquatic environmental monitoring. The freshwater catfish Rita rita of the family Bagridae inhabiting the tropical rivers and estuaries is an important food fish with high nutritive value and is also considered a species of choice in riverine pollution monitoring. Omics information that could enhance utility of this species in molecular research is meager. Therefore, in the present study, proteomic analysis of Rita rita muscle has been carried out and functional genomics data have been generated. A reference muscle proteome has been developed, and 23 protein spots, representing 18 proteins, have been identified by MALDI-TOF/TOF-MS and LC-MS/MS. Besides, transcript information on a battery of heat shock proteins (Hsps) has been generated. The functional genomics information generated could act as the baseline data for further molecular research on this species.

Proteomics: from hypothesis to quantitative assay on a single platform. Guidelines for developing MRM assays using ion trap mass spectrometers.

PubMed

Han, Bomie; Higgs, Richard E

2008-09-01

High-throughput HPLC-mass spectrometry (HPLC-MS) is routinely used to profile biological samples for potential protein markers of disease, drug efficacy and toxicity. The discovery technology has advanced to the point where translating hypotheses from proteomic profiling studies into clinical use is the bottleneck to realizing the full potential of these approaches. The first step in this translation is the development and analytical validation of a higher throughput assay with improved sensitivity and selectivity relative to typical profiling assays. Multiple reaction monitoring (MRM) assays are an attractive approach for this stage of biomarker development given their improved sensitivity and specificity, the speed at which the assays can be developed and the quantitative nature of the assay. While the profiling assays are performed with ion trap mass spectrometers, MRM assays are traditionally developed in quadrupole-based mass spectrometers. Development of MRM assays from the same instrument used in the profiling analysis enables a seamless and rapid transition from hypothesis generation to validation. This report provides guidelines for rapidly developing an MRM assay using the same mass spectrometry platform used for profiling experiments (typically ion traps) and reviews methodological and analytical validation considerations. The analytical validation guidelines presented are drawn from existing practices on immunological assays and are applicable to any mass spectrometry platform technology.

Proteomic Analysis of Serum from Patients with Major Depressive Disorder to Compare Their Depressive and Remission Statuses

PubMed Central

Lee, Jiyeong; Joo, Eun-Jeong; Lim, Hee-Joung; Park, Jong-Moon; Lee, Kyu Young; Park, Arum; Seok, AeEun

2015-01-01

Objective Currently, there are a few biological markers to aid in the diagnosis and treatment of depression. However, it is not sufficient for diagnosis. We attempted to identify differentially expressed proteins during depressive moods as putative diagnostic biomarkers by using quantitative proteomic analysis of serum. Methods Blood samples were collected twice from five patients with major depressive disorder (MDD) at depressive status before treatment and at remission status during treatment. Samples were individually analyzed by liquid chromatography-tandem mass spectrometry for protein profiling. Differentially expressed proteins were analyzed by label-free quantification. Enzyme-linked immunosorbent assay (ELISA) results and receiver-operating characteristic (ROC) curves were used to validate the differentially expressed proteins. For validation, 8 patients with MDD including 3 additional patients and 8 matched normal controls were analyzed. Results The quantitative proteomic studies identified 10 proteins that were consistently upregulated or downregulated in 5 MDD patients. ELISA yielded results consistent with the proteomic analysis for 3 proteins. Expression levels were significantly different between normal controls and MDD patients. The 3 proteins were ceruloplasmin, inter-alpha-trypsin inhibitor heavy chain H4 and complement component 1qC, which were upregulated during the depressive status. The depressive status could be distinguished from the euthymic status from the ROC curves for these proteins, and this discrimination was enhanced when all 3 proteins were analyzed together. Conclusion This is the first proteomic study in MDD patients to compare intra-individual differences dependent on mood. This technique could be a useful approach to identify MDD biomarkers, but requires additional proteomic studies for validation. PMID:25866527

Cell- and Tissue-based Proteome Profiling and Bioimaging with the Probes Derived from a Potent AXL Kinase Inhibitor.

PubMed

Li, Zhengqiu; Zheng, Binbin; Guo, Haijun; Xu, Jiaqian; Ma, Nan; Ni, Yun; Li, Lin; Hao, Piliang; Ding, Ke

2018-06-25

AXL has been defined as a novel target for cancer therapeutics. However, only a few potent and selective inhibitors targeting AXL are available to date. Our group has developed a lead compound, 9im, capable of excellent inhibition against AXL. With the aim of understanding its cellular and tissue mechanism of actions and direct subsequent structure optimization, a study on competitive affinity-based proteome profiling and bioimaging was carried out. A series of unknown cellular and tissue targets, including RYK, PCK, ATP1A3, EIF4A, Ptprn and Cox5b were discovered. In addition, trans-cyclooctene (TCO) and acedan-containing probes were developed to image the binding between 9im and its target proteins inside live cells and tumor tissues. These probes would be useful tools in the detection of expression and activity of AXL. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Proteomic profiling of an undefined microbial consortium cultured in fermented dairy manure: Methods development.

PubMed

Hanson, Andrea J; Paszczynski, Andrzej J; Coats, Erik R

2016-03-01

The production of polyhydroxyalkanoates (PHA; bioplastics) from waste or surplus feedstocks using mixed microbial consortia (MMC) and aerobic dynamic feeding (ADF) is a growing field within mixed culture biotechnology. This study aimed to optimize a 2DE workflow to investigate the proteome dynamics of an MMC synthesizing PHA from fermented dairy manure. To mitigate the challenges posed to effective 2DE by this complex sample matrix, the bacterial biomass was purified using Accudenz gradient centrifugation (AGC) before protein extraction. The optimized 2DE method yielded high-quality gels suitable for quantitative comparative analysis and subsequent protein identification by LC-MS/MS. The optimized 2DE method could be adapted to other proteomic investigations involving MMC in complex organic or environmental matrices. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

PROTEOMICS IN ECOTOXICOLOGY: PROTEIN EXPRESSION PROFILING TO SCREEN CHEMICALS FOR ENDOCRINE ACTIVITY

EPA Science Inventory

Abstract for poster.

Current endocrine testing methods are animal intensive and lack the throughput necessary to screen large numbers of environmental chemicals for adverse effects. In this study, Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry...

Proteomic profiling of mitochondria: what does it tell us about the ageing brain?

PubMed

Ingram, Thomas; Chakrabarti, Lisa

2016-12-13

Mitochondrial dysfunction is evident in numerous neurodegenerative and age-related disorders. It has also been linked to cellular ageing, however our current understanding of the mitochondrial changes that occur are unclear. Functional studies have made some progress reporting reduced respiration, dynamic structural modifications and loss of membrane potential, though there are conflicts within these findings. Proteomic analyses, together with functional studies, are required in order to profile the mitochondrial changes that occur with age and can contribute to unravelling the complexity of the ageing phenotype. The emergence of improved protein separation techniques, combined with mass spectrometry analyses has allowed the identification of age and cell-type specific mitochondrial changes in energy metabolism, antioxidants, fusion and fission machinery, chaperones, membrane proteins and biosynthesis pathways. Here, we identify and review recent data from the analyses of mitochondria from rodent brains. It is expected that knowledge gained from understanding age-related mitochondrial changes of the brain should lead to improved biomarkers of normal ageing and also age-related disease progression.

Proteomic Profile of Unstable Atheroma Plaque: Increased Neutrophil Defensin 1, Clusterin, and Apolipoprotein E Levels in Carotid Secretome.

PubMed

Aragonès, Gemma; Auguet, Teresa; Guiu-Jurado, Esther; Berlanga, Alba; Curriu, Marta; Martinez, Salomé; Alibalic, Ajla; Aguilar, Carmen; Hernández, Esteban; Camara, María-Luisa; Canela, Núria; Herrero, Pol; Ruyra, Xavier; Martín-Paredero, Vicente; Richart, Cristóbal

2016-03-04

Because of the clinical significance of carotid atherosclerosis, the search for novel biomarkers has become a priority. The aim of the present study was to compare the protein secretion profile of the carotid atherosclerotic plaque (CAP, n = 12) and nonatherosclerotic mammary artery (MA, n = 10) secretomes. We used a nontargeted proteomic approach that incorporated tandem immunoaffinity depletion, iTRAQ labeling, and nanoflow liquid chromatography coupled to high-resolution mass spectrometry. In total, 162 proteins were quantified, of which 25 showed statistically significant differences in secretome levels between carotid atherosclerotic plaque and nondiseased mammary artery. We found increased levels of neutrophil defensin 1, apolipoprotein E, clusterin, and zinc-alpha-2-glycoprotein in CAP secretomes. Results were validated by ELISA assays. Also, differentially secreted proteins are involved in pathways such as focal adhesion and leukocyte transendothelial migration. In conclusion, this study provides a subset of identified proteins that are differently expressed in secretomes of clinical significance.

Proteomic profiling reveals dopaminergic regulation of progenitor cell functions of goldfish radial glial cells in vitro.

PubMed

Xing, Lei; Martyniuk, Christopher J; Esau, Crystal; Da Fonte, Dillon F; Trudeau, Vance L

2016-07-20

Radial glial cells (RGCs) are stem-like cells found in the developing and adult central nervous system. They function as both a scaffold to guide neuron migration and as progenitor cells that support neurogenesis. Our previous study revealed a close anatomical relationship between dopamine neurons and RGCs in the telencephalon of female goldfish. In this study, label-free proteomics was used to identify the proteins in a primary RGC culture and to determine the proteome response to the selective dopamine D1 receptor agonist SKF 38393 (10μM), in order to better understand dopaminergic regulation of RGCs. A total of 689 unique proteins were identified in the RGCs and these were classified into biological and pathological pathways. Proteins such as nucleolin (6.9-fold) and ependymin related protein 1 (4.9-fold) were increased in abundance while proteins triosephosphate isomerase (10-fold) and phosphoglycerate dehydrogenase (5-fold) were decreased in abundance. Pathway analysis revealed that proteins that consistently changed in abundance across biological replicates were related to small molecules such as ATP, lipids and steroids, hormones, glucose, cyclic AMP and Ca(2+). Sub-network enrichment analysis suggested that estrogen receptor signaling, among other transcription factors, is regulated by D1 receptor activation. This suggests that these signaling pathways are correlated to dopaminergic regulation of radial glial cell functions. Most proteins down-regulated by SKF 38393 were involved in cell cycle/proliferation, growth, death, and survival, which suggests that dopamine inhibits the progenitor-related processes of radial glial cells. Examples of differently expressed proteins including triosephosphate isomerase, nucleolin, phosphoglycerate dehydrogenase and capping protein (actin filament) muscle Z-line beta were validated by qPCR and western blot, which were consistent with MS/MS data in the direction of change. This is the first study to characterize the RGC proteome on a large scale in a vertebrate species. These data provide novel insight into glial protein networks that are associated with neuroendocrine function and neurogenesis in the teleost brain. While the role of radial glial cells in organizing brain structure and neurogenesis has been well studied, protein profiling experiments in this unique cell type has not been conducted. This study is the first to profile the proteome of goldfish radial glial cells in culture and to study the regulation of progenitor functions of radial glial cells by the neurotransmitter dopamine. This study provides the foundation for molecular network analysis in fish radial glial cells, and identifies cellular processes and signaling pathways in these cells with roles in neurogenesis and neuroendocrine function. Lastly, this study begins to characterize signatures and biomarkers for specific neuroendocrine and neurogenesis disruptors. Copyright © 2016 Elsevier B.V. All rights reserved.

N-terminal Proteomics Assisted Profiling of the Unexplored Translation Initiation Landscape in Arabidopsis thaliana *

PubMed Central

Ndah, Elvis; Jonckheere, Veronique

2017-01-01

Proteogenomics is an emerging research field yet lacking a uniform method of analysis. Proteogenomic studies in which N-terminal proteomics and ribosome profiling are combined, suggest that a high number of protein start sites are currently missing in genome annotations. We constructed a proteogenomic pipeline specific for the analysis of N-terminal proteomics data, with the aim of discovering novel translational start sites outside annotated protein coding regions. In summary, unidentified MS/MS spectra were matched to a specific N-terminal peptide library encompassing protein N termini encoded in the Arabidopsis thaliana genome. After a stringent false discovery rate filtering, 117 protein N termini compliant with N-terminal methionine excision specificity and indicative of translation initiation were found. These include N-terminal protein extensions and translation from transposable elements and pseudogenes. Gene prediction provided supporting protein-coding models for approximately half of the protein N termini. Besides the prediction of functional domains (partially) contained within the newly predicted ORFs, further supporting evidence of translation was found in the recently released Araport11 genome re-annotation of Arabidopsis and computational translations of sequences stored in public repositories. Most interestingly, complementary evidence by ribosome profiling was found for 23 protein N termini. Finally, by analyzing protein N-terminal peptides, an in silico analysis demonstrates the applicability of our N-terminal proteogenomics strategy in revealing protein-coding potential in species with well- and poorly-annotated genomes. PMID:28432195

N-terminal Proteomics Assisted Profiling of the Unexplored Translation Initiation Landscape in Arabidopsis thaliana.

PubMed

Willems, Patrick; Ndah, Elvis; Jonckheere, Veronique; Stael, Simon; Sticker, Adriaan; Martens, Lennart; Van Breusegem, Frank; Gevaert, Kris; Van Damme, Petra

2017-06-01

Proteogenomics is an emerging research field yet lacking a uniform method of analysis. Proteogenomic studies in which N-terminal proteomics and ribosome profiling are combined, suggest that a high number of protein start sites are currently missing in genome annotations. We constructed a proteogenomic pipeline specific for the analysis of N-terminal proteomics data, with the aim of discovering novel translational start sites outside annotated protein coding regions. In summary, unidentified MS/MS spectra were matched to a specific N-terminal peptide library encompassing protein N termini encoded in the Arabidopsis thaliana genome. After a stringent false discovery rate filtering, 117 protein N termini compliant with N-terminal methionine excision specificity and indicative of translation initiation were found. These include N-terminal protein extensions and translation from transposable elements and pseudogenes. Gene prediction provided supporting protein-coding models for approximately half of the protein N termini. Besides the prediction of functional domains (partially) contained within the newly predicted ORFs, further supporting evidence of translation was found in the recently released Araport11 genome re-annotation of Arabidopsis and computational translations of sequences stored in public repositories. Most interestingly, complementary evidence by ribosome profiling was found for 23 protein N termini. Finally, by analyzing protein N-terminal peptides, an in silico analysis demonstrates the applicability of our N-terminal proteogenomics strategy in revealing protein-coding potential in species with well- and poorly-annotated genomes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

LC-MS Proteomics Analysis of the Insulin/IGF-1 Deficient Caenorhabditis elegans daf-2(e1370) Mutant Reveals Extensive Restructuring of Intermediary Metabolism

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

Depuydt, Geert G.; Xie, Fang; Petyuk, Vladislav A.

2014-02-20

The insulin/IGF-1 receptor is a major known determinant of dauer formation, stress resistance, longevity and metabolism in C. elegans. In the past, whole-genome transcript profiling was used extensively to study differential gene expression in response to reduced insulin/IGF-1 signaling, including expression levels of metabolism-associated genes. Taking advantage of the recent developments in quantitative liquid chromatography mass-spectrometry (LC-MS) based proteomics, we profiled the proteomic changes that occur in response to activation of the DAF-16 transcription factor in the germline-less glp-4(bn2); daf-2(e1370) receptor mutant. Strikingly, the daf-2 profile suggests extensive reorganization of intermediary metabolism, characterized by the up-regulation of many core intermediarymore » metabolic pathways. These include, glycolysis/gluconeogenesis, glycogenesis, pentose phosphate cycle, citric acid cycle, glyoxylate shunt, fatty acid β-oxidation, one-carbon metabolism, propionate and tyrosine catabolism, and complex I, II, III and V of the electron transport chain. Interestingly, we found simultaneous activation of reciprocally regulated metabolic pathways, which is indicative for spatio-temporal coordination of energy metabolism and/or extensive post-translational regulation of these enzymes. This restructuring of daf-2 metabolism is reminiscent to that of hypometabolic dauers, allowing the efficient and economical utilization of internal nutrient reserves, possibly also shunting metabolites through alternative energy-generating pathways, in order to sustain longevity.« less

LC–MS Proteomics Analysis of the Insulin/IGF-1-Deficient Caenorhabditis elegans daf-2(e1370) Mutant Reveals Extensive Restructuring of Intermediary Metabolism

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

Depuydt, Geert; Xie, Fang; Petyuk, Vladislav A.

2014-04-04

The insulin/IGF-1 receptor is a major known determinant of dauer formation, stress resistance, longevity, and metabolism in Caenorhabditis elegans. In the past, whole-genome transcript profiling was used extensively to study differential gene expression in response to reduced insulin/IGF-1 signaling, including the expression levels of metabolism-associated genes. Taking advantage of the recent developments in quantitative liquid chromatography mass spectrometry (LC–MS)-based proteomics, we profiled the proteomic changes that occur in response to activation of the DAF-16 transcription factor in the germline-less glp-4(bn2);daf-2(e1370) receptor mutant. Strikingly, the daf-2 profile suggests extensive reorganization of intermediary metabolism, characterized by the upregulation of many core intermediarymore » metabolic pathways. These include glycolysis/gluconeogenesis, glycogenesis, pentose phosphate cycle, citric acid cycle, glyoxylate shunt, fatty acid β-oxidation, one-carbon metabolism, propionate and tyrosine catabolism, and complexes I, II, III, and V of the electron transport chain. Interestingly, we found simultaneous activation of reciprocally regulated metabolic pathways, which is indicative of spatiotemporal coordination of energy metabolism and/or extensive post-translational regulation of these enzymes. Finally, this restructuring of daf-2 metabolism is reminiscent to that of hypometabolic dauers, allowing the efficient and economical utilization of internal nutrient reserves and possibly also shunting metabolites through alternative energy-generating pathways to sustain longevity.« less

Salt stress induces changes in the proteomic profile of micropropagated sugarcane shoots

PubMed Central

Reis, Ricardo S.; Heringer, Angelo S.; Rangel, Patricia L.; Santa-Catarina, Claudete; Grativol, Clícia; Veiga, Carlos F. M.; Souza-Filho, Gonçalo A.

2017-01-01

Salt stress is one of the most common stresses in agricultural regions worldwide. In particular, sugarcane is affected by salt stress conditions, and no sugarcane cultivar presently show high productivity accompanied by a tolerance to salt stress. Proteomic analysis allows elucidation of the important pathways involved in responses to various abiotic stresses at the biochemical and molecular levels. Thus, this study aimed to analyse the proteomic effects of salt stress in micropropagated shoots of two sugarcane cultivars (CB38-22 and RB855536) using a label-free proteomic approach. The mass spectrometry proteomics data are available via ProteomeXchange with identifier PXD006075. The RB855536 cultivar is more tolerant to salt stress than CB38-22. A quantitative label-free shotgun proteomic analysis identified 1172 non-redundant proteins, and 1160 of these were observed in both cultivars in the presence or absence of NaCl. Compared with CB38-22, the RB855536 cultivar showed a greater abundance of proteins involved in non-enzymatic antioxidant mechanisms, ion transport, and photosynthesis. Some proteins, such as calcium-dependent protein kinase, photosystem I, phospholipase D, and glyceraldehyde-3-phosphate dehydrogenase, were more abundant in the RB855536 cultivar under salt stress. Our results provide new insights into the response of sugarcane to salt stress, and the changes in the abundance of these proteins might be important for the acquisition of ionic and osmotic homeostasis during exposure to salt stress. PMID:28419154

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.

Proteomic Analysis of Whole Human Saliva Detects Enhanced Expression of Interleukin-1 Receptor Antagonist, Thioredoxin and Lipocalin-1 in Cigarette Smokers Compared to Non-Smokers

PubMed Central

Jessie, Kala; Pang, Wei Wei; Haji, Zubaidah; Rahim, Abdul; Hashim, Onn Haji

2010-01-01

A gel-based proteomics approach was used to screen for proteins of differential abundance between the saliva of smokers and those who had never smoked. Subjecting precipitated proteins from whole human saliva of healthy non-smokers to two-dimensional electrophoresis (2-DE) generated typical profiles comprising more than 50 proteins. While 35 of the proteins were previously established by other researchers, an additional 22 proteins were detected in the 2-DE saliva protein profiles generated in the present study. When the 2-DE profiles were compared to those obtained from subjects considered to be heavy cigarette smokers, three saliva proteins, including interleukin-1 receptor antagonist, thioredoxin and lipocalin-1, showed significant enhanced expression. The distribution patterns of lipocalin-1 isoforms were also different between cigarette smokers and non-smokers. The three saliva proteins have good potential to be used as biomarkers for the adverse effects of smoking and the risk for inflammatory and chronic diseases that are associated with it. PMID:21151451

Global iTRAQ-based proteomic profiling of Toxoplasma gondii oocysts during sporulation.

PubMed

Zhou, Chun-Xue; Zhu, Xing-Quan; Elsheikha, Hany M; He, Shuai; Li, Qian; Zhou, Dong-Hui; Suo, Xun

2016-10-04

Toxoplasma gondii is a medically and economically important protozoan parasite. However, the molecular mechanisms of its sporulation remain largely unknown. Here, we applied iTRAQ coupled with 2D LC-MS/MS proteomic analysis to investigate the proteomic expression profile of T. gondii oocysts during sporulation. Of the 2095 non-redundant proteins identified, 587 were identified as differentially expressed proteins (DEPs). Based on Gene Ontology enrichment and KEGG pathway analyses the majority of these DEPs were found related to the metabolism of amino acids, carbon and energy. Protein interaction network analysis generated by STRING identified ATP-citrate lyase (ACL), GMP synthase, IMP dehydrogenase (IMPDH), poly (ADP-ribose) glycohydrolase (PARG), and bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) as the top five hubs. We also identified 25 parasite virulence factors that were expressed at relatively high levels in sporulated oocysts compared to non-sporulated oocysts, which might contribute to the infectivity of mature oocysts. Considering the importance of oocysts in the dissemination of toxoplasmosis these findings may help in the search of protein targets with a key role in infectiousness and ecological success of oocysts, creating new opportunities for the development of better means for disease prevention. The development of new preventative interventions against T. gondii infection relies on an improved understanding of the proteome and chemical pathways of this parasite. To identify proteins required for the development of environmentally resistant and infective T. gondii oocysts, we compared the proteome of non-sporulated (immature) oocysts with the proteome of sporulated (mature, infective) oocysts. iTRAQ 2D-LC-MS/MS analysis revealed proteomic changes that distinguish non-sporulated from sporulated oocysts. Many of the differentially expressed proteins were involved in metabolic pathways and 25 virulence factors were identified upregulated in the sporulated oocysts. This work provides the first quantitative characterization of the proteomic variations that occur in T. gondii oocyst stage during sporulation. Copyright © 2016. Published by Elsevier B.V.

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.

Combined analysis of transcriptome and proteome data as a tool for the identification of candidate biomarkers in renal cell carcinoma

PubMed Central

Seliger, Barbara; Dressler, Sven P.; Wang, Ena; Kellner, Roland; Recktenwald, Christian V.; Lottspeich, Friedrich; Marincola, Francesco M.; Baumgärtner, Maja; Atkins, Derek; Lichtenfels, Rudolf

2012-01-01

Results obtained from expression profilings of renal cell carcinoma using different “ome”-based approaches and comprehensive data analysis demonstrated that proteome-based technologies and cDNA microarray analyses complement each other during the discovery phase for disease-related candidate biomarkers. The integration of the respective data revealed the uniqueness and complementarities of the different technologies. While comparative cDNA microarray analyses though restricted to upregulated targets largely revealed genes involved in controlling gene/protein expression (19%) and signal transduction processes (13%), proteomics/PROTEOMEX-defined candidate biomarkers include enzymes of the cellular metabolism (36%), transport proteins (12%) and cell motility/structural molecules (10%). Candidate biomarkers defined by proteomics and PROTEOMEX are frequently shared, whereas the sharing rate between cDNA microarray and proteome-based profilings is limited. Putative candidate biomarkers provide insights into their cellular (dys)function and their diagnostic/prognostic value but still warrant further validation in larger patient numbers. Based on the fact that merely 3 candidate biomarkers were shared by all applied technologies, namely annexin A4, tubulin alpha-1A chain and ubiquitin carboxyl-terminal hydrolase L1 the analysis at a single hierarchical level of biological regulation seems to provide only limited results thus emphasizing the importance and benefit of performing rather combinatorial screenings which can complement the standard clinical predictors. PMID:19235166

Advancing the global proteome survey platform by using an oriented single chain antibody fragment immobilization approach.

PubMed

Säll, Anna; Persson, Helena; Ohlin, Mats; Borrebaeck, Carl A K; Wingren, Christer

2016-09-25

Increasing the understanding of a proteome and how its protein composition is affected by for example different diseases, such as cancer, has the potential to improve strategies for early diagnosis and therapeutics. The Global Proteome Survey or GPS is a method that combines mass spectrometry and affinity enrichment with the use of antibodies. The technology enables profiling of complex proteomes in a species independent manner. The sensitivity of GPS, and other methods relying on affinity enrichment, is largely affected by the activity of the exploited affinity reagent. We here present an improvement of the GPS platform by utilizing an antibody immobilization approach which ensures a controlled immobilization process of the antibody to the magnetic bead support. More specifically, we make use of an antibody format that enables site-directed biotinylation and use this in combination with streptavidin coated magnetic beads. The performance of the expanded GPS platform was evaluated by profiling yeast proteome samples. We demonstrate that the oriented antibody immobilization strategy increases the ability of the GPS platform and results in larger fraction of functional antibodies. Additionally, we show that this new antibody format enabled in-solution capture, i.e. immobilization of the antibodies after sample incubation. A workflow has been established that permit the use of an oriented immobilization strategy for the GPS platform. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

The rat red blood cell proteome is altered by priming with 2-butoxyethanol

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

Palkar, Prajakta S.; Kakhniashvili, David G.; Goodman, Steven R.

2008-08-01

Administration of a low priming dose of 2-butoxyethanol (BE, 500 mg/kg, p.o.) 7 days prior to a larger LD{sub 90} dose (1500 mg BE/kg, p.o.) offers protection against the lethal dose-induced hemolysis and death in female Sprague Dawley rats because of prompt and efficient replacement of red blood cells (RBCs) with new resilient RBCs. The objective of the present work was to analyze the altered proteome of RBCs upon priming with BE in order to identify the potential anti-hemolytic survival proteins induced in the primed rat RBCs (P-RBCs) as opposed to vehicle-treated RBCs (V-RBCs). The RBCs from the two groupsmore » were fractionated into membrane and cytosolic fractions. The cytosolic fractions were further fractionated for proteomic analysis into 3 fractions. The fractions were labeled with Cy3 and Cy5 fluorescent dyes and subjected to 2-dimensional differential gel electrophoresis (DIGE) to analyze the protein profiles. Seven membrane and 8 cytosolic proteins were found to be significantly increased ({>=} 2.5 fold) in P-RBCs as compared to V-RBCs. The identified proteins can be classified into antioxidant, membrane skeleton, protein turnover, lipid raft, and energy metabolism components. Increased levels of the proteins from antioxidant and membrane skeleton groups were confirmed by Western blot analysis. The study provides the first report on protein profiling of rat RBCs as well as on alteration of the proteome upon exposure to a priming dose of hemotoxicant. Further studies are needed to prove the protective role of the identified proteins and will initiate the field of survival/protective/anti-hemolytic proteins in RBCs.« less

Integration of Transcriptome, Proteome and Metabolism Data Reveals the Alkaloids Biosynthesis in Macleaya cordata and Macleaya microcarpa

PubMed Central

Liu, Fuqing; Huang, Peng; Zhu, Pengcheng; Chen, Jinjun; Shi, Mingming; Guo, Fang; Cheng, Pi; Zeng, Jing; Liao, Yifang; Gong, Jing; Zhang, Hong-Mei; Wang, Depeng; Guo, An-Yuan; Xiong, Xingyao

2013-01-01

Background The Macleaya spp., including Macleaya cordata and Macleaya microcarpa, are traditional anti-virus, inflammation eliminating, and insecticide herb medicines for their isoquinoline alkaloids. They are also known as the basis of the popular natural animal food addictive in Europe. However, few studies especially at genomics level were conducted on them. Hence, we performed the Macleaya spp. transcriptome and integrated it with iTRAQ proteome analysis in order to identify potential genes involved in alkaloids biosynthesis. Methodology and Principal Findings We elaborately designed the transcriptome, proteome and metabolism profiling for 10 samples of both species to explore their alkaloids biosynthesis. From the transcriptome data, we obtained 69367 and 78255 unigenes for M. cordata and M. microcarpa, in which about two thirds of them were similar to sequences in public databases. By metabolism profiling, reverse patterns for alkaloids sanguinarine, chelerythrine, protopine, and allocryptopine were observed in different organs of two species. We characterized the expressions of enzymes in alkaloid biosynthesis pathways. We also identified more than 1000 proteins from iTRAQ proteome data. Our results strongly suggest that the root maybe the organ for major alkaloids biosynthesis of Macleaya spp. Except for biosynthesis, the alkaloids storage and transport were also important for their accumulation. The ultrastructure of laticifers by SEM helps us to prove the alkaloids maybe accumulated in the mature roots. Conclusions/Significance To our knowledge this is the first study to elucidate the genetic makeup of Macleaya spp. This work provides clues to the identification of the potential modulate genes involved in alkaloids biosynthesis in Macleaya spp., and sheds light on researches for non-model medicinal plants by integrating different high-throughput technologies. PMID:23326424

Proteomic profiling of the brain of mice with experimental cerebral malaria.

PubMed

Moussa, Ehab; Huang, Honglei; Ahras, Malika; Lall, Amar; Thezenas, Marie L; Fischer, Roman; Kessler, Benedikt M; Pain, Arnab; Billker, Oliver; Casals-Pascual, Climent

2018-05-30

Cerebral malaria (CM) is a severe neurological complication of malaria infection in both adults and children. In pursuit of effective treatment of CM, clinical studies, postmortem analysis and animal models have been employed to understand the pathology and identify effective interventions. In this study, a shotgun proteomics analysis was conducted to profile the proteomic signature of the brain tissue of mice with experimental cerebral malaria (ECM) in order to further understand the underlying pathology. To identify CM-associated response, proteomic signatures of the brains of C57/Bl6N mice infected with P. berghei ANKA that developed neurological syndrome were compared to those of mice infected with P. berghei NK65 that developed equally high parasite burdens without neurological signs, and to those of non-infected mice. The results show that the CM-associated response in mice that developed neurological signs comprise mainly acute-phase reaction and coagulation cascade activation, and indicate the leakage of plasma proteins into the brain parenchyma. Cerebral malaria (CM) remains a major cause of death in children. The majority of these deaths occur in sub-Saharan Africa. Even with adequate access to treatment, mortality remains high and neurological sequelae can be found in up to 20% of survivors. No adjuvant treatment to date has been shown to reduce mortality and the pathophysiology of CM is largely unknown. Experimental cerebral malaria (ECM) is a well-established model that may contribute to identify and test druggable targets. In this study we have identified the disruption of the blood-brain barrier following inflammatory and vascular injury as a mechanism of disease. In this study we report a number of proteins that could be validated as potential biomarkers of ECM. Further studies, will be required to validate the clinical relevance of these biomarkers in human CM. Copyright © 2017 Elsevier B.V. All rights reserved.

A Comprehensive Proteomics Analysis Reveals a Secretory Path- and Status-Dependent Signature of Exosomes Released from Tumor-Associated Macrophages.

PubMed

Zhu, Yinghui; Chen, Xianwei; Pan, Qingfei; Wang, Yang; Su, Siyuan; Jiang, Cuicui; Li, Yang; Xu, Ningzhi; Wu, Lin; Lou, Xiaomin; Liu, Siqi

2015-10-02

Exosomes are 30-120 nm-sized membrane vesicles of endocytic origin that are released into the extracellular environment and play roles in cell-cell communication. Tumor-associated macrophages (TAMs) are important constituents of the tumor microenvironment; thus, it is critical to study the features and complex biological functions of TAM-derived exosomes. Here, we constructed a TAM cell model from a mouse macrophage cell line, Ana-1, and performed comparative proteomics on exosomes, exosome-free media, and cells between TAMs and Ana-1. Proteomic analysis between exosome and exosome-free fractions indicated that the functions of exosome dominant proteins were mainly enriched in RNA processing and proteolysis. TAM status dramatically affected the abundances of 20S proteasome subunits and ribosomal proteins in their exosomes. The 20S proteasome activity assay strongly indicated that TAM exosomes possessed higher proteolytic activity. In addition, Ana-1- and TAM-derived exosomes have different RNA profiles, which may result from differential RNA processing proteins. Taken together, our comprehensive proteomics study provides novel views for understanding the complicated roles of macrophage-derived exosomes in the tumor microenvironment.

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.

Cassava root membrane proteome reveals activities during storage root maturation.

PubMed

Naconsie, Maliwan; Lertpanyasampatha, Manassawe; Viboonjun, Unchera; Netrphan, Supatcharee; Kuwano, Masayoshi; Ogasawara, Naotake; Narangajavana, Jarunya

2016-01-01

Cassava (Manihot esculenta Crantz) is one of the most important crops of Thailand. Its storage roots are used as food, feed, starch production, and be the important source for biofuel and biodegradable plastic production. Despite the importance of cassava storage roots, little is known about the mechanisms involved in their formation. This present study has focused on comparison of the expression profiles of cassava root proteome at various developmental stages using two-dimensional gel electrophoresis and LC-MS/MS. Based on an anatomical study using Toluidine Blue, the secondary growth was confirmed to be essential during the development of cassava storage root. To investigate biochemical processes occurring during storage root maturation, soluble and membrane proteins were isolated from storage roots harvested from 3-, 6-, 9-, and 12-month-old cassava plants. The proteins with differential expression pattern were analysed and identified to be associated with 8 functional groups: protein folding and degradation, energy, metabolism, secondary metabolism, stress response, transport facilitation, cytoskeleton, and unclassified function. The expression profiling of membrane proteins revealed the proteins involved in protein folding and degradation, energy, and cell structure were highly expressed during early stages of development. Integration of these data along with the information available in genome and transcriptome databases is critical to expand knowledge obtained solely from the field of proteomics. Possible role of identified proteins were discussed in relation with the activities during storage root maturation in cassava.

Medullospheres from DAOY, UW228 and ONS-76 cells: increased stem cell population and proteomic modifications.

PubMed

Zanini, Cristina; Ercole, Elisabetta; Mandili, Giorgia; Salaroli, Roberta; Poli, Alice; Renna, Cristiano; Papa, Valentina; Cenacchi, Giovanna; Forni, Marco

2013-01-01

Medulloblastoma (MB) is an aggressive pediatric tumor of the Central Nervous System (CNS) usually treated according to a refined risk stratification. The study of cancer stem cells (CSC) in MB is a promising approach aimed at finding new treatment strategies. The CSC compartment was studied in three characterized MB cell lines (DAOY, UW228 and ONS-76) grown in standard adhesion as well as being grown as spheres, which enables expansion of the CSC population. MB cell lines, grown in adherence and as spheres, were subjected to morphologic analysis at the light and electron microscopic level, as well as cytofluorimetric determinations. Medullospheres (MBS) were shown to express increasingly immature features, along with the stem cells markers: CD133, Nestin and β-catenin. Proteomic analysis highlighted the differences between MB cell lines, demonstrating a unique protein profile for each cell line, and minor differences when grown as spheres. In MBS, MALDI-TOF also identified some proteins, that have been linked to tumor progression and resistance, such as Nucleophosmin (NPM). In addition, immunocytochemistry detected Sox-2 as a stemness marker of MBS, as well as confirming high NPM expression. Culture conditioning based on low attachment flasks and specialized medium may provide new data on the staminal compartment of CNS tumors, although a proteomic profile of CSC is still elusive for MB.

Medullospheres from DAOY, UW228 and ONS-76 Cells: Increased Stem Cell Population and Proteomic Modifications

PubMed Central

Zanini, Cristina; Ercole, Elisabetta; Mandili, Giorgia; Salaroli, Roberta; Poli, Alice; Renna, Cristiano; Papa, Valentina; Cenacchi, Giovanna; Forni, Marco

2013-01-01

Background Medulloblastoma (MB) is an aggressive pediatric tumor of the Central Nervous System (CNS) usually treated according to a refined risk stratification. The study of cancer stem cells (CSC) in MB is a promising approach aimed at finding new treatment strategies. Methodology/Principal Findings The CSC compartment was studied in three characterized MB cell lines (DAOY, UW228 and ONS-76) grown in standard adhesion as well as being grown as spheres, which enables expansion of the CSC population. MB cell lines, grown in adherence and as spheres, were subjected to morphologic analysis at the light and electron microscopic level, as well as cytofluorimetric determinations. Medullospheres (MBS) were shown to express increasingly immature features, along with the stem cells markers: CD133, Nestin and β-catenin. Proteomic analysis highlighted the differences between MB cell lines, demonstrating a unique protein profile for each cell line, and minor differences when grown as spheres. In MBS, MALDI-TOF also identified some proteins, that have been linked to tumor progression and resistance, such as Nucleophosmin (NPM). In addition, immunocytochemistry detected Sox-2 as a stemness marker of MBS, as well as confirming high NPM expression. Conclusions/Significance Culture conditioning based on low attachment flasks and specialized medium may provide new data on the staminal compartment of CNS tumors, although a proteomic profile of CSC is still elusive for MB. PMID:23717474

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

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.

Comparative quantitative proteomics analysis of the ABA response of roots of drought-sensitive and drought-tolerant wheat varieties identifies proteomic signatures of drought adaptability.

PubMed

Alvarez, Sophie; Roy Choudhury, Swarup; Pandey, Sona

2014-03-07

Wheat is one of the most highly cultivated cereals in the world. Like other cultivated crops, wheat production is significantly affected by abiotic stresses such as drought. Multiple wheat varieties suitable for different geographical regions of the world have been developed that are adapted to different environmental conditions; however, the molecular basis of such adaptations remains unknown in most cases. We have compared the quantitative proteomics profile of the roots of two different wheat varieties, Nesser (drought-tolerant) and Opata (drought-sensitive), in the absence and presence of abscisic acid (ABA, as a proxy for drought). A labeling LC-based quantitative proteomics approach using iTRAQ was applied to elucidate the changes in protein abundance levels. Quantitative differences in protein levels were analyzed for the evaluation of inherent differences between the two varieties as well as the overall and variety-specific effect of ABA on the root proteome. This study reveals the most elaborate ABA-responsive root proteome identified to date in wheat. A large number of proteins exhibited inherently different expression levels between Nesser and Opata. Additionally, significantly higher numbers of proteins were ABA-responsive in Nesser roots compared with Opata roots. Furthermore, several proteins showed variety-specific regulation by ABA, suggesting their role in drought adaptation.

Protein expression profiles of human lymph and plasma mapped by 2D-DIGE and 1D SDS–PAGE coupled with nanoLC–ESI–MS/MS bottom-up proteomics

PubMed Central

Clement, Cristina C.; Aphkhazava, David; Nieves, Edward; Callaway, Myrasol; Olszewski, Waldemar; Rotzschke, Olaf; Santambrogio, Laura

2013-01-01

In this study a proteomic approach was used to define the protein content of matched samples of afferent prenodal lymph and plasma derived from healthy volunteers. The analysis was performed using two analytical methodologies coupled with nanoliquid chromatography-tandem mass spectrometry: one-dimensional gel electrophoresis (1DEF nanoLC Orbitrap–ESI–MS/MS), and two-dimensional fluorescence difference-in-gel electrophoresis (2D-DIGE nanoLC–ESI–MS/MS). The 253 significantly identified proteins (p<0.05), obtained from the tandem mass spectrometry data, were further analyzed with pathway analysis (IPA) to define the functional signature of prenodal lymph and matched plasma. The 1DEF coupled with nanoLC–MS–MS revealed that the common proteome between the two biological fluids (144 out of 253 proteins) was dominated by complement activation and blood coagulation components, transporters and protease inhibitors. The enriched proteome of human lymph (72 proteins) consisted of products derived from the extracellular matrix, apoptosis and cellular catabolism. In contrast, the enriched proteome of human plasma (37 proteins) consisted of soluble molecules of the coagulation system and cell–cell signaling factors. The functional networks associated with both common and source-distinctive proteomes highlight the principal biological activity of these immunologically relevant body fluids. PMID:23202415

Quantitative proteomic profiling for clarification of the crucial roles of lysosomes in microbial infections.

PubMed

Xu, Benhong; Gao, Yanpan; Zhan, Shaohua; Ge, Wei

2017-07-01

Lysosomes play vital roles in both innate and adaptive immunity. It is widely accepted that lysosomes do not function exclusively as a digestive organelle. It is also involved in the process of immune cells against pathogens. However, the changes in the lysosomal proteome caused by infection with various microbes are still largely unknown, and our understanding of the proteome of the purified lysosome is another obstacle that needs to be resolved. Here, we performed a proteomic study on lysosomes enriched from THP1 cells after infection with Listeria monocytogenes (L.m), Herpes Simplex Virus 1 (HSV-1) and Vesicular Stomatitis Virus (VSV). In combination with the gene ontology (GO) analysis, we identified 284 lysosomal-related proteins from a total of 4560 proteins. We also constructed the protein-protein interaction networks for the differentially expressed proteins and revealed the core lysosomal proteins, including SRC in the L. m treated group, SRC, GLB1, HEXA and HEXB in the HSV-1 treated group and GLB1, CTSA, CTSB, HEXA and HEXB in the VSV treated group, which are involved in responding to diverse microbial infections. This study not only reveals variable lysosome responses depending on the bacterial or virus infection, but also provides the evidence based on which we propose a novel approach to proteome research for investigation of the function of the enriched organelles. Copyright © 2017 Elsevier Ltd. All rights reserved.

Comparative shotgun proteomic analysis of Clostridium acetobutylicum from butanol fermentation using glucose and xylose

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

Sivagnanam, Kumaran; Raghavan, Vijaya G. S.; Shah, Manesh B

2011-01-01

Background: Butanol is a second generation biofuel produced by Clostridium acetobutylicum through acetonebutanol- ethanol (ABE) fermentation process. Shotgun proteomics provides a direct approach to study the whole proteome of an organism in depth. This paper focuses on shotgun proteomic profiling of C. acetobutylicum from ABE fermentation using glucose and xylose to understand the functional mechanisms of C. acetobutylicum proteins involved in butanol production. Results: We identified 894 different proteins in C. acetobutylicum from ABE fermentation process by two dimensional - liquid chromatography - tandem mass spectrometry (2D-LC-MS/MS) method. This includes 717 proteins from glucose and 826 proteins from the xylosemore » substrate. A total of 649 proteins were found to be common and 22 significantly differentially expressed proteins were identified between glucose and xylose substrates. Conclusion: Our results demonstrate that flagellar proteins are highly up-regulated with glucose compared to xylose substrate during ABE fermentation. Chemotactic activity was also found to be lost with the xylose substrate due to the absence of CheW and CheV proteins. This is the first report on the shotgun proteomic analysis of C. acetobutylicum ATCC 824 in ABE fermentation between glucose and xylose substrate from a single time data point and the number of proteins identified here is more than any other study performed on this organism up to this report.« less

Proteome profiling in the aorta and kidney of type 1 diabetic rats

PubMed Central

Zhu, Rui; Jaffa, Miran A.; Zhao, Jingfu; Mirzaei, Parvin; Ahmed, Adnan; Kobeissy, Firas; Ziyadeh, Fuad N.; Mechref, Yehia

2017-01-01

Diabetes is associated with a number of metabolic and cardiovascular risk factors that contribute to a high rate of microvascular and macrovascular complications. The risk factors and mechanisms that contribute to the development of micro- and macrovascular disease in diabetes are not fully explained. In this study, we employed mass spectrometric analysis using tandem LC-MS/MS to generate a proteomic profile of protein abundance and post-translational modifications (PTM) in the aorta and kidney of diabetic rats. In addition, systems biology analyses were employed to identify key protein markers that can provide insights into molecular pathways and processes that are differentially regulated in the aorta and kidney of type 1 diabetic rats. Our results indicated that 188 (111 downregulated and 77 upregulated) proteins were significantly identified in the aorta of diabetic rats compared to normal controls. A total of 223 (109 downregulated and 114 upregulated) proteins were significantly identified in the kidney of diabetic rats compared to normal controls. When the protein profiles from the kidney and aorta of diabetic and control rats were analyzed by principal component analysis, a distinct separation of the groups was observed. In addition, diabetes resulted in a significant increase in PTM (oxidation, phosphorylation, and acetylation) of proteins in the kidney and aorta and this effect was partially reversed by insulin treatment. Ingenuity pathway analysis performed on the list of differentially expressed proteins depicted mitochondrial dysfunction, oxidative phosphorylation and acute phase response signaling to be among the altered canonical pathways by diabetes in both tissues. The findings of the present study provide a global proteomics view of markers that highlight the mechanisms and putative processes that modulate renal and vascular injury in diabetes. PMID:29121074

Proteome profiling in the aorta and kidney of type 1 diabetic rats.

PubMed

Al Hariri, Moustafa; Elmedawar, Mohamad; Zhu, Rui; Jaffa, Miran A; Zhao, Jingfu; Mirzaei, Parvin; Ahmed, Adnan; Kobeissy, Firas; Ziyadeh, Fuad N; Mechref, Yehia; Jaffa, Ayad A

2017-01-01

Diabetes is associated with a number of metabolic and cardiovascular risk factors that contribute to a high rate of microvascular and macrovascular complications. The risk factors and mechanisms that contribute to the development of micro- and macrovascular disease in diabetes are not fully explained. In this study, we employed mass spectrometric analysis using tandem LC-MS/MS to generate a proteomic profile of protein abundance and post-translational modifications (PTM) in the aorta and kidney of diabetic rats. In addition, systems biology analyses were employed to identify key protein markers that can provide insights into molecular pathways and processes that are differentially regulated in the aorta and kidney of type 1 diabetic rats. Our results indicated that 188 (111 downregulated and 77 upregulated) proteins were significantly identified in the aorta of diabetic rats compared to normal controls. A total of 223 (109 downregulated and 114 upregulated) proteins were significantly identified in the kidney of diabetic rats compared to normal controls. When the protein profiles from the kidney and aorta of diabetic and control rats were analyzed by principal component analysis, a distinct separation of the groups was observed. In addition, diabetes resulted in a significant increase in PTM (oxidation, phosphorylation, and acetylation) of proteins in the kidney and aorta and this effect was partially reversed by insulin treatment. Ingenuity pathway analysis performed on the list of differentially expressed proteins depicted mitochondrial dysfunction, oxidative phosphorylation and acute phase response signaling to be among the altered canonical pathways by diabetes in both tissues. The findings of the present study provide a global proteomics view of markers that highlight the mechanisms and putative processes that modulate renal and vascular injury in diabetes.

Circulating proteins as predictors of incident heart failure in the elderly.

PubMed

Stenemo, Markus; Nowak, Christoph; Byberg, Liisa; Sundström, Johan; Giedraitis, Vilmantas; Lind, Lars; Ingelsson, Erik; Fall, Tove; Ärnlöv, Johan

2018-01-01

To identify novel risk markers for incident heart failure using proteomic profiling of 80 proteins previously associated with cardiovascular pathology. Proteomic profiling (proximity extension assay) was performed in two community-based prospective cohorts of elderly individuals without heart failure at baseline: the Prospective Investigation of the Vasculature in Uppsala Seniors [PIVUS, n = 901, median age 70.2 (interquartile range 70.0-70.3) years, 80 events]; and the Uppsala Longitudinal Study of Adult Men [ULSAM, n = 685, median age 77.8 (interquartile range 76.9-78.1) years, 90 events]. Twenty-nine proteins were associated with incident heart failure in the discovery cohort PIVUS after adjustment for age and sex, and correction for multiple testing. Eighteen associations replicated in ULSAM. In pooled analysis of both cohorts, higher levels of nine proteins were associated with incident heart failure after adjustment for established risk factors: growth differentiation factor 15 (GDF-15), T-cell immunoglobulin and mucin domain 1 (TIM-1), tumour necrosis factor-related apoptosis-inducing ligand receptor 2 (TRAIL-R2), spondin-1 (SPON1), matrix metalloproteinase-12 (MMP-12), follistatin (FS), urokinase-type plasminogen activator surface receptor (U-PAR), osteoprotegerin (OPG), and suppression of tumorigenicity 2 (ST2). Of these, GDF-15, U-PAR, MMP-12, TRAIL-R2, SPON1 and FS were associated with worsened echocardiographic left ventricular systolic function at baseline, while only TIM-1 was positively associated with worsened diastolic function (P < 0.02 for all). Proteomic profiling identified several novel associations between proteins involved in apoptosis, inflammation, matrix remodelling, and fibrinolysis with incident heart failure in elderly individuals. Our results encourage additional studies investigating the underlying mechanisms and the clinical utility of our findings. © 2017 The Authors. European Journal of Heart Failure © 2017 European Society of Cardiology.

Proteomic profile of culture filtrate from the Brazilian vaccine strain Mycobacterium bovis BCG Moreau compared to M. bovis BCG Pasteur

PubMed Central

2011-01-01

Background Bacille Calmette-Guerin (BCG) is currently the only available vaccine against tuberculosis (TB) and comprises a heterogeneous family of sub-strains with genotypic and phenotypic differences. The World Health Organization (WHO) affirms that the characterization of BCG sub-strains, both on genomic and proteomic levels, is crucial for a better comprehension of the vaccine. In addition, these studies can contribute in the development of a more efficient vaccine against TB. Here, we combine two-dimensional electrophoresis (2DE) and mass spectrometry to analyse the proteomic profile of culture filtrate proteins (CFPs) from M. bovis BCG Moreau, the Brazilian vaccine strain, comparing it to that of BCG Pasteur. CFPs are considered of great importance given their dominant immunogenicity and role in pathogenesis, being available for interaction with host cells since early infection. Results The 2DE proteomic map of M. bovis BCG Moreau CFPs in the pH range 3 - 8 allowed the identification of 158 spots corresponding to 101 different proteins, identified by MS/MS. Comparison to BCG Pasteur highlights the great similarity between these BCG strains. However, quantitative analysis shows a higher expression of immunogenic proteins such as Rv1860 (BCG1896, Apa), Rv1926c (BCG1965c, Mpb63) and Rv1886c (BCG1923c, Ag85B) in BCG Moreau when compared to BCG Pasteur, while some heat shock proteins, such as Rv0440 (BCG0479, GroEL2) and Rv0350 (BCG0389, DnaK), show the opposite pattern. Conclusions Here we report the detailed 2DE profile of CFPs from M. bovis BCG Moreau and its comparison to BCG Pasteur, identifying differences that may provide relevant information on vaccine efficacy. These findings contribute to the detailed characterization of the Brazilian vaccine strain against TB, revealing aspects that may lead to a better understanding of the factors leading to BCG's variable protective efficacy against TB. PMID:21507239

Proteomic profile of culture filtrate from the Brazilian vaccine strain Mycobacterium bovis BCG Moreau compared to M. bovis BCG Pasteur.

PubMed

Berrêdo-Pinho, Marcia; Kalume, Dario E; Correa, Paloma R; Gomes, Leonardo H F; Pereira, Melissa P; da Silva, Renata F; Castello-Branco, Luiz R R; Degrave, Wim M; Mendonça-Lima, Leila

2011-04-20

Bacille Calmette-Guerin (BCG) is currently the only available vaccine against tuberculosis (TB) and comprises a heterogeneous family of sub-strains with genotypic and phenotypic differences. The World Health Organization (WHO) affirms that the characterization of BCG sub-strains, both on genomic and proteomic levels, is crucial for a better comprehension of the vaccine. In addition, these studies can contribute in the development of a more efficient vaccine against TB. Here, we combine two-dimensional electrophoresis (2DE) and mass spectrometry to analyse the proteomic profile of culture filtrate proteins (CFPs) from M. bovis BCG Moreau, the Brazilian vaccine strain, comparing it to that of BCG Pasteur. CFPs are considered of great importance given their dominant immunogenicity and role in pathogenesis, being available for interaction with host cells since early infection. The 2DE proteomic map of M. bovis BCG Moreau CFPs in the pH range 3-8 allowed the identification of 158 spots corresponding to 101 different proteins, identified by MS/MS. Comparison to BCG Pasteur highlights the great similarity between these BCG strains. However, quantitative analysis shows a higher expression of immunogenic proteins such as Rv1860 (BCG1896, Apa), Rv1926c (BCG1965c, Mpb63) and Rv1886c (BCG1923c, Ag85B) in BCG Moreau when compared to BCG Pasteur, while some heat shock proteins, such as Rv0440 (BCG0479, GroEL2) and Rv0350 (BCG0389, DnaK), show the opposite pattern. Here we report the detailed 2DE profile of CFPs from M. bovis BCG Moreau and its comparison to BCG Pasteur, identifying differences that may provide relevant information on vaccine efficacy. These findings contribute to the detailed characterization of the Brazilian vaccine strain against TB, revealing aspects that may lead to a better understanding of the factors leading to BCG's variable protective efficacy against TB.

Large-scale inference of protein tissue origin in gram-positive sepsis plasma using quantitative targeted proteomics

PubMed Central

Malmström, Erik; Kilsgård, Ola; Hauri, Simon; Smeds, Emanuel; Herwald, Heiko; Malmström, Lars; Malmström, Johan

2016-01-01

The plasma proteome is highly dynamic and variable, composed of proteins derived from surrounding tissues and cells. To investigate the complex processes that control the composition of the plasma proteome, we developed a mass spectrometry-based proteomics strategy to infer the origin of proteins detected in murine plasma. The strategy relies on the construction of a comprehensive protein tissue atlas from cells and highly vascularized organs using shotgun mass spectrometry. The protein tissue atlas was transformed to a spectral library for highly reproducible quantification of tissue-specific proteins directly in plasma using SWATH-like data-independent mass spectrometry analysis. We show that the method can determine drastic changes of tissue-specific protein profiles in blood plasma from mouse animal models with sepsis. The strategy can be extended to several other species advancing our understanding of the complex processes that contribute to the plasma proteome dynamics. PMID:26732734

Diagnosis of major depressive disorder by combining multimodal information from heart rate dynamics and serum proteomics using machine-learning algorithm.

PubMed

Kim, Eun Young; Lee, Min Young; Kim, Se Hyun; Ha, Kyooseob; Kim, Kwang Pyo; Ahn, Yong Min

2017-06-02

Major depressive disorder (MDD) is a systemic and multifactorial disorder that involves abnormalities in multiple biochemical pathways and the autonomic nervous system. This study applied a machine-learning method to classify MDD and control groups by incorporating data from serum proteomic analysis and heart rate variability (HRV) analysis for the identification of novel peripheral biomarkers. The study subjects consisted of 25 drug-free female MDD patients and 25 age- and sex-matched healthy controls. First, quantitative serum proteome profiles were analyzed by liquid chromatography-tandem mass spectrometry using pooled serum samples from 10 patients and 10 controls. Next, candidate proteins were quantified with multiple reaction monitoring (MRM) in 50 subjects. We also analyzed 22 linear and nonlinear HRV parameters in 50 subjects. Finally, we identified a combined biomarker panel consisting of proteins and HRV indexes using a support vector machine with recursive feature elimination. A separation between MDD and control groups was achieved using five parameters (apolipoprotein B, group-specific component, ceruloplasmin, RMSSD, and SampEn) at 80.1% classification accuracy. A combination of HRV and proteomic data achieved better classification accuracy. A high classification accuracy can be achieved by combining multimodal information from heart rate dynamics and serum proteomics in MDD. Our approach can be helpful for accurate clinical diagnosis of MDD. Further studies using larger, independent cohorts are needed to verify the role of these candidate biomarkers for MDD diagnosis. Copyright © 2017 Elsevier Inc. All rights reserved.

Isobaric Tags for Relative and Absolute Quantification (iTRAQ)-Based Untargeted Quantitative Proteomic Approach To Identify Change of the Plasma Proteins by Salbutamol Abuse in Beef Cattle.

PubMed

Zhang, Kai; Tang, Chaohua; Liang, Xiaowei; Zhao, Qingyu; Zhang, Junmin

2018-01-10

Salbutamol, a selective β 2 -agonist, endangers the safety of animal products as a result of illegal use in food animals. In this study, an iTRAQ-based untargeted quantitative proteomic approach was applied to screen potential protein biomarkers in plasma of cattle before and after treatment with salbutamol for 21 days. A total of 62 plasma proteins were significantly affected by salbutamol treatment, which can be used as potential biomarkers to screen for the illegal use of salbutamol in beef cattle. Enzyme-linked immunosorbent assay measurements of five selected proteins demonstrated the reliability of iTRAQ-based proteomics in screening of candidate biomarkers among the plasma proteins. The plasma samples collected before and after salbutamol treatment were well-separated by principal component analysis (PCA) using the differentially expressed proteins. These results suggested that an iTRAQ-based untargeted quantitative proteomic strategy combined with PCA pattern recognition methods can discriminate differences in plasma protein profiles collected before and after salbutamol treatment.

Evaluation of hepatitis B viral replication and proteomic analysis of HepG2.2.15 cell line after knockdown of HBx.

PubMed

Xie, Hai-Yang; Cheng, Jun; Xing, Chun-Yang; Wang, Jin-Jin; Su, Rong; Wei, Xu-Yong; Zhou, Lin; Zheng, Shu-Sen

2011-06-01

Hepatitis B virus (HBV) is one of the major pathogens of human liver disease. Studies have shown that HBV X protein (HBx) plays an important role in promoting viral gene expression and replication. In this study we performed a global proteomic profiling to identify the downstream functional proteins of HBx, thereby detecting the mechanisms of action of HBx on virion replication. HBx in the HepG2.2.15 cell line was knocked down by the transfection of small interfering RNA (siRNA). The replication level of HBV was evaluated by microparticle enzyme immunoassay analysis of HBsAg and HBeAg in the culture supernatant, and real-time quantitative PCR analysis of HBV DNA. Two-dimensional electrophoresis combined with MALDI-TOF/TOF was performed to analyze the changes in protein expression profile after treatment with HBx siRNA. Knockdown of HBx disturbed HBV replication in vitro. HBx target siRNA significantly inhibited the expression of HBsAg, HBeAg and the replication of HBV DNA. Twelve significantly changed proteins (7 upregulated and 5 downregulated) were successfully identified by MALDI-TOF/TOF using proteomics differential expression analysis after the knockdown of HBx. Among these identified proteins, HSP70 was validated by Western blotting. The results of the study indicated the positive effect of HBx on HBV replication, and a group of downstream target proteins of HBx may be responsible for this effect.

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

Proteomic analysis of heat treated bitter gourd (Momordica charantia L. var. Hong Kong Green) using 2D-DIGE.

PubMed

Ng, Zhi Xiang; Chua, Kek Heng; Kuppusamy, Umah Rani

2014-04-01

This study aimed to investigate the changes in the proteome of bitter gourd prior to and after subjecting to boiling and microwaving. A comparative analysis of the proteome profiles of raw and thermally treated bitter gourds was performed using 2D-DIGE. The protein content and number of protein spots in raw sample was higher when compared to the cooked samples. Qualitative analysis revealed that 103 (boiled sample) and 110 (microwaved sample) protein spots were up regulated whereas 120 (boiled sample) and 107 (microwaved sample) protein spots were down regulated. Ten protein spots with the highest significant fold change in the cooked samples were involved in carbohydrate/energy metabolisms and stress responses. Small heat shock proteins, superoxide dismutase, quinone oxidoreductase, UDP-glucose pyrophosphorylase and phosphoglycerate kinase play a role in heat-stress-mediated protection of bitter gourd. This study suggests that appropriate heat treatment (cooking methods) can lead to induction of selected proteins in bitter gourd. Copyright © 2013 Elsevier Ltd. All rights reserved.

Proteomic features of delayed ocular symptoms caused by exposure to sulfur mustard: As studied by protein profiling of corneal epithelium.

PubMed

Pajoohesh, Maryam; Naderi, Mostafa; Naderi-Manesh, Hossein

2017-11-01

Exposure to mustard gas can lead to variations in the proteome of corneal epithelium cells and after a latency period produces delayed symptoms in the eyes of chemical victims. Hence, a comparative proteome analysis was conducted between the corneal epithelial cells of chemical victims from Iran-Iraq war (1980-1988) and healthy donors. To this end, corneal epithelium samples from victims and healthy individuals were collected, and the proteome of these samples were prepared for two-dimensional electrophoresis and the analysis of spots by statistical software. Selected spots were further analyzed by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry. Twenty four proteins were identified of which eighteen proteins showed downregulation while six proteins were upregulated in the victims in comparison to the normal individuals. Also, six protein spots were confirmed by western-blot analysis. In conclusion, all the twenty-four identified proteins are involved in pathways which their up- or down-regulation leads to the accumulation of undesired substrates, cell death and apoptosis. Bioinformatics' tools indicated that these identified proteins were involved in various metabolic processes, DNA damage response, immune response and etc. The present study provides a suitable platform for further clinical studies. Copyright © 2017 Elsevier B.V. All rights reserved.

Differential proteomics profiling of the ova between healthy and Rice stripe virus-infected female insects of Laodelphax striatellus.

PubMed

Liu, Beibei; Qin, Faliang; Liu, Wenwen; Wang, Xifeng

2016-06-09

Rice stripe virus-infected females of the small brown planthopper (SBPH, Laodelphax striatellus) usually lay fewer eggs with a longer hatch period, low hatchability, malformation and retarded or defective development compared with healthy females. To explore the molecular mechanism of those phenomena, we analyzed the differential proteomics profiling of the ova between viruliferous and healthy female insects using an isobaric tag for relative and absolute quantitation (iTRAQ) approach. We obtained 147 differentially accumulated proteins: 98 (66.7%) proteins increased, but 49 (33.3%) decreased in the ova of the viruliferous females. RT-qPCR was used to verify the 12 differential expressed proteins from iTRAQ, finding that trends in the transcriptional change for the 12 genes were consistent with those at the proteomic level. Differentially expressed proteins that were associated with meiosis (serine/threonine-protein phosphatase 2B and cyclin B3) and mitosis (cyclin B3 and dynein heavy chain) in viruliferous ova may contribute to low hatchability and defective or retarded development. Alterations in the abundance of proteins involved in the respiratory chain and nutrition metabolism may affect embryonic development. Our study begins to explain macroscopical developmental phenomena and explore the mechanisms by which Rice stripe virus impacts the development of SBPH.

Differential proteomics profiling of the ova between healthy and Rice stripe virus-infected female insects of Laodelphax striatellus

PubMed Central

Liu, Beibei; Qin, Faliang; Liu, Wenwen; Wang, Xifeng

2016-01-01

Rice stripe virus-infected females of the small brown planthopper (SBPH, Laodelphax striatellus) usually lay fewer eggs with a longer hatch period, low hatchability, malformation and retarded or defective development compared with healthy females. To explore the molecular mechanism of those phenomena, we analyzed the differential proteomics profiling of the ova between viruliferous and healthy female insects using an isobaric tag for relative and absolute quantitation (iTRAQ) approach. We obtained 147 differentially accumulated proteins: 98 (66.7%) proteins increased, but 49 (33.3%) decreased in the ova of the viruliferous females. RT-qPCR was used to verify the 12 differential expressed proteins from iTRAQ, finding that trends in the transcriptional change for the 12 genes were consistent with those at the proteomic level. Differentially expressed proteins that were associated with meiosis (serine/threonine-protein phosphatase 2B and cyclin B3) and mitosis (cyclin B3 and dynein heavy chain) in viruliferous ova may contribute to low hatchability and defective or retarded development. Alterations in the abundance of proteins involved in the respiratory chain and nutrition metabolism may affect embryonic development. Our study begins to explain macroscopical developmental phenomena and explore the mechanisms by which Rice stripe virus impacts the development of SBPH. PMID:27277140

Proteomic profile of mouse fibroblasts exposed to pure magnesium extract.

PubMed

Zhen, Zhen; Luthringer, Bérengère; Yang, Li; Xi, Tingfei; Zheng, Yufeng; Feyerabend, Frank; Willumeit, Regine; Lai, Chen; Ge, Zigang

2016-12-01

Magnesium and its alloys gain wide attention as degradable biomaterials. In order to reveal the molecular mechanism of the influence of biodegradable magnesium on cells, proteomics analysis was performed in this work. After mouse fibroblasts (L929) were cultured with or without Mg degradation products (Mg-extract) for 8, 24, and 48h, changes in protein expression profiles were obtained using isobaric tags for relative and absolute quantitation (iTRAQ) coupled two dimensional liquid chromatography-tandem mass spectrometry (2D LC MS/MS). A total of 867 proteins were identified (relying on at least two peptides). Compared to the control group, 205, 282, and 217 regulated proteins were identified at 8, 24, and 48h, respectively. 65 common proteins were up or down- regulated within all the three time points, which were involved in various physiological and metabolic activities. Consistent with viability, proliferation, and cell cycle analysis, stimulated energy metabolism as well as protein synthesis pathways were discussed, indicating a possible effect of Mg-extract on L929 proliferation. Furthermore, endocytosis and focal adhesion processes were also discussed. This proteomics study uncovers early cellular mechanisms triggered by Mg degradation products and highlights the cytocompatibility of biodegradable metallic materials for biomedical applications such as stents or orthopaedic implants. Copyright © 2016. Published by Elsevier B.V.

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

PubMed Central

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

2015-01-01

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

The proteomic profile of hair damage.

PubMed

Sinclair, R; Flagler, M J; Jones, L; Rufaut, N; Davis, M G

2012-06-01

Monilethrix is a congenital hair shaft disorder with associated fragility. Many of the changes seen in monilethrix hair on light microscopy and scanning electron microscopy are also seen in hair weathering and cosmetic damage to hair. We used monilethrix as a model to investigate the relationship between hair protein structure and hair strength and resistance to cosmetic insult. We applied proteomic techniques to identify novel peptide damage markers for chemical oxidative damage to hair. The findings suggest that specific sites in the protein structure of hair are targeted during oxidative damage from bleaching, a unique insight into how chemical damage compromises the structural integrity of the hair shaft at the molecular level. Applying proteomics to the study of congenital and acquired hair shaft disorders can deliver new insights into hair damage and novel strategies to strengthen hair. © 2012 The Authors. BJD © 2012 British Association of Dermatologists.

Time Series Proteome Profiling

PubMed Central

Formolo, Catherine A.; Mintz, Michelle; Takanohashi, Asako; Brown, Kristy J.; Vanderver, Adeline; Halligan, Brian; Hathout, Yetrib

2014-01-01

This chapter provides a detailed description of a method used to study temporal changes in the endoplasmic reticulum (ER) proteome of fibroblast cells exposed to ER stress agents (tunicamycin and thapsigargin). Differential stable isotope labeling by amino acids in cell culture (SILAC) is used in combination with crude ER fractionation, SDS–PAGE and LC-MS/MS to define altered protein expression in tunicamycin or thapsigargin treated cells versus untreated cells. Treated and untreated cells are harvested at different time points, mixed at a 1:1 ratio and processed for ER fractionation. Samples containing labeled and unlabeled proteins are separated by SDS–PAGE, bands are digested with trypsin and the resulting peptides analyzed by LC-MS/MS. Proteins are identified using Bioworks software and the Swiss-Prot data-base, whereas ratios of protein expression between treated and untreated cells are quantified using ZoomQuant software. Data visualization is facilitated by GeneSpring software. proteomics PMID:21082445

Proteomic Analysis of Grape Berry Cell Cultures Reveals that Developmentally Regulated Ripening Related Processes Can Be Studied Using Cultured Cells

PubMed Central

Sharathchandra, Ramaschandra G.; Stander, Charmaine; Jacobson, Dan; Ndimba, Bongani; Vivier, Melané A.

2011-01-01

Background This work describes a proteomics profiling method, optimized and applied to berry cell suspensions to evaluate organ-specific cultures as a platform to study grape berry ripening. Variations in berry ripening within a cluster(s) on a vine and in a vineyard are a major impediment towards complete understanding of the functional processes that control ripening, specifically when a characterized and homogenous sample is required. Berry cell suspensions could overcome some of these problems, but their suitability as a model system for berry development and ripening needs to be established first. Methodology/Principal Findings In this study we report on the proteomic evaluation of the cytosolic proteins obtained from synchronized cell suspension cultures that were established from callus lines originating from green, véraison and ripe Vitis vinifera berry explants. The proteins were separated using liquid phase IEF in a Microrotofor cell and SDS PAGE. This method proved superior to gel-based 2DE. Principal component analysis confirmed that biological and technical repeats grouped tightly and importantly, showed that the proteomes of berry cultures originating from the different growth/ripening stages were distinct. A total of twenty six common bands were selected after band matching between different growth stages and twenty two of these bands were positively identified. Thirty two % of the identified proteins are currently annotated as hypothetical. The differential expression profile of the identified proteins, when compared with published literature on grape berry ripening, suggested common trends in terms of relative abundance in the different developmental stages between real berries and cell suspensions. Conclusions The advantages of having suspension cultures that accurately mimic specific developmental stages are profound and could significantly contribute to the study of the intricate regulatory and signaling networks responsible for berry development and ripening. PMID:21379583

Proteomics Perspectives in Rotator Cuff Research: A Systematic Review of Gene Expression and Protein Composition in Human Tendinopathy

PubMed Central

Sejersen, Maria Hee Jung; Frost, Poul; Hansen, Torben Bæk; Deutch, Søren Rasmussen; Svendsen, Susanne Wulff

2015-01-01

Background Rotator cuff tendinopathy including tears is a cause of significant morbidity. The molecular pathogenesis of the disorder is largely unknown. This review aimed to present an overview of the literature on gene expression and protein composition in human rotator cuff tendinopathy and other tendinopathies, and to evaluate perspectives of proteomics – the comprehensive study of protein composition - in tendon research. Materials and Methods We conducted a systematic search of the literature published between 1 January 1990 and 18 December 2012 in PubMed, Embase, and Web of Science. We included studies on objectively quantified differential gene expression and/or protein composition in human rotator cuff tendinopathy and other tendinopathies as compared to control tissue. Results We identified 2199 studies, of which 54 were included; 25 studies focussed on rotator cuff or biceps tendinopathy. Most of the included studies quantified prespecified mRNA molecules and proteins using polymerase chain reactions and immunoassays, respectively. There was a tendency towards an increase of collagen I (11 of 15 studies) and III (13 of 14), metalloproteinase (MMP)-1 (6 of 12), -9 (7 of 7), -13 (4 of 7), tissue inhibitor of metalloproteinase (TIMP)-1 (4 of 7), and vascular endothelial growth factor (4 of 7), and a decrease in MMP-3 (10 of 12). Fourteen proteomics studies of tendon tissues/cells failed inclusion, mostly because they were conducted in animals or in vitro. Conclusions Based on methods, which only allowed simultaneous quantification of a limited number of prespecified mRNA molecules or proteins, several proteins appeared to be differentially expressed/represented in rotator cuff tendinopathy and other tendinopathies. No proteomics studies fulfilled our inclusion criteria, although proteomics technologies may be a way to identify protein profiles (including non-prespecified proteins) that characterise specific tendon disorders or stages of tendinopathy. Thus, our results suggested an untapped potential for proteomics in tendon research. PMID:25879758

Proteomics perspectives in rotator cuff research: a systematic review of gene expression and protein composition in human tendinopathy.

PubMed

Sejersen, Maria Hee Jung; Frost, Poul; Hansen, Torben Bæk; Deutch, Søren Rasmussen; Svendsen, Susanne Wulff

2015-01-01

Rotator cuff tendinopathy including tears is a cause of significant morbidity. The molecular pathogenesis of the disorder is largely unknown. This review aimed to present an overview of the literature on gene expression and protein composition in human rotator cuff tendinopathy and other tendinopathies, and to evaluate perspectives of proteomics--the comprehensive study of protein composition--in tendon research. We conducted a systematic search of the literature published between 1 January 1990 and 18 December 2012 in PubMed, Embase, and Web of Science. We included studies on objectively quantified differential gene expression and/or protein composition in human rotator cuff tendinopathy and other tendinopathies as compared to control tissue. We identified 2199 studies, of which 54 were included; 25 studies focussed on rotator cuff or biceps tendinopathy. Most of the included studies quantified prespecified mRNA molecules and proteins using polymerase chain reactions and immunoassays, respectively. There was a tendency towards an increase of collagen I (11 of 15 studies) and III (13 of 14), metalloproteinase (MMP)-1 (6 of 12), -9 (7 of 7), -13 (4 of 7), tissue inhibitor of metalloproteinase (TIMP)-1 (4 of 7), and vascular endothelial growth factor (4 of 7), and a decrease in MMP-3 (10 of 12). Fourteen proteomics studies of tendon tissues/cells failed inclusion, mostly because they were conducted in animals or in vitro. Based on methods, which only allowed simultaneous quantification of a limited number of prespecified mRNA molecules or proteins, several proteins appeared to be differentially expressed/represented in rotator cuff tendinopathy and other tendinopathies. No proteomics studies fulfilled our inclusion criteria, although proteomics technologies may be a way to identify protein profiles (including non-prespecified proteins) that characterise specific tendon disorders or stages of tendinopathy. Thus, our results suggested an untapped potential for proteomics in tendon research.

A two-dimensional proteome map of the aflatoxigenic fungus Aspergillus flavus.

PubMed

Pechanova, Olga; Pechan, Tibor; Rodriguez, Jose M; Williams, W Paul; Brown, Ashli E

2013-05-01

The filamentous fungus Aspergillus flavus is an opportunistic soil-borne pathogen that produces aflatoxins, the most potent naturally occurring carcinogenic compounds known. This work represents the first gel-based profiling analysis of A. flavus proteome and establishes a 2D proteome map. Using 2DE and MALDI-TOF-MS/MS, we identified 538 mycelial proteins of the aflatoxigenic strain NRRL 3357, the majority of which were functionally annotated as related to various cellular metabolic and biosynthetic processes. Additionally, a few enzymes from the aflatoxin synthesis pathway were also identified. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Proteomics analyses for the global proteins in the brain tissues of different human prion diseases.

PubMed

Shi, Qi; Chen, Li-Na; Zhang, Bao-Yun; Xiao, Kang; Zhou, Wei; Chen, Cao; Zhang, Xiao-Mei; Tian, Chan; Gao, Chen; Wang, Jing; Han, Jun; Dong, Xiao-Ping

2015-04-01

Proteomics changes of brain tissues have been described in different neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. However, the brain proteomics of human prion disease remains less understood. In the study, the proteomics patterns of cortex and cerebellum of brain tissues of sporadic Creutzfeldt-Jakob disease, fatal familial insomnia, and G114V genetic CJD were analyzed with isobaric tags for relative and absolute quantitation combined with multidimensional liquid chromatography and MS analysis, with the brains from three normal individuals as controls. Global protein profiling, significant pathway, and functional categories were analyzed. In total, 2287 proteins were identified with quantitative information both in cortex and cerebellum regions. Cerebellum tissues appeared to contain more up- and down-regulated proteins (727 proteins) than cortex regions (312 proteins) of Creutzfeldt-Jakob disease, fatal familial insomnia, and G114V genetic CJD. Viral myocarditis, Parkinson's disease, Alzheimer's disease, lysosome, oxidative phosphorylation, protein export, and drug metabolism-cytochrome P450 were the most commonly affected pathways of the three kinds of diseases. Almost coincident biological functions were identified in the brain tissues of the three diseases. In all, data here demonstrate that the brain tissues of Creutzfeldt-Jakob disease, fatal familial insomnia, and G114V genetic CJD have obvious proteomics changes at their terminal stages, which show the similarities not only among human prion diseases but also with other neurodegeneration diseases. This is the first study to provide a reference proteome map for human prion diseases and will be helpful for future studies focused on potential biomarkers for the diagnosis and therapy of human prion diseases. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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.

Toxicogenomic analysis of N-nitrosomorpholine induced changes in rat liver: Comparison of genomic and proteomic responses and anchoring to histopathological parameters

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

Oberemm, A., E-mail: axel.oberemm@bfr.bund.d; Ahr, H.-J.; Bannasch, P.

2009-12-01

A common animal model of chemical hepatocarcinogenesis was used to examine the utility of transcriptomic and proteomic data to identify early biomarkers related to chemically induced carcinogenesis. N-nitrosomorpholine, a frequently used genotoxic model carcinogen, was applied via drinking water at 120 mg/L to male Wistar rats for 7 weeks followed by an exposure-free period of 43 weeks. Seven specimens of each treatment group (untreated control and 120 mg/L N-nitrosomorpholine in drinking water) were sacrificed at nine time points during and after N-nitrosomorpholine treatment. Individual samples from the liver were prepared for histological and toxicogenomic analyses. For histological detection of preneoplasticmore » and neoplastic tissue areas, sections were stained using antibodies against the placental form of glutathione-S-transferase (GST-P). Gene and protein expression profiles of liver tissue homogenates were analyzed using RG-U34A Affymetrix rat gene chips and two-dimensional gel electrophoresis-based proteomics, respectively. In order to compare results obtained by histopathology, transcriptomics and proteomics, GST-P-stained liver sections were evaluated morphometrically, which revealed a parallel time course of the area fraction of preneoplastic lesions and gene plus protein expression patterns. On the transcriptional level, an increase of hepatic GST-P expression was detectable as early as 3 weeks after study onset. Comparing deregulated genes and proteins, eight species were identified which showed a corresponding expression profile on both expression levels. Functional analysis suggests that these genes and corresponding proteins may be useful as biomarkers of early hepatocarcinogenesis.« less

Synthesis and Evaluation of a Novel Adenosine-Ribose Probe for Global-Scale Profiling of Nucleoside and Nucleotide-Binding Proteins

PubMed Central

Mahajan, Shikha; Manetsch, Roman; Merkler, David J.; Stevens Jr., Stanley M.

2015-01-01

Proteomics is a powerful approach used for investigating the complex molecular mechanisms of disease pathogenesis and progression. An important challenge in modern protein profiling approaches involves targeting of specific protein activities in order to identify altered molecular processes associated with disease pathophysiology. Adenosine-binding proteins represent an important subset of the proteome where aberrant expression or activity changes of these proteins have been implicated in numerous human diseases. Herein, we describe an affinity-based approach for the enrichment of adenosine-binding proteins from a complex cell proteome. A novel N 6-biotinylated-8-azido-adenosine probe (AdoR probe) was synthesized, which contains a reactive group that forms a covalent bond with the target proteins, as well as a biotin tag for affinity enrichment using avidin chromatography. Probe specificity was confirmed with protein standards prior to further evaluation in a complex protein mixture consisting of a lysate derived from mouse neuroblastoma N18TG2 cells. Protein identification and relative quantitation using mass spectrometry allowed for the identification of small variations in abundance of nucleoside- and nucleotide-binding proteins in these samples where a significant enrichment of AdoR-binding proteins in the labeled proteome from the neuroblastoma cells was observed. The results from this study demonstrate the utility of this method to enrich for nucleoside- and nucleotide-binding proteins in a complex protein mixture, pointing towards a unique set of proteins that can be examined in the context of further understanding mechanisms of disease, or fundamental biological processes in general. PMID:25671571

Capillary nano-immunoassays: advancing quantitative proteomics analysis, biomarker assessment, and molecular diagnostics.

PubMed

Chen, Jin-Qiu; Wakefield, Lalage M; Goldstein, David J

2015-06-06

There is an emerging demand for the use of molecular profiling to facilitate biomarker identification and development, and to stratify patients for more efficient treatment decisions with reduced adverse effects. In the past decade, great strides have been made to advance genomic, transcriptomic and proteomic approaches to address these demands. While there has been much progress with these large scale approaches, profiling at the protein level still faces challenges due to limitations in clinical sample size, poor reproducibility, unreliable quantitation, and lack of assay robustness. A novel automated capillary nano-immunoassay (CNIA) technology has been developed. This technology offers precise and accurate measurement of proteins and their post-translational modifications using either charge-based or size-based separation formats. The system not only uses ultralow nanogram levels of protein but also allows multi-analyte analysis using a parallel single-analyte format for increased sensitivity and specificity. The high sensitivity and excellent reproducibility of this technology make it particularly powerful for analysis of clinical samples. Furthermore, the system can distinguish and detect specific protein post-translational modifications that conventional Western blot and other immunoassays cannot easily capture. This review will summarize and evaluate the latest progress to optimize the CNIA system for comprehensive, quantitative protein and signaling event characterization. It will also discuss how the technology has been successfully applied in both discovery research and clinical studies, for signaling pathway dissection, proteomic biomarker assessment, targeted treatment evaluation and quantitative proteomic analysis. Lastly, a comparison of this novel system with other conventional immuno-assay platforms is performed.

Protein profile in HBx transfected cells: a comparative iTRAQ-coupled 2D LC-MS/MS analysis.

PubMed

Feng, Huixing; Li, Xi; Niu, Dandan; Chen, Wei Ning

2010-06-16

The x protein of HBV (HBx) has been involved in the development of hepatocellular carcinoma (HCC), with a possible link to individual genotypes. Nevertheless, the underlying mechanism remains obscure. In this study, we aim to identify the HBx-induced protein profile in HepG2 cells by LC-MS/MS proteomics analysis. Our results indicated that proteins were differentially expressed in HepG2 cells transfected by HBx of various genotypes. Proteins associated with cytoskeleton were found to be either up-regulated (MACF1, HMGB1, Annexin A2) or down-regulated (Lamin A/C). These may in turn result in the decrease of focal adhesion and increase of cell migration in response to HBx. Levels of other cellular proteins with reported impact on the function of extracellular matrix (ECM) proteins and cell migration, including Ca(2+)-binding proteins (S100A11, S100A6, and S100A4) and proteasome protein (PSMA3), were affected by HBx. The differential protein profile identified in this study was also supported by our functional assay which indicated that cell migration was enhanced by HBx. Our preliminary study provided a new platform to establish a comprehensive cellular protein profile by LC-MS/MS proteomics analysis. Further downstream functional assays, including our reported cell migration assay, should provide new insights in the association between HCC and HBx. Copyright 2009 Elsevier B.V. All rights reserved.

Unbiased and targeted mass spectrometry for the HDL proteome.

PubMed

Singh, Sasha A; Aikawa, Masanori

2017-02-01

Mass spectrometry is an ever evolving technology that is equipped with a variety of tools for protein research. Some lipoprotein studies, especially those pertaining to HDL biology, have been exploiting the versatility of mass spectrometry to understand HDL function through its proteome. Despite the role of mass spectrometry in advancing research as a whole, however, the technology remains obscure to those without hands on experience, but still wishing to understand it. In this review, we walk the reader through the coevolution of common mass spectrometry workflows and HDL research, starting from the basic unbiased mass spectrometry methods used to profile the HDL proteome to the most recent targeted methods that have enabled an unprecedented view of HDL metabolism. Unbiased global proteomics have demonstrated that the HDL proteome is organized into subgroups across the HDL size fractions providing further evidence that HDL functional heterogeneity is in part governed by its varying protein constituents. Parallel reaction monitoring, a novel targeted mass spectrometry method, was used to monitor the metabolism of HDL apolipoproteins in humans and revealed that apolipoproteins contained within the same HDL size fraction exhibit diverse metabolic properties. Mass spectrometry provides a variety of tools and strategies to facilitate understanding, through its proteins, the complex biology of HDL.

Cross-platform method for identifying candidate network biomarkers for prostate cancer.

PubMed

Jin, G; Zhou, X; Cui, K; Zhang, X-S; Chen, L; Wong, S T C

2009-11-01

Discovering biomarkers using mass spectrometry (MS) and microarray expression profiles is a promising strategy in molecular diagnosis. Here, the authors proposed a new pipeline for biomarker discovery that integrates disease information for proteins and genes, expression profiles in both genomic and proteomic levels, and protein-protein interactions (PPIs) to discover high confidence network biomarkers. Using this pipeline, a total of 474 molecules (genes and proteins) related to prostate cancer were identified and a prostate-cancer-related network (PCRN) was derived from the integrative information. Thus, a set of candidate network biomarkers were identified from multiple expression profiles composed by eight microarray datasets and one proteomics dataset. The network biomarkers with PPIs can accurately distinguish the prostate patients from the normal ones, which potentially provide more reliable hits of biomarker candidates than conventional biomarker discovery methods.

Proteome-wide analysis of Anopheles culicifacies mosquito midgut: new insights into the mechanism of refractoriness.

PubMed

Vijay, Sonam; Rawal, Ritu; Kadian, Kavita; Singh, Jagbir; Adak, Tridibesh; Sharma, Arun

2018-05-08

Midgut invasion, a major bottleneck for malaria parasites transmission is considered as a potential target for vector-parasite interaction studies. New intervention strategies are required to explore the midgut proteins and their potential role in refractoriness for malaria control in Anopheles mosquitoes. To better understand the midgut functional proteins of An. culicifacies susceptible and refractory species, proteomic approaches coupled with bioinformatics analysis is an effective means in order to understand the mechanism of refractoriness. In the present study, an integrated in solution- in gel trypsin digestion approach, along with Isobaric tag for relative and absolute quantitation (iTRAQ)-Liquid chromatography/Mass spectrometry (LC/MS/MS) and data mining were performed to identify the proteomic profile and differentially expressed proteins in Anopheles culicifacies susceptible species A and refractory species B. Shot gun proteomics approaches led to the identification of 80 proteins in An. culicifacies susceptible species A and 92 in refractory species B and catalogue was prepared. iTRAQ based proteomic analysis identified 48 differentially expressed proteins from total 130 proteins. Of these, 41 were downregulated and 7 were upregulated in refractory species B in comparison to susceptible species A. We report that the altered midgut proteins identified in naturally refractory mosquitoes are involved in oxidative phosphorylation, antioxidant and proteolysis process that may suggest their role in parasite growth inhibition. Furthermore, real time polymerase chain reaction (PCR) analysis of few proteins indicated higher expression of iTRAQ upregulated protein in refractory species than susceptible species. This study elucidates the first proteome of the midguts of An. culicifacies sibling species that attempts to analyze unique proteogenomic interactions to provide insights for better understanding of the mechanism of refractoriness. Functional implications of these upregulated proteins in refractory species may reflect the phenotypic characteristics of the mosquitoes and will improve our understandings of blood meal digestion process, parasite vector interactions and proteomes of other vectors of human diseases for development of novel vector control strategies.

Spermatogenesis in mammals: proteomic insights.

PubMed

Chocu, Sophie; Calvel, Pierre; Rolland, Antoine D; Pineau, Charles

2012-08-01

Spermatogenesis is a highly sophisticated process involved in the transmission of genetic heritage. It includes halving ploidy, repackaging of the chromatin for transport, and the equipment of developing spermatids and eventually spermatozoa with the advanced apparatus (e.g., tightly packed mitochondrial sheat in the mid piece, elongating of the tail, reduction of cytoplasmic volume) to elicit motility once they reach the epididymis. Mammalian spermatogenesis is divided into three phases. In the first the primitive germ cells or spermatogonia undergo a series of mitotic divisions. In the second the spermatocytes undergo two consecutive divisions in meiosis to produce haploid spermatids. In the third the spermatids differentiate into spermatozoa in a process called spermiogenesis. Paracrine, autocrine, juxtacrine, and endocrine pathways all contribute to the regulation of the process. The array of structural elements and chemical factors modulating somatic and germ cell activity is such that the network linking the various cellular activities during spermatogenesis is unimaginably complex. Over the past two decades, advances in genomics have greatly improved our knowledge of spermatogenesis, by identifying numerous genes essential for the development of functional male gametes. Large-scale analyses of testicular function have deepened our insight into normal and pathological spermatogenesis. Progress in genome sequencing and microarray technology have been exploited for genome-wide expression studies, leading to the identification of hundreds of genes differentially expressed within the testis. However, although proteomics has now come of age, the proteomics-based investigation of spermatogenesis remains in its infancy. Here, we review the state-of-the-art of large-scale proteomic analyses of spermatogenesis, from germ cell development during sex determination to spermatogenesis in the adult. Indeed, a few laboratories have undertaken differential protein profiling expression studies and/or systematic analyses of testicular proteomes in entire organs or isolated cells from various species. We consider the pros and cons of proteomics for studying the testicular germ cell gene expression program. Finally, we address the use of protein datasets, through integrative genomics (i.e., combining genomics, transcriptomics, and proteomics), bioinformatics, and modelling.

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.

Nanodroplet processing platform for deep and quantitative proteome profiling of 10–100 mammalian cells

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

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

Nanoscale or single cell technologies are critical for biomedical applications. However, current mass spectrometry (MS)-based proteomic approaches require samples comprising a minimum of thousands of cells to provide in-depth profiling. Here, we report the development of a nanoPOTS (Nanodroplet Processing in One pot for Trace Samples) platform as a major advance in overall sensitivity. NanoPOTS dramatically enhances the efficiency and recovery of sample processing by downscaling processing volumes to <200 nL to minimize surface losses. When combined with ultrasensitive LC-MS, nanoPOTS allows identification of ~1500 to ~3,000 proteins from ~10 to ~140 cells, respectively. By incorporating the Match Between Runsmore » algorithm of MaxQuant, >3000 proteins were consistently identified from as few as 10 cells. Furthermore, we demonstrate robust quantification of ~2400 proteins from single human pancreatic islet thin sections from type 1 diabetic and control donors, illustrating the application of nanoPOTS for spatially resolved proteome measurements from clinical tissues.« less

Mining the human plasma proteome with three-dimensional strategies by high-resolution Quadrupole Orbitrap Mass Spectrometry.

PubMed

Zhao, Yan; Chang, Cheng; Qin, Peibin; Cao, Qichen; Tian, Fang; Jiang, Jing; Li, Xianyu; Yu, Wenfeng; Zhu, Yunping; He, Fuchu; Ying, Wantao; Qian, Xiaohong

2016-01-21

Human plasma is a readily available clinical sample that reflects the status of the body in normal physiological and disease states. Although the wide dynamic range and immense complexity of plasma proteins are obstacles, comprehensive proteomic analysis of human plasma is necessary for biomarker discovery and further verification. Various methods such as immunodepletion, protein equalization and hyper fractionation have been applied to reduce the influence of high-abundance proteins (HAPs) and to reduce the high level of complexity. However, the depth at which the human plasma proteome has been explored in a relatively short time frame has been limited, which impedes the transfer of proteomic techniques to clinical research. Development of an optimal strategy is expected to improve the efficiency of human plasma proteome profiling. Here, five three-dimensional strategies combining HAP depletion (the 1st dimension) and protein fractionation (the 2nd dimension), followed by LC-MS/MS analysis (the 3rd dimension) were developed and compared for human plasma proteome profiling. Pros and cons of the five strategies are discussed for two issues: HAP depletion and complexity reduction. Strategies A and B used proteome equalization and tandem Seppro IgY14 immunodepletion, respectively, as the first dimension. Proteome equalization (strategy A) was biased toward the enrichment of basic and low-molecular weight proteins and had limited ability to enrich low-abundance proteins. By tandem removal of HAPs (strategy B), the efficiency of HAP depletion was significantly increased, whereas more off-target proteins were subtracted simultaneously. In the comparison of complexity reduction, strategy D involved a deglycosylation step before high-pH RPLC separation. However, the increase in sequence coverage did not increase the protein number as expected. Strategy E introduced SDS-PAGE separation of proteins, and the results showed oversampling of HAPs and identification of fewer proteins. Strategy C combined single Seppro IgY14 immunodepletion, high-pH RPLC fractionation and LC-MS/MS analysis. It generated the largest dataset, containing 1544 plasma protein groups and 258 newly identified proteins in a 30-h-machine-time analysis, making it the optimum three-dimensional strategy in our study. Further analysis of the integrated data from the five strategies showed identical distribution patterns in terms of sequence features and GO functional analysis with the 1929-plasma-protein dataset, further supporting the reliability of our plasma protein identifications. The characterization of 20 cytokines in the concentration range from sub-nanograms/milliliter to micrograms/milliliter demonstrated the sensitivity of the current strategies. Copyright © 2015 Elsevier B.V. All rights reserved.

Mapping the Small Molecule Interactome by Mass Spectrometry.

PubMed

Flaxman, Hope A; Woo, Christina M

2018-01-16

Mapping small molecule interactions throughout the proteome provides the critical structural basis for functional analysis of their impact on biochemistry. However, translation of mass spectrometry-based proteomics methods to directly profile the interaction between a small molecule and the whole proteome is challenging because of the substoichiometric nature of many interactions, the diversity of covalent and noncovalent interactions involved, and the subsequent computational complexity associated with their spectral assignment. Recent advances in chemical proteomics have begun fill this gap to provide a structural basis for the breadth of small molecule-protein interactions in the whole proteome. Innovations enabling direct characterization of the small molecule interactome include faster, more sensitive instrumentation coupled to chemical conjugation, enrichment, and labeling methods that facilitate detection and assignment. These methods have started to measure molecular interaction hotspots due to inherent differences in local amino acid reactivity and binding affinity throughout the proteome. Measurement of the small molecule interactome is producing structural insights and methods for probing and engineering protein biochemistry. Direct structural characterization of the small molecule interactome is a rapidly emerging area pushing new frontiers in biochemistry at the interface of small molecules and the proteome.

A proteomics approach to study synergistic and antagonistic interactions of the fungal-bacterial consortium Fusarium oxysporum wild-type MSA 35.

PubMed

Moretti, Marino; Grunau, Alexander; Minerdi, Daniela; Gehrig, Peter; Roschitzki, Bernd; Eberl, Leo; Garibaldi, Angelo; Gullino, Maria Lodovica; Riedel, Kathrin

2010-09-01

Fusarium oxysporum is an important plant pathogen that causes severe damage of many economically important crop species. Various microorganisms have been shown to inhibit this soil-borne plant pathogen, including non-pathogenic F. oxysporum strains. In this study, F. oxysporum wild-type (WT) MSA 35, a biocontrol multispecies consortium that consists of a fungus and numerous rhizobacteria mainly belonging to gamma-proteobacteria, was analyzed by two complementary metaproteomic approaches (2-DE combined with MALDI-Tof/Tof MS and 1-D PAGE combined with LC-ESI-MS/MS) to identify fungal or bacterial factors potentially involved in antagonistic or synergistic interactions between the consortium members. Moreover, the proteome profiles of F. oxysporum WT MSA 35 and its cured counter-part CU MSA 35 (WT treated with antibiotics) were compared with unravel the bacterial impact on consortium functioning. Our study presents the first proteome mapping of an antagonistic F. oxysporum strain and proposes candidate proteins that might play an important role for the biocontrol activity and the close interrelationship between the fungus and its bacterial partners.

iTRAQ-based proteome profiling of Saccharomyces cerevisiae and cryotolerant species Saccharomyces uvarum and Saccharomyces kudriavzevii during low-temperature wine fermentation.

PubMed

García-Ríos, Estéfani; Querol, Amparo; Guillamón, José Manuel

2016-09-02

Temperature is one of the most important parameters to affect the duration and rate of alcoholic fermentation and final wine quality. Some species of the Saccharomyces genus have shown better adaptation at low temperature than Saccharomyces cerevisiae, which was the case of cryotolerant yeasts Saccharomyces uvarum and Saccharomyces kudriavzevii. In an attempt to detect inter-specific metabolic differences, we characterized the proteomic landscape of these cryotolerant species grown at 12°C and 28°C, which we compared with the proteome of S. cerevisiae (poorly adapted at low temperature). Our results showed that the main differences among the proteomic profiling of the three Saccharomyces strains grown at 12°C and 28°C lay in translation, glycolysis and amino acid metabolism. Our data corroborate previous transcriptomic results, which suggest that S. kudriavzevii is better adapted to grow at low temperature as a result of enhanced more efficient translation. Fitter amino acid biosynthetic pathways can also be mechanisms that better explain biomass yield in cryotolerant strains. Yet even at low temperature, S. cerevisiae is the most fermentative competitive species. A higher concentration of glycolytic and alcoholic fermentation enzymes in the S. cerevisiae strain might explain such greater fermentation activity. Temperature is one of the main relevant environmental variables that microorganisms have to cope with and it is also a key factor in some industrial processes that involve microorganisms. However, we are still far from understanding the molecular and physiological mechanisms of adaptation at low temperatures. The results obtained in this study provided a global atlas of the proteome changes triggered by temperature in three different species of the genus Saccharomyces with different degree of cryotolerance. These results would facilitate a better understanding of mechanisms for how yeast could adapt at the low temperature of growth. Copyright © 2016 Elsevier B.V. All rights reserved.

Protein profile of Beta vulgaris leaf apoplastic fluid and changes induced by Fe deficiency and Fe resupply

PubMed Central

Ceballos-Laita, Laura; Gutierrez-Carbonell, Elain; Lattanzio, Giuseppe; Vázquez, Saul; Contreras-Moreira, Bruno; Abadía, Anunciación; Abadía, Javier; López-Millán, Ana-Flor

2015-01-01

The fluid collected by direct leaf centrifugation has been used to study the proteome of the sugar beet apoplastic fluid as well as the changes induced by Fe deficiency and Fe resupply to Fe-deficient plants in the protein profile. Plants were grown in Fe-sufficient and Fe-deficient conditions, and Fe resupply was carried out with 45 μM Fe(III)-EDTA for 24 h. Protein extracts of leaf apoplastic fluid were analyzed by two-dimensional isoelectric focusing-SDS-PAGE electrophoresis. Gel image analysis revealed 203 consistent spots, and proteins in 81% of them (164) were identified by nLC-MS/MS using a custom made reference repository of beet protein sequences. When redundant UniProt entries were deleted, a non-redundant leaf apoplastic proteome consisting of 109 proteins was obtained. TargetP and SecretomeP algorithms predicted that 63% of them were secretory proteins. Functional classification of the non-redundant proteins indicated that stress and defense, protein metabolism, cell wall and C metabolism accounted for approximately 75% of the identified proteome. The effects of Fe-deficiency on the leaf apoplast proteome were limited, with only five spots (2.5%) changing in relative abundance, thus suggesting that protein homeostasis in the leaf apoplast fluid is well-maintained upon Fe shortage. The identification of three chitinase isoforms among proteins increasing in relative abundance with Fe-deficiency suggests that one of the few effects of Fe deficiency in the leaf apoplast proteome includes cell wall modifications. Iron resupply to Fe deficient plants changed the relative abundance of 16 spots when compared to either Fe-sufficient or Fe-deficient samples. Proteins identified in these spots can be broadly classified as those responding to Fe-resupply, which included defense and cell wall related proteins, and non-responsive, which are mainly protein metabolism related proteins and whose changes in relative abundance followed the same trend as with Fe-deficiency. PMID:25852707

Proteomic characterization of an isolated fraction of synthetic proteasome inhibitor (PSI)-induced inclusions in PC12 cells might offer clues to aggresomes as a cellular defensive response against proteasome inhibition by PSI

PubMed Central

2010-01-01

Background Cooperation of constituents of the ubiquitin proteasome system (UPS) with chaperone proteins in degrading proteins mediate a wide range of cellular processes, such as synaptic function and neurotransmission, gene transcription, protein trafficking, mitochondrial function and metabolism, antioxidant defence mechanisms, and apoptotic signal transduction. It is supposed that constituents of the UPS and chaperone proteins are recruited into aggresomes where aberrant and potentially cytotoxic proteins may be sequestered in an inactive form. Results To determinate the proteomic pattern of synthetic proteasome inhibitor (PSI)-induced inclusions in PC12 cells after proteasome inhibition by PSI, we analyzed a fraction of PSI-induced inclusions. A proteomic feature of the isolated fraction was characterized by identification of fifty six proteins including twenty previously reported protein components of Lewy bodies, twenty eight newly identified proteins and eight unknown proteins. These proteins, most of which were recognized as a profile of proteins within cellular processes mediated by the UPS, a profile of constituents of the UPS and a profile of chaperone proteins, are classed into at least nine accepted categories. In addition, prolyl-4-hydroxylase beta polypeptide, an endoplasmic reticulum member of the protein disulfide isomerase family, was validated in the developmental process of PSI-induced inclusions in the cells. Conclusions It is speculated that proteomic characterization of an isolated fraction of PSI-induced inclusions in PC12 cells might offer clues to appearance of aggresomes serving as a cellular defensive response against proteasome inhibition. PMID:20704702

Proteomic profile of dormant Trichophyton Rubrum conidia

PubMed Central

Leng, Wenchuan; Liu, Tao; Li, Rui; Yang, Jian; Wei, Candong; Zhang, Wenliang; Jin, Qi

2008-01-01

Background Trichophyton rubrum is the most common dermatophyte causing fungal skin infections in humans. Asexual sporulation is an important means of propagation for T. rubrum, and conidia produced by this way are thought to be the primary cause of human infections. Despite their importance in pathogenesis, the conidia of T. rubrum remain understudied. We intend to intensively investigate the proteome of dormant T. rubrum conidia to characterize its molecular and cellular features and to enhance the development of novel therapeutic strategies. Results The proteome of T. rubrum conidia was analyzed by combining shotgun proteomics with sample prefractionation and multiple enzyme digestion. In total, 1026 proteins were identified. All identified proteins were compared to those in the NCBI non-redundant protein database, the eukaryotic orthologous groups database, and the gene ontology database to obtain functional annotation information. Functional classification revealed that the identified proteins covered nearly all major biological processes. Some proteins were spore specific and related to the survival and dispersal of T. rubrum conidia, and many proteins were important to conidial germination and response to environmental conditions. Conclusion Our results suggest that the proteome of T. rubrum conidia is considerably complex, and that the maintenance of conidial dormancy is an intricate and elaborate process. This data set provides the first global framework for the dormant T. rubrum conidia proteome and is a stepping stone on the way to further study of the molecular mechanisms of T. rubrum conidial germination and the maintenance of conidial dormancy. PMID:18578874

A comparative proteomic characterization and nutritional assessment of naturally- and artificially-cultivated Cordyceps sinensis.

PubMed

Zhang, Xu; Liu, Qun; Zhou, Wei; Li, Ping; Alolga, Raphael N; Qi, Lian-Wen; Yin, Xiaojian

2018-06-15

Cordyceps sinensis has gained increasing attention due to its nutritional and medicinal properties. Herein, we employed label-free quantitative mass spectrometry to explore the proteome differences between naturally- and artificially-cultivated C. sinensis. A total of 22,829 peptides with confidence ≥95%, corresponding to 2541 protein groups were identified from the caterpillar bodies/stromata of 12 naturally- and artificially-cultivated samples of C. sinensis. Among them, 165 proteins showed significant differences between the samples of natural and artificial cultivation. These proteins were mainly involved in energy production/conversion, amino acid transport/metabolism, and transcription regulation. The proteomic results were confirmed by the identification of 4 significantly changed metabolites, thus, lysine, threonine, serine, and arginine via untargeted metabolomics. The change tendencies of these metabolites were partly in accordance with changes in abundance of the proteins, which was upstream of their synthetic pathways. In addition, the nutritional value in terms of the levels of nucleosides, nucleotides, and adenosine between the artificially- and naturally-cultivated samples was virtually same. These proteomic data will be useful for understanding the medicinal value of C. sinensis and serve as reference for its artificial cultivation. C. sinensis is a precious and valued medicinal product, the current basic proteome dataset would provide useful information to understand its development/infection processes as well as help to artificially cultivate it. This work would also provide basic proteome profile for further study of C. sinensis. Copyright © 2018. Published by Elsevier B.V.

Mass Spectrometry-Based Proteomics for Pre-Eclampsia and Preterm Birth

PubMed Central

Law, Kai P.; Han, Ting-Li; Tong, Chao; Baker, Philip N.

2015-01-01

Pregnancy-related complications such as pre-eclampsia and preterm birth now represent a notable burden of adverse health. Pre-eclampsia is a hypertensive disorder unique to pregnancy. It is an important cause of maternal death worldwide and a leading cause of fetal growth restriction and iatrogenic prematurity. Fifteen million infants are born preterm each year globally, but more than one million of those do not survive their first month of life. Currently there are no predictive tests available for diagnosis of these pregnancy-related complications and the biological mechanisms of the diseases have not been fully elucidated. Mass spectrometry-based proteomics have all the necessary attributes to provide the needed breakthrough in understanding the pathophysiology of complex human diseases thorough the discovery of biomarkers. The mass spectrometry methodologies employed in the studies for pregnancy-related complications are evaluated in this article. Top-down proteomic and peptidomic profiling by laser mass spectrometry, liquid chromatography or capillary electrophoresis coupled to mass spectrometry, and bottom-up quantitative proteomics and targeted proteomics by liquid chromatography mass spectrometry have been applied to elucidate protein biomarkers and biological mechanism of pregnancy-related complications. The proteomes of serum, urine, amniotic fluid, cervical-vaginal fluid, placental tissue, and cytotrophoblastic cells have all been investigated. Numerous biomarkers or biomarker candidates that could distinguish complicated pregnancies from healthy controls have been proposed. Nevertheless, questions as to the clinically utility and the capacity to elucidate the pathogenesis of the pre-eclampsia and preterm birth remain to be answered. PMID:26006232

Quantitative Molecular Phenotyping of Gill Remodeling in a Cichlid Fish Responding to Salinity Stress*

PubMed Central

Kültz, Dietmar; Li, Johnathon; Gardell, Alison; Sacchi, Romina

2013-01-01

A two-tiered label-free quantitative (LFQ) proteomics workflow was used to elucidate how salinity affects the molecular phenotype, i.e. proteome, of gills from a cichlid fish, the euryhaline tilapia (Oreochromis mossambicus). The workflow consists of initial global profiling of relative tryptic peptide abundances in treated versus control samples followed by targeted identification (by MS/MS) and quantitation (by chromatographic peak area integration) of validated peptides for each protein of interest. Fresh water acclimated tilapia were independently exposed in separate experiments to acute short-term (34 ppt) and gradual long-term (70 ppt, 90 ppt) salinity stress followed by molecular phenotyping of the gill proteome. The severity of salinity stress can be deduced with high technical reproducibility from the initial global label-free quantitative profiling step alone at both peptide and protein levels. However, an accurate regulation ratio can only be determined by targeted label-free quantitative profiling because not all peptides used for protein identification are also valid for quantitation. Of the three salinity challenges, gradual acclimation to 90 ppt has the most pronounced effect on gill molecular phenotype. Known salinity effects on tilapia gills, including an increase in the size and number of mitochondria-rich ionocytes, activities of specific ion transporters, and induction of specific molecular chaperones are reflected in the regulation of abundances of the corresponding proteins. Moreover, specific protein isoforms that are responsive to environmental salinity change are resolved and it is revealed that salinity effects on the mitochondrial proteome are nonuniform. Furthermore, protein NDRG1 has been identified as a novel key component of molecular phenotype restructuring during salinity-induced gill remodeling. In conclusion, besides confirming known effects of salinity on gills of euryhaline fish, molecular phenotyping reveals novel insight into proteome changes that underlie the remodeling of tilapia gill epithelium in response to environmental salinity change. PMID:24065692

Noninvasive metabolic profiling for painless diagnosis of human diseases and disorders.

PubMed

Mal, Mainak

2016-06-01

Metabolic profiling provides a powerful diagnostic tool complementary to genomics and proteomics. The pain, discomfort and probable iatrogenic injury associated with invasive or minimally invasive diagnostic methods, render them unsuitable in terms of patient compliance and participation. Metabolic profiling of biomatrices like urine, breath, saliva, sweat and feces, which can be collected in a painless manner, could be used for noninvasive diagnosis. This review article covers the noninvasive metabolic profiling studies that have exhibited diagnostic potential for diseases and disorders. Their potential applications are evident in different forms of cancer, metabolic disorders, infectious diseases, neurodegenerative disorders, rheumatic diseases and pulmonary diseases. Large scale clinical validation of such diagnostic methods is necessary in future.

Noninvasive metabolic profiling for painless diagnosis of human diseases and disorders

PubMed Central

Mal, Mainak

2016-01-01

Metabolic profiling provides a powerful diagnostic tool complementary to genomics and proteomics. The pain, discomfort and probable iatrogenic injury associated with invasive or minimally invasive diagnostic methods, render them unsuitable in terms of patient compliance and participation. Metabolic profiling of biomatrices like urine, breath, saliva, sweat and feces, which can be collected in a painless manner, could be used for noninvasive diagnosis. This review article covers the noninvasive metabolic profiling studies that have exhibited diagnostic potential for diseases and disorders. Their potential applications are evident in different forms of cancer, metabolic disorders, infectious diseases, neurodegenerative disorders, rheumatic diseases and pulmonary diseases. Large scale clinical validation of such diagnostic methods is necessary in future. PMID:28031956

Proteome profile and biological activity of caprine, bovine and human milk fat globules.

PubMed

Spertino, Stefano; Cipriani, Valentina; De Angelis, Chiara; Giuffrida, Maria Gabriella; Marsano, Francesco; Cavaletto, Maria

2012-04-01

Upon combining bidimensional electrophoresis with monodimensional separation, a more comprehensive analysis of the milk fat globule membrane has been obtained. The proteomic profile of caprine milk fat globules revealed the presence of butyrophilin, lactadherin and perilipin as the major proteins, they were also associated to bovine and human milk fat globule membranes. Xanthine dehydrogenase/oxidase has been detected only in monodimensional gels. Biological activity of milk fat globules has been evaluated in Caco2-cells, as a representative model of the intestinal barrier. The increase of cell viability was indicative of a potential nutraceutical role for the whole milk fat globule, suggesting a possible employment in milk formula preparation.

Proteomics in investigation of cancer metastasis: functional and clinical consequences and methodological challenges.

PubMed

Maryáš, Josef; Faktor, Jakub; Dvořáková, Monika; Struhárová, Iva; Grell, Peter; Bouchal, Pavel

2014-03-01

Metastases are responsible for most of the cases of death in patients with solid tumors. There is thus an urgent clinical need of better understanding the exact molecular mechanisms and finding novel therapeutics targets and biomarkers of metastatic disease of various tumors. Metastases are formed in a complicated biological process called metastatic cascade. Up to now, proteomics has enabled the identification of number of metastasis-associated proteins and potential biomarkers in cancer tissues, microdissected cells, model systems, and secretomes. Expression profiles and biological role of key proteins were confirmed in verification and functional experiments. This communication reviews these observations and analyses the methodological aspects of the proteomics approaches used. Moreover, it reviews contribution of current proteomics in the field of functional characterization and interactome analysis of proteins involved in various events in metastatic cascade. It is evident that ongoing technical progress will further increase proteome coverage and sample capacity of proteomics technologies, giving complex answers to clinical and functional questions asked. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Straightforward and Highly Efficient Precipitation/On-pellet Digestion Procedure Coupled to a Long Gradient Nano-LC Separation and Orbitrap Mass Spectrometry for Label-free Expression Profiling of the Swine Heart Mitochondrial Proteome

PubMed Central

Duan, Xiaotao; Young, Rebecca; Straubinger, Robert M.; Page, Brian J.; Cao, Jin; Wang, Hao; Yu, Haoying; Canty, John M.; Qu, Jun

2009-01-01

For label-free expression profiling of tissue proteomes, efficient protein extraction, thorough and quantitative sample cleanup and digestion procedures, as well as sufficient and reproducible chromatographic separation, are highly desirable but remain challenging. However, optimal methodology has remained elusive, especially for proteomes that are rich in membrane proteins, such as the mitochondria. Here we describe a straightforward and reproducible sample preparation procedure, coupled with a highly selective and sensitive nano-LC/Orbitrap analysis, which enables reliable and comprehensive expression profiling of tissue mitochondria. The mitochondrial proteome of swine heart was selected as a test system. Efficient protein extraction was accomplished using a strong buffer containing both ionic and non-ionic detergents. Overnight precipitation was used for cleanup of the extract, and the sample was subjected to an optimized 2-step, on-pellet digestion approach. In the first step, the protein pellet was dissolved via a 4 h tryptic digestion under vigorous agitation, which nano-LC/LTQ/ETD showed to produce large and incompletely cleaved tryptic peptides. The mixture was then reduced, alkylated, and digested into its full complement of tryptic peptides with additional trypsin. This solvent precipitation/on-pellet digestion procedure achieved significantly higher and more reproducible peptide recovery of the mitochondrial preparation, than observed using a prevalent alternative procedure for label-free expression profiling, SDS-PAGE/in-gel digestion (87% vs. 54%). Furthermore, uneven peptide losses were lower than observed with SDS-PAGE/in-gel digestion. The resulting peptides were sufficiently resolved by a 5 h gradient using a nano-LC configuration that features a low-void-volume, high chromatographic reproducibility, and an LTQ/Orbitrap analyzer for protein identification and quantification. The developed method was employed for label-free comparison of the mitochondrial proteomes of myocardium from healthy animals vs. those with hibernating myocardium. Each experimental group consisted of a relatively large number of animals (n=10), and samples were analyzed in random order to minimize quantitative false-positives. Using this approach, 904 proteins were identified and quantified with high confidence, and those mitochondrial proteins that were altered significantly between groups were compared with the results of a parallel 2D-DIGE analysis. The sample preparation and analytical strategy developed here represents an advancement that can be adapted to analyze other tissue proteomes. PMID:19290621

Microbial profiling, neural network and semantic web: an integrated information system for human pathogen risk management, prevention and surveillance in food safety

USDA-ARS?s Scientific Manuscript database

It is estimated that food-borne pathogens cause approximately 76 million cases of gastrointestinal illnesses, 325,000 hospitalizations, and 5,000 deaths in the United States annually. Genomic, proteomic, and metabolomic studies, particularly, genome sequencing projects are providing valuable inform...

Deciphering the proteomic profile of rice (Oryza sativa) bran: a pilot study.

PubMed

Ferrari, Fabio; Fumagalli, Marco; Profumo, Antonella; Viglio, Simona; Sala, Alberto; Dolcini, Lorenzo; Temporini, Caterina; Nicolis, Stefania; Merli, Daniele; Corana, Federica; Casado, Begona; Iadarola, Paolo

2009-12-01

The exact knowledge of the qualitative and quantitative protein components of rice bran is an essential aspect to be considered for a better understanding of the functional properties of this resource. Aim of the present investigation was to extract the largest number of rice bran proteins and to obtain their qualitative characterization. For this purpose, three different extraction protocols have been applied either on full-fat or on defatted rice bran. Likewise, to identify the highest number of proteins, MS data collected from 1-DE, 2-DE and gel-free procedures have been combined. These approaches allowed to unambiguously identify 43 proteins that were classified as signalling/regulation proteins (30%), proteins with enzymatic activity (30%), storage proteins (30%), transfer (5%) and structural (5%) proteins. The fact that all extraction and identification procedures have been performed in triplicate with an excellent reproducibility provides a rationale for considering the platform of proteins shown in this study as the potential proteome profile of rice bran. It also represents a source of information to evaluate better the qualities of rice bran as food resource.

Identification of Potential Biomarkers for Rhegmatogenous Retinal Detachment Associated with Choroidal Detachment by Vitreous iTRAQ-Based Proteomic Profiling

PubMed Central

Wu, Zhifeng; Ding, Nannan; Yu, Mengxi; Wang, Ke; Luo, Shasha; Zou, Wenjun; Zhou, Ying; Yan, Biao; Jiang, Qin

2016-01-01

Rhegmatogenous retinal detachment associated with choroidal detachment (RRDCD) is a complicated and serious type of rhegmatogenous retinal detachment (RRD). In this study, we identified differentially expressed proteins in the vitreous humors of RRDCD and RRD using isobaric tags for relative and absolute quantitation (iTRAQ) combined with nano-liquid chromatography-electrospray ion trap-mass spectrometry-mass spectrometry (nano-LC-ESI-MS/MS) and bioinformatic analysis. Our result shows that 103 differentially expressed proteins, including 54 up-regulated and 49 down-regulated proteins were identified in RRDCD. Gene ontology (GO) analysis suggested that most of the differentially expressed proteins were extracellular．The Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis suggested that proteins related to complement and coagulation cascades were significantly enriched. iTRAQ-based proteomic profiling reveals that complement and coagulation cascades and inflammation may play important roles in the pathogenesis of RRDCD. This study may provide novel insights into the pathogenesis of RRDCD and offer potential opportunities for the diagnosis and treatment of RRDCD. PMID:27941623

Chromatography/Mass Spectrometry-Based Biomarkers in the Field of Obstructive Sleep Apnea

PubMed Central

Xu, Huajun; Zheng, Xiaojiao; Jia, Wei; Yin, Shankai

2015-01-01

Abstract Biomarker assessment is based on quantifying several proteins and metabolites. Recent developments in proteomics and metabolomics have enabled detection of these small molecules in biological samples and exploration of the underlying disease mechanisms in obstructive sleep apnea (OSA). This systemic review was performed to identify biomarkers, which were only detected by chromatography and/or mass spectrometry (MS) and to discuss the role of these biomarkers in the field of OSA. We systemically reviewed relevant articles from PubMed and EMBASE referring to proteins and metabolite profiles of biological samples in patients with OSA. The analytical platforms in this review were focused on chromatography and/or MS. In total, 30 studies evaluating biomarkers in patients with OSA using chromatography and/or MS methods were included. Numerous proteins and metabolites, including lipid profiles, adrenergic/dopaminergic biomarkers and derivatives, amino acids, oxidative stress biomarkers, and other micromolecules were identified in patients with OSA. Applying chromatography and/or MS methods to detect biomarkers helps develop an understanding of OSA mechanisms. More proteomic and metabolomic studies are warranted to develop potential diagnostic and clinical monitoring methods for OSA. PMID:26448002

Proteomic investigation of protein profile changes and amino acid residue-level modification in cooked lamb longissimus thoracis et lumborum: The effect of roasting.

PubMed

Yu, Tzer-Yang; Morton, James D; Clerens, Stefan; Dyer, Jolon M

2016-09-01

Protein modifications of meat cooked by typical dry-heat methods (e.g., roasting) are currently not well understood. The present study utilised a shotgun proteomic approach to examine the molecular-level effect of roasting on thin lamb longissimus thoracis et lumborum patties, in terms of changes to both the protein profile and amino acid residue side-chain modifications. Cooking caused aggregation of actin, myosin heavy chains and sarcoplasmic proteins. Longer roasting time resulted in significantly reduced protein extractability as well as protein truncation involving particularly a number of myofibrillar and sarcoplasmic proteins, e.g., 6-phosphofructokinase, beta-enolase, l-lactate dehydrogenase A chain, alpha-actinin-3, actin and possibly myosin heavy chains. Modifications that have potential influence on nutritional properties, including carboxyethyllysine and a potentially glucose-derived N-terminal Amadori compound, were observed in actin and myoglobin after roasting. This study provided new insights into molecular changes resulting from the dry-heat treatment of meat, such as commonly used in food preparation. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

PubMed

Wang, Xin; Komatsu, Setsuko

2016-06-30

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

Functional Proteomic Profiling of Secreted Serine Proteases in Health and Inflammatory Bowel Disease.

PubMed

Denadai-Souza, Alexandre; Bonnart, Chrystelle; Tapias, Núria Solà; Marcellin, Marlène; Gilmore, Brendan; Alric, Laurent; Bonnet, Delphine; Burlet-Schiltz, Odile; Hollenberg, Morley D; Vergnolle, Nathalie; Deraison, Céline

2018-05-18

While proteases are essential in gastrointestinal physiology, accumulating evidence indicates that dysregulated proteolysis plays a pivotal role in the pathophysiology of inflammatory bowel disease (IBD). Nonetheless, the identity of overactive proteases released by human colonic mucosa remains largely unknown. Studies of protease abundance have primarily investigated expression profiles, not taking into account their enzymatic activity. Herein we have used serine protease-targeted activity-based probes (ABPs) coupled with mass spectral analysis to identify active forms of proteases secreted by the colonic mucosa of healthy controls and IBD patients. Profiling of (Pro-Lys)-ABP bound proteases revealed that most of hyperactive proteases from IBD secretome are clustered at 28-kDa. We identified seven active proteases: the serine proteases cathepsin G, plasma kallikrein, plasmin, tryptase, chymotrypsin-like elastase 3 A, and thrombin and the aminopeptidase B. Only cathepsin G and thrombin were overactive in supernatants from IBD patient tissues compared to healthy controls. Gene expression analysis highlighted the transcription of genes encoding these proteases into intestinal mucosae. The functional ABP-targeted proteomic approach that we have used to identify active proteases in human colonic samples bears directly on the understanding of the role these enzymes may play in the pathophysiology of IBD.

Comparative proteomic profiling and possible toxicological mechanism of acute injury induced by carbon ion radiation in pubertal mice testes

NASA Astrophysics Data System (ADS)

Zhang, Hong

2016-07-01

We investigated potential mechanisms of acute injury in pubertal mice testes after exposure to carbon ion radiation (CIR). Serum testosterone was measured following whole-body irradiation with a 2Gy carbon ion beam. Comparative proteomic profiling and Western blotting were applied to identify potential biomarkers and measure protein expression, and terminal dUTP nick end-labeling (TUNEL) was performed to detect apoptotic cells. Immunohistochemistry and immunofluorescence were used to investigate protein localization. Serum testosterone was lowest at 24h after CIR, and 10 differentially expressed proteins were identified at this time point that included eIF4E, an important regulator of initiation that combines with mTOR and 4EBP1 to control protein synthesis via the mTOR signalling pathway during proliferation and apoptosis. Protein expression and localization studies confirmed their association with acute injury following exposure to CIR. These three proteins may be useful molecular markers for detecting abnormal spermatogenesis following exposure to environmental and cosmic radiation

Cell- and Tissue-based Proteome Profiling and Dual Imaging of Apoptosis Markers with Probes Derived from Venetoclax and Idasanutlin.

PubMed

Li, Zhengqiu; Zhu, Dongsheng; Guo, Haijun; Chang, Yu; Ni, Yun; Li, Lin; Hao, Piliang; Xu, Yong; Ding, Ke

2018-05-16

Venetoclax (ABT-199) and idasanutlin (RG7388) are efficient anticancer drugs targeting two essential apoptosis markers, Bcl2 and MDM2, respectively. Recent studies have shown that the combination of these two drugs leads to remarkable enhancement of anticancer efficacy, both in vitro and in vivo. In an attempt to understand the mechanism of this synergistic effect, competitive affinity-based proteome profiling coupled with bioimaging was employed to characterize their protein targets in the same cancer cell line and tumor tissue. A series of protein hits, including ITPR1, GSR, RER1, PDIA3, Apoa1 and Tnfrsf17 were simultaneously identified by pull-down/LC-MS/MS with the two sets of affinity-based probes. Dual imaging was successfully carried out, simultaneously detecting Bcl2 and MDM2 expression in various cancer cells. This could facilitate the novel diagnostic and therapeutic strategies of dual targeting of Bcl2/MDM2. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Functional proteomics outlines the complexity of breast cancer molecular subtypes.

PubMed

Gámez-Pozo, Angelo; Trilla-Fuertes, Lucía; Berges-Soria, Julia; Selevsek, Nathalie; López-Vacas, Rocío; Díaz-Almirón, Mariana; Nanni, Paolo; Arevalillo, Jorge M; Navarro, Hilario; Grossmann, Jonas; Gayá Moreno, Francisco; Gómez Rioja, Rubén; Prado-Vázquez, Guillermo; Zapater-Moros, Andrea; Main, Paloma; Feliú, Jaime; Martínez Del Prado, Purificación; Zamora, Pilar; Ciruelos, Eva; Espinosa, Enrique; Fresno Vara, Juan Ángel

2017-08-30

Breast cancer is a heterogeneous disease comprising a variety of entities with various genetic backgrounds. Estrogen receptor-positive, human epidermal growth factor receptor 2-negative tumors typically have a favorable outcome; however, some patients eventually relapse, which suggests some heterogeneity within this category. In the present study, we used proteomics and miRNA profiling techniques to characterize a set of 102 either estrogen receptor-positive (ER+)/progesterone receptor-positive (PR+) or triple-negative formalin-fixed, paraffin-embedded breast tumors. Protein expression-based probabilistic graphical models and flux balance analyses revealed that some ER+/PR+ samples had a protein expression profile similar to that of triple-negative samples and had a clinical outcome similar to those with triple-negative disease. This probabilistic graphical model-based classification had prognostic value in patients with luminal A breast cancer. This prognostic information was independent of that provided by standard genomic tests for breast cancer, such as MammaPrint, OncoType Dx and the 8-gene Score.

Cochliobolus lunatus down-regulates proteome at late stage of colonization and transiently alters StNPR1 expression in Solanum tuberosum L.

PubMed

Louis, Bengyella; Waikhom, Sayanika D; Jose, Robinson C; Goyari, Sailendra; Bhardwaj, Pardeep Kumar; Talukdar, Narayan C; Roy, Pranab

2017-03-01

Cochliobolus lunatus abundantly produces four-celled conidia at high temperatures (>30 °C) and under suitable conditions; the fungus colonizes potato (Solanum tuberosum L.) cultivars by adopting different invasion strategies at the microscopic level. Long-lasting defence during infection requires an upsurge in proteome changes particularly pathogenesis-related proteins chiefly under the control of nonexpresser of pathogenesis-related proteins. In order to gain molecular insights, we profiled the changes in proteome and potato nonexpresser of pathogenesis-related proteins (StNPR1) during the infection process. It is found that C. lunatus significantly (P < 0.05) suppressed the host functional proteome by 96 h after infection (hai), principally, affecting the expression of ribulose bisphosphate carboxylase enzyme, plastidic aldolase enzyme, alcohol dehydrogenase 2 and photosystem II protein prior to the formation of brown-to-black leaf spot disease. Strongest host response was observed at 24 hai hallmarked by 307 differentially expressed peptide spots concurring with the active phase of production of penetrating hyphae. Additionally, C. lunatus differentially down-regulated StNPR1 transcript by 8.19 fold by 24 hai. This study is the first to elucidate that C. lunatus transiently down-regulates the expression of StNPR1 at the onset of infection, and as a whole, infection negatively affects the expression of proteome components involved in photosynthesis, carbon fixation and light assimilation. This study contributes towards better understanding of the mechanism underlining the invasion strategies of C. lunatus.

The day/night proteome in the murine heart.

PubMed

Podobed, Peter; Pyle, W Glen; Ackloo, Suzanne; Alibhai, Faisal J; Tsimakouridze, Elena V; Ratcliffe, William F; Mackay, Allison; Simpson, Jeremy; Wright, David C; Kirby, Gordon M; Young, Martin E; Martino, Tami A

2014-07-15

Circadian rhythms are essential to cardiovascular health and disease. Temporal coordination of cardiac structure and function has focused primarily at the physiological and gene expression levels, but these analyses are invariably incomplete, not the least because proteins underlie many biological processes. The purpose of this study was to reveal the diurnal cardiac proteome and important contributions to cardiac function. The 24-h day-night murine cardiac proteome was assessed by two-dimensional difference in gel electrophoresis (2D-DIGE) and liquid chromatography-mass spectrometry. Daily variation was considerable, as ∼7.8% (90/1,147) of spots exhibited statistical changes at paired times across the 24-h light- (L) dark (D) cycle. JTK_CYCLE was used to investigate underlying diurnal rhythms in corresponding mRNA. We next revealed that disruption of the L:D cycle altered protein profiles and diurnal variation in cardiac function in Langendorff-perfused hearts, relative to the L:D cycle. To investigate the role of the circadian clock mechanism, we used cardiomyocyte clock mutant (CCM) mice. CCM myofilaments exhibited a loss of time-of-day-dependent maximal calcium-dependent ATP consumption, and altered phosphorylation rhythms. Moreover, the cardiac proteome was significantly altered in CCM hearts, especially enzymes regulating vital metabolic pathways. Lastly, we used a model of pressure overload cardiac hypertrophy to demonstrate the temporal proteome during heart disease. Our studies demonstrate that time of day plays a direct role in cardiac protein abundance and indicate a novel mechanistic contribution of circadian biology to cardiovascular structure and function.

Proteomic profiling of bone marrow mesenchymal stem cells upon TGF-beta stimulation

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

Wang, Daojing; Park, Jennifer S.; Chu, Julia S.F.

Bone marrow mesenchymal stem cells (MSCs) can differentiate into different types of cells, and have tremendous potential for cell therapy and tissue engineering. Transforming growth factor {beta}1 (TGF-{beta}) plays an important role in cell differentiation and vascular remodeling. We showed that TGF-{beta} induced cell morphology change and an increase in actin fibers in MSCs. To determine the global effects of TGF-{beta} on MSCs, we employed a proteomic strategy to analyze the effect of TGF-{beta} on the human MSC proteome. By using two-dimensional gel electrophoresis and electrospray ionization coupled to Quadrupole/time-of-flight tandem mass spectrometers, we have generated a proteome reference mapmore » of MSCs, and identified {approx}30 proteins with an increase or decrease in expression or phosphorylation in response to TGF-{beta}. The proteins regulated by TGF-{beta} included cytoskeletal proteins, matrix synthesis proteins, membrane proteins, metabolic enzymes, etc. TGF-{beta} increased the expression of smooth muscle (SM) {alpha}-actin and decreased the expression of gelsolin. Over-expression of gelsolin inhibited TGF-{beta}-induced assembly of SM {alpha}-actin; on the other hand, knocking down gelsolin expression enhanced the assembly of {alpha}-actin and actin filaments without significantly affecting {alpha}-actin expression. These results suggest that TGF-{beta} coordinates the increase of {alpha}-actin and the decrease of gelsolin to promote MSC differentiation. This study demonstrates that proteomic tools are valuable in studying stem cell differentiation and elucidating the underlying molecular mechanisms.« less

The day/night proteome in the murine heart

PubMed Central

Podobed, Peter; Pyle, W. Glen; Ackloo, Suzanne; Alibhai, Faisal J.; Tsimakouridze, Elena V.; Ratcliffe, William F.; Mackay, Allison; Simpson, Jeremy; Wright, David C.; Kirby, Gordon M.; Young, Martin E.

2014-01-01

Circadian rhythms are essential to cardiovascular health and disease. Temporal coordination of cardiac structure and function has focused primarily at the physiological and gene expression levels, but these analyses are invariably incomplete, not the least because proteins underlie many biological processes. The purpose of this study was to reveal the diurnal cardiac proteome and important contributions to cardiac function. The 24-h day-night murine cardiac proteome was assessed by two-dimensional difference in gel electrophoresis (2D-DIGE) and liquid chromatography-mass spectrometry. Daily variation was considerable, as ∼7.8% (90/1,147) of spots exhibited statistical changes at paired times across the 24-h light- (L) dark (D) cycle. JTK_CYCLE was used to investigate underlying diurnal rhythms in corresponding mRNA. We next revealed that disruption of the L:D cycle altered protein profiles and diurnal variation in cardiac function in Langendorff-perfused hearts, relative to the L:D cycle. To investigate the role of the circadian clock mechanism, we used cardiomyocyte clock mutant (CCM) mice. CCM myofilaments exhibited a loss of time-of-day-dependent maximal calcium-dependent ATP consumption, and altered phosphorylation rhythms. Moreover, the cardiac proteome was significantly altered in CCM hearts, especially enzymes regulating vital metabolic pathways. Lastly, we used a model of pressure overload cardiac hypertrophy to demonstrate the temporal proteome during heart disease. Our studies demonstrate that time of day plays a direct role in cardiac protein abundance and indicate a novel mechanistic contribution of circadian biology to cardiovascular structure and function. PMID:24789993

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

Statistical issues in the design and planning of proteomic profiling experiments.

PubMed

Cairns, David A

2015-01-01

The statistical design of a clinical proteomics experiment is a critical part of well-undertaken investigation. Standard concepts from experimental design such as randomization, replication and blocking should be applied in all experiments, and this is possible when the experimental conditions are well understood by the investigator. The large number of proteins simultaneously considered in proteomic discovery experiments means that determining the number of required replicates to perform a powerful experiment is more complicated than in simple experiments. However, by using information about the nature of an experiment and making simple assumptions this is achievable for a variety of experiments useful for biomarker discovery and initial validation.

Comprehensive Analysis of the Triterpenoid Saponins Biosynthetic Pathway in Anemone flaccida by Transcriptome and Proteome Profiling

PubMed Central

Zhan, Chuansong; Li, Xiaohua; Zhao, Zeying; Yang, Tewu; Wang, Xuekui; Luo, Biaobiao; Zhang, Qiyun; Hu, Yanru; Hu, Xuebo

2016-01-01

Background: Anemone flaccida Fr. Shmidt (Ranunculaceae), commonly known as ‘Di Wu’ in China, is a perennial herb with limited distribution. The rhizome of A. flaccida has long been used to treat arthritis as a tradition in China. Studies disclosed that the plant contains a rich source of triterpenoid saponins. However, little is known about triterpenoid saponins biosynthesis in A. flaccida. Results: In this study, we conducted the tandem transcriptome and proteome profiling of a non-model medicinal plant, A. flaccida. Using Illumina HiSeq 2000 sequencing and iTRAQ technique, a total of 46,962 high-quality unigenes were obtained with an average sequence length of 1,310 bp, along with 1473 unique proteins from A. flaccida. Among the A. flaccida transcripts, 36,617 (77.97%) showed significant similarity (E-value < 1e-5) to the known proteins in the public database. Of the total 46,962 unigenes, 36,617 open reading frame (ORFs) were predicted. By the fragments per kilobases per million reads (FPKM) statistics, 14,004 isoforms/unigenes were found to be upregulated, and 14,090 isoforms/unigenes were down-regulated in the rhizomes as compared to those in the leaves. Based on the bioinformatics analysis, all possible enzymes involved in the triterpenoid saponins biosynthetic pathway of A. flaccida were identified, including cytosolic mevalonate pathway (MVA) and the plastidial methylerythritol pathway (MEP). Additionally, a total of 126 putative cytochrome P450 (CYP450) and 32 putative UDP glycosyltransferases were selected as the candidates of triterpenoid saponins modifiers. Among them, four of them were annotated as the gene of CYP716A subfamily, the key enzyme in the oleanane-type triterpenoid saponins biosynthetic pathway. Furthermore, based on RNA-Seq and proteome analysis, as well as quantitative RT-PCR verification, the expression level of gene and protein committed to triterpenoids biosynthesis in the leaf versus the rhizome was compared. Conclusion: A combination of the de novo transcriptome and proteome profiling based on the Illumina HiSeq 2000 sequencing platform and iTRAQ technique was shown to be a powerful method for the discovery of candidate genes, which encoded enzymes that were responsible for the biosynthesis of novel secondary metabolites in a non-model plant. The transcriptome data of our study provides a very important resource for the understanding of the triterpenoid saponins biosynthesis of A. flaccida. PMID:27504115

Cumulative effects of the ApoE genotype and gender on the synaptic proteome and oxidative stress in the mouse brain.

PubMed

Shi, Lv; Du, Xin; Zhou, Hong; Tao, Changlu; Liu, Yuntao; Meng, Fantao; Wu, Gao; Xiong, Ying; Xia, Chun; Wang, Yu; Bi, Guoqiang; Zhou, Jiang-Ning

2014-11-01

Elderly females, particularly those carrying the apolipoprotein E (ApoE)-ε4 allele, have a higher risk of developing Alzheimer's disease (AD). However, the underlying mechanism for this increased susceptibility remains unclear. In this study, we investigated the effects of the ApoE genotype and gender on the proteome of synaptosomes. We isolated synaptosomes and used label-free quantitative proteomics, to report, for the first time, that the synaptosomal proteomic profiles in the cortex of female human-ApoE4 mice exhibited significantly reduced expression of proteins related to energy metabolism, which was accompanied by increased levels of oxidative stress. In addition, we also first demonstrated that the proteomic response in synaptic termini was more susceptible than that in the soma to the adverse effects induced by genders and genotypes. This suggests that synaptic mitochondria might be 'older' than mitochondria in the soma of neurons; therefore, they might contain increased cumulative damage from oxidative stress. Furthermore, female human-ApoE4 mice had much lower oestrogen levels in the cortex and treatment with oestrogen protected ApoE3 stable transfected C6 neurons from oxidative stress. Overall, this study reveals complex ApoE- and gender-dependent effects on synaptic function and also provides a basis for future studies of candidates based on specific pathways involved in the pathogenesis of AD. The lack of oestrogen-mediated protection regulated by the ApoE genotype led to synaptic mitochondrial dysfunction and increased oxidative stress, which might make older females more susceptible to AD.

Long-term in vivo polychlorinated biphenyl 126 exposure induces oxidative stress and alters proteomic profile on islets of Langerhans

NASA Astrophysics Data System (ADS)

Loiola, Rodrigo Azevedo; Dos Anjos, Fabyana Maria; Shimada, Ana Lúcia; Cruz, Wesley Soares; Drewes, Carine Cristiane; Rodrigues, Stephen Fernandes; Cardozo, Karina Helena Morais; Carvalho, Valdemir Melechco; Pinto, Ernani; Farsky, Sandra Helena

2016-06-01

It has been recently proposed that exposure to polychlorinated biphenyls (PCBs) is a risk factor to type 2 diabetes mellitus (DM2). We investigated this hypothesis using long-term in vivo PCB126 exposure to rats addressing metabolic, cellular and proteomic parameters. Male Wistar rats were exposed to PCB126 (0.1, 1 or 10 μg/kg of body weight/day; for 15 days) or vehicle by intranasal instillation. Systemic alterations were quantified by body weight, insulin and glucose tolerance, and blood biochemical profile. Pancreatic toxicity was measured by inflammatory parameters, cell viability and cycle, free radical generation, and proteomic profile on islets of Langerhans. In vivo PCB126 exposure enhanced the body weight gain, impaired insulin sensitivity, reduced adipose tissue deposit, and elevated serum triglycerides, cholesterol, and insulin levels. Inflammatory parameters in the pancreas and cell morphology, viability and cycle were not altered in islets of Langerhans. Nevertheless, in vivo PCB126 exposure increased free radical generation and modified the expression of proteins related to oxidative stress on islets of Langerhans, which are indicative of early β-cell failure. Data herein obtained show that long-term in vivo PCB126 exposure through intranasal route induced alterations on islets of Langerhans related to early end points of DM2.

Highly multiplexed and quantitative cell-surface protein profiling using genetically barcoded antibodies.

PubMed

Pollock, Samuel B; Hu, Amy; Mou, Yun; Martinko, Alexander J; Julien, Olivier; Hornsby, Michael; Ploder, Lynda; Adams, Jarrett J; Geng, Huimin; Müschen, Markus; Sidhu, Sachdev S; Moffat, Jason; Wells, James A

2018-03-13

Human cells express thousands of different surface proteins that can be used for cell classification, or to distinguish healthy and disease conditions. A method capable of profiling a substantial fraction of the surface proteome simultaneously and inexpensively would enable more accurate and complete classification of cell states. We present a highly multiplexed and quantitative surface proteomic method using genetically barcoded antibodies called phage-antibody next-generation sequencing (PhaNGS). Using 144 preselected antibodies displayed on filamentous phage (Fab-phage) against 44 receptor targets, we assess changes in B cell surface proteins after the development of drug resistance in a patient with acute lymphoblastic leukemia (ALL) and in adaptation to oncogene expression in a Myc-inducible Burkitt lymphoma model. We further show PhaNGS can be applied at the single-cell level. Our results reveal that a common set of proteins including FLT3, NCR3LG1, and ROR1 dominate the response to similar oncogenic perturbations in B cells. Linking high-affinity, selective, genetically encoded binders to NGS enables direct and highly multiplexed protein detection, comparable to RNA-sequencing for mRNA. PhaNGS has the potential to profile a substantial fraction of the surface proteome simultaneously and inexpensively to enable more accurate and complete classification of cell states. Copyright © 2018 the Author(s). Published by PNAS.

Proteomic analysis of hair shafts from monozygotic twins: Expression profiles and genetically variant peptides.

PubMed

Wu, Pei-Wen; Mason, Katelyn E; Durbin-Johnson, Blythe P; Salemi, Michelle; Phinney, Brett S; Rocke, David M; Parker, Glendon J; Rice, Robert H

2017-07-01

Forensic association of hair shaft evidence with individuals is currently assessed by comparing mitochondrial DNA haplotypes of reference and casework samples, primarily for exclusionary purposes. Present work tests and validates more recent proteomic approaches to extract quantitative transcriptional and genetic information from hair samples of monozygotic twin pairs, which would be predicted to partition away from unrelated individuals if the datasets contain identifying information. Protein expression profiles and polymorphic, genetically variant hair peptides were generated from ten pairs of monozygotic twins. Profiling using the protein tryptic digests revealed that samples from identical twins had typically an order of magnitude fewer protein expression differences than unrelated individuals. The data did not indicate that the degree of difference within twin pairs increased with age. In parallel, data from the digests were used to detect genetically variant peptides that result from common nonsynonymous single nucleotide polymorphisms in genes expressed in the hair follicle. Compilation of the variants permitted sorting of the samples by hierarchical clustering, permitting accurate matching of twin pairs. The results demonstrate that genetic differences are detectable by proteomic methods and provide a framework for developing quantitative statistical estimates of personal identification that increase the value of hair shaft evidence. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

MEGGASENSE - The Metagenome/Genome Annotated Sequence Natural Language Search Engine: A Platform for 
the Construction of Sequence Data Warehouses.

PubMed

Gacesa, Ranko; Zucko, Jurica; Petursdottir, Solveig K; Gudmundsdottir, Elisabet Eik; Fridjonsson, Olafur H; Diminic, Janko; Long, Paul F; Cullum, John; Hranueli, Daslav; Hreggvidsson, Gudmundur O; Starcevic, Antonio

2017-06-01

The MEGGASENSE platform constructs relational databases of DNA or protein sequences. The default functional analysis uses 14 106 hidden Markov model (HMM) profiles based on sequences in the KEGG database. The Solr search engine allows sophisticated queries and a BLAST search function is also incorporated. These standard capabilities were used to generate the SCATT database from the predicted proteome of Streptomyces cattleya . The implementation of a specialised metagenome database (AMYLOMICS) for bioprospecting of carbohydrate-modifying enzymes is described. In addition to standard assembly of reads, a novel 'functional' assembly was developed, in which screening of reads with the HMM profiles occurs before the assembly. The AMYLOMICS database incorporates additional HMM profiles for carbohydrate-modifying enzymes and it is illustrated how the combination of HMM and BLAST analyses helps identify interesting genes. A variety of different proteome and metagenome databases have been generated by MEGGASENSE.

Global Membrane Protein Interactome Analysis using In vivo Crosslinking and Mass Spectrometry-based Protein Correlation Profiling*

PubMed Central

Larance, Mark; Kirkwood, Kathryn J.; Tinti, Michele; Brenes Murillo, Alejandro; Ferguson, Michael A. J.; Lamond, Angus I.

2016-01-01

We present a methodology using in vivo crosslinking combined with HPLC-MS for the global analysis of endogenous protein complexes by protein correlation profiling. Formaldehyde crosslinked protein complexes were extracted with high yield using denaturing buffers that maintained complex solubility during chromatographic separation. We show this efficiently detects both integral membrane and membrane-associated protein complexes,in addition to soluble complexes, allowing identification and analysis of complexes not accessible in native extracts. We compare the protein complexes detected by HPLC-MS protein correlation profiling in both native and formaldehyde crosslinked U2OS cell extracts. These proteome-wide data sets of both in vivo crosslinked and native protein complexes from U2OS cells are freely available via a searchable online database (www.peptracker.com/epd). Raw data are also available via ProteomeXchange (identifier PXD003754). PMID:27114452

Oxidative stress in aging: advances in proteomic approaches.

PubMed

Ortuño-Sahagún, Daniel; Pallàs, Mercè; Rojas-Mayorquín, Argelia E

2014-01-01

Aging is a gradual, complex process in which cells, tissues, organs, and the whole organism itself deteriorate in a progressive and irreversible manner that, in the majority of cases, implies pathological conditions that affect the individual's Quality of Life (QOL). Although extensive research efforts in recent years have been made, the anticipation of aging and prophylactic or treatment strategies continue to experience major limitations. In this review, the focus is essentially on the compilation of the advances generated by cellular expression profile analysis through proteomics studies (two-dimensional [2D] electrophoresis and mass spectrometry [MS]), which are currently used as an integral approach to study the aging process. Additionally, the relevance of the oxidative stress factors is discussed. Emphasis is placed on postmitotic tissues, such as neuronal, muscular, and red blood cells, which appear to be those most frequently studied with respect to aging. Additionally, models for the study of aging are discussed in a number of organisms, such as Caenorhabditis elegans, senescence-accelerated probe-8 mice (SAMP8), naked mole-rat (Heterocephalus glaber), and the beagle canine. Proteomic studies in specific tissues and organisms have revealed the extensive involvement of reactive oxygen species (ROS) and oxidative stress in aging.

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

Two independent proteomic approaches provide a comprehensive analysis of the synovial fluid proteome response to Autologous Chondrocyte Implantation.

PubMed

Hulme, Charlotte H; Wilson, Emma L; Fuller, Heidi R; Roberts, Sally; Richardson, James B; Gallacher, Pete; Peffers, Mandy J; Shirran, Sally L; Botting, Catherine H; Wright, Karina T

2018-05-02

Autologous chondrocyte implantation (ACI) has a failure rate of approximately 20%, but it is yet to be fully understood why. Biomarkers are needed that can pre-operatively predict in which patients it is likely to fail, so that alternative or individualised therapies can be offered. We previously used label-free quantitation (LF) with a dynamic range compression proteomic approach to assess the synovial fluid (SF) of ACI responders and non-responders. However, we were able to identify only a few differentially abundant proteins at baseline. In the present study, we built upon these previous findings by assessing higher-abundance proteins within this SF, providing a more global proteomic analysis on the basis of which more of the biology underlying ACI success or failure can be understood. Isobaric tagging for relative and absolute quantitation (iTRAQ) proteomic analysis was used to assess SF from ACI responders (mean Lysholm improvement of 33; n = 14) and non-responders (mean Lysholm decrease of 14; n = 13) at the two stages of surgery (cartilage harvest and chondrocyte implantation). Differentially abundant proteins in iTRAQ and combined iTRAQ and LF datasets were investigated using pathway and network analyses. iTRAQ proteomic analysis confirmed our previous finding that there is a marked proteomic shift in response to cartilage harvest (70 and 54 proteins demonstrating ≥ 2.0-fold change and p < 0.05 between stages I and II in responders and non-responders, respectively). Further, it highlighted 28 proteins that were differentially abundant between responders and non-responders to ACI, which were not found in the LF study, 16 of which were altered at baseline. The differential expression of two proteins (complement C1s subcomponent and matrix metalloproteinase 3) was confirmed biochemically. Combination of the iTRAQ and LF proteomic datasets generated in-depth SF proteome information that was used to generate interactome networks representing ACI success or failure. Functional pathways that are dysregulated in ACI non-responders were identified, including acute-phase response signalling. Several candidate biomarkers for baseline prediction of ACI outcome were identified. A holistic overview of the SF proteome in responders and non-responders to ACI  has been profiled, providing a better understanding of the biological pathways underlying clinical outcome, particularly the differential response to cartilage harvest in non-responders.

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.

Proteomics in pharmaceutical research and development.

PubMed

Cutler, Paul; Voshol, Hans

2015-08-01

In the 20 years since its inception, the evolution of proteomics in pharmaceutical industry has mirrored the developments within academia and indeed other industries. From initial enthusiasm and subsequent disappointment in global protein expression profiling, pharma research saw the biggest impact when relating to more focused approaches, such as those exploring the interaction between proteins and drugs. Nowadays, proteomics technologies have been integrated in many areas of pharmaceutical R&D, ranging from the analysis of therapeutic proteins to the monitoring of clinical trials. Here, we review the development of proteomics in the drug discovery process, placing it in a historical context as well as reviewing the current status in light of the contributions to this special issue, which reflect some of the diverse demands of the drug and biomarker pipelines. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Feed efficiency and the liver proteome of fattening lambs are modified by feed restriction during the suckling period.

PubMed

Santos, A; Valdés, C; Giráldez, F J; López, S; France, J; Frutos, J; Fernández, M; Andrés, S

2018-01-24

The present study was designed to describe the effects of early feed restriction of Merino lambs on feed efficiency during the fattening period by examining ruminal microbiota and fermentation parameters, gastrointestinal morphology, digestibility or liver proteome. In total, 24 male Merino lambs were randomly assigned to two experimental treatments (n=12 per treatment). Lambs of the first group (ad libitum (ADL)) were kept permanently with the dams, whereas the other 12 lambs (restricted (RES)) were milk restricted. When lambs reached a live BW (LBW) of 15 kg, all the animals were offered the same complete pelleted diet (35 g dry matter/kg LBW per day) until slaughter at a LBW of 27 kg. The RES lambs showed poorer feed efficiency during the fattening period when compared with the ADL group (feed to gain ratio, 3.69 v. 3.05, P<0.001). No differences were observed in ruminal microbiota, fermentation parameters or apparent digestibility. However, the proportion of the small intestine and the length of ileal villi were reduced in the RES lambs. In total, 26 spots/proteins were identified in the liver proteomic profile, with significant differences (P<0.05) between experimental treatments, suggesting a higher catabolism of proteins and a reduction in β-oxidation of fatty acids in RES lambs when compared with the ADL animals. In conclusion, early feed restriction of Merino lambs during the suckling period promotes long-term effects on the small intestine and the proteomic profile of the liver, which may influence the metabolic use of nutrients, thus negatively affecting feed efficiency during the fattening phase.

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.

Quantitative proteomic analysis of human lung tumor xenografts treated with the ectopic ATP synthase inhibitor citreoviridin.

PubMed

Wu, Yi-Hsuan; Hu, Chia-Wei; Chien, Chih-Wei; Chen, Yu-Ju; Huang, Hsuan-Cheng; Juan, Hsueh-Fen

2013-01-01

ATP synthase is present on the plasma membrane of several types of cancer cells. Citreoviridin, an ATP synthase inhibitor, selectively suppresses the proliferation and growth of lung cancer without affecting normal cells. However, the global effects of targeting ectopic ATP synthase in vivo have not been well defined. In this study, we performed quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ) and provided a comprehensive insight into the complicated regulation by citreoviridin in a lung cancer xenograft model. With high reproducibility of the quantitation, we obtained quantitative proteomic profiling with 2,659 proteins identified. Bioinformatics analysis of the 141 differentially expressed proteins selected by their relative abundance revealed that citreoviridin induces alterations in the expression of glucose metabolism-related enzymes in lung cancer. The up-regulation of enzymes involved in gluconeogenesis and storage of glucose indicated that citreoviridin may reduce the glycolytic intermediates for macromolecule synthesis and inhibit cell proliferation. Using comprehensive proteomics, the results identify metabolic aspects that help explain the antitumorigenic effect of citreoviridin in lung cancer, which may lead to a better understanding of the links between metabolism and tumorigenesis in cancer therapy.

MAPA distinguishes genotype-specific variability of highly similar regulatory protein isoforms in potato tuber.

PubMed

Hoehenwarter, Wolfgang; Larhlimi, Abdelhalim; Hummel, Jan; Egelhofer, Volker; Selbig, Joachim; van Dongen, Joost T; Wienkoop, Stefanie; Weckwerth, Wolfram

2011-07-01

Mass Accuracy Precursor Alignment is a fast and flexible method for comparative proteome analysis that allows the comparison of unprecedented numbers of shotgun proteomics analyses on a personal computer in a matter of hours. We compared 183 LC-MS analyses and more than 2 million MS/MS spectra and could define and separate the proteomic phenotypes of field grown tubers of 12 tetraploid cultivars of the crop plant Solanum tuberosum. Protein isoforms of patatin as well as other major gene families such as lipoxygenase and cysteine protease inhibitor that regulate tuber development were found to be the primary source of variability between the cultivars. This suggests that differentially expressed protein isoforms modulate genotype specific tuber development and the plant phenotype. We properly assigned the measured abundance of tryptic peptides to different protein isoforms that share extensive stretches of primary structure and thus inferred their abundance. Peptides unique to different protein isoforms were used to classify the remaining peptides assigned to the entire subset of isoforms based on a common abundance profile using multivariate statistical procedures. We identified nearly 4000 proteins which we used for quantitative functional annotation making this the most extensive study of the tuber proteome to date.

Quantitative Proteomic Analysis of Human Lung Tumor Xenografts Treated with the Ectopic ATP Synthase Inhibitor Citreoviridin

PubMed Central

Wu, Yi-Hsuan; Hu, Chia-Wei; Chien, Chih-Wei; Chen, Yu-Ju; Huang, Hsuan-Cheng; Juan, Hsueh-Fen

2013-01-01

ATP synthase is present on the plasma membrane of several types of cancer cells. Citreoviridin, an ATP synthase inhibitor, selectively suppresses the proliferation and growth of lung cancer without affecting normal cells. However, the global effects of targeting ectopic ATP synthase in vivo have not been well defined. In this study, we performed quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ) and provided a comprehensive insight into the complicated regulation by citreoviridin in a lung cancer xenograft model. With high reproducibility of the quantitation, we obtained quantitative proteomic profiling with 2,659 proteins identified. Bioinformatics analysis of the 141 differentially expressed proteins selected by their relative abundance revealed that citreoviridin induces alterations in the expression of glucose metabolism-related enzymes in lung cancer. The up-regulation of enzymes involved in gluconeogenesis and storage of glucose indicated that citreoviridin may reduce the glycolytic intermediates for macromolecule synthesis and inhibit cell proliferation. Using comprehensive proteomics, the results identify metabolic aspects that help explain the antitumorigenic effect of citreoviridin in lung cancer, which may lead to a better understanding of the links between metabolism and tumorigenesis in cancer therapy. PMID:23990911

Systematic Proteomic Approach to Characterize the Impacts of ...

EPA Pesticide Factsheets

Chemical interactions have posed a big challenge in toxicity characterization and human health risk assessment of environmental mixtures. To characterize the impacts of chemical interactions on protein and cytotoxicity responses to environmental mixtures, we established a systems biology approach integrating proteomics, bioinformatics, statistics, and computational toxicology to measure expression or phosphorylation levels of 21 critical toxicity pathway regulators and 445 downstream proteins in human BEAS-28 cells treated with 4 concentrations of nickel, 2 concentrations each of cadmium and chromium, as well as 12 defined binary and 8 defined ternary mixtures of these metals in vitro. Multivariate statistical analysis and mathematical modeling of the metal-mediated proteomic response patterns showed a high correlation between changes in protein expression or phosphorylation and cellular toxic responses to both individual metals and metal mixtures. Of the identified correlated proteins, only a small set of proteins including HIF-1a is likely to be responsible for selective cytotoxic responses to different metals and metals mixtures. Furthermore, support vector machine learning was utilized to computationally predict protein responses to uncharacterized metal mixtures using experimentally generated protein response profiles corresponding to known metal mixtures. This study provides a novel proteomic approach for characterization and prediction of toxicities of

Proteomics analysis of melanoma metastases: association between S100A13 expression and chemotherapy resistance

PubMed Central

Azimi, A; Pernemalm, M; Frostvik Stolt, M; Hansson, J; Lehtiö, J; Egyházi Brage, S; Hertzman Johansson, C

2014-01-01

Background: Disseminated cutaneous malignant melanoma (CMM) is commonly unresponsive to standard chemotherapies, and there are as yet no predictive markers of therapy response. Methods: In the present study we collected fresh-frozen pretreatment lymph-node metastasis samples (n=14) from melanoma patients with differential response to dacarbazine (DTIC) or temozolomide (TMZ) chemotherapy, to identify proteins with an impact on treatment response. We performed quantitative protein profiling using tandem mass spectrometry and compared the proteome differences between responders (R) and non-responders (NR), matched for age, gender and histopathological type of CMM. Results: Biological pathway analyses showed several signalling pathways differing between R vs NR, including Rho signalling. Gene expression profiling data was available for a subset of the samples, and the results were compared with the proteomics data. Four proteins with differential expression between R and NR were selected for technical validation by immunoblotting (ISYNA1, F13A1, CSTB and S100A13), and CSTB and S100A13 were further validated on a larger sample set by immunohistochemistry (n=48). The calcium binding protein S100A13 was found to be significantly overexpressed in NR compared with R in all analyses performed. Conclusions: Our results suggest that S100A13 is involved in CMM resistance to DTIC/TMZ. PMID:24722184

Integrating Proteomics and Enzyme Kinetics Reveals Tissue-Specific Types of the Glycolytic and Gluconeogenic Pathways.

PubMed

Wiśniewski, Jacek R; Gizak, Agnieszka; Rakus, Dariusz

2015-08-07

Glycolysis is the core metabolic pathway supplying energy to cells. Whereas the vast majority of studies focus on specific aspects of the process, global analyses characterizing simultaneously all enzymes involved in the process are scarce. Here, we demonstrate that quantitative label- and standard-free proteomics allows accurate determination of titers of metabolic enzymes and enables simultaneous measurements of titers and maximal enzymatic activities (Amax) of all glycolytic enzymes and the gluconeogenic fructose 1,6-bisphosphatase in mouse brain, liver and muscle. Despite occurrence of tissue-specific isoenzymes bearing different kinetic properties, the enzyme titers often correlated well with the Amax values. To provide a more general picture of energy metabolism, we analyzed titers of the enzymes in additional 7 mouse organs and in human cells. Across the analyzed samples, we identified two basic profiles: a "fast glucose uptake" one in brain and heart, and a "gluconeogenic rich" one occurring in liver. In skeletal muscles and other organs, we found intermediate profiles. Obtained data highlighted the glucose-flux-limiting role of hexokinase which activity was always 10- to 100-fold lower than the average activity of all other glycolytic enzymes. A parallel determination of enzyme titers and maximal enzymatic activities allowed determination of kcat values without enzyme purification. Results of our in-depth proteomic analysis of the mouse organs did not support the concepts of regulation of glycolysis by lysine acetylation.

Surface modified capillary electrophoresis combined with in solution isoelectric focusing and MALDI-TOF/TOF MS: a gel-free multidimensional electrophoresis approach for proteomic profiling--exemplified on human follicular fluid.

PubMed

Hanrieder, Jörg; Zuberovic, Aida; Bergquist, Jonas

2009-04-24

Development of miniaturized analytical tools continues to be of great interest to face the challenges in proteomic analysis of complex biological samples such as human body fluids. In the light of these challenges, special emphasis is put on the speed and simplicity of newly designed technological approaches as well as the need for cost efficiency and low sample consumption. In this study, we present an alternative multidimensional bottom-up approach for proteomic profiling for fast, efficient and sensitive protein analysis in complex biological matrices. The presented setup was based on sample pre-fractionation using microscale in solution isoelectric focusing (IEF) followed by tryptic digestion and subsequent capillary electrophoresis (CE) coupled off-line to matrix assisted laser desorption/ionization time of flight tandem mass spectrometry (MALDI TOF MS/MS). For high performance CE-separation, PolyE-323 modified capillaries were applied to minimize analyte-wall interactions. The potential of the analytical setup was demonstrated on human follicular fluid (hFF) representing a typical complex human body fluid with clinical implication. The obtained results show significant identification of 73 unique proteins (identified at 95% significance level), including mostly acute phase proteins but also protein identities that are well known to be extensively involved in follicular development.

SELDI-TOF MS of quadruplicate urine and serum samples to evaluate changes related to storage conditions.

PubMed

Traum, Avram Z; Wells, Meghan P; Aivado, Manuel; Libermann, Towia A; Ramoni, Marco F; Schachter, Asher D

2006-03-01

Proteomic profiling with SELDI-TOF MS has facilitated the discovery of disease-specific protein profiles. However, multicenter studies are often hindered by the logistics required for prompt deep-freezing of samples in liquid nitrogen or dry ice within the clinic setting prior to shipping. We report high concordance between MS profiles within sets of quadruplicate split urine and serum samples deep-frozen at 0, 2, 6, and 24 h after sample collection. Gage R&R results confirm that deep-freezing times are not a statistically significant source of SELDI-TOF MS variability for either blood or urine.

Proteomics and Metabolomics: Two Emerging Areas for Legume Improvement

PubMed Central

Ramalingam, Abirami; Kudapa, Himabindu; Pazhamala, Lekha T.; Weckwerth, Wolfram; Varshney, Rajeev K.

2015-01-01

The crop legumes such as chickpea, common bean, cowpea, peanut, pigeonpea, soybean, etc. are important sources of nutrition and contribute to a significant amount of biological nitrogen fixation (>20 million tons of fixed nitrogen) in agriculture. However, the production of legumes is constrained due to abiotic and biotic stresses. It is therefore imperative to understand the molecular mechanisms of plant response to different stresses and identify key candidate genes regulating tolerance which can be deployed in breeding programs. The information obtained from transcriptomics has facilitated the identification of candidate genes for the given trait of interest and utilizing them in crop breeding programs to improve stress tolerance. However, the mechanisms of stress tolerance are complex due to the influence of multi-genes and post-transcriptional regulations. Furthermore, stress conditions greatly affect gene expression which in turn causes modifications in the composition of plant proteomes and metabolomes. Therefore, functional genomics involving various proteomics and metabolomics approaches have been obligatory for understanding plant stress tolerance. These approaches have also been found useful to unravel different pathways related to plant and seed development as well as symbiosis. Proteome and metabolome profiling using high-throughput based systems have been extensively applied in the model legume species, Medicago truncatula and Lotus japonicus, as well as in the model crop legume, soybean, to examine stress signaling pathways, cellular and developmental processes and nodule symbiosis. Moreover, the availability of protein reference maps as well as proteomics and metabolomics databases greatly support research and understanding of various biological processes in legumes. Protein-protein interaction techniques, particularly the yeast two-hybrid system have been advantageous for studying symbiosis and stress signaling in legumes. In this review, several studies on proteomics and metabolomics in model and crop legumes have been discussed. Additionally, applications of advanced proteomics and metabolomics approaches have also been included in this review for future applications in legume research. The integration of these “omics” approaches will greatly support the identification of accurate biomarkers in legume smart breeding programs. PMID:26734026

Molecular Profiles for Lung Cancer Pathogenesis and Detection in US Veterans

DTIC Science & Technology

2012-10-01

will be further strengthened via Multiple Reaction Monitoring ( MRM ) performed on the remaining samples by the Vanderbilt group. MRM using mass...proteomics detects all protein changes in the sample in an unfocused fashion, MRM is targeted and highly selective, allowing us to specifically look for...proteins of interest. To this end, we have generated a list of candidate proteins for MRM utilizing shotgun proteomic, mRNA array, and miRNA array

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 investigation of the effect of salicylic acid on Arabidopsis seed germination and establishment of early defense mechanisms.

PubMed

Rajjou, Loïc; Belghazi, Maya; Huguet, Romain; Robin, Caroline; Moreau, Adrien; Job, Claudette; Job, Dominique

2006-07-01

The influence of salicylic acid (SA) on elicitation of defense mechanisms in Arabidopsis (Arabidopsis thaliana) seeds and seedlings was assessed by physiological measurements combined with global expression profiling (proteomics). Parallel experiments were carried out using the NahG transgenic plants expressing the bacterial gene encoding SA hydroxylase, which cannot accumulate the active form of this plant defense elicitor. SA markedly improved germination under salt stress. Proteomic analyses disclosed a specific accumulation of protein spots regulated by SA as inferred by silver-nitrate staining of two-dimensional gels, detection of carbonylated (oxidized) proteins, and neosynthesized proteins with [35S]-methionine. The combined results revealed several processes potentially affected by SA. This molecule enhanced the reinduction of the late maturation program during early stages of germination, thereby allowing the germinating seeds to reinforce their capacity to mount adaptive responses in environmental water stress. Other processes affected by SA concerned the quality of protein translation, the priming of seed metabolism, the synthesis of antioxidant enzymes, and the mobilization of seed storage proteins. All the observed effects are likely to improve seed vigor. Another aspect revealed by this study concerned the oxidative stress entailed by SA in germinating seeds, as inferred from a characterization of the carbonylated (oxidized) proteome. Finally, the proteomic data revealed a close interplay between abscisic signaling and SA elicitation of seed vigor.

Population-specific plasma proteomes of marine and freshwater three-spined sticklebacks (Gasterosteus aculeatus).

PubMed

Kültz, Dietmar; Li, Johnathon; Zhang, Xuezhen; Villarreal, Fernando; Pham, Tuan; Paguio, Darlene

2015-12-01

Molecular phenotypes that distinguish resident marine (Bodega Harbor) from landlocked freshwater (FW, Lake Solano) three-spined sticklebacks were revealed by label-free quantitative proteomics. Secreted plasma proteins involved in lipid transport, blood coagulation, proteolysis, plasminogen-activating cascades, extracellular stimulus responses, and immunity are most abundant in this species. Globulins and albumins are much less abundant than in mammalian plasma. Unbiased quantitative proteome profiling identified 45 highly population-specific plasma proteins. Population-specific abundance differences were validated by targeted proteomics based on data-independent acquisition. Gene ontology enrichment analyses and known functions of population-specific plasma proteins indicate enrichment of processes controlling cell adhesion, tissue remodeling, proteolytic processing, and defense signaling in marine sticklebacks. Moreover, fetuin B and leukocyte cell derived chemotaxin 2 are much more abundant in marine fish. These proteins promote bone morphogenesis and likely contribute to population-specific body armor differences. Plasma proteins enriched in FW fish promote translation, heme biosynthesis, and lipid transport, suggesting a greater presence of plasma microparticles. Many prominent population-specific plasma proteins (e.g. apoptosis-associated speck-like protein containing a CARD) lack any homolog of known function or adequate functional characterization. Their functional characterization and the identification of population-specific environmental contexts and selective pressures that cause plasma proteome diversification are future directions emerging from this study. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Comparative Analysis of Computational Approaches to Relative Protein Quantification Using Peptide Peak Intensities in Label-free LC-MS Proteomics Experiments

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

Matzke, Melissa M.; Brown, Joseph N.; Gritsenko, Marina A.

2013-02-01

Liquid chromatography coupled with mass spectrometry (LC-MS) is widely used to identify and quantify peptides in complex biological samples. In particular, label-free shotgun proteomics is highly effective for the identification of peptides and subsequently obtaining a global protein profile of a sample. As a result, this approach is widely used for discovery studies. Typically, the objective of these discovery studies is to identify proteins that are affected by some condition of interest (e.g. disease, exposure). However, for complex biological samples, label-free LC-MS proteomics experiments measure peptides and do not directly yield protein quantities. Thus, protein quantification must be inferred frommore » one or more measured peptides. In recent years, many computational approaches to relative protein quantification of label-free LC-MS data have been published. In this review, we examine the most commonly employed quantification approaches to relative protein abundance from peak intensity values, evaluate their individual merits, and discuss challenges in the use of the various computational approaches.« less

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.

Proteomics boosts translational and clinical microbiology.

PubMed

Del Chierico, F; Petrucca, A; Vernocchi, P; Bracaglia, G; Fiscarelli, E; Bernaschi, P; Muraca, M; Urbani, A; Putignani, L

2014-01-31

The application of proteomics to translational and clinical microbiology is one of the most advanced frontiers in the management and control of infectious diseases and in the understanding of complex microbial systems within human fluids and districts. This new approach aims at providing, by dedicated bioinformatic pipelines, a thorough description of pathogen proteomes and their interactions within the context of human host ecosystems, revolutionizing the vision of infectious diseases in biomedicine and approaching new viewpoints in both diagnostic and clinical management of the patient. Indeed, in the last few years, many laboratories have matured a series of advanced proteomic applications, aiming at providing individual proteome charts of pathogens, with respect to their morph and/or cell life stages, antimicrobial or antimycotic resistance profiling, epidemiological dispersion. Herein, we aim at reviewing the current state-of-the-art on proteomic protocols designed and set-up for translational and diagnostic microbiological purposes, from axenic pathogens' characterization to microbiota ecosystems' full description. The final goal is to describe applications of the most common MALDI-TOF MS platforms to advanced diagnostic issues related to emerging infections, increasing of fastidious bacteria, and generation of patient-tailored phylotypes. This article is part of a Special Issue entitled: Trends in Microbial Proteomics. © 2013. Published by Elsevier B.V. All rights reserved.

Demonstration of protein-based human identification using the hair shaft proteome [Protein-based human identification: A proof of concept using the hair shaft proteome

DOE PAGES

Parker, Glendon J.; Leppert, Tami; Anex, Deon S.; ...

2016-09-07

Human identification from biological material is largely dependent on the ability to characterize genetic polymorphisms in DNA. Unfortunately, DNA can degrade in the environment, sometimes below the level at which it can be amplified by PCR. Protein however is chemically more robust than DNA and can persist for longer periods. Protein also contains genetic variation in the form of single amino acid polymorphisms. These can be used to infer the status of non-synonymous single nucleotide polymorphism alleles. To demonstrate this, we used mass spectrometry-based shotgun proteomics to characterize hair shaft proteins in 66 European-American subjects. A total of 596 singlemore » nucleotide polymorphism alleles were correctly imputed in 32 loci from 22 genes of subjects’ DNA and directly validated using Sanger sequencing. Estimates of the probability of resulting individual non-synonymous single nucleotide polymorphism allelic profiles in the European population, using the product rule, resulted in a maximum power of discrimination of 1 in 12,500. Imputed non-synonymous single nucleotide polymorphism profiles from European–American subjects were considerably less frequent in the African population (maximum likelihood ratio = 11,000). The converse was true for hair shafts collected from an additional 10 subjects with African ancestry, where some profiles were more frequent in the African population. Genetically variant peptides were also identified in hair shaft datasets from six archaeological skeletal remains (up to 260 years old). Furthermore, this study demonstrates that quantifiable measures of identity discrimination and biogeographic background can be obtained from detecting genetically variant peptides in hair shaft protein, including hair from bioarchaeological contexts.« less

Differential Expression of In Vivo and In Vitro Protein Profile of Outer Membrane of Acidovorax avenae Subsp. avenae

PubMed Central

Qiu, Hui; Li, Bin; Jabeen, Amara; Li, Liping; Liu, He; Kube, Michael; Xie, Guanlin; Wang, Yanli; Sun, Guochang

2012-01-01

Outer membrane (OM) proteins play a significant role in bacterial pathogenesis. In this work, we examined and compared the expression of the OM proteins of the rice pathogen Acidovorax avenae subsp. avenae strain RS-1, a Gram-negative bacterium, both in an in vitro culture medium and in vivo rice plants. Global proteomic profiling of A. avenae subsp. avenae strain RS-1 comparing in vivo and in vitro conditions revealed the differential expression of proteins affecting the survival and pathogenicity of the rice pathogen in host plants. The shotgun proteomics analysis of OM proteins resulted in the identification of 97 proteins in vitro and 62 proteins in vivo by mass spectrometry. Among these OM proteins, there is a high number of porins, TonB-dependent receptors, lipoproteins of the NodT family, ABC transporters, flagellins, and proteins of unknown function expressed under both conditions. However, the major proteins such as phospholipase and OmpA domain containing proteins were expressed in vitro, while the proteins such as the surface anchored protein F, ATP-dependent Clp protease, OmpA and MotB domain containing proteins were expressed in vivo. This may indicate that these in vivo OM proteins have roles in the pathogenicity of A. avenae subsp. avenae strain RS-1. In addition, the LC-MS/MS identification of OmpA and MotB validated the in silico prediction of the existance of Type VI secretion system core components. To the best of our knowledge, this is the first study to reveal the in vitro and in vivo protein profiles, in combination with LC-MS/MS mass spectra, in silico OM proteome and in silico genome wide analysis, of pathogenicity or plant host required proteins of a plant pathogenic bacterium. PMID:23166741

Demonstration of protein-based human identification using the hair shaft proteome [Protein-based human identification: A proof of concept using the hair shaft proteome

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

Parker, Glendon J.; Leppert, Tami; Anex, Deon S.

Human identification from biological material is largely dependent on the ability to characterize genetic polymorphisms in DNA. Unfortunately, DNA can degrade in the environment, sometimes below the level at which it can be amplified by PCR. Protein however is chemically more robust than DNA and can persist for longer periods. Protein also contains genetic variation in the form of single amino acid polymorphisms. These can be used to infer the status of non-synonymous single nucleotide polymorphism alleles. To demonstrate this, we used mass spectrometry-based shotgun proteomics to characterize hair shaft proteins in 66 European-American subjects. A total of 596 singlemore » nucleotide polymorphism alleles were correctly imputed in 32 loci from 22 genes of subjects’ DNA and directly validated using Sanger sequencing. Estimates of the probability of resulting individual non-synonymous single nucleotide polymorphism allelic profiles in the European population, using the product rule, resulted in a maximum power of discrimination of 1 in 12,500. Imputed non-synonymous single nucleotide polymorphism profiles from European–American subjects were considerably less frequent in the African population (maximum likelihood ratio = 11,000). The converse was true for hair shafts collected from an additional 10 subjects with African ancestry, where some profiles were more frequent in the African population. Genetically variant peptides were also identified in hair shaft datasets from six archaeological skeletal remains (up to 260 years old). Furthermore, this study demonstrates that quantifiable measures of identity discrimination and biogeographic background can be obtained from detecting genetically variant peptides in hair shaft protein, including hair from bioarchaeological contexts.« less

Do cultural conditions induce differential protein expression: Profiling of extracellular proteome of Aspergillus terreus CM20.

PubMed

M, Saritha; Singh, Surender; Tiwari, Rameshwar; Goel, Renu; Nain, Lata

2016-11-01

The present study reports the diversity in extracellular proteins expressed by the filamentous fungus, Aspergillus terreus CM20 with respect to differential hydrolytic enzyme production profiles in submerged fermentation (SmF) and solid-state fermentation (SSF) conditions, and analysis of the extracellular proteome. The SSF method was superior in terms of increase in enzyme activities resulting in 1.5-3 fold enhancement as compared to SmF, which was explained by the difference in growth pattern of the fungus under the two culture conditions. As revealed by zymography, multiple isoforms of endo-β-glucanase, β-glucosidase and xylanase were expressed in SSF, but not in SmF. Extracellular proteome profiling of A. terreus CM20 under SSF condition using liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) identified 63 proteins. Functional classification revealed the hydrolytic system to be composed of glycoside hydrolases (56%), proteases (16%), oxidases and dehydrogenases (6%), decarboxylases (3%), esterases (3%) and other proteins (16%). Twenty families of glycoside hydrolases (GH) (1, 3, 5, 7, 10, 11, 12, 15, 16, 28, 30, 32, 35, 43, 54, 62, 67, 72, 74 and 125), and one family each of auxiliary activities (AA7) and carbohydrate esterase (CE1) were detected, unveiling the vast diversity of synergistically acting biomass-cleaving enzymes expressed by the fungus. Saccharification of alkali-pretreated paddy straw with A. terreus CM20 proteins released high amounts of glucose (439.63±1.50mg/gds), xylose (121.04±1.25mg/gds) and arabinose (56.13±0.56mg/gds), thereby confirming the potential of the enzyme cocktail in bringing about considerable conversion of lignocellulosic polysaccharides to sugar monomers. Copyright © 2016 Elsevier GmbH. All rights reserved.

Subcellular proteome profiles of different latex fractions revealed washed solutions from rubber particles contain crucial enzymes for natural rubber biosynthesis.

PubMed

Wang, Dan; Sun, Yong; Chang, Lili; Tong, Zheng; Xie, Quanliang; Jin, Xiang; Zhu, Liping; He, Peng; Li, Hongbin; Wang, Xuchu

2018-06-30

Rubber particle (RP) is a specific organelle for natural rubber biosynthesis (NRB) and storage in rubber tree Hevea brasiliensis. NRB is processed by RP membrane-localized proteins, which were traditionally purified by repeated washing. However, we noticed many proteins in the discarded washing solutions (WS) from RP. Here, we compared the proteome profiles of WS, C-serum (CS) and RP by 2-DE, and identified 233 abundant proteins from WS by mass spectrometry. Many spots on 2-DE gels were identified as different protein species. We further performed shotgun analysis of CS, WS and RP and identified 1837, 1799 and 1020 unique proteins, respectively. Together with 2-DE, we finally identified 1825 proteins from WS, 246 were WS-specific. These WS-specific proteins were annotated in Gene Ontology, indicating most abundant pathways are organic substance metabolic process, protein degradation, primary metabolic process, and energy metabolism. Protein-protein interaction analysis revealed these WS-specific proteins are mainly involved in ribosomal metabolism, proteasome system, vacuolar protein sorting and endocytosis. Label free and Western blotting revealed many WS-specific proteins and protein complexes are crucial for NRB initiation. These findings not only deepen our understanding of WS proteome, but also provide new evidences on the roles of RP membrane proteins in NRB. Natural rubber is stored in rubber particle from the rubber tree. Rubber particles were traditionally purified by repeated washing, but many proteins were identified from the washing solutions (WS). We obtained the first visualization proteome profiles with 1825 proteins from WS, including 246 WS-specific ones. These WS proteins contain almost all enzymes for polyisoprene initiation and may play important roles in rubber biosynthesis. Copyright © 2018 Elsevier B.V. All rights reserved.

In-depth 2-DE reference map of Aspergillus fumigatus and its proteomic profiling on exposure to itraconazole.

PubMed

Gautam, Poonam; Mushahary, Dolly; Hassan, Wazid; Upadhyay, Santosh Kumar; Madan, Taruna; Sirdeshmukh, Ravi; Sundaram, Curam Sreenivasacharlu; Sarma, Puranam Usha

2016-07-01

Aspergillus fumigatus (A. fumigatus) is a medically important opportunistic fungus that may lead to invasive aspergillosis in humans with weak immune system. Proteomic profiling of this fungus on exposure to itraconazole (ITC), an azole antifungal drug, may lead to identification of its molecular targets and better understanding on the development of drug resistance against ITC in A. fumigatus. Here, proteome analysis was performed using 2-DE followed by mass spectrometric analysis which resulted in identification of a total of 259 unique proteins. Further, proteome profiling of A. fumigatus was carried out on exposure to ITC, 0.154 μg/ml, the minimum inhibitory concentration (MIC50). Image analysis showed altered levels of 175 proteins (66 upregulated and 109 downregulated) of A. fumigatus treated with ITC as compared to the untreated control. Peptide mass fingerprinting led to the identification of 54 proteins (12 up-regulated and 42 down-regulated). The differentially expressed proteins include proteins related to cell stress, carbohydrate metabolism and amino acid metabolism. We also observed four proteins, including nucleotide phosphate kinase (NDK), that are reported to interact with calcineurin, a protein involved in regulation of cell morphology and fungal virulence. Comparison of differentially expressed proteins on exposure to ITC with artemisinin (ART), an antimalarial drug with antifungal activity(1), revealed a total of 26 proteins to be common among them suggesting that common proteins and pathways are targeted by these two antifungal agents. The proteins targeted by ITC may serve as important leads for development of new antifungal drugs. © The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A Targeted Quantitative Proteomics Strategy for Global Kinome Profiling of Cancer Cells and Tissues*

PubMed Central

Xiao, Yongsheng; Guo, Lei; Wang, Yinsheng

2014-01-01

Kinases are among the most intensively pursued enzyme superfamilies as targets for anti-cancer drugs. Large data sets on inhibitor potency and selectivity for more than 400 human kinases became available recently, offering the opportunity to design rationally novel kinase-based anti-cancer therapies. However, the expression levels and activities of kinases are highly heterogeneous among different types of cancer and even among different stages of the same cancer. The lack of effective strategy for profiling the global kinome hampers the development of kinase-targeted cancer chemotherapy. Here, we introduced a novel global kinome profiling method, based on our recently developed isotope-coded ATP-affinity probe and a targeted proteomic method using multiple-reaction monitoring (MRM), for assessing simultaneously the expression of more than 300 kinases in human cells and tissues. This MRM-based assay displayed much better sensitivity, reproducibility, and accuracy than the discovery-based shotgun proteomic method. Approximately 250 kinases could be routinely detected in the lysate of a single cell line. Additionally, the incorporation of iRT into MRM kinome library rendered our MRM kinome assay easily transferrable across different instrument platforms and laboratories. We further employed this approach for profiling kinase expression in two melanoma cell lines, which revealed substantial kinome reprogramming during cancer progression and demonstrated an excellent correlation between the anti-proliferative effects of kinase inhibitors and the expression levels of their target kinases. Therefore, this facile and accurate kinome profiling assay, together with the kinome-inhibitor interaction map, could provide invaluable knowledge to predict the effectiveness of kinase inhibitor drugs and offer the opportunity for individualized cancer chemotherapy. PMID:24520089

Combined proteomic and metallomic analyses in Scrobicularia plana clams to assess environmental pollution of estuarine ecosystems.

PubMed

González-Domínguez, Raúl; Santos, Hugo Miguel; Bebianno, Maria João; García-Barrera, Tamara; Gómez-Ariza, José Luis; Capelo, José Luis

2016-12-15

Estuaries are very important ecosystems with great ecological and economic value, but usually highly impacted by anthropogenic pressure. Thus, the assessment of pollution levels in these habitats is critical in order to evaluate their environmental quality. In this work, we combined complementary metallomic and proteomic approaches with the aim to monitor the effects of environmental pollution on Scrobicularia plana clams captured in three estuarine systems from the south coast of Portugal; Arade estuary, Ria Formosa and Guadiana estuary. Multi-elemental profiling of digestive glands was carried out to evaluate the differential pollution levels in the three study areas. Then, proteomic analysis by means of two-dimensional gel electrophoresis and mass spectrometry revealed twenty-one differential proteins, which could be associated with multiple toxicological mechanisms induced in environmentally stressed organisms. Accordingly, it could be concluded that the combination of different omic approaches presents a great potential in environmental research. Copyright © 2016 Elsevier Ltd. All rights reserved.

Data on xylem sap proteins from Mn- and Fe-deficient tomato plants obtained using shotgun proteomics.

PubMed

Ceballos-Laita, Laura; Gutierrez-Carbonell, Elain; Takahashi, Daisuke; Abadía, Anunciación; Uemura, Matsuo; Abadía, Javier; López-Millán, Ana Flor

2018-04-01

This article contains consolidated proteomic data obtained from xylem sap collected from tomato plants grown in Fe- and Mn-sufficient control, as well as Fe-deficient and Mn-deficient conditions. Data presented here cover proteins identified and quantified by shotgun proteomics and Progenesis LC-MS analyses: proteins identified with at least two peptides and showing changes statistically significant (ANOVA; p ≤ 0.05) and above a biologically relevant selected threshold (fold ≥ 2) between treatments are listed. The comparison between Fe-deficient, Mn-deficient and control xylem sap samples using a multivariate statistical data analysis (Principal Component Analysis, PCA) is also included. Data included in this article are discussed in depth in the research article entitled "Effects of Fe and Mn deficiencies on the protein profiles of tomato ( Solanum lycopersicum) xylem sap as revealed by shotgun analyses" [1]. This dataset is made available to support the cited study as well to extend analyses at a later stage.

Proteomic Analysis Reveals Inflammation Modulation of κ/ι-Carrageenan Hexaoses in Lipopolysaccharide-Induced RAW264.7 Macrophages.

PubMed

Guo, Juanjuan; Chen, Jinghao; Lu, Xu; Guo, Zebin; Huang, Zhiwei; Zeng, Shaoxiao; Zhang, Yi; Zheng, Baodong

2018-05-09

κ/ι-Carrageenan hexaoses (κ/ι-neocarrahexaoses, KCO-4) are a type of carrageenan oligosaccharide that have a broad spectrum of bioactivities due to the presence of sulfate groups. However, the anti-inflammatory capacity of purified carrageenan oligosaccharides and the underlying mechanism has not been completely elucidated. The present study aimed to investigate inflammatory signaling modulation of KCO-4 in LPS-induced macrophages using a quantitative proteomic strategy. KCO-4 inhibited the oversecretion of inflammatory mediators (i.e., NO, TNF-α, IL-1β, IL-8, iNOS, and COX-2). KCO-4 treatment altered proteome profile, and metabolic processes in mitochondria were significantly disrupted. The IPA network analysis proposed that KCO-4 triggered the NF-κB signaling pathway-dependent anti-inflammation process through the inhibition of CD14/Rel@p50 in LPS-induced RAW264.7 macrophages. These data improve our understanding of the anti-inflammatory mechanism and contribute to exposure biomarker screening of κ-carrageenan oligosaccharides.

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.

Combining Patient-Reprogrammed Neural Cells and Proteomics as a Model to Study Psychiatric Disorders.

PubMed

Zuccoli, Giuliana S; Martins-de-Souza, Daniel; Guest, Paul C; Rehen, Stevens K; Nascimento, Juliana Minardi

2017-01-01

The mechanisms underlying the pathophysiology of psychiatric disorders are still poorly known. Most of the studies about these disorders have been conducted on postmortem tissue or in limited preclinical models. The development of human induced pluripotent stem cells (iPSCs) has helped to increase the translational capacity of molecular profiling studies of psychiatric disorders through provision of human neuronal-like tissue. This approach consists of generation of pluripotent cells by genetically reprogramming somatic cells to produce the multiple neural cell types as observed within the nervous tissue. The finding that iPSCs can recapitulate the phenotype of the donor also affords the possibility of using this approach to study both the disease and control states in a given medical area. Here, we present a protocol for differentiation of human pluripotent stem cells to neural progenitor cells followed by subcellular fractionation which allows the study of specific cellular organelles and proteomic analysis.

Unmasking molecular profiles of bladder cancer.

PubMed

Piao, Xuan-Mei; Byun, Young Joon; Kim, Wun-Jae; Kim, Jayoung

2018-03-01

Precision medicine is designed to tailor treatments for individual patients by factoring in each person's specific biology and mechanism of disease. This paradigm shifted from a "one size fits all" approach to "personalized and precision care" requires multiple layers of molecular profiling of biomarkers for accurate diagnosis and prediction of treatment responses. Intensive studies are also being performed to understand the complex and dynamic molecular profiles of bladder cancer. These efforts involve looking bladder cancer mechanism at the multiple levels of the genome, epigenome, transcriptome, proteome, lipidome, metabolome etc. The aim of this short review is to outline the current technologies being used to investigate molecular profiles and discuss biomarker candidates that have been investigated as possible diagnostic and prognostic indicators of bladder cancer.

HPLC-Chip/MS Technology in Proteomic Profiling

NASA Astrophysics Data System (ADS)

Vollmer, Martin; van de Goor, Tom

HPLC-chip/MS is a novel nanoflow analytical technology conducted on a microfabricated chip that allows for highly efficient HPLC separation and superior sensitive MS detection of complex proteomic mixtures. This is possible through on-chip preconcentration and separation with fluidic connection made automatically in a leak-tight fashion. Minimum precolumn and postcolumn peak dispersion and uncompromised ease of use result in compounds eluting in bands of only a few nanoliters. The chip is fabricated out of bio-inert polyimide-containing channels and integrated chip structures, such as an electrospray emitter, columns, and frits manufactured by laser ablation technology. Meanwhile, a variety of HPLC-chips differing in design and stationary phase are commercially available, which provide a comprehensive solution for applications in proteomics, glycomics, biomarker, and pharmaceutical discovery. The HPLC-chip can also be easily integrated into a multidimensional separation workflow where different orthogonal separation techniques are combined to solve a highly complex separation problems. In this chapter, we describe in detail the methodological chip usage and functionality and its application in the elucidation of the protein profile of human nucleoli.

Disclosure of the differences of Mesorhizobium loti under the free-living and symbiotic conditions by comparative proteome analysis without bacteroid isolation.

PubMed

Tatsukami, Yohei; Nambu, Mami; Morisaka, Hironobu; Kuroda, Kouichi; Ueda, Mitsuyoshi

2013-07-31

Rhizobia are symbiotic nitrogen-fixing soil bacteria that show a symbiotic relationship with their host legume. Rhizobia have 2 different physiological conditions: a free-living condition in soil, and a symbiotic nitrogen-fixing condition in the nodule. The lifestyle of rhizobia remains largely unknown, although genome and transcriptome analyses have been carried out. To clarify the lifestyle of bacteria, proteome analysis is necessary because the protein profile directly reflects in vivo reactions of the organisms. In proteome analysis, high separation performance is required to analyze complex biological samples. Therefore, we used a liquid chromatography-tandem mass spectrometry system, equipped with a long monolithic silica capillary column, which is superior to conventional columns. In this study, we compared the protein profile of Mesorhizobium loti MAFF303099 under free-living condition to that of symbiotic conditions by using small amounts of crude extracts. We identified 1,533 and 847 proteins for M. loti under free-living and symbiotic conditions, respectively. Pathway analysis by Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that many of the enzymes involved in the central carbon metabolic pathway were commonly detected under both conditions. The proteins encoded in the symbiosis island, the transmissible chromosomal region that includes the genes that are highly upregulated under the symbiotic condition, were uniquely detected under the symbiotic condition. The features of the symbiotic condition that have been reported by transcriptome analysis were confirmed at the protein level by proteome analysis. In addition, the genes of the proteins involved in cell surface structure were repressed under the symbiotic nitrogen-fixing condition. Furthermore, farnesyl pyrophosphate (FPP) was found to be biosynthesized only in rhizobia under the symbiotic condition. The obtained protein profile appeared to reflect the difference in phenotypes under the free-living and symbiotic conditions. In addition, KEGG pathway analysis revealed that the cell surface structure of rhizobia was largely different under each condition, and surprisingly, rhizobia might provided FPP to the host as a source of secondary metabolism. M. loti changed its metabolism and cell surface structure in accordance with the surrounding conditions.

Disclosure of the differences of Mesorhizobium loti under the free-living and symbiotic conditions by comparative proteome analysis without bacteroid isolation

PubMed Central

2013-01-01

Background Rhizobia are symbiotic nitrogen-fixing soil bacteria that show a symbiotic relationship with their host legume. Rhizobia have 2 different physiological conditions: a free-living condition in soil, and a symbiotic nitrogen-fixing condition in the nodule. The lifestyle of rhizobia remains largely unknown, although genome and transcriptome analyses have been carried out. To clarify the lifestyle of bacteria, proteome analysis is necessary because the protein profile directly reflects in vivo reactions of the organisms. In proteome analysis, high separation performance is required to analyze complex biological samples. Therefore, we used a liquid chromatography-tandem mass spectrometry system, equipped with a long monolithic silica capillary column, which is superior to conventional columns. In this study, we compared the protein profile of Mesorhizobium loti MAFF303099 under free-living condition to that of symbiotic conditions by using small amounts of crude extracts. Result We identified 1,533 and 847 proteins for M. loti under free-living and symbiotic conditions, respectively. Pathway analysis by Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that many of the enzymes involved in the central carbon metabolic pathway were commonly detected under both conditions. The proteins encoded in the symbiosis island, the transmissible chromosomal region that includes the genes that are highly upregulated under the symbiotic condition, were uniquely detected under the symbiotic condition. The features of the symbiotic condition that have been reported by transcriptome analysis were confirmed at the protein level by proteome analysis. In addition, the genes of the proteins involved in cell surface structure were repressed under the symbiotic nitrogen-fixing condition. Furthermore, farnesyl pyrophosphate (FPP) was found to be biosynthesized only in rhizobia under the symbiotic condition. Conclusion The obtained protein profile appeared to reflect the difference in phenotypes under the free-living and symbiotic conditions. In addition, KEGG pathway analysis revealed that the cell surface structure of rhizobia was largely different under each condition, and surprisingly, rhizobia might provided FPP to the host as a source of secondary metabolism. M. loti changed its metabolism and cell surface structure in accordance with the surrounding conditions. PMID:23898917

Comparison of protein expression between human livers and the hepatic cell lines HepG2, Hep3B, and Huh7 using SWATH and MRM-HR proteomics: Focusing on drug-metabolizing enzymes.

PubMed

Shi, Jian; Wang, Xinwen; Lyu, Lingyun; Jiang, Hui; Zhu, Hao-Jie

2018-04-01

Human hepatic cell lines are widely used as an in vitro model for the study of drug metabolism and liver toxicity. However, the validity of this model is still a subject of debate because the expressions of various proteins in the cell lines, including drug-metabolizing enzymes (DMEs), can differ significantly from those in human livers. In the present study, we first conducted an untargeted proteomics analysis of the microsomes of the cell lines HepG2, Hep3B, and Huh7, and compared them to human livers using a sequential window acquisition of all theoretical mass spectra (SWATH) method. Furthermore, high-resolution multiple reaction monitoring (MRM-HR), a targeted proteomic approach, was utilized to compare the expressions of pre-selected DMEs between human livers and the cell lines. In general, the SWATH quantifications were in good agreement with the MRM-HR analysis. Over 3000 protein groups were quantified in the cells and human livers, and the proteome profiles of human livers significantly differed from the cell lines. Among the 101 DMEs quantified with MRM-HR, most were expressed at substantially lower levels in the cell lines. Thus, appropriate caution must be exercised when using these cell lines for the study of hepatic drug metabolism and toxicity. Copyright © 2018 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

Global analysis of the rat and human platelet proteome – the molecular blueprint for illustrating multi-functional platelets and cross-species function evolution

PubMed Central

Yu, Yanbao; Leng, Taohua; Yun, Dong; Liu, Na; Yao, Jun; Dai, Ying; Yang, Pengyuan; Chen, Xian

2013-01-01

Emerging evidences indicate that blood platelets function in multiple biological processes including immune response, bone metastasis and liver regeneration in addition to their known roles in hemostasis and thrombosis. Global elucidation of platelet proteome will provide the molecular base of these platelet functions. Here, we set up a high throughput platform for maximum exploration of the rat/human platelet proteome using integrated proteomics technologies, and then applied to identify the largest number of the proteins expressed in both rat and human platelets. After stringent statistical filtration, a total of 837 unique proteins matched with at least two unique peptides were precisely identified, making it the first comprehensive protein database so far for rat platelets. Meanwhile, quantitative analyses of the thrombin-stimulated platelets offered great insights into the biological functions of platelet proteins and therefore confirmed our global profiling data. A comparative proteomic analysis between rat and human platelets was also conducted, which revealed not only a significant similarity, but also an across-species evolutionary link that the orthologous proteins representing ‘core proteome’, and the ‘evolutionary proteome’ is actually a relatively static proteome. PMID:20443191

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.

A Shotgun Proteomic Approach Reveals That Fe Deficiency Causes Marked Changes in the Protein Profiles of Plasma Membrane and Detergent-Resistant Microdomain Preparations from Beta vulgaris Roots.

PubMed

Gutierrez-Carbonell, Elain; Takahashi, Daisuke; Lüthje, Sabine; González-Reyes, José Antonio; Mongrand, Sébastien; Contreras-Moreira, Bruno; Abadía, Anunciación; Uemura, Matsuo; Abadía, Javier; López-Millán, Ana Flor

2016-08-05

In the present study we have used label-free shotgun proteomic analysis to examine the effects of Fe deficiency on the protein profiles of highly pure sugar beet root plasma membrane (PM) preparations and detergent-resistant membranes (DRMs), the latter as an approach to study microdomains. Altogether, 545 proteins were detected, with 52 and 68 of them changing significantly with Fe deficiency in PM and DRM, respectively. Functional categorization of these proteins showed that signaling and general and vesicle-related transport accounted for approximately 50% of the differences in both PM and DRM, indicating that from a qualitative point of view changes induced by Fe deficiency are similar in both preparations. Results indicate that Fe deficiency has an impact in phosphorylation processes at the PM level and highlight the involvement of signaling proteins, especially those from the 14-3-3 family. Lipid profiling revealed Fe-deficiency-induced decreases in phosphatidic acid derivatives, which may impair vesicle formation, in agreement with the decreases measured in proteins related to intracellular trafficking and secretion. The modifications induced by Fe deficiency in the relative enrichment of proteins in DRMs revealed the existence of a group of cytoplasmic proteins that appears to be more attached to the PM in conditions of Fe deficiency.

Mass spectrometry-assisted gel-based proteomics in cancer biomarker discovery: approaches and application

PubMed Central

Huang, Rongrong; Chen, Zhongsi; He, Lei; He, Nongyue; Xi, Zhijiang; Li, Zhiyang; Deng, Yan; Zeng, Xin

2017-01-01

There is a critical need for the discovery of novel biomarkers for early detection and targeted therapy of cancer, a major cause of deaths worldwide. In this respect, proteomic technologies, such as mass spectrometry (MS), enable the identification of pathologically significant proteins in various types of samples. MS is capable of high-throughput profiling of complex biological samples including blood, tissues, urine, milk, and cells. MS-assisted proteomics has contributed to the development of cancer biomarkers that may form the foundation for new clinical tests. It can also aid in elucidating the molecular mechanisms underlying cancer. In this review, we discuss MS principles and instrumentation as well as approaches in MS-based proteomics, which have been employed in the development of potential biomarkers. Furthermore, the challenges in validation of MS biomarkers for their use in clinical practice are also reviewed. PMID:28912895

Global profiling of lysine reactivity and ligandability in the human proteome

NASA Astrophysics Data System (ADS)

Hacker, Stephan M.; Backus, Keriann M.; Lazear, Michael R.; Forli, Stefano; Correia, Bruno E.; Cravatt, Benjamin F.

2017-12-01

Nucleophilic amino acids make important contributions to protein function, including performing key roles in catalysis and serving as sites for post-translational modification. Electrophilic groups that target amino-acid nucleophiles have been used to create covalent ligands and drugs, but have, so far, been mainly limited to cysteine and serine. Here, we report a chemical proteomic platform for the global and quantitative analysis of lysine residues in native biological systems. We have quantified, in total, more than 9,000 lysines in human cell proteomes and have identified several hundred residues with heightened reactivity that are enriched at protein functional sites and can frequently be targeted by electrophilic small molecules. We have also discovered lysine-reactive fragment electrophiles that inhibit enzymes by active site and allosteric mechanisms, as well as disrupt protein-protein interactions in transcriptional regulatory complexes, emphasizing the broad potential and diverse functional consequences of liganding lysine residues throughout the human proteome.

Proteomic Profiling of Mitochondrial Enzymes during Skeletal Muscle Aging.

PubMed

Staunton, Lisa; O'Connell, Kathleen; Ohlendieck, Kay

2011-03-07

Mitochondria are of central importance for energy generation in skeletal muscles. Expression changes or functional alterations in mitochondrial enzymes play a key role during myogenesis, fibre maturation, and various neuromuscular pathologies, as well as natural fibre aging. Mass spectrometry-based proteomics suggests itself as a convenient large-scale and high-throughput approach to catalogue the mitochondrial protein complement and determine global changes during health and disease. This paper gives a brief overview of the relatively new field of mitochondrial proteomics and discusses the findings from recent proteomic surveys of mitochondrial elements in aged skeletal muscles. Changes in the abundance, biochemical activity, subcellular localization, and/or posttranslational modifications in key mitochondrial enzymes might be useful as novel biomarkers of aging. In the long term, this may advance diagnostic procedures, improve the monitoring of disease progression, help in the testing of side effects due to new drug regimes, and enhance our molecular understanding of age-related muscle degeneration.

Modulating the protein content of complex proteomes using acetonitrile.

PubMed

Prates, João; Martins, Gonçalo; López-Fernández, Hugo; Lodeiro, Carlos; Capelo, J L; Santos, Hugo M

2018-05-15

In this work we present acetonitrile as a tool to modulate the dynamic range of the proteome of complex samples. Different concentrations of acetonitrile ranging from 15% v/v to 65% v/v were used to modulate the protein content of serum samples from healthy people and patients with lymphoma and myeloma. We show that the proteome above 70 kDa is pelleted as a function of the concentration of acetonitrile and that profiling with PCA or Clustering is only possible using the supernatants obtained for concentrations of acetonitrile higher than 45% v/v or the pellets for concentrations of acetonitrile of 35% and 45%. The differentiation and classification of the three groups of sera samples (healthy, lymphoma and myeloma) were possible using acetonitrile at 55% v/v concentration. This work opens new avenues for the application of acetonitrile as a cost-effective tool in proteomics applications. Copyright © 2018 Elsevier B.V. All rights reserved.

Global profiling of lysine reactivity and ligandability in the human proteome.

PubMed

Hacker, Stephan M; Backus, Keriann M; Lazear, Michael R; Forli, Stefano; Correia, Bruno E; Cravatt, Benjamin F

2017-12-01

Nucleophilic amino acids make important contributions to protein function, including performing key roles in catalysis and serving as sites for post-translational modification. Electrophilic groups that target amino-acid nucleophiles have been used to create covalent ligands and drugs, but have, so far, been mainly limited to cysteine and serine. Here, we report a chemical proteomic platform for the global and quantitative analysis of lysine residues in native biological systems. We have quantified, in total, more than 9,000 lysines in human cell proteomes and have identified several hundred residues with heightened reactivity that are enriched at protein functional sites and can frequently be targeted by electrophilic small molecules. We have also discovered lysine-reactive fragment electrophiles that inhibit enzymes by active site and allosteric mechanisms, as well as disrupt protein-protein interactions in transcriptional regulatory complexes, emphasizing the broad potential and diverse functional consequences of liganding lysine residues throughout the human proteome.

Quantitative analysis of cellular proteome alterations in human influenza A virus-infected mammalian cell lines.

PubMed

Vester, Diana; Rapp, Erdmann; Gade, Dörte; Genzel, Yvonne; Reichl, Udo

2009-06-01

Over the last years virus-host cell interactions were investigated in numerous studies. Viral strategies for evasion of innate immune response, inhibition of cellular protein synthesis and permission of viral RNA and protein production were disclosed. With quantitative proteome technology, comprehensive studies concerning the impact of viruses on the cellular machinery of their host cells at protein level are possible. Therefore, 2-D DIGE and nanoHPLC-nanoESI-MS/MS analysis were used to qualitatively and quantitatively determine the dynamic cellular proteome responses of two mammalian cell lines to human influenza A virus infection. A cell line used for vaccine production (MDCK) was compared with a human lung carcinoma cell line (A549) as a reference model. Analyzing 2-D gels of the proteomes of uninfected and influenza-infected host cells, 16 quantitatively altered protein spots (at least +/-1.7-fold change in relative abundance, p<0.001) were identified for both cell lines. Most significant changes were found for keratins, major components of the cytoskeleton system, and for Mx proteins, interferon-induced key components of the host cell defense. Time series analysis of infection processes allowed the identification of further proteins that are described to be involved in protein synthesis, signal transduction and apoptosis events. Most likely, these proteins are required for supporting functions during influenza viral life cycle or host cell stress response. Quantitative proteome-wide profiling of virus infection can provide insights into complexity and dynamics of virus-host cell interactions and may accelerate antiviral research and support optimization of vaccine manufacturing processes.

Consistency of the Proteome in Primary Human Keratinocytes With Respect to Gender, Age, and Skin Localization*

PubMed Central

Sprenger, Adrian; Weber, Sebastian; Zarai, Mostafa; Engelke, Rudolf; Nascimento, Juliana M.; Gretzmeier, Christine; Hilpert, Martin; Boerries, Melanie; Has, Cristina; Busch, Hauke; Bruckner-Tuderman, Leena; Dengjel, Jörn

2013-01-01

Keratinocytes account for 95% of all cells of the epidermis, the stratified squamous epithelium forming the outer layer of the skin, in which a significant number of skin diseases takes root. Immortalized keratinocyte cell lines are often used as research model systems providing standardized, reproducible, and homogenous biological material. Apart from that, primary human keratinocytes are frequently used for medical studies because the skin provides an important route for drug administration and is readily accessible for biopsies. However, comparability of these cell systems is not known. Cell lines may undergo phenotypic shifts and may differ from the in vivo situation in important aspects. Primary cells, on the other hand, may vary in biological functions depending on gender and age of the donor and localization of the biopsy specimen. Here we employed metabolic labeling in combination with quantitative mass spectrometry-based proteomics to assess A431 and HaCaT cell lines for their suitability as model systems. Compared with cell lines, comprehensive profiling of the primary human keratinocyte proteome with respect to gender, age, and skin localization identified an unexpected high proteomic consistency. The data were analyzed by an improved ontology enrichment analysis workflow designed for the study of global proteomics experiments. It enables a quick, comprehensive and unbiased overview of altered biological phenomena and links experimental data to literature. We guide through our workflow, point out its advantages compared with other methods and apply it to visualize differences of cell lines compared with primary human keratinocytes. PMID:23722187

Nutrigenomics in human intervention studies: current status, lessons learned and future perspectives.

PubMed

Wittwer, Jonas; Rubio-Aliaga, Isabel; Hoeft, Birgit; Bendik, Igor; Weber, Peter; Daniel, Hannelore

2011-03-01

Nutrigenomics applications comprise transcript-, proteome- and metabolome-profiling techniques in which responses to diets or individual ingredients are assessed in biological samples. They may also include the characterization of heterogeneity in relevant genes that affect the biological processes. This review explores various areas of nutrition and food sciences in which transcriptome-, proteome- and metabolome-analyses have been applied in human intervention studies, including nutrigenetics aspects and discusses the advantages and limitations of the methodologies. Despite the power of the profiling techniques to generate huge data sets, a critical assessment of the study outcomes emphasizes the current constraints in data interpretation, including huge knowledge gaps, the need for improved study designs and more comprehensive phenotyping of volunteers before selection for study participation. In this respect, nutrigenomics faces the same problems as all other areas of the life sciences, employing the same tools. However, there is a growing trend toward systemic approaches in which different technologies are combined and applied to the same sample, allowing physiological changes to be assessed more robustly throughout all molecular layers of mRNA, protein and metabolite changes. Nutrigenomics is thereby maturing as a branch of the life sciences and is gaining significant recognition in the scientific community. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rapid Myeloid Cell Transcriptional and Proteomic Responses to Periodontopathogenic Porphyromonas gingivalis

PubMed Central

Nares, Salvador; Moutsopoulos, Niki M.; Angelov, Nikola; Rangel, Zoila G.; Munson, Peter J.; Sinha, Neha; Wahl, Sharon M.

2009-01-01

Long-lived monocytes, macrophages, and dendritic cells (DCs) are Toll-like receptor-expressing, antigen-presenting cells derived from a common myeloid lineage that play key roles in innate and adaptive immune responses. Based on immunohistochemical and molecular analyses of inflamed tissues from patients with chronic destructive periodontal disease, these cells, found in the inflammatory infiltrate, may drive the progressive periodontal pathogenesis. To investigate early transcriptional signatures and subsequent proteomic responses to the periodontal pathogen, Porphyromonas gingivalis, donor-matched human blood monocytes, differentiated DCs, and macrophages were exposed to P. gingivalis lipopolysaccharide (LPS) and gene expression levels were measured by oligonucleotide microarrays. In addition to striking differences in constitutive transcriptional profiles between these myeloid populations, we identify a P. gingivalis LPS-inducible convergent, transcriptional core response of more than 400 annotated genes/ESTs among these populations, reflected by a shared, but quantitatively distinct, proteomic response. Nonetheless, clear differences emerged between the monocytes, DCs, and macrophages. The finding that long-lived myeloid inflammatory cells, particularly DCs, rapidly and aggressively respond to P. gingivalis LPS by generating chemokines, proteases, and cytokines capable of driving T-helper cell lineage polarization without evidence of corresponding immunosuppressive pathways highlights their prominent role in host defense and progressive tissue pathogenesis. The shared, unique, and/or complementary transcriptional and proteomic profiles may frame the context of the host response to P. gingivalis, contributing to the destructive nature of periodontal inflammation. PMID:19264901

Effects of sub-lethal high-pressure homogenization treatment on the outermost cellular structures and the volatile-molecule profiles of two strains of probiotic lactobacilli.

PubMed

Tabanelli, Giulia; Vernocchi, Pamela; Patrignani, Francesca; Del Chierico, Federica; Putignani, Lorenza; Vinderola, Gabriel; Reinheimer, Jorge A; Gardini, Fausto; Lanciotti, Rosalba

2015-01-01

Applying sub-lethal levels of high-pressure homogenization (HPH) to lactic acid bacteria has been proposed as a method of enhancing some of their functional properties. Because the principal targets of HPH are the cell-surface structures, the aim of this study was to examine the effect of sub-lethal HPH treatment on the outermost cellular structures and the proteomic profiles of two known probiotic bacterial strains. Moreover, the effect of HPH treatment on the metabolism of probiotic cells within a dairy product during its refrigerated storage was investigated using SPME-GC-MS. Transmission electron microscopy was used to examine the microstructural changes in the outermost cellular structures due to HPH treatment. These alterations may be involved in the changes in some of the technological and functional properties of the strains that were observed after pressure treatment. Moreover, the proteomic profiles of the probiotic strains treated with HPH and incubated at 37°C for various periods showed different peptide patterns compared with those of the untreated cells. In addition, there were differences in the peaks that were observed in the low-mass spectral region (2000-3000 Da) of the spectral profiles of the control and treated samples. Due to pressure treatment, the volatile-molecule profiles of buttermilk inoculated with treated or control cells and stored at 4°C for 30 days exhibited overall changes in the aroma profile and in the production of molecules that improved its sensory profile, although the two different species imparted specific fingerprints to the product. The results of this study will contribute to understanding the changes that occur in the outermost cellular structures and the metabolism of LAB in response to HPH treatment. The findings of this investigation may contribute to elucidating the relationships between these changes and the alterations of the technological and functional properties of LAB induced by pressure treatment.

"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.

Comparative Salivary Proteome of Hepatitis B- and C-Infected Patients

PubMed Central

Gonçalves, Lorena Da Rós; Campanhon, Isabele Batista; Domingues, Romênia R.; Paes Leme, Adriana F.; Soares da Silva, Márcia Regina

2014-01-01

Hepatitis B and C virus (HBV and HCV) infections are an important cause of cirrhosis and hepatocellular carcinoma. The natural history has a prominent latent phase, and infected patients may remain undiagnosed; this situation may lead to the continuing spread of these infections in the community. Compelling reasons exist for using saliva as a diagnostic fluid because it meets the demands of being an inexpensive, noninvasive and easy-to-use diagnostic method. Indeed, comparative analysis of the salivary proteome using mass spectrometry is a promising new strategy for identifying biomarkers. Our goal is to apply an Orbitrap-based quantitative approach to explore the salivary proteome profile in HBV- and HCV-infected patients. In the present study, whole saliva was obtained from 20 healthy, (control) 20 HBV-infected and 20 HCV-infected subjects. Two distinct pools containing saliva from 10 subjects of each group were obtained. The samples were ultracentrifuged and fractionated, and all fractions were hydrolyzed (trypsin) and injected into an LTQ-VELOS ORBITRAP. The identification and analyses of peptides were performed using Proteome Discoverer1.3 and ScaffoldQ + v.3.3.1. From a total of 362 distinct proteins identified, 344 proteins were identified in the HBV, 326 in the HCV and 303 in the control groups. Some blood proteins, such as flavin reductase (which converts biliverdin to bilirubin), were detected only in the HCV group. The data showed a reduced presence of complement C3, ceruloplasmin, alpha(1)-acid glycoprotein and alpha(2)-acid glycoprotein in the hepatitis-infected patients. Peptides of serotransferrin and haptoglobin were less detected in the HCV group. This study provides an integrated perspective of the salivary proteome, which should be further explored in future studies targeting specific disease markers for HBV and HCV infection. PMID:25423034

Proteomics to assess the role of phenotypic plasticity in aquatic organisms exposed to pollution and global warming.

PubMed

Silvestre, Frédéric; Gillardin, Virginie; Dorts, Jennifer

2012-11-01

Nowadays, the unprecedented rates of anthropogenic changes in ecosystems suggest that organisms have to migrate to new distributional ranges or to adapt commensurately quickly to new conditions to avoid becoming extinct. Pollution and global warming are two of the most important threats aquatic organisms will have to face in the near future. If genetic changes in a population in response to natural selection are extensively studied, the role of acclimation through phenotypic plasticity (the property of a given genotype to produce different phenotypes in response to particular environmental conditions) in a species to deal with new environmental conditions remains largely unknown. Proteomics is the extensive study of the protein complement of a genome. It is dynamic and depends on the specific tissue, developmental stage, and environmental conditions. As the final product of gene expression, it is subjected to several regulatory steps from gene transcription to the functional protein. Consequently, there is a discrepancy between the abundance of mRNA and the abundance of the corresponding protein. Moreover, proteomics is closer to physiology and gives a more functional knowledge of the regulation of gene expression than does transcriptomics. The study of protein-expression profiles, however, gives a better portrayal of the cellular phenotype and is considered as a key link between the genotype and the organismal phenotype. Under new environmental conditions, we can observe a shift of the protein-expression pattern defining a new cellular phenotype that can possibly improve the fitness of the organism. It is now necessary to define a proteomic norm of reaction for organisms acclimating to environmental stressors. Its link to fitness will give new insights into how organisms can evolve in a changing environment. The proteomic literature bearing on chronic exposure to pollutants and on acclimation to heat stress in aquatic organisms, as well as potential application of proteomics in evolutionary issues, are outlined. While the transcriptome responses are commonly investigated, proteomics approaches now need to be intensified, with the new perspective of integrating the cellular phenotype with the organismal phenotype and with the mechanisms of the regulation of gene expression, such as epigenetics.

Comparative evaluation of electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) and high-pH reversed phase (Hp-RP) chromatography in profiling of rat kidney proteome.

PubMed

Hao, Piliang; Ren, Yan; Dutta, Bamaprasad; Sze, Siu Kwan

2013-04-26

ERLIC and high-pH RP (Hp-RP) have been reported to be promising alternatives to strong cation exchange (SCX) in proteome fractionation. Here we compared the performance of ERLIC, concatenated ERLIC and concatenated Hp-RP in proteome profiling. The protein identification is comparable in these three strategies, but significantly more unique peptides are identified by the two concatenation methods, resulting in a significant increase of the average protein sequence coverage. The pooling of fractions from spaced intervals results in more uniform distribution of peptides in each fraction compared with the chromatogram-based pooling of adjacent fractions. ERLIC fractionates peptides according to their pI and GRAVY values. These properties remains but becomes less remarkable in concatenated ERLIC. In contrast, the average pI and GRAVY values of the peptides are comparable in each fraction in concatenated Hp-RP. ERLIC performs the best in identifying peptides with pI>9 among the three strategies, while concatenated Hp-RP is good at identifying peptides with pI<4. These advantages are useful when either basic or acidic peptides/proteins are analytical targets. The power of ERLIC in identification of basic peptides seems to be due to their efficient separation from acidic peptides. This study facilitates the choice of proper fractionation strategies based on specific objectives. For in-depth proteomic analysis of a cell, tissue and plasma, multidimensional liquid chromatography (MDLC) is still necessary to reduce sample complexity for improving analytical dynamic range and proteome coverage. This work conducts a direct comparison of three promising first-dimensional proteome fractionation methods. They are comparable in identifying proteins, but concatenated ERLIC and concatenated Hp-RP identify significantly more unique peptides than ERLIC. ERLIC is good at analyzing basic peptides, while concatenated Hp-RP performs the best in analyzing acidic peptides with pI<4. This will facilitate the choice of the proper peptide fractionation strategy based on a specific need. A combination of different fractionation strategies can be used to increase the sequence coverage and number of protein identification due to the complementary effect between different methods. Copyright © 2013 Elsevier B.V. All rights reserved.

Proteomic Profiling of β-hCG-Induced Spheres in BRCA1 Defective Triple Negative Breast Cancer Cells.

PubMed

Sengodan, Satheesh Kumar; Rajan, Arathi; Hemalatha, Sreelatha Krishnakumar; Nadhan, Revathy; Jaleel, Abdul; Srinivas, Priya

2018-01-05

Previously, we identified that β-hCG is expressed by BRCA1 mutated but not wild type breast cancers in vitro/in vivo and exhibited a novel event in β-hCG overexpressing BRCA1 mutated HCC1937 cells where the cells were able to form spheres (HCC1937 β spheres) in adherent cell culture plates even in the absence of any growth factors. These spheres express stem cell and EMT markers. In the present study, we carried out the total proteomic profiling of these HCC1937 β spheres obtained from BRCA1 defective β-hCG expressing stable breast cancer cells to analyze the cell signaling pathways that are active in these cells. Functional annotation revealed proteins (164 cellular and 97 secretory) predominantly involved in oxygen binding, nucleosome assembly, cytoskeleton organization, protein folding, etc. Many of the proteins identified from HCC1937 β spheres in this study are also up regulated in breast cancers, which are directly linked with poor prognosis in human cancer samples as analyzed using TCGA data set. Survival analysis shows that β-hCG expressing cancer patients are linked with poor survival rate. Interestingly, hemoglobins were identified at both cellular and secretory level in HCC1937 β spheres and experiments after treating with ROS inducers revealed that β-hCG induces hemoglobin and protects the cancer cells during oxidative stress. Our proteomic data strongly propose β-hCG as an oncogenic molecule associated with BRCA1 mutation, and hence, targeting β-hCG could be a strategy to treat BRCA1 defective breast cancers.

Genes and pathways co-associated with the exposure to multiple drugs of abuse, including alcohol, amphetamine/methamphetamine, cocaine, marijuana, morphine, and/or nicotine: a review of proteomics analyses.

PubMed

Wang, Ju; Yuan, Wenji; Li, Ming D

2011-12-01

Drug addiction is a chronic neuronal disease. In recent years, proteomics technology has been widely used to assess the protein expression in the brain tissues of both animals and humans exposed to addictive drugs. Through this approach, a large number of proteins potentially involved in the etiology of drug addictions have been identified, which provide a valuable resource to study protein function, biochemical pathways, and networks related to the molecular mechanisms underlying drug dependence. In this article, we summarize the recent application of proteomics to profiling protein expression patterns in animal or human brain tissues after the administration of alcohol, amphetamine/methamphetamine, cocaine, marijuana, morphine/heroin/butorphanol, or nicotine. From available reports, we compiled a list of 497 proteins associated with exposure to one or more addictive drugs, with 160 being related to exposure to at least two abused drugs. A number of biochemical pathways and biological processes appear to be enriched among these proteins, including synaptic transmission and signaling pathways related to neuronal functions. The data included in this work provide a summary and extension of the proteomics studies on drug addiction. Furthermore, the proteins and biological processes highlighted here may provide valuable insight into the cellular activities and biological processes in neurons in the development of drug addiction.

Proteomic evaluation of sheep serum proteins

PubMed Central

2012-01-01

Background The applications of proteomic strategies to ovine medicine remain limited. The definition of serum proteome may be a good tool to identify useful protein biomarkers for recognising sub-clinical conditions and overt disease in sheep. Findings from bovine species are often directly translated for use in ovine medicine. In order to characterize normal protein patterns and improve knowledge of molecular species-specific characteristics, we generated a two-dimensional reference map of sheep serum. The possible application of this approach was tested by analysing serum protein patterns in ewes with mild broncho-pulmonary disease, which is very common in sheep and in the peripartum period which is a stressful time, with a high incidence of infectious and parasitic diseases. Results This study generated the first reference 2-DE maps of sheep serum. Overall, 250 protein spots were analyzed, and 138 identified. Compared with healthy sheep, serum protein profiles of animals with rhino-tracheo-bronchitis showed a significant decrease in protein spots identified as transthyretin, apolipoprotein A1 and a significant increase in spots identified as haptoglobin, endopin 1b and alpha1B glycoprotein. In the peripartum period, haptoglobin, alpha-1-acid glycoprotein, apolipoprotein A1 levels rose, while transthyretin content dropped. Conclusions This study describes applications of proteomics in putative biomarker discovery for early diagnosis as well as for monitoring the physiological and metabolic situations critical for ovine welfare. PMID:22630135

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.

Genome-wide identification of the subcellular localization of the Escherichia coli B proteome using experimental and computational methods.

PubMed

Han, Mee-Jung; Yun, Hongseok; Lee, Jeong Wook; Lee, Yu Hyun; Lee, Sang Yup; Yoo, Jong-Shin; Kim, Jin Young; Kim, Jihyun F; Hur, Cheol-Goo

2011-04-01

Escherichia coli K-12 and B strains have most widely been employed for scientific studies as well as industrial applications. Recently, the complete genome sequences of two representative descendants of E. coli B strains, REL606 and BL21(DE3), have been determined. Here, we report the subproteome reference maps of E. coli B REL606 by analyzing cytoplasmic, periplasmic, inner and outer membrane, and extracellular proteomes based on the genome information using experimental and computational approaches. Among the total of 3487 spots, 651 proteins including 410 non-redundant proteins were identified and characterized by 2-DE and LC-MS/MS; they include 440 cytoplasmic, 45 periplasmic, 50 inner membrane, 61 outer membrane, and 55 extracellular proteins. In addition, subcellular localizations of all 4205 ORFs of E. coli B were predicted by combined computational prediction methods. The subcellular localizations of 1812 (43.09%) proteins of currently unknown function were newly assigned. The results of computational prediction were also compared with the experimental results, showing that overall precision and recall were 92.16 and 92.16%, respectively. This work represents the most comprehensive analyses of the subproteomes of E. coli B, and will be useful as a reference for proteome profiling studies under various conditions. The complete proteome data are available online (http://ecolib.kaist.ac.kr). Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Proteomic profiling of mature leaves from oil palm (Elaeis guineensis Jacq.).

PubMed

Tan, Hooi Sin; Jacoby, Richard P; Ong-Abdullah, Meilina; Taylor, Nicolas L; Liddell, Susan; Chee, Wong Wei; Chin, Chiew Foan

2017-04-01

Oil palm is one of the most productive oil bearing crops grown in Southeast Asia. Due to the dwindling availability of agricultural land and increasing demand for high yielding oil palm seedlings, clonal propagation is vital to the oil palm industry. Most commonly, leaf explants are used for in vitro micropropagation of oil palm and to optimize this process it is important to unravel the physiological and molecular mechanisms underlying somatic embryo production from leaves. In this study, a proteomic approach was used to determine protein abundance of mature oil palm leaves. To do this, leaf proteins were extracted using TCA/acetone precipitation protocol and separated by 2DE. A total of 191 protein spots were observed on the 2D gels and 67 of the most abundant protein spots that were consistently observed were selected for further analysis with 35 successfully identified using MALDI TOF/TOF MS. The majority of proteins were classified as being involved in photosynthesis, metabolism, cellular biogenesis, stress response, and transport. This study provides the first proteomic assessment of oil palm leaves in this important oil crop and demonstrates the successful identification of selected proteins spots using the Malaysian Palm Oil Board (MPOB) Elaeis guineensis EST and NCBI-protein databases. The MS data have been deposited in the ProteomeXchange Consortium database with the data set identifier PXD001307. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Nasal protein profiles in work-related asthma caused by different exposures.

PubMed

Suojalehto, H; Lindström, I; Wolff, H; Puustinen, A

2018-03-01

The mechanisms of work-related asthma (WRA) are incompletely delineated. Nasal cell samples may be informative about processes in the lower airways. Our aim was to determine the nasal protein expression profiles of WRA caused by different kind of exposures. We collected nasal brush samples from 82 nonsmoking participants, including healthy controls and WRA patients exposed to (i) protein allergens, (ii) isocyanates and (iii) welding fumes the day after relevant exposure. The proteome changes in samples were analysed by two-dimensional difference gel electrophoresis, and the differentially regulated proteins found were identified by mass spectrometry. Immunological comparison was carried out using Western blot. We detected an average of 2500 spots per protein gel. Altogether, 228 protein spots were chosen for identification, yielding 77 different proteins. Compared to the controls, exposure to protein allergens had the largest effects on the proteome. Hierarchical clustering revealed that protein allergen- and isocyanate-related asthma had similar profiles, whereas asthma related to welding fumes differed. The highly overrepresented functional categories in the asthma groups were defence response, protease inhibitor activity, inflammatory and calcium signalling, complement activation and cellular response to oxidative stress. Immunological analysis confirmed the found abundance differences in galectin 10 and protein S100-A9 between the groups. Work-related asthma patients exposed to protein allergens and isocyanates elicit similar nasal proteome responses and the profiles of welders and healthy controls were alike. Revealed biological activities of the protein expression changes are associated with allergic inflammation and asthma. © 2017 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.

Standardized Profiling of The Membrane-Enriched Proteome of Mouse Dorsal Root Ganglia (DRG) Provides Novel Insights Into Chronic Pain.

PubMed

Rouwette, Tom; Sondermann, Julia; Avenali, Luca; Gomez-Varela, David; Schmidt, Manuela

2016-06-01

Chronic pain is a complex disease with limited treatment options. Several profiling efforts have been employed with the aim to dissect its molecular underpinnings. However, generated results are often inconsistent and nonoverlapping, which is largely because of inherent technical constraints. Emerging data-independent acquisition (DIA)-mass spectrometry (MS) has the potential to provide unbiased, reproducible and quantitative proteome maps - a prerequisite for standardization among experiments. Here, we designed a DIA-based proteomics workflow to profile changes in the abundance of dorsal root ganglia (DRG) proteins in two mouse models of chronic pain, inflammatory and neuropathic. We generated a DRG-specific spectral library containing 3067 DRG proteins, which enables their standardized quantification by means of DIA-MS in any laboratory. Using this resource, we profiled 2526 DRG proteins in each biological replicate of both chronic pain models and respective controls with unprecedented reproducibility. We detected numerous differentially regulated proteins, the majority of which exhibited pain model-specificity. Our approach recapitulates known biology and discovers dozens of proteins that have not been characterized in the somatosensory system before. Functional validation experiments and analysis of mouse pain behaviors demonstrate that indeed meaningful protein alterations were discovered. These results illustrate how the application of DIA-MS can open new avenues to achieve the long-awaited standardization in the molecular dissection of pathologies of the somatosensory system. Therefore, our findings provide a valuable framework to qualitatively extend our understanding of chronic pain and somatosensation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Dynamic proteome profiling of individual proteins in human skeletal muscle after a high-fat diet and resistance exercise.

PubMed

Camera, Donny M; Burniston, Jatin G; Pogson, Mark A; Smiles, William J; Hawley, John A

2017-12-01

It is generally accepted that muscle adaptation to resistance exercise (REX) training is underpinned by contraction-induced, increased rates of protein synthesis and dietary protein availability. By using dynamic proteome profiling (DPP), we investigated the contribution of both synthesis and breakdown to changes in abundance on a protein-by-protein basis in human skeletal muscle. Age-matched, overweight males consumed 9 d of a high-fat, low-carbohydrate diet during which time they either undertook 3 sessions of REX or performed no exercise. Precursor enrichment and the rate of incorporation of deuterium oxide into newly synthesized muscle proteins were determined by mass spectrometry. Ninety proteins were included in the DPP, with 28 proteins exhibiting significant responses to REX. The most common pattern of response was an increase in turnover, followed by an increase in abundance with no detectable increase in protein synthesis. Here, we provide novel evidence that demonstrates that the contribution of synthesis and breakdown to changes in protein abundance induced by REX differ on a protein-by-protein basis. We also highlight the importance of the degradation of individual muscle proteins after exercise in human skeletal muscle.-Camera, D. M., Burniston, J. G., Pogson, M. A., Smiles, W. J., Hawley, J. A. Dynamic proteome profiling of individual proteins in human skeletal muscle after a high-fat diet and resistance exercise. © FASEB.

Corra: Computational framework and tools for LC-MS discovery and targeted mass spectrometry-based proteomics

PubMed Central

Brusniak, Mi-Youn; Bodenmiller, Bernd; Campbell, David; Cooke, Kelly; Eddes, James; Garbutt, Andrew; Lau, Hollis; Letarte, Simon; Mueller, Lukas N; Sharma, Vagisha; Vitek, Olga; Zhang, Ning; Aebersold, Ruedi; Watts, Julian D

2008-01-01

Background Quantitative proteomics holds great promise for identifying proteins that are differentially abundant between populations representing different physiological or disease states. A range of computational tools is now available for both isotopically labeled and label-free liquid chromatography mass spectrometry (LC-MS) based quantitative proteomics. However, they are generally not comparable to each other in terms of functionality, user interfaces, information input/output, and do not readily facilitate appropriate statistical data analysis. These limitations, along with the array of choices, present a daunting prospect for biologists, and other researchers not trained in bioinformatics, who wish to use LC-MS-based quantitative proteomics. Results We have developed Corra, a computational framework and tools for discovery-based LC-MS proteomics. Corra extends and adapts existing algorithms used for LC-MS-based proteomics, and statistical algorithms, originally developed for microarray data analyses, appropriate for LC-MS data analysis. Corra also adapts software engineering technologies (e.g. Google Web Toolkit, distributed processing) so that computationally intense data processing and statistical analyses can run on a remote server, while the user controls and manages the process from their own computer via a simple web interface. Corra also allows the user to output significantly differentially abundant LC-MS-detected peptide features in a form compatible with subsequent sequence identification via tandem mass spectrometry (MS/MS). We present two case studies to illustrate the application of Corra to commonly performed LC-MS-based biological workflows: a pilot biomarker discovery study of glycoproteins isolated from human plasma samples relevant to type 2 diabetes, and a study in yeast to identify in vivo targets of the protein kinase Ark1 via phosphopeptide profiling. Conclusion The Corra computational framework leverages computational innovation to enable biologists or other researchers to process, analyze and visualize LC-MS data with what would otherwise be a complex and not user-friendly suite of tools. Corra enables appropriate statistical analyses, with controlled false-discovery rates, ultimately to inform subsequent targeted identification of differentially abundant peptides by MS/MS. For the user not trained in bioinformatics, Corra represents a complete, customizable, free and open source computational platform enabling LC-MS-based proteomic workflows, and as such, addresses an unmet need in the LC-MS proteomics field. PMID:19087345

Comprehensive proteomic profiling of adult Angiostrongylus costaricensis, a human parasitic nematode.

PubMed

Rebello, Karina M; Barros, Juliana S L; Mota, Ester M; Carvalho, Paulo C; Perales, Jonas; Lenzi, Henrique L; Neves-Ferreira, Ana G C

2011-08-24

Angiostrongylus costaricensis is a nematode helminth that causes an intestinal acute inflammatory process known as abdominal angiostrongyliasis, which is a poorly understood human disease occurring in Latin America. Our aim was to study the proteomic profiles of adult parasites focusing on immunogenic proteins. Total cellular extracts from both genders showed similar 2-DE profiles, with 60% of all protein spots focused between pH 5-7 and presenting molecular masses from 20.1 to 66 kDa. A total of 53 different dominant proteins were identified in our dataset and were mainly associated with the following over-represented Gene Ontology Biological Process terms: "macromolecule metabolic process", "developmental process", "response to stress", and "biological regulation". Female and male immunoblots showed similar patterns of reactive proteins. Immunoreactive spots identified by MALDI-PSD were found to represent heat shock proteins, a putative abnormal DAuer Formation family member, and galectins. To date, very few biochemical analyses have focused on the nematode Angiostrongylus costaricensis. As such, our results contribute to a better understanding of its biology and the mechanisms underlying the host-parasite relationship associated with this species. Moreover, our findings represent a first step in the search for candidate proteins for diagnostic assays and the treatment of this parasitic infection. Copyright © 2011 Elsevier B.V. All rights reserved.

New horizon for breast cancer biomarker discoveries: What might the liquid biopsy of nipple aspirate fluid hold?

PubMed

Mannello, Ferdinando

2017-09-01

The existence of cellular, molecular and biochemical heterogeneity of human breast cancers reveals the intricacy of biomarkers complexity, stimulating studies on new approaches (like "liquid biopsies") for the improvements in precision medicine. Breast cancer is recognized as a leading cause of morbidity and mortality worldwide with tumors significantly diverse and containing many types of cells showing different genetic and epigenetic profiles. In this field, the technology of liquid biopsy (applied to a fluid produced by breast gland, named nipple aspirate fluids, NAF) highlights the power of combining basic and clinical research. NAF is the mirror of the entire ductal/alveolar breast tree providing almost complete proteomic profile and a valuable source for biomarker discovery, in non-invasive manner than tissue biopsies. The liquid biopsy technology using NAF may represent the outstanding breakthrough of proteomic cancer research revealing novel diagnostic and prognostic applications. In conjunction to metabolomic and degradome profiling, the use of NAF as liquid biopsy approach will improve the detection of changes in the cellular microenvironment of the breast tumors, understanding molecular and biochemical mechanisms which drive breast tumor initiation, maintenance and progression, and finally enhancing the development of novel drug targets and new treatment strategies. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Integrative FourD omics approach profiles the target network of the carbon storage regulatory system

PubMed Central

Sowa, Steven W.; Gelderman, Grant; Leistra, Abigail N.; Buvanendiran, Aishwarya; Lipp, Sarah; Pitaktong, Areen; Vakulskas, Christopher A.; Romeo, Tony; Baldea, Michael

2017-01-01

Abstract Multi-target regulators represent a largely untapped area for metabolic engineering and anti-bacterial development. These regulators are complex to characterize because they often act at multiple levels, affecting proteins, transcripts and metabolites. Therefore, single omics experiments cannot profile their underlying targets and mechanisms. In this work, we used an Integrative FourD omics approach (INFO) that consists of collecting and analyzing systems data throughout multiple time points, using multiple genetic backgrounds, and multiple omics approaches (transcriptomics, proteomics and high throughput sequencing crosslinking immunoprecipitation) to evaluate simultaneous changes in gene expression after imposing an environmental stress that accentuates the regulatory features of a network. Using this approach, we profiled the targets and potential regulatory mechanisms of a global regulatory system, the well-studied carbon storage regulatory (Csr) system of Escherichia coli, which is widespread among bacteria. Using 126 sets of proteomics and transcriptomics data, we identified 136 potential direct CsrA targets, including 50 novel ones, categorized their behaviors into distinct regulatory patterns, and performed in vivo fluorescence-based follow up experiments. The results of this work validate 17 novel mRNAs as authentic direct CsrA targets and demonstrate a generalizable strategy to integrate multiple lines of omics data to identify a core pool of regulator targets. PMID:28126921

Characterising the enzymatic profile of crude tentacle extracts from the South Atlantic jellyfish Olindias sambaquiensis (Cnidaria: Hydrozoa).

PubMed

Knittel, Paloma S; Long, Paul F; Brammall, Lucas; Marques, Antonio C; Almeida, Michelle T; Padilla, Gabriel; Moura-da-Silva, Ana M

2016-09-01

Jellyfish venoms are of medical and biotechnological importance, with toxins displaying antimicrobial, analgesic and anti-tumor activities. Although proteolytic enzymes have also been described, detailed characterisation of these proteins is scant in Olindias spp. High throughput mass spectrometry profiling of cnidarian venoms has become increasingly popular since the first description of the proteomic profile of putative toxins isolated from nematocysts of the hydrozoan jellyfish Olindias sambaquiensis describing the presence of orthologous enzymes as presented in venoms of advanced species as snakes. Rigorous bioinformatics analyses can aid functional annotation, but biochemical assays are prerequisite to unambiguously assign toxic function to a peptide or protein. Here we present results that experimentally confirm previously predicted proteomic analysis that crude venom extracts from tentacles of O. sambaquiensis are composed of polypeptides with metalloproteinase, serine proteinase and phospholipases A2 activities. Surprisingly, levels of serine proteinase and phospholipase A2 activities were comparable to those observed in venoms of Bothrops snakes which were used as positive controls in this study. Hence, these data offer new opportunities to explore serine proteinase and phospholipase A2 activities in the clinical sequelae following O. sambaquiensis envenomation, with future possible biopharmaceutical applications. Copyright © 2016 Elsevier Ltd. All rights reserved.

Proteomic profiling reveals DNA damage, nucleolar and ribosomal stress are the main responses to oxaliplatin treatment in cancer cells.

PubMed

Ozdian, Tomas; Holub, Dusan; Maceckova, Zuzana; Varanasi, Lakshman; Rylova, Gabriela; Rehulka, Jiri; Vaclavkova, Jana; Slavik, Hanus; Moudry, Pavel; Znojek, Pawel; Stankova, Jarmila; de Sanctis, Juan Bautista; Hajduch, Marian; Dzubak, Petr

2017-06-06

Oxaliplatin is widely used to treat colorectal cancer in both palliative and adjuvant settings. It is also being tested for use in treating hematological, esophageal, biliary tract, pancreatic, gastric, and hepatocellular cancers. Despite its routine clinical use, little is known about the responses it induces in cancer cells. Therefore the whole-cell proteomics study was conducted to characterize the cellular response induced by oxaliplatin. Chemosensitive CCRF-CEM cells were treated with oxaliplatin at 29.3μM (5×IC 50 ) for 240min (half-time to caspase activation). The proteomes of un-/treated cells were then compared by high-resolution mass spectrometry, revealing 4049 proteins expressed over 3 biological replicates. Among these proteins, 76 were significantly downregulated and 31 significantly upregulated in at least two replicates. In agreement with the DNA-damaging effects of platinum drugs, proteins involved in DNA damage responses were present in both the upregulated and downregulated groups. The downregulated proteins were divided into three subgroups; i) centrosomal proteins, ii) RNA processing and iii) ribosomal proteins, which indicates nucleolar and ribosomal stress. In conclusion, our data supported by further validation experiments indicate the initial cellular response to oxaliplatin is the activation of DNA damage response, which in turn or in parallel triggers nucleolar and ribosomal stress. We have performed a whole-cell proteomic study of cellular response to oxaliplatin treatment, which is the drug predominantly used in the treatment of colorectal cancer. Compared to its predecessors, cisplatin and carboplatin, there is only a small fraction of studies dedicated to oxaliplatin. From those studies, most of them are focused on modification of treatment regimens or study of oxaliplatin in new cancer diagnoses. Cellular response hasn't been studied deeply and to our best knowledge, this is the first whole-cell proteomics study focused exclusively to this important topic, which can help to understand molecular mechanisms of action. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

Proteomic profiling reveals candidate markers for arsenic-induced skin keratosis.

PubMed

Guo, Zhiling; Hu, Qin; Tian, Jijing; Yan, Li; Jing, Chuanyong; Xie, Heidi Qunhui; Bao, Wenjun; Rice, Robert H; Zhao, Bin; Jiang, Guibin

2016-11-01

Proteomics technology is an attractive biomarker candidate discovery tool that can be applied to study large sets of biological molecules. To identify novel biomarkers and molecular targets in arsenic-induced skin lesions, we have determined the protein profile of arsenic-affected human epidermal stratum corneum by shotgun proteomics. Samples of palm and foot sole from healthy subjects were analyzed, demonstrating similar protein patterns in palm and sole. Samples were collected from the palms of subjects with arsenic keratosis (lesional and adjacent non-lesional samples) and arsenic-exposed subjects without lesions (normal). Samples from non-exposed healthy individuals served as controls. We found that three proteins in arsenic-exposed lesional epidermis were consistently distinguishably expressed from the unaffected epidermis. One of these proteins, the cadherin-like transmembrane glycoprotein, desmoglein 1 (DSG1) was suppressed. Down-regulation of DSG1 may lead to reduced cell-cell adhesion, resulting in abnormal epidermal differentiation. The expression of keratin 6c (KRT6C) and fatty acid binding protein 5 (FABP5) were significantly increased. FABP5 is an intracellular lipid chaperone that plays an essential role in fatty acid metabolism in human skin. This raises a possibility that overexpression of FABP5 may affect the proliferation or differentiation of keratinocytes by altering lipid metabolism. KRT6C is a constituent of the cytoskeleton that maintains epidermal integrity and cohesion. Abnormal expression of KRT6C may affect its structural role in the epidermis. Our findings suggest an important approach for future studies of arsenic-mediated toxicity and skin cancer, where certain proteins may represent useful biomarkers of early diagnoses in high-risk populations and hopefully new treatment targets. Further studies are required to understand the biological role of these markers in skin pathogenesis from arsenic exposure. Copyright © 2016 Elsevier Ltd. All rights reserved.

Comparison of protein profiles of beech bark disease-resistant or beech bark disease-susceptible American beech

Treesearch

Mary E. Mason; Marek Krasowski; Judy Loo; Jennifer. Koch

2011-01-01

Proteomic analysis of beech bark proteins from trees resistant and susceptible to beech bark disease (BBD) was conducted. Sixteen trees from eight geographically isolated stands, 10 resistant (healthy) and 6 susceptible (diseased/infested) trees, were studied. The genetic complexity of the sample unit, the sampling across a wide geographic area, and the complexity of...

Root responses of Medicago truncatula plants grown in two different iron deficiency conditions: changes in root protein profile and riboflavin biosynthesis

USDA-ARS?s Scientific Manuscript database

Iron deficiency is a yield-limiting factor with major implications for field crop production in one-third of the world's agricultural areas, especially those with high soil CaCO3. A two-dimensional gel electrophoresis proteomic approach was combined with a study on the riboflavin synthesis pathway, ...

Compartment-resolved Proteomic Analysis of Mouse Aorta during Atherosclerotic Plaque Formation Reveals Osteoclast-specific Protein Expression.

PubMed

Wierer, Michael; Prestel, Matthias; Schiller, Herbert B; Yan, Guangyao; Schaab, Christoph; Azghandi, Sepiede; Werner, Julia; Kessler, Thorsten; Malik, Rainer; Murgia, Marta; Aherrahrou, Zouhair; Schunkert, Heribert; Dichgans, Martin; Mann, Matthias

2018-02-01

Atherosclerosis leads to vascular lesions that involve major rearrangements of the vascular proteome, especially of the extracellular matrix (ECM). Using single aortas from ApoE knock out mice, we quantified formation of plaques by single-run, high-resolution mass spectrometry (MS)-based proteomics. To probe localization on a proteome-wide scale we employed quantitative detergent solubility profiling. This compartment- and time-resolved resource of atherogenesis comprised 5117 proteins, 182 of which changed their expression status in response to vessel maturation and atherosclerotic plaque development. In the insoluble ECM proteome, 65 proteins significantly changed, including relevant collagens, matrix metalloproteinases and macrophage derived proteins. Among novel factors in atherosclerosis, we identified matrilin-2, the collagen IV crosslinking enzyme peroxidasin as well as the poorly characterized MAM-domain containing 2 (Mamdc2) protein as being up-regulated in the ECM during atherogenesis. Intriguingly, three subunits of the osteoclast specific V-ATPase complex were strongly increased in mature plaques with an enrichment in macrophages thus implying an active de-mineralization function. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Compartment-resolved Proteomic Analysis of Mouse Aorta during Atherosclerotic Plaque Formation Reveals Osteoclast-specific Protein Expression*

PubMed Central

Wierer, Michael; Prestel, Matthias; Schiller, Herbert B.; Yan, Guangyao; Schaab, Christoph; Azghandi, Sepiede; Werner, Julia; Kessler, Thorsten; Malik, Rainer; Murgia, Marta; Aherrahrou, Zouhair; Schunkert, Heribert; Dichgans, Martin; Mann, Matthias

2018-01-01

Atherosclerosis leads to vascular lesions that involve major rearrangements of the vascular proteome, especially of the extracellular matrix (ECM). Using single aortas from ApoE knock out mice, we quantified formation of plaques by single-run, high-resolution mass spectrometry (MS)-based proteomics. To probe localization on a proteome-wide scale we employed quantitative detergent solubility profiling. This compartment- and time-resolved resource of atherogenesis comprised 5117 proteins, 182 of which changed their expression status in response to vessel maturation and atherosclerotic plaque development. In the insoluble ECM proteome, 65 proteins significantly changed, including relevant collagens, matrix metalloproteinases and macrophage derived proteins. Among novel factors in atherosclerosis, we identified matrilin-2, the collagen IV crosslinking enzyme peroxidasin as well as the poorly characterized MAM-domain containing 2 (Mamdc2) protein as being up-regulated in the ECM during atherogenesis. Intriguingly, three subunits of the osteoclast specific V-ATPase complex were strongly increased in mature plaques with an enrichment in macrophages thus implying an active de-mineralization function. PMID:29208753

Saliva Proteomics Analysis Offers Insights on Type 1 Diabetes Pathology in a Pediatric Population

PubMed Central

Pappa, Eftychia; Vastardis, Heleni; Mermelekas, George; Gerasimidi-Vazeou, Andriani; Zoidakis, Jerome; Vougas, Konstantinos

2018-01-01

The composition of the salivary proteome is affected by pathological conditions. We analyzed by high resolution mass spectrometry approaches saliva samples collected from children and adolescents with type 1 diabetes and healthy controls. The list of more than 2000 high confidence protein identifications constitutes a comprehensive characterization of the salivary proteome. Patients with good glycemic regulation and healthy individuals have comparable proteomic profiles. In contrast, a significant number of differentially expressed proteins were identified in the saliva of patients with poor glycemic regulation compared to patients with good glycemic control and healthy children. These proteins are involved in biological processes relevant to diabetic pathology such as endothelial damage and inflammation. Moreover, a putative preventive therapeutic approach was identified based on bioinformatic analysis of the deregulated salivary proteins. Thus, thorough characterization of saliva proteins in diabetic pediatric patients established a connection between molecular changes and disease pathology. This proteomic and bioinformatic approach highlights the potential of salivary diagnostics in diabetes pathology and opens the way for preventive treatment of the disease. PMID:29755368

Proteome profiling of virus-host interactions of wild type and attenuated measles virus strains.

PubMed

Billing, Anja M; Kessler, Julia R; Revets, Dominique; Sausy, Aurélie; Schmitz, Stephanie; Barra, Claire; Muller, Claude P

2014-08-28

Quantitative gel-based proteomics (2D DIGE coupled to MALDI-TOF/TOF MS) has been used to investigate the effects of different measles virus (MV) strains on the host cell proteome. A549/hSLAM cells were infected either with wild type MV strains, an attenuated vaccine or a multiple passaged Vero cell adapted strain. By including interferon beta treatment as a control it was possible to distinguish between the classical antiviral response and changes induced specifically by the different strains. Of 38 differentially expressed proteins in total (p-value ≤0.05, fold change ≥2), 18 proteins were uniquely modulated following MV infection with up to 9 proteins specific per individual strain. Interestingly, wt strains displayed distinct protein patterns particularly during the late phase of infection. Proteins were grouped into cytoskeleton, metabolism, transcription/translation, immune response and mitochondrial proteins. Bioinformatics analysis revealed mostly changes in proteins regulating cell death and apoptosis. Surprisingly, wt strains affected the cytokeratin system much stronger than the vaccine strain. To our knowledge, this is the first study on the MV-host proteome addressing interstrain differences. In the present study we investigated the host cell proteome upon measles virus (MV) infection. The novelty about this study is the side-by side comparison of different strains from the same virus, which has not been done at the proteome level for any other virus including MV. We used different virus strains including a vaccine strain, wild type isolates derived from MV-infected patients as well as a Vero cell adapted strain, which serves as an intermediate between vaccine and wild type strain. We observed differences between vaccine and wild type strains as well as common features between different wild type strains. Perhaps one of the most surprising findings was that differences did not only occur between wild type and vaccine or Vero cell adapted strains but also between different wild type strains. In fact our study suggests that besides the cytokeratin and the IFN system wild type viruses seem to differ as much among each other than from vaccine strains. Thus our results are suggestive of complex and diverse virus-host interactions which differ considerably between different wild type strains. Our data indicate that interstrain differences are prominent and have so far been neglected by proteomics studies. Copyright © 2014 Elsevier B.V. All rights reserved.

Proteomic Approaches to Enable Point-of-Care Testing and Personalized Medicine for Psychiatric Disorders.

PubMed

Guest, Francesca L; Guest, Paul C

2017-01-01

This chapter describes how innovations that are driven by proteomic biomarker techniques can facilitate earlier and better treatment of patients who suffer from psychiatric disorders. The application of new micro-fluidic devices along with miniaturized biosensors and transducers will enable the development of handheld point-of-care testing instruments which can analyse a drop of a blood within the time span of a single visit to the doctor's office. It is anticipated that these approaches will incorporate both biochemical and clinical information, resulting in unique profiles for each test subject. These profiles can in turn be used to drive personalized medicine approaches in this devastating disease area. In addition, smartphone applications (apps) for self-monitoring will see increasing use for improved patient outcomes.

Proteomic Profile of Brucella abortus-Infected Bovine Chorioallantoic Membrane Explants

PubMed Central

Mol, Juliana P. S.; Pires, Simone F.; Chapeaurouge, Alexander D.; Perales, Jonas; Santos, Renato L.; Andrade, Hélida M.; Lage, Andrey P.

2016-01-01

Brucella abortus is the etiological agent of bovine brucellosis, a zoonotic disease that causes significant economic losses worldwide. The differential proteomic profile of bovine chorioallantoic membrane (CAM) explants at early stages of infection with B. abortus (0.5, 2, 4, and 8 h) was determined. Analysis of CAM explants at 0.5 and 4 h showed the highest differences between uninfected and infected CAM explants, and therefore were used for the Differential Gel Electrophoresis (DIGE). A total of 103 spots were present in only one experimental group and were selected for identification by mass spectrometry (MALDI/ToF-ToF). Proteins only identified in extracts of CAM explants infected with B. abortus were related to recognition of PAMPs by TLR, production of reactive oxygen species, intracellular trafficking, and inflammation. PMID:27104343

Systems biology of human atherosclerosis.

PubMed

Shalhoub, Joseph; Sikkel, Markus B; Davies, Kerry J; Vorkas, Panagiotis A; Want, Elizabeth J; Davies, Alun H

2014-01-01

Systems biology describes a holistic and integrative approach to understand physiology and pathology. The "omic" disciplines include genomics, transcriptomics, proteomics, and metabolic profiling (metabonomics and metabolomics). By adopting a stance, which is opposing (yet complimentary) to conventional research techniques, systems biology offers an overview by assessing the "net" biological effect imposed by a disease or nondisease state. There are a number of different organizational levels to be understood, from DNA to protein, metabolites, cells, organs and organisms, even beyond this to an organism's context. Systems biology relies on the existence of "nodes" and "edges." Nodes are the constituent part of the system being studied (eg, proteins in the proteome), while the edges are the way these constituents interact. In future, it will be increasingly important to collaborate, collating data from multiple studies to improve data sets, making them freely available and undertaking integrative analyses.

Molecular targets of alcohol action: translational research for pharmacotherapy development and screening

PubMed Central

Gorini, Giorgio; Bell, Richard L.; Mayfield, R. Dayne

2016-01-01

Summary Alcohol abuse and dependence are multifaceted disorders with neurobiological, psychological, and environmental components. Research on other complex neuropsychiatric diseases suggests that genetically influenced intermediate characteristics affect the risk for heavy alcohol consumption and its consequences. Diverse therapeutic interventions can be developed through identification of reliable biomarkers for this disorder and new pharmacological targets for its treatment. Advances in the fields of genomics and proteomics offer a number of possible targets for the development of new therapeutic approaches. This brain-focused review highlights studies identifying neurobiological systems associated with these targets and possible pharmacotherapies, summarizing evidence from clinically relevant animal and human studies, as well as sketching improvements and challenges facing the fields of proteomics and genomics. Concluding thoughts on using results from these profiling technologies for medication development are also presented. PMID:21199775

[Plasma proteomic analysis in children with infectious mononucleosis].

PubMed

Ran, Zhi-Ling; Xiao, Bin; Liu, Hong-Rui; Liu, You-Ping; Sheng, Qiao-Ni

2015-03-01

To explore the abnormal expression of plasma proteins by analysis of proteomic expression profile in children with infectious mononucleosis (IM). Two dimensional gel electrophoresis (2-DE) followed by the mass spectrometry was used to examine important protein spots with different expression levels between children with IM and normal controls. Seven differential proteins were obtained: hemopexin, vitamin D binding protein, fetuin A, C-reactive protein, apolipoprotein A, haptoglobin and transthyretin. Compared with the control group, haptoglobin showed a higher expression level in children with IM, and the expression levels of the other proteins were obviously down-regulated. The expression changes of differential proteins identified in this study are all related with the liver acute injury, suggesting that children with IM are associated with acute liver injury. Further studies on the characteristics of above proteins will contribute to the diagnosis and treatment of pediatric IM.

Identification of phosphoproteins associated with maintenance of transformed state in temperature-sensitive Rous sarcoma-virus infected cells by proteomic analysis

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

Yamaoka, Kazuko; Imajoh-Ohmi, Shinobu; Fukuda, Hiroyuki

2006-07-07

To identify phosphotyrosine-containing proteins essential for maintaining the transformed state, we studied the tyrosine phosphorylation profile of temperature-sensitive mutant of Rous sarcoma virus, tsNY68, infected cells (68N7). Shifting the temperature from 39 {sup o}C (nonpermissive) to 32 {sup o}C (permissive) markedly increased the expression of phosphotyrosine-containing cell membrane proteins of {approx}40 kDa, as assessed by SDS-PAGE. Membrane and nuclear proteins were separated by two-dimensional gel electrophoresis and immunoblotted with anti-phosphotyrosine antibody. Proteins showing temperature-dependent changes in phosphorylation profile were subjected to in-gel digestion with trypsin and analyzed by mass spectrometry. Five proteins were identified: heterogeneous nuclear ribonucleoprotein (hnRNP) A3, hnRNPmore » A2, annexin II, phosphoglycerate mutase 1, and triosephosphate isomerase 1. hnRNP A3 was phosphorylated at serine residues and had both serine and tyrosine phosphorylated sites. These results suggest an important complementary role for proteomics in identifying molecular abnormalities associated with tumor progression that may be attractive candidates for tumor diagnosis.« less

Sulfur alleviates arsenic toxicity by reducing its accumulation and modulating proteome, amino acids and thiol metabolism in rice leaves

NASA Astrophysics Data System (ADS)

Dixit, Garima; Singh, Amit Pal; Kumar, Amit; Dwivedi, Sanjay; Deeba, Farah; Kumar, Smita; Suman, Shankar; Adhikari, Bijan; Shukla, Yogeshwar; Trivedi, Prabodh Kumar; Pandey, Vivek; Tripathi, Rudra Deo

2015-11-01

Arsenic (As) contamination of water is a global concern and rice consumption is the biggest dietary exposure to human posing carcinogenic risks, predominantly in Asia. Sulfur (S) is involved in di-sulfide linkage in many proteins and plays crucial role in As detoxification. Present study explores role of variable S supply on rice leaf proteome, its inclination towards amino acids (AA) profile and non protein thiols under arsenite exposure. Analysis of 282 detected proteins on 2-DE gel revealed 113 differentially expressed proteins, out of which 80 were identified by MALDI-TOF-TOF. The identified proteins were mostly involved in glycolysis, TCA cycle, AA biosynthesis, photosynthesis, protein metabolism, stress and energy metabolism. Among these, glycolytic enzymes play a major role in AA biosynthesis that leads to change in AAs profiling. Proteins of glycolytic pathway, photosynthesis and energy metabolism were also validated by western blot analysis. Conclusively S supplementation reduced the As accumulation in shoot positively skewed thiol metabolism and glycolysis towards AA accumulation under AsIII stress.

The Combined Use of Proteomics and Transcriptomics Reveals a Complex Secondary Metabolite Network in Peperomia obtusifolia.

PubMed

Batista, Andrea N L; Santos-Pinto, José Roberto A Dos; Batista, João M; Souza-Moreira, Tatiana M; Santoni, Mariana M; Zanelli, Cleslei F; Kato, Massuo J; López, Silvia N; Palma, Mario S; Furlan, Maysa

2017-05-26

Peperomia obtusifolia, an ornamental plant from the Piperaceae family, accumulates a series of secondary metabolites with interesting biological properties. From a biosynthesis standpoint, this species produces several benzopyrans derived from orsellinic acid, which is a polyketide typically found in fungi. Additionally, the chiral benzopyrans were reported as racemic and/or as diastereomeric mixtures, which raises questions about the level of enzymatic control in the cyclization step for the formation of the 3,4-dihydro-2H-pyran moiety. Therefore, this article describes the use of shotgun proteomic and transcriptome studies as well as phytochemical profiling for the characterization of the main biosynthesis pathways active in P. obtusifolia. This combined approach resulted in the identification of a series of proteins involved in its secondary metabolism, including tocopherol cyclase and prenyltransferases. The activity of these enzymes was supported by the phytochemical profiling performed in different organs of P. obtusifolia. However, the polyketide synthases possibly involved in the production of orsellinic acid could not be identified, suggesting that orsellinic acid may be produced by endophytes intimately associated with the plant.

Comparative proteomic expression profile in all-trans retinoic acid differentiated neuroblastoma cell line.

PubMed

Cimmino, Flora; Spano, Daniela; Capasso, Mario; Zambrano, Nicola; Russo, Roberta; Zollo, Massimo; Iolascon, Achille

2007-07-01

Neuroblastoma (NB) is an infant tumor which frequently differentiates into neurons. We used two-dimensional differential in-gel electrophoresis (2D-DIGE) to analyze the cytosolic and nuclear protein expression patterns of LAN-5 cells following neuronal differentiating agent all-trans-retinoic acid treatment. We identified several candidate proteins, from which G beta2 and Prefoldin 3 may have a role on NB development. These results strength the use of proteomics to discover new putative protein targets in cancer.

Analysis of the functional aspects and seminal plasma proteomic profile of sperm from smokers.

PubMed

Antoniassi, Mariana Pereira; Intasqui, Paula; Camargo, Mariana; Zylbersztejn, Daniel Suslik; Carvalho, Valdemir Melechco; Cardozo, Karina H M; Bertolla, Ricardo Pimenta

2016-11-01

To evaluate the effect of smoking on sperm functional quality and seminal plasma proteomic profile. Sperm functional tests were performed in 20 non-smoking men with normal semen quality, according to the World Health Organization (2010) and in 20 smoking patients. These included: evaluation of DNA fragmentation by alkaline Comet assay; analysis of mitochondrial activity using DAB staining; and acrosomal integrity evaluation by PNA binding. The remaining semen was centrifuged and seminal plasma was used for proteomic analysis (liquid chromatography-tandem mass spectrometry). The quantified proteins were used for Venn diagram construction in Cytoscape 3.2.1 software, using the PINA4MS plug-in. Then, differentially expressed proteins were used for functional enrichment analysis of Gene Ontology categories, Kyoto Encyclopedia of Genes and Genomes and Reactome, using Cytoscape software and the ClueGO 2.2.0 plug-in. Smokers had a higher percentage of sperm DNA damage (Comet classes III and IV; P < 0.01), partially and fully inactive mitochondria (DAB classes III and IV; P = 0.001 and P = 0.006, respectively) and non-intact acrosomes (P < 0.01) when compared with the control group. With respect to proteomic analysis, 422 proteins were identified and quantified, of which one protein was absent, 27 proteins were under-represented and six proteins were over-represented in smokers. Functional enrichment analysis showed the enrichment of antigen processing and presentation, positive regulation of prostaglandin secretion involved in immune response, protein kinase A signalling and arachidonic acid secretion, complement activation, regulation of the cytokine-mediated signalling pathway and regulation of acute inflammatory response in the study group (smokers). In conclusion, cigarette smoking was associated with an inflammatory state in the accessory glands and in the testis, as shown by enriched proteomic pathways. This state causes an alteration in sperm functional quality, which is characterized by decreased acrosome integrity and mitochondrial activity, as well as by increased nuclear DNA fragmentation. © 2016 The Authors BJU International © 2016 BJU International Published by John Wiley & Sons Ltd.

Proteomic analysis of enterotoxigenic Escherichia coli (ETEC) in neutral and alkaline conditions.

PubMed

Gonzales-Siles, Lucia; Karlsson, Roger; Kenny, Diarmuid; Karlsson, Anders; Sjöling, Åsa

2017-01-07

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea in children and travelers to endemic areas. Secretion of the heat labile AB 5 toxin (LT) is induced by alkaline conditions. In this study, we determined the surface proteome of ETEC exposed to alkaline conditions (pH 9) as compared to neutral conditions (pH 7) using a LPI Hexalane FlowCell combined with quantitative proteomics. Relative quantitation with isobaric labeling (TMT) was used to compare peptide abundance and their corresponding proteins in multiple samples at MS/MS level. For protein identification and quantification samples were analyzed using either a 1D-LCMS or a 2D-LCMS approach. Strong up-regulation of the ATP synthase operon encoding F1Fo ATP synthase and down-regulation of proton pumping proteins NuoF, NuoG, Ndh and WrbA were detected among proteins involved in regulating the proton and electron transport under alkaline conditions. Reduced expression of proteins involved in osmotic stress was found at alkaline conditions while the Sec-dependent transport over the inner membrane and outer membrane protein proteins such as OmpA and the β-Barrel Assembly Machinery (BAM) complex were up-regulated. ETEC exposed to alkaline environments express a specific proteome profile characterized by up-regulation of membrane proteins and secretion of LT toxin. Alkaline microenvironments have been reported close to the intestinal epithelium and the alkaline proteome may hence represent a better view of ETEC during infection.

A draft map of the human ovarian proteome for tissue engineering and clinical applications.

PubMed

Ouni, Emna; Vertommen, Didier; Chiti, Maria Costanza; Dolmans, Marie-Madeleine; Amorim, Christiani Andrade

2018-02-23

Fertility preservation research in women today is increasingly taking advantage of bioengineering techniques to develop new biomimetic materials and solutions to safeguard ovarian cell function and microenvironment in vitro and in vivo. However, available data on the human ovary are limited and fundamental differences between animal models and humans are hampering researchers in their quest for more extensive knowledge of human ovarian physiology and key reproductive proteins that need to be preserved. We therefore turned to multi-dimensional label-free mass spectrometry to analyze human ovarian cortex, as it is a high-throughput and conclusive technique providing information on the proteomic composition of complex tissues like the ovary. In-depth proteomic profiling through two-dimensional liquid chromatography-mass spectrometry, western blot, histological and immunohistochemical analyses, and data mining helped us to confidently identify 1,508 proteins. Moreover, our method allowed us to chart the most complete representation so far of the ovarian matrisome, defined as the ensemble of extracellular matrix proteins and associated factors, including more than 80 proteins. In conclusion, this study will provide a better understanding of ovarian proteomics, with a detailed characterization of the ovarian follicle microenvironment, in order to enable bioengineers to create biomimetic scaffolds for transplantation and three-dimensional in vitro culture. By publishing our proteomic data, we also hope to contribute to accelerating biomedical research into ovarian health and disease in general. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Isolation of Polysomal RNA for Analyzing Stress-Responsive Genes Regulated at the Translational Level in Plants.

PubMed

Li, Yong-Fang; Mahalingam, Ramamurthy; Sunkar, Ramanjulu

2017-01-01

Alteration of gene expression is an essential mechanism, which allows plants to respond and adapt to adverse environmental conditions. Transcriptome and proteome analyses in plants exposed to abiotic stresses revealed that protein levels are not correlated with the changes in corresponding mRNAs, indicating regulation at translational level is another major regulator for gene expression. Analysis of translatome, which refers to all mRNAs associated with ribosomes, thus has the potential to bridge the gap between transcriptome and proteome. Polysomal RNA profiling and recently developed ribosome profiling (Ribo-seq) are two main methods for translatome analysis at global level. Here, we describe the classical procedure for polysomal RNA isolation by sucrose gradient ultracentrifugation followed by highthroughput RNA-seq to identify genes regulated at translational level. Polysomal RNA can be further used for a variety of downstream applications including Northern blot analysis, qRT-PCR, RNase protection assay, and microarray-based gene expression profiling.

Urine Collected From Diapers Can Be Used for 2-Dimensional Polyacrylamide Gel Electrophoresis (2D-PAGE) in Infants and Young Children

PubMed Central

Kennedy, Mary Jayne; Griffin, Angela; Su, Ruifeng; Merchant, Michael; Klein, Jon

2011-01-01

Urinary proteomic profiling has potential to identify candidate biomarkers of renal injury in infants provided an adequate urine sample can be obtained. Although diapers are used to obtain urine for clinical evaluation, their use for proteomic analysis has not been investigated. We therefore performed feasibility studies on the use of diaper-extracted urine for 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Pediatric waste urine (2–20 mL) was applied to gel-containing, non-gel and cotton-gauze diapers and then mechanically expressed. Urine volume and total protein were measured pre- and post-extraction. Proteins were separated via 2D-PAGE following application of urine (20–40 mL) to each matrix. 2D-PAGE was also performed on clinical specimens collected using each diaper type. Differences in the adsorption and retention of urine volume and protein were noted between matrices. Non-gel and cotton-gauze diapers provided the best protein/volume recovery and the lowest interference with the Bradford assay. 2D-PAGE was also successfully completed using urine samples from both cotton fiber matrices. Conversely, samples from low-gel diapers demonstrated poor protein separation and reproducibility. Diapers containing cotton-fiber matrices appear adequate for 2D-PAGE. Qualitative and quantitative analyses of resolved proteins using replicate, high resolution gels will be required, however, before diaper-extracted urine can be applied in proteomic profiling. PMID:21137001

Differential proteome profiling in the hippocampus of amnesic mice.

PubMed

Baghel, Meghraj Singh; Thakur, Mahendra Kumar

2017-08-01

Amnesia or memory loss is associated with brain aging and several neurodegenerative pathologies including Alzheimer's disease (AD). This can be induced by a cholinergic antagonist scopolamine but the underlying molecular mechanism is poorly understood. This study of proteome profiling in the hippocampus could provide conceptual insights into the molecular mechanisms involved in amnesia. To reveal this, mice were administered scopolamine to induce amnesia and memory impairment was validated by novel object recognition test. Using two-dimensional gel electrophoresis coupled with MALDI-MS/MS, we have analyzed the hippocampal proteome and identified 18 proteins which were differentially expressed. Out of these proteins, 11 were downregulated and 7 were upregulated in scopolamine-treated mice as compared to control. In silico analysis showed that the majority of identified proteins are involved in metabolism, catalytic activity, and cytoskeleton architectural functions. STRING interaction network analysis revealed that majority of identified proteins exhibit common association with Actg1 cytoskeleton and Vdac1 energy transporter protein. Furthermore, interaction map analysis showed that Fascin1 and Coronin 1b individually interact with Actg1 and regulate the actin filament dynamics. Vdac1 was significantly downregulated in amnesic mice and showed interaction with other proteins in interaction network. Therefore, we silenced Vdac1 in the hippocampus of normal young mice and found similar impairment in recognition memory of Vdac1 silenced and scopolamine-treated mice. Thus, these findings suggest that Vdac1-mediated disruption of energy metabolism and cytoskeleton architecture might be involved in scopolamine-induced amnesia. © 2017 Wiley Periodicals, Inc.

Mass spectral analysis of urine proteomic profiles of dairy cows suffering from clinical ketosis.

PubMed

Xu, Chuang; Shu, Shi; Xia, Cheng; Wang, Pengxian; Sun, Yuhang; Xu, Chuchu; Li, Changsheng

2015-01-01

Ketosis is an important metabolic disorder in dairy cows during the transition period. The urine proteomics of ketosis has not been investigated using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). The aim is to determine differences between urine proteomic profiles of healthy cows and those with clinical ketosis, and facilitate studies of the underlying physiological and biochemical mechanisms that lead to liver pathology in ketosis. We analyzed the urine samples of 20 cows with clinical ketosis (group 1) and 20 control cows (group 2) using SELDI-TOF-MS. Thirty-nine peptide peaks differed between both groups. Polypeptides corresponding to 26 of these differential peptide peaks were identified using the SWISS-PROT protein database. We found that the peaks of 11 distinct polypeptides from the urine samples of the ketosis group were significantly reduced, compared with those of the control group as based on the Wilcoxon rank sum test. Among these were VGF (non-acronymic) protein, amyloid precursor protein, serum amyloid A (SAA), fibrinogen, C1INH, apolipoprotein C-III, cystatin C, transthyretin, hepcidin, human neutrophil peptides, and osteopontin. These proteins may represent novel biomarkers of the metabolic changes that occur in dairy cows with ketosis. Our results will help to better understand the physiological changes and pathogenesis observed in cows with ketosis. The SELDI-TOF-MS can be used to understand the physiological and biochemical mechanisms of ketosis and identify biomarkers of the disease.

Proteomic peptide profiling for preemptive diagnosis of acute graft-versus-host disease after allogeneic stem cell transplantation.

PubMed

Weissinger, E M; Metzger, J; Dobbelstein, C; Wolff, D; Schleuning, M; Kuzmina, Z; Greinix, H; Dickinson, A M; Mullen, W; Kreipe, H; Hamwi, I; Morgan, M; Krons, A; Tchebotarenko, I; Ihlenburg-Schwarz, D; Dammann, E; Collin, M; Ehrlich, S; Diedrich, H; Stadler, M; Eder, M; Holler, E; Mischak, H; Krauter, J; Ganser, A

2014-04-01

Allogeneic hematopoietic stem cell transplantation is one curative treatment for hematological malignancies, but is compromised by life-threatening complications, such as severe acute graft-versus-host disease (aGvHD). Prediction of severe aGvHD as early as possible is crucial to allow timely initiation of treatment. Here we report on a multicentre validation of an aGvHD-specific urinary proteomic classifier (aGvHD_MS17) in 423 patients. Samples (n=1106) were collected prospectively between day +7 and day +130 and analyzed using capillary electrophoresis coupled on-line to mass spectrometry. Integration of aGvHD_MS17 analysis with demographic and clinical variables using a logistic regression model led to correct classification of patients developing severe aGvHD 14 days before any clinical signs with 82.4% sensitivity and 77.3% specificity. Multivariate regression analysis showed that aGvHD_MS17 positivity was the only strong predictor for aGvHD grade III or IV (P<0.0001). The classifier consists of 17 peptides derived from albumin, β2-microglobulin, CD99, fibronectin and various collagen α-chains, indicating inflammation, activation of T cells and changes in the extracellular matrix as early signs of GvHD-induced organ damage. This study is currently the largest demonstration of accurate and investigator-independent prediction of patients at risk for severe aGvHD, thus allowing preemptive therapy based on proteomic profiling.

Light of DNA-alkylating agents in castration-resistant prostate cancer cells: a novel mixed EGFR/DNA targeting combi-molecule.

PubMed

Liang, Guan-Can; Zheng, Hao-Feng; Chen, Yan-Xiong; Li, Teng-Cheng; Liu, Wei; Fang, You-Qiang

2017-01-01

The mechanism underlying the therapeutic effects of combi-molecule JDF12 on prostate cancer (PCa) DU145 cells remains still unclear. This study aimed to investigate the proteomic profile after JDF12 treatment in DU145 cells by comparing with that in Iressa treated cells and untreated cells. MTT was used to evaluate drug cytotoxicity, DAPI staining was done to assess apoptosis of cells, and flow cytometry was used to analyze cell cycle. iTRAQ and qPCR were employed to obtain the proteomic profiles of JDF12 treated, Iressa treated, and untreated DU145 cells, and validate the expression of selected differentially expressed proteins, respectively. JDF12 could significantly inhibit the proliferation and increase the apoptosis of DU145 cells when compared with Iressa or blank group. In total, 5071 proteins were obtained, out of which, 42, including 21 up-regulated and 21 down-regulated proteins, were differentially expressed in JDF12 group when compared with Iressa and blank groups. The up-regulated proteins were mainly involved in DNA damage/repair and energy metabolism; while the down-regulated proteins were mainly associated with cell apoptosis. qPCR confirmed the expression of several biologically important proteins in DU145 cells after JDF12 treatment. The molecular mechanisms of DNA alkylating agents on PCa therapy that with the assistant of EGFR-blocker were revealed on proteomic level, which may increase the possible applications of DNA alkylating agents and JDF12 on PCa therapy.

Streptococcus iniae SF1: Complete Genome Sequence, Proteomic Profile, and Immunoprotective Antigens

PubMed Central

Zhang, Bao-cun; Zhang, Jian; Sun, Li

2014-01-01

Streptococcus iniae is a Gram-positive bacterium that is reckoned one of the most severe aquaculture pathogens. It has a broad host range among farmed marine and freshwater fish and can also cause zoonotic infection in humans. Here we report for the first time the complete genome sequence as well as the host factor-induced proteomic profile of a pathogenic S. iniae strain, SF1, a serotype I isolate from diseased fish. SF1 possesses a single chromosome of 2,149,844 base pairs, which contains 2,125 predicted protein coding sequences (CDS), 12 rRNA genes, and 45 tRNA genes. Among the protein-encoding CDS are genes involved in resource acquisition and utilization, signal sensing and transduction, carbohydrate metabolism, and defense against host immune response. Potential virulence genes include those encoding adhesins, autolysins, toxins, exoenzymes, and proteases. In addition, two putative prophages and a CRISPR-Cas system were found in the genome, the latter containing a CRISPR locus and four cas genes. Proteomic analysis detected 21 secreted proteins whose expressions were induced by host serum. Five of the serum-responsive proteins were subjected to immunoprotective analysis, which revealed that two of the proteins were highly protective against lethal S. iniae challenge when used as purified recombinant subunit vaccines. Taken together, these results provide an important molecular basis for future study of S. iniae in various aspects, in particular those related to pathogenesis and disease control. PMID:24621602

Unveiling the elusive and exotic: Venomics of the Malayan blue coral snake (Calliophis bivirgata flaviceps).

PubMed

Tan, Choo Hock; Fung, Shin Yee; Yap, Michelle Khai Khun; Leong, Poh Kuan; Liew, Jia Lee; Tan, Nget Hong

2016-01-30

The venom proteome of the Malayan blue coral snake, Calliophis bivirgata flaviceps from west Malaysia was investigated by 1D-SDS-PAGE and shotgun-LCMS/MS. A total of 23 proteins belonging to 11 protein families were detected from the venom proteome. For the toxin proteins, the venom consists mainly of phospholipase A2 (41.1%), cytotoxin (22.6%), SVMPs (18.7%) and vespryns (14.6%). However, in contrast to the venoms of New World coral snakes and most elapids, there was no post-synaptic α-neurotoxin detected. The proteome also revealed a relatively high level of phosphodiesterase (1.3%), which may be associated with the reported high level of adenosine in the venom. Also detected were 5'-nucleotidase (0.3%), hyaluronidase (0.1%) and cysteine-type endopeptide inhibitor (0.6%). Enzymatic studies confirmed the presence of phospholipase A2, phosphodiesterase, 5'-nucleotidase and acetylcholinesterase activities but not l-amino acid oxidase activity. The venom exhibited moderate cytotoxic activity against CRL-2648 fibroblast cell lines (IC50=62.14±0.87 μg/mL) and myotoxicity in mice, presumably due to the action of its cytotoxin or its synergistic action with phospholipase A2. Interestingly, the venom lethality could be cross-neutralized by a neurotoxic bivalent antivenom from Taiwan. Together, the findings provide insights into the composition and functions of the venom of this exotic oriental elapid snake. While venoms of the New World coral snake have been extensively studied, literature pertaining to the Old World or Asiatic coral snake venoms remains lacking. This could be partly due to the inaccessibility to the venom of this rare species and infrequent cases of envenomation reported. This study identified and profiled the venom proteome of the Malayan blue coral snake (C. b. flaviceps) through SDS-PAGE and a high-resolution nano-LCMS/MS method, detailing the types and abundance of proteins found in the venom. The biological and toxic activities of the venom were also investigated, offering functional correlation to the venom proteome studied. Of note, the venom contains a unique toxin profile predominated with phospholipase A2 and cytotoxin with no detectable post-synaptic neurotoxin. The venom is moderately lethal to mice and the fatal effect could be cross-neutralized by a heterologous elapid bivalent antivenom from Taiwan. The findings enrich snake toxin databases and provide insights into the composition and pathogenesis of the venom of this exotic species. Copyright © 2015 Elsevier B.V. All rights reserved.

Subpopulation-proteomics in prokaryotic populations.

PubMed

Jahn, Michael; Seifert, Jana; von Bergen, Martin; Schmid, Andreas; Bühler, Bruno; Müller, Susann

2013-02-01

Clonal microbial cells do not behave in an identical manner and form subpopulations during cultivation. Besides varying micro-environmental conditions, cell inherent features like cell cycle dependent localization and concentration of regulatory proteins as well as epigenetic properties are well accepted mechanisms creating cell heterogeneity. Another suspected reason is molecular noise on the transcriptional and translational level. A promising tool to unravel reasons for cell heterogeneity is the combination of cell sorting and subpopulation proteomics. This review summarizes recent developments in prokaryotic single-cell analytics and provides a workflow for selection of single cells, low cell number mass spectrometry, and proteomics evaluation. This approach is useful for understanding the dependency of individual cell decisions on inherent protein profiles. Copyright © 2012 Elsevier Ltd. All rights reserved.

Respiratory Proteomics Today: Are Technological Advances for the Identification of Biomarker Signatures Catching up with Their Promise? A Critical Review of the Literature in the Decade 2004-2013.

PubMed

Viglio, Simona; Stolk, Jan; Iadarola, Paolo; Giuliano, Serena; Luisetti, Maurizio; Salvini, Roberta; Fumagalli, Marco; Bardoni, Anna

2014-01-22

To improve the knowledge on a variety of severe disorders, research has moved from the analysis of individual proteins to the investigation of all proteins expressed by a tissue/organism. This global proteomic approach could prove very useful: (i) for investigating the biochemical pathways involved in disease; (ii) for generating hypotheses; or (iii) as a tool for the identification of proteins differentially expressed in response to the disease state. Proteomics has not been used yet in the field of respiratory research as extensively as in other fields, only a few reproducible and clinically applicable molecular markers, which can assist in diagnosis, having been currently identified. The continuous advances in both instrumentation and methodology, which enable sensitive and quantitative proteomic analyses in much smaller amounts of biological material than before, will hopefully promote the identification of new candidate biomarkers in this area. The aim of this report is to critically review the application over the decade 2004-2013 of very sophisticated technologies to the study of respiratory disorders. The observed changes in protein expression profiles from tissues/fluids of patients affected by pulmonary disorders opens the route for the identification of novel pathological mediators of these disorders.

Mass spectrometry-based targeted quantitative proteomics: achieving sensitive and reproducible detection of proteins.

PubMed

Boja, Emily S; Rodriguez, Henry

2012-04-01

Traditional shotgun proteomics used to detect a mixture of hundreds to thousands of proteins through mass spectrometric analysis, has been the standard approach in research to profile protein content in a biological sample which could lead to the discovery of new (and all) protein candidates with diagnostic, prognostic, and therapeutic values. In practice, this approach requires significant resources and time, and does not necessarily represent the goal of the researcher who would rather study a subset of such discovered proteins (including their variations or posttranslational modifications) under different biological conditions. In this context, targeted proteomics is playing an increasingly important role in the accurate measurement of protein targets in biological samples in the hope of elucidating the molecular mechanism of cellular function via the understanding of intricate protein networks and pathways. One such (targeted) approach, selected reaction monitoring (or multiple reaction monitoring) mass spectrometry (MRM-MS), offers the capability of measuring multiple proteins with higher sensitivity and throughput than shotgun proteomics. Developing and validating MRM-MS-based assays, however, is an extensive and iterative process, requiring a coordinated and collaborative effort by the scientific community through the sharing of publicly accessible data and datasets, bioinformatic tools, standard operating procedures, and well characterized reagents. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Proteomic Analysis of the Human Olfactory Bulb.

PubMed

Dammalli, Manjunath; Dey, Gourav; Madugundu, Anil K; Kumar, Manish; Rodrigues, Benvil; Gowda, Harsha; Siddaiah, Bychapur Gowrishankar; Mahadevan, Anita; Shankar, Susarla Krishna; Prasad, Thottethodi Subrahmanya Keshava

2017-08-01

The importance of olfaction to human health and disease is often underappreciated. Olfactory dysfunction has been reported in association with a host of common complex diseases, including neurological diseases such as Alzheimer's disease and Parkinson's disease. For health, olfaction or the sense of smell is also important for most mammals, for optimal engagement with their environment. Indeed, animals have developed sophisticated olfactory systems to detect and interpret the rich information presented to them to assist in day-to-day activities such as locating food sources, differentiating food from poisons, identifying mates, promoting reproduction, avoiding predators, and averting death. In this context, the olfactory bulb is a vital component of the olfactory system receiving sensory information from the axons of the olfactory receptor neurons located in the nasal cavity and the first place that processes the olfactory information. We report in this study original observations on the human olfactory bulb proteome in healthy subjects, using a high-resolution mass spectrometry-based proteomic approach. We identified 7750 nonredundant proteins from human olfactory bulbs. Bioinformatics analysis of these proteins showed their involvement in biological processes associated with signal transduction, metabolism, transport, and olfaction. These new observations provide a crucial baseline molecular profile of the human olfactory bulb proteome, and should assist the future discovery of biomarker proteins and novel diagnostics associated with diseases characterized by olfactory dysfunction.

Proteomic analysis of the phytopathogenic soilborne fungus Verticillium dahliae reveals differential protein expression in isolates that differ in aggressiveness.

PubMed

El-Bebany, Ahmed F; Rampitsch, Christof; Daayf, Fouad

2010-01-01

Verticillium dahliae is a soilborne fungus that causes a vascular wilt disease of plants and losses in a broad range of economically important crops worldwide. In this study, we compared the proteomes of highly (Vd1396-9) and weakly (Vs06-14) aggressive isolates of V. dahliae to identify protein factors that may contribute to pathogenicity. Twenty-five protein spots were consistently observed as differential in the proteome profiles of the two isolates. The protein sequences in the spots were identified by LC-ESI-MS/MS and MASCOT database searches. Some of the identified sequences shared homology with fungal proteins that have roles in stress response, colonization, melanin biosynthesis, microsclerotia formation, antibiotic resistance, and fungal penetration. These are important functions for infection of the host and survival of the pathogen in soil. One protein found only in the highly aggressive isolate was identified as isochorismatase hydrolase, a potential plant-defense suppressor. This enzyme may inhibit the production of salicylic acid, which is important for plant defense response signaling. Other sequences corresponding to potential pathogenicity factors were identified in the highly aggressive isolate. This work indicates that, in combination with functional genomics, proteomics-based analyses can provide additional insights into pathogenesis and potential management strategies for this disease.

In vivo Assessment and Potential Diagnosis of Xenobiotics that Perturb the Thyroid Pathway: Proteomic Analysis of Xenopus laevis Brain Tissue following Exposure to Model T4 Inhibitors

EPA Science Inventory

As part of a multi-endpoint systems approach to develop comprehensive methods for assessing endocrine stressors in vertebrates, differential protein profiling was used to investigate expression profiles in the brain of an amphibian model (Xenopus laevis) following in vivo exposur...

Evaluation of a genome-scale in silico metabolic model for Geobacter metallireducens by using proteomic data from a field biostimulation experiment.

PubMed

Fang, Yilin; Wilkins, Michael J; Yabusaki, Steven B; Lipton, Mary S; Long, Philip E

2012-12-01

Accurately predicting the interactions between microbial metabolism and the physical subsurface environment is necessary to enhance subsurface energy development, soil and groundwater cleanup, and carbon management. This study was an initial attempt to confirm the metabolic functional roles within an in silico model using environmental proteomic data collected during field experiments. Shotgun global proteomics data collected during a subsurface biostimulation experiment were used to validate a genome-scale metabolic model of Geobacter metallireducens-specifically, the ability of the metabolic model to predict metal reduction, biomass yield, and growth rate under dynamic field conditions. The constraint-based in silico model of G. metallireducens relates an annotated genome sequence to the physiological functions with 697 reactions controlled by 747 enzyme-coding genes. Proteomic analysis showed that 180 of the 637 G. metallireducens proteins detected during the 2008 experiment were associated with specific metabolic reactions in the in silico model. When the field-calibrated Fe(III) terminal electron acceptor process reaction in a reactive transport model for the field experiments was replaced with the genome-scale model, the model predicted that the largest metabolic fluxes through the in silico model reactions generally correspond to the highest abundances of proteins that catalyze those reactions. Central metabolism predicted by the model agrees well with protein abundance profiles inferred from proteomic analysis. Model discrepancies with the proteomic data, such as the relatively low abundances of proteins associated with amino acid transport and metabolism, revealed pathways or flux constraints in the in silico model that could be updated to more accurately predict metabolic processes that occur in the subsurface environment.

Aptamer-based multiplexed proteomic technology for biomarker discovery.

PubMed

Gold, Larry; Ayers, Deborah; Bertino, Jennifer; Bock, Christopher; Bock, Ashley; Brody, Edward N; Carter, Jeff; Dalby, Andrew B; Eaton, Bruce E; Fitzwater, Tim; Flather, Dylan; Forbes, Ashley; Foreman, Trudi; Fowler, Cate; Gawande, Bharat; Goss, Meredith; Gunn, Magda; Gupta, Shashi; Halladay, Dennis; Heil, Jim; Heilig, Joe; Hicke, Brian; Husar, Gregory; Janjic, Nebojsa; Jarvis, Thale; Jennings, Susan; Katilius, Evaldas; Keeney, Tracy R; Kim, Nancy; Koch, Tad H; Kraemer, Stephan; Kroiss, Luke; Le, Ngan; Levine, Daniel; Lindsey, Wes; Lollo, Bridget; Mayfield, Wes; Mehan, Mike; Mehler, Robert; Nelson, Sally K; Nelson, Michele; Nieuwlandt, Dan; Nikrad, Malti; Ochsner, Urs; Ostroff, Rachel M; Otis, Matt; Parker, Thomas; Pietrasiewicz, Steve; Resnicow, Daniel I; Rohloff, John; Sanders, Glenn; Sattin, Sarah; Schneider, Daniel; Singer, Britta; Stanton, Martin; Sterkel, Alana; Stewart, Alex; Stratford, Suzanne; Vaught, Jonathan D; Vrkljan, Mike; Walker, Jeffrey J; Watrobka, Mike; Waugh, Sheela; Weiss, Allison; Wilcox, Sheri K; Wolfson, Alexey; Wolk, Steven K; Zhang, Chi; Zichi, Dom

2010-12-07

The interrogation of proteomes ("proteomics") in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology and medicine. We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 µL of serum or plasma). Our current assay measures 813 proteins with low limits of detection (1 pM median), 7 logs of overall dynamic range (~100 fM-1 µM), and 5% median coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding signature of DNA aptamer concentrations, which is quantified on a DNA microarray. Our assay takes advantage of the dual nature of aptamers as both folded protein-binding entities with defined shapes and unique nucleotide sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD). We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to rapidly discover unique protein signatures characteristic of various disease states. We describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine.

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.

Absolute Quantification of Selected Proteins in the Human Osteoarthritic Secretome

PubMed Central

Peffers, Mandy J.; Beynon, Robert J.; Clegg, Peter D.

2013-01-01

Osteoarthritis (OA) is characterized by a loss of extracellular matrix which is driven by catabolic cytokines. Proteomic analysis of the OA cartilage secretome enables the global study of secreted proteins. These are an important class of molecules with roles in numerous pathological mechanisms. Although cartilage studies have identified profiles of secreted proteins, quantitative proteomics techniques have been implemented that would enable further biological questions to be addressed. To overcome this limitation, we used the secretome from human OA cartilage explants stimulated with IL-1β and compared proteins released into the media using a label-free LC-MS/MS-based strategy. We employed QconCAT technology to quantify specific proteins using selected reaction monitoring. A total of 252 proteins were identified, nine were differentially expressed by IL-1 β stimulation. Selected protein candidates were quantified in absolute amounts using QconCAT. These findings confirmed a significant reduction in TIMP-1 in the secretome following IL-1β stimulation. Label-free and QconCAT analysis produced equivocal results indicating no effect of cytokine stimulation on aggrecan, cartilage oligomeric matrix protein, fibromodulin, matrix metalloproteinases 1 and 3 or plasminogen release. This study enabled comparative protein profiling and absolute quantification of proteins involved in molecular pathways pertinent to understanding the pathogenesis of OA. PMID:24132152

Transcriptome and proteome data reveal candidate genes for pollinator attraction in sexually deceptive orchids.

PubMed

Sedeek, Khalid E M; Qi, Weihong; Schauer, Monica A; Gupta, Alok K; Poveda, Lucy; Xu, Shuqing; Liu, Zhong-Jian; Grossniklaus, Ueli; Schiestl, Florian P; Schlüter, Philipp M

2013-01-01

Sexually deceptive orchids of the genus Ophrys mimic the mating signals of their pollinator females to attract males as pollinators. This mode of pollination is highly specific and leads to strong reproductive isolation between species. This study aims to identify candidate genes responsible for pollinator attraction and reproductive isolation between three closely related species, O. exaltata, O. sphegodes and O. garganica. Floral traits such as odour, colour and morphology are necessary for successful pollinator attraction. In particular, different odour hydrocarbon profiles have been linked to differences in specific pollinator attraction among these species. Therefore, the identification of genes involved in these traits is important for understanding the molecular basis of pollinator attraction by sexually deceptive orchids. We have created floral reference transcriptomes and proteomes for these three Ophrys species using a combination of next-generation sequencing (454 and Solexa), Sanger sequencing, and shotgun proteomics (tandem mass spectrometry). In total, 121 917 unique transcripts and 3531 proteins were identified. This represents the first orchid proteome and transcriptome from the orchid subfamily Orchidoideae. Proteome data revealed proteins corresponding to 2644 transcripts and 887 proteins not observed in the transcriptome. Candidate genes for hydrocarbon and anthocyanin biosynthesis were represented by 156 and 61 unique transcripts in 20 and 7 genes classes, respectively. Moreover, transcription factors putatively involved in the regulation of flower odour, colour and morphology were annotated, including Myb, MADS and TCP factors. Our comprehensive data set generated by combining transcriptome and proteome technologies allowed identification of candidate genes for pollinator attraction and reproductive isolation among sexually deceptive orchids. This includes genes for hydrocarbon and anthocyanin biosynthesis and regulation, and the development of floral morphology. These data will serve as an invaluable resource for research in orchid floral biology, enabling studies into the molecular mechanisms of pollinator attraction and speciation.

Transcriptome and Proteome Data Reveal Candidate Genes for Pollinator Attraction in Sexually Deceptive Orchids

PubMed Central

Sedeek, Khalid E. M.; Qi, Weihong; Schauer, Monica A.; Gupta, Alok K.; Poveda, Lucy; Xu, Shuqing; Liu, Zhong-Jian; Grossniklaus, Ueli; Schiestl, Florian P.; Schlüter, Philipp M.

2013-01-01

Background Sexually deceptive orchids of the genus Ophrys mimic the mating signals of their pollinator females to attract males as pollinators. This mode of pollination is highly specific and leads to strong reproductive isolation between species. This study aims to identify candidate genes responsible for pollinator attraction and reproductive isolation between three closely related species, O. exaltata, O. sphegodes and O. garganica. Floral traits such as odour, colour and morphology are necessary for successful pollinator attraction. In particular, different odour hydrocarbon profiles have been linked to differences in specific pollinator attraction among these species. Therefore, the identification of genes involved in these traits is important for understanding the molecular basis of pollinator attraction by sexually deceptive orchids. Results We have created floral reference transcriptomes and proteomes for these three Ophrys species using a combination of next-generation sequencing (454 and Solexa), Sanger sequencing, and shotgun proteomics (tandem mass spectrometry). In total, 121 917 unique transcripts and 3531 proteins were identified. This represents the first orchid proteome and transcriptome from the orchid subfamily Orchidoideae. Proteome data revealed proteins corresponding to 2644 transcripts and 887 proteins not observed in the transcriptome. Candidate genes for hydrocarbon and anthocyanin biosynthesis were represented by 156 and 61 unique transcripts in 20 and 7 genes classes, respectively. Moreover, transcription factors putatively involved in the regulation of flower odour, colour and morphology were annotated, including Myb, MADS and TCP factors. Conclusion Our comprehensive data set generated by combining transcriptome and proteome technologies allowed identification of candidate genes for pollinator attraction and reproductive isolation among sexually deceptive orchids. This includes genes for hydrocarbon and anthocyanin biosynthesis and regulation, and the development of floral morphology. These data will serve as an invaluable resource for research in orchid floral biology, enabling studies into the molecular mechanisms of pollinator attraction and speciation. PMID:23734209

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

Dual-color Proteomic Profiling of Complex Samples with a Microarray of 810 Cancer-related Antibodies*

PubMed Central

Schröder, Christoph; Jacob, Anette; Tonack, Sarah; Radon, Tomasz P.; Sill, Martin; Zucknick, Manuela; Rüffer, Sven; Costello, Eithne; Neoptolemos, John P.; Crnogorac-Jurcevic, Tatjana; Bauer, Andrea; Fellenberg, Kurt; Hoheisel, Jörg D.

2010-01-01

Antibody microarrays have the potential to enable comprehensive proteomic analysis of small amounts of sample material. Here, protocols are presented for the production, quality assessment, and reproducible application of antibody microarrays in a two-color mode with an array of 1,800 features, representing 810 antibodies that were directed at 741 cancer-related proteins. In addition to measures of array quality, we implemented indicators for the accuracy and significance of dual-color detection. Dual-color measurements outperform a single-color approach concerning assay reproducibility and discriminative power. In the analysis of serum samples, depletion of high-abundance proteins did not improve technical assay quality. On the contrary, depletion introduced a strong bias in protein representation. In an initial study, we demonstrated the applicability of the protocols to proteins derived from urine samples. We identified differences between urine samples from pancreatic cancer patients and healthy subjects and between sexes. This study demonstrates that biomedically relevant data can be produced. As demonstrated by the thorough quality analysis, the dual-color antibody array approach proved to be competitive with other proteomic techniques and comparable in performance to transcriptional microarray analyses. PMID:20164060

Effect of sex and RYR1 gene mutation on the muscle proteomic profile and main physiological biomarkers in pigs at slaughter.

PubMed

Oliván, Mamen; González, Joel; Bassols, Anna; Díaz, Fernando; Carreras, Ricard; Mainau, Eva; Arroyo, Laura; Peña, Raquel; Potes, Yaiza; Coto-Montes, Ana; Hollung, Kristin; Velarde, Antonio

2018-07-01

Gender and RYR1 gene mutation might have an effect on the muscle metabolic characteristics and on the animal's stress at slaughter, which could influence the process of muscle-to-meat conversion. Forty-eight pigs were distributed in a design including two factors: sex (male/female) and RYR1 genotype (NN/Nn). At slaughter, physiological blood biomarkers and muscle proteome were analyzed and carcass and meat quality traits were registered. Females had higher serum levels of glucose, urea, C-reactive protein "CRP", Pig-MAP and glutation-peroxidase "GPx" and lower levels of lactate, showed faster muscle pH decline and higher meat exudation. RYR1 mutation increased serum creatinine, creatine kinase and CRP and decreased GPx. The proteomic study highlighted significant effects of gender and RYR1 genotype on proteins related to fibre composition, antioxidant defense and post mortem glycolytic pathway, which correlate to differences of meat quality. This study provides interesting information on muscle biomarkers of the ultimate meat quality that are modulated by the animal's individual susceptibility to stress at slaughter. Copyright © 2018 Elsevier Ltd. All rights reserved.

Osmotic stress adaptation of Paracoccidioides lutzii, Pb01, monitored by proteomics.

PubMed

Rodrigues, Leandro Nascimento da Silva; Brito, Wesley de Almeida; Parente, Ana Flávia Alves; Weber, Simone Schneider; Bailão, Alexandre Melo; Casaletti, Luciana; Borges, Clayton Luiz; Soares, Célia Maria de Almeida

2016-10-01

The ability to respond to stressful conditions is essential for most living organisms. In pathogenic organisms, this response is required for effective transition from a saprophytic lifestyle to the establishment of pathogenic interactions within a susceptible host. Hyperosmotic stress has been used as a model to study signal transduction and seems to cause many cellular adaptations, including the alteration of protein expression and cellular volume as well as size regulation. In this work, we evaluated the proteomic profile of Paracoccidioides lutzii Pb01 yeast cells during osmotic stress induced by potassium chloride. We performed a high accuracy proteomic technique (NanoUPLC-MS(E)) to identify differentially expressed proteins during osmotic shock. The data describe an osmoadaptative response of this fungus when subjected to this treatment. Proteins involved in the synthesis of cell wall components were modulated, which suggested cell wall remodeling. In addition, alterations in the energy metabolism were observed. Furthermore, proteins involved in amino acid metabolism and hydrogen peroxide detoxification were modulated during osmotic stress. Our study suggests that P. lutzii Pb01. presents a vast osmoadaptative response that is composed of different proteins that act together to minimize the effects caused by osmotic stress. Copyright © 2016 Elsevier Inc. All rights reserved.

Quantitative proteomics and systems analysis of cultured H9C2 cardiomyoblasts during differentiation over time supports a 'function follows form' model of differentiation.

PubMed

Kankeu, Cynthia; Clarke, Kylie; Van Haver, Delphi; Gevaert, Kris; Impens, Francis; Dittrich, Anna; Roderick, H Llewelyn; Passante, Egle; Huber, Heinrich J

2018-05-17

The rat cardiomyoblast cell line H9C2 has emerged as a valuable tool for studying cardiac development, mechanisms of disease and toxicology. We present here a rigorous proteomic analysis that monitored the changes in protein expression during differentiation of H9C2 cells into cardiomyocyte-like cells over time. Quantitative mass spectrometry followed by gene ontology (GO) enrichment analysis revealed that early changes in H9C2 differentiation are related to protein pathways of cardiac muscle morphogenesis and sphingolipid synthesis. These changes in the proteome were followed later in the differentiation time-course by alterations in the expression of proteins involved in cation transport and beta-oxidation. Studying the temporal profile of the H9C2 proteome during differentiation in further detail revealed eight clusters of co-regulated proteins that can be associated with early, late, continuous and transient up- and downregulation. Subsequent reactome pathway analysis based on these eight clusters further corroborated and detailed the results of the GO analysis. Specifically, this analysis confirmed that proteins related to pathways in muscle contraction are upregulated early and transiently, and proteins relevant to extracellular matrix organization are downregulated early. In contrast, upregulation of proteins related to cardiac metabolism occurs at later time points. Finally, independent validation of the proteomics results by immunoblotting confirmed hereto unknown regulators of cardiac structure and ionic metabolism. Our results are consistent with a 'function follows form' model of differentiation, whereby early and transient alterations of structural proteins enable subsequent changes that are relevant to the characteristic physiology of cardiomyocytes.

Proteomic Changes in Cerebrospinal Fluid of Presymptomatic and Affected Persons Carrying Familial Alzheimer Disease Mutations

PubMed Central

Ringman, John M.; Schulman, Howard; Becker, Chris; Jones, Ted; Bai, Yuchen; Immermann, Fred; Cole, Gregory; Sokolow, Sophie; Gylys, Karen; Geschwind, Daniel H.; Cummings, Jeffrey L.; Wan, Hong I.

2013-01-01

Objective To identify cerebrospinal fluid (CSF) protein changes in persons who will develop familial Alzheimer disease (FAD) due to PSEN1 and APP mutations, using unbiased proteomics. Design We compared proteomic profiles of CSF from individuals with FAD who were mutation carriers (MCs) and related noncarriers (NCs). Abundant proteins were depleted and samples were analyzed using liquid chromatography– electrospray ionization–mass spectrometry on a high-resolution time-of-flight instrument. Tryptic peptides were identified by tandem mass spectrometry. Proteins differing in concentration between the MCs and NCs were identified. Setting A tertiary dementia referral center and a proteomic biomarker discovery laboratory. Participants Fourteen FAD MCs (mean age, 34.2 years; 10 are asymptomatic, 12 have presenilin-1 [PSEN1] gene mutations, and 2 have amyloid precursor protein [APP] gene mutations) and 5 related NCs (mean age, 37.6 years). Results Fifty-six proteins were identified, represented by multiple tryptic peptides showing significant differences between MCs and NCs (46 upregulated and 10 downregulated); 40 of these proteins differed when the analysis was restricted to asymptomatic individuals. Fourteen proteins have been reported in prior proteomic studies in late-onset AD, including amyloid precursor protein, transferrin, α1β-glycoprotein, complement components, afamin precursor, spondin 1, plasminogen, hemopexin, and neuronal pentraxin receptor. Many other proteins were unique to our study, including calsyntenin 3, AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) 4 glutamate receptor, CD99 antigen, di-N-acetyl-chitobiase, and secreted phosphoprotein 1. Conclusions We found much overlap in CSF protein changes between individuals with presymptomatic and symptomatic FAD and those with late-onset AD. Our results are consistent with inflammation and synaptic loss early in FAD and suggest new presymptomatic biomarkers of potential usefulness in drug development. PMID:22232349

Impact of nanoscale topography on genomics and proteomics of adherent bacteria.

PubMed

Rizzello, Loris; Sorce, Barbara; Sabella, Stefania; Vecchio, Giuseppe; Galeone, Antonio; Brunetti, Virgilio; Cingolani, Roberto; Pompa, Pier Paolo

2011-03-22

Bacterial adhesion onto inorganic/nanoengineered surfaces is a key issue in biotechnology and medicine, because it is one of the first necessary steps to determine a general pathogenic event. Understanding the molecular mechanisms of bacteria-surface interaction represents a milestone for planning a new generation of devices with unanimously certified antibacterial characteristics. Here, we show how highly controlled nanostructured substrates impact the bacterial behavior in terms of morphological, genomic, and proteomic response. We observed by atomic force microscopy (AFM) and scanning electron microscopy (SEM) that type-1 fimbriae typically disappear in Escherichia coli adherent onto nanostructured substrates, as opposed to bacteria onto reference glass or flat gold surfaces. A genetic variation of the fimbrial operon regulation was consistently identified by real time qPCR in bacteria interacting with the nanorough substrates. To gain a deeper insight into the molecular basis of the interaction mechanisms, we explored the entire proteomic profile of E. coli by 2D-DIGE, finding significant changes in the bacteria adherent onto the nanorough substrates, such as regulations of proteins involved in stress processes and defense mechanisms. We thus demonstrated that a pure physical stimulus, that is, a nanoscale variation of surface topography, may play per se a significant role in determining the morphological, genetic, and proteomic profile of bacteria. These data suggest that in depth investigations of the molecular processes of microorganisms adhering to surfaces are of great importance for the design of innovative biomaterials with active biological functionalities.

S-Nitrosylation Proteome Profile of Peripheral Blood Mononuclear Cells in Human Heart Failure

PubMed Central

Spratt, Heidi M.; Gupta, Shivali; Petersen, John R.; Kuyumcu-Martinez, Muge N.

2016-01-01

Nitric oxide (NO) protects the heart against ischemic injury; however, NO- and superoxide-dependent S-nitrosylation (S-NO) of cysteines can affect function of target proteins and play a role in disease outcome. We employed 2D-GE with thiol-labeling FL-maleimide dye and MALDI-TOF MS/MS to capture the quantitative changes in abundance and S-NO proteome of HF patients (versus healthy controls, n = 30/group). We identified 93 differentially abundant (59-increased/34-decreased) and 111 S-NO-modified (63-increased/48-decreased) protein spots, respectively, in HF subjects (versus controls, fold-change | ≥1.5|, p ≤ 0.05). Ingenuity pathway analysis of proteome datasets suggested that the pathways involved in phagocytes' migration, free radical production, and cell death were activated and fatty acid metabolism was decreased in HF subjects. Multivariate adaptive regression splines modeling of datasets identified a panel of proteins that will provide >90% prediction success in classifying HF subjects. Proteomic profiling identified ATP-synthase, thrombospondin-1 (THBS1), and vinculin (VCL) as top differentially abundant and S-NO-modified proteins, and these proteins were verified by Western blotting and ELISA in different set of HF subjects. We conclude that differential abundance and S-NO modification of proteins serve as a mechanism in regulating cell viability and free radical production, and THBS1 and VCL evaluation will potentially be useful in the prediction of heart failure. PMID:27635260

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.

Altered proteomic polymorphisms in the caterpillar body and stroma of natural Cordyceps sinensis during maturation.

PubMed

Dong, Yun-Zi; Zhang, Li-Juan; Wu, Zi-Mei; Gao, Ling; Yao, Yi-Sang; Tan, Ning-Zhi; Wu, Jian-Yong; Ni, Luqun; Zhu, Jia-Shi

2014-01-01

To examine the maturational changes in proteomic polymorphisms resulting from differential expression by multiple intrinsic fungi in the caterpillar body and stroma of natural Cordyceps sinensis (Cs), an integrated micro-ecosystem. The surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) biochip technique was used to profile the altered protein compositions in the caterpillar body and stroma of Cs during its maturation. The MS chromatograms were analyzed using density-weighted algorithms to examine the similarities and cluster relationships among the proteomic polymorphisms of the Cs compartments and the mycelial products Hirsutella sinensis (Hs) and Paecilomyces hepiali (Ph). SELDI-TOF MS chromatograms displayed dynamic proteomic polymorphism alterations among samples from the different Cs compartments during maturation. More than 1,900 protein bands were analyzed using density-weighted ZUNIX similarity equations and clustering methods, revealing integral polymorphism similarities of 57.4% between the premature and mature stromata and 42.8% between the premature and mature caterpillar bodies. The across-compartment similarity was low, ranging from 10.0% to 18.4%. Consequently, each Cs compartment (i.e., the stroma and caterpillar body) formed a clustering clade, and the 2 clades formed a Cs cluster. The polymorphic similarities ranged from 0.51% to 1.04% between Hs and the Cs compartments and were 2.8- to 4.8-fold higher (1.92%-4.34%) between Ph and the Cs compartments. The Hs and Ph mycelial samples formed isolated clades outside of the Cs cluster. Proteomic polymorphisms in the caterpillar body and stroma of Cs change dynamically during maturation. The proteomic polymorphisms in Hs and Ph differ from those in Cs, suggesting the presence of multiple Cs-associated fungi and multiple Ophiocordyceps sinensis genotypes with altered differential protein expression in the Cs compartments during maturation. In conjunction with prior mycological and molecular observations, the findings from this proteomic study support the integrated micro-ecosystem hypothesis for natural Cs.

Altered Proteomic Polymorphisms in the Caterpillar Body and Stroma of Natural Cordyceps sinensis during Maturation

PubMed Central

Wu, Zi-Mei; Gao, Ling; Yao, Yi-Sang; Tan, Ning-Zhi; Wu, Jian-Yong; Ni, Luqun; Zhu, Jia-Shi

2014-01-01

Objective To examine the maturational changes in proteomic polymorphisms resulting from differential expression by multiple intrinsic fungi in the caterpillar body and stroma of natural Cordyceps sinensis (Cs), an integrated micro-ecosystem. Methods The surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) biochip technique was used to profile the altered protein compositions in the caterpillar body and stroma of Cs during its maturation. The MS chromatograms were analyzed using density-weighted algorithms to examine the similarities and cluster relationships among the proteomic polymorphisms of the Cs compartments and the mycelial products Hirsutella sinensis (Hs) and Paecilomyces hepiali (Ph). Results: SELDI-TOF MS chromatograms displayed dynamic proteomic polymorphism alterations among samples from the different Cs compartments during maturation. More than 1,900 protein bands were analyzed using density-weighted ZUNIX similarity equations and clustering methods, revealing integral polymorphism similarities of 57.4% between the premature and mature stromata and 42.8% between the premature and mature caterpillar bodies. The across-compartment similarity was low, ranging from 10.0% to 18.4%. Consequently, each Cs compartment (i.e., the stroma and caterpillar body) formed a clustering clade, and the 2 clades formed a Cs cluster. The polymorphic similarities ranged from 0.51% to 1.04% between Hs and the Cs compartments and were 2.8- to 4.8-fold higher (1.92%–4.34%) between Ph and the Cs compartments. The Hs and Ph mycelial samples formed isolated clades outside of the Cs cluster. Conclusion Proteomic polymorphisms in the caterpillar body and stroma of Cs change dynamically during maturation. The proteomic polymorphisms in Hs and Ph differ from those in Cs, suggesting the presence of multiple Cs-associated fungi and multiple Ophiocordyceps sinensis genotypes with altered differential protein expression in the Cs compartments during maturation. In conjunction with prior mycological and molecular observations, the findings from this proteomic study support the integrated micro-ecosystem hypothesis for natural Cs. PMID:25310818

In-depth proteomic profiling of left ventricular tissues in human end-stage dilated cardiomyopathy.

PubMed

Liu, Shanshan; Xia, Yan; Liu, Xiaohui; Wang, Yi; Chen, Zhangwei; Xie, Juanjuan; Qian, Juying; Shen, Huali; Yang, Pengyuan

2017-07-18

Dilated cardiomyopathy (DCM) is caused by reduced left ventricular (LV) myocardial function, which is one of the most common causes of heart failure (HF). We performed iTRAQ-coupled 2D-LC-MS/MS to profile the cardiac proteome of LV tissues from healthy controls and patients with end-stage DCM. We identified 4263 proteins, of which 125 were differentially expressed in DCM tissues compared to LV controls. The majority of these were membrane proteins related to cellular junctions and neuronal metabolism. In addition, these proteins were involved in membrane organization, mitochondrial organization, translation, protein transport, and cell death process. Four key proteins involved in the cell death process were also detected by western blotting, indicated that cell death was activated in DCM tissues. Furthermore, S100A1 and eEF2 were enriched in the "cellular assembly and organization" and "cell cycle" networks, respectively. We verified decreases in these two proteins in end-stage DCM LV samples through multiple reaction monitoring (MRM). These observations demonstrate that our understanding of the mechanisms underlying DCM can be deepened through comparison of the proteomes of normal LV tissues with that from end-stage DCM in humans.

Display technologies: application for the discovery of drug and gene delivery agents

PubMed Central

Sergeeva, Anna; Kolonin, Mikhail G.; Molldrem, Jeffrey J.; Pasqualini, Renata; Arap, Wadih

2007-01-01

Recognition of molecular diversity of cell surface proteomes in disease is essential for the development of targeted therapies. Progress in targeted therapeutics requires establishing effective approaches for high-throughput identification of agents specific for clinically relevant cell surface markers. Over the past decade, a number of platform strategies have been developed to screen polypeptide libraries for ligands targeting receptors selectively expressed in the context of various cell surface proteomes. Streamlined procedures for identification of ligand-receptor pairs that could serve as targets in disease diagnosis, profiling, imaging and therapy have relied on the display technologies, in which polypeptides with desired binding profiles can be serially selected, in a process called biopanning, based on their physical linkage with the encoding nucleic acid. These technologies include virus/phage display, cell display, ribosomal display, mRNA display and covalent DNA display (CDT), with phage display being by far the most utilized. The scope of this review is the recent advancements in the display technologies with a particular emphasis on molecular mapping of cell surface proteomes with peptide phage display. Prospective applications of targeted compounds derived from display libraries in the discovery of targeted drugs and gene therapy vectors are discussed. PMID:17123658

Lens proteome map and alpha-crystallin profile of the catfish Rita rita.

PubMed

Mohanty, Bimal Prasanna; Bhattacharjee, Soma; Das, Manas Kumar

2011-02-01

Crystallins are a diverse group of proteins that constitute nearly 90% of the total soluble proteins of the vertebrate eye lens and these tightly packed crystallins are responsible for transparency of the lens. These proteins have been studied in different model and non-model species for understanding the modifications they undergo with ageing that lead to cataract, a disease of protein aggregation. In the present investigation, we studied the lens crystallin profile of the tropical freshwater catfish Rita rita. Profiles of lens crystallins were analyzed and crystallin proteome maps of Rita rita were generated for the first time. alphaA-crystallins, member of the alpha-crystallin family, which are molecular chaperons and play crucial role in maintaining lens transparency were identified by 1- and 2-D immunoblot analysis with anti-alphaA-crystallin antibody. Two protein bands of 19-20 kDa were identified as alphaA-crystallins on 1-D immunoblots and these bands separated into 10 discrete spots on 2-D immunoblot. However, anti-alphaB-crystallin and antiphospho-alphaB-crystallin antibodies were not able to detect any immunoreactive bands on 1- and 2-D immunoblots, indicating alphaB-crystallin was either absent or present in extremely low concentration in Rita rita lens. Thus, Rita rita alpha-crystallins are more like that of the catfish Clarias batrachus and the mammal kangaroo in its alphaA- and alphaB-crystallin content (contain low amount from 5-9% of alphaB-crystallin) and unlike the dogfish, zebrafish, human, bovine and mouse alpha-crystallins (contain higher amount of alphaB-crystallin from 25% in mouse and bovine to 85% in dogfish). Results of the present study can be the baseline information for stimulating further investigation on Rita rita lens crystallins for comparative lens proteomics. Comparing and contrasting the alpha-crystallins of the dogfish and Rita rita may provide valuable information on the functional attributes of alphaA- and alphaB-isoforms, as they are at the two extremes in terms of alphaA-and alphaB-crystallin content.

Profiling plasma extracellular vesicle by pluronic block-copolymer based enrichment method unveils features associated with breast cancer aggression, metastasis and invasion

PubMed Central

Rosenow, Matthew; Xiao, Nick; Spetzler, David

2018-01-01

ABSTRACT Extracellular vesicle (EV)-based liquid biopsies have been proposed to be a readily obtainable biological substrate recently for both profiling and diagnostics purposes. Development of a fast and reliable preparation protocol to enrich such small particles could accelerate the discovery of informative, disease-related biomarkers. Though multiple EV enrichment protocols are available, in terms of efficiency, reproducibility and simplicity, precipitation-based methods are most amenable to studies with large numbers of subjects. However, the selectivity of the precipitation becomes critical. Here, we present a simple plasma EV enrichment protocol based on pluronic block copolymer. The enriched plasma EV was able to be verified by multiple platforms. Our results showed that the particles enriched from plasma by the copolymer were EV size vesicles with membrane structure; proteomic profiling showed that EV-related proteins were significantly enriched, while high-abundant plasma proteins were significantly reduced in comparison to other precipitation-based enrichment methods. Next-generation sequencing confirmed the existence of various RNA species that have been observed in EVs from previous studies. Small RNA sequencing showed enriched species compared to the corresponding plasma. Moreover, plasma EVs enriched from 20 advanced breast cancer patients and 20 age-matched non-cancer controls were profiled by semi-quantitative mass spectrometry. Protein features were further screened by EV proteomic profiles generated from four breast cancer cell lines, and then selected in cross-validation models. A total of 60 protein features that highly contributed in model prediction were identified. Interestingly, a large portion of these features were associated with breast cancer aggression, metastasis as well as invasion, consistent with the advanced clinical stage of the patients. In summary, we have developed a plasma EV enrichment method with improved precipitation selectivity and it might be suitable for larger-scale discovery studies. PMID:29696079

Protein extraction and gel-based separation methods to analyze responses to pathogens in carnation (Dianthus caryophyllus L).

PubMed

Ardila, Harold Duban; Fernández, Raquel González; Higuera, Blanca Ligia; Redondo, Inmaculada; Martínez, Sixta Tulia

2014-01-01

We are currently using a 2-DE-based proteomics approach to study plant responses to pathogenic fungi by using the carnation (Dianthus caryophyllus L)-Fusarium oxysporum f. sp. dianthi pathosystem. It is clear that the protocols for the first stages of a standard proteomics workflow must be optimized to each biological system and objectives of the research. The optimization procedure for the extraction and separation of proteins by 1-DE and 2-DE in the indicated system is reported. This strategy can be extrapolated to other plant-pathogen interaction systems in order to perform an evaluation of the changes in the host protein profile caused by the pathogen and to identify proteins which, at early stages, are involved or implicated in the plant defense response.

Exposure to an extremely low-frequency electromagnetic field only slightly modifies the proteome of Chromobacterium violaceumATCC 12472.

PubMed

Baraúna, Rafael A; Santos, Agenor V; Graças, Diego A; Santos, Daniel M; Ghilardi, Rubens; Pimenta, Adriano M C; Carepo, Marta S P; Schneider, Maria P C; Silva, Artur

2015-05-01

Several studies of the physiological responses of different organisms exposed to extremely low-frequency electromagnetic fields (ELF-EMF) have been described. In this work, we report the minimal effects of in situ exposure to ELF-EMF on the global protein expression of Chromobacterium violaceum using a gel-based proteomic approach. The protein expression profile was only slightly altered, with five differentially expressed proteins detected in the exposed cultures; two of these proteins (DNA-binding stress protein, Dps, and alcohol dehydrogenase) were identified by MS/MS. The enhanced expression of Dps possibly helped to prevent physical damage to DNA. Although small, the changes in protein expression observed here were probably beneficial in helping the bacteria to adapt to the stress generated by the electromagnetic field.

Proteomic analysis of acute responses to copper sulfate stress in larvae of the brine shrimp, Artemia sinica

NASA Astrophysics Data System (ADS)

Zhou, Qian; Wu, Changgong; Dong, Bo; Li, Fuhua; Liu, Fengqi; Xiang, Jianhai

2010-03-01

Proteomics was used to reveal the differential protein expression profiles of acute responses to copper sulfate exposure in larvae of Artemia sinica. Fourteen differentially displayed protein spots were detected and seven of them were identified. Three spots were up-expressed and identified: actin, heat shock protein 70, and chaperone subunit 1; three down-regulated proteins were identified: arginine kinase, elongation factor-2, and glycine-rich protein; and a newly expressed protein was identified as peroxiredoxin. The study indicates the involvement of all the differentially expressed proteins in the early responses of protein expression, and in the survival of A. sinica in the presence of copper and other heavy metals; the findings improve understanding of the organism’s adaptive responses and resistance.

Deep Proteome Profiling Reveals Common Prevalence of MZB1-Positive Plasma B Cells in Human Lung and Skin Fibrosis.

PubMed

Schiller, Herbert B; Mayr, Christoph H; Leuschner, Gabriela; Strunz, Maximilian; Staab-Weijnitz, Claudia; Preisendörfer, Stefan; Eckes, Beate; Moinzadeh, Pia; Krieg, Thomas; Schwartz, David A; Hatz, Rudolf A; Behr, Jürgen; Mann, Matthias; Eickelberg, Oliver

2017-11-15

Analyzing the molecular heterogeneity of different forms of organ fibrosis may reveal common and specific factors and thus identify potential future therapeutic targets. We sought to use proteome-wide profiling of human tissue fibrosis to (1) identify common and specific signatures across end-stage interstitial lung disease (ILD) cases, (2) characterize ILD subgroups in an unbiased fashion, and (3) identify common and specific features of lung and skin fibrosis. We collected samples of ILD tissue (n = 45) and healthy donor control samples (n = 10), as well as fibrotic skin lesions from localized scleroderma and uninvolved skin (n = 6). Samples were profiled by quantitative label-free mass spectrometry, Western blotting, or confocal imaging. We determined the abundance of more than 7,900 proteins and stratified these proteins according to their detergent solubility profiles. Common protein regulations across all ILD cases, as well as distinct ILD subsets, were observed. Proteomic comparison of lung and skin fibrosis identified a common upregulation of marginal zone B- and B1-cell-specific protein (MZB1), the expression of which identified MZB1 + /CD38 + /CD138 + /CD27 + /CD45 - /CD20 - plasma B cells in fibrotic lung and skin tissue. MZB1 levels correlated positively with tissue IgG and negatively with diffusing capacity of the lung for carbon monoxide. Despite the presumably high molecular and cellular heterogeneity of ILD, common protein regulations are observed, even across organ boundaries. The surprisingly high prevalence of MZB1-positive plasma B cells in tissue fibrosis warrants future investigations regarding the causative role of antibody-mediated autoimmunity in idiopathic cases of organ fibrosis, such as idiopathic pulmonary fibrosis.

The NEDD8 inhibitor MLN4924 increases the size of the nucleolus and activates p53 through the ribosomal-Mdm2 pathway.

PubMed

Bailly, A; Perrin, A; Bou Malhab, L J; Pion, E; Larance, M; Nagala, M; Smith, P; O'Donohue, M-F; Gleizes, P-E; Zomerdijk, J; Lamond, A I; Xirodimas, D P

2016-01-28

The ubiquitin-like molecule NEDD8 is essential for viability, growth and development, and is a potential target for therapeutic intervention. We found that the small molecule inhibitor of NEDDylation, MLN4924, alters the morphology and increases the surface size of the nucleolus in human and germline cells of Caenorhabditis elegans in the absence of nucleolar fragmentation. SILAC proteomics and monitoring of rRNA production, processing and ribosome profiling shows that MLN4924 changes the composition of the nucleolar proteome but does not inhibit RNA Pol I transcription. Further analysis demonstrates that MLN4924 activates the p53 tumour suppressor through the RPL11/RPL5-Mdm2 pathway, with characteristics of nucleolar stress. The study identifies the nucleolus as a target of inhibitors of NEDDylation and provides a mechanism for p53 activation upon NEDD8 inhibition. It also indicates that targeting the nucleolar proteome without affecting nucleolar transcription initiates the required signalling events for the control of cell cycle regulators.

Proteomic profiling of ATM kinase proficient and deficient cell lines upon blockage of proteasome activity☆

PubMed Central

Marzano, Valeria; Santini, Simonetta; Rossi, Claudia; Zucchelli, Mirco; D'Alessandro, Annamaria; Marchetti, Carlo; Mingardi, Michele; Stagni, Venturina; Barilà, Daniela; Urbani, Andrea

2012-01-01

Ataxia Telangiectasia Mutated (ATM) protein kinase is a key effector in the modulation of the functionality of some important stress responses, including DNA damage and oxidative stress response, and its deficiency is the hallmark of Ataxia Telangiectasia (A-T), a rare genetic disorder. ATM modulates the activity of hundreds of target proteins, essential for the correct balance between proliferation and cell death. The aim of this study is to evaluate the phenotypic adaptation at the protein level both in basal condition and in presence of proteasome blockage in order to identify the molecules whose level and stability are modulated through ATM expression. We pursued a comparative analysis of ATM deficient and proficient lymphoblastoid cells by label-free shotgun proteomic experiments comparing the panel of proteins differentially expressed. Through a non-supervised comparative bioinformatic analysis these data provided an insight on the functional role of ATM deficiency in cellular carbohydrate metabolism's regulation. This hypothesis has been demonstrated by targeted metabolic fingerprint analysis SRM (Selected Reaction Monitoring) on specific thermodynamic checkpoints of glycolysis. This article is part of a Special Issue entitled: Translational Proteomics. PMID:22641158

Comparative proteomic analysis reveals heart toxicity induced by chronic arsenic exposure in rats.

PubMed

Huang, Qingyu; Xi, Guochen; Alamdar, Ambreen; Zhang, Jie; Shen, Heqing

2017-10-01

Arsenic is a widespread metalloid in the environment, which poses a broad spectrum of adverse effects on human health. However, a global view of arsenic-induced heart toxicity is still lacking, and the underlying molecular mechanisms remain unclear. By performing a comparative quantitative proteomic analysis, the present study aims to investigate the alterations of proteome profile in rat heart after long-term exposure to arsenic. As a result, we found that the abundance of 81 proteins were significantly altered by arsenic treatment (35 up-regulated and 46 down-regulated). Among these, 33 proteins were specifically associated with cardiovascular system development and function, including heart development, heart morphology, cardiac contraction and dilation, and other cardiovascular functions. It is further proposed that the aberrant regulation of 14 proteins induced by arsenic would disturb cardiac contraction and relaxation, impair heart morphogenesis and development, and induce thrombosis in rats, which is mediated by the Akt/p38 MAPK signaling pathway. Overall, these findings will augment our knowledge of the involved mechanisms and develop useful biomarkers for cardiotoxicity induced by environmental arsenic exposure. Copyright © 2017 Elsevier Ltd. All rights reserved.

Proteomic Data Resources for EDRN Ovary Cancer Researchers within the EDRN — EDRN Public Portal

Cancer.gov

This project will generate a highly valuable data resource and make it available to all EDRN ovarian cancer researchers. The resource will include comprehensive proteomic (tandem mass spectrometry, MS/MS) data generated from plasma samples that have been collected between four months and four years prior to clinical detection of ovarian cancer. These pre-clinical samples, provided from the Beta Carotene and Retinol Efficacy Trial (CARET) prospective study, will be interrogated using IPAS, the proteomic profiling method developed by the Hanash Laboratory and with the quantitative methods developed by the McIntosh laboratory. In addition, we will combine these pre-clinical data with already completed IPAS interrogations of plasma collected at the time of ovarian cancer diagnosis. Thus together we will provide information on both pre-clinical and clinical behavior of a large number of proteins. Based on our preliminary work we are able to quantify over 500 plasma proteins in each of these experiments, many of which are putative ovarian cancer biomarkers, showing the platform is capable of providing useful information regarding biomarker candidates.

Proteomic profiling of occupational medicamentosa-like dermatitis induced by trichloroethylene in serum based on MALDI-TOF MS.

PubMed

Liu, Wei; Hong, Wen-Xu; Zhang, Yanfang; Huang, Peiwu; Yang, Xifei; Ren, Xiaohu; Huang, Haiyan; Liu, Jianjun

2015-11-01

Trichloroethylene (TCE) has long been well known as a major pollutant that affects both occupational and general environments. Occupational medicamentosa-like dermatitis induced by TCE (OMLDT) is an autoimmune disease, which has become one of the critical occupational health issues in China. In this study, we analyzed 18 OMLDT patients and 29 professional TCE contact people on serum proteomic analysis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and ClinProTools bioinformatics software. The intensities of 35 protein/peptide peaks were significantly different between TCE contact controls and OMLDT patients. A pattern of six peaks (m/z 1,450.33, 1,866.16, 3,262.39, 4,109.55, 5,064.85 and 5,956.57) were selected to construct a diagnostic model to discriminate the OMLDT patients from controls with sensitivity and specificity of both 93.8 %. Our findings provide an alternative proteomic approach to differentiate the OMLDT patients from TCE contact workers with high sensitivity and high specificity, which will be of potential value in clinical diagnosis for occupational disease.

Similarities and Differences of Blood N-Glycoproteins in Five Solid Carcinomas at Localized Clinical Stage Analyzed by SWATH-MS.

PubMed

Sajic, Tatjana; Liu, Yansheng; Arvaniti, Eirini; Surinova, Silvia; Williams, Evan G; Schiess, Ralph; Hüttenhain, Ruth; Sethi, Atul; Pan, Sheng; Brentnall, Teresa A; Chen, Ru; Blattmann, Peter; Friedrich, Betty; Niméus, Emma; Malander, Susanne; Omlin, Aurelius; Gillessen, Silke; Claassen, Manfred; Aebersold, Ruedi

2018-05-29

Cancer is mostly incurable when diagnosed at a metastatic stage, making its early detection via blood proteins of immense clinical interest. Proteomic changes in tumor tissue may lead to changes detectable in the protein composition of circulating blood plasma. Using a proteomic workflow combining N-glycosite enrichment and SWATH mass spectrometry, we generate a data resource of 284 blood samples derived from patients with different types of localized-stage carcinomas and from matched controls. We observe whether the changes in the patient's plasma are specific to a particular carcinoma or represent a generic signature of proteins modified uniformly in a common, systemic response to many cancers. A quantitative comparison of the resulting N-glycosite profiles discovers that proteins related to blood platelets are common to several cancers (e.g., THBS1), whereas others are highly cancer-type specific. Available proteomics data, including a SWATH library to study N-glycoproteins, will facilitate follow-up biomarker research into early cancer detection. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Comparative analysis of methicillin-sensitive and resistant Staphylococcus aureus exposed to emodin based on proteomic profiling.

PubMed

Ji, Xiaoyu; Liu, Xiaoqiang; Peng, Yuanxia; Zhan, Ruoting; Xu, Hui; Ge, Xijin

2017-12-09

Emodin has a strong antibacterial activity, including methicillin-resistant Staphylococcus aureus (MRSA). However, the mechanism by which emodin induces growth inhibition against MRSA remains unclear. In this study, the isobaric tags for relative and absolute quantitation (iTRAQ) proteomics approach was used to investigate the modes of action of emodin on a MRSA isolate and methicillin-sensitive S. aureus ATCC29213(MSSA). Proteomic analysis showed that expression levels of 145 and 122 proteins were changed significantly in MRSA and MSSA, respectively, after emodin treatment. Comparative analysis of the functions of differentially expressed proteins between the two strains was performed via bioinformatics tools blast2go and STRING database. Proteins related to pyruvate pathway imbalance induction, protein synthesis inhibition, and DNA synthesis suppression were found in both methicillin-sensitive and resistant strains. Moreover, Interference proteins related to membrane damage mechanism were also observed in MRSA. Our findings indicate that emodin is a potential antibacterial agent targeting MRSA via multiple mechanisms. Copyright © 2017 Elsevier Inc. All rights reserved.

Pathogen profiling for disease management and surveillance.

PubMed

Sintchenko, Vitali; Iredell, Jonathan R; Gilbert, Gwendolyn L

2007-06-01

The usefulness of rapid pathogen genotyping is widely recognized, but its effective interpretation and application requires integration into clinical and public health decision-making. How can pathogen genotyping data best be translated to inform disease management and surveillance? Pathogen profiling integrates microbial genomics data into communicable disease control by consolidating phenotypic identity-based methods with DNA microarrays, proteomics, metabolomics and sequence-based typing. Sharing data on pathogen profiles should facilitate our understanding of transmission patterns and the dynamics of epidemics.

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

PubMed

Maver, Ales; Medica, Igor; Peterlin, Borut

2009-12-01

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

Evolutionary Proteomics Uncovers Ancient Associations of Cilia with Signaling Pathways.

PubMed

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

2017-12-18

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

Interaction Analysis through Proteomic Phage Display

PubMed Central

2014-01-01

Phage display is a powerful technique for profiling specificities of peptide binding domains. The method is suited for the identification of high-affinity ligands with inhibitor potential when using highly diverse combinatorial peptide phage libraries. Such experiments further provide consensus motifs for genome-wide scanning of ligands of potential biological relevance. A complementary but considerably less explored approach is to display expression products of genomic DNA, cDNA, open reading frames (ORFs), or oligonucleotide libraries designed to encode defined regions of a target proteome on phage particles. One of the main applications of such proteomic libraries has been the elucidation of antibody epitopes. This review is focused on the use of proteomic phage display to uncover protein-protein interactions of potential relevance for cellular function. The method is particularly suited for the discovery of interactions between peptide binding domains and their targets. We discuss the largely unexplored potential of this method in the discovery of domain-motif interactions of potential biological relevance. PMID:25295249