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Sample records for differential expression proteomics

  1. Differential expression of the skeletal muscle proteome in grazed cattle.

    PubMed

    Shibata, M; Matsumoto, K; Oe, M; Ohnishi-Kameyama, M; Ojima, K; Nakajima, I; Muroya, S; Chikuni, K

    2009-08-01

    The objective of this study was to investigate the differences in the muscle proteome of grass-fed and grain-fed cattle. Eight Japanese Black Cattle 10 mo of age were separated randomly into 2 groups: 1) grazing (grass-fed) and 2) concentrate (grain-fed) groups. All cattle were first housed individually in a stall barn and fed a combination of concentrate ad libitum and Italian ryegrass hay until 21 mo of age. After this control period, the 4 grass-fed cattle were placed on outdoor pasture, whereas the other 4 grain-fed cattle continued on the concentrate diet. The cattle were slaughtered at 27 mo of age, and tissues from the semitendinosus muscle were obtained for use in proteome analysis. Differential expression of muscle proteins in the 2 groups was carried out using 2-dimensional gel electrophoresis (2DE) and Western blot analyses, with subsequent mass spectrometry. Approximately 200 individual protein spots were detected and compared in each group using 2DE, of which 20 and 9 spots, respectively, showed differences in the spot intensity for the sarcoplasmic fraction and myofibrillar fraction. In the grazing group, the relative intensity of spots was significantly greater for adenylate kinase 1 and myoglobin in the sarcoplasmic fraction, and for slow-twitch myosin light chain 2 in the myofibrillar fraction (P < 0.05), than the concentrate group. The relative spot intensity of several glycolytic enzymes was significantly greater in the grazing group, such as beta-enolase 3, fructose-1,6-bisphosphate aldolase A, triosephosphate isomerase, and heat shock 27 kDa protein (P < 0.05). Moreover, significantly greater slow twitch of troponin T, troponin I, and myosin heavy chain of semitendinosus muscle was detected in the grazing group than in the concentrate group using Western blot analysis (P < 0.05). Several previous reports have described that the slow-twitch muscle contents affect elements of nutrition, flavor, and food texture of meat. This study revealed muscle

  2. Differential expression of proteins in renal cortex and medulla: a proteomic approach.

    PubMed

    Arthur, John M; Thongboonkerd, Visith; Scherzer, Janice A; Cai, Jian; Pierce, William M; Klein, Jon B

    2002-10-01

    Western blotting has previously been used to identify changes in protein expression in renal tissue. However, only a few proteins can be studied in each experiment by Western blot. We have used proteomic tools to construct protein maps of rat kidney cortex and medulla. Expression of proteins was determined by silver stain after two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Protein spots were excised and digested with trypsin. Peptide masses were identified by MALDI-TOF mass spectrometry. The Mascot search engine was used to analyze the peptide masses and identify the proteins. Seventy-two proteins were identified (54 unique proteins) out of approximately 1000 spots visualized on each gel. Most of the spots were expressed both in cortex and medulla. Of the identified proteins, three were expressed only in medulla and one only in cortex. Nine proteins were expressed in both regions but to a greater extent in cortex and three proteins were expressed more in medulla. Differential expression was confirmed for three proteins by Western blot. A large group of proteins and their relative expression levels from cortical and medullary portions of rat kidneys were found. Sixteen proteins are differentially expressed. Proteomics can be used to identify differential expression of proteins in the kidney on a large scale. Proteomics should be useful to detect changes in renal protein expression in response to a large range of physiological and pathophysiological stimuli.

  3. Detecting differential protein expression in large-scale population proteomics

    SciTech Connect

    Ryu, Soyoung; Qian, Weijun; Camp, David G.; Smith, Richard D.; Tompkins, Ronald G.; Davis, Ronald W.; Xiao, Wenzhong

    2014-06-17

    Mass spectrometry-based high-throughput quantitative proteomics shows great potential in clinical biomarker studies, identifying and quantifying thousands of proteins in biological samples. However, methods are needed to appropriately handle issues/challenges unique to mass spectrometry data in order to detect as many biomarker proteins as possible. One issue is that different mass spectrometry experiments generate quite different total numbers of quantified peptides, which can result in more missing peptide abundances in an experiment with a smaller total number of quantified peptides. Another issue is that the quantification of peptides is sometimes absent, especially for less abundant peptides and such missing values contain the information about the peptide abundance. Here, we propose a Significance Analysis for Large-scale Proteomics Studies (SALPS) that handles missing peptide intensity values caused by the two mechanisms mentioned above. Our model has a robust performance in both simulated data and proteomics data from a large clinical study. Because varying patients’ sample qualities and deviating instrument performances are not avoidable for clinical studies performed over the course of several years, we believe that our approach will be useful to analyze large-scale clinical proteomics data.

  4. Differential proteomic and tissue expression analyses identify valuable diagnostic biomarkers of hepatocellular differentiation and hepatoid adenocarcinomas.

    PubMed

    Reis, Henning; Padden, Juliet; Ahrens, Maike; Pütter, Carolin; Bertram, Stefanie; Pott, Leona L; Reis, Anna-Carinna; Weber, Frank; Juntermanns, Benjamin; Hoffmann, Andreas-C; Eisenacher, Martin; Schlaak, Joörg F; Canbay, Ali; Meyer, Helmut E; Sitek, Barbara; Baba, Hideo A

    2015-10-01

    The exact discrimination of lesions with true hepatocellular differentiation from secondary tumours and neoplasms with hepatocellular histomorphology like hepatoid adenocarcinomas (HAC) is crucial. Therefore, we aimed to identify ancillary protein biomarkers by using complementary proteomic techniques (2D-DIGE, label-free MS). The identified candidates were immunohistochemically validated in 14 paired samples of hepatocellular carcinoma (HCC) and non-tumourous liver tissue (NT). The candidates and HepPar1/Arginase1 were afterwards tested for consistency in a large cohort of hepatocellular lesions and NT (n = 290), non-hepatocellular malignancies (n = 383) and HAC (n = 13). Eight non-redundant, differentially expressed proteins were suitable for further immunohistochemical validation and four (ABAT, BHMT, FABP1, HAOX1) for further evaluation. Sensitivity and specificity rates for HCC/HAC were as follows: HepPar1 80.2%, 94.3% / 80.2%, 46.2%; Arginase1 82%, 99.4% / 82%, 69.2%; BHMT 61.4%, 93.8% / 61.4%, 100%; ABAT 84.4%, 33.7% / 84.4%, 30.8%; FABP1 87.2%, 95% / 87.2%, 69.2%; HAOX1 95.5%, 36.3% / 95.5%, 46.2%. The best 2×/3× biomarker panels for the diagnosis of HCC consisted of Arginase1/HAOX1 and BHMT/Arginase1/HAOX1 and for HAC consisted of Arginase1/FABP1 and BHMT/Arginase1/FABP1. In summary, we successfully identified, validated and benchmarked protein biomarker candidates of hepatocellular differentiation. BHMT in particular exhibited superior diagnostic characteristics in hepatocellular lesions and specifically in HAC. BHMT is therefore a promising (panel based) biomarker candidate in the differential diagnostic process of lesions with hepatocellular aspect.

  5. Biological network module-based model for the analysis of differential expression in shotgun proteomics.

    PubMed

    Xu, Jia; Wang, Lily; Li, Jing

    2014-12-05

    Protein differential expression analysis plays an important role in the understanding of molecular mechanisms as well as the pathogenesis of complex diseases. With the rapid development of mass spectrometry, shotgun proteomics using spectral counts has become a prevailing method for the quantitative analysis of complex protein mixtures. Existing methods in differential proteomics expression typically carry out analysis at the single-protein level. However, it is well-known that proteins interact with each other when they function in biological processes. In this study, focusing on biological network modules, we proposed a negative binomial generalized linear model for differential expression analysis of spectral count data in shotgun proteomics. In order to show the efficacy of the model in protein expression analysis at the level of protein modules, we conducted two simulation studies using synthetic data sets generated from theoretical distribution of count data and a real data set with shuffled counts. Then, we applied our method to a colorectal cancer data set and a nonsmall cell lung cancer data set. When compared with single-protein analysis methods, the results showed that module-based statistical model which takes account of the interactions among proteins led to more effective identification of subtle but coordinated changes at the systems level.

  6. A Hybrid Approach to Protein Differential Expression in Mass Spectrometry-Based Proteomics

    SciTech Connect

    Wang, Xuan; Anderson, Gordon A.; Smith, Richard D.; Dabney, Alan R.

    2012-04-19

    Motivation: Quantitative mass spectrometry-based proteomics involves statistical inference on protein abundance, based on the intensities of each protein's associated spectral peaks. However, typical MS-based proteomics data sets have substantial proportions of missing observations, due at least in part to censoring of low intensities. This complicates intensity-based differential expression analysis. Results: We outline a statistical method for protein differential expression, based on a simple Binomial likelihood. By modeling peak intensities as binary, in terms of 'presence/ absence,' we enable the selection of proteins not typically amendable to quantitative analysis; e.g., 'one-state' proteins that are present in one condition but absent in another. In addition, we present an analysis protocol that combines quantitative and presence/ absence analysis of a given data set in a principled way, resulting in a single list of selected proteins with a single associated FDR.

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

    PubMed

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

    2013-04-05

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

  8. Quantitative Proteomic Analysis of Differentially Expressed Protein Profiles Involved in Pancreatic Ductal Adenocarcinoma

    PubMed Central

    Kuo, Kung-Kai; Kuo, Chao-Jen; Chiu, Chiang-Yen; Liang, Shih-Shin; Huang, Chun-Hao; Chi, Shu-Wen; Tsai, Kun-Bow; Chen, Chiao-Yun; Hsi, Edward; Cheng, Kuang-Hung; Chiou, Shyh-Horng

    2016-01-01

    Objectives The aim of this study was to identify differentially expressed proteins among various stages of pancreatic ductal adenocarcinoma (PDAC) by shotgun proteomics using nano-liquid chromatography coupled tandem mass spectrometry and stable isotope dimethyl labeling. Methods Differentially expressed proteins were identified and compared based on the mass spectral differences of their isotope-labeled peptide fragments generated from protease digestion. Results Our quantitative proteomic analysis of the differentially expressed proteins with stable isotope (deuterium/hydrogen ratio, ≥2) identified a total of 353 proteins, with at least 5 protein biomarker proteins that were significantly differentially expressed between cancer and normal mice by at least a 2-fold alteration. These 5 protein biomarker candidates include α-enolase, α-catenin, 14-3-3 β, VDAC1, and calmodulin with high confidence levels. The expression levels were also found to be in agreement with those examined by Western blot and histochemical staining. Conclusions The systematic decrease or increase of these identified marker proteins may potentially reflect the morphological aberrations and diseased stages of pancreas carcinoma throughout progressive developments leading to PDAC. The results would form a firm foundation for future work concerning validation and clinical translation of some identified biomarkers into targeted diagnosis and therapy for various stages of PDAC. PMID:26262590

  9. Differential Expression of Proteins Associated with the Hair Follicle Cycle - Proteomics and Bioinformatics Analyses

    PubMed Central

    Tian, Tian; Yang, Mifang; Li, Zhongming; Ping, Fengfeng; Fan, Weixin

    2016-01-01

    Hair follicle cycling can be divided into the following three stages: anagen, catagen, and telogen. The molecular signals that orchestrate the follicular transition between phases are still unknown. To better understand the detailed protein networks controlling this process, proteomics and bioinformatics analyses were performed to construct comparative protein profiles of mouse skin at specific time points (0, 8, and 20 days). Ninety-five differentially expressed protein spots were identified by MALDI-TOF/TOF as 44 proteins, which were found to change during hair follicle cycle transition. Proteomics analysis revealed that these changes in protein expression are involved in Ca2+-regulated biological processes, migration, and regulation of signal transduction, among other processes. Subsequently, three proteins were selected to validate the reliability of expression patterns using western blotting. Cluster analysis revealed three expression patterns, and each pattern correlated with specific cell processes that occur during the hair cycle. Furthermore, bioinformatics analysis indicated that the differentially expressed proteins impacted multiple biological networks, after which detailed functional analyses were performed. Taken together, the above data may provide insight into the three stages of mouse hair follicle morphogenesis and provide a solid basis for potential therapeutic molecular targets for this hair disease. PMID:26752403

  10. Proteomic analyses of methamphetamine (METH)-induced differential protein expression by immature dendritic cells (IDC).

    PubMed

    Reynolds, Jessica L; Mahajan, Supriya D; Sykes, Donald E; Schwartz, Stanley A; Nair, Madhavan P N

    2007-04-01

    In the US, the increase in methamphetamine (METH) use has been associated with increased human immunodeficiency virus (HIV-1) infection. Dendritic cells (DC) are the first line of defense against HIV-1. DC play a critical role in harboring HIV-1 and facilitate the infection of neighboring T cells. However, the role of METH on HIV-1 infectivity and the expression of the proteome of immature dendritic cells (IDC) has not been elucidated. We hypothesize that METH modulates the expression of a number of proteins by IDC that foster the immunopathogenesis of HIV-1 infection. We utilized LTR amplification, p24 antigen assay and the proteomic method of difference gel electrophoresis (DIGE) combined with protein identification through high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to analyze the effects of METH on HIV-1 infectivity (HIV-1 IIIB; CXCR4-tropic, X4 strain) and the proteomic profile of IDC. Our results demonstrate that METH potentiates HIV-1 replication in IDC. Furthermore, METH significantly differentially regulates the expression of several proteins including CXCR3, protein disulfide isomerase, procathepsin B, peroxiredoxin and galectin-1. Identification of unique, METH-induced proteins may help to develop novel markers for diagnostic, preventive and therapeutic targeting in METH using subjects.

  11. f-divergence cutoff index to simultaneously identify differential expression in the integrated transcriptome and proteome

    PubMed Central

    Tang, Shaojun; Hemberg, Martin; Cansizoglu, Ertugrul; Belin, Stephane; Kosik, Kenneth; Kreiman, Gabriel; Steen, Hanno; Steen, Judith

    2016-01-01

    Abstract The ability to integrate ‘omics’ (i.e. transcriptomics and proteomics) is becoming increasingly important to the understanding of regulatory mechanisms. There are currently no tools available to identify differentially expressed genes (DEGs) across different ‘omics’ data types or multi-dimensional data including time courses. We present fCI (f-divergence Cut-out Index), a model capable of simultaneously identifying DEGs from continuous and discrete transcriptomic, proteomic and integrated proteogenomic data. We show that fCI can be used across multiple diverse sets of data and can unambiguously find genes that show functional modulation, developmental changes or misregulation. Applying fCI to several proteogenomics datasets, we identified a number of important genes that showed distinctive regulation patterns. The package fCI is available at R Bioconductor and http://software.steenlab.org/fCI/. PMID:26980280

  12. Analysis of protein expression in zebrafish during gonad differentiation by targeted proteomics.

    PubMed

    Groh, Ksenia J; Schönenberger, René; Eggen, Rik I L; Segner, Helmut; Suter, Marc J-F

    2013-11-01

    The molecular mechanisms governing sex determination and differentiation in the zebrafish (Danio rerio) are not fully understood. To gain more insights into the function of specific genes in these complex processes, the expression of multiple candidates needs to be assessed, preferably on the protein level. Here, we developed a targeted proteomics method based on selected reaction monitoring (SRM) to study the candidate sex-related proteins in zebrafish which were selected based on a global proteomics analysis of adult gonads and representational difference analysis of male and female DNA, as well as on published information on zebrafish and other vertebrates. We employed the developed SRM protocols to acquire time-resolved protein expression profiles during the gonad differentiation period in vas::EGFP transgenic zebrafish. Evidence on protein expression was obtained for the first time for several candidate genes previously studied only on the mRNA level or suggested by bioinformatic predictions. Tuba1b (tubulin alpha 1b), initially included in the study as one of the potential housekeeping proteins, was found to be preferentially expressed in the adult testis with nearly absent expression in the ovary. The revealed changes in protein expression patterns associated with gonad differentiation suggest that several of the examined proteins, especially Ilf2 and Ilf3 (interleukin enhancer-binding factors 2 and 3), Raldh3 (retinaldehyde dehydrogenase type 3), Zgc:195027 (low density lipoprotein-related receptor protein 3) and Sept5a (septin 5a), may play a specific role in the sexual differentiation in zebrafish. Copyright © 2013 Elsevier Inc. All rights reserved.

  13. Comparative analysis of statistical methods used for detecting differential expression in label-free mass spectrometry proteomics.

    PubMed

    Langley, Sarah R; Mayr, Manuel

    2015-11-03

    Label-free LC-MS/MS proteomics has proven itself to be a powerful method for evaluating protein identification and quantification from complex samples. For comparative proteomics, several methods have been used to detect the differential expression of proteins from such data. We have assessed seven methods used across the literature for detecting differential expression from spectral count quantification: Student's t-test, significance analysis of microarrays (SAM), normalised spectral abundance factor (NSAF), normalised spectral abundance factor-power law global error model (NSAF-PLGEM), spectral index (SpI), DESeq and QSpec. We used 2000 simulated datasets as well as publicly available data from a proteomic standards study to assess the ability of these methods to detect differential expression in varying effect sizes and proportions of differentially expressed proteins. At two false discovery rate (FDR) levels, we find that several of the methods detect differential expression within the data with reasonable precision, others detect differential expression at the expense of low precision, and finally, others which fail to identify any differentially expressed proteins. The inability of these seven methods to fully capture the differential landscape, even at the largest effect size, illustrates some of the limitations of the existing technologies and the statistical methodologies. In label-free mass spectrometry experiments, protein identification and quantification have always been important, but there is now a growing focus on comparative proteomics. Detecting differential expression in protein levels can inform on important biological mechanisms and provide direction for further study. Given the high cost and labour intensive nature of validation experiments, statistical methods are important for prioritising proteins of interest. Here, we have performed a comparative analysis to investigate the statistical methodologies for detecting differential expression

  14. Detecting Differential and Correlated Protein Expression in Label-Free Shotgun Proteomics

    SciTech Connect

    Zhang, Bing; Verberkmoes, Nathan C; Langston, Michael A; Uberbacher, Edward C; Hettich, Robert {Bob} L; Samatova, Nagiza F

    2006-01-01

    Recent studies have revealed a relationship between protein abundance and sampling statistics, such as sequence coverage, peptide count, and spectral count, in label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) shotgun proteomics. The use of sampling statistics offers a promising method of measuring relative protein abundance and detecting differentially expressed or coexpressed proteins. We performed a systematic analysis of various approaches to quantifying differential protein expression in eukaryotic Saccharomycescerevisiaeand prokaryotic Rhodopseudomonaspalustrislabel free LC-MS/MS data. First, we showed that, among three sampling statistics, the spectral count has the highest technical reproducibility, followed by the less-reproducible peptide count and relatively nonreproducible sequence coverage. Second, we used spectral count statistics to measure differential protein expression in pairwise experiments using five statistical tests: Fisher's exact test, G-test, AC test, t-test, and LPE test. Given the S.cerevisiaedata set with spiked proteins as a benchmark and the false positive rate as a metric, our evaluation suggested that the Fisher's exact test, G-test, and AC test can be used when the number of replications is limited (one or two), whereas the t-test is useful with three or more replicates available. Third, we generalized the G-test to increase the sensitivity of detecting differential protein expression under multiple experimental conditions. Out of 1622 identified R.palustris proteins in the LC-MS/MS experiment, the generalized G-test detected 1119 differentially expressed proteins under six growth conditions. Finally, we studied correlated expression of these 1119 proteins by analyzing pairwise expression correlations and by delineating protein clusters according to expression patterns. Through pairwise expression correlation analysis, we demonstrated that proteins co-located in the same operon were much more strongly coexpressed

  15. Identification of differentially expressed peptides in high-throughput proteomics data.

    PubMed

    van Ooijen, Michiel P; Jong, Victor L; Eijkemans, Marinus J C; Heck, Albert J R; Andeweg, Arno C; Binai, Nadine A; van den Ham, Henk-Jan

    2017-03-23

    With the advent of high-throughput proteomics, the type and amount of data pose a significant challenge to statistical approaches used to validate current quantitative analysis. Whereas many studies focus on the analysis at the protein level, the analysis of peptide-level data provides insight into changes at the sub-protein level, including splice variants, isoforms and a range of post-translational modifications. Statistical evaluation of liquid chromatography-mass spectrometry/mass spectrometry peptide-based label-free differential data is most commonly performed using a t-test or analysis of variance, often after the application of data imputation to reduce the number of missing values. In high-throughput proteomics, statistical analysis methods and imputation techniques are difficult to evaluate, given the lack of gold standard data sets. Here, we use experimental and resampled data to evaluate the performance of four statistical analysis methods and the added value of imputation, for different numbers of biological replicates. We find that three or four replicates are the minimum requirement for high-throughput data analysis and confident assignment of significant changes. Data imputation does increase sensitivity in some cases, but leads to a much higher actual false discovery rate. Additionally, we find that empirical Bayes method (limma) achieves the highest sensitivity, and we thus recommend its use for performing differential expression analysis at the peptide level.

  16. Quantitative Analysis of Differential Proteome Expression in Bladder Cancer vs. Normal Bladder Cells Using SILAC Method

    PubMed Central

    Yang, Ganglong; Xu, Zhipeng; Lu, Wei; Li, Xiang; Sun, Chengwen; Guo, Jia; Xue, Peng; Guan, Feng

    2015-01-01

    The best way to increase patient survival rate is to identify patients who are likely to progress to muscle-invasive or metastatic disease upfront and treat them more aggressively. The human cell lines HCV29 (normal bladder epithelia), KK47 (low grade nonmuscle invasive bladder cancer, NMIBC), and YTS1 (metastatic bladder cancer) have been widely used in studies of molecular mechanisms and cell signaling during bladder cancer (BC) progression. However, little attention has been paid to global quantitative proteome analysis of these three cell lines. We labeled HCV29, KK47, and YTS1 cells by the SILAC method using three stable isotopes each of arginine and lysine. Labeled proteins were analyzed by 2D ultrahigh-resolution liquid chromatography LTQ Orbitrap mass spectrometry. Among 3721 unique identified and annotated proteins in KK47 and YTS1 cells, 36 were significantly upregulated and 74 were significantly downregulated with >95% confidence. Differential expression of these proteins was confirmed by western blotting, quantitative RT-PCR, and cell staining with specific antibodies. Gene ontology (GO) term and pathway analysis indicated that the differentially regulated proteins were involved in DNA replication and molecular transport, cell growth and proliferation, cellular movement, immune cell trafficking, and cell death and survival. These proteins and the advanced proteome techniques described here will be useful for further elucidation of molecular mechanisms in BC and other types of cancer. PMID:26230496

  17. Proteomic analysis of differentially expressed Chlamydia pneumoniae genes during persistent infection of HEp-2 cells.

    PubMed

    Molestina, Robert E; Klein, Jon B; Miller, Richard D; Pierce, William H; Ramirez, Julio A; Summersgill, James T

    2002-06-01

    Recent data have shown that the respiratory pathogen Chlamydia pneumoniae expresses an altered gene transcription profile during gamma interferon (IFN-gamma)-induced persistent infection in vitro. In the present study, we examined, by proteomics, expression of C. pneumoniae proteins labeled intracellularly with [(35)S]methionine/cysteine under normal conditions or IFN-gamma-mediated persistence. The identity of differentially expressed proteins during persistent infection was determined by matching spots to those of proteins identified in C. pneumoniae elementary bodies by matrix-assisted laser desorption ionization mass spectrometry. Upon treatment with 50 U of IFN-gamma per ml, a marked upregulation of major outer membrane protein (MOMP), heat shock protein 60 (Hsp-60/GroEL), and proteins with functions in DNA replication (GyrA), transcription (RpoA, PnP), translation (Rrf), glycolysis (PgK, GlgP), and type III secretion (SctN) was observed at 24 h of infection. In contrast, no significant decreases in bacterial protein expression were found in C. pneumoniae-infected cells due to IFN-gamma treatment. Upregulation of C. pneumoniae proteins involved in diverse functions during persistent infection may allow the organism to resist the inhibitory effects of IFN-gamma while retaining basic functions. Future studies should examine the differential expression of chlamydial proteins during the developmental cycle under IFN-gamma pressure to obtain a finer representation of the gene products involved in establishing persistence.

  18. Proteomics identification of differentially expressed proteins associated with pollen germination and tube growth reveals characteristics of germinated Oryza sativa pollen.

    PubMed

    Dai, Shaojun; Chen, Taotao; Chong, Kang; Xue, Yongbiao; Liu, Siqi; Wang, Tai

    2007-02-01

    Mature pollen from most plant species is metabolically quiescent; however, after pollination, it germinates quickly and gives rise to a pollen tube to transport sperms into the embryo sac. Because methods for collecting a large amount of in vitro germinated pollen grains for transcriptomics and proteomics studies from model plants of Arabidopsis and rice are not available, molecular information about the germination developmental process is lacking. Here we describe a method for obtaining a large quantity of in vitro germinating rice pollen for proteomics study. Two-dimensional electrophoresis of approximately 2300 protein spots revealed 186 that were differentially expressed in mature and germinated pollen. Most showed a changed level of expression, and only 66 appeared to be specific to developmental stages. Furthermore 160 differentially expressed protein spots were identified on mass spectrometry to match 120 diverse protein species. These proteins involve different cellular and metabolic processes with obvious functional skew toward wall metabolism, protein synthesis and degradation, cytoskeleton dynamics, and carbohydrate/energy metabolism. Wall metabolism-related proteins are prominently featured in the differentially expressed proteins and the pollen proteome as compared with rice sporophytic proteomes. Our study also revealed multiple isoforms and differential expression patterns between isoforms of a protein. These results provide novel insights into pollen function specialization.

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

    PubMed

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

    2015-11-03

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

  20. f-divergence cutoff index to simultaneously identify differential expression in the integrated transcriptome and proteome.

    PubMed

    Tang, Shaojun; Hemberg, Martin; Cansizoglu, Ertugrul; Belin, Stephane; Kosik, Kenneth; Kreiman, Gabriel; Steen, Hanno; Steen, Judith

    2016-06-02

    The ability to integrate 'omics' (i.e. transcriptomics and proteomics) is becoming increasingly important to the understanding of regulatory mechanisms. There are currently no tools available to identify differentially expressed genes (DEGs) across different 'omics' data types or multi-dimensional data including time courses. We present fCI (f-divergence Cut-out Index), a model capable of simultaneously identifying DEGs from continuous and discrete transcriptomic, proteomic and integrated proteogenomic data. We show that fCI can be used across multiple diverse sets of data and can unambiguously find genes that show functional modulation, developmental changes or misregulation. Applying fCI to several proteogenomics datasets, we identified a number of important genes that showed distinctive regulation patterns. The package fCI is available at R Bioconductor and http://software.steenlab.org/fCI/. Published by Oxford University Press on behalf of Nucleic Acids Research 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  1. Proteomic analysis reveals differentially expressed proteins in the rat frontal cortex after methamphetamine treatment.

    PubMed

    Faure, J J; Hattingh, S M; Stein, D J; Daniels, W M

    2009-12-01

    Methamphetamine (MA) is an addictive psycho-stimulant and the illicit use of the drug is escalating. In the present study, we examined protein expression profiles in the rat frontal cortex exposed to a total of eight MA injections (1 mg/kg, intraperitoneal) using 2-DE based proteomics. We investigated protein changes occurring in both the cytosolic fraction and the membrane fraction. 2-DE analysis resulted in 62 cytosolic and 44 membrane protein spots that were differentially regulated in the frontal cortex of rats exposed to MA when compared to control animals. Of these spots, 47 cytosolic and 42 membrane proteins were identified respectively, using ESI-Quad-TOF, which included ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCH-L1), beta-synuclein, 78 kDa glucose-regulated protein (GRP 78), gamma-enolase, dihydropyrimidase-related protein 2 (DRP 2), complexin 2 and synapsin II. These proteins are associated with protein degradation, redox regulation, energy metabolism, cellular growth, cytoskeletal modifications and synaptic function. Proteomic research may be useful in exploring the complex underlying molecular mechanisms of MA dependence.

  2. Comparative proteomic analysis of differentially expressed proteins between peripheral sensory and motor nerves.

    PubMed

    He, Qianru; Man, Lili; Ji, Yuhua; Zhang, Shuqiang; Jiang, Maorong; Ding, Fei; Gu, Xiaosong

    2012-06-01

    Peripheral sensory and motor nerves have different functions and different approaches to regeneration, especially their distinct ability to accurately reinervate terminal nerve pathways. To understand the molecular aspects underlying these differences, the proteomics technique by coupling isobaric tags for relative and absolute quantitation (iTRAQ) with online two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS) was used to investigate the protein profile of sensory and motor nerve samples from rats. A total of 1472 proteins were identified in either sensory or motor nerve. Of them, 100 proteins showed differential expressions between both nerves, and some of them were validated by quantitative real time RT-PCR, Western blot analysis, and immunohistochemistry. In the light of functional categorization, the differentially expressed proteins in sensory and motor nerves, belonging to a broad range of classes, were related to a diverse array of biological functions, which included cell adhesion, cytoskeleton, neuronal plasticity, neurotrophic activity, calcium-binding, signal transduction, transport, enzyme catalysis, lipid metabolism, DNA-binding, synaptosome function, actin-binding, ATP-binding, extracellular matrix, and commitment to other lineages. The relatively higher expressed proteins in either sensory or motor nerve were tentatively discussed in combination with their specific molecular characteristics. It is anticipated that the database generated in this study will provide a solid foundation for further comprehensive investigation of functional differences between sensory and motor nerves, including the specificity of their regeneration.

  3. 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.

  4. Identification and validation of differentially expressed proteins in epithelial ovarian cancers using quantitative proteomics

    PubMed Central

    Cao, Guangming; Liu, Chongdong; Xu, Jiatong; Deng, Haiteng; Zhang, Zhenyu

    2016-01-01

    Ovarian cancer is the most lethal gynecological malignant tumor because of its high recurrence rate. In the present work, in order to find new therapeutic targets, we identified 8480 proteins in thirteen pairs of ovarian cancer tissues and normal ovary tissues through quantitative proteomics. 498 proteins were found to be differentially expressed in ovarian cancer, which involved in various cellular processes, including metabolism, response to stimulus and biosynthetic process. The expression levels of chloride intracellular channel protein 1 (CLIC1) and lectin galactoside-binding soluble 3 binding protein (LGALS3BP) in epithelial ovarian cancer tissues were significantly higher than those in normal ovary tissues as confirmed by western blotting and immunohistochemistry. The knockdown of CLIC1 in A2780 cell line downregulated expression of CTPS1, leading to the decrease of CTP and an arrest of cell cycle G1 phase, which results into a slower proliferation. CLIC1-knockdown can also slow down the tumor growth in vivo. Besides, CLIC1-knockdown cells showed an increased sensitivity to hydrogen peroxide and cisplatin, suggesting that CLIC1 was involved in regulation of redox and drug resistance in ovarian cancer cells. These results indicate CLIC1 promotes tumorgenesis, and is a potential therapeutic target in epithelial ovarian cancer treatment. PMID:27825122

  5. Quantitative proteomics reveals differential regulation of protein expression in recipient myocardium after trilineage cardiovascular cell transplantation

    PubMed Central

    Chang, Ying-Hua; Ye, Lei; Cai, Wenxuan; Lee, Yoonkyu; Guner, Huseyin; Lee, Youngsook; Kamp, Timothy J.; Zhang, Jianyi; Ge, Ying

    2015-01-01

    Intramyocardial transplantation of cardiomyocytes (CMs), endothelial cells (ECs), and smooth muscle cells (SMCs) derived from human induced pluripotent stem cells (hiPSCs) has beneficial effects on the post-infarction heart. However, the mechanisms underlying the functional improvements remain undefined. We employed large-scale label-free quantitative proteomics to identify proteins that were differentially regulated following cellular transplantation in a swine model of myocardial infarction (MI). We identified 22 proteins that were significantly up-regulated after trilineage cell transplantation compared to both MI and Sham groups. Among them, 12 proteins, including adenylyl cyclase-associated protein 1 and tropomodulin-1, are associated with positive regulation of muscular contraction whereas 11 proteins, such as desmoplakin and zyxin, are involved in embryonic and muscular development and regeneration. Moreover, we identified 21 proteins up-regulated and another 21 down-regulated in MI, but reversed after trilineage cell transplantation. Proteins up-regulated after MI but reversed by transplantation are related to fibrosis and apoptosis. Conversely, proteins down-regulated in MI but restored after cell therapy are regulators of protein nitrosylation. Our results show that the functionally beneficial effects of trilineage cell therapy are accompanied by differential regulation of protein expression in the recipient myocardium, which may contribute to the improved cardiac function. PMID:26033914

  6. Quantitative proteomics reveals differential regulation of protein expression in recipient myocardium after trilineage cardiovascular cell transplantation.

    PubMed

    Chang, Ying-Hua; Ye, Lei; Cai, Wenxuan; Lee, Yoonkyu; Guner, Huseyin; Lee, Youngsook; Kamp, Timothy J; Zhang, Jianyi; Ge, Ying

    2015-08-01

    Intramyocardial transplantation of cardiomyocytes (CMs), endothelial cells (ECs), and smooth muscle cells (SMCs) derived from human induced pluripotent stem cells (hiPSCs) has beneficial effects on the post-infarction heart. However, the mechanisms underlying the functional improvements remain undefined. We employed large-scale label-free quantitative proteomics to identify proteins that were differentially regulated following cellular transplantation in a swine model of myocardial infarction (MI). We identified 22 proteins that were significantly up-regulated after trilineage cell transplantation compared to both MI and Sham groups. Among them, 12 proteins, including adenylyl cyclase-associated protein 1 and tropomodulin-1, are associated with positive regulation of muscular contraction whereas 11 proteins, such as desmoplakin and zyxin, are involved in embryonic and muscular development and regeneration. Moreover, we identified 21 proteins up-regulated and another 21 down-regulated in MI, but reversed after trilineage cell transplantation. Proteins up-regulated after MI but reversed by transplantation are related to fibrosis and apoptosis. Conversely, proteins down-regulated in MI but restored after cell therapy are regulators of protein nitrosylation. Our results show that the functionally beneficial effects of trilineage cell therapy are accompanied by differential regulation of protein expression in the recipient myocardium, which may contribute to the improved cardiac function.

  7. Comparative proteomic changes of differentially expressed whey proteins in clinical mastitis and healthy yak cows.

    PubMed

    Li, X; Ding, X Z; Wan, Y L; Liu, Y M; Du, G Z

    2014-08-28

    Under the traditional grazing system on the Qinghai-Tibetan Plateau, the amount of milk in domesticated yak (Bos grunniens) with clinical mastitis decreases and the milk composition is altered. To understand the mechanisms of mammary gland secreted milk and disease infection, changes in the protein composition of milk during clinical mastitis were investigated using a proteomic approach. Milk whey from yak with clinical mastitis was compared to whey from healthy animals with two-dimensional gel electrophoresis using a mass spectrometer. Thirteen protein spots were identified to be four differentially expressed proteins. Increases in the concentrations of proteins of blood serum origin, including lactoferrin, were identified in mastitic whey compared to normal whey, while concentrations of the major whey proteins, casocidin-I, a-lactalbumin, and b-lactoglobulin, were downregulated in mastitic whey. These results indicated significant differences in protein expression between healthy yaks and those with clinical mastitis, and they may provide valuable information for finding new regulation markers and potential protein targets for the treatment of mastitis.

  8. Proteomic analysis identifies differentially expressed proteins after red propolis treatment in Hep-2 cells.

    PubMed

    Frozza, Caroline Olivieri da Silva; Ribeiro, Tanara da Silva; Gambato, Gabriela; Menti, Caroline; Moura, Sidnei; Pinto, Paulo Marcos; Staats, Charley Christian; Padilha, Francine Ferreira; Begnini, Karine Rech; de Leon, Priscila Marques Moura; Borsuk, Sibele; Savegnago, Lucielli; Dellagostin, Odir; Collares, Tiago; Seixas, Fabiana Kömmling; Henriques, João Antonio Pêgas; Roesch-Ely, Mariana

    2014-01-01

    Here we investigated alterations in the protein profile of Hep-2 treated with red propolis using two-dimensional electrophoresis associated to mass spectrometry and apoptotic rates of cells treated with and without red propolis extracts through TUNEL and Annexin-V assays. A total of 325 spots were manually excised from the two-dimensional gel electrophoresis and 177 proteins were identified using LC-MS-MS. Among all proteins identified that presented differential expression, most were down-regulated in presence of red propolis extract at a concentration of 120 μg/mL (IC50): GRP78, PRDX2, LDHB, VIM and TUBA1A. Only two up-regulated proteins were identified in this study in the non-cytotoxic (6 μg/mL) red propolis treated group: RPLP0 and RAD23B. TUNEL staining assay showed a markedly increase in the mid- to late-stage apoptosis of Hep-2 cells induced by red propolis at concentrations of 60 and 120 μg/mL when compared with non-treated cells. The increase of late apoptosis was confirmed by in situ Annexin-V analysis in which red propolis extract induced late apoptosis in a dose-dependent manner. The differences in tumor cell protein profiles warrant further investigations including isolation of major bioactive compounds of red propolis in different cell lines using proteomics and molecular tests to validate the protein expression here observed. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. Proteomic Identification of Differentially Expressed Proteins during Alfalfa (Medicago sativa L.) Flower Development.

    PubMed

    Chen, Lingling; Chen, Quanzhu; Zhu, Yanqiao; Hou, Longyu; Mao, Peisheng

    2016-01-01

    Flower development, pollination, and fertilization are important stages in the sexual reproduction process of plants; they are also critical steps in the control of seed formation and development. During alfalfa (Medicago sativa L.) seed production, some distinct phenomena such as a low seed setting ratio, serious flower falling, and seed abortion commonly occur. However, the causes of these phenomena are complicated and largely unknown. An understanding of the mechanisms that regulate alfalfa flowering is important in order to increase seed yield. Hence, proteomic technology was used to analyze changes in protein expression during the stages of alfalfa flower development. Flower samples were collected at pre-pollination (S1), pollination (S2), and the post-pollination senescence period (S3). Twenty-four differentially expressed proteins were successfully identified, including 17 down-regulated in pollinated flowers, one up-regulated in pollinated and senesced flowers, and six up-regulated in senesced flowers. The largest proportions of the identified proteins were involved in metabolism, signal transduction, defense response, oxidation reduction, cell death, and programmed cell death (PCD). Their expression profiles demonstrated that energy metabolism, carbohydrate metabolism, and amino acid metabolism provided the nutrient foundation for pollination in alfalfa. Furthermore, there were three proteins involved in multiple metabolic pathways: dual specificity kinase splA-like protein (kinase splALs), carbonic anhydrase, and NADPH: quinone oxidoreductase-like protein. Expression patterns of these proteins indicated that MAPK cascades regulated multiple processes, such as signal transduction, stress response, and cell death. PCD also played an important role in the alfalfa flower developmental process, and regulated both pollination and flower senescence. The current study sheds some light on protein expression profiles during alfalfa flower development and

  10. Proteomic Identification of Differentially Expressed Proteins during Alfalfa (Medicago sativa L.) Flower Development

    PubMed Central

    Chen, Lingling; Chen, Quanzhu; Zhu, Yanqiao; Hou, Longyu; Mao, Peisheng

    2016-01-01

    Flower development, pollination, and fertilization are important stages in the sexual reproduction process of plants; they are also critical steps in the control of seed formation and development. During alfalfa (Medicago sativa L.) seed production, some distinct phenomena such as a low seed setting ratio, serious flower falling, and seed abortion commonly occur. However, the causes of these phenomena are complicated and largely unknown. An understanding of the mechanisms that regulate alfalfa flowering is important in order to increase seed yield. Hence, proteomic technology was used to analyze changes in protein expression during the stages of alfalfa flower development. Flower samples were collected at pre-pollination (S1), pollination (S2), and the post-pollination senescence period (S3). Twenty-four differentially expressed proteins were successfully identified, including 17 down-regulated in pollinated flowers, one up-regulated in pollinated and senesced flowers, and six up-regulated in senesced flowers. The largest proportions of the identified proteins were involved in metabolism, signal transduction, defense response, oxidation reduction, cell death, and programmed cell death (PCD). Their expression profiles demonstrated that energy metabolism, carbohydrate metabolism, and amino acid metabolism provided the nutrient foundation for pollination in alfalfa. Furthermore, there were three proteins involved in multiple metabolic pathways: dual specificity kinase splA-like protein (kinase splALs), carbonic anhydrase, and NADPH: quinone oxidoreductase-like protein. Expression patterns of these proteins indicated that MAPK cascades regulated multiple processes, such as signal transduction, stress response, and cell death. PCD also played an important role in the alfalfa flower developmental process, and regulated both pollination and flower senescence. The current study sheds some light on protein expression profiles during alfalfa flower development and

  11. Proteomic analysis of differential protein expression and processing induced modifications in peanuts and peanut skins

    USDA-ARS?s Scientific Manuscript database

    Peanut (Arachis hypogaea L.) is grown extensively worldwide for its edible seed and oil. Proteomics has become a powerful tool in plant research; however, studies involving legumes, and especially peanuts, are in their infancy. Furthermore, protein expression in the peanut seed coat (skin), which is...

  12. Comparative proteomic study for profiling differentially expressed proteins between Chinese left- and right-sided colon cancers.

    PubMed

    Shen, Hong; Huang, Jinlin; Pei, Haiping; Zeng, Shan; Tao, Yiming; Shen, Liangfang; Zeng, Liang; Zhu, Hong

    2013-01-01

    The aim of the present study is to profile differentially expressed protein markers between left-sided colon cancer (LSCC) and right-sided colon cancer (RSCC). Fresh tumor tissue samples from LSCC (n = 7) and RSCC (n = 7) groups were analyzed by two-dimensional electrophoresis coupled with MALDI-TOF-MS, followed by Western blotting. In 50 paraffin embedded samples from each group, levels of four differentially expressed proteins (identified by proteomics analysis) were measured by tissue microarray with immunohistochemistry staining to compare the different protein markers between LSCC and RSCC. Sixteen proteins were found to be differentially expressed between LSCC and RSCC. Ten proteins including HSP-60 and PDIA1 were identified to be highly expressed in LSCC (P < 0.01 or P < 0.05), while the expression of six proteins including EEF1D and HSP-27 were higher in RSCC (P < 0.01 or P < 0.05). Virtually all of the indentified proteins were involved in cellular energy metabolism, protein folding/unfolding, and/or oxidative stress. Human colon tumors at various locations have different proteomic biomarkers. Differentially expressed proteins associated with energy metabolism, protein folding/unfolding and oxidative stress contribute to different tumorigenesis, tumor progression, and prognosis between left- and right-sided colon cancer.

  13. Identification of stromal differentially expressed proteins in the colon carcinoma by quantitative proteomics.

    PubMed

    Mu, Yibing; Chen, Yongheng; Zhang, Guiying; Zhan, Xianquan; Li, Yuanyuan; Liu, Ting; Li, Guoqing; Li, Maoyu; Xiao, Zhefeng; Gong, Xiaoxiang; Chen, Zhuchu

    2013-06-01

    Tumor microenvironment plays very important roles in the carcinogenesis. A variety of stromal cells in the microenvironment have been modified to support the unique needs of the malignant state. This study was to discover stromal differentially expressed proteins (DEPs) that were involved in colon carcinoma carcinogenesis. Laser capture microdissection (LCM) was captured and isolated the stromal cells from colon adenocarcinoma (CAC) and non-neoplastic colon mucosa (NNCM) tissues, respectively. Seventy DEPs were identified between the pooled LCM-enriched CAC and NNCM stroma samples by iTRAQ-based quantitative proteomics. Gene Ontology (GO) relationship analysis revealed that DEPs were hierarchically grouped into 10 clusters, and were involved in multiple biological functions that were altered during carcinogenesis, including extracellular matrix organization, cytoskeleton, transport, metabolism, inflammatory response, protein polymerization, and cell motility. Pathway network analysis revealed 6 networks and 56 network eligible proteins with Ingenuity pathway analysis. Four significant networks functioned in digestive system development and its function, inflammatory disease, and developmental disorder. Eight DEPs (DCN, FN1, PKM2, HSP90B1, S100A9, MYH9, TUBB, and YWHAZ) were validated by Western blotting, and four DEPs (DCN, FN1, PKM2, and HSP90B1) were validated by immunohistochemical analysis. It is the first report of stromal DEPs between CAC and NNCM tissues. It will be helpful to recognize the roles of stromas in the colon carcinoma microenvironment, and improve the understanding of carcinogenesis in colon carcinoma. The present data suggest that DCN, FN1, PKM2, HSP90B1, S100A9, MYH9, TUBB, and YWHAZ might be the potential targets for colon cancer prevention and therapy.

  14. [Application of differential proteomics in mechanism research of acupuncture].

    PubMed

    Yu, Lin-na; Pei, Jian

    2011-08-01

    Proteomics, a new branch of science, has been used to study protein expressions on the molecular level with a dynamic perspective. Organisms under varying states may express different proteins, which results in the set-up of differential proteomics. Research methods of differential proteomics include the separation and identification of proteins. Differential proteomics has a rapid development in recent years. In the study of acupuncture, researchers have reached certain achievements using differential proteomics to investigate the mechanisms of acupuncture treatment for some diseases, including acute spinal cord injury, ischemic cerebrovascular disease, Parkinson's disease and neuralgia.

  15. Maize (Zea mays L.) seedling leaf nuclear proteome and differentially expressed proteins between a hybrid and its parental lines.

    PubMed

    Guo, Baojian; Chen, Yanhong; Li, Chuan; Wang, Tianya; Wang, Rui; Wang, Bo; Hu, Sha; Du, Xiaofen; Xing, Hongyan; Song, Xiao; Yao, Yingyin; Sun, Qixin; Ni, Zhongfu

    2014-05-01

    To better understand the underlying molecular basis of leaf development in maize, a reference map of nuclear proteins in basal region of seedling leaf was established using a combination of 2DE and MALDI-TOF-MS. In total, 441 reproducible protein spots in nuclear proteome of maize leaf basal region were detected with silver staining in a pH range of 3-10, among which 203 spots corresponding to 163 different proteins were identified. As expected, proteins implicated in RNA and protein-associated functions were overrepresented in nuclear proteome. Remarkably, a high percentage (10%) of proteins was identified to be involved in cell division and growth. In addition, comparative nuclear proteomic analysis in leaf basal region of highly heterotic hybrid Mo17/B73 and its parental lines was also performed and 52 of 445 (11.69%) detected protein spots were differentially expressed between the hybrid and its parental lines, among which 16 protein spots displayed nonadditively expressed pattern. These results indicated that hybridization between two parental lines can cause changes in the expression of a variety of nuclear proteins, which may be responsible for the observed leaf size heterosis.

  16. Proteomic analysis from haploid and diploid embryos of Quercus suber L. identifies qualitative and quantitative differential expression patterns.

    PubMed

    Gómez, Aranzazu; López, Juan Antonio; Pintos, Beatriz; Camafeita, Emilio; Bueno, Ma Angeles

    2009-09-01

    Quercus suber L. is a Mediterranean forest species with ecological, social and economic value. Clonal propagation of Q. suber elite trees has been successfully obtained from in vitro-derived somatic and gametic embryos. These clonal lines play a main role in breeding and genetic studies of Q. suber. To aid in unravelling diverse genetic and biological unknowns, a proteomic approach is proposed. The proteomic analysis of Q. suber somatic and gametic in vitro culture-derived embryos, based on DIGE and MALDI-MS, has produced for the first time proteomic data on this species. Seventeen differentially expressed proteins have been identified which display significantly altered levels between gametic and somatic embryos. These proteins are involved in a variety of cellular processes, most of which had been neither previously associated with embryo development nor identified in the genus Quercus. Some of these proteins are involved in stress and pollen development and others play a role in the metabolism of tannins and phenylpropanoids, which represent two of the major pathways for the synthesis of cork chemical components. Furthermore, the augmented expression levels found for specific proteins are probably related to the homozygous state of a doubled-haploid sample. Proteins involved in synthesis of cork components can be detected at such early stages of development, showing the potential of the method to be useful in searching for biomarkers related to cork quality.

  17. Proteomics-based study on asthenozoospermia: differential expression of proteasome alpha complex.

    PubMed

    Siva, Archana Bharadwaj; Kameshwari, Duvvuri Butchi; Singh, Vaibhav; Pavani, Kadupu; Sundaram, Curam Sreenivasacharlu; Rangaraj, Nandini; Deenadayal, Mamata; Shivaji, Sisinthy

    2010-07-01

    With a view to understand the molecular basis of sperm motility, we have tried to establish the human sperm proteome by two-dimensional PAGE MALDI MS/MS analysis. We report identification of 75 different proteins in the human spermatozoa. Comparative proteome analysis was carried out for asthenozoospermic and normozoospermic patients to understand the molecular basis of sperm motility. Analysis revealed eight proteins (including one unidentified) with altered intensity between the groups. Differential proteins distributed into three functional groups: 'energy and metabolism' (triose-phosphate isomerase, glycerol kinase 2, testis specific isoform and succinyl-CoA:3-ketoacid co-enzyme A transferase 1, mitochondrial precursor); 'movement and organization' (tubulin beta 2C and tektin 1) and 'protein turnover, folding and stress response' (proteasome alpha 3 subunit and heat shock-related 70 kDa protein 2). It was interesting to note that although the proteins falling in the functional group of 'energy and metabolism' are higher in the asthenozoospermic patients, the other two functional groups contain proteins, which are higher in the normozoospermic samples. Validation of results carried out for proteasome alpha 3 subunit by immunoblotting and confocal microscopy, confirmed significant changes in intensity of proteasome alpha 3 subunit in asthenozoospermic samples when compared with normozoospermic controls. Significant positive correlation too was found between proteasome alpha 3 subunit levels and rapid, linear progressive motility of the spermatozoa. In our understanding, this data would contribute appreciably to the presently limited information available about the proteins implicated in human sperm motility.

  18. Unraveling the Equine Lymphocyte Proteome: Differential Septin 7 Expression Associates with Immune Cells in Equine Recurrent Uveitis

    PubMed Central

    Degroote, Roxane L.; Hauck, Stefanie M.; Amann, Barbara; Hirmer, Sieglinde; Ueffing, Marius; Deeg, Cornelia A.

    2014-01-01

    Equine recurrent uveitis is a spontaneous, lymphocyte-driven autoimmune disease. It affects horses worldwide and presents with painful remitting-relapsing inflammatory attacks of inner eye structures eventually leading to blindness. Since lymphocytes are the key players in equine recurrent uveitis, we were interested in potential changes of their protein repertoire which may be involved in disease pathogenesis. To create a reference for differential proteome analysis, we first unraveled the equine lymphocyte proteome by two-dimensional sodium dodecyl sulfate - polyacrylamide gel electrophoresis and subsequently identified 352 protein spots. Next, we compared lymphocytes from ERU cases and healthy horses with a two-dimensional fluorescence difference in gel electrophoresis approach. With this technique, we identified seven differentially expressed proteins between conditions. One of the significantly lower expressed candidates, septin 7, plays a role in regulation of cell shape, motility and migration. Further analyses revealed T cells as the main cell type with decreased septin 7 abundance in equine recurrent uveitis. These findings point to a possible pathogenetic role of septin 7 in this sight-threatening disease. PMID:24614191

  19. Unraveling the equine lymphocyte proteome: differential septin 7 expression associates with immune cells in equine recurrent uveitis.

    PubMed

    Degroote, Roxane L; Hauck, Stefanie M; Amann, Barbara; Hirmer, Sieglinde; Ueffing, Marius; Deeg, Cornelia A

    2014-01-01

    Equine recurrent uveitis is a spontaneous, lymphocyte-driven autoimmune disease. It affects horses worldwide and presents with painful remitting-relapsing inflammatory attacks of inner eye structures eventually leading to blindness. Since lymphocytes are the key players in equine recurrent uveitis, we were interested in potential changes of their protein repertoire which may be involved in disease pathogenesis. To create a reference for differential proteome analysis, we first unraveled the equine lymphocyte proteome by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently identified 352 protein spots. Next, we compared lymphocytes from ERU cases and healthy horses with a two-dimensional fluorescence difference in gel electrophoresis approach. With this technique, we identified seven differentially expressed proteins between conditions. One of the significantly lower expressed candidates, septin 7, plays a role in regulation of cell shape, motility and migration. Further analyses revealed T cells as the main cell type with decreased septin 7 abundance in equine recurrent uveitis. These findings point to a possible pathogenetic role of septin 7 in this sight-threatening disease.

  20. Proteomic Analysis of Signaling Network Regulation in Renal Cell Carcinomas with Differential Hypoxia-Inducible Factor-2α Expression

    PubMed Central

    Nagaprashantha, Lokesh Dalasanur; Talamantes, Tatjana; Singhal, Jyotsana; Guo, Jia; Vatsyayan, Rit; Rauniyar, Navin; Awasthi, Sanjay

    2013-01-01

    Background The loss of von Hippel–Lindau (VHL) protein function leads to highly vascular renal tumors characterized by an aggressive course of disease and refractoriness to chemotherapy and radiotherapy. Loss of VHL in renal tumors also differs from tumors of other organs in that the oncogenic cascade is mediated by an increase in the levels of hypoxia-inducible factor-2α (HIF2α) instead of hypoxia-inducible factor-1α (HIF1α). Methods and Principal Findings We used renal carcinoma cell lines that recapitulate the differences between mutant VHL and wild-type VHL genotypes. Utilizing a method relying on extracted peptide intensities as a label-free approach for quantitation by liquid chromatography–mass spectrometry, our proteomics study revealed regulation of key proteins important for cancer cell survival, proliferation and stress-resistance, and implicated differential regulation of signaling networks in VHL-mutant renal cell carcinoma. We also observed upregulation of cellular energy pathway enzymes and the stress-responsive mitochondrial 60-kDa heat shock protein. Finding reliance on glutaminolysis in VHL-mutant renal cell carcinoma was of particular significance, given the generally predominant dependence of tumors on glycolysis. The data have been deposited to the ProteomeXchange with identifier PXD000335. Conclusions and Significance Pathway analyses provided corroborative evidence for differential regulation of molecular and cellular functions influencing cancer energetics, metabolism and cell proliferation in renal cell carcinoma with distinct VHL genotype. Collectively, the differentially regulated proteome characterized by this study can potentially guide translational research specifically aimed at effective clinical interventions for advanced VHL-mutant, HIF2α-over-expressing tumors. PMID:23940778

  1. Proteomic Study of Differential Protein Expression in Mouse Lung Tissues after Aerosolized Ricin Poisoning

    PubMed Central

    Guo, Zhendong; Han, Chao; Du, Jiajun; Zhao, Siyan; Fu, Yingying; Zheng, Guanyu; Sun, Yucheng; Zhang, Yi; Liu, Wensen; Wan, Jiayu; Qian, Jun; Liu, Linna

    2014-01-01

    Ricin is one of the most poisonous natural toxins from plants and is classified as a Class B biological threat pathogen by the Centers for Disease Control and Prevention (CDC) of U.S.A. Ricin exposure can occur through oral or aerosol routes. Ricin poisoning has a rapid onset and a short incubation period. There is no effective treatment for ricin poisoning. In this study, an aerosolized ricin-exposed mouse model was developed and the pathology was investigated. The protein expression profile in the ricin-poisoned mouse lung tissue was analyzed using proteomic techniques to determine the proteins that were closely related to the toxicity of ricin. 2D gel electrophoresis, mass spectrometry and subsequent biological functional analysis revealed that six proteins including Apoa1 apolipoprotein, Ywhaz 14-3-3 protein, Prdx6 Uncharacterized Protein, Selenium-binding protein 1, HMGB1, and DPYL-2, were highly related to ricin poisoning. PMID:24786090

  2. Proteomic study of differential protein expression in mouse lung tissues after aerosolized ricin poisoning.

    PubMed

    Guo, Zhendong; Han, Chao; Du, Jiajun; Zhao, Siyan; Fu, Yingying; Zheng, Guanyu; Sun, Yucheng; Zhang, Yi; Liu, Wensen; Wan, Jiayu; Qian, Jun; Liu, Linna

    2014-04-28

    Ricin is one of the most poisonous natural toxins from plants and is classified as a Class B biological threat pathogen by the Centers for Disease Control and Prevention (CDC) of U.S.A. Ricin exposure can occur through oral or aerosol routes. Ricin poisoning has a rapid onset and a short incubation period. There is no effective treatment for ricin poisoning. In this study, an aerosolized ricin-exposed mouse model was developed and the pathology was investigated. The protein expression profile in the ricin-poisoned mouse lung tissue was analyzed using proteomic techniques to determine the proteins that were closely related to the toxicity of ricin. 2D gel electrophoresis, mass spectrometry and subsequent biological functional analysis revealed that six proteins including Apoa1 apolipoprotein, Ywhaz 14-3-3 protein, Prdx6 Uncharacterized Protein, Selenium-binding protein 1, HMGB1, and DPYL-2, were highly related to ricin poisoning.

  3. Using Peptide-Level Proteomics Data for Detecting Differentially Expressed Proteins.

    PubMed

    Suomi, Tomi; Corthals, Garry L; Nevalainen, Olli S; Elo, Laura L

    2015-11-06

    The expression of proteins can be quantified in high-throughput means using different types of mass spectrometers. In recent years, there have emerged label-free methods for determining protein abundance. Although the expression is initially measured at the peptide level, a common approach is to combine the peptide-level measurements into protein-level values before differential expression analysis. However, this simple combination is prone to inconsistencies between peptides and may lose valuable information. To this end, we introduce here a method for detecting differentially expressed proteins by combining peptide-level expression-change statistics. Using controlled spike-in experiments, we show that the approach of averaging peptide-level expression changes yields more accurate lists of differentially expressed proteins than does the conventional protein-level approach. This is particularly true when there are only few replicate samples or the differences between the sample groups are small. The proposed technique is implemented in the Bioconductor package PECA, and it can be downloaded from http://www.bioconductor.org.

  4. Differentially expressed proteins underlying childhood cortical dysplasia with epilepsy identified by iTRAQ proteomic profiling

    PubMed Central

    Liu, Shiyong; Liu, Yi; Yang, Yixuan; Yang, Hui; Chen, Yangmei; Chen, Lifen

    2017-01-01

    Cortical dysplasia accounts for at least 14% of epilepsy cases, and is mostly seen in children. However, the understanding of molecular mechanisms and pathogenesis underlying cortical dysplasia is limited. The aim of this cross-sectional study is to identify potential key molecules in the mechanisms of cortical dysplasia by screening the proteins expressed in brain tissues of childhood cortical dysplasia patients with epilepsy using isobaric tags for relative and absolute quantitation-based tandem mass spectrometry compared to controls, and several differentially expressed proteins that are not reported to be associated with cortical dysplasia previously were selected for validation using real-time polymerase chain reaction, immunoblotting and immunohistochemistry. 153 out of 3340 proteins were identified differentially expressed between childhood cortical dysplasia patients and controls. And FSCN1, CRMP1, NDRG1, DPYSL5, MAP4, and FABP3 were selected for validation and identified to be increased in childhood cortical dysplasia patients, while PRDX6 and PSAP were identified decreased. This is the first report on differentially expressed proteins in childhood cortical dysplasia. We identified differential expression of FSCN1, CRMP1, NDRG1, DPYSL5, MAP4, FABP3, PRDX6 and PSAP in childhood cortical dysplasia patients, these proteins are involved in various processes and have various function. These results may provide new directions or targets for the research of childhood cortical dysplasia, and may be helpful in revealing molecular mechanisms and pathogenesis and/or pathophysiology of childhood cortical dysplasia if further investigated. PMID:28222113

  5. A differentially expressed proteomic analysis in placental tissues in relation to pungency during the pepper fruit development.

    PubMed

    Lee, Je Min; Kim, Seyoon; Lee, Ji Young; Yoo, Eun Young; Cho, Myeong Cheoul; Cho, Min Rae; Kim, Byung-Dong; Bahk, Young Yil

    2006-10-01

    Using proteomic analysis including 2-DE, image analysis, and protein identification with LC-MS/MS, an investigation aimed at a better understanding of the differentially expressed proteins and/or gene products was carried out with total cell extracts from placental tissues in nonpungent (Capsicum annuum cv. Saeng-Ryeog #213) and pungent peppers (C. annuum cv. Saeng-Ryeog #211). Mobilization of the most abundant proteins, which were on the gels of pH ranges of 4-7, 4.5-5.5, 5.5-6.7, and 6-9, and showed very similar profiles in the two tissues, revealing approximately 2600 protein spots consisting of 1200 on pH 4-7, 600 on 4.5-5.5, 550 on 5.5-6.7, 250 on 6-9. Of these, 37 protein spots, which appeared in only pungent tissues but not in nonpungent tissues or markedly increased in their staining intensities on the gels from pungent tissue, were selected, excised, in-gel trypsin digested, and analyzed by LC-ESI-MS/MS. Peptide MS/MS data were searched against publicly available protein and EST databases, and 22 proteins were identified. Based on this result, we tested and compared the differential expression during fruit development on the 2-DE gels with total cell extracts from placental tissues of pungent and nonpungent peppers at an interval of 10 days from 10 to 40 days after flowering. In addition, this differential protein expression was further confirmed for some subsets of candidates by Northern-blot analysis with RNA samples from placental tissues harvested from each pepper fruit at the same sampling intervals. In this study, the physiological implications, revealed from the experimental data in the levels of proteome and transcripts, are discussed in the context of a complex biosynthesis network of capsaicinoids in pepper cells responsive to pungency.

  6. Comparative proteomic analysis of differentially expressed proteins between K562 and K562/ADM cells.

    PubMed

    Shen, Shao-hua; Gu, Long-jun; Liu, Pei-qing; Ye, Xin; Chang, Wei-shan; Li, Ben-shang

    2008-03-05

    Multidrug resistance to chemotherapeutic agents is an important clinical problem during the treatment of leukemia. The resistance process is multifactorial. To realize the total factors involved in multidrug resistance, we analyzed the differentially expressed proteins of K562 and K562/ADM cells and we investigated one of the up-regulated proteins (CRKL) using siRNA to determine its role in K562/ADM cells. Altered protein expressions between K562/S (K562 ADM-sensitive cell line) and K562/ADM (K562 multidrug resistant cell line induced by adriamycin) were identified by 2D-DIGE coupled with mass spectrometry. Meanwhile, we confirmed the differential expression of CRKL and Stathmin in both K562 and K562/ADM cells by Western blot analysis. Furthermore, we used RNA interference to silence the CRKL gene expression. Among the 9 differentially expressed proteins, 3 were up-regulated in K562/ADM cells, while 6 were down-regulated in the K562/ADM cells compared with its parent cell line. The expression of CRKL was up-regulated significantly in K562/ADM cells, and it can be decreased by recombinant lentivirus. Moreover, the multidrug resistance of K562/ADM cells was efficiently reversed by silence of CRKL gene expression. The data provided the differentially expressed proteins in K562 and its resistant cell line and highlights the power of 2D-DIGE for the discovery of resistance markers in cancer. We found CRKL may be a new protein involved in the multidrug resistance of leukaemia cells.

  7. Differential proteome analysis of tonsils from children with chronic tonsillitis or with hyperplasia reveals disease-associated protein expression differences.

    PubMed

    Just, Tino; Gafumbegete, Evariste; Gramberg, Jan; Prüfer, Ines; Mikkat, Stefan; Ringel, Bruno; Pau, Hans Wilhelm; Glocker, Michael O

    2006-03-01

    A proteomic approach has been used to establish a proteome map and differentiate between the protein composition of tonsils from patients with chronic tonsillitis (CT) and that of tonsils with hyperplasia (HPL). Two-dimensional gel analysis was performed with material from four patients with HPL and five patients with CT. An average of approximately 600 spots were detected in each gel. A total of 127 different proteins were identified in 158 spots analyzed by mass spectrometry. Our study revealed disease-associated differences between protein abundance for two protein spots, an HSP27 isoform and UMP-CMP kinase. Both protein spots were more abundant in the CT group. HSP27 ELISA was performed for 32 patients, 12 belonging to the HPL group and 20 to the CT group. ELISA could not be used to differentiate HSP27 isoforms nor to distinguish CT from HPL. HSP27 was found to migrate to two further protein spots in the 2D gels. The differently expressed HSP27 isoform migrated as the most acidic of all the HSP27 isoforms detected, indicating the highest degree of phosphorylation. The sum of all three HSP27 abundances in the gels from the CT group was not different from that of the HPL group, consistent with the ELISA results. Our results suggest that phosphorylation differences caused the observed migration differences of HSP27. Together with the UMP-CMP kinase abundance differences, we conclude that kinase and/or phosphatase activity are different in CT and HPL.

  8. Differential expression of equine muscle biopsy proteins during normal training and intensified training in young standardbred horses using proteomics technology.

    PubMed

    Bouwman, Freek G; van Ginneken, Mireille M E; Noben, Jean-Paul; Royackers, Erik; de Graaf-Roelfsema, Ellen; Wijnberg, Inge D; van der Kolk, Johannes H; Mariman, Edwin C M; van Breda, Eric

    2010-03-01

    The major aim of the present study was to investigate the proteome of standardbred horses at different stages of training and intensified training. We searched for biomarkers using small skeletal muscle biopsies of live animals. 2D gel electrophoresis and mass spectrometry were successfully applied to investigate training-induced differential expression of equine muscle biopsy proteins. Despite the poor resolution of the equine genome and proteome, we were able to identify the proteins of 20 differential spots representing 16 different proteins. Evaluation of those proteins complies with adaptation of the skeletal muscle after normal training involving structural changes towards a higher oxidative capacity, an increased capacity to take up long-chain fatty acids, and to store energy in the form of glycogen. Intensified training leads to additional changed spots. Alpha-1-antitrypsin was found increased after intensified training but not after normal training. This protein may thus be considered as a marker for overtraining in horses and also linked to overtraining in human athletes. Copyright 2009 Elsevier Inc. All rights reserved.

  9. Proteomic analysis of differentially expressed proteins in bovine milk during experimentally induced Escherichia coli mastitis

    USDA-ARS?s Scientific Manuscript database

    The objectives of the current study were to profile changes in protein composition using 2-dimensional gel electrophoresis (2D-GE) on whey samples from a group of 8 cows prior to and 18 hours after infection with Escherichia coli, and to identify differentially expressed milk proteins by peptide seq...

  10. Proteomic analysis of human brain microvascular endothelial cells reveals differential protein expression in response to enterovirus 71 infection.

    PubMed

    Luo, Wenying; Zhong, Jiayu; Zhao, Wei; Liu, Jianjun; Zhang, Renli; Peng, Liang; Hong, Wenxu; Huang, Sheng He; Cao, Hong

    2015-01-01

    2D DIGE technology was employed on proteins prepared from human brain microvascular endothelial cells (HBMEC), to study the differentially expressed proteins in cells at 0 h, 1 h, 16 h, and 24 h after infection. Proteins found to be differentially expressed were identified with matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDITOF/TOF MS) analysis. We identified 43 spots showing changes of at least 2.5 fold up- or downregulated expressions in EV71-infected cells at different time when comparing to control, and 28 proteins could be successfully identified by MALDI TOF/TOF mass spectrometry analysis. 4 proteins were significantly upregulated, and 6 proteins were downregulated, another 18 proteins were different expression at different incubation time. We identified changes in the expression of 12 cellular metabolism-related proteins, 5 molecules involved in cytoskeleton, 3 molecules involved in energy metabolism, 2 molecules involved in signal transduction, 1 molecule involved in the ubiquitin-proteasome pathway, 1 molecule involved in cell cycle, 1 molecule involved in apoptosis-related protein, 1 molecular chaperone, and 2 unknown proteins. These findings build up a comprehensive profile of the HBMEC proteome and provide a useful basis for further analysis of the pathogenic mechanism that underlies EV71 infections to induce severe neural complications.

  11. Proteomic Profiling and Differential Messenger RNA Expression Correlate HSP27 and Serpin Family B Member 1 to Apical Periodontitis Outcomes.

    PubMed

    Cavalla, Franco; Biguetti, Claudia; Jain, Sameer; Johnson, Cleverick; Letra, Ariadne; Garlet, Gustavo Pompermaier; Silva, Renato Menezes

    2017-09-01

    Understanding protein expression profiles of apical periodontitis may contribute to the discovery of novel diagnostic or therapeutic molecular targets. Periapical tissue samples (n = 5) of patients with lesions characterized as nonhealing were submitted for proteomic analysis. Two differentially expressed proteins (heat shock protein 27 [HSP27] and serpin family B member 1 [SERPINB1]) were selected for characterization, localization by immunofluorescence, and association with known biomarkers of acute inflammatory response in human apical periodontitis (n = 110) and healthy periodontal ligaments (n = 26). Apical periodontitis samples were categorized as stable/inactive (n = 70) or progressive/active (n = 40) based on the ratio of expression of receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG). Next, the expression of HSP27, SERPINB1, C-X-C motif Chemokine Receptor 1 (CXCR1), matrix metalloproteinase 8 (MMP8), myeloperoxidase (MPO), and cathepsin G (CTSG) messenger RNA was evaluated using real-time polymerase chain reaction. Data analysis was performed using the Shapiro-Wilk test, analysis of variance, and the Pearson test. P values <.05 were considered statistically significant. Proteomic analysis revealed 48 proteins as differentially expressed in apical periodontitis compared with a healthy periodontium, with 30 of these proteins found to be expressed in all 4 lesions. The expression of HSP27 and SERPINB1 was ∼2-fold higher in apical periodontitis. Next, an increased expression of HSP27 was detected in epithelial cells, whereas SERPINB1 expression was noted in neutrophils and epithelial cells. HSP27 and SERPINB1 transcripts were highly expressed in stable/inactive lesions (P < .05). Significant negative correlations were found between the expression of HSP27 and SERPINB1 with biomarkers of acute inflammation including CXCR1, MPO, and CTSG. Our data suggest HSP27 and SERPINB1 as potential regulators of the inflammatory

  12. Comparative proteome analysis of embryo and endosperm reveals central differential expression proteins involved in wheat seed germination.

    PubMed

    He, Miao; Zhu, Chong; Dong, Kun; Zhang, Ting; Cheng, Zhiwei; Li, Jiarui; Yan, Yueming

    2015-04-08

    Wheat seeds provide a staple food and an important protein source for the world's population. Seed germination is vital to wheat growth and development and directly affects grain yield and quality. In this study, we performed the first comparative proteomic analysis of wheat embryo and endosperm during seed germination. The proteomic changes in embryo and endosperm during the four different seed germination stages of elite Chinese bread wheat cultivar Zhengmai 9023 were first investigated. In total, 74 and 34 differentially expressed protein (DEP) spots representing 63 and 26 unique proteins were identified in embryo and endosperm, respectively. Eight common DEP were present in both tissues, and 55 and 18 DEP were specific to embryo and endosperm, respectively. These identified DEP spots could be sorted into 13 functional groups, in which the main group was involved in different metabolism pathways, particularly in the reserves necessary for mobilization in preparation for seed germination. The DEPs from the embryo were mainly related to carbohydrate metabolism, proteometabolism, amino acid metabolism, nucleic acid metabolism, and stress-related proteins, whereas those from the endosperm were mainly involved in protein storage, carbohydrate metabolism, inhibitors, stress response, and protein synthesis. During seed germination, both embryo and endosperm had a basic pattern of oxygen consumption, so the proteins related to respiration and energy metabolism were up-regulated or down-regulated along with respiration of wheat seeds. When germination was complete, most storage proteins from the endosperm began to be mobilized, but only a small amount was degraded during germination. Transcription expression of six representative DEP genes at the mRNA level was consistent with their protein expression changes. Wheat seed germination is a complex process with imbibition, stirring, and germination stages, which involve a series of physiological, morphological, and

  13. Proteome Analysis of the Surface of Trichomonas vaginalis Reveals Novel Proteins and Strain-dependent Differential Expression*

    PubMed Central

    de Miguel, Natalia; Lustig, Gil; Twu, Olivia; Chattopadhyay, Arnab; Wohlschlegel, James A.; Johnson, Patricia J.

    2010-01-01

    The identification of surface proteins on the plasma membrane of pathogens is of fundamental importance in understanding host-pathogen interactions. Surface proteins of the extracellular parasite Trichomonas are implicated in the initial adherence to mucosal tissue and are likely to play a critical role in the long term survival of this pathogen in the urogenital tract. In this study, we used cell surface biotinylation and multidimensional protein identification technology to identify the surface proteome of six strains of Trichomonas vaginalis with differing adherence capacities to vaginal epithelial cells. A combined total of 411 proteins were identified, and of these, 11 were found to be more abundant in adherent strains relative to less adherent parasites. The mRNA levels of five differentially expressed proteins selected for quantitative RT-PCR analysis mirrored their observed protein levels, confirming their up-regulation in highly adherent strains. As proof of principle and to investigate a possible role in pathogenesis for differentially expressed proteins, gain of function experiments were performed using two novel proteins that were among the most highly expressed surface proteins in adherent strains. Overexpression of either of these proteins, TVAG_244130 or TVAG_166850, in a relatively non-adherent strain increased attachment of transfected parasites to vaginal epithelial cells ∼2.2-fold. These data support a role in adhesion for these abundant surface proteins. Our analyses demonstrate that comprehensive profiling of the cell surface proteome of different parasite strains is an effective approach to identify potential new adhesion factors as well as other surface molecules that may participate in establishing and maintaining infection by this extracellular pathogen. PMID:20467041

  14. Comparative Proteomic Analysis of Differentially Expressed Proteins Induced by Hydrogen Sulfide in Spinacia oleracea Leaves

    PubMed Central

    Chen, Juan; Liu, Ting-Wu; Hu, Wen-Jun; Simon, Martin; Wang, Wen-Hua; Chen, Juan; Liu, Xiang; Zheng, Hai-Lei

    2014-01-01

    Hydrogen sulfide (H2S), as a potential gaseous messenger molecule, has been suggested to play important roles in a wide range of physiological processes in plants. The aim of present study was to investigate which set of proteins is involved in H2S-regulated metabolism or signaling pathways. Spinacia oleracea seedlings were treated with 100 µM NaHS, a donor of H2S. Changes in protein expression profiles were analyzed by 2-D gel electrophoresis coupled with MALDI-TOF MS. Over 1000 protein spots were reproducibly resolved, of which the abundance of 92 spots was changed by at least 2-fold (sixty-five were up-regulated, whereas 27 were down-regulated). These proteins were functionally divided into 9 groups, including energy production and photosynthesis, cell rescue, development and cell defense, substance metabolism, protein synthesis and folding, cellular signal transduction. Further, we found that these proteins were mainly localized in cell wall, plasma membrane, chloroplast, mitochondria, nucleus, peroxisome and cytosol. Our results demonstrate that H2S is involved in various cellular and physiological activities and has a distinct influence on photosynthesis, cell defense and cellular signal transduction in S. oleracea leaves. These findings provide new insights into proteomic responses in plants under physiological levels of H2S. PMID:25181351

  15. Comparative proteomic analysis of differentially expressed proteins induced by hydrogen sulfide in Spinacia oleracea leaves.

    PubMed

    Chen, Juan; Liu, Ting-Wu; Hu, Wen-Jun; Simon, Martin; Wang, Wen-Hua; Chen, Juan; Liu, Xiang; Zheng, Hai-Lei

    2014-01-01

    Hydrogen sulfide (H2S), as a potential gaseous messenger molecule, has been suggested to play important roles in a wide range of physiological processes in plants. The aim of present study was to investigate which set of proteins is involved in H2S-regulated metabolism or signaling pathways. Spinacia oleracea seedlings were treated with 100 µM NaHS, a donor of H2S. Changes in protein expression profiles were analyzed by 2-D gel electrophoresis coupled with MALDI-TOF MS. Over 1000 protein spots were reproducibly resolved, of which the abundance of 92 spots was changed by at least 2-fold (sixty-five were up-regulated, whereas 27 were down-regulated). These proteins were functionally divided into 9 groups, including energy production and photosynthesis, cell rescue, development and cell defense, substance metabolism, protein synthesis and folding, cellular signal transduction. Further, we found that these proteins were mainly localized in cell wall, plasma membrane, chloroplast, mitochondria, nucleus, peroxisome and cytosol. Our results demonstrate that H2S is involved in various cellular and physiological activities and has a distinct influence on photosynthesis, cell defense and cellular signal transduction in S. oleracea leaves. These findings provide new insights into proteomic responses in plants under physiological levels of H2S.

  16. Proteomic analysis of differentially expressed proteins in the two developmental stages of Ichthyophthirius multifiliis.

    PubMed

    Yao, Jia-Yun; Xu, Yang; Yuan, Xue-Mei; Yin, Wen-Lin; Yang, Gui-Lian; Lin, Ling-Yun; Pan, Xiao-Yi; Wang, Chun-Feng; Shen, Jin-Yu

    2017-02-01

    Ichthyophthirius is a severe disease of farmed freshwater fish caused by the parasitic ciliate Ichthyophthirius multifiliis (Ich). This disease can lead to considerable economic loss, but the protein profiles in different developmental stages of the parasite remain unknown. In the present study, proteins from trophonts and theronts of Ich were identified by isobaric tags for relative and absolute quantitation (iTRAQ). A total of 2300 proteins were identified in the two developmental stages, of which 1520 proteins were differentially expressed. Among them, 84 proteins were uniquely expressed in the theronts stage, while 656 proteins were expressed only in trophonts. The differentially expressed proteins were catalogued (assorted) to various functions of Ich life cycle, including biological process, cellular component, and molecular function that occur at distinct stages. Using a 1.5-fold change in expression as a physiologically significant benchmark, a lot of differentially expressed proteins were reliably quantified by iTRAQ analysis. Two hundred forty upregulated and 57 downregulated proteins in the trophonts stage were identified as compared with theronts. The identified proteins were involved in various functions of the I. multifiliis life cycle, including binding, catalytic activity, structural molecule activity, and transporter activity. Further investigation of the transcriptional levels of periplasmic immunogenic protein, transketolase, zinc finger, isocitrate dehydrogenase, etc., from the different protein profiles using quantitative RT-PCR showed identical results to the iTRAQ analysis. This work provides an effective resource to further our understanding of Ich biology, and lays the groundwork for the identification of potential drug targets and vaccines candidates for the control of this devastating fish pathogen.

  17. Proteomics identifies differentially expressed proteins in neonatal murine thymus compared with adults

    PubMed Central

    2012-01-01

    Background The thymus is an immune organ essential for life and plays a crucial role in the development of T cells. It undergoes a fetal to adult developmental maturation process occurring in mouse during the postnatal months. The molecular modifications underlying these ontogenic changes are essentially unknown. Here we used a differential proteomic-based technique (2D-Difference Gel Electrophoresis) coupled with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry to search for key proteins in the postnatal development of the thymus. Eight different BALB/c mice were used in the study: four mice aged of 1 day (neonatal) and four mice aged of 60 days (adult). Protein samples derived from thymus were labeled and run in 2D-PAGE (Two-Dimensional Polyacrylamide Gel Electrophoresis). One whole-thymus tissue from each mouse was run on gels and each gel containing a pooled sample of the eight mice was run in parallel. The pooled sample was set as the internal pool, containing equal amount of each protein extract used in the experiment. Gels were matched and compared with Difference In-gel Analysis software. Differential spots were picked, in-gel digested and peptide mass fingerprints were obtained. Results Among the differentially regulated proteins in neonatal thymus group, 111 proteins were identified by mass spectrometry, of which 95 proteins were up-regulated and 16 proteins were down-regulated. The identified proteins belong to several functional categories, including cell proliferation, cycle and apoptosis, transcription regulation, signal transduction, nucleotide processing, proteolysis and translation, protein folding, metabolism, oxidoreduction, cytoskeleton, immune response, and embryonic development. The major interaction networks comprised of cellular function and maintenance, cellular assembly and organization, and metabolism were also identified by STRING analysis. Conclusions The demonstrated molecular changes are

  18. The use of proteomics in identifying differentially expressed serum proteins in humans with type 2 diabetes

    PubMed Central

    Sundsten, Tea; Eberhardson, Michael; Göransson, Michael; Bergsten, Peter

    2006-01-01

    Background The aim of the study was to optimize protocols for finding and identifying serum proteins that are differentially expressed in persons with normal glucose tolerance (NGT) compared to individuals with type 2 diabetes mellitus (T2DM). Serum from persons with NGT and persons with T2DM was profiled using ProteinChip arrays and time-of-flight mass spectra were generated by surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). Results Mass spectra from NGT- and T2DM-groups were compared. Fifteen proteins ranging from 5 to 79 kDa were differentially expressed (p < 0.05). Five of these proteins showed decreased and ten showed increased serum levels in individuals with T2DM. To be able to identify the proteins, the complexity of the sample was reduced by fractionation approaches. Subsequently, the purified fractions containing biomarkers were separated by one-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in two identical lanes. Protein bands of the first lane were excised and subjected to passive elution to recapture the biomarkers on ProteinChip arrays. The corresponding bands of the second lane were subjected to peptide-mass fingerprinting (PMF). Using this approach four of the differentially expressed proteins were identified as apolipoprotein C3 (9.4 kDa), transthyretin (13.9 kDa), albumin (66 kDa) and transferrin (79 kDa). Whereas apolipoprotein C3 and transthyretin were up-regulated, albumin and transferrin were down-regulated in T2DM. Conclusion Protocols for protein profiling by SELDI-TOF MS and protein identification by fractionation, SDS-PAGE and PMF were optimized for serum from humans with T2DM. With these protocols differentially expressed proteins were discovered and identified when serum from NGT- and T2DM-individuals was analyzed. PMID:17163994

  19. Comparative proteomic analysis of differentially expressed proteins in the early milky stage of rice grains during high temperature stress

    PubMed Central

    Liao, Jiang-Lin; Zhou, Hui-Wen; Huang, Ying-Jin

    2014-01-01

    Rice yield and quality are adversely affected by high temperatures, and these effects are more pronounced at the ‘milky stage’ of the rice grain ripening phase. Identifying the functional proteins involved in the response of rice to high temperature stress may provide the basis for improving heat tolerance in rice. In the present study, a comparative proteomic analysis of paired, genetically similar heat-tolerant and heat-sensitive rice lines was conducted. Two-dimensional electrophoresis (2-DE) revealed a total of 27 differentially expressed proteins in rice grains, predominantly from the heat-tolerant lines. The protein profiles clearly indicated variations in protein expression between the heat-tolerant and heat-sensitive rice lines. Matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS) analysis revealed that 25 of the 27 differentially displayed proteins were homologous to known functional proteins. These homologous proteins were involved in biosynthesis, energy metabolism, oxidation, heat shock metabolism, and the regulation of transcription. Seventeen of the 25 genes encoding the differentially displayed proteins were mapped to rice chromosomes according to the co-segregating conditions between the simple sequence repeat (SSR) markers and the target genes in recombinant inbred lines (RILs). The proteins identified in the present study provide a basis to elucidate further the molecular mechanisms underlying the adaptation of rice to high temperature stress. PMID:24376254

  20. Proteomic identification of differentially expressed proteins during the acquisition of somatic embryogenesis in oil palm (Elaeis guineensis Jacq.).

    PubMed

    Silva, Rafael de Carvalho; Carmo, Lilian Silveira Travassos; Luis, Zanderluce Gomes; Silva, Luciano Paulino; Scherwinski-Pereira, Jonny Everson; Mehta, Angela

    2014-06-02

    In the present study we have identified and characterized the proteins expressed during different developmental stages of Elaeis guineensis calli obtained from zygotic embryos. We were interested in the possible proteomic changes that would occur during the acquisition of somatic embryogenesis and therefore samples were collected from zygotic embryos (E1), swollen explants 14days (E2) in induction medium, primary callus (E3), and pro-embryogenic callus (E4). The samples were grinded in liquid nitrogen, followed by total protein extraction using phenol and extraction buffer. Proteins were analyzed by two-dimensional electrophoresis (2-DE) and the differentially expressed protein spots were analyzed by MALDI-TOF mass spectrometry (MS and MS/MS). Interestingly, we have identified proteins, which can be used as potential candidates for future studies aiming at the development of biomarkers for embryogenesis acquisition and for the different stages leading to pro-embryogenic callus formation such as type IIIa membrane protein cp-wap13, fructokinase and PR proteins. The results obtained shed some light on the biochemical events involved in the process of somatic embryogenesis of E. guineensis obtained from zygotic embryos. The use of stage-specific protein markers can help monitor cell differentiation and contribute to improve the protocols for successfully cloning the species. Understanding the fate and dynamics of cells and tissues during callus formation is essential to understand totipotency and the mechanisms involved during acquisition of somatic embryogenesis (SE). In this study we have investigated the early stages of somatic embryogenesis induction in oil palm and have identified potential markers as well as proteins potentially involved in embryogenic competence acquisition. The use of these proteins can help improve tissue culture protocols in order to increase regeneration rates. This article is part of a Special Issue entitled: Environmental and structural

  1. Comparative proteomic analysis reveals differentially expressed proteins correlated with fuzz fiber initiation in diploid cotton (Gossypium arboreum L.).

    PubMed

    Du, Shao-Jun; Dong, Chun-Juan; Zhang, Bing; Lai, Tong-Fei; Du, Xiong-Ming; Liu, Jin-Yuan

    2013-04-26

    In this study, a comparative proteomic analysis was employed to identify fuzz fiber initiation-related proteins in wild-type diploid cotton (Gossypium arboreum L.) and its fuzzless mutant. Temporal changes in global proteomes were examined using 2-DE at five developmental time points for fuzz fiber initiation, and 71 differentially expressed protein species were identified by MS, 45 of which were preferentially accumulated in the wild-type. These proteins were assigned to several functional categories, mainly in cell response/signal transduction, redox homeostasis, protein metabolism and energy/carbohydrate metabolism. It was remarkable that more than ten key proteins with high-abundance were involved in gibberellic acid (GA) signaling and ROS scavenging, and increasing concentrations of active GAs and H2O2 were also detected approximately 5dpa in wild type ovules. Furthermore, in vivo GA and H2O2 treatments of ovules inside young bolls showed that these compounds can synergistically promote fuzz fiber initiation. Our findings not only described a dynamic protein network supporting fuzz initiation in diploid cotton fiber ovules, but also deepened our understanding of the molecular basis of cotton fiber initiation. Our study reported the identification of differentially expressed proteins in wild-type diploid cotton (G. arboreum L.) and its fuzzless mutant by comparative proteomic approach. In total, 71 protein species related to fuzz initiation were identified by MS. These proteins were assigned to several functional categories, mainly in energy/carbohydrate metabolism, protein metabolism, signal transduction, redox homeostasis etc. Importantly, a number of key proteins were found to be associated with GA signaling and ROS scavenging. In consistence with these findings, we detected the increase of GAs and H2O2 concentrations during fiber initiation, and our in vivo ovule experiments with GA and H2O2 injection and following microscopy observation of fuzz fiber

  2. Proteomic identification of differentially expressed proteins in Gossypium thurberi inoculated with cotton Verticillium dahliae.

    PubMed

    Zhao, Fu'an; Fang, Weiping; Xie, Deyi; Zhao, Yuanming; Tang, Zhongjie; Li, Wu; Nie, Lihong; Lv, Shuping

    2012-04-01

    Thurber's cotton (Gossypium thurberi) is the wild relative of cultivated cotton. It is highly resistant to cotton Verticillium wilt, a disease that significantly affects cotton yield and quality. To reveal the mechanism of disease resistance in G. thurberi and to clone resistance-related genes, we used two-dimensional electrophoresis (2-DE) and tandem time-of-flight mass spectrometry (MALDI-TOF-MS) to identify differentially expressed proteins in Thurber's cotton after inoculation with Verticillium dahliae. A total of 57 different protein spots were upregulated, including 52 known proteins representing 11% of the total protein spots. These proteins are involved in resistance to stress and disease, transcriptional regulation, signal transduction, protein processing and degradation, photosynthesis, production capacity, basic metabolism, and other processes. In addition, five disease resistance proteins showed intense upregulation, indicating that resistance genes (R genes) may play a critical role in resistance to Verticillium wilt in Thurber's cotton. Our results suggest that disease and stress resistance are the combined effects of multiple co-expressed genes. This provides a basis for further, detailed investigation into the mechanisms underlying Verticillium wilt resistance of G. thurberi and for cloning essential genes into cotton cultivars to produce Verticillium wilt resistant plants.

  3. Differential proteome and gene expression for testis of mice exposed to carbon ion radiation

    NASA Astrophysics Data System (ADS)

    Zhang, Hong; Li, Hongyan

    Objective To investigate the effect and mechanism of high linear energy transfer (LET) carbon ion irradiation (CIR) on reproduction in the testis of male Swiss Webster mice, and assess the risk associated with space environment. Methods Male mice underwent whole-body irradiation with CIR (0.5, 1 and 4Gy), and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF) analysis was used to determine the alteration in protein expression in 2-DE (two-dimensional gel electrophoresis) gels of testes caused by irradiation after 7, 14 days. Results 15 differentially expressed proteins, such as glucose-regulated protein(GRP78), aconitate hydratase-mitochondrial precursor (ACO), pyruvate kinase isozymes M1/M2 (PKM1/M2), glutathione-S-transferaseA3 (GSTA3), glutathione S-transferase Pi 1 (GSTP1), Cu/Zn super-oxide dismutase (SOD1), Peptidyl-prolyl cis-trans isomerase (Pin1) and Heat shock 70 kDa protein 4L (HSPa4L), were identified and these proteins were mainly involved in energy supply, the endoplasmic reticulum, cell proliferation, cell cycle, antioxidant capacity and mitochondrial respiration, which play important roles in the inhibition of testicular function in response to CIR. Furthermore, we confirmed the relationship between transcription of mRNA and the abundance of proteins. Conclusion The findings of the present study demonstrated that these proteins may lead to new insights into the molecular mechanism of CIR toxicity, and suggested that the gene expression response to CIR involves diverse regulatory mechanisms from transcription of mRNA to the formation of functional proteins. These data also may provide a scientific basis for protecting astronauts and space traveler’s health and safety.

  4. Proteomic Analysis of Differentially Expressed Proteins Involved in Peel Senescence in Harvested Mandarin Fruit.

    PubMed

    Li, Taotao; Zhang, Jingying; Zhu, Hong; Qu, Hongxia; You, Shulin; Duan, Xuewu; Jiang, Yueming

    2016-01-01

    Mandarin (Citrus reticulata), a non-climacteric fruit, is an economically important fruit worldwide. The mechanism underlying senescence of non-climacteric fruit is poorly understood. In this study, a gel-based proteomic study followed by LC-ESI-MS/MS analysis was carried out to investigate the proteomic changes involved in peel senescence in harvested mandarin "Shatangju" fruit stored for 18 days. Over the course of the storage period, the fruit gradually senesced, accompanied by a decreased respiration rate and increased chlorophyll degradation and disruption of membrane integrity. Sixty-three proteins spots that showed significant differences in abundance were identified. The up-regulated proteins were mainly associated with cell wall degradation, lipid degradation, protein degradation, senescence-related transcription factors, and transcription-related proteins. In contrast, most proteins associated with ATP synthesis and scavenging of reactive oxygen species were significantly down-regulated during peel senescence. Three thioredoxin proteins and three Ca(2+) signaling-related proteins were significantly up-regulated during peel senescence. It is suggested that mandarin peel senescence is associated with energy supply efficiency, decreased antioxidant capability, and increased protein and lipid degradation. In addition, activation of Ca(2+) signaling and transcription factors might be involved in cell wall degradation and primary or secondary metabolism.

  5. Proteomic Analysis of Differentially Expressed Proteins Involved in Peel Senescence in Harvested Mandarin Fruit

    PubMed Central

    Li, Taotao; Zhang, Jingying; Zhu, Hong; Qu, Hongxia; You, Shulin; Duan, Xuewu; Jiang, Yueming

    2016-01-01

    Mandarin (Citrus reticulata), a non-climacteric fruit, is an economically important fruit worldwide. The mechanism underlying senescence of non-climacteric fruit is poorly understood. In this study, a gel-based proteomic study followed by LC-ESI-MS/MS analysis was carried out to investigate the proteomic changes involved in peel senescence in harvested mandarin “Shatangju” fruit stored for 18 days. Over the course of the storage period, the fruit gradually senesced, accompanied by a decreased respiration rate and increased chlorophyll degradation and disruption of membrane integrity. Sixty-three proteins spots that showed significant differences in abundance were identified. The up-regulated proteins were mainly associated with cell wall degradation, lipid degradation, protein degradation, senescence-related transcription factors, and transcription-related proteins. In contrast, most proteins associated with ATP synthesis and scavenging of reactive oxygen species were significantly down-regulated during peel senescence. Three thioredoxin proteins and three Ca2+ signaling-related proteins were significantly up-regulated during peel senescence. It is suggested that mandarin peel senescence is associated with energy supply efficiency, decreased antioxidant capability, and increased protein and lipid degradation. In addition, activation of Ca2+ signaling and transcription factors might be involved in cell wall degradation and primary or secondary metabolism. PMID:27303420

  6. Proteomic analysis of Pteris vittata fronds: two arbuscular mycorrhizal fungi differentially modulate protein expression under arsenic contamination.

    PubMed

    Bona, Elisa; Cattaneo, Chiara; Cesaro, Patrizia; Marsano, Francesco; Lingua, Guido; Cavaletto, Maria; Berta, Graziella

    2010-11-01

    Arbuscular mycorrhizae (AM) are the most widespread mutualistic symbioses between the roots of most land plants and a phylum of soil fungi. AM are known to influence plant performance by improving mineral nutrition, protecting against pathogens and enhancing resistance or tolerance to biotic and abiotic stresses. The aim of this study was to investigate the frond proteome of the arsenic hyperaccumulator fern Pteris vittata in plants that had been inoculated with one of the two AM fungi (Glomus mosseae or Gigaspora margarita) with and without arsenic treatment. A protective role for AM fungi colonisation in the absence of arsenic was indicated by the down-regulation of oxidative damage-related proteins. Arsenic treatment of mycorrhizal ferns induced the differential expression of 130 leaf proteins with specific responses in G. mosseae- and Gi. margarita-colonised plants. Up-regulation of multiple forms of glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and enolase, primarily in G. mosseae-inoculated plants, suggests a central role for glycolytic enzymes in arsenic metabolism. Moreover, a putative arsenic transporter, PgPOR29, has been identified as an up-regulated protein by arsenic treatment.

  7. Grape berry plasma membrane proteome analysis and its differential expression during ripening.

    PubMed

    Zhang, Jiangwei; Ma, Huiqin; Feng, Jidong; Zeng, Lei; Wang, Zeng; Chen, Shangwu

    2008-01-01

    High purity berry plasma membranes (PMs) of Vitis vinifera L. cv. Cabernet Sauvignon were isolated by two-phase partitioning of microsome fractions at different stages of berry ripening. PM proteins resolvable by the detergent cocktail of CHAPS and ASB-14 were separated by two-dimensional electrophoresis. A total of 119 protein spots from pre-véraison berry PMs on 2-D gels detected with silver staining were subjected to MALDI-TOF mass spectrometry analysis. Sixty-two spots were identified as putative PM proteins, with 1-6 predicted transmembrane helices, including true PM proteins such as ATP synthase, ABC transporters, and GTP-binding proteins reported in plants. They were then grouped into eight functional categories, mainly involved in transport, metabolism, signal transduction, and protein synthesis. Another 11 spots were identified as proteins of unknown function. The véraison and post-véraison samples stained 98 and 86 spots on the gels, respectively. During the berry ripening process, total PM protein content gradually decreased. Among all identified proteins, 12 showed significant differences in terms of their relative abundance. Increasing ubiquitin proteolysis and cytoskeleton proteins were observed from pre-véraison to post-véraison. Zeatin O-glucosyltransferase peaked at véraison, while ubiquitin-conjugating enzyme E2-21 was down-regulated at this stage. This proteome research provides the first information on PM protein characterization during the grape berry ripening process.

  8. Differential protein expression using proteomics from a crustacean brine shrimp (Artemia sinica) under CO2-driven seawater acidification.

    PubMed

    Chang, Xue-Jiao; Zheng, Chao-Qun; Wang, Yu-Wei; Meng, Chuang; Xie, Xiao-Lu; Liu, Hai-Peng

    2016-11-01

    Gradually increasing atmospheric CO2 partial pressure (pCO2) has caused an imbalance in carbonate chemistry and resulted in decreased seawater pH in marine ecosystems, termed seawater acidification. Anthropogenic seawater acidification is postulated to affect the physiology of many marine calcifying organisms. To understand the possible effects of seawater acidification on the proteomic responses of a marine crustacean brine shrimp (Artemia sinica) three groups of cysts were hatched and further raised in seawater at different pH levels (8.2 as control and 7.8 and 7.6 as acidification stress levels according to the predicted levels at the end of this century and next century, respectively) for 1, 7 and 14 days followed by examination of the protein expression changes via two-dimensional gel electrophoresis. Searches of protein databases revealed that 67 differential protein spots were altered due to lower pH level (7.6 and 7.8) stress in comparison to control groups (pH 8.2) by mass spectrometry. Generally, these differentially expressed proteins included the following: 1) metabolic process-related proteins involved in glycolysis and glucogenesis, nucleotide/amino acid/fatty acid metabolism, protein biosynthesis, DNA replication and apoptosis; 2) stress response-related proteins, such as peroxiredoxin, thioredoxin peroxidase, 70-kDa heat shock protein, Na/K ATPase, and ubiquinol-cytochrome c reductase; 3) immune defence-related proteins, such as prophenoloxidase and ferritin; 4) cytoskeletal-related proteins, such as myosin light chain, TCP1 subunit 2, tropomyosin and tubulin alpha chain; and 5) signal transduction-related proteins, such as phospholipase C-like protein, 14-3-3 zeta, translationally controlled tumour protein and RNA binding motif protein. Taken together, these data support the idea that CO2-driven seawater acidification may affect protein expression in the crustacean A. sinica and possibly also in other species that feed on brine shrimp in the

  9. Differential serum proteomic analysis in a model of metabolic disease.

    PubMed

    Matsumura, Takayoshi; Suzuki, Toru; Kada, Nanae; Aizawa, Kenichi; Munemasa, Yoshiko; Nagai, Ryozo

    2006-12-29

    Protein profiling would aid in better understanding the pathophysiology of metabolic disease. Here, we report on differential proteomic analysis using an animal model of diabetes mellitus and associated metabolic disorders (Otsuka Long-Evans Tokushima Fatty rat). Serum was analyzed by a new two-dimensional liquid chromatography system which separated proteins by chromatofocusing and subsequent reversed-phase chromatography. This is the first application of this approach to differential serum proteomics. Differentially expressed proteins, identified with MALDI-TOF mass spectrometry, included apolipoproteins and alpha2-HS-glycoprotein. These findings add to our understanding of the underlying pathophysiology. This new proteomic analysis is a promising tool to elucidate disease mechanisms.

  10. Comparative Proteomic Analysis of saccharopolyspora spinosa SP06081 and PR2 strains reveals the differentially expressed proteins correlated with the increase of spinosad yield

    PubMed Central

    2011-01-01

    Background Saccharopolyspora spinosa produces the environment-friendly biopesticide spinosad, a mixture of two polyketide-derived macrolide active ingredients called spinosyns A and D. Therefore considerable interest is in the improvement of spinosad production because of its low yield in wild-type S. spinosa. Recently, a spinosad-hyperproducing PR2 strain with stable heredity was obtained from protoplast regeneration of the wild-type S. spinosa SP06081 strain. A comparative proteomic analysis was performed on the two strains during the first rapid growth phase (RG1) in seed medium (SM) by using label-free quantitative proteomics to investigate the underlying mechanism leading to the enhancement of spinosad yield. Results In total, 224 proteins from the SP06081 strain and 204 proteins from the PR2 strain were unambiguously identified by liquid chromatography-tandem mass spectrometry analysis, sharing 140 proteins. A total of 12 proteins directly related to spinosad biosynthesis were identified from the two strains in RG1. Comparative analysis of the shared proteins revealed that approximately 31% of them changed their abundance significantly and fell in all of the functional groups, such as tricarboxylic acid cycles, glycolysis, biosynthetic processes, catabolic processes, transcription, translation, oxidation and reduction. Several key enzymes involved in the synthesis of primary metabolic intermediates used as precursors for spinosad production, energy supply, polyketide chain assembly, deoxysugar methylation, and antioxidative stress were differentially expressed in the same pattern of facilitating spinosad production by the PR2 strain. Real-time reverse transcriptase polymerase chain reaction analysis revealed that four of five selected genes showed a positive correlation between changes at the translational and transcriptional expression level, which further confirmed the proteomic analysis. Conclusions The present study is the first comprehensive and

  11. Proteomic Profiling for Identification of Novel Biomarkers Differentially Expressed in Human Ovaries from Polycystic Ovary Syndrome Patients

    PubMed Central

    Li, Li; Zhang, Jiangyu; Deng, Qingshan; Li, Jieming; Li, Zhengfen; Xiao, Yao; Hu, Shuiwang; Li, Tiantian; Tan, Qiuxiao; Li, Xiaofang; Luo, Bingshu; Mo, Hui

    2016-01-01

    Objectives To identify differential protein expression pattern associated with polycystic ovary syndrome (PCOS). Methods Twenty women were recruited for the study, ten with PCOS as a test group and ten without PCOS as a control group. Differential in-gel electrophoresis (DIGE) analysis and mass spectroscopy were employed to identify proteins that were differentially expressed between the PCOS and normal ovaries. The differentially expressed proteins were further validated by western blot (WB) and immunohistochemistry (IHC). Results DIGE analysis revealed eighteen differentially expressed proteins in the PCOS ovaries of which thirteen were upregulated, and five downregulated. WB and IHC confirmed the differential expression of membrane-associated progesterone receptor component 1 (PGRMC1), retinol-binding protein 1 (RBP1), heat shock protein 90B1, calmodulin 1, annexin A6, and tropomyosin 2. Also, WB analysis revealed significantly (P<0.05) higher expression of PGRMC1 and RBP1 in PCOS ovaries as compared to the normal ovaries. The differential expression of the proteins was also validated by IHC. Conclusions The present study identified novel differentially expressed proteins in the ovarian tissues of women with PCOS that can serve as potential biomarkers for the diagnosis and development of novel therapeutics for the treatment of PCOS using molecular interventions. PMID:27846214

  12. Proteomic Analysis of an Immortalized Mouse Pancreatic Stellate Cell Line Identifies Differentially-Expressed Proteins in Activated vs. Non-Proliferating Cell States

    PubMed Central

    Paulo, Joao A.; Urrutia, Raul; Banks, Peter A.; Conwell, Darwin L.; Steen, Hanno

    2011-01-01

    Pancreatic stellate cells (PaSC) are mediators in chronic pancreatitis and pancreatic cancer pathogenesis. Proteins regulating the biomolecular pathways involved in the conversion of activated to quiescent PaSC may have a significant influence in the development of chronic pancreatitis. We aim to compare differentially expressed proteins from an immortalized cell line of mouse PaSC in the activated and serum-starved cell states using mass spectrometry-based proteomics. PaSC cultured in media supplemented with fetal bovine serum (FBS) proliferate in the activated state, while serum starvation promotes the cellular transition to a “pseudo-quiescent” state. Using these two cell states, we performed a comparative mass spectrometry (GeLC-MS/MS) proteomic analysis. We identified over 2000 non-redundant proteins in PaSC. Qualitative and label-free quantitative analysis revealed several hundred proteins that were differentially abundant between the cell states. Proteins that were more abundant in activated PaSC included cytoskeletal proteins and ribosomal proteins, while those more abundant in pseudo-quiescent PaSC included proteins involved in protein degradation-related pathways (lysosome, ubiquitin-mediated proteolysis, and the proteasome). Investigation of the role of PaSC in the pathogenesis of chronic pancreatitis using the mass spectrometry-based proteomics strategy described herein will lead to further insights into the molecular mechanisms associated with the disease. PMID:21838295

  13. Transcriptomic and proteomic approach to identify differentially expressed genes and proteins in Arabidopsis thaliana mutants lacking chloroplastic 1 and cytosolic FBPases reveals several levels of metabolic regulation.

    PubMed

    Soto-Suárez, Mauricio; Serrato, Antonio J; Rojas-González, José A; Bautista, Rocío; Sahrawy, Mariam

    2016-12-01

    During the photosynthesis, two isoforms of the fructose-1,6-bisphosphatase (FBPase), the chloroplastidial (cFBP1) and the cytosolic (cyFBP), catalyse the first irreversible step during the conversion of triose phosphates (TP) to starch or sucrose, respectively. Deficiency in cyFBP and cFBP1 isoforms provokes an imbalance of the starch/sucrose ratio, causing a dramatic effect on plant development when the plastidial enzyme is lacking. We study the correlation between the transcriptome and proteome profile in rosettes and roots when cFBP1 or cyFBP genes are disrupted in Arabidopsis thaliana knock-out mutants. By using a 70-mer oligonucleotide microarray representing the genome of Arabidopsis we were able to identify 1067 and 1243 genes whose expressions are altered in the rosettes and roots of the cfbp1 mutant respectively; whilst in rosettes and roots of cyfbp mutant 1068 and 1079 genes are being up- or down-regulated respectively. Quantitative real-time PCR validated 100% of a set of 14 selected genes differentially expressed according to our microarray analysis. Two-dimensional (2-D) gel electrophoresis-based proteomic analysis revealed quantitative differences in 36 and 26 proteins regulated in rosettes and roots of cfbp1, respectively, whereas the 18 and 48 others were regulated in rosettes and roots of cyfbp mutant, respectively. The genes differentially expressed and the proteins more or less abundant revealed changes in protein metabolism, RNA regulation, cell signalling and organization, carbon metabolism, redox regulation, and transport together with biotic and abiotic stress. Notably, a significant set (25%) of the proteins identified were also found to be regulated at a transcriptional level. This transcriptomic and proteomic analysis is the first comprehensive and comparative study of the gene/protein re-adjustment that occurs in photosynthetic and non-photosynthetic organs of Arabidopsis mutants lacking FBPase isoforms.

  14. Quantitative proteomics analysis of varicose veins: identification of a set of differentially expressed proteins related to ATP generation and utilization.

    PubMed

    Kuo, Chao-Jen; Liang, Shih-Shin; Hsi, Edward; Chiou, Shyh-Horng; Lin, Sin-Daw

    2013-11-01

    Although morphological and anatomical studies indicate that varicose veins are characterized by venous wall weakening and subendothelial fibrosis, the exact underlying biochemical mechanism of their development remains unknown. Additionally, no quantitative proteomic study of venous proteins leading to decreased contractility of varicose veins has been reported to date. Therefore, to elucidate the molecular mechanism of altered vascular contractility, this study performed shotgun proteomic analysis to obtain protein expression profiles in patients with varicose veins. Stable isotope dimethyl labeling coupled with nanoLC-MS/MS revealed downregulation in 12 polypeptides, including myosin light chain kinase, creatine kinase B-type, ATP synthase, phosphoglycerate kinase, and pyruvate kinase. However, analyses of protein species associated with cytoskeletal assembly or with cellular morphology showed no clear up- or down-regulation. These results indicate that defects in ATP generation and utilization may account for the dysfunction of vascular smooth muscle following formation of varicose veins. Collectively, the severity of varicose veins depends on the regulatory roles of various protein factors in the metabolic coordination of physiological functions. This pilot study improves understanding of the pathogenesis of varicose veins and lays the foundation for further validation and clinical translation of biomarkers for targeted therapies in treating this disease. Copyright © 2013. Published by Elsevier B.V.

  15. Proteomic analysis of the nuclear matrix in the early stages of rat liver carcinogenesis: Identification of differentially expressed and MAR-binding proteins

    SciTech Connect

    Barboro, Paola; D'Arrigo, Cristina; Repaci, Erica; Bagnasco, Luca; Orecchia, Paola; Carnemolla, Barbara; Patrone, Eligio; Balbi, Cecilia

    2009-01-15

    Tumor progression is characterized by definite changes in the protein composition of the nuclear matrix (NM). The interactions of chromatin with the NM occur via specific DNA sequences called MARs (matrix attachment regions). In the present study, we applied a proteomic approach along with a Southwestern assay to detect both differentially expressed and MAR-binding NM proteins, in persistent hepatocyte nodules (PHN) in respect with normal hepatocytes (NH). In PHN, the NM undergoes changes both in morphology and in protein composition. We detected over 500 protein spots in each two dimensional map and 44 spots were identified. Twenty-three proteins were differentially expressed; among these, 15 spots were under-expressed and 8 spots were over-expressed in PHN compared to NH. These changes were synchronous with several modifications in both NM morphology and the ability of NM proteins to bind nuclear RNA and/or DNA containing MARs sequences. In PHN, we observed a general decrease in the expression of the basic proteins that bound nuclear RNA and the over-expression of two species of Mw 135 kDa and 81 kDa and pI 6.7-7.0 and 6.2-7.4, respectively, which exclusively bind to MARs. These results suggest that the deregulated expression of these species might be related to large-scale chromatin reorganization observed in the process of carcinogenesis by modulating the interaction between MARs and the scaffold structure.

  16. Proteomic Analysis of Frankliniella occidentalis and Differentially Expressed Proteins in Response to Tomato Spotted Wilt Virus Infection

    PubMed Central

    Badillo-Vargas, I. E.; Rotenberg, D.; Schneweis, D. J.; Hiromasa, Y.; Tomich, J. M.

    2012-01-01

    Tomato spotted wilt virus (TSWV) is transmitted by Frankliniella occidentalis in a persistent propagative manner. Despite the extensive replication of TSWV in midgut and salivary glands, there is little to no pathogenic effect on F. occidentalis. We hypothesize that the first-instar larva (L1) of F. occidentalis mounts a response to TSWV that protects it from pathogenic effects caused by virus infection and replication in various insect tissues. A partial thrips transcriptome was generated using 454-Titanium sequencing of cDNA generated from F. occidentalis exposed to TSWV. Using these sequences, the L1 thrips proteome that resolved on a two-dimensional gel was characterized. Forty-seven percent of the resolved protein spots were identified using the thrips transcriptome. Real-time quantitative reverse transcriptase PCR (RT-PCR) analysis of virus titer in L1 thrips revealed a significant increase in the normalized abundance of TSWV nucleocapsid RNA from 2 to 21 h after a 3-h acquisition access period on virus-infected plant tissue, indicative of infection and accumulation of virus. We compared the proteomes of infected and noninfected L1s to identify proteins that display differential abundances in response to virus. Using four biological replicates, 26 spots containing 37 proteins were significantly altered in response to TSWV. Gene ontology assignments for 32 of these proteins revealed biological roles associated with the infection cycle of other plant- and animal-infecting viruses and antiviral defense responses. Our findings support the hypothesis that L1 thrips display a complex reaction to TSWV infection and provide new insights toward unraveling the molecular basis of this interaction. PMID:22696645

  17. A proteomic approach to identifying proteins differentially expressed in conidia and mycelium of the entomopathogenic fungus Metarhizium acridum.

    PubMed

    Barros, Bruno H R; da Silva, Sérgio H; dos ReisMarques, Everaldo Dos Reis; Rosa, José C; Yatsuda, Ana Patrícia; Roberts, Donald W; Braga, Gilberto U L

    2010-07-01

    Metarhizium spp. is an important worldwide group of entomopathogenic fungi used as an interesting alternative to chemical insecticides in programs of agricultural pest and disease vector control. Metarhizium conidia are important in fungal propagation and also are responsible for host infection. Despite their importance, several aspects of conidial biology, including their proteome, are still unknown. We have established conidial and mycelial proteome reference maps for Metarhizium acridum using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF MS). In all, 1130±102 and 1200±97 protein spots were detected in ungerminated conidia and fast-growing mycelia, respectively. Comparison of the two protein-expression profiles reveled that only 35% of the protein spots were common to both developmental stages. Out of 94 2-DE protein spots (65 from conidia, 25 from mycelia and two common to both) analyzed using mass spectrometry, seven proteins from conidia, 15 from mycelia and one common to both stages were identified. The identified protein spots exclusive to conidia contained sequences similar to known fungal stress-protector proteins (such as heat shock proteins (HSP) and 6-phosphogluconate dehydrogenase) plus the fungal allergen Alt a 7, actin and the enzyme cobalamin-independent methionine synthase. The identified protein spots exclusive to mycelia included proteins involved in several cell housekeeping biological processes. Three proteins (HSP 90, 6-phosphogluconate dehydrogenase and allergen Alt a 7) were present in spots in conidial and mycelial gels, but they differed in their locations on the two gels.

  18. Proteomic analysis of differential protein expression of achilles tendon in a rabbit model by two-dimensional polyacrylamide gel electrophoresis at 21 days postoperation.

    PubMed

    Jielile, Jiasharete; Jialili, Ainuer; Sabirhazi, Gulnur; Shawutali, Nuerai; Redati, Darebai; Chen, Jiangtao; Tang, Bin; Bai, Jingping; Aldyarhan, Kayrat

    2011-10-01

    Postoperative early kinesitherapy has been advocated as an optimal method for treating Achilles tendon rupture. However, an insight into the rationale of how early kinesitherapy contributes to healing of Achilles tendon remains to be achieved, and research in the area of proteomic analysis of Achilles tendon has so far been lacking. Forty-two rabbits were randomized into control group, immobilization group, and early motion group, and received postoperative cast immobilization and early motion treatments. Achilles tendon samples were prepared 21 days following microsurgery, and the proteins were separated with two-dimensional polyacrylamide gel electrophoresis. Differentially expressed proteins were first recognized by PDQuest software, and then identified using peptide mass fingerprinting, tandem mass spectrometry, and database searching. A total of 463  ±  12, 511  ±  39, and 513  ±  80 protein spots were successfully detected in the two-dimensional polyacrylamide gels for the Achilles tendon samples of rabbits in the control group, immobilization group, and early motion group, respectively. There were 15, 8, and 9 unique proteins in these three groups, respectively, and some differentially expressed proteins were also identified in each group. It was indicated that some of the differentially expressed proteins were involved in various metabolism pathways and may play an important role in healing of Achilles tendon rupture. Postoperative early kinesitherapy resulted in differentially expressed proteins in ruptured Achilles tendon compared with those treated with postoperative cast immobilization. These differentially expressed proteins may contribute to healing of Achilles tendon rupture through a mechanobiological mechanism due to the application of postoperative early kinesitherapy.

  19. Proteomic approaches to bacterial differentiation

    SciTech Connect

    Norbeck, Angela D.; Callister, Stephen J.; Monroe, Matthew E.; Jaitly, Navdeep; Elias, Dwayne A.; Lipton, Mary S.; Smith, Richard D.

    2006-12-01

    While genomic approaches have been applied for the detection and identification of individual bacteria within microbial communities, analogous proteomics approaches have been effectively precluded due to their inherent complexity. An in silico assessment of peptides that could potentially be present in the proteomes of artificial simple and complex communities was performed to evaluate the effect of proteome complexity on species detection. A mass spectrometry-based proteomics approach was employed to experimentally detect and validate the predicted tryptic peptides initially identified as distinctive within the simple community. An assessment of peptide distinctiveness and the potential for mapping to a particular bacterium within a community was made throughout each step of the study. A second in silico assessment of peptide distinctiveness for a complex community of 25 microorganisms was conducted to investigate the levels of instrumental performance that would be required to experimentally detect these peptides, as well as how performance varied with complexity (e.g., the number of different microorganisms). The experimental data for a simple community showed that it is feasible to predict, observe, and to quantify distinctive peptides from one organism in the presence of at least a 100-fold greater abundance of another, thus yielding putative markers for identifying a bacterium of interest. This work represents a first step towards quantitative proteomic characterization of complex microbial communities and the possible development of community wide markers of perturbations to such communities.

  20. Quantitative proteomics analysis of differential protein expression and oxidative modification of specific proteins in the brains of old mice.

    PubMed

    Poon, H Fai; Vaishnav, Radhika A; Getchell, Thomas V; Getchell, Marilyn L; Butterfield, D Allan

    2006-07-01

    The brain is susceptible to oxidative stress, which is associated with age-related brain dysfunction, because of its high content of peroxidizable unsaturated fatty acids, high oxygen consumption per unit weight, high content of key components for oxidative damage, and the relative scarcity of antioxidant defense systems. Protein oxidation, which results in functional disruption, is not random but appears to be associated with increased oxidation in specific proteins. By using a proteomics approach, we have compared the protein levels and specific protein carbonyl levels, an index of oxidative damage in the brains of old mice, to these parameters in the brains of young mice and have identified specific proteins that are altered as a function of aging. We show here that the expression levels of dihydropyrimidinase-like 2 (DRP2), alpha-enolase (ENO1), dynamin-1 (DNM1), and lactate dehydrogenase 2 (LDH2) were significantly increased in the brains of old versus young mice; the expression levels of three unidentified proteins were significantly decreased. The specific carbonyl levels of beta-actin (ACTB), glutamine synthase (GS), and neurofilament 66 (NF-66) as well as a novel protein were significantly increased, indicating protein oxidation, in the brains of old versus young mice. These results were validated by immunochemistry. In addition, enzyme activity assays demonstrated that oxidation was associated with decreased GS activity, while the activity of lactate dehydrogenase was unchanged in spite of an up-regulation of LDH2 levels. Several of the up-regulated and oxidized proteins in the brains of old mice identified in this report are known to be oxidized in neurodegenerative diseases as well, suggesting that these proteins may be particularly susceptible to processes associated with neurodegeneration. Our results establish an initial basis for understanding protein alterations that may lead to age-related cellular dysfunction in the brain.

  1. Comparative proteomic analysis of differentially expressed proteins in the earthworm Eisenia fetida during Escherichia coli O157:H7 stress.

    PubMed

    Wang, Xing; Chang, Li; Sun, Zhenjun; Zhang, Yufeng

    2010-12-03

    Escherichia coli O157:H7 is an intestine-inhabiting bacterium associated with many severe disease outbreaks worldwide. It may enter the soil environment with the excreta of infected animals (e.g., horses, cattle, chickens) and humans. Earthworms can protect themselves against invading pathogens because of their efficient innate defense system. Identification of differential proteomic responses to E. coli O157:H7 may provide a better understanding of the survival mechanisms of the earthworm Eisenia fetida that lives in E. coli O157:H7-polluted environments. Whole earthworm extracts, collected at days 7, 14, 21, and 28 after E. coli O157:H7 stress, were analyzed by two-dimensional gel electrophoresis and quantitative image analysis. In total, 124 proteins demonstrated significant regulation at least at one time point, and 52 proteins were identified by matrix-assisted laser desorption/ionization-tandem time-of-flight mass spectrometry and database searching. Compared with control samples, 11 protein spots were up-regulated and 41 were down-regulated for at least one time point. The identified proteins, including heat shock protein 90, fibrinolytic protease 0, gelsolin-like protein, lombricine kinase, coelomic cytolytic factor-1, manganous superoxide dismutase, catalase, triosephosphate isomerase, extracellular globin-4, lysenin, intermediate filament protein, and glyceraldehyde-3-phosphate dehydrogenase, are involved in several processes, including transcription, translation, the tricarboxylic acid cycle, and the glucose metabolic process. Thus, our study provides a functional profile of the E. coli O157:H7-responsive proteins in earthworms. We suggest that the variable levels and trends in these spots on the gel may be useful as biomarker profiles to investigate E. coli O157:H7 contamination levels in soils.

  2. Increased cell proliferation and differential protein expression induced by low-level Er:YAG laser irradiation in human gingival fibroblasts: proteomic analysis.

    PubMed

    Ogita, Mayumi; Tsuchida, Sachio; Aoki, Akira; Satoh, Mamoru; Kado, Sayaka; Sawabe, Masanori; Nanbara, Hiromi; Kobayashi, Hiroaki; Takeuchi, Yasuo; Mizutani, Koji; Sasaki, Yoshiyuki; Nomura, Fumio; Izumi, Yuichi

    2015-09-01

    Erbium-doped yttrium aluminum garnet (Er:YAG) laser treatment has demonstrated favorable wound healing effect after periodontal therapy. One of the reasons may be the positive biological effect of the low-level laser on the irradiated tissues, although the mechanism remains unclear. The aim of this study was to investigate the effect of low-level Er:YAG laser irradiation on cell proliferation and laser-induced differential expression of proteins in human gingival fibroblasts (HGFs) by proteomic analysis. In the first experiment, HGFs were exposed to low-level Er:YAG laser irradiation and the laser-induced cell proliferation and damage were evaluated on day 3. In the second experiment, proteomic analysis was performed on day 1 after irradiation. The peptides prepared from HGFs were analyzed by a hybrid ion trap-Fourier transform mass spectrometer, Mascot search engine, and UniProtKB database. A significant increase in cell proliferation without cell damage after irradiation was observed. Among the total identified 377 proteins, 59 proteins, including galectin-7, which was associated with the process of wound healing, were upregulated and 15 proteins were downregulated in laser-treated HGFs. In the third experiment, the increase in messenger RNA (mRNA) and protein expression of galectin-7 in the irradiated HGFs was validated by various analytical techniques. In addition, the effect of recombinant human galectin-7 on the modulation of HGFs proliferation was confirmed. The results indicate that low-level Er:YAG laser irradiation can promote HGF proliferation and induce a significant change in protein expression and the upregulation of galectin-7 expression may partly contribute to the increase in cell proliferation.

  3. Proteomic analysis of differentially expressed protein in hemocytes of wild giant freshwater prawn Macrobrachium rosenbergii infected with infectious hypodermal and hematopoietic necrosis virus (IHHNV)

    PubMed Central

    Alinejad, T.; Bin, Kwong Q.; Vejayan, J.; Othman, R.Y.; Bhassu, S.

    2015-01-01

    Epizootic diseases cause huge mortality and economical loses at post larvae stages in freshwater prawn aquaculture industry. These prawns seem less susceptible to viral diseases except for infectious hypodermal and hematopoietic necrosis virus (IHHNV). During viral infection in prawns, hemocytes are the primary organ that shows immunological response within the early stages of infection. We applied proteomic approaches to understand differential expression of the proteins in hemocytes during the viral disease outbreak. To aid the goal, we collected Macrobrachium rosenbergii broodstocks from the local grow out hatchery which reported the first incidence of IHHNV viral outbreak during larvae stage. Primarily, application of the OIE primer targeting 389 bp fragments of IHHNV virus was used in identification of the infected and non-infected samples of the prawn breeding line. Analysis of two-dimensional gel electrophoresis showed specific down-regulation of Arginine kinase and Sarcoplasmic calcium-binding protein and up/down-regulation of Prophenoloxidase1 and hemocyanin isoforms. These proteins were validated using semi quantitative RT-PCR and gene transcripts at mRNA level. These identified proteins can be used as biomarkers, providing a powerful approach to better understanding of the immunity pathway of viral disease with applications in analytic and observational epidemiology diagnosis. Proteomic profiling allows deep insight into the pathogenesis of IHHNV molecular regulation and mechanism of hemocyte in freshwater prawns. PMID:26106581

  4. Proteomic analysis of stipe explants reveals differentially expressed proteins involved in early direct somatic embryogenesis of the tree fern Cyathea delgadii Sternb.

    PubMed

    Domżalska, Lucyna; Kędracka-Krok, Sylwia; Jankowska, Urszula; Grzyb, Małgorzata; Sobczak, Mirosław; Rybczyński, Jan J; Mikuła, Anna

    2017-05-01

    Using cyto-morphological analysis of somatic embryogenesis (SE) in the tree fern Cyathea delgadii as a guide, we performed a comparative proteomic analysis in stipe explants undergoing direct SE. Plant material was cultured on hormone-free medium supplemented with 2% sucrose. Phenol extracted proteins were separated using two-dimensional gel electrophoresis (2-DE) and mass spectrometry was performed for protein identification. A total number of 114 differentially regulated proteins was identified during early SE, i.e. when the first cell divisions started and several-cell pro-embryos were formed. Proteins were assigned to seven functional categories: carbohydrate metabolism, protein metabolism, cell organization, defense and stress responses, amino acid metabolism, purine metabolism, and fatty acid metabolism. Carbohydrate and protein metabolism were found to be the most sensitive SE functions with the greatest number of alterations in the intensity of spots in gel. Differences, especially in non-enzymatic and structural protein abundance, are indicative for cell organization, including cytoskeleton rearrangement and changes in cell wall components. The highest induced changes concern those enzymes related to fatty acid metabolism. Global analysis of the proteome reveals several proteins that can represent markers for the first 16days of SE induction and expression in fern. The findings of this research improve the understanding of molecular processes involved in direct SE in C. delgadii. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Bottom-up proteomics suggests an association between differential expression of mitochondrial proteins and chronic fatigue syndrome

    PubMed Central

    Ciregia, F; Kollipara, L; Giusti, L; Zahedi, R P; Giacomelli, C; Mazzoni, M R; Giannaccini, G; Scarpellini, P; Urbani, A; Sickmann, A; Lucacchini, A; Bazzichi, L

    2016-01-01

    Chronic fatigue syndrome (CFS) is a debilitating and complex disorder characterized by unexplained fatigue not improved by rest. An area of investigation is the likely connection of CFS with defective mitochondrial function. In a previous work, we investigated the proteomic salivary profile in a couple of monozygotic twins discordant for CFS. Following this work, we analyzed mitochondrial proteins in the same couple of twins. Nano-liquid chromatography electrospray ionization mass spectrometry (nano-LC-MS) was used to study the mitochondria extracted from platelets of the twins. Subsequently, we selected three proteins that were validated using western blot analysis in a big cohort of subjects (n=45 CFS; n=45 healthy), using whole saliva (WS). The selected proteins were as follows: aconitate hydratase (ACON), ATP synthase subunit beta (ATPB) and malate dehydrogenase (MDHM). Results for ATPB and ACON confirmed their upregulation in CFS. However, the MDHM alteration was not confirmed. Thereafter, seeing the great variability of clinical features of CFS patients, we decided to analyze the expression of our proteins after splitting patients according to clinical parameters. For each marker, the values were actually higher in the group of patients who had clinical features similar to the ill twin. In conclusion, these results suggest that our potential markers could be one of the criteria to be taken into account for helping in diagnosis. Furthermore, the identification of biomarkers present in particular subgroups of CFS patients may help in shedding light upon the complex entity of CFS. Moreover, it could help in developing tailored treatments. PMID:27676445

  6. Proteomic analysis of differentially expressed proteins in vitreous humor of patients with retinoblastoma using iTRAQ-coupled ESI-MS/MS approach.

    PubMed

    Naru, Jasmine; Aggarwal, Ritu; Singh, Usha; Mohanty, Ashok Kumar; Bansal, Deepak; Mangat, Navdeep; Kakkar, Nandita; Agnihotri, Navneet

    2016-10-01

    There is close proximity of vitreous humor with the tumor bulk in eyes with retinoblastoma. This renders vitreous humor a promising source to evaluate disease-specific protein targets in retinoblastoma. We studied the differential proteome of vitreous fluid in retinoblastoma tumors (n = 4) as compared to controls (n = 4). The vitreous humor was depleted off the high abundant fraction using MARS-6 affinity column. Subsequently, the tryptic peptides were derivatised with iTRAQ labels. The labelled peptides were pooled and subjected to fractionation using bRPLC. This was followed by protein identification and quantification using electrospray ionisation mass spectrometry (ESI-MS/MS) approach. The identified proteins were subjected to bioinformatics analysis utilizing PANTHER 7.0 and IPA software. Four hundred and thirty-one non-redundant (362 upregulated and 69 downregulated) proteins (≥2 unique peptides, ± 1.5 folds, p < 0.05) were identified. The majority of the proteins were cytoplasmic (40 %), majorly involved in catalytic (32.7 %) and binding activities (26.3 %). Highly deregulated proteins included MMP2, TNC, CD44, SUZ12 and CRABP1. The protein expression of GFAP, CRABP1, MMP2 and TNC was validated by western blotting. Pathway and network analyses revealed p38MAPK and Akt signalling to be the most significantly regulated pathways in retinoblastoma. This is the first report of differential vitreous proteome of retinoblastoma and highlights novel protein targets, such as MMP2, TNC and CRABP1. Further investigations into unravelling the biological role of the proteins and their prospects of being utilised as potential candidates in therapeutics are warranted.

  7. A proteomic study of the differential protein expression in MDBK cells after bovine herpesvirus type 1 infection (BHV-1) strain treatment

    PubMed Central

    Guo, Li; Yang, Yanling; Liu, Linna; Liao, Peng; Wen, Yongjun; Wu, Hua; Cheng, Shipeng

    2015-01-01

    Different BHV-1 strains, such as the virulent IBRV LN01/08 strains and the attenuated vaccine strain IBRV LNM, produces different clinical immune responses; however, the study of the differential protein expression in Madin-Darby bovine kidney (MDBK) cells after BHV-1-infection still remains unclear. Here, we applied a comparative proteomic strategy, based on 2D and MALDI-TOF/MS platforms, to examine the differential expression of proteins in MDBK cells that were treated and not treated with virulent IBRV LN01/08 and attenuated IBRV LNM strains. A total of eight differential proteins, including pyruvate kinase, heat shock protein (HSP) 90 (HSP90AA1 and HSP90AB1), annexin A, albumin (ALB), scinderin (SCIN), tubulin (alpha 1a) and vimentin (VIM), were identified. Among these proteins, pyruvate kinase, and HSP90 (HSP90AB1), tubulin and vimentin were identified in the virulent IBRV LN01/08 strain group, but were not identified in the attenuated IBRV LNM group. These results play an important role in tumor formation and development, cell migration, tumor cell line apoptosis, cell invasion and viral infection. The HSP90 (HSP90AA1) protein was identified in the control group and the attenuated IBRV LNM-infected group. Most studies have shown that HSP90 proteins were more of a cancer gene target, and inhibiting its function would result to oncogene degradation during cancer treatment. On the other hand, ALB is associated to cell differentiation, apoptosis, necrosis, cell death, viral infection, autophagy, interstitial tissue inflammation, and cell survival. These results provide a theoretical basis for the systematic understanding of BHV-1-infection mechanisms and BHV-1-induced immune responses. PMID:26064331

  8. Capillary Isoelectric Focusing Immunoassay for Fat Cell Differentiation Proteomics

    PubMed Central

    Johlfs, Mary G.; Gorjala, Priyatham; Urasaki, Yasuyo; Le, Thuc T.; Fiscus, Ronald R.

    2015-01-01

    Profiling cellular proteome is critical to understanding signal integration during cell fate determination. In this study, the capability of capillary isoelectric focusing (cIEF) immunoassays to detect post-translational modifications (PTM) of protein isoforms is demonstrated. cIEF immunoassays exhibit protein detection sensitivity at up to 5 orders of magnitude higher than traditional methods. This detection ultra-sensitivity permits proteomic profiling of several nanograms of tissue samples. cIEF immunoassays are employed to simultaneously profile three protein kinases during fat cell differentiation: cGMP-dependent protein kinase type I (PKG-I) of the nitric oxide (NO) signaling pathway, protein kinase B (Akt) of the insulin signaling pathway, and extracellular signal-regulated kinase (ERK) of the mitogen-activated protein kinase (MAPK) signaling pathway. Interestingly, a switch in the expression level of PKG- isoforms is observed during fat cell differentiation. While both PKG-Iα and PKG-Iβ isoforms are present in preadipocytes, only PKG-Iβ isoform is expressed in adipocytes. On the other hand, the phosphorylation level increases for Akt while decreases for ERK1 and ERK2 following the maturation of preadipocytes into adipocytes. Taken together, cIEF immunoassay provides a highly sensitive means to study fat cell differentiation proteomics. cIEF immunoassay should be a powerful proteomics tool to study complex protein signal integration in biological systems. PMID:26132171

  9. Capillary Isoelectric Focusing Immunoassay for Fat Cell Differentiation Proteomics.

    PubMed

    Johlfs, Mary G; Gorjala, Priyatham; Urasaki, Yasuyo; Le, Thuc T; Fiscus, Ronald R

    2015-01-01

    Profiling cellular proteome is critical to understanding signal integration during cell fate determination. In this study, the capability of capillary isoelectric focusing (cIEF) immunoassays to detect post-translational modifications (PTM) of protein isoforms is demonstrated. cIEF immunoassays exhibit protein detection sensitivity at up to 5 orders of magnitude higher than traditional methods. This detection ultra-sensitivity permits proteomic profiling of several nanograms of tissue samples. cIEF immunoassays are employed to simultaneously profile three protein kinases during fat cell differentiation: cGMP-dependent protein kinase type I (PKG-I) of the nitric oxide (NO) signaling pathway, protein kinase B (Akt) of the insulin signaling pathway, and extracellular signal-regulated kinase (ERK) of the mitogen-activated protein kinase (MAPK) signaling pathway. Interestingly, a switch in the expression level of PKG- isoforms is observed during fat cell differentiation. While both PKG-Iα and PKG-Iβ isoforms are present in preadipocytes, only PKG-Iβ isoform is expressed in adipocytes. On the other hand, the phosphorylation level increases for Akt while decreases for ERK1 and ERK2 following the maturation of preadipocytes into adipocytes. Taken together, cIEF immunoassay provides a highly sensitive means to study fat cell differentiation proteomics. cIEF immunoassay should be a powerful proteomics tool to study complex protein signal integration in biological systems.

  10. Differential proteomics of Helicobacter pylori associated with autoimmune atrophic gastritis.

    PubMed

    Repetto, Ombretta; Zanussi, Stefania; Casarotto, Mariateresa; Canzonieri, Vincenzo; De Paoli, Paolo; Cannizzaro, Renato; De Re, Valli

    2014-02-28

    Atrophic autoimmune gastritis (AAG) is a condition of chronic inflammation and atrophy of stomach mucosa, for which development can be partially triggered by the bacterial pathogen Helicobacter pylori (HP). HP can cause a variety of gastric diseases, such as duodenal ulcer (DU) or gastric cancer (GC). In this study, a comparative proteomic approach was used by two-dimensional fluorescence difference gel electrophoresis (DIGE) to identify differentially expressed proteins of HP strains isolated from patients with AAG, to identify markers of HP strain associated with AAG. Proteome profiles of HP isolated from GC or DU were used as a reference to compare proteomic levels. Proteomics analyses revealed 27 differentially expressed spots in AAG-associated HP in comparison with GC, whereas only 9 differential spots were found in AAG-associated HP profiles compared with DU. Proteins were identified after matrix-assisted laser desorption ionization (MALDI)-TOF and peptide mass fingerprinting. Some AAG-HP differential proteins were common between DU- and GC-HP (peroxiredoxin, heat shock protein 70 [HSP70], adenosine 5'-triphosphate [ATP] synthase subunit α, flagellin A). Our results presented here may suggest that comparative proteomes of HP isolated from AAG and DU share more common protein expression than GC and provide subsets of putative AAG-specific upregulated or downregulated proteins that could be proposed as putative markers of AAG-associated HP. Other comparative studies by two-dimensional maps integrated with functional genomics of candidate proteins will undoubtedly contribute to better decipher the biology of AAG-associated HP strains.

  11. Quantitative proteomics identifies 38 proteins that are differentially expressed in cucumber in response to cucumber green mottle mosaic virus infection.

    PubMed

    Liu, Hua-Wei; Liang, Chao-Qiong; Liu, Peng-Fei; Luo, Lai-Xin; Li, Jian-Qiang

    2015-12-15

    Since it was first reported in 1935, Cucumber green mottle mosaic virus (CGMMV) has become a serious pathogen in a range of cucurbit crops. The virus is generally transmitted by propagation materials, and to date no effective chemical or cultural methods of control have been developed to combat its spread. The current study presents a preliminary analysis of the pathogenic mechanisms from the perspective of protein expression levels in an infected cucumber host, with the objective of elucidating the infection process and potential strategies to reduce both the economic and yield losses associated with CGMMV. Isobaric tags for relative and absolute quantitation (iTRAQ) technology coupled with liquid chromatography-tandem mass spectrometric (LC-MS/MS) were used to identify the differentially expressed proteins in cucumber plants infected with CGMMV compared with mock-inoculated plants. The functions of the proteins were deduced by functional annotation and their involvement in metabolic processes explored by KEGG pathway analysis to identify their interactions during CGMMV infection, while their in vivo expression was further verified by qPCR. Infection by CGMMV altered both the expression level and absolute quantity of 38 proteins (fold change >0.6) in cucumber hosts. Of these, 23 were found to be up-regulated, while 15 were down-regulated. Gene ontology (GO) analysis revealed that 22 of the proteins had a combined function and were associated with molecular function (MF), biological process (BP) and cellular component (CC). Several other proteins had a dual function with 1, 7, and 2 proteins being associated with BP/CC, BP/MF, CC/MF, respectively. The remaining 3 proteins were only involved in MF. In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis identified 18 proteins that were involved in 13 separate metabolic pathways. These pathways were subsequently merged to generate three network diagrams illustrating the interactions between the different

  12. Differential expression of proteins in the wild type and 7B-1 male-sterile mutant anthers of tomato (Solanum lycopersicum): a proteomic analysis.

    PubMed

    Sheoran, Inder S; Ross, Andrew R S; Olson, Douglas J H; Sawhney, Vipen K

    2009-01-30

    In the 7B-1 male-sterile mutant of tomato, pollen development breaks down prior to meiosis in microspore mother cells (MMCs). We have used the proteomic approach to identify differentially expressed proteins in the wild type (WT) and mutant anthers with the objective of analyzing their roles in normal pollen development and in male sterility. By using 2-DE and DIGE technologies, over 1800 spots were detected and of these 215 spots showed 1.5-fold or higher volume ratio in either WT or 7B-1 anthers. Seventy spots, either up-regulated in WT, or in 7B-1, were subjected to mass spectrometry and 59 spots representing 48 distinct proteins were identified. The proteins up-regulated in WT anthers included proteases, e.g., subtilase, proteasome subunits, and 5B-protein with potential roles in tapetum degeneration, FtsZ protein, leucine-rich repeat proteins, translational and transcription factors. In 7B-1 anthers, aspartic protease, superoxide dismutase, ACP reductase, ribonucleoprotein and diphosphate kinase were up-regulated. Also, cystatin inhibitory activity was high in the mutant and correlated with the expression of male sterility. Other proteins including calreticulin, Heat shock protein 70, glucoside hydrolase, and ATPase, were present in both genotypes. The function of identified proteins in tapetum and normal pollen development, and in male sterility is discussed.

  13. Proteomic analysis of differentially expressed proteins involved in ethylene-induced chilling tolerance in harvested banana fruit.

    PubMed

    Li, Taotao; Yun, Ze; Zhang, Dandan; Yang, Chengwei; Zhu, Hong; Jiang, Yueming; Duan, Xuewu

    2015-01-01

    To better understand the mechanism involved in ethylene-induced chilling tolerance in harvested banana fruit, a gel-based proteomic study followed by MALDI-TOF-TOF MS was carried out. Banana fruit were treated with 500 ppm ethylene for 12 h and then stored at 6°C. During cold storage, the chilling tolerance was assessed and the proteins from the peel were extracted for proteomic analysis. It was observed that ethylene pretreatment significantly induced the chilling tolerance in harvested banana fruit, manifesting as increases in maximal chlorophyll fluorescence (Fv/Fm) and decreased electrolyte leakage. Sixty-four proteins spots with significant differences in abundance were identified, most of which were induced by ethylene pretreatment during cold storage. The up-regulated proteins induced by ethylene pretreatment were mainly related to energy metabolism, stress response and defense, methionine salvage cycle and protein metabolism. These proteins were involved in ATP synthesis, ROS scavenging, protective compounds synthesis, protein refolding and degradation, and polyamine biosynthesis. It is suggested that these up-regulated proteins might play a role in the ethylene-induced chilling tolerance in harvested banana fruit.

  14. Proteomic analysis of differentially expressed proteins involved in ethylene-induced chilling tolerance in harvested banana fruit

    PubMed Central

    Li, Taotao; Yun, Ze; Zhang, Dandan; Yang, Chengwei; Zhu, Hong; Jiang, Yueming; Duan, Xuewu

    2015-01-01

    To better understand the mechanism involved in ethylene-induced chilling tolerance in harvested banana fruit, a gel-based proteomic study followed by MALDI-TOF-TOF MS was carried out. Banana fruit were treated with 500 ppm ethylene for 12 h and then stored at 6°C. During cold storage, the chilling tolerance was assessed and the proteins from the peel were extracted for proteomic analysis. It was observed that ethylene pretreatment significantly induced the chilling tolerance in harvested banana fruit, manifesting as increases in maximal chlorophyll fluorescence (Fv/Fm) and decreased electrolyte leakage. Sixty-four proteins spots with significant differences in abundance were identified, most of which were induced by ethylene pretreatment during cold storage. The up-regulated proteins induced by ethylene pretreatment were mainly related to energy metabolism, stress response and defense, methionine salvage cycle and protein metabolism. These proteins were involved in ATP synthesis, ROS scavenging, protective compounds synthesis, protein refolding and degradation, and polyamine biosynthesis. It is suggested that these up-regulated proteins might play a role in the ethylene-induced chilling tolerance in harvested banana fruit. PMID:26528309

  15. Quantitative proteome analysis of colorectal cancer-related differential proteins.

    PubMed

    Zhang, Yanbin; Liu, Yue; Ye, Yingjiang; Shen, Danhua; Zhang, Hui; Huang, Hongyan; Li, Sha; Wang, Shan; Ren, Jun

    2017-02-01

    To evaluate a new strategy for profiling proteomic changes in colorectal cancer (CRC). We used laser capture microdissection (LCM) to obtain cells from 20 CRC and paired normal mucosal tissues. The differential proteins between the microdissected tumor cells and normal mucosa epithelia were analyzed by acetylation stable isotopic labeling coupled with L linear ion trap Fourier transform ion cyclotron resonance mass spectrometry (LTQ-FT MS). Western blotting was used to assess the differential expression of proteins. We used bioinformatics tools for cluster and ingenuity pathway analysis of the differential proteins. In total, 798 confident proteins were quantified and 137 proteins were differentially expressed by at least twofold, including 67 that were upregulated and 70 that were downregulated in cancer. Two differential proteins, solute carrier family 12 member 2 (SLC12A2) and Ras-related protein Rab-10, were validated by Western blotting, and the results were consistent with acetylation stable isotopic labeling analysis. According to gene ontology analysis, CRC-related differential proteins covered a wide range of subcellular locations and were involved in many biological processes. According to ingenuity pathway analysis of the differential proteins, the most relevant canonical pathway associated with CRC was the 14-3-3-mediated signaling pathway, and seven reliable functional networks including cellular growth and proliferation, amino acid metabolism, inflammatory response, embryonic development, carbohydrate metabolism, cellular assembly and organization, and cell morphology were obtained. Combination of LCM, acetylation stable isotopic labeling analysis and LTQ-FT MS is effective for profiling proteomic changes in CRC cells.

  16. Comparative proteomic analysis of differentially expressed proteins in β-aminobutyric acid enhanced Arabidopsis thaliana tolerance to simulated acid rain.

    PubMed

    Liu, Tingwu; Jiang, Xinwu; Shi, Wuliang; Chen, Juan; Pei, Zhenming; Zheng, Hailei

    2011-05-01

    Acid rain is a worldwide environmental issue that has seriously destroyed forest ecosystems. As a highly effective and broad-spectrum plant resistance-inducing agent, β-aminobutyric acid could elevate the tolerance of Arabidopsis when subjected to simulated acid rain. Using comparative proteomic strategies, we analyzed 203 significantly varied proteins of which 175 proteins were identified responding to β-aminobutyric acid in the absence and presence of simulated acid rain. They could be divided into ten groups according to their biological functions. Among them, the majority was cell rescue, development and defense-related proteins, followed by transcription, protein synthesis, folding, modification and destination-associated proteins. Our conclusion is β-aminobutyric acid can lead to a large-scale primary metabolism change and simultaneously activate antioxidant system and salicylic acid, jasmonic acid, abscisic acid signaling pathways. In addition, β-aminobutyric acid can reinforce physical barriers to defend simulated acid rain stress. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Proteomic expression profiling of thyroid neoplasms.

    PubMed

    Braunschweig, Till; Kaserer, Klaus; Chung, Joon-Yong; Bilke, Sven; Krizman, David; Knezevic, Vladimir; Hewitt, Stephen M

    2007-03-01

    Thyroid cancer is the most common endocrine neoplasm with multiple histologic subtypes, each associated with different treatments and outcomes. Differentiating benign neoplasms such as follicular adenomas from malignant entities such as follicular carcinomas and papillary carcinoma can be challenging. To define the proteomic profile of different thyroid tumors, we screened an antibody array of 330 features against five thyroid neoplasms: follicular adenoma, follicular carcinoma, papillary carcinoma, anaplastic carcinoma, and medullary carcinoma as well as normal thyroid epithelium. Eight candidate biomarkers; c-erbB-2, Stat5a, Annexin IV, IL-11, RARα, FGF7, Caspase 9, and phospho-c-myc were identified as differentially expressed on the antibody array, and validated with immunohistochemistry on tissue microarrays, with a total of 144 samples of the same variety of thyroid neoplasms. Analysis revealed c-erbB-2, Annexin IV, and Stat5a have potential clinical utility to differentiate follicular adenoma, follicular carcinoma, and papillary carcinoma from each other. By using an antibody array as a discovery platform and a tissue microarray as a first step in validation on a large number of specimens, we have identified new markers that have potential utility in the diagnosis of thyroid neoplasms. Copyright © 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Differential expression and redox proteomics analyses of an Alzheimer disease transgenic mouse model: effects of the amyloid-β peptide of amyloid precursor protein.

    PubMed

    Robinson, R A S; Lange, M B; Sultana, R; Galvan, V; Fombonne, J; Gorostiza, O; Zhang, J; Warrier, G; Cai, J; Pierce, W M; Bredesen, D E; Butterfield, D A

    2011-03-17

    Among the pathological factors known to be associated with Alzheimer disease (AD), oxidative stress induced by the amyloid-β peptide (Aβ) has been demonstrated to play a key role in human brain and animal models of AD. Recently, we reported elevated levels of oxidative damage in the brain of a transgenic (Tg) AD mouse model with Swedish and Indiana familial AD mutations in human amyloid precursor protein (APP) [PDAPP mice, line J20], as evidenced by increased levels of protein carbonyls, 3-nitrotyrosine, and protein-bound 4-hydroxy-2-nonenal. This oxidative damage was dependent on the methionine 35 residue within the Aβ peptide. Further insight into the molecular pathways affected in this Tg model of AD may be gained with discovery-based proteomics studies; therefore, two-dimensional gel-based expression proteomics was performed to compare differences in brain protein levels of J20 Tg mice with non-transgenic (NTg) littermate controls. Based on our studies, we identified six proteins that had significantly increased levels in J20 Tg relative to NTg mice: calcineurin subunit B type 1, ρ GDP-dissociation inhibitor 1, T-complex protein 1 subunit α A, α-enolase, peptidyl-prolyl cis-trans isomerase (Pin-1), and ATP synthase subunit α mitochondrial. Several of these proteins have previously been implicated in in vitro and in vivo models and subjects with AD. Additionally, using redox proteomics analyses we identified two oxidatively-modified proteins: phosphatidylethanolamine-binding protein 1 and Pin-1 with decreased levels of protein 3-nitrotyrosine in J20 Tg mice relative to NTg. Western blotting and immunoprecipitation analyses were used to validate proteomics results. Overall, these studies provide information about changes in the brain proteome as a result of Aβ deposition and clues with which to further direct studies on elucidating AD pathogenesis. Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

  19. Directed Shotgun Proteomics Guided by Saturated RNA-seq Identifies a Complete Expressed Prokaryotic Proteome

    SciTech Connect

    Omasits, U.; Quebatte, Maxime; Stekhoven, Daniel J.; Fortes, Claudia; Roschitzki, Bernd; Robinson, Mark D.; Dehio, Christoph; Ahrens, Christian H.

    2013-11-01

    Prokaryotes, due to their moderate complexity, are particularly amenable to the comprehensive identification of the protein repertoire expressed under different conditions. We applied a generic strategy to identify a complete expressed prokaryotic proteome, which is based on the analysis of RNA and proteins extracted from matched samples. Saturated transcriptome profiling by RNA-seq provided an endpoint estimate of the protein-coding genes expressed under two conditions which mimic the interaction of Bartonella henselae with its mammalian host. Directed shotgun proteomics experiments were carried out on four subcellular fractions. By specifically targeting proteins which are short, basic, low abundant, and membrane localized, we could eliminate their initial underrepresentation compared to the estimated endpoint. A total of 1250 proteins were identified with an estimated false discovery rate below 1%. This represents 85% of all distinct annotated proteins and ~90% of the expressed protein-coding genes. Genes that were detected at the transcript but not protein level, were found to be highly enriched in several genomic islands. Furthermore, genes that lacked an ortholog and a functional annotation were not detected at the protein level; these may represent examples of overprediction in genome annotations. A dramatic membrane proteome reorganization was observed, including differential regulation of autotransporters, adhesins, and hemin binding proteins. Particularly noteworthy was the complete membrane proteome coverage, which included expression of all members of the VirB/D4 type IV secretion system, a key virulence factor.

  20. Directed shotgun proteomics guided by saturated RNA-seq identifies a complete expressed prokaryotic proteome

    PubMed Central

    Omasits, Ulrich; Quebatte, Maxime; Stekhoven, Daniel J.; Fortes, Claudia; Roschitzki, Bernd; Robinson, Mark D.; Dehio, Christoph; Ahrens, Christian H.

    2013-01-01

    Prokaryotes, due to their moderate complexity, are particularly amenable to the comprehensive identification of the protein repertoire expressed under different conditions. We applied a generic strategy to identify a complete expressed prokaryotic proteome, which is based on the analysis of RNA and proteins extracted from matched samples. Saturated transcriptome profiling by RNA-seq provided an endpoint estimate of the protein-coding genes expressed under two conditions which mimic the interaction of Bartonella henselae with its mammalian host. Directed shotgun proteomics experiments were carried out on four subcellular fractions. By specifically targeting proteins which are short, basic, low abundant, and membrane localized, we could eliminate their initial underrepresentation compared to the estimated endpoint. A total of 1250 proteins were identified with an estimated false discovery rate below 1%. This represents 85% of all distinct annotated proteins and ∼90% of the expressed protein-coding genes. Genes that were detected at the transcript but not protein level, were found to be highly enriched in several genomic islands. Furthermore, genes that lacked an ortholog and a functional annotation were not detected at the protein level; these may represent examples of overprediction in genome annotations. A dramatic membrane proteome reorganization was observed, including differential regulation of autotransporters, adhesins, and hemin binding proteins. Particularly noteworthy was the complete membrane proteome coverage, which included expression of all members of the VirB/D4 type IV secretion system, a key virulence factor. PMID:23878158

  1. Proteomic Characterization Reveals a Molecular Portrait of Nasopharyngeal Carcinoma Differentiation

    PubMed Central

    Xiao, Zhefeng; Li, Maoyu; Li, Guoqing; Fu, Ying; Peng, Fang; Chen, Yongheng; Chen, Zhuchu

    2017-01-01

    Nasopharyngeal carcinoma (NPC) is categorized into three different differentiated subtypes by World Health Organization (WHO). Based on an earlier comparative proteomic database of the three histological subtypes, the study was to deepen our understanding of molecular mechanisms associated with NPC differentiation through bio-information mining. Among the three subtypes were 194 differentially expressed proteins (DEPs) of 725 identified proteins. Two DEPs, heat shock protein family B (small) member 1 (HSPB1) and keratin 5 (KRT5), were validated in a series of NPC tissue samples by using immunohistochemistry. Quantified protein families including keratins, S100 proteins (S100s) and heat shock proteins exhibited characteristic expression alterations. Comparisons of predicted bio-function activation states among different subtypes, including formation of cellular protrusion, metastasis, cell death, and viral infections, were conducted. Canonical pathway analysis inferred that Rho GTPases related signaling pathways regulated the motility and invasion of dedifferentiated NPC. In conclusion, the study explored the proteomic characteristics of NPC differentiation, which could deepen our knowledge of NPC tumorigenesis and allow the development of novel targets of therapeutic and prognostic value in NPC. PMID:28367237

  2. Quantitative proteomics of fractionated membrane and lumen exosome proteins from isogenic metastatic and nonmetastatic bladder cancer cells reveal differential expression of EMT factors.

    PubMed

    Jeppesen, Dennis Kjølhede; Nawrocki, Arkadiusz; Jensen, Steffen Grann; Thorsen, Kasper; Whitehead, Bradley; Howard, Kenneth A; Dyrskjøt, Lars; Ørntoft, Torben Falck; Larsen, Martin R; Ostenfeld, Marie Stampe

    2014-03-01

    Cancer cells secrete soluble factors and various extracellular vesicles, including exosomes, into their tissue microenvironment. The secretion of exosomes is speculated to facilitate local invasion and metastatic spread. Here, we used an in vivo metastasis model of human bladder carcinoma cell line T24 without metastatic capacity and its two isogenic derivate cell lines SLT4 and FL3, which form metastases in the lungs and liver of mice, respectively. Cultivation in CLAD1000 bioreactors rather than conventional culture flasks resulted in a 13- to 16-fold increased exosome yield and facilitated quantitative proteomics of fractionated exosomes. Exosomes from T24, SLT4, and FL3 cells were partitioned into membrane and luminal fractions and changes in protein abundance related to the gain of metastatic capacity were identified by quantitative iTRAQ proteomics. We identified several proteins linked to epithelial-mesenchymal transition, including increased abundance of vimentin and hepatoma-derived growth factor in the membrane, and casein kinase II α and annexin A2 in the lumen of exosomes, respectively, from metastatic cells. The change in exosome protein abundance correlated little, although significant for FL3 versus T24, with changes in cellular mRNA expression. Our proteomic approach may help identification of proteins in the membrane and lumen of exosomes potentially involved in the metastatic process.

  3. Leaf proteome analysis of transgenic plants expressing antiviral antibodies.

    PubMed

    Di Carli, Mariasole; Villani, Maria Elena; Renzone, Giovanni; Nardi, Luca; Pasquo, Alessandra; Franconi, Rosella; Scaloni, Andrea; Benvenuto, Eugenio; Desiderio, Angiola

    2009-02-01

    The expression of exogenous antibodies in plant is an effective strategy to confer protection against viral infection or to produce molecules with pharmaceutical interest. However, the acceptance of the transgenic technology to obtain self-protecting plants depends on the assessment of their substantial equivalence compared to non-modified crops with an established history of safe use. In fact, the possibility exists that the introduction of transgenes in plants may alter expression of endogenous genes and/or normal production of metabolites. In this study, we investigated whether the expression in plant of recombinant antibodies directed against viral proteins may influence the host leaf proteome. Two transgenic plant models, generated by Agrobacterium tumefaciens-mediated transformation, were analyzed for this purpose, namely, Lycopersicon esculentum cv. MicroTom and Nicotiana benthamiana, expressing recombinant antibodies against cucumber mosaic virus and tomato spotted wilt virus, respectively. To obtain a significant representation of plant proteomes, optimized extraction procedures have been devised for each plant species. The proteome repertoire of antibody-expressing and control plants was compared by 2-DE associated to DIGE technology. Among the 2000 spots detected within the gels, about 10 resulted differentially expressed in each transgenic model and were identified by MALDI-TOF PMF and muLC-ESI-IT-MS/MS procedures. Protein variations were restricted to a limited number of defined differences with an average ratio below 2.4. Most of the differentially expressed proteins were related to photosynthesis or defense function. The overall results suggest that the expression of recombinant antibodies in both systems does not significantly alter the leaf proteomic profile, contributing to assess the biosafety of resistant plants expressing antiviral antibodies.

  4. Proteomic analysis of placentas from cloned cat embryos identifies a set of differentially expressed proteins related to oxidative damage, senescence and apoptosis.

    PubMed

    Bang, Jae-Il; Bae, Dong-Won; Lee, Hyo-Sang; Deb, Gautam K; Kim, Myeong-Ok; Sohn, Sea-Hwan; Han, Chang-Hee; Kong, Il-Keun

    2011-12-01

    Production of cloned mammals by somatic cell nuclear transfer is associated with functional and structural abnormalities of placentation and with abnormal fetal development. A proteomic analysis was performed in domestic cats (Felis catus) to compare cloned term placentas (CTP) obtained from cesarean section (CS) to control placentas obtained from CS or vaginal delivery. The expression of 20 proteins was altered in CTP (p<0.05) compared to control placentas. The two control groups showed that the method of delivery, vaginal delivery or CS, did not affect protein expression (p>0.05). A total of 13 proteins were up-regulated in CTP, including apoptosis-related cathepsin D (CD), annexin A1 and heat shock protein 27 (HSP 27), and seven proteins were down-regulated in CTP, including prohibitin (PHB). The expression of PHB and CD was confirmed by Western blotting and immunofluorescence staining. The abnormal expression of PHB and CD correlated with the generation of reactive oxygen species, leading to decreased mitochondrial membrane potential and telomeric DNA, which are associated with cellular senescence and apoptosis. In summary, a specific pattern of abnormal protein expression is associated with the impaired development and functions of cloned placentas and hence with decreased fetal viability. Strategies aimed at restoring normal placental protein expression may increase the efficiency of somatic cell nuclear transfer and transgenic cat production and help restore endangered species.

  5. Comparative proteomic study of arsenic-induced differentially expressed proteins in rice roots reveals glutathione plays a central role during As stress.

    PubMed

    Ahsan, Nagib; Lee, Dong-Gi; Alam, Iftekhar; Kim, Pil Joo; Lee, Jeung Joo; Ahn, Young-Ock; Kwak, Sang-Soo; Lee, In-Jung; Bahk, Jeong Dong; Kang, Kyu Young; Renaut, Jenny; Komatsu, Setsuko; Lee, Byung-Hyun

    2008-09-01

    While the phytotoxic responses of arsenic (As) on plants have been studied extensively, based on physiological and biochemical aspects, very little is known about As stress-elicited changes in plants at the proteome level. Hydroponically grown 2-wk-old rice seedlings were exposed to different doses of arsenate, and roots were collected after 4 days of treatment, as well as after a recovery period. To gain a comprehensive understanding of the precise mechanisms underlying As toxicity, metabolism, and the defense reactions in plants, a comparative proteomic analysis of rice roots has been conducted in combination with physiological and biochemical analyses. Arsenic treatment resulted in increases of As accumulation, lipid peroxidation, and in vivo H(2)O(2) contents in roots. A total of 23 As-regulated proteins including predicted and novel ones were identified using 2-DE coupled with MS analyses. The expression levels of S-adenosylmethionine synthetase (SAMS), GSTs, cysteine synthase (CS), GST-tau, and tyrosine-specific protein phosphatase proteins (TSPP) were markedly up-regulated in response to arsenate, whereas treatment by H(2)O(2) also regulated the levels of CS suggesting that its expression was certainly regulated by As or As-induced oxidative stress. In addition, an omega domain containing GST was induced only by arsenate. However, it was not altered by treatment of arsenite, copper, or aluminum, suggesting that it may play a particular role in arsenate stress. Analysis of the total glutathione (GSH) content and enzymatic activity of glutathione reductase (GR) in rice roots during As stress revealed that their activities respond in a dose-dependent manner of As. These results suggest that SAMS, CS, GSTs, and GR presumably work synchronously wherein GSH plays a central role in protecting cells against As stress.

  6. Image analysis tools and emerging algorithms for expression proteomics

    PubMed Central

    English, Jane A.; Lisacek, Frederique; Morris, Jeffrey S.; Yang, Guang-Zhong; Dunn, Michael J.

    2012-01-01

    Since their origins in academic endeavours in the 1970s, computational analysis tools have matured into a number of established commercial packages that underpin research in expression proteomics. In this paper we describe the image analysis pipeline for the established 2-D Gel Electrophoresis (2-DE) technique of protein separation, and by first covering signal analysis for Mass Spectrometry (MS), we also explain the current image analysis workflow for the emerging high-throughput ‘shotgun’ proteomics platform of Liquid Chromatography coupled to MS (LC/MS). The bioinformatics challenges for both methods are illustrated and compared, whilst existing commercial and academic packages and their workflows are described from both a user’s and a technical perspective. Attention is given to the importance of sound statistical treatment of the resultant quantifications in the search for differential expression. Despite wide availability of proteomics software, a number of challenges have yet to be overcome regarding algorithm accuracy, objectivity and automation, generally due to deterministic spot-centric approaches that discard information early in the pipeline, propagating errors. We review recent advances in signal and image analysis algorithms in 2-DE, MS, LC/MS and Imaging MS. Particular attention is given to wavelet techniques, automated image-based alignment and differential analysis in 2-DE, Bayesian peak mixture models and functional mixed modelling in MS, and group-wise consensus alignment methods for LC/MS. PMID:21046614

  7. Melanogenesis stimulation in B16-F10 melanoma cells induces cell cycle alterations, increased ROS levels and a differential expression of proteins as revealed by proteomic analysis

    SciTech Connect

    Cunha, Elizabeth S.; Kawahara, Rebeca; Kadowaki, Marina K.; Amstalden, Hudson G.; Noleto, Guilhermina R.; Cadena, Silvia Maria S.C.; Winnischofer, Sheila M.B.; Martinez, Glaucia R.

    2012-09-10

    Considering that stimulation of melanogenesis may lead to alterations of cellular responses, besides melanin production, our main goal was to study the cellular effects of melanogenesis stimulation of B16-F10 melanoma cells. Our results show increased levels of the reactive oxygen species after 15 h of melanogenesis stimulation. Following 48 h of melanogenesis stimulation, proliferation was inhibited (by induction of cell cycle arrest in the G1 phase) and the expression levels of p21 mRNA were increased. In addition, melanogenesis stimulation did not induce cellular senescence. Proteomic analysis demonstrated the involvement of proteins from other pathways besides those related to the cell cycle, including protein disulfide isomerase A3, heat-shock protein 70, and fructose biphosphate aldolase A (all up-regulated), and lactate dehydrogenase (down-regulated). In RT-qPCR experiments, the levels of pyruvate kinase M2 mRNA dropped, whereas the levels of ATP synthase (beta-F1) mRNA increased. These data indicate that melanogenesis stimulation of B16-F10 cells leads to alterations in metabolism and cell cycle progression that may contribute to an induction of cell quiescence, which may provide a mechanism of resistance against cellular injury promoted by melanin synthesis. -- Highlights: Black-Right-Pointing-Pointer Melanogenesis stimulation by L-tyrosine+NH{sub 4}Cl in B16-F10 melanoma cells increases ROS levels. Black-Right-Pointing-Pointer Melanogenesis inhibits cell proliferation, and induced cell cycle arrest in the G1 phase. Black-Right-Pointing-Pointer Proteomic analysis showed alterations in proteins of the cell cycle and glucose metabolism. Black-Right-Pointing-Pointer RT-qPCR analysis confirmed alterations of metabolic targets after melanogenesis stimulation.

  8. Two-dimensional liquid chromatography protein expression mapping for differential proteomic analysis of normal and O157:H7 Escherichia coli.

    PubMed

    Zheng, Suping; Schneider, Kimberly A; Barder, Timothy J; Lubman, David M

    2003-12-01

    A multidimensional chromatographic method has been applied for the differential analysis of proteins from different strains of Escherichia coli bacteria. Proteins are separated in the first dimension using chromatofocusing (CF) and further separated by nonporous reversed-phase high-performance liquid chromatography (NPS-RP-HPLC) in the second dimension. A 2-dimensional (2-D) expression map of bacterial protein content is created for virulent O157:H7 and nonvirulent E. coli strains depicting protein isoelectric point (pI) versus protein hydrophobicity. Differentially expressed proteins are further characterized using electrospray/ionization time-of-flight mass spectrometry (ESI-TOF-MS) for intact protein molecular weight (MW) determination and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) peptide mass fingerprinting for protein identification. Using this method, no significant differential protein expression is exhibited between the two O157:H7 strains examined over a pH range of 4.0-7.0, and O157:H7 strains could be distinguished from nonvirulent E. coli. Several proteins differentially expressed between O157:H7 and nonvirulent E. coli are identified as potential markers for detection and treatment of O157:H7 infection.

  9. Eukaryotic expression: developments for structural proteomics.

    PubMed

    Aricescu, A R; Assenberg, R; Bill, R M; Busso, D; Chang, V T; Davis, S J; Dubrovsky, A; Gustafsson, L; Hedfalk, K; Heinemann, U; Jones, I M; Ksiazek, D; Lang, C; Maskos, K; Messerschmidt, A; Macieira, S; Peleg, Y; Perrakis, A; Poterszman, A; Schneider, G; Sixma, T K; Sussman, J L; Sutton, G; Tarboureich, N; Zeev-Ben-Mordehai, T; Jones, E Yvonne

    2006-10-01

    The production of sufficient quantities of protein is an essential prelude to a structure determination, but for many viral and human proteins this cannot be achieved using prokaryotic expression systems. Groups in the Structural Proteomics In Europe (SPINE) consortium have developed and implemented high-throughput (HTP) methodologies for cloning, expression screening and protein production in eukaryotic systems. Studies focused on three systems: yeast (Pichia pastoris and Saccharomyces cerevisiae), baculovirus-infected insect cells and transient expression in mammalian cells. Suitable vectors for HTP cloning are described and results from their use in expression screening and protein-production pipelines are reported. Strategies for co-expression, selenomethionine labelling (in all three eukaryotic systems) and control of glycosylation (for secreted proteins in mammalian cells) are assessed.

  10. Uncovering stem cell differentiation factors for salivary gland regeneration by quantitative analysis of differential proteomes

    PubMed Central

    Park, Yun-Jong; Koh, Jin; Kwon, Jin Teak; Park, Yong-Seok; Yang, Lijun; Cha, Seunghee

    2017-01-01

    Severe xerostomia (dry mouth) compromises the quality of life in patients with Sjögren’s syndrome or radiation therapy for head and neck cancer. A clinical management of xerostomia is often unsatisfactory as most interventions are palliative with limited efficacy. Following up our previous study demonstrating that mouse BM-MSCs are capable of differentiating into salivary epithelial cells in a co-culture system, we further explored the molecular basis that governs the MSC reprogramming by utilizing high-throughput iTRAQ-2D-LC-MS/MS-based proteomics. Our data revealed the novel induction of pancreas-specific transcription factor 1a (PTF1α), muscle, intestine and stomach expression-1 (MIST-1), and achaete-scute complex homolog 3 (ASCL3) in 7 day co-cultured MSCs but not in control MSCs. More importantly, a common notion of pancreatic-specific expression of PTF1 α was challenged for the first time by our verification of PTF1 α expression in the mouse salivary glands. Furthermore, a molecular network simulation of our selected putative MSC reprogramming factors demonstrated evidence for their perspective roles in salivary gland development. In conclusion, quantitative proteomics with extensive data analyses narrowed down a set of MSC reprograming factors potentially contributing to salivary gland regeneration. Identification of their differential/synergistic impact on MSC conversion warrants further investigation. PMID:28158262

  11. Uncovering stem cell differentiation factors for salivary gland regeneration by quantitative analysis of differential proteomes.

    PubMed

    Park, Yun-Jong; Koh, Jin; Kwon, Jin Teak; Park, Yong-Seok; Yang, Lijun; Cha, Seunghee

    2017-01-01

    Severe xerostomia (dry mouth) compromises the quality of life in patients with Sjögren's syndrome or radiation therapy for head and neck cancer. A clinical management of xerostomia is often unsatisfactory as most interventions are palliative with limited efficacy. Following up our previous study demonstrating that mouse BM-MSCs are capable of differentiating into salivary epithelial cells in a co-culture system, we further explored the molecular basis that governs the MSC reprogramming by utilizing high-throughput iTRAQ-2D-LC-MS/MS-based proteomics. Our data revealed the novel induction of pancreas-specific transcription factor 1a (PTF1α), muscle, intestine and stomach expression-1 (MIST-1), and achaete-scute complex homolog 3 (ASCL3) in 7 day co-cultured MSCs but not in control MSCs. More importantly, a common notion of pancreatic-specific expression of PTF1 α was challenged for the first time by our verification of PTF1 α expression in the mouse salivary glands. Furthermore, a molecular network simulation of our selected putative MSC reprogramming factors demonstrated evidence for their perspective roles in salivary gland development. In conclusion, quantitative proteomics with extensive data analyses narrowed down a set of MSC reprograming factors potentially contributing to salivary gland regeneration. Identification of their differential/synergistic impact on MSC conversion warrants further investigation.

  12. iTRAQ-MS/MS Proteomic Analysis Reveals Differentially Expressed Proteins During Post-harvest Maturation of the White Button Mushroom Agaricus bisporus.

    PubMed

    Chen, Meiyuan; Liao, Jianhua; Li, Hongrong; Cai, Zhixin; Guo, Zhongjie; Wach, Mark P; Wang, Zesheng

    2017-05-01

    Agaricus bisporus (J. E. Lange) Imbach, well known as the common cultivated mushroom or white button mushroom, is widely cultivated all over the world. We used iTRAQ-MS/MS (i.e., isobaric tags for relative and absolute quantification-coupled two-dimensional liquid chromatography-tandem mass spectrometry) to identify protein expression changes that occur during the post-harvest maturation of Agaricus bisporus fruitbodies at 0, 6, 12, and 48 h after harvest. A total of 5878 unique peptides, representing 1063 proteins, were identified. Quantitative data were obtained from 1012 out of the 1063 proteins. A total of 102, 106, and 160 differentially expressed proteins (>twofolds differences) were identified from samples collected at 6, 12, and 48 h compared to sample from 0 h post harvest, accounting for 10.1, 10.5, and 15.8% of the total proteins identified, respectively. All identified proteins including the differentially expressed proteins among different post-harvest stages were subjected to bioinformatic analysis. Furthermore, seven representative proteins either up or down-regulated at different post-harvest stages were analyzed by real-time PCR. Three out of the seven proteins, the mismatched base pair and cruciform DNA recognition protein, hydrophobin-B and protein transporter, exhibited similar expression patterns as their corresponding proteins. The 260 differentially expressed proteins identified from our study laid a foundation for future studies aiming to understand the post-harvest maturation process of A. bisporus and eventually, might help in the development of breeding program to identify strains with extended shelf life.

  13. 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

    2017-05-17

    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.

  14. Discovery and verification of gelsolin as a potential biomarker of colorectal adenocarcinoma in a Chinese population: Examining differential protein expression using an iTRAQ labelling-based proteomics approach

    PubMed Central

    Fan, Nai-Jun; Gao, Chun-Fang; Wang, Chang-Song; Lv, Jing-Jing; Zhao, Guang; Sheng, Xin-Hua; Wang, Xiu-Li; Li, Dong-Hui; Liu, Qing-Yin; Yin, Jian

    2012-01-01

    OBJECTIVE: To identify and validate potential biomarkers of colorectal adenocarcinoma using a proteomic approach. METHODS: Multidimensional liquid chromatography/mass spectrometry was used to analyze biological samples labelled with isobaric mass tags for relative and absolute quantitation to identify differentially expressed proteins in human colorectal adenocarcinoma and paired normal mucosa for the discovery of cancerous biomarkers. Cancerous and noncancerous samples were compared using online and offline separation. Protein identification was performed using mass spectrometry. The downregulation of gelsolin protein in colorectal adenocarcinoma samples was confirmed by Western blot analysis and validated using immunohistochemistry. RESULTS: A total of 802 nonredundant proteins were identified in colorectal adenocarcinoma samples, 82 of which fell outside the expression range of 0.8 to 1.2, and were considered to be potential cancer-specific proteins. Immunohistochemistry revealed a complete absence of gelsolin expression in 86.89% of samples and a reduction of expression in 13.11% of samples, yielding a sensitivity of 86.89% and a specificity of 100% for distinguishing colorectal adenocarcinoma from normal tissue. CONCLUSIONS: These findings suggest that decreased expression of gelsolin is a potential biomarker of colorectal adenocarcinoma. PMID:22288069

  15. Identification of proteins differentially expressed in adriamycin-resistant (pumc-91/ADM) and parental (pumc-91) human bladder cancer cell lines by proteome analysis.

    PubMed

    Meng, Qian; Lei, Ting; Zhang, Min; Zhao, Jin; Zhao, Xu-Hong; Zhang, Man

    2013-03-01

    Resistance to chemotherapy drugs remains a difficult problem in bladder cancer treatment. Protein expression is an important factor underlying multidrug resistance (MDR) in bladder cancer. The aim of the study was to explore differentially expressed proteins responsible for MDR between an adriamycin-resistant human bladder cancer cell line (pumc-91/ADM) and its parental cell line (pumc-91). Two-dimensional gel electrophoresis (2-DE) combining image analysis was used to screen the differentially expressed protein spots between the pumc-91/ADM and pumc-91 cell lines. Then, the protein spots were identified using MALDI-TOF/TOF mass spectrometry. Among the identified proteins, annexin A2 (ANXA2) and nucleophosmin (NPM1) were then further verified using RT-PCR and Western blot analysis. A total of 30 proteins, including 19 up-regulated and 11 down-regulated proteins, were successfully identified in pumc-91/ADM. According to their different functions, these 30 proteins were classified into 12 categories. Annexin A2 (ANXA2) and nucleophosmin (NPM1) were up-regulated in pumc-91/ADM compared with pumc-91. The proteins identified may have an important clinical significance in MDR, and ANXA2 and NPM1 may take part in mechanism of MDR in bladder cancer.

  16. Proteomic analysis identifies differentially expressed proteins in AML1/ETO acute myeloid leukemia cells treated with DNMT inhibitors azacitidine and decitabine.

    PubMed

    Buchi, Francesca; Spinelli, Elena; Masala, Erico; Gozzini, Antonella; Sanna, Alessandro; Bosi, Alberto; Ferrari, Germano; Santini, Valeria

    2012-05-01

    Azacitidine and decitabine are DNA methyltransferase inhibitors used to treat myelodysplastic syndromes and acute myeloid leukemias. To further characterize different mechanisms between these two agents, cellular extracts from leukemic cells untreated or treated with either drug were analyzed using 2D electrophoresis. Numerous differentially expressed proteins were identified with MALDI-TOF/TOF-MS. Cyclophilin A, Catalase, Nucleophosmin and PCNA were decreased exclusively by azacitidine, TCP1 and hnRNP A2/B1 by both drugs; alpha-Enolase and Peroxiredoxin-1 by decitabine. Interestingly, the expression of the proinflammatory protein Cyclophilin A, also suggested as marker of cell necrosis, was stimulated by decitabine. Finally, a comprehensive pathway analysis of data highlighted a relationship between the identified proteins and potential effectors.

  17. Technique appraisement of comparative proteomics and screening of differentiation-related protein in gastric carcinoma.

    PubMed

    Liu, Yu; Li, Yong; Tan, Bi-bo; Zhao, Qun; Fan, Li-qiao; Zhang, Zhi-dong; Li, Zhao-xing

    2013-05-01

    Different differentiations of cancer have resulted in its unique biological characteristics. We screen and appraise differentially expressed proteins in different differentiated gastric adenocarcinoma with comparative proteomics technology in order to find regulatory factors of tumor differentiation related and finally reach the purpose of tumor differentiation reversal. With two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) and liquid chromatography in conjunction with tandem mass spectrometry (LC–MS/MS), the differentially expressed proteins from 8 patients with different differentiated gastric adenocarcinoma were identified and some factors identified were verified with application of QPCR and Western blot techniques. Significant differences in 35 protein spots were found and 48 kinds of proteins were identified. Other than structural proteins and non-specific protein, six possible proteins associated with tumor differentiation were determined - the serine protease inhibitor B1 (serine protease inhibitor, clade B, member 1, SERPINB1), calcium-phospholipid binding protein III (annexin A3), transcription factor Nm23-H1, adenine phosphoribosyl-transferase enzyme APRT (Adenine Phosphoribosyltransferase in APO and AMP), glutathione S-transferase P1-1 (GST-π-1), antimicrobial peptides Dermcidin-lL. The identified SERPINB1, annexin A3, Nm23-H1 and APRT were verified, confirming the expression of these factors was in line with proteomics identification. Protein expression in different differentiated gastric adenocarcinoma was varied.

  18. Reproducibility of Differential Proteomic Technologies in CPTAC Fractionated Xenografts

    SciTech Connect

    Tabb, David L.; Wang, Xia; Carr, Steven A.; Clauser, Karl R.; Mertins, Philipp; Chambers, Matthew C.; Holman, Jerry D.; Wang, Jing; Zhang, Bing; Zimmerman, Lisa J.; Chen, Xian; Gunawardena, Harsha P.; Davies, Sherri R.; Ellis, Matthew J. C.; Li, Shunqiang; Townsend, R. Reid; Boja, Emily S.; Ketchum, Karen A.; Kinsinger, Christopher R.; Mesri, Mehdi; Rodriguez, Henry; Liu, Tao; Kim, Sangtae; McDermott, Jason E.; Payne, Samuel H.; Petyuk, Vladislav A.; Rodland, Karin D.; Smith, Richard D.; Yang, Feng; Chan, Daniel W.; Zhang, Bai; Zhang, Hui; Zhang, Zhen; Zhou, Jian-Ying; Liebler, Daniel C.

    2016-03-04

    The NCI Clinical Proteomic Tumor Analysis Consortium (CPTAC) employed a pair of reference xenograft proteomes for initial platform validation and ongoing quality control of its data collection for The Cancer Genome Atlas (TCGA) tumors. These two xenografts, representing basal and luminal-B human breast cancer, were fractionated and analyzed on six mass spectrometers in a total of 46 replicates divided between iTRAQ and label-free technologies, spanning a total of 1095 LC-MS/MS experiments. These data represent a unique opportunity to evaluate the stability of proteomic differentiation by mass spectrometry over many months of time for individual instruments or across instruments running dissimilar workflows. We evaluated iTRAQ reporter ions, label-free spectral counts, and label-free extracted ion chromatograms as strategies for data interpretation. From these assessments we found that differential genes from a single replicate were confirmed by other replicates on the same instrument from 61-93% of the time. When comparing across different instruments and quantitative technologies, differential genes were reproduced by other data sets from 67-99% of the time. Projecting gene differences to biological pathways and networks increased the similarities. These overlaps send an encouraging message about the maturity of technologies for proteomic differentiation.

  19. Reproducibility of Differential Proteomic Technologies in CPTAC Fractionated Xenografts

    PubMed Central

    2015-01-01

    The NCI Clinical Proteomic Tumor Analysis Consortium (CPTAC) employed a pair of reference xenograft proteomes for initial platform validation and ongoing quality control of its data collection for The Cancer Genome Atlas (TCGA) tumors. These two xenografts, representing basal and luminal-B human breast cancer, were fractionated and analyzed on six mass spectrometers in a total of 46 replicates divided between iTRAQ and label-free technologies, spanning a total of 1095 LC–MS/MS experiments. These data represent a unique opportunity to evaluate the stability of proteomic differentiation by mass spectrometry over many months of time for individual instruments or across instruments running dissimilar workflows. We evaluated iTRAQ reporter ions, label-free spectral counts, and label-free extracted ion chromatograms as strategies for data interpretation (source code is available from http://homepages.uc.edu/~wang2x7/Research.htm). From these assessments, we found that differential genes from a single replicate were confirmed by other replicates on the same instrument from 61 to 93% of the time. When comparing across different instruments and quantitative technologies, using multiple replicates, differential genes were reproduced by other data sets from 67 to 99% of the time. Projecting gene differences to biological pathways and networks increased the degree of similarity. These overlaps send an encouraging message about the maturity of technologies for proteomic differentiation. PMID:26653538

  20. Identification of differentially expressed proteins of Arthrospira (Spirulina) plantensis-YZ under salt-stress conditions by proteomics and qRT-PCR analysis

    PubMed Central

    2013-01-01

    Arthrospira (Spirulina) platensis as a representative species of cyanobacteria has been recognized and used worldwide as a source of protein in the food, which possesses some unusual and valuable physiological characteristics, such as alkali and salt tolerance. Based on complete genome sequencing of Arthrospira (Spirulina) plantensis-YZ, we compared the protein expression profiles of this organism under different salt-stress conditions (i.e. 0.02 M, 0.5 M and 1.0 M NaCl, respectively), using 2-D electrophoresis and peptide mass fingerprinting, and retrieved 141 proteins showing significantly differential expression in response to salt-stress. Of the 141 proteins, 114 Arthrospira (Spirulina) plantensis-YZ proteins were found with significant homology to those found in Arthrospira (76 proteins in Arthrospira platensis str. Paraca and 38 in Arthrospira maxima CS-328). The remaining 27 proteins belong to other bacteria. Subsequently, we determined the transcriptional level of 29 genes in vivo in response to NaCl treatments and verified them by qRT-PCR. We found that 12 genes keep consistency at both transcription and protein levels, and transcription of all of them but one were up-regulated. We classified the 141 differentially expressed proteins into 18 types of function categories using COG database, and linked them to their respective KEGG metabolism pathways. These proteins are involved in 31 metabolism pathways, such as photosynthesis, glucose metabolism, cysteine and methionine metabolism, lysine synthesis, fatty acid metabolism, glutathione metabolism. Additionally, the SRPs, heat shock protein and ABC transporter proteins were identified, which probably render Arthrospira (Spirulina) plantensis’s resistance against high salt stress. PMID:23363438

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-02-01

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

  3. Proteomic Analysis of Differential Expression of Cellular Proteins in Response to Avian H9N2 Virus Infection of A549 Cells

    PubMed Central

    Yu, Guanliu; Liang, Wei; Liu, Jiyuan; Meng, Dan; Wei, Liangmeng; Chai, Tongjie; Cai, Yumei

    2016-01-01

    In this study, differentially expressed proteins in A549 cells (human lung adenocarcinoma epithelial cell line) infected with H9N2 avian influenza virus (AIV) were investigated by two-dimensional electrophoresis (2-DE). Sixteen different spots between the groups (ratio > 2, p < 0.05) were identified with mass spectrometry identification. Proteins located in the downstream of the NF-κB and IFN transcription factor pathways were identified, e.g., ISG15. Actin and keratin were also identified, suggesting that the cytoskeleton may plays an important role in the AIV infection of mammalian cells. These findings could provide insights into the interaction between host and influenza viruses and might provide valuable information for clarifying the pathogenesis of viral infections as well. PMID:28018302

  4. [FUNCTIONAL DIFFERENTIATION IN BRYOZOAN COLONY: A PROTEOMIC ANALYSIS].

    PubMed

    Kutyumov, V A; Maltseva, A L; Kotenko, N; Ostrovsky, A N

    2016-01-01

    Bryozoans are typical modular organisms. They consist of repetitive structural units, the zooids. Bryozoan colonies grow by zooidal budding, with the distribution pattern of the budding loci underlying the diversity of colony forms. Budding is usually restricted to the zooids at the periphery of the colony, which form a "growing edge" or local terminal growth zones. Non-budding parts of the colony can be functionally subdivided, too. In many species colonies consists of regular, often repetitive zones of feeding and non-feeding modules, associated with a periodical degeneration and regeneration of the polypide, retractile tentacle crown with a gut and the accompanying musculature. So, there is functional differentiation in bryozoan colonies but its mechanisms are unknown. Presumably, budding and/or polypide recycling in different colony parts are induced or inhibited by certain determinants of functional specialization. An effective tool of their identification is the comparison of proteomes of functionally different zones. Here we report the results of proteomic analysis of three bryozoan species from the White Sea, which have a different colony form: Flustrellidra hispida, Terminoflustra membranaceotruncata and Securiflustra securifrons. Using differential two-dimensional electrophoresis (2D-DIGE), we compared proteomes of the growing edge and the zones consisting of feeding and non-feeding zooids in these species. We estimated the overall proteome variability, revealed proteins whose relative abundance gradually changed along the proximal-distal colony axis and suggested that they might be involved in the functional differentiation of the colony.

  5. Differential Secreted Proteome Approach in Murine Model for Candidate Biomarker Discovery in Colon Cancer

    PubMed Central

    Rangiah, Kannan; Tippornwong, Montri; Sangar, Vineet; Austin, David; Tétreault, Marie-Pier; Rustgi, Anil K.; Blair, Ian A.; Yu, Kenneth H.

    2009-01-01

    The complexity and heterogeneity of the plasma proteome have presented significant challenges in the identification of protein changes associated with tumor development. We used cell culture as a model system and identified differentially expressed, secreted proteins which may constitute serological biomarkers. A stable isotope labeling by amino acids in cell culture (SILAC) approach was used to label the entire secreted proteomes of the CT26 murine colon cancer cell line and normal young adult mouse colon (YAMC) cell line, thereby creating a stable isotope labeled proteome (SILAP) standard. This SILAP standard was added to unlabeled murine CT26 colon cancer cell or normal murine YAMC colon epithelial cell secreted proteome samples. A multidimensional approach combining isoelectric focusing (IEF), strong cation exchange (SCX) followed by reversed phase liquid chromatography was used for extensive protein and peptide separation. A total of 614 and 929 proteins were identified from the YAMC and CT26 cell lines, with 418 proteins common to both cell lines. Twenty highly abundant differentially expressed proteins from these groups were selected for liquid chromatography-multiple reaction monitoring/mass spectrometry (LC-MRM/MS) analysis in sera. Differential secretion into the serum was observed for several proteins when Apcmin mice were compared with control mice. These findings were then confirmed by Western blot analysis. PMID:19769411

  6. Beef quality with different intramuscular fat content and proteomic analysis using isobaric tag for relative and absolute quantitation of differentially expressed proteins.

    PubMed

    Mao, Yanwei; Hopkins, David L; Zhang, Yimin; Li, Peng; Zhu, Lixian; Dong, Pengcheng; Liang, Rongrong; Dai, Jin; Wang, Xiaoyun; Luo, Xin

    2016-08-01

    Intramuscular fat (IMF) is an important trait for beef eating quality. The mechanism of how IMF is deposited in beef cattle muscle is not clear at the molecular level. The muscle (M. longissimus lumborum: LL) of a group of Xiangxi yellow×Angus cattle with high fat levels (HF), was compared to the muscle of a low fat group (LF). The meat quality and the expressed protein patterns were compared. It was shown that LL from the HF animals had a greater fat content (P<0.05) and lower moisture content (P<0.05) than LL from LF animals. Forty seven sarcoplasmic proteins were differentially expressed and identified between the two groups. These proteins are involved in 6 molecular functions and 16 biological processes, and affect the Mitogen-activated protein kinases pathway, insulin pathway and c-Jun N-terminal kinases leading to greater IMF deposition. Cattle in the HF group had greater oxidative capacity and lower glycolytic levels suggesting a greater energetic efficiency. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Differential expression of prognostic proteomic markers in primary tumour, venous tumour thrombus and metastatic renal cell cancer tissue and correlation with patient outcome.

    PubMed

    Laird, Alexander; O'Mahony, Fiach C; Nanda, Jyoti; Riddick, Antony C P; O'Donnell, Marie; Harrison, David J; Stewart, Grant D

    2013-01-01

    Renal cell carcinoma (RCC) is the most deadly of urological malignancies. Metastatic disease affects one third of patients at diagnosis with a further third developing metastatic disease after extirpative surgery. Heterogeneity in the clinical course ensures predicting metastasis is notoriously difficult, despite the routine use of prognostic clinico-pathological parameters in risk stratification. With greater understanding of pathways involved in disease pathogenesis, a number of biomarkers have been shown to have prognostic significance, including Ki67, p53, vascular endothelial growth factor receptor 1 (VEGFR1) and ligand D (VEGFD), SNAIL and SLUG. Previous pathway analysis has been from study of the primary tumour, with little attention to the metastatic tumours which are the focus of targeted molecular therapies. As such, in this study a tissue microarray from 177 patients with primary renal tumour, renal vein tumour thrombus and/or RCC metastasis has been created and used with Automated Quantitative Analysis (AQUA) of immunofluorescence to study the prognostic significance of these markers in locally advanced and metastatic disease. Furthermore, this has allowed assessment of differential protein expression between the primary tumours, renal vein tumour thrombi and metastases. The results demonstrate that clinico-pathological parameters remain the most significant predictors of cancer specific survival; however, high VEGFR1 or VEGFD can predict poor cancer specific survival on univariate analysis for locally advanced and metastatic disease. There was significantly greater expression of Ki67, p53, VEGFR1, SLUG and SNAIL in the metastases compared with the primary tumours and renal vein tumour thrombi. With the exception of p53, these differences in protein expression have not been shown previously in RCC. This confirms the importance of proliferation, angiogenesis and epithelial to mesenchymal transition in the pathogenesis and metastasis of RCC. Importantly

  8. Proteomic analysis of human nasal mucosa: different expression profile in rhino-pathologic states.

    PubMed

    Gelardi, M; Siciliano, R A; Papa, F; Mazzeo, M F; De Nitto, E; Quaranta, N; Lippolis, R

    2014-09-01

    Rhinitis comprises several diseases with varying causes and different clinical manifestations and pathological features, but treated as a single clinical disorder. As heterogeneous disease, proper differential diagnosis is useful to delineate appropriate therapeutic intervention. Comparative proteomic investigation was aimed to provide information for specific differentially expressed proteins in rhino pathologic state, that could be used for diagnostic purpose and therapeutic monitoring. Proteins extracted from nasal mucosa cells of patients with different features of rhinitis and from control subjects, were separated by 2-DE. Proteins differentially expressed were identified by mass spectrometry (MS). Comparative proteomic analyses led to the identification of eighteen proteins differentially expressed in patients with rhinitis, mainly related to cell defense and innate and acquired immunity. From that, at least one protein can be a possible candidate as biomarker of disease.

  9. Proteome analysis reveals differential expression of proteins involved in triacylglycerol accumulation by Rhodococcus jostii RHA1 after addition of methyl viologen.

    PubMed

    Dávila Costa, José Sebastián; Silva, Roxana A; Leichert, Lars; Alvarez, Héctor M

    2017-03-01

    Rhodococcus jostii RHA1 is able to degrade toxic compounds and accumulate high amounts of triacylglycerols (TAG) upon nitrogen starvation. These NADPH-dependent processes are essential for the adaptation of rhodococci to fluctuating environmental conditions. In this study, we used an MS-based, label-free and quantitative proteomic approach to better understand the integral response of R. jostii RHA1 to the presence of methyl viologen (MV) in relation to the synthesis and accumulation of TAG. The addition of MV promoted a decrease of TAG accumulation in comparison to cells cultivated under nitrogen-limiting conditions in the absence of this pro-oxidant. Proteomic analyses revealed that the abundance of key proteins of fatty acid biosynthesis, the Kennedy pathway, glyceroneogenesis and methylmalonyl-CoA pathway, among others, decreased in the presence of MV. In contrast, some proteins involved in lipolysis and β-oxidation of fatty acids were upregulated. Some metabolic pathways linked to the synthesis of NADPH remained activated during oxidative stress as well as under nitrogen starvation conditions. Additionally, exposure to MV resulted in the activation of complete antioxidant machinery comprising superoxide dismutases, catalases, mycothiol biosynthesis, mycothione reductase and alkyl hydroperoxide reductases, among others. Our study suggests that oxidative stress response affects TAG accumulation under nitrogen-limiting conditions through programmed molecular mechanisms when both stresses occur simultaneously.

  10. Temporal profiling of the adipocyte proteome during differentiation using a 5-plex SILAC based strategy

    PubMed Central

    Ruch, Travis; Kim, Jae-Woo; Mortensen, Peter; Otto, Tamara; Nalli, Anuradha; Tang, Qi-Qun; Lane, M. Daniel; Chaerkady, Raghothama; Pandey, Akhilesh

    2008-01-01

    The adipose tissue has important secretory and endocrine functions in humans. The regulation of adipocyte differentiation has been actively pursued using transcriptomic methods over the last several years. Quantitative proteomics has emerged as a promising approach to obtain temporal profiles of biological processes such as differentiation. Stable isotope labeling with amino acids in cell culture (SILAC) is a simple and robust method for labeling proteins in vivo. Here, we describe the development and application of a five-plex SILAC experiment using four different heavy stable isotopic forms of arginine to study the nuclear proteome and the secretome during the course of adipocyte differentiation. Tandem mass spectrometry analysis using a quadrupole time-of-flight instrument resulted in identification of a total 882 proteins from these two proteomes. Of these proteins, 427 were identified on the basis of one or more arginine containing peptides that allowed quantitation. In addition to previously reported molecules that are differentially expressed during the process of adipogenesis (e.g. adiponectin and lipoprotein lipase), we identified several proteins whose differential expression during adipocyte differentiation has not been documented previously. For example, THO complex 4, a context-dependent transcriptional activator in the T-cell receptor alpha enhancer complex, showed highest expression at middle stage of adipogenesis while SNF2 alpha, a chromatin remodeling protein, was downregulated upon initiation of adipogenesis and remained so during subsequent time points. This study using a 5-plex SILAC to investigate dynamics illustrates the power of this approach to identify differentially expressed proteins in a temporal fashion. PMID:18947249

  11. 2D electrophoresis-based expression proteomics: a microbiologist's perspective.

    PubMed

    Sá-Correia, Isabel; Teixeira, Miguel C

    2010-12-01

    Quantitative proteomics based on 2D electrophoresis (2-DE) coupled with peptide mass fingerprinting is still one of the most widely used quantitative proteomics approaches in microbiology research. Our view on the exploitation of this global expression analysis technique and its contribution and potential to push forward the field of molecular microbial physiology towards a molecular systems microbiology perspective is discussed in this article. The advances registered in 2-DE-based quantitative proteomic analysis leading to increased protein resolution, sensitivity and accuracy, and the promising use of 2-DE to gain insights into post-translational modifications at a proteome-wide level (considering all the proteins/protein forms expressed by the genome) are focused on. Given the progress made in this field, it is foreseen that the 2-DE-based approach to quantitative proteomics will continue to be a fundamental tool for microbiologists working at a genome-wide scale. Guidelines are also provided for the exploitation of expression proteomics data, based on useful computational tools, and for the integration of these data with other genome-wide expression information. The advantages and limitations of a complete 2-DE-based expression proteomics analysis, envisaging the quantification of the global changes occurring in the proteome of a given cell depending on environmental or genetic manipulations, are discussed from the microbiologist's perspective. Particular focus is given to the emerging field of toxicoproteomics, a new systems toxicity approach that offers a powerful tool to directly monitor the earliest stages of the toxicological response by identifying critical proteins and pathways that are affected by, and respond to, a chemical stress. The experimental design and the bioinformatics analysis of data used in our laboratory to gain mechanistic insights through expression proteomics into the responses of the eukaryotic model Saccharomyces cerevisiae or of

  12. Differential proteomics analysis of Bacillus amyloliquefaciens and its genome-shuffled mutant for improving surfactin production.

    PubMed

    Zhao, Junfeng; Cao, Lin; Zhang, Chong; Zhong, Lei; Lu, Jing; Lu, Zhaoxin

    2014-10-31

    Genome shuffling technology was used as a novel whole-genome engineering approach to rapidly improve the antimicrobial lipopeptide yield of Bacillus amyloliquefaciens. Comparative proteomic analysis of the parental ES-2-4 and genome-shuffled FMB38 strains was conducted to examine the differentially expressed proteins. The proteome was separated by 2-DE (two dimensional electrophoresis) and analyzed by MS (mass spectrum). In the shuffled strain FMB38, 51 differentially expressed protein spots with higher than two-fold spot density were detected by gel image comparison. Forty-six protein spots were detectable by silver staining and further MS analysis. The results demonstrated that among the 46 protein spots expressed particularly induced in the genome-shuffled mutant, 15 were related to metabolism, five to DNA replication, recombination and repair, six to translation and post-translational modifications, one to cell secretion and signal transduction mechanisms, three to surfactin synthesis, two to energy production and conversion, and 14 to others. All these indicated that the metabolic capability of the mutant was improved by the genome shuffling. The study will enable future detailed investigation of gene expression and function linked with surfactin synthesis. The results of proteome analysis may provide information for metabolic engineering of Bacillus amyloliquefaciens for overproduction of surfactin.

  13. Differential alkylation-based redox proteomics--Lessons learnt.

    PubMed

    Wojdyla, Katarzyna; Rogowska-Wrzesinska, Adelina

    2015-12-01

    Cysteine is one of the most reactive amino acids. This is due to the electronegativity of sulphur atom in the side chain of thiolate group. It results in cysteine being present in several distinct redox forms inside the cell. Amongst these, reversible oxidations, S-nitrosylation and S-sulfenylation are crucial mediators of intracellular redox signalling, with known associations to health and disease. Study of their functionalities has intensified thanks to the development of various analytical strategies, with particular contribution from differential alkylation-based proteomics methods. Presented here is a critical evaluation of differential alkylation-based strategies for the analysis of S-nitrosylation and S-sulfenylation. The aim is to assess the current status and to provide insights for future directions in the dynamically evolving field of redox proteomics. To achieve that we collected 35 original research articles published since 2010 and analysed them considering the following parameters, (i) resolution of modification site, (ii) quantitative information, including correction of modification levels by protein abundance changes and determination of modification site occupancy, (iii) throughput, including the amount of starting material required for analysis. The results of this meta-analysis are the core of this review, complemented by issues related to biological models and sample preparation in redox proteomics, including conditions for free thiol blocking and labelling of target cysteine oxoforms.

  14. Quantitative temporal proteomic analysis of human embryonic stem cell differentiation into oligodendrocyte progenitor cells

    PubMed Central

    Chaerkady, Raghothama; Letzen, Brian; Renuse, Santosh; Sahasrabuddhe, Nandini A.; Kumar, Praveen; All, Angelo H.; Thakor, Nitish V.; Delanghe, Bernard; Gearhart, John D.; Pandey, Akhilesh; Kerr, Candace L.

    2013-01-01

    Oligodendrocytes (OLs) are glial cells of the central nervous system which produce myelin. Cultured OLs provide immense therapeutic opportunities for treating a variety of neurological conditions. One of the most promising sources for such therapies is human embryonic stem cells (ESCs), as well as providing a model to study human oligodendrocyte development. For these purposes, an investigation of proteome level changes is critical for understanding the process of OL differentiation. In this report, an iTRAQ-based quantitative proteomic approach was used to study multiple steps during oligodendrocyte differentiation including neural precursors (NPCs), glial precursors (GPCs), and oligodendrocyte progenitors (OPCs) compared to undifferentiated embryonic stem cells. Using a 1% false discovery rate cutoff, ~3,145 proteins were quantitated and several demonstrated progressive stage-specific expression. Proteins such as TF, NCAM1, APOE, and WNT5A showed increased expression from the NPC to OPC stage. Several proteins that have demonstrated evidence or been suspected in OL maturation were also found upregulated in OPCs including FABP4, THBS1, BMP1, CRYAB, TF, TNC, COL3A1, TGFBI and EPB41L3. Thus, by providing the first extensive proteomic profiling of human embryonic stem cell differentiation into oligodendrocyte progenitor cells, this study provides many novel proteins that are potentially involved in OL development. PMID:21770034

  15. Proteomic Profile in Glomeruli of Type-2 Diabetic KKAy Mice using 2-Dimensional Differential Gel Electrophoresis

    PubMed Central

    Liu, Xiaodan; Yang, Gang; Fan, Qiuling; Wang, Lining

    2014-01-01

    Background Diabetic nephropathy (DN) is a leading cause of end-stage renal disease. To search for glomerular proteins associated with early-stage DN, glomeruli of spontaneous type 2 diabetic KKAy mice were analyzed by 2-dimensional differential gel electrophoresis (2D-DIGE). Material/Methods Glomeruli of 20-week spontaneous type 2 diabetic KKAy mice and age-matched C57BL/6 mice were isolated by kidney perfusion with magnetic beads. Proteomic profiles of glomeruli were investigated by using 2D-DIGE and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Western blot analysis was used to confirm the results of proteomics. Immunohistochemical and semi-quantitative analysis were used to confirm the differential expression of prohibitin and annexin A2 in glomeruli. Results We identified 19 differentially expressed proteins – 17 proteins were significantly up-regulated and 2 proteins were significantly down-regulated in glomeruli of diabetic KKAy mice. Among them, prohibitin and annexin A2 were up-regulated and Western blot analysis validated the same result in proteomics. Immunohistochemical analysis also revealed up-regulation of prohibitin and annexin A2 in glomeruli of KKAy mice. Conclusions Our findings suggest that prohibitin and annexin A2 may be associated with early-stage DN. Further functional research might help to reveal the pathogenesis of DN. PMID:25515740

  16. Proteomic analysis of nipple aspirate fluid from women with early-stage breast cancer using isotope-coded affinity tags and tandem mass spectrometry reveals differential expression of vitamin D binding protein

    PubMed Central

    Pawlik, Timothy M; Hawke, David H; Liu, Yanna; Krishnamurthy, Savitri; Fritsche, Herbert; Hunt, Kelly K; Kuerer, Henry M

    2006-01-01

    Background Isotope-coded affinity tag (ICAT) tandem mass spectrometry (MS) allows for qualitative and quantitative analysis of paired protein samples. We sought to determine whether ICAT technology could quantify and identify differential expression of tumor-specific proteins in nipple aspirate fluid (NAF) from the tumor-bearing and contralateral disease-free breasts of patients with unilateral early-stage breast cancer. Methods Paired NAF samples from 18 women with stage I or II unilateral invasive breast carcinoma and 4 healthy volunteers were analyzed using ICAT labeling, sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), liquid chromatography, and MS. Proteins were identified by sequence database analysis. Western blot analysis of NAF from an independent sample set from 12 women (8 with early-stage breast cancer and 4 healthy volunteers) was also performed. Results 353 peptides were identified from tandem mass spectra and matched to peptide sequences in the National Center for Biotechnology Information database. Equal numbers of peptides were up- versus down-regulated. Alpha2HS-glycoprotein [Heavy:Light (H:L) ratio 0.63] was underexpressed in NAF from tumor-bearing breasts, while lipophilin B (H:L ratio 1.42), beta-globin (H:L ratio 1.98), hemopexin (H:L ratio 1.73), and vitamin D-binding protein precursor (H:L ratio 1.82) were overexpressed. Western blot analysis of pooled samples of NAF from healthy volunteers versus NAF from women with breast cancer confirmed the overexpression of vitamin D-binding protein in tumor-bearing breasts. Conclusion ICAT tandem MS was able to identify and quantify differences in specific protein expression between NAF samples from tumor-bearing and disease-free breasts. Proteomic screening techniques using ICAT and NAF may be used to find markers for diagnosis of breast cancer. PMID:16542425

  17. Differential proteomics analysis of mononuclear cells in cerebrospinal fluid of Parkinson's disease.

    PubMed

    Xing, Lifei; Wang, Dongtao; Wang, Lihong; Lan, Wenjie; Pan, Suyue

    2015-01-01

    Parkinson's disease (PD) is one common neurodegenerative disease featured with degeneration of dopaminergic neurons in substantia nigra. Multiple factors participate in the pathogenesis and progression of PD. In this study, we investigated the proteomics profiles of mononuclear cells in cerebrospinal fluids from both PD patients and normal people, in order to explore the correlation between disease factors and PD. Cerebrospinal fluid samples were collected from both PD and normal people and were separated for mononuclear cells in vitro. Proteins were then extracted and separated by 2-dimensional gel electrophoresis. Proteins with differential expressions were identified by comparison to standard proteome expression profile map, followed by software and database analysis. In PD patients, there were 8 proteins with consistent expression profile and 16 proteins with differential expressions. Those differential proteins identified include cytoskeleton proteins (actin, myosin), signal transduction proteins (adenosine cyclase binding protein 1, calcium binding protein, talin) and anti-oxidation factor (thioredoxin peroxide reductase). PD patients had differential protein expressional profiles in the mononuclear cells of cerebrospinal fluids compared to normal people, suggesting the potential involvement of cytoskeleton and signal transduction proteins in apoptosis of neuronal apoptosis and PD pathogenesis.

  18. Proteome Dynamics during Plastid Differentiation in Rice1[W

    PubMed Central

    Kleffmann, Torsten; von Zychlinski, Anne; Russenberger, Doris; Hirsch-Hoffmann, Matthias; Gehrig, Peter; Gruissem, Wilhelm; Baginsky, Sacha

    2007-01-01

    We have analyzed proteome dynamics during light-induced development of rice (Oryza sativa) chloroplasts from etioplasts using quantitative two-dimensional gel electrophoresis and tandem mass spectrometry protein identification. In the dark, the etioplast allocates the main proportion of total protein mass to carbohydrate and amino acid metabolism and a surprisingly high number of proteins to the regulation and expression of plastid genes. Chaperones, proteins for photosynthetic energy metabolism, and enzymes of the tetrapyrrole pathway were identified among the most abundant etioplast proteins. The detection of 13 N-terminal acetylated peptides allowed us to map the exact localization of the transit peptide cleavage site, demonstrating good agreement with the prediction for most proteins. Based on the quantitative etioplast proteome map, we examined early light-induced changes during chloroplast development. The transition from heterotrophic metabolism to photosynthesis-supported autotrophic metabolism was already detectable 2 h after illumination and affected most essential metabolic modules. Enzymes in carbohydrate metabolism, photosynthesis, and gene expression were up-regulated, whereas enzymes in amino acid and fatty acid metabolism were significantly decreased in relative abundance. Enzymes involved in nucleotide metabolism, tetrapyrrole biosynthesis, and redox regulation remained unchanged. Phosphoprotein-specific staining at different time points during chloroplast development revealed light-induced phosphorylation of a nuclear-encoded plastid RNA-binding protein, consistent with changes in plastid RNA metabolism. Quantitative information about all identified proteins and their regulation by light is available in plprot, the plastid proteome database (http://www.plprot.ethz.ch). PMID:17189339

  19. Dissecting the Escherichia coli periplasmic chaperone network using differential proteomics

    PubMed Central

    Vertommen, Didier; Silhavy, Thomas J.; Collet, Jean-Francois

    2013-01-01

    β-barrel proteins, or outer membrane proteins (OMPs), perform many essential functions in Gram-negative bacteria, but questions remain about the mechanism by which they are assembled into the outer membrane (OM). In Escherichia coli, β-barrels are escorted across the periplasm by chaperones, most notably SurA and Skp. However, the contributions of these two chaperones to the assembly of the OM proteome remained unclear. We used differential proteomics to determine how the elimination of Skp and SurA affects the assembly of many OMPs. We have shown that removal of Skp has no impact on the levels of the 63 identified OM proteins. However, depletion of SurA in the skp strain has a marked impact on the OM proteome, diminishing the levels of almost all β-barrel proteins. Our results are consistent with a model in which SurA plays a primary chaperone role in E. coli. Furthermore, they suggest that while no OMPs prefer the Skp chaperone pathway in wild-type cells, most can use Skp efficiently when SurA is absent. Our data, which provide a unique glimpse into the protein content of the non-viable surA skp mutant, clarify the roles of the periplasmic chaperones in E. coli. PMID:22589188

  20. Soybean proteome database: a data resource for plant differential omics.

    PubMed

    Sakata, Katsumi; Ohyanagi, Hajime; Nobori, Hiroya; Nakamura, Takuji; Hashiguchi, Akiko; Nanjo, Yohei; Mikami, Yoji; Yunokawa, Harunobu; Komatsu, Setsuko

    2009-07-01

    The Soybean Proteome Database aims to be a data repository for functional analyses of soybean responses to flooding injury, recognized as a major constraint for establishment and production of this plant. The current release contains 21 reference maps of soybean (Glycine max cv. Enrei) proteins electrophoresed on two-dimensional polyacrylamide gels of which the samples were collected from several organs, tissues and organelles. These reference maps include 7311 detected proteins and 532 identified proteins, or proteins for which a sequence or peptide peak has been determined. The database is searchable by protein properties such as accession number, description and isoelectric point and molecular weight range. The Soybean Proteome Database also integrates multiple "omes". An omics table reveals relationships among 106 mRNAs, 51 proteins and 89 metabolites that vary over time under flooding stress. The tabulated metabolites are anchored to a metabolome network. A unified temporal-profile tag attached to the mRNAs, proteins and metabolites facilitates retrieval of the data based on the temporal expression profiles. A graphical user interface based on dynamic HTML facilitates viewing the metabolome network as well as the profiles of multiple omes in a uniform manner. The entire database is available at http://proteome.dc.affrc.go.jp/Soybean/.

  1. Differential Expression Analysis for Pathways

    PubMed Central

    Haynes, Winston A.; Higdon, Roger; Stanberry, Larissa; Collins, Dwayne; Kolker, Eugene

    2013-01-01

    Life science technologies generate a deluge of data that hold the keys to unlocking the secrets of important biological functions and disease mechanisms. We present DEAP, Differential Expression Analysis for Pathways, which capitalizes on information about biological pathways to identify important regulatory patterns from differential expression data. DEAP makes significant improvements over existing approaches by including information about pathway structure and discovering the most differentially expressed portion of the pathway. On simulated data, DEAP significantly outperformed traditional methods: with high differential expression, DEAP increased power by two orders of magnitude; with very low differential expression, DEAP doubled the power. DEAP performance was illustrated on two different gene and protein expression studies. DEAP discovered fourteen important pathways related to chronic obstructive pulmonary disease and interferon treatment that existing approaches omitted. On the interferon study, DEAP guided focus towards a four protein path within the 26 protein Notch signalling pathway. PMID:23516350

  2. Differential proteomics of Picrorhiza kurrooa Royle ex Benth. in response to dark stress.

    PubMed

    Parkash, Jai; Kashyap, Sanjeeta; Kalita, Prakash Jyoti; Devi, Manjula; Ahuja, Paramvir Singh; Dutt, Som

    2014-09-01

    Picrorhiza (Picrorhiza kurrooa Royle ex Benth.) an important medicinal herb of western Himalayan region has been used to treat various diseases and disorders. Over-harvesting and lack of cultivation has led to its entry in Red Data Book as an endangered species. Further, its very restrictive habitat and lesser biomass production are major limitations for bringing it under commercial cultivation. All these issues necessitate deeper insights into mechanisms governing its growth and interaction with the environmental cues. Light may be one of the important factors to be studied for its role in regulating growth and adaptation of Picrorhiza as in natural habitat it prefers shady niches. Keeping this in view, proteome of Picrorhiza kept under light vis-à-vis under dark was analysed and compared. Leaf as well as root proteome of Picrorhiza was studied. Denaturing two dimensional gel electrophoresis and mass spectrometry techniques were used to detect and identify differentially expressed proteins, respectively. Twenty two proteins from leaf and 25 proteins from root showed differential expression levels under dark and light conditions. Among the differentially expressed proteins, majority were those involved in metabolism, protein synthesis, and stress and defense response. Other differentially expressed proteins were those involved in photosynthetic process, photorespiration and few proteins were with unknown function indicating that many different processes work together to establish a new cellular homeostasis in response to dark and light conditions. Proteins found to be differentially expressed under light vis-à-vis dark conditions suggested a range of biochemical pathways and processes being associated with response of plant to dark conditions. The identified proteins may be utilized for developing strategies for improving the biomass production/performance of Picrorhiza under varied light/dark habitats.

  3. Identification of putative fertility markers in seminal plasma of crossbred bulls through differential proteomics.

    PubMed

    Aslam, M K Muhammad; Kumaresan, A; Sharma, Vinay K; Tajmul, Md; Chhillar, Shivani; Chakravarty, A K; Manimaran, A; Mohanty, T K; Srinivasan, A; Yadav, Savita

    2014-12-01

    Sub-fertility is a major problem in crossbred bulls leading to disintegration of breeding systems and huge economic loss. Identification of some potential biomarkers to determine the latent fertility of bulls accurately has long been the interest of researchers. In this study, we analyzed the proteome of seminal plasma (SP) from bulls with varying fertility to identify the fertility-associated proteins. The proteomic profile of high- and low-fertile bulls was compared by two-dimensional difference gel electrophoresis and differentially expressed proteins were identified through matrix-assisted laser desorption/ionization-time of flight/mass spectrometry. Out of the 18 differentially expressed proteins (P < 0.05), 9 were overexpressed in SP of high-fertile bulls and 9 were overexpressed in SP of low-fertile bulls. The differential expressions ranged from 1.5- to 5.5-fold between the two groups, where protection of telomeres-1 protein (POT1) was highly overexpressed (2.9-fold) in high-fertile group and prostaglandin E2 receptor EP3 (PTGER3) was highly abundant (5.5-fold) in low-fertile group. The protein interaction network was elucidated using STRING software tool, and the functional bioinformatics analysis was done using Blast2Go software. Most of the differentially expressed proteins were found to be involved in cellular processes and biological regulation with binding and catalytic function. It is inferred that the expression of certain proteins in the SP varied with bull fertility, and concurrent appraisal of their expression along with other fertility assays may help in determining bull fertility. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Proteomic analysis of MG132-treated germinating pollen reveals expression signatures associated with proteasome inhibition.

    PubMed

    Vannini, Candida; Bracale, Marcella; Crinelli, Rita; Marconi, Valerio; Campomenosi, Paola; Marsoni, Milena; Scoccianti, Valeria

    2014-01-01

    Chemical inhibition of the proteasome has been previously found to effectively impair pollen germination and tube growth in vitro. However, the mediators of these effects at the molecular level are unknown. By performing 2DE proteomic analysis, 24 differentially expressed protein spots, representing 14 unique candidate proteins, were identified in the pollen of kiwifruit (Actinidia deliciosa) germinated in the presence of the MG132 proteasome inhibitor. qPCR analysis revealed that 11 of these proteins are not up-regulated at the mRNA level, but are most likely stabilized by proteasome inhibition. These differentially expressed proteins are predicted to function in various pathways including energy and lipid metabolism, cell wall synthesis, protein synthesis/degradation and stress responses. In line with this evidence, the MG132-induced changes in the proteome were accompanied by an increase in ATP and ROS content and by an alteration in fatty acid composition.

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

    PubMed Central

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

    2015-01-01

    Finding the minimal set of gene functions needed to sustain life is of both fundamental and practical importance. Minimal gene lists have been proposed by using comparative genomics-based core proteome definitions. A definition of a core proteome that is supported by empirical data, is understood at the systems-level, and provides a basis for computing essential cell functions is lacking. Here, we use a systems biology-based genome-scale model of metabolism and expression to define a functional core proteome consisting of 356 gene products, accounting for 44% of the Escherichia coli proteome by mass based on proteomics data. This systems biology core proteome includes 212 genes not found in previous comparative genomics-based core proteome definitions, accounts for 65% of known essential genes in E. coli, and has 78% gene function overlap with minimal genomes (Buchnera aphidicola and Mycoplasma genitalium). Based on transcriptomics data across environmental and genetic backgrounds, the systems biology core proteome is significantly enriched in nondifferentially expressed genes and depleted in differentially expressed genes. Compared with the noncore, core gene expression levels are also similar across genetic backgrounds (two times higher Spearman rank correlation) and exhibit significantly more complex transcriptional and posttranscriptional regulatory features (40% more transcription start sites per gene, 22% longer 5′UTR). Thus, genome-scale systems biology approaches rigorously identify a functional core proteome needed to support growth. This framework, validated by using high-throughput datasets, facilitates a mechanistic understanding of systems-level core proteome function through in silico models; it de facto defines a paleome. PMID:26261351

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

    PubMed

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

    2015-08-25

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

  7. Dissecting Escherichia coli Outer Membrane Biogenesis Using Differential Proteomics

    PubMed Central

    Martorana, Alessandra M.; Motta, Sara; Di Silvestre, Dario; Falchi, Federica; Dehò, Gianni; Mauri, Pierluigi; Sperandeo, Paola; Polissi, Alessandra

    2014-01-01

    The cell envelope of Gram-negative bacteria is a complex multi-layered structure comprising an inner cytoplasmic membrane and an additional asymmetric lipid bilayer, the outer membrane, which functions as a selective permeability barrier and is essential for viability. Lipopolysaccharide, an essential glycolipid located in the outer leaflet of the outer membrane, greatly contributes to the peculiar properties exhibited by the outer membrane. This complex molecule is transported to the cell surface by a molecular machine composed of seven essential proteins LptABCDEFG that form a transenvelope complex and function as a single device. While advances in understanding the mechanisms that govern the biogenesis of the cell envelope have been recently made, only few studies are available on how bacterial cells respond to severe envelope biogenesis defects on a global scale. Here we report the use of differential proteomics based on Multidimensional Protein Identification Technology (MudPIT) to investigate how Escherichia coli cells respond to a block of lipopolysaccharide transport to the outer membrane. We analysed the envelope proteome of a lptC conditional mutant grown under permissive and non permissive conditions and identified 123 proteins whose level is modulated upon LptC depletion. Most such proteins belong to pathways implicated in cell envelope biogenesis, peptidoglycan remodelling, cell division and protein folding. Overall these data contribute to our understanding on how E. coli cells respond to LPS transport defects to restore outer membrane functionality. PMID:24967819

  8. Differential proteomic screening and identification for non-traumatic necrotic femoral osseous tissue.

    PubMed

    Yan, Peng; Zhu, Yeping; Zhao, Hui; Lu, Yanyan; Gao, Yuzhong

    2017-06-01

    Currently, there is a lack of effective early screening and detection methods for femoral head necrosis. Current research on most orthopedic diseases focuses on proteomics in the preliminary stage. The recent fluorescence differential in gel electrophoresis (DIGE) has advantages such as a high reproducibility, high sensitivity, high throughput, and high dynamic range. It is currently one of the most widely used quantitative proteomic research means. We conducted this study to investigate the pathogenesis of non-traumatic femoral head necrosis using the fluorescence DIGE to screen non-traumatic femoral head necrosis based on proteomics and provide a theoretical basis for screening possible biomarkers and molecular targeted treatment. The DIGE technique was used to separate the protein. An electrophoretogram was established on the basis of scanning and analysis. Identification and a bioinformatics analysis were conducted for the differential protein. The protein with differential expression of over 2-fold was excavated and ionized by means of substrate assisted laser desorption. The flight time was identified with a mass spectrometer (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, MALDI-TOF/TOF). The formation on sequences, structures and functions of these proteins were obtained through database retrieval. Western blot analysis was used to verify the differential protein expression and the reliability of the DIGE result was verified. DIGE was used to successfully separate 1,500±40 protein spots. There were 252 significant differential protein spots. The Ettan™ Spot Picker automatic work station was used to excavate 49 significant differential protein spots with expression difference over 2-fold. The MALDI-TOF/TOF mass spectrometer was used to identify these differential protein spots. Six proteins were identified in total, which include apolipoprotein A1 (APOA1), fibrous protein original chain, fibrous protein original chain, serum

  9. Differential Proteomics Analysis of Colonic Tissues in Patients of Slow Transit Constipation

    PubMed Central

    Wan, Songlin; Liu, Weicheng; Tian, Cuiping; Ren, Xianghai; Ding, Zhao; Qian, Qun; Jiang, Congqing; Wu, Yunhua

    2016-01-01

    Objective. To investigate and screen the different expression of proteins in STC and normal group with a comparative proteomic approach. Methods. Two-dimensional electrophoresis was applied to separate the proteins in specimens from both 5 STC patients and 5 normal controls. The proteins with statistically significant differential expression between two groups were identified by computer aided image analysis and matrix assisted laser desorption ionization tandem time of flight mass spectrometry (MALDI-TOF-MS). Results. A total of 239 protein spots were identified in the average gel of the normal control and 215 in patients with STC. A total of 197 protein spots were matched and the mean matching rate was 82%. There were 14 protein spots which were expressed with statistically significant differences from others. Of those 14 protein spots, the expression of 12 spots increased markedly, while that of 2 spots decreased significantly. Conclusion. The proteomics expression in colonic specimens of STC patients is statistically significantly different from that of normal control, which may be associated with the pathogenesis of STC. PMID:27239471

  10. Impact of replicate types on proteomic expression analysis.

    PubMed

    Karp, Natasha A; Spencer, Matthew; Lindsay, Helen; O'Dell, Kevin; Lilley, Kathryn S

    2005-01-01

    In expression proteomics, the samples utilized within an experimental design may include technical, biological, or pooled replicates. This manuscript discusses various experimental designs and the conclusions that can be drawn from them. Specifically, it addresses the impact of mixing replicate types on the statistical analysis which can be performed. This study focuses on difference gel electrophoresis (DiGE), but the issues are equally applicable to all quantitative methodologies assessing relative changes in protein expression.

  11. Comparative Proteomics for the Evaluation of Protein Expression and Modifications in Neurodegenerative Diseases.

    PubMed

    Conti, Antonio; Alessio, Massimo

    2015-01-01

    Together with hypothesis-driven approaches, high-throughput differential proteomic analysis performed primarily not only in human cerebrospinal fluid and serum but also on protein content of other tissues (blood cells, muscles, peripheral nerves, etc.) has been used in the last years to investigate neurodegenerative diseases. Even if the goal for these analyses was mainly the discovery of neurodegenerative disorders biomarkers, the characterization of specific posttranslational modifications (PTMs) and the differential protein expression resulted in being very informative to better define the pathological mechanisms. In this chapter are presented and discussed the positive aspects and challenges of the outcomes of some of our investigations on neurological and neurodegenerative disease, in order to highlight the important role of protein PTMs studies in proteomics-based approaches.

  12. Exploration of Serum Proteomic Profiling and Diagnostic Model That Differentiate Crohn's Disease and Intestinal Tuberculosis

    PubMed Central

    Zhang, Fenming; Xu, Chengfu; Ning, Longgui; Hu, Fengling; Shan, Guodong; Chen, Hongtan; Yang, Ming; Chen, Wenguo; Yu, Jiekai; Xu, Guoqiang

    2016-01-01

    Aim To explore the diagnostic models of Crohn’s disease (CD), Intestinal tuberculosis (ITB) and the differential diagnostic model between CD and ITB by analyzing serum proteome profiles. Methods Serum proteome profiles from 30 CD patients, 21 ITB patients and 30 healthy controls (HCs) were analyzed by using weak cationic magnetic beads combined with MALDI-TOF-MS technique to detect the differentially expressed proteins of serum samples. Three groups were made and compared accordingly: group of CD patients and HCs, group of ITB patients and HCs, group of CD patients and ITB patients. Wilcoxon rank sum test was used to screen the ten most differentiated protein peaks (P < 0.05). Genetic algorithm combining with support vector machine (SVM) was utilized to establish the optimal diagnostic models for CD, ITB and the optimal differential diagnostic model between CD and ITB. The predictive effects of these models were evaluated by Leave one out (LOO) cross validation method. Results There were 236 protein peaks differently expressed between group of CD patients and HCs, 305 protein peaks differently expressed between group of ITB patients and HCs, 332 protein peaks differently expressed between group of CD patients and ITB patients. Ten most differentially expressed peaks were screened out between three groups respectively (P < 0.05) to establish diagnostic models and differential diagnostic model. A diagnostic model comprising of four protein peaks (M/Z 4964, 3029, 2833, 2900) can well distinguish CD patients and HCs, with a specificity and sensitivity of 96.7% and 96.7% respectively. A diagnostic model comprising four protein peaks (M/Z 3030, 2105, 2545, 4210) can well distinguish ITB patients and HCs, with a specificity and sensitivity of 93.3% and 95.2% respectively. A differential diagnostic model comprising three potential biomarkers protein peaks (M/Z 4267, 4223, 1541) can well distinguish CD patients and ITB patients, with a specificity and sensitivity of 76

  13. Morphological and proteomic analysis of early stage of osteoblast differentiation in osteoblastic progenitor cells

    SciTech Connect

    Hong, Dun; Chen, Hai-Xiao; Yu, Hai-Qiang; Liang, Yong; Wang, Carrie; Lian, Qing-Quan; Deng, Hai-Teng; Ge, Ren-Shan

    2010-08-15

    Bone remodeling relies on a dynamic balance between bone formation and resorption, mediated by osteoblasts and osteoclasts, respectively. Under certain stimuli, osteoprogenitor cells may differentiate into premature osteoblasts and further into mature osteoblasts. This process is marked by increased alkaline phosphatase (ALP) activity and mineralized nodule formation. In this study, we induced osteoblast differentiation in mouse osteoprogenitor MC3T3-E1 cells and divided the process into three stages. In the first stage (day 3), the MC3T3-E1 cell under osteoblast differentiation did not express ALP or deposit a mineralized nodule. In the second stage, the MC3T3-E1 cell expressed ALP but did not form a mineralized nodule. In the third stage, the MC3T3-E1 cell had ALP activity and formed mineralized nodules. In the present study, we focused on morphological and proteomic changes of MC3T3-E1 cells in the early stage of osteoblast differentiation - a period when premature osteoblasts transform into mature osteoblasts. We found that mean cell area and mean stress fiber density were increased in this stage due to enhanced cell spreading and decreased cell proliferation. We further analyzed the proteins in the signaling pathway of regulation of the cytoskeleton using a proteomic approach and found upregulation of IQGAP1, gelsolin, moesin, radixin, and Cfl1. After analyzing the focal adhesion signaling pathway, we found the upregulation of FLNA, LAMA1, LAMA5, COL1A1, COL3A1, COL4A6, and COL5A2 as well as the downregulation of COL4A1, COL4A2, and COL4A4. In conclusion, the signaling pathway of regulation of the cytoskeleton and focal adhesion play critical roles in regulating cell spreading and actin skeleton formation in the early stage of osteoblast differentiation.

  14. Genomic and proteomic expression analysis of Leishmania promastigote and amastigote life stages: the Leishmania genome is constitutively expressed.

    PubMed

    Leifso, Kirk; Cohen-Freue, Gabriela; Dogra, Nisha; Murray, Angus; McMaster, W Robert

    2007-03-01

    Leishmania are protozoan parasites that cause a wide spectrum of clinical diseases in humans and are a major public health risk in several countries. Leishmania life cycle consists of an extracellular flagellated promastigote stage within the midgut of a sandfly vector, and a morphological distinct intracellular amastigote stage within macrophages of a mammalian host. This study reports the use of DNA oligonucleotide genome microarrays representing 8160 genes to analyze the mRNA expression profiles of L. major promastigotes and lesion derived amastigotes. Over 94% of the genes were expressed in both life stages. Advanced statistical analysis identified a surprisingly low degree of differential mRNA expression: 1.4% of the total genes in amastigotes and 1.5% in promastigotes. These microarray results demonstrate that the L. major genome is essentially constitutively expressed in both life stages and suggest that Leishmania is constitutively adapted for survival and replication in either the sandfly vector or macrophage host utilizing an appropriate set of genes for each vastly different environment. Quantitative proteomics, using the isotope coded affinity tag (ICAT) technology and mass spectrometry, was used to identify L. infantum promastigote and axenic amastigote differentially expressed proteins. Of the 91 distinct proteins identified, 8% were differentially expressed in the amastigote stage, 20% were differentially expressed in the promastigote stage, and the remaining 72% were considered constitutively expressed. The differential expression was validated by the identification of previously reported stage specific proteins and identified several amastigote and promastigote novel stage specific proteins.

  15. Proteomic analysis of hyperadhesive Candida glabrata clinical isolates reveals a core wall proteome and differential incorporation of adhesins.

    PubMed

    Gómez-Molero, Emilia; de Boer, Albert D; Dekker, Henk L; Moreno-Martínez, Ana; Kraneveld, Eef A; Ichsan; Chauhan, Neeraj; Weig, Michael; de Soet, Johannes J; de Koster, Chris G; Bader, Oliver; de Groot, Piet W J

    2015-12-01

    Attachment to human host tissues or abiotic medical devices is a key step in the development of infections by Candida glabrata. The genome of this pathogenic yeast codes for a large number of adhesins, but proteomic work using reference strains has shown incorporation of only few adhesins in the cell wall. By making inventories of the wall proteomes of hyperadhesive clinical isolates and reference strain CBS138 using mass spectrometry, we describe the cell wall proteome of C. glabrata and tested the hypothesis that hyperadhesive isolates display differential incorporation of adhesins. Two clinical strains (PEU382 and PEU427) were selected, which both were hyperadhesive to polystyrene and showed high surface hydrophobicity. Cell wall proteome analysis under biofilm-forming conditions identified a core proteome of about 20 proteins present in all C. glabrata strains. In addition, 12 adhesin-like wall proteins were identified in the hyperadherent strains, including six novel adhesins (Awp8-13) of which only Awp12 was also present in CBS138. We conclude that the hyperadhesive capacity of these two clinical C. glabrata isolates is correlated with increased and differential incorporation of cell wall adhesins. Future studies should elucidate the role of the identified proteins in the establishment of C. glabrata infections.

  16. Differential proteomics analysis of liver failure in peripheral blood mononuclear cells using isobaric tags for relative and absolute quantitation

    PubMed Central

    Lin, Hua; Tan, Qiu-Pei; Sui, Wei-Guo; Chen, Wen-Biao; Peng, Wu-Jian; Liu, Xing-Chao; Dai, Yong

    2017-01-01

    The aim of the present study was to examine differentially expressed proteome profiles for candidate biomarkers in peripheral blood mononuclear cells (PBMCs) of liver failure (LF) patients. Ten patients were diagnosed as LF and 10 age- and gender-matched subjects were recruited as healthy controls. Isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomic technology is efficiently applicable for identification and relative quantitation of the proteomes of PBMCs. Eight-plex iTRAQ coupled with strong cation exchange chromatography, and liquid chromatography coupled with tandem mass spectrometry were used to analyze total proteins in LF patients and healthy control subjects. Molecular variations were detected using the iTRAQ method, and western blotting was used to verify the results. LF is a complex type of medical emergency that evolves following a catastrophic insult to the liver, and its outcome remains the most ominous of all gastroenterologic diseases. Serious complications tend to occur during the course of the disease and further exacerbate the problems. Using the iTRAQ method, differentially expressed proteome profiles of LF patients were determined. In the present study, 627 proteins with different expression levels were identified in LF patients compared with the control subjects; with 409 proteins upregulated and 218 proteins downregulated. Among them, four proteins were significantly differentially expressed; acylaminoacyl-peptide hydrolase and WW domain binding protein 2 were upregulated, and resistin and tubulin β 2A class IIa were downregulated. These proteins demonstrated differences in their expression levels compared with other proteins with normal expression levels and the significant positive correlation with LF. The western blot results were consistent with the results from iTRAQ. Thus, investigation of the molecular mechanism of the proteins involved in LF may facilitate an improved understanding of the

  17. Differential proteomic analysis of synovial fluid from rheumatoid arthritis and osteoarthritis patients

    PubMed Central

    2014-01-01

    Background Rheumatoid arthritis and osteoarthritis are two common musculoskeletal disorders that affect the joints. Despite high prevalence rates, etiological factors involved in these disorders remain largely unknown. Dissecting the molecular aspects of these disorders will significantly contribute to improving their diagnosis and clinical management. In order to identify proteins that are differentially expressed between these two conditions, a quantitative proteomic profiling of synovial fluid obtained from rheumatoid arthritis and osteoarthritis patients was carried out by using iTRAQ labeling followed by high resolution mass spectrometry analysis. Results We have identified 575 proteins out of which 135 proteins were found to be differentially expressed by ≥3-fold in the synovial fluid of rheumatoid arthritis and osteoarthritis patients. Proteins not previously reported to be associated with rheumatoid arthritis including, coronin-1A (CORO1A), fibrinogen like-2 (FGL2), and macrophage capping protein (CAPG) were found to be upregulated in rheumatoid arthritis. Proteins such as CD5 molecule-like protein (CD5L), soluble scavenger receptor cysteine-rich domain-containing protein (SSC5D), and TTK protein kinase (TTK) were found to be upregulated in the synovial fluid of osteoarthritis patients. We confirmed the upregulation of CAPG in rheumatoid arthritis synovial fluid by multiple reaction monitoring assay as well as by Western blot. Pathway analysis of differentially expressed proteins revealed a significant enrichment of genes involved in glycolytic pathway in rheumatoid arthritis. Conclusions We report here the largest identification of proteins from the synovial fluid of rheumatoid arthritis and osteoarthritis patients using a quantitative proteomics approach. The novel proteins identified from our study needs to be explored further for their role in the disease pathogenesis of rheumatoid arthritis and osteoarthritis. Sartaj Ahmad and Raja Sekhar Nirujogi

  18. Proteomic Profiling Reveals Upregulated Protein Expression of Hsp70 in Keloids

    PubMed Central

    Lee, Ju Hee; Shin, Jung U.; Lee, Hemin; Rah, Dong Kyun; Jung, Jin Young; Lee, Won Jai

    2013-01-01

    Background. The biochemical characteristics of keloid-derived fibroblasts differ from those of adjacent normal fibroblasts, and these differences are thought to be the cause of abnormal fibrosis. Therefore, we investigated the characteristic proteins that are differentially expressed in keloid-derived fibroblasts using proteomics tools. Objective. We attempted to investigate the novel proteins that play important roles in the pathophysiology of keloids. Methods. Proteomics analysis was performed to identify differentially expressed proteins in keloid-derived fibroblasts. Keloid-derived fibroblasts and adjacent normal fibroblasts were analyzed with 2-DAGE. We validated these proteins with immunoblot analysis, real-time RT-PCR, and immunohistochemistry. Results. Sixteen differentially expressed protein spots were identified in keloid-derived fibroblasts. Among them, heat shock protein 70 (Hsp70) was specifically upregulated in keloid-derived fibroblasts. Also, immunohistochemistry and western blot analysis revealed increased Hsp70, TGF-β, and PCNA expressions in keloids compared to normal tissue. Conclusion. Hsp70 is overexpressed in keloid fibroblasts and tissue. The overexpression of Hsp70 may be involved in the pathogenesis of keloids, and the inhibition of Hsp70 could be a new therapeutic tool for the treatment of keloids. PMID:24260741

  19. Comparative proteomic analysis of four Bacillus clausii strains: proteomic expression signature distinguishes protein profile of the strains.

    PubMed

    Lippolis, Rosa; Gnoni, Antonio; Abbrescia, Anna; Panelli, Damiano; Maiorano, Stefania; Paternoster, Maria Stefania; Sardanelli, Anna Maria; Papa, Sergio; Gaballo, Antonio

    2011-11-18

    A comparative proteomic approach, using two dimensional gel electrophoresis and mass spectrometry, has been developed to compare and elucidate the differences among the cellular proteomes of four closely related isogenic O/C, SIN, N/R and T, B. clausii strains during both exponential and stationary phases of growth. Image analysis of the electropherograms reveals a high degree of concordance among the four proteomes, some proteins result, however, differently expressed. The proteins spots exhibiting high different expression level were identified, by mass-spectrometry analysis, as alcohol dehydrogenase (ADHA, EC1.2.1.3; ABC0046 isoform) aldehyde dehydrogenase (DHAS, EC 1.2.1.3; ABC0047 isoform) and flagellin-protein of B. clausii KSM-k16. The different expression levels of the two dehydrogenases were confirmed by quantitative RT-PCR and dehydrogenases enzymatic activity. The different patterns of protein expression can be considered as cell proteome signatures of the different strains.

  20. Quantitative proteomic analysis for high-throughput screening of differential glycoproteins in hepatocellular carcinoma serum

    PubMed Central

    Gao, Hua-Jun; Chen, Ya-Jing; Zuo, Duo; Xiao, Ming-Ming; Li, Ying; Guo, Hua; Zhang, Ning; Chen, Rui-Bing

    2015-01-01

    Objective Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths. Novel serum biomarkers are required to increase the sensitivity and specificity of serum screening for early HCC diagnosis. This study employed a quantitative proteomic strategy to analyze the differential expression of serum glycoproteins between HCC and normal control serum samples. Methods Lectin affinity chromatography (LAC) was used to enrich glycoproteins from the serum samples. Quantitative mass spectrometric analysis combined with stable isotope dimethyl labeling and 2D liquid chromatography (LC) separations were performed to examine the differential levels of the detected proteins between HCC and control serum samples. Western blot was used to analyze the differential expression levels of the three serum proteins. Results A total of 2,280 protein groups were identified in the serum samples from HCC patients by using the 2D LC-MS/MS method. Up to 36 proteins were up-regulated in the HCC serum, whereas 19 proteins were down-regulated. Three differential glycoproteins, namely, fibrinogen gamma chain (FGG), FOS-like antigen 2 (FOSL2), and α-1,6-mannosylglycoprotein 6-β-N-acetylglucosaminyltransferase B (MGAT5B) were validated by Western blot. All these three proteins were up-regulated in the HCC serum samples. Conclusion A quantitative glycoproteomic method was established and proven useful to determine potential novel biomarkers for HCC. PMID:26487969

  1. Differential proteome analysis of human embryonic kidney cell line (HEK-293) following mycophenolic acid treatment

    PubMed Central

    2011-01-01

    Background Mycophenolic acid (MPA) is widely used as a post transplantation medicine to prevent acute organ rejection. In the present study we used proteomics approach to identify proteome alterations in human embryonic kidney cells (HEK-293) after treatment with therapeutic dose of MPA. Following 72 hours MPA treatment, total protein lysates were prepared, resolved by two dimensional gel electrophoresis and differentially expressed proteins were identified by QTOF-MS/MS analysis. Expressional regulations of selected proteins were further validated by real time PCR and Western blotting. Results The proliferation assay demonstrated that therapeutic MPA concentration causes a dose dependent inhibition of HEK-293 cell proliferation. A significant apoptosis was observed after MPA treatment, as revealed by caspase 3 activity. Proteome analysis showed a total of 12 protein spots exhibiting differential expression after incubation with MPA, of which 7 proteins (complement component 1 Q subcomponent-binding protein, electron transfer flavoprotein subunit beta, cytochrome b-c1 complex subunit, peroxiredoxin 1, thioredoxin domain-containing protein 12, myosin regulatory light chain 2, and profilin 1) showed significant increase in their expression. The expression of 5 proteins (protein SET, stathmin, 40S ribosomal protein S12, histone H2B type 1 A, and histone H2B type 1-C/E/F/G/I) were down-regulated. MPA mainly altered the proteins associated with the cytoskeleton (26%), chromatin structure/dynamics (17%) and energy production/conversion (17%). Both real time PCR and Western blotting confirmed the regulation of myosin regulatory light chain 2 and peroxiredoxin 1 by MPA treatment. Furthermore, HT-29 cells treated with MPA and total kidney cell lysate from MMF treated rats showed similar increased expression of myosin regulatory light chain 2. Conclusion The emerging use of MPA in diverse pathophysiological conditions demands in-depth studies to understand molecular basis of

  2. A combined proteomic and transcriptomic approach to the study of stage differentiation in Leishmania infantum.

    PubMed

    McNicoll, François; Drummelsmith, Jolyne; Müller, Michaela; Madore, Eric; Boilard, Nathalie; Ouellette, Marc; Papadopoulou, Barbara

    2006-06-01

    Protozoan parasites of the genus Leishmania are found as promastigotes in the sandfly vector and as amastigotes in mammalian macrophages. Mechanisms controlling stage-regulated gene expression in these organisms are poorly understood. Here, we applied a comprehensive approach consisting of protein prefractionation, global proteomics and targeted DNA microarray analysis to the study of stage differentiation in Leishmania. By excluding some abundant structural proteins and reducing complexity, we detected and identified numerous novel differentially expressed protein isoforms in L. infantum. Using 2-D gels, over 2200 protein isoforms were visualized in each developmental stage. Of these, 6.1% were strongly increased or appeared unique in the promastigote stage, while the relative amounts of 12.4% were increased in amastigotes. Amastigote-specific protein isoform and mRNA expression trends correlated modestly (53%), while no correlation was found for promastigote-specific spots. Even where direction of regulation was similar, fold-changes were more modest at the RNA than protein level. Many proteins were present in multiple spots, suggesting that PTM is extensive in this organism. In several cases, different isoforms appeared to be specific to different life stages. Our results suggest that post-transcriptional controls at translational and post-translational levels could play major roles in differentiation in Leishmania parasites.

  3. Differential proteomics analysis of female and male adults of Angiostrongylus cantonensis.

    PubMed

    Song, Zengmei; Huang, Huicong; Tan, Feng; Zhang, Erpeng; Hu, Jianwen; Pan, Changwang

    2012-06-01

    In this study, we identified the differentially expressed proteins of female and male adults of Angiostrongylus cantonensis through differential proteomics. We extracted and purified total proteins from male and female adults, separated proteins by two-dimensional difference gel electrophoresis (2D-DIGE) in pH 4-7, analyzed the gel images by DeCyder 7.0 software, and sacrificed the infected rats to count the number of male and female adults. It was found 28 protein spots that were differentially expressed; seven protein spots were then identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Five proteins were up-regulated and two proteins down-regulated in male adults compared with female adults. Three of the five up-regulated proteins with known functions ascribed to them were identified as galectin-1, proteasome alpha subunit and peroxiredoxin. The two down-regulated proteins were identified as indoleamine dioxygenase like-myoglobin and galectin. Furthermore, the female was significantly greater than male adults (P<0.01) in the rats. The findings demonstrate the differences in protein expression profiles and the ability to survive in the final host between female and male adults of A. cantonensis, and may provide a theoretical basis to study their developmental biology further.

  4. 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

  5. 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

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

    SciTech Connect

    Takahashi, Shoko; Masuda, Junko; Shimagami, Hiroshi; Ohta, Yutaka; Kanda, Tomomasa; Saito, Kenji; Kato, Hisanori

    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, despite 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.

  7. Differential proteomic profiling to study the mechanism of cardiac pharmacological preconditioning by resveratrol

    PubMed Central

    Bezstarosti, Karel; Das, Samarjit; Lamers, Jos M J; Das, Dipak K

    2006-01-01

    Recent studies demonstrated that resveratrol, a grape-derived polyphenolic phytoalexin, provides pharmacological preconditioning of the heart through a NO-dependent mechanism. To further explore the molecular mechanisms involved in resveratrol-mediated cardioprotection, we monitored the effects of resveratrol treatment after ischemia-reperfusion on the protein profile by implementation of proteomic analysis. Two groups of rats were studied; one group of animals was fed resveratrol for 7 days, while the other group was given vehicle only. The rats were sacrificed for the isolated working heart preparation and for isolation of cytoplasmic fraction from left ventricle homogenates to carry out the proteomic as well as immunoblot at baseline and at the end of 30 min ischemia/2-h perfusion. The results demonstrate significant cardiopro-tection with resveratrol evidenced by improved ventricular recovery and reduced infarct size and cardiomyocyte apopto-sis. The left ventricular cytoplasmic fractions were separated by two-dimensional electrophoresis (2-DE). Differentially regulated proteins were detected with quantitative computer analysis of the Coomassie blue stained 2-DE images and identified by MALDI-TOF (MS) and nanoLC-ESI-Q-TOF mass spectrometry (MS/MS). Five redox-regulated and precondi-tioning-related proteins were identified that were all upregulated by resveratrol: MAPKK, two different aB-crystallin species, HSP 27 and PE binding protein. Another HSP27 species and aldose reductase were downregulated and peroxire-doxin-2 remained constant. The results of the immunoblot analysis of phosphorylated MAPKK, -HSP27 and -aB-crys-tallin and PE binding protein were consistent with the proteomic findings, but not with peroxiredoxin-2. The proteomic analysis showed also downregulation of some proteins in the mitochondrial respiratory chain and matrix and the myofila-ment regulating protein MLC kinase-2. The results of the present study demonstrate that proteomic profiling

  8. Differential proteome and secretome analysis during rice-pathogen interaction.

    PubMed

    Wang, Yiming; Kim, Sang Gon; Wu, Jingni; Kim, Sun Tae; Kang, Kyu Young

    2014-01-01

    Substantial evidences implicate that sample preparation and protein extraction in proteomic studies of plant-pathogen interactions are critical to understand cross talk between host and pathogen. Therefore, interest is growing in applying proteomics techniques to investigate simultaneously secreted proteins from rice and pathogen. We have found, however, that most proteins of interest are low abundant so that proper prefractionation or extraction of secreted proteins from extracellular space (ECS) in the rice leaf is required to excavate relevant protein. This chapter describes the preparation of sample and extraction procedure to enrich the proteins interested before separation by 2-DE or LC-MS/MS. This method significantly increases the sensitivity of proteomic comparisons.

  9. Human Amniotic Fluid Mesenchymal Stem Cells from Second- and Third-Trimester Amniocentesis: Differentiation Potential, Molecular Signature, and Proteome Analysis.

    PubMed

    Savickiene, Jurate; Treigyte, Grazina; Baronaite, Sandra; Valiuliene, Giedre; Kaupinis, Algirdas; Valius, Mindaugas; Arlauskiene, Audrone; Navakauskiene, Ruta

    2015-01-01

    Human amniotic fluid stem cells have become an attractive stem cell source for potential applications in regenerative medicine and tissue engineering. The aim of this study was to characterize amniotic fluid-derived mesenchymal stem cells (AF-MSCs) from second- and third-trimester of gestation. Using two-stage protocol, MSCs were successfully cultured and exhibited typical stem cell morphological, specific cell surface, and pluripotency markers characteristics. AF-MSCs differentiated into adipocytes, osteocytes, chondrocytes, myocytes, and neuronal cells, as determined by morphological changes, cell staining, and RT-qPCR showing the tissue-specific gene presence for differentiated cell lineages. Using SYNAPT G2 High Definition Mass Spectrometry technique approach, we performed for the first time the comparative proteomic analysis between undifferentiated AF-MSCs from late trimester of gestation and differentiated into myogenic, adipogenic, osteogenic, and neurogenic lineages. The analysis of the functional and expression patterns of 250 high abundance proteins selected from more than 1400 demonstrated the similar proteome of cultured and differentiated AF-MSCs but the unique changes in their expression profile during cell differentiation that may help the identification of key markers in differentiated cells. Our results provide evidence that human amniotic fluid of second- and third-trimester contains stem cells with multilineage potential and may be attractive source for clinical applications.

  10. Human Amniotic Fluid Mesenchymal Stem Cells from Second- and Third-Trimester Amniocentesis: Differentiation Potential, Molecular Signature, and Proteome Analysis

    PubMed Central

    Savickiene, Jurate; Treigyte, Grazina; Baronaite, Sandra; Valiuliene, Giedre; Kaupinis, Algirdas; Valius, Mindaugas; Arlauskiene, Audrone; Navakauskiene, Ruta

    2015-01-01

    Human amniotic fluid stem cells have become an attractive stem cell source for potential applications in regenerative medicine and tissue engineering. The aim of this study was to characterize amniotic fluid-derived mesenchymal stem cells (AF-MSCs) from second- and third-trimester of gestation. Using two-stage protocol, MSCs were successfully cultured and exhibited typical stem cell morphological, specific cell surface, and pluripotency markers characteristics. AF-MSCs differentiated into adipocytes, osteocytes, chondrocytes, myocytes, and neuronal cells, as determined by morphological changes, cell staining, and RT-qPCR showing the tissue-specific gene presence for differentiated cell lineages. Using SYNAPT G2 High Definition Mass Spectrometry technique approach, we performed for the first time the comparative proteomic analysis between undifferentiated AF-MSCs from late trimester of gestation and differentiated into myogenic, adipogenic, osteogenic, and neurogenic lineages. The analysis of the functional and expression patterns of 250 high abundance proteins selected from more than 1400 demonstrated the similar proteome of cultured and differentiated AF-MSCs but the unique changes in their expression profile during cell differentiation that may help the identification of key markers in differentiated cells. Our results provide evidence that human amniotic fluid of second- and third-trimester contains stem cells with multilineage potential and may be attractive source for clinical applications. PMID:26351462

  11. Differential gene expression in glaucoma.

    PubMed

    Jakobs, Tatjana C

    2014-07-01

    In glaucoma, regardless of its etiology, retinal ganglion cells degenerate and eventually die. Although age and elevated intraocular pressure (IOP) are the main risk factors, there are still many mysteries in the pathogenesis of glaucoma. The advent of genome-wide microarray expression screening together with the availability of animal models of the disease has allowed analysis of differential gene expression in all parts of the eye in glaucoma. This review will outline the findings of recent genome-wide expression studies and discuss their commonalities and differences. A common finding was the differential regulation of genes involved in inflammation and immunity, including the complement system and the cytokines transforming growth factor β (TGFβ) and tumor necrosis factor α (TNFα). Other genes of interest have roles in the extracellular matrix, cell-matrix interactions and adhesion, the cell cycle, and the endothelin system.

  12. Differential Gene Expression in Glaucoma

    PubMed Central

    Jakobs, Tatjana C.

    2014-01-01

    In glaucoma, regardless of its etiology, retinal ganglion cells degenerate and eventually die. Although age and elevated intraocular pressure (IOP) are the main risk factors, there are still many mysteries in the pathogenesis of glaucoma. The advent of genome-wide microarray expression screening together with the availability of animal models of the disease has allowed analysis of differential gene expression in all parts of the eye in glaucoma. This review will outline the findings of recent genome-wide expression studies and discuss their commonalities and differences. A common finding was the differential regulation of genes involved in inflammation and immunity, including the complement system and the cytokines transforming growth factor β (TGFβ) and tumor necrosis factor α (TNFα). Other genes of interest have roles in the extracellular matrix, cell–matrix interactions and adhesion, the cell cycle, and the endothelin system. PMID:24985133

  13. iTRAQ-based differential proteomic analysis in Mongolian gerbil brains chronically infected with Toxoplasma gondii.

    PubMed

    Lv, Lin; Wang, Yapei; Feng, Weili; Hernandez, Jorge A; Huang, Wanyi; Zheng, Yuxiang; Zhou, Xue; Lv, Shumei; Chen, Yajun; Yuan, Zi-Guo

    2017-03-17

    The aim of our study was to detect differentially regulated proteins and specific signaling pathways in Mongolian gerbil brains during chronic Toxoplasma gondii (T.gondii) PRU strain infection. We use a iTRAQ-based strategy to detecte 4935 proteins, out of which 110 proteins were differentially expressed (>/=2.0-fold, p value <0.05) when the brain of gerbils infected with T.gondii was compared to control brain tissues. We confirmed the authenticity and the accuracy of iTRAQ results through quantitative real-time PCR and western blot (WB), which was consistent with mass spectrometry analysis. Pathway analysis and GO (Gene Ontology) annotations indicated the deregulation of several pathways related to immune response, metabolism and neurological processes, like neuronal growth and neurotransmitter transport. Through the iTRAQ-based strategy, we obtained a comparative proteome profile of brain tissues from Mongolian gerbils with chronic infection of T.gondii. Several differentially expressed proteins involved in neurological pathways, like Parvalbumin, Drebrin or Synaptotagmin, can be further investigated to enhance our understanding of central nervous system (CNS) injury caused by T.gondii.

  14. Transcriptome and proteome characterization of surface ectoderm cells differentiated from human iPSCs

    PubMed Central

    Qu, Ying; Zhou, Bo; Yang, Wei; Han, Bingchen; Yu-Rice, Yi; Gao, Bowen; Johnson, Jeffery; Svendsen, Clive N.; Freeman, Michael R.; Giuliano, Armando E.; Sareen, Dhruv; Cui, Xiaojiang

    2016-01-01

    Surface ectoderm (SE) cells give rise to structures including the epidermis and ectodermal associated appendages such as hair, eye, and the mammary gland. In this study, we validate a protocol that utilizes BMP4 and the γ-secretase inhibitor DAPT to induce SE differentiation from human induced pluripotent stem cells (hiPSCs). hiPSC-differentiated SE cells expressed markers suggesting their commitment to the SE lineage. Computational analyses using integrated quantitative transcriptomic and proteomic profiling reveal that TGFβ superfamily signaling pathways are preferentially activated in SE cells compared with hiPSCs. SE differentiation can be enhanced by selectively blocking TGFβ-RI signaling. We also show that SE cells and neural ectoderm cells possess distinct gene expression patterns and signaling networks as indicated by functional Ingenuity Pathway Analysis. Our findings advance current understanding of early human SE cell development and pave the way for modeling of SE-derived tissue development, studying disease pathogenesis, and development of regenerative medicine approaches. PMID:27550649

  15. High-Throughput Proteomics Reveals the Unicellular Roots of Animal Phosphosignaling and Cell Differentiation.

    PubMed

    Sebé-Pedrós, Arnau; Peña, Marcia Ivonne; Capella-Gutiérrez, Salvador; Antó, Meritxell; Gabaldón, Toni; Ruiz-Trillo, Iñaki; Sabidó, Eduard

    2016-10-24

    Cell-specific regulation of protein levels and activity is essential for the distribution of functions among multiple cell types in animals. The finding that many genes involved in these regulatory processes have a premetazoan origin raises the intriguing possibility that the mechanisms required for spatially regulated cell differentiation evolved prior to the appearance of animals. Here, we use high-throughput proteomics in Capsaspora owczarzaki, a close unicellular relative of animals, to characterize the dynamic proteome and phosphoproteome profiles of three temporally distinct cell types in this premetazoan species. We show that life-cycle transitions are linked to extensive proteome and phosphoproteome remodeling and that they affect key genes involved in animal multicellularity, such as transcription factors and tyrosine kinases. The observation of shared features between Capsaspora and metazoans indicates that elaborate and conserved phosphosignaling and proteome regulation supported temporal cell-type differentiation in the unicellular ancestor of animals. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Differential proteomic analysis during the vegetative phase change and the floral transition in Malus domestica.

    PubMed

    Zeng, Guang J; Li, Chun M; Zhang, Xin Z; Han, Zhen H; Yang, Feng Q; Gao, Yan; Chen, Dong M; Zhao, Yong B; Wang, Yi; Teng, Yun L; Dong, Wen X

    2010-09-01

    In order to identify the proteomic changes of apple (Malus domestica Borkh.) during the vegetative phase change and the floral transition, leaf protein of juvenile, adult vegetative and reproductive phase in a seedling ('Jonathan' × 'Golden Delicious') was extracted and analyzed by 2-D electrophoresis and Matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Seventy two gel spots with significant expression differences between ontogenetic phases were obtained. Five protein spots were only detected in leaves of juvenile phase and 11 were not; 17 spots were found exclusively in adult vegetative leaves; and only one spot solely appeared in reproductive leaves while 12 did not. Twenty six of the differentially expressed proteins identified were involved in photosynthesis. Seven enzymes were related to respiration and carbohydrate metabolism. Fifteen other proteins also presented qualitative or quantitative differences among developmental phases. The spatial distribution of one differentially expressed protein, serine hydroxymethyltransferase, was confirmed by enzyme linked immunosorbent assay and immunohistochemistry. These results strongly support the idea that the vegetative phase change and the floral transition are regulated independently during developmental process.

  17. Differential proteomics analysis of low- and high-grade of astrocytoma using iTRAQ quantification

    PubMed Central

    Ren, Tong; Lin, Shide; Wang, Zhongfeng; Shang, Aijia

    2016-01-01

    Astrocytoma is one of the most common types of brain tumor, which is histologically and clinically classified into four grades (I–IV): I (pilocytic astrocytoma), II (diffuse astrocytoma), III (anaplastic astrocytoma), and IV (glioblastoma multiforme). A higher grade astrocytoma represents a worse prognosis and is more aggressive. In this study, we compared the differential proteome profile of astrocytoma from grades I to IV. The protein samples from clinical specimens of grades I, II, III, and IV astrocytoma were analyzed by two-dimensional liquid chromatography–tandem mass spectrometry and isobaric tags for relative and absolute quantitation and quantification. A total of 2,190 proteins were identified. Compared to grade I astrocytoma, 173 (12.4%), 304 (14%), and 462 (21.2%) proteins were aberrantly expressed in grades II, III, and IV, respectively. By bioinformatics analysis, the cell proliferation, invasion, and angiogenesis-related pathways increase from low- to high-grade of astrocytoma. Five differentially expressed proteins were validated by Western blot. Within them, matrix metalloproteinase-9 and metalloproteinase inhibitor 1 were upregulated in glioblastoma multiforme group; whereas fibulin-2 and -5 were downregulated in grade II/III/IV astrocytoma, and the negative expression was significantly associated with advanced clinical stage. Functional analysis showed that both fibulin-2 and -5 may exert an antitumor effect by inhibiting cell proliferation, in vitro migration/invasion in glioma cells. New molecular biomarkers are likely to be used for accurate classification of astrocytoma and likely to be the target for drug development. PMID:27713642

  18. Novel markers of osteogenic and adipogenic differentiation of human bone marrow stromal cells identified using a quantitative proteomics approach.

    PubMed

    Granéli, Cecilia; Thorfve, Anna; Ruetschi, Ulla; Brisby, Helena; Thomsen, Peter; Lindahl, Anders; Karlsson, Camilla

    2014-01-01

    Today, the tool that is most commonly used to evaluate the osteogenic differentiation of bone marrow stromal cells (BMSCs) in vitro is the demonstration of the expression of multiple relevant markers, such as ALP, RUNX2 and OCN. However, as yet, there is no single surface marker or panel of markers which clearly defines human BMSCs (hBMSCs) differentiating towards the osteogenic lineage. The aim of this study was therefore to examine this issue. Stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics was utilized to investigate differently expressed surface markers in osteogenically differentiated and undifferentiated hBMSCs. Labeled membrane proteins were analyzed by mass spectrometry (MS) and 52 proteins with an expression ratio above 2, between osteogenically differentiated and undifferentiated cells, were identified. Subsequent validation, by flow cytometry and ELISA, of the SILAC expression ratios for a number of these proteins and investigations of the lineage specificity of three candidate markers were performed. The surface markers, CD10 and CD92, demonstrated significantly increased expression in hBMSCs differentiated towards the osteogenic and adipogenic lineages. In addition, there was a slight increase in CD10 expression during chondrogenic differentiation. Furthermore, the expression of the intracellular protein, crystalline-αB (CRYaB), was only significantly increased in osteogenically differentiated hBMSCs and not affected during differentiation towards the chondrogenic or adipogenic lineages. It has been concluded from the present results that CD10 and CD92 are potential markers of osteogenic and adipogenic differentiation and that CRYaB is a potential novel osteogenic marker specifically expressed during the osteogenic differentiation of hBMSCs in vitro.

  19. Differential Proteomic Analysis Using iTRAQ Reveals Alterations in Hull Development in Rice (Oryza sativa L.).

    PubMed

    Wang, Shuzhen; Chen, Wenyue; Xiao, Wenfei; Yang, Changdeng; Xin, Ya; Qiu, Jieren; Hu, Weimin; Ying, Wu; Fu, Yaping; Tong, Jianxin; Hu, Guocheng; Chen, Zhongzhong; Fang, Xianping; Yu, Hong; Lai, Wenguo; Ruan, Songlin; Ma, Huasheng

    2015-01-01

    Rice hull, the outer cover of the rice grain, determines grain shape and size. Changes in the rice hull proteome in different growth stages may reflect the underlying mechanisms involved in grain development. To better understand these changes, isobaric tags for relative and absolute quantitative (iTRAQ) MS/MS was used to detect statistically significant changes in the rice hull proteome in the booting, flowering, and milk-ripe growth stages. Differentially expressed proteins were analyzed to predict their potential functions during development. Gene ontology (GO) terms and pathways were used to evaluate the biological mechanisms involved in rice hull at the three growth stages. In total, 5,268 proteins were detected and characterized, of which 563 were differentially expressed across the development stages. The results showed that the flowering and milk-ripe stage proteomes were more similar to each other (r=0.61) than either was to the booting stage proteome. A GO enrichment analysis of the differentially expressed proteins was used to predict their roles during rice hull development. The potential functions of 25 significantly differentially expressed proteins were used to evaluate their possible roles at various growth stages. Among these proteins, an unannotated protein (Q7X8A1) was found to be overexpressed especially in the flowering stage, while a putative uncharacterized protein (B8BF94) and an aldehyde dehydrogenase (Q9FPK6) were overexpressed only in the milk-ripe stage. Pathways regulated by differentially expressed proteins were also analyzed. Magnesium-protoporphyrin IX monomethyl ester [oxidative] cyclase (Q9SDJ2), and two magnesium-chelatase subunits, ChlD (Q6ATS0), and ChlI (Q53RM0), were associated with chlorophyll biosynthesis at different developmental stages. The expression of Q9SDJ2 in the flowering and milk-ripe stages was validated by qRT-PCR. The 25 candidate proteins may be pivotal markers for controlling rice hull development at various

  20. Differential Peripheral Proteomic Biosignature of Fluoxetine Response in a Mouse Model of Anxiety/Depression

    PubMed Central

    Mendez-David, Indira; Boursier, Céline; Domergue, Valérie; Colle, Romain; Falissard, Bruno; Corruble, Emmanuelle; Gardier, Alain M.; Guilloux, Jean-Philippe; David, Denis J.

    2017-01-01

    The incorporation of peripheral biomarkers in the treatment of major depressive disorders (MDD) could improve the efficiency of treatments and increase remission rate. Peripheral blood mononuclear cells (PBMCs) represent an attractive biological substrate allowing the identification of a drug response signature. Using a proteomic approach with high-resolution mass spectrometry, the present study aimed to identify a biosignature of antidepressant response (fluoxetine, a Selective Serotonin Reuptake Inhibitor) in PBMCs in a mouse model of anxiety/depression. Following determination of an emotionality score, using complementary behavioral analysis of anxiety/depression across three different tests (Elevated Plus Maze, Novelty Suppressed Feeding, Splash Test), we showed that a 4-week corticosterone treatment (35 μg/ml, CORT model) in C57BL/6NTac male mice induced an anxiety/depressive-like behavior. Then, chronic fluoxetine treatment (18 mg/kg/day for 28 days in the drinking water) reduced corticosterone-induced increase in emotional behavior. However, among 46 fluoxetine-treated mice, only 30 of them presented a 50% decrease in emotionality score, defining fluoxetine responders (CORT/Flx-R). To determine a peripheral biological signature of fluoxetine response, proteomic analysis was performed from PBMCs isolated from the “most” affected corticosterone/vehicle (CORT/V), corticosterone/fluoxetine responders and non-responders (CORT/Flx-NR) animals. In comparison to CORT/V, a total of 263 proteins were differently expressed after fluoxetine exposure. Expression profile of these proteins showed a strong similarity between CORT/Flx-R and CORT/Flx-NR (R = 0.827, p < 1e-7). Direct comparison of CORT/Flx-R and CORT/Flx-NR groups revealed 100 differently expressed proteins, representing a combination of markers associated either with the maintenance of animals in a refractory state, or associated with behavioral improvement. Finally, 19 proteins showed a differential

  1. Proteomic and gene expression analyses during bolting-related leaf color change in Brassica rapa.

    PubMed

    Zhang, Y W; Guo, M H; Tang, X B; Jin, D; Fang, Z Y

    2016-08-12

    Bolting and flowering are key processes during the growth and development of Chinese cabbage (Brassica rapa L. ssp pekinensis). Understanding the molecular mechanisms underlying bolting and flowering is of significance for improving production of the vegetable. A leaf-color change from bright green to gray-green has been observed following differentiation of the flowering stem and before bolting in the vegetable, and is considered to be a signal for bolting. Proteomics in meristem tissues of an inbred line (C30) were analyzed by two-dimensional electrophoresis during the transition period. We found that some proteins were specifically expressed while others were differentially expressed. Among these, 17 proteins were specifically expressed before the color change, 18 were specifically expressed after the color change, 21 were downregulated during the color change, and 29 were upregulated. Mass spectrometric analysis (MALDI-TOF-TOF/MS) was used to analyze 17 protein spots, and four proteins (subunit E1 of vacuolar-type H+ transporter ATPase, the large subunit of Rubicon, S-adenosylmethionine synthetase, and tubulin α-2) were identified. qPCR analysis was conducted to quantify the expression of genes encoding these proteins during the transitional period. The expression of BrVHA-E1, BrSAMS, BrrbcL, and BrTUA6 was significantly different before and after the leaf-color change, suggesting that these genes might be involved in regulating flower differentiation and bolting.

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

    ERIC Educational Resources Information Center

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

    2009-01-01

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

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

    ERIC Educational Resources Information Center

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

    2009-01-01

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

  4. Differential Aminoacylase Expression in Neuroblastoma

    PubMed Central

    Long, Patrick M.; Stradecki, Holly M.; Minturn, Jane E.; Wesley, Umadevi V.; Jaworski, Diane M.

    2012-01-01

    Neuroblastoma, a cancer of the sympathetic nervous system, is the most common extracranial solid tumor in children. MYCN amplification and increased BDNF/TrkB signaling are features of high-risk tumors; yet, only ~25% of malignant tumors display these features. Thus, the identification of additional biomarkers and therapeutic targets is essential. Since aminoacylase 1 (ACY1), an amino acid deacetylase, is a putative tumor suppressor in small cell lung and renal cell carcinomas, we investigated whether it or the other family members aspartoacylase (ASPA, aminoacylase 2) or aminoacylase 3 (ACY3) could serve a similar function in neuroblastoma. Aminoacylase expression was examined in TrkB-positive, MYCN-amplified (SMS-KCNR and SK-N-BE) and TrkB-negative, non-MYCN amplified (SK-N-AS, SK-N-SH, SH-SY5Y, and SH-EP) neuroblastoma cell lines. Each aminoacylase exhibited distinct spatial localization (i.e., cytosolic ACY1, membrane-associated ASPA, and nuclear ACY3). When SK-N-SH cells were treated with neural differentiation agents (e.g., retinoic acid, cAMP) in media containing 10% serum ACY1 was the only aminoacylase whose expression was up-regulated. ASPA was primarily expressed in SH-EP cells of a glial sublineage. ACY3 was more highly expressed in the TrkB-positive, MYCN-amplified lines. All three aminoacylases were expressed in normal human adrenal gland, a common site of neuroblastoma origin, but only ACY1 and ACY3 displayed detectable expression in primary neuroblastoma tumor. Bioinformatics data mining of Kaplan-Meier survival revealed that high ACY3 expression is correlated with poor prognosis; while, low expression of ACY1 or ASPA is correlated with poor prognosis. These data suggest that aminoacylase expression is dysregulated in neuroblastoma. PMID:21128244

  5. Shotgun proteomics in neuroscience.

    PubMed

    Liao, Lujian; McClatchy, Daniel B; Yates, John R

    2009-07-16

    Mass spectrometry-based proteomics is increasingly used to address basic and clinical questions in biomedical research through studies of differential protein expression, protein-protein interactions, and posttranslational modifications. The complex structural and functional organization of the human brain warrants the application of high-throughput, systematic approaches to understand the functional alterations under normal physiological conditions and the perturbations of neurological diseases. This primer focuses on shotgun-proteomics-based tandem mass spectrometry for the identification of proteins in a complex mixture. It describes the basic concepts of protein differential expression analysis and posttranslational modification analysis and discusses several strategies to improve the coverage of the proteome.

  6. Expressed proteins of Herbaspirillum seropedicae in maize (DKB240) roots-bacteria interaction revealed using proteomics.

    PubMed

    Ferrari, Cibele Santos; Amaral, Fernanda Plucani; Bueno, Jessica Cavalheiro Ferreira; Scariot, Mirella Christine; Valentim-Neto, Pedro Alexandre; Arisi, Ana Carolina Maisonnave

    2014-11-01

    Several molecular tools have been used to clarify the basis of plant-bacteria interaction; however, the mechanism behind the association is still unclear. In this study, we used a proteomic approach to investigate the root proteome of Zea mays (cv. DKB240) inoculated with Herbaspirillum seropedicae strain SmR1 grown in vitro and harvested 7 days after inoculation. Eighteen differentially accumulated proteins were observed in root samples, ten of which were identified by MALDI-TOF mass spectrometry peptide mass fingerprint. Among the identified proteins, we observed three proteins present exclusively in inoculated root samples and six upregulated proteins and one downregulated protein relative to control. Differentially expressed maize proteins were identified as hypothetical protein ZEAMMB73_483204, hypothetical protein ZEAMMB73_269466, and tubulin beta-7 chain. The following were identified as H. seropedicae proteins: peroxiredoxin protein, EF-Tu elongation factor protein, cation transport ATPase, NADPH:quinone oxidoreductase, dinitrogenase reductase, and type III secretion ATP synthase. Our results presented the first evidence of type III secretion ATP synthase expression during H. seropedicae-maize root interaction.

  7. Identification of prostate-enriched proteins by in-depth proteomic analyses of expressed prostatic secretions in urine

    PubMed Central

    Principe, Simona; Kim, Yunee; Fontana, Simona; Ignatchenko, Vladimir; Nyalwidhe, Julius O.; Lance, Raymond S.; Troyer, Dean A.; Alessandro, Riccardo; Semmes, O. John; Kislinger, Thomas; Drake, Richard R.; Medin, Jeffrey A.

    2013-01-01

    Urinary expressed prostatic secretion or “EPS-urine” is proximal tissue fluid that is collected after a digital rectal exam (DRE). EPS-urine is a rich source of prostate-derived proteins that can be used for biomarker discovery for prostate cancer (PCa) and other prostatic diseases. We previously conducted a comprehensive proteome analysis of direct prostatic excretion. In the current study we defined the proteome of EPS-urine employing Multidimensional Protein Identification Technology (MudPIT) and providing a comprehensive catalogue of this body fluid for future biomarker studies. We identified 1022 unique proteins in a heterogeneous cohort of 11 EPS-urines derived from biopsy negative non-cancer diagnoses with some benign prostatic diseases (BPH) and low-grade PCa, representative of secreted prostate and immune system-derived proteins in a urine background. We further applied MudPIT-based proteomics to generate and compare the differential proteome from a subset of pooled urines (pre-DRE) and EPS-urines (post-DRE) from non-cancer and PCa patients. The direct proteomic comparison of these highly controlled patient sample pools enabled us to define a list of prostate-enriched proteins detectable in EPS-urine and distinguishable from a complex urine protein background. A combinatorial analysis of both proteomics datasets and systematic integration with publicly-available proteomics data of related body fluids, human tissue transcriptomic data, and immunohistochemistry images from the Human Protein Atlas database allowed us to demarcate a robust panel of 49 prostate-derived proteins in EPS-urine. Finally, we validated the expression of seven of these proteins using Western blotting, supporting the likelihood that they originate from the prostate. The definition of these prostatic proteins in EPS-urine samples provides a reference for future investigations for prostatic-disease biomarker studies. PMID:22339264

  8. 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

  9. Cardioinductive network guiding stem cell differentiation revealed by proteomic cartography of tumor necrosis factor alpha-primed endodermal secretome.

    PubMed

    Arrell, D Kent; Niederländer, Nicolas J; Faustino, Randolph S; Behfar, Atta; Terzic, Andre

    2008-02-01

    In the developing embryo, instructive guidance from the ventral endoderm secures cardiac program induction within the anterolateral mesoderm. Endoderm-guided cardiogenesis, however, has yet to be resolved at the proteome level. Here, through cardiopoietic priming of the endoderm with the reprogramming cytokine tumor necrosis factor alpha (TNFalpha), candidate effectors of embryonic stem cell cardiac differentiation were delineated by comparative proteomics. Differential two-dimensional gel electrophoretic mapping revealed that more than 75% of protein species increased >1.5-fold in the TNFalpha-primed versus unprimed endodermal secretome. Protein spot identification by linear ion trap quadrupole (LTQ) tandem mass spectrometry (MS/MS) and validation by shotgun LTQ-Fourier transform MS/MS following multidimensional chromatography mapped 99 unique proteins from 153 spot assignments. A definitive set of 48 secretome proteins was deduced by iterative bioinformatic screening using algorithms for detection of canonical and noncanonical indices of secretion. Protein-protein interaction analysis, in conjunction with respective expression level changes, revealed a nonstochastic TNFalpha-centric secretome network with a scale-free hierarchical architecture. Cardiovascular development was the primary developmental function of the resolved TNFalpha-anchored network. Functional cooperativity of the derived cardioinductive network was validated through direct application of the TNFalpha-primed secretome on embryonic stem cells, potentiating cardiac commitment and sarcomerogenesis. Conversely, inhibition of primary network hubs negated the procardiogenic effects of TNFalpha priming. Thus, proteomic cartography establishes a systems biology framework for the endodermal secretome network guiding stem cell cardiopoiesis.

  10. Comparative proteomics reveals novel components at the plasma membrane of differentiated HepaRG cells and different distribution in hepatocyte- and biliary-like cells.

    PubMed

    Petrareanu, Catalina; Macovei, Alina; Sokolowska, Izabela; Woods, Alisa G; Lazar, Catalin; Radu, Gabriel L; Darie, Costel C; Branza-Nichita, Norica

    2013-01-01

    Hepatitis B virus (HBV) is a human pathogen causing severe liver disease and eventually death. Despite important progress in deciphering HBV internalization, the early virus-cell interactions leading to infection are not known. HepaRG is a human bipotent liver cell line bearing the unique ability to differentiate towards a mixture of hepatocyte- and biliary-like cells. In addition to expressing metabolic functions normally found in liver, differentiated HepaRG cells support HBV infection in vitro, thus resembling cultured primary hepatocytes more than other hepatoma cells. Therefore, extensive characterization of the plasma membrane proteome from HepaRG cells would allow the identification of new cellular factors potentially involved in infection. Here we analyzed the plasma membranes of non-differentiated and differentiated HepaRG cells using nanoliquid chromatography-tandem mass spectrometry to identify the differences between the proteomes and the changes that lead to differentiation of these cells. We followed up on differentially-regulated proteins in hepatocytes- and biliary-like cells, focusing on Cathepsins D and K, Cyclophilin A, Annexin 1/A1, PDI and PDI A4/ERp72. Major differences between the two proteomes were found, including differentially regulated proteins, protein-protein interactions and intracellular localizations following differentiation. The results advance our current understanding of HepaRG differentiation and the unique properties of these cells.

  11. Comparative Proteomics Reveals Novel Components at the Plasma Membrane of Differentiated HepaRG Cells and Different Distribution in Hepatocyte- and Biliary-Like Cells

    PubMed Central

    Woods, Alisa G.; Lazar, Catalin; Radu, Gabriel L.; Darie, Costel C.; Branza-Nichita, Norica

    2013-01-01

    Hepatitis B virus (HBV) is a human pathogen causing severe liver disease and eventually death. Despite important progress in deciphering HBV internalization, the early virus-cell interactions leading to infection are not known. HepaRG is a human bipotent liver cell line bearing the unique ability to differentiate towards a mixture of hepatocyte- and biliary-like cells. In addition to expressing metabolic functions normally found in liver, differentiated HepaRG cells support HBV infection in vitro, thus resembling cultured primary hepatocytes more than other hepatoma cells. Therefore, extensive characterization of the plasma membrane proteome from HepaRG cells would allow the identification of new cellular factors potentially involved in infection. Here we analyzed the plasma membranes of non-differentiated and differentiated HepaRG cells using nanoliquid chromatography-tandem mass spectrometry to identify the differences between the proteomes and the changes that lead to differentiation of these cells. We followed up on differentially-regulated proteins in hepatocytes- and biliary-like cells, focusing on Cathepsins D and K, Cyclophilin A, Annexin 1/A1, PDI and PDI A4/ERp72. Major differences between the two proteomes were found, including differentially regulated proteins, protein-protein interactions and intracellular localizations following differentiation. The results advance our current understanding of HepaRG differentiation and the unique properties of these cells. PMID:23977166

  12. Inoculation with endophytic Bacillus megaterium 1Y31 increases Mn accumulation and induces the growth and energy metabolism-related differentially-expressed proteome in Mn hyperaccumulator hybrid pennisetum.

    PubMed

    Zhang, Wen-hui; He, Lin-yan; Wang, Qi; Sheng, Xia-Fang

    2015-12-30

    In this study, a hydroponic culture experiment was conducted in a greenhouse to investigate the molecular and microbial mechanisms involved in the endophytic Bacillus megaterium 1Y31-enhanced Mn tolerance and accumulation in Mn hyperaccumulator hybrid pennisetum. Strain 1Y31 significantly increased the dry weights (ranging from 28% to 94%) and total Mn uptake (ranging from 23% to 112%) of hybrid pennisetum treated with 0, 2, and 10mM Mn compared to the control. Total 98 leaf differentially expressed proteins were identified between the live and dead bacterial inoculated hybrid pennisetum. The major leaf differentially expressed proteins were involved in energy generation, photosynthesis, response to stimulus, metabolisms, and unknown function. Furthermore, most of the energy generation and photosynthesis-related proteins were up-regulated, whereas most of the response to stimulus and metabolism-related proteins were down-regulated under Mn stress. Notably, the proportion of indole-3-acetic acid (IAA)-producing endophytic bacteria was significantly higher in the bacterial inoculated plants under Mn stress. The results suggested that strain 1Y31 increased the growth and Mn uptake of hybrid pennisetum through increasing the efficiency of photosynthesis and energy metabolism as well as the proportion of plant growth-promoting endophytic bacteria in the plants. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Stromal proteome expression profile and muscle-invasive bladder cancer research

    PubMed Central

    2012-01-01

    Background To globally characterize the cancer stroma expression profile of muscle-invasive transitional cell carcinoma and to discuss the cancer biology as well as biomarker discovery from stroma. Laser capture micro dissection was used to harvest purified muscle-invasive bladder cancer stromal cells and normal urothelial stromal cells from 4 paired samples. Two-dimensional liquid chromatography tandem mass spectrometry was used to identify the proteome expression profile. The differential proteins were further analyzed using bioinformatics tools and compared with the published literature. Results We identified 868/872 commonly expressed proteins and 978 differential proteins from 4 paired cancer and normal stromal samples using laser capture micro dissection coupled with two-dimensional liquid chromatography tandem mass spectrometry. 487/491 proteins uniquely expressed in cancer/normal stroma. Differential proteins were compared with the entire list of the international protein index (IPI), and there were 42/42 gene ontology (GO) terms exhibited as enriched and 8/5 exhibited as depleted in cellular Component, respectively. Significantly altered pathways between cancer/normal stroma mainly include metabolic pathways, ribosome, focal adhesion, etc. Finally, descriptive statistics show that the stromal proteins with extremes of PI and MW have the same probability to be a biomarker. Conclusions Based on our results, stromal cells are essential component of the cancer, biomarker discovery and network based multi target therapy should consider neoplastic cells itself and corresponding stroma as whole one. PMID:22920603

  14. Differential proteomic analysis of embryogenic lines in oil palm (Elaeis guineensis Jacq).

    PubMed

    Tan, Hooi Sin; Liddell, Susan; Ong Abdullah, Meilina; Wong, Wei Chee; Chin, Chiew Foan

    2016-06-30

    Oil palm tissue culture is one way to produce superior oil palm planting materials. However, the low rate of embryogenesis is a major hindrance for the adoption of this technology in oil palm tissue culture laboratories. In this study, we use proteomic technologies to compare differential protein profiles in leaves from palms of high and low proliferation rates in tissue culture in order to understand the underlying biological mechanism for the low level of embryogenesis. Two protein extraction methods, namely trichloroacetic acid/acetone precipitation and polyethylene glycol fractionation were used to produce total proteins and fractionated protein extracts respectively, with the aim of improving the resolution of protein species using two-dimensional gel electrophoresis. A total of 40 distinct differential abundant protein spots were selected from leaf samples collected from palms with proven high and low proliferation rates. The variant proteins were subsequently identified using mass spectrometric analysis. Twelve prominent protein spots were then characterised using real-time polymerase chain reaction to compare the mRNA expression and protein abundant profiles. Three proteins, namely triosephosphate isomerase, l-ascorbate peroxidase, and superoxide dismutase were identified to be potential biomarker candidates at both the protein abundant and mRNA expression levels. In this study, proteomic analysis was used to identify abundant proteins from total protein extracts. PEG fractionation was used to reveal lower abundant proteins from both high and low proliferation embryogenic lines of oil palm samples in tissue culture. A total of 40 protein spots were found to be significant in abundance and the mRNA levels of 12 of these were assessed using real time PCR. Three proteins namely, triosephosphate isomerase, l-ascorbate peroxidase and superoxide dismutase were found to be concordant in their mRNA expression and protein abundance. Triosephosphate isomerase is a

  15. Functional Differentiation of Bundle Sheath and Mesophyll Maize Chloroplasts Determined by Comparative ProteomicsW⃞

    PubMed Central

    Majeran, Wojciech; Cai, Yang; Sun, Qi; van Wijk, Klaas J.

    2005-01-01

    Chloroplasts of maize (Zea mays) leaves differentiate into specific bundle sheath (BS) and mesophyll (M) types to accommodate C4 photosynthesis. Consequences for other plastid functions are not well understood but are addressed here through a quantitative comparative proteome analysis of purified M and BS chloroplast stroma. Three independent techniques were used, including cleavable stable isotope coded affinity tags. Enzymes involved in lipid biosynthesis, nitrogen import, and tetrapyrrole and isoprenoid biosynthesis are preferentially located in the M chloroplasts. By contrast, enzymes involved in starch synthesis and sulfur import preferentially accumulate in BS chloroplasts. The different soluble antioxidative systems, in particular peroxiredoxins, accumulate at higher levels in M chloroplasts. We also observed differential accumulation of proteins involved in expression of plastid-encoded proteins (e.g., EF-Tu, EF-G, and mRNA binding proteins) and thylakoid formation (VIPP1), whereas others were equally distributed. Enzymes related to the C4 shuttle, the carboxylation and regeneration phase of the Calvin cycle, and several regulators (e.g., CP12) distributed as expected. However, enzymes involved in triose phosphate reduction and triose phosphate isomerase are primarily located in the M chloroplasts, indicating that the M-localized triose phosphate shuttle should be viewed as part of the BS-localized Calvin cycle, rather than a parallel pathway. PMID:16243905

  16. Differential proteomic analysis of an engineered Streptomyces coelicolor strain reveals metabolic pathways supporting growth on n-hexadecane.

    PubMed

    Gallo, Giuseppe; Lo Piccolo, Luca; Renzone, Giovanni; La Rosa, Ruggero; Scaloni, Andrea; Quatrini, Paola; Puglia, Anna Maria

    2012-06-01

    The alkB gene, encoding an alkane monooxygenase in the actinomycete Gordonia sp. SoCg, was expressed in the non-alkane-degrading actinomycete Streptomyces coelicolor M145. The resulting engineered strain, M145-AH, can grow on n-hexadecane as sole carbon source. To unravel proteins associated with growth on n-alkanes, proteome of M145-AH after 6, 24, and 48 h of incubation in the Bushnell-Haas (BH) mineral medium containing n-hexadecane as sole carbon source (H condition) and in BH without any carbon source (0 condition) were compared using 2D-differential gel electrophoresis. Proteome analysis revealed significant changes only at 48 h, showing 48 differentially abundant proteins identified by mass spectrometry procedures. To asses if these proteins were specifically related to n-hexadecane metabolism, their expression was investigated, comparing H proteome with that of M145-AH incubated in BH with glucose as sole carbon source (G condition). Thus, protein expression profiles at 6, 24, and 48 h under H, 0, and G conditions were combined, revealing that M145-AH regulates in a temporally- and carbon source-dependent manner the expression of proteins involved in regulatory events, central carbon metabolism, respiration, β-oxidation, membrane transport, and amino acid and protein metabolism. Interestingly, 21 % of them, mostly involved in membrane transport and protein metabolism, showed a n-hexadecane-dependent regulation with regulatory proteins such as CRP likely to have a key role in M145-AH n-hexadecane growth. These results, expanding the knowledge on n-alkane utilization in Gram-positive bacteria, reveal genes to be targeted to develop an efficient S. coelicolor M145-AH-based bioremediation system.

  17. 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

  18. 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

  19. Optimal control of gene expression for fast proteome adaptation to environmental change.

    PubMed

    Pavlov, Michael Y; Ehrenberg, Måns

    2013-12-17

    Bacterial populations growing in a changing world must adjust their proteome composition in response to alterations in the environment. Rapid proteome responses to growth medium changes are expected to increase the average growth rate and fitness value of these populations. Little is known about the dynamics of proteome change, e.g., whether bacteria use optimal strategies of gene expression for rapid proteome adjustments and if there are lower bounds to the time of proteome adaptation in response to growth medium changes. To begin answering these types of questions, we modeled growing bacteria as stoichiometrically coupled networks of metabolic pathways. These are balanced during steady-state growth in a constant environment but are initially unbalanced after rapid medium shifts due to a shortage of enzymes required at higher concentrations in the new environment. We identified an optimal strategy for rapid proteome adjustment in the absence of protein degradation and found a lower bound to the time of proteome adaptation after medium shifts. This minimal time is determined by the ratio between the Kullback-Leibler distance from the pre- to the postshift proteome and the postshift steady-state growth rate. The dynamics of optimally controlled proteome adaptation has a simple analytical solution. We used detailed numerical modeling to demonstrate that realistic bacterial control systems can emulate this optimal strategy for rapid proteome adaptation. Our results may provide a conceptual link between the physiology and population genetics of growing bacteria.

  20. Optimal control of gene expression for fast proteome adaptation to environmental change

    PubMed Central

    Pavlov, Michael Y.; Ehrenberg, Måns

    2013-01-01

    Bacterial populations growing in a changing world must adjust their proteome composition in response to alterations in the environment. Rapid proteome responses to growth medium changes are expected to increase the average growth rate and fitness value of these populations. Little is known about the dynamics of proteome change, e.g., whether bacteria use optimal strategies of gene expression for rapid proteome adjustments and if there are lower bounds to the time of proteome adaptation in response to growth medium changes. To begin answering these types of questions, we modeled growing bacteria as stoichiometrically coupled networks of metabolic pathways. These are balanced during steady-state growth in a constant environment but are initially unbalanced after rapid medium shifts due to a shortage of enzymes required at higher concentrations in the new environment. We identified an optimal strategy for rapid proteome adjustment in the absence of protein degradation and found a lower bound to the time of proteome adaptation after medium shifts. This minimal time is determined by the ratio between the Kullback–Leibler distance from the pre- to the postshift proteome and the postshift steady-state growth rate. The dynamics of optimally controlled proteome adaptation has a simple analytical solution. We used detailed numerical modeling to demonstrate that realistic bacterial control systems can emulate this optimal strategy for rapid proteome adaptation. Our results may provide a conceptual link between the physiology and population genetics of growing bacteria. PMID:24297927

  1. 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.

  2. Protein solubility and differential proteomic profiling of recombinant Escherichia coli overexpressing double-tagged fusion proteins.

    PubMed

    Cheng, Chung-Hsien; Lee, Wen-Chien

    2010-08-28

    Overexpression of recombinant proteins usually triggers the induction of heat shock proteins that regulate aggregation and solubility of the overexpressed protein. The two-dimensional gel electrophoresis (2-DE)-mass spectrometry approach was used to profile the proteome of Escherichia coli overexpressing N-acetyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase) and N-acetyl-D-neuraminic acid aldolase (Neu5Ac aldolase), both fused to glutathione S-transferase (GST) and polyionic peptide (5D or 5R). Overexpression of fusion proteins by IPTG induction caused significant differential expression of numerous cellular proteins; most of these proteins were down-regulated, including enzymes connected to the pentose phosphate pathway and the enzyme LuxS that could lead to an inhibition of tRNA synthesis. Interestingly, when plasmid-harboring cells were cultured in LB medium, gluconeogenesis occurred mainly through MaeB, while in the host strain, gluconeogenesis occurred by a different pathway (by Mdh and PckA). Significant up-regulation of the chaperones ClpB, HslU and GroEL and high-level expression of two protective small heat shock proteins (IbpA and IbpB) were found in cells overexpressing GST-GlcNAc 2-epimerase-5D but not in GST-Neu5Ac aldolase-5R-expressing E. coli. Although most of the recombinant protein was present in insoluble aggregates, the soluble fraction of GST-GlcNAc 2-epimerase-5D was higher than that of GST-Neu5Ac aldolase-5R. Also, in cells overexpressing recombinant GST-GlcNAc 2-epimerase-5D, the expression of σ32 was maintained at a higher level following induction. Differential expression of metabolically functional proteins, especially those in the gluconeogenesis pathway, was found between host and recombinant cells. Also, the expression patterns of chaperones/heat shock proteins differed among the plasmid-harboring bacteria in response to overproduction of recombinant proteins. In conclusion, the solubility of overexpressed recombinant proteins could

  3. Protein solubility and differential proteomic profiling of recombinant Escherichia coli overexpressing double-tagged fusion proteins

    PubMed Central

    2010-01-01

    Background Overexpression of recombinant proteins usually triggers the induction of heat shock proteins that regulate aggregation and solubility of the overexpressed protein. The two-dimensional gel electrophoresis (2-DE)-mass spectrometry approach was used to profile the proteome of Escherichia coli overexpressing N-acetyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase) and N-acetyl-D-neuraminic acid aldolase (Neu5Ac aldolase), both fused to glutathione S-transferase (GST) and polyionic peptide (5D or 5R). Results Overexpression of fusion proteins by IPTG induction caused significant differential expression of numerous cellular proteins; most of these proteins were down-regulated, including enzymes connected to the pentose phosphate pathway and the enzyme LuxS that could lead to an inhibition of tRNA synthesis. Interestingly, when plasmid-harboring cells were cultured in LB medium, gluconeogenesis occurred mainly through MaeB, while in the host strain, gluconeogenesis occurred by a different pathway (by Mdh and PckA). Significant up-regulation of the chaperones ClpB, HslU and GroEL and high-level expression of two protective small heat shock proteins (IbpA and IbpB) were found in cells overexpressing GST-GlcNAc 2-epimerase-5D but not in GST-Neu5Ac aldolase-5R-expressing E. coli. Although most of the recombinant protein was present in insoluble aggregates, the soluble fraction of GST-GlcNAc 2-epimerase-5D was higher than that of GST-Neu5Ac aldolase-5R. Also, in cells overexpressing recombinant GST-GlcNAc 2-epimerase-5D, the expression of σ32 was maintained at a higher level following induction. Conclusions Differential expression of metabolically functional proteins, especially those in the gluconeogenesis pathway, was found between host and recombinant cells. Also, the expression patterns of chaperones/heat shock proteins differed among the plasmid-harboring bacteria in response to overproduction of recombinant proteins. In conclusion, the solubility of

  4. Visualization and Differential Analysis of Protein Expression Data Using R.

    PubMed

    Silva, Tomé S; Richard, Nadège

    2016-01-01

    Data analysis is essential to derive meaningful conclusions from proteomic data. This chapter describes ways of performing common data visualization and differential analysis tasks on gel-based proteomic datasets using a freely available statistical software package (R). A workflow followed is illustrated using a synthetic dataset as example.

  5. Differential proteomics of human seminal plasma: A potential target for searching male infertility marker proteins.

    PubMed

    Tomar, Anil Kumar; Sooch, Balwinder Singh; Singh, Sarman; Yadav, Savita

    2012-04-01

    The clinical fertility tests, available in the market, fail to define the exact cause of male infertility in almost half of the cases and point toward a crucial need of developing better ways of infertility investigations. The protein biomarkers may help us toward better understanding of unknown cases of male infertility that, in turn, can guide us to find better therapeutic solutions. Many clinical attempts have been made to identify biomarkers of male infertility in sperm proteome but only few studies have targeted seminal plasma. Human seminal plasma is a rich source of proteins that are essentially required for development of sperm and successful fertilization. This viewpoint article highlights the importance of human seminal plasma proteome in reproductive physiology and suggests that differential proteomics integrated with functional analysis may help us in searching potential biomarkers of male infertility.

  6. Evaluation of differential protein expression in Haliclona aquarius and sponge-associated microorganisms under cadmium stress.

    PubMed

    Wanick, Rodrigo Cunha; de Sousa Barbosa, Herbert; Frazão, Leonardo Revoredo; Santelli, Ricardo Erthal; Arruda, Marco Aurélio Zezzi; Coutinho, Cristiano Carvalho

    2013-09-01

    A comparative proteomic approach was used to assess differentially expressed proteins in marine sponges after 36 h of exposure to cadmium (Cd). After separation performed by 2-D polyacrylamide gel electrophoresis, 46 protein spots indicated differential expression, and 17 of these proteins were identified by electrospray ionization quadrupole time-of-flight mass spectrometry. From the proteins identified, 76% were attributed to sponge-associated microorganisms (fungi and bacteria), and 24% were attributed to Haliclona aquarius. Some of the proteins that were identified may be related to cell proliferation and differentiation or processes of oxidative stress repair and energy procurement. An integrated evaluation based on spot expression levels and the postulated functions of these proteins allowed a more accurate evaluation of the stress caused to the sponge holobiont system by cadmium exposure. This study could provide new insights into the use of a proteomic approach in the marine sponge to assess the effects of Cd pollution in a marine environment.

  7. DNA microarray analysis of genes differentially expressed in adipocyte differentiation.

    PubMed

    Yin, Chunyan; Xiao, Yanfeng; Zhang, Wei; Xu, Erdi; Liu, Weihua; Yi, Xiaoqing; Chang, Ming

    2014-06-01

    In the present study, the human liposarcoma cell line SW872 was used to identify global changes in gene expression profiles occurring during adipogenesis. We further explored some of the genes expressed during the late phase of adipocyte differentiation. These genes may play a major role in promoting excessive proliferation and accumulation of lipid droplets, which contribute to the development of obesity. By using microarray-based technology, we examined differential gene expression in early differentiated adipocytes and late differentiated adipocytes. Validated genes exhibited a greater than or equal to 10-fold increase in the late phase of adipocyte differentiation by polymerase chain reaction (RT-PCR). Compared with undifferentiated preadipocytes, we found that 763 genes were increased in early differentiated adipocytes, and 667 genes were increased in later differentiated adipocytes. Furthermore, 21 genes were found being expressed 10-fold higher in the late phase of adipocyte differentiation. The results were in accordance with the RTPCR test, which validated 11 genes, namely, CIDEC, PID1, LYRM1, ADD1, PPAR?2, ANGPTL4, ADIPOQ, ACOX1, FIP1L1, MAP3K2 and PEX14. Most of these genes were found being expressed in the later phase of adipocyte differentiation involved in obesity-related diseases. The findings may help to better understand the mechanism of obesity and related diseases.

  8. Differential proteome analysis of hippocampus and temporal cortex using label-free based 2D-LC-MS/MS.

    PubMed

    Liu, Xiaoyan; Guo, Zhengguang; Liu, Wei; Sun, Wei; Ma, Chao

    2017-08-08

    Hippocampus and temporal cortex are important brain regions, which play distinct, but complimentary roles in mediating learning and memory. Herein, we utilized label-free differential proteome strategy to explore function of normal human hippocampus and temporal cortex in learning and memory. As a result, a total of 5529 and 5702 proteins were identified in hippocampus and temporal cortex, respectively, 516 of which were significantly differential expressed, with abundance span 5 orders of magnitudes. Pathways analysis showed that temporal cortex was involved in growth of axons growth and synapse density regulation, through which could regulate long-term potentiation and long-term retention of trace memory. Hippocampus was involved in regulation of cell survival and cell viability, and regulates neurons proliferation by actin dynamics changes, through which involved in both short-term memory and long-term memory. Four selected differential proteins were further validated by Western blot and immunohistochemistry. For the first time, we identified proteins and associated pathways of hippocampus and temporal cortex in human cognition process using proteomic strategy, which would provide references for generating corresponding insights in hippocampus and temporal cortex-related cognitive function. The original data files can be downloaded at http://211.102.209.254/page/PSV023.html;?url=1489542083729AFHp (password: kYxh). This study explored the potential molecular mechanism of hippocampus and temporal cortex in human cognition function using proteomics strategy, which will offer a baseline reference for further cognitive disorders study and reveal insights into physiology of temporal cortex and hippocampus. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Differential proteome profile of skin mucus of gilthead seabream (Sparus aurata) after probiotic intake and/or overcrowding stress.

    PubMed

    Cordero, Héctor; Morcillo, Patricia; Cuesta, Alberto; Brinchmann, Monica F; Esteban, María A

    2016-01-30

    Gilthead seabream (Sparus aurata L.) is the major cultured fish species in the Mediterranean area. High density stocking causes stress and increases the impact of diseases leading to economic losses. Probiotics could represent a solution to prevent diseases through several mechanisms such as improving the immune status and/or mucosal microbiota or competing with pathogens. The probiotic Shewanella putrefaciens, also known as Pdp11, was firstly isolated from the skin of healthy gilthead seabream. Our study focuses on the skin mucus proteome after dietary probiotic Pdp11 intake in fish maintained under normal or overcrowding conditions. 2-DE of skin mucus followed by LC-MS/MS analysis was done for each experimental group and differentially expressed proteins were identified. The results showed differentially expressed proteins especially involved in immune processes, such as lysozyme, complement C3, natural killer cell enhancing factor and nonspecific cytotoxic cell receptor protein 1, whose transcript profiles were studied by qPCR. A consistency between lysozyme protein levels in the mucus and lysozyme mRNA levels in skin was found. Further research is necessary to unravel the implications of skin mucosal immunity on fish welfare and disease. The present work reveals the proteomic changes, which are taking place in the skin mucus of stressed and non-stressed gilthead seabream after Pdp11 probiotic intake. The study contributes to improving the knowledge on skin mucosal immunology of this relevant farmed fish species. Furthermore, the paper shows for the first time how a suitable proteomic methodology, in this case 2-DE followed by LC-MS/MS is useful to perform a comparative study with a non-invasive technique of skin mucus of gilthead seabream. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Proteome analysis of functionally differentiated bovine (Bos indicus) mammary epithelial cells isolated from milk.

    PubMed

    Janjanam, Jagadeesh; Jamwal, Manu; Singh, Surender; Kumar, Saravanan; Panigrahi, Aswini K; Hariprasad, Gururao; Jena, Manoj K; Anand, Vijay; Kumar, Sudarshan; Kaushik, Jai K; Dang, Ajay K; Mukesh, Manishi; Mishra, Bishnu P; Srinivasan, Alagiri; Reddy, Vanga S; Mohanty, Ashok K

    2013-11-01

    Mammary gland is made up of a branching network of ducts that end in alveoli. Terminally differentiated mammary epithelial cells (MECs) constitute the innermost layer of aveoli. They are milk-secreting cuboidal cells that secrete milk proteins during lactation. Little is known about the expression profile of proteins in the metabolically active MECs during lactation or their functional role in the lactation process. In the present investigation, we have reported the proteome map of MECs in lactating cows using 2DE MALDI-TOF/TOF MS and 1D-Gel-LC-MS/MS. MECs were isolated from milk using immunomagnetic beads and confirmed by RT-PCR and Western blotting. The 1D-Gel-LC-MS/MS and 2DE-MS/MS based approaches led to identification of 431 and 134 proteins, respectively, with a total of 497 unique proteins. Proteins identified in this study were clustered into functional groups using bioinformatics tools. Pathway analysis of the identified proteins revealed 28 pathways (p < 0.05) providing evidence for involvement of various proteins in lactation function. This study further provides experimental evidence for the presence of many proteins that have been predicted in annotated bovine genome. The data generated further provide a set of bovine MEC-specific proteins that will help the researchers to understand the molecular events taking place during lactation.

  11. Differential chromatin proteomics of the MMS-induced DNA damage response in yeast

    PubMed Central

    2011-01-01

    Background Protein enrichment by sub-cellular fractionation was combined with differential-in-gel-electrophoresis (DIGE) to address the detection of the low abundance chromatin proteins in the budding yeast proteome. Comparisons of whole-cell extracts and chromatin fractions were used to provide a measure of the degree of chromatin association for individual proteins, which could be compared across sample treatments. The method was applied to analyze the effect of the DNA damaging agent methyl methanesulfonate (MMS) on levels of chromatin-associated proteins. Results Up-regulation of several previously characterized DNA damage checkpoint-regulated proteins, such as Rnr4, Rpa1 and Rpa2, was observed. In addition, several novel DNA damage responsive proteins were identified and assessed for genotoxic sensitivity using either DAmP (decreased abundance by mRNA perturbation) or knockout strains, including Acf2, Arp3, Bmh1, Hsp31, Lsp1, Pst2, Rnr4, Rpa1, Rpa2, Ste4, Ycp4 and Yrb1. A strain in which the expression of the Ran-GTPase binding protein Yrb1 was reduced was found to be hypersensitive to genotoxic stress. Conclusion The described method was effective at unveiling chromatin-associated proteins that are less likely to be detected in the absence of fractionation. Several novel proteins with altered chromatin abundance were identified including Yrb1, pointing to a role for this nuclear import associated protein in DNA damage response. PMID:21967861

  12. Diabetic retinopathy: Proteomic approaches to help the differential diagnosis and to understand the underlying molecular mechanisms.

    PubMed

    Csősz, Éva; Deák, Eszter; Kalló, Gergő; Csutak, Adrienne; Tőzsér, József

    2017-01-06

    Diabetic retinopathy is the most common diabetic eye disease and a leading cause of blindness among patients with diabetes. The appearance and the severity of the symptoms correlate with the duration of diabetes and poor blood glucose level management. Diabetic retinopathy is also categorized as a chronic low-level inflammatory disease; the high blood glucose level promotes the accumulation of the advanced glycation end products and leads to the stimulation of monocytes and macrophages. Examination of protein level alterations in tears using state-of the art proteomics techniques have identified several proteins as possible biomarkers for the different stages of the diabetic retinopathy. Some of the differentially expressed tear proteins have a role in the barrier function of tears linking the diabetic retinopathy with another eye complication of diabetes, namely the diabetic keratopathy resulting in impaired wound healing. Understanding the molecular events leading to the eye complications caused by hyperglycemia may help the identification of novel biomarkers as well as therapeutic targets in order to improve quality of life of diabetic patients.

  13. Differential Proteomic Analysis of Human Saliva using Tandem Mass Tags Quantification for Gastric Cancer Detection

    PubMed Central

    Xiao, Hua; Zhang, Yan; Kim, Yong; Kim, Sung; Kim, Jae Joon; Kim, Kyoung Mee; Yoshizawa, Janice; Fan, Liu-Yin; Cao, Cheng-Xi; Wong, David T. W.

    2016-01-01

    Novel biomarkers and non-invasive diagnostic methods are urgently needed for the screening of gastric cancer to reduce its high mortality. We employed quantitative proteomics approach to develop discriminatory biomarker signatures from human saliva for the detection of gastric cancer. Salivary proteins were analyzed and compared between gastric cancer patients and matched control subjects by using tandem mass tags (TMT) technology. More than 500 proteins were identified with quantification, and 48 of them showed significant difference expression (p < 0.05) between normal controls and gastric cancer patients, including 7 up-regulated proteins and 41 down-regulated proteins. Five proteins were selected for initial verification by ELISA and three were successfully verified, namely cystatin B (CSTB), triosephosphate isomerase (TPI1), and deleted in malignant brain tumors 1 protein (DMBT1). All three proteins could differentiate gastric cancer patients from normal control subjects, dramatically (p < 0.05). The combination of these three biomarkers could reach 85% sensitivity and 80% specificity for the detection of gastric cancer with accuracy of 0.93. This study provides the proof of concept of salivary biomarkers for the non-invasive detection of gastric cancer. It is highly encouraging to turn these biomarkers into an applicable clinical test after large scale validation. PMID:26911362

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

    PubMed

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

    2015-07-20

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

  15. Mass spectrometry-based proteomics identifies UPF1 as a critical gene expression regulator in MonoMac 6 cells.

    PubMed

    Ochs, Meike J; Ossipova, Elena; Oliynyk, Ganna; Steinhilber, Dieter; Suess, Beatrix; Jakobsson, Per-Johan

    2013-06-07

    5-Lipoxygenase (5-LO) catalyzes the two initial steps in the biosynthesis of leukotrienes, a group of inflammatory lipid mediators derived from arachidonic acid. Recently, we have demonstrated that 5-LO mRNA expression is regulated by alternative splicing and nonsense-mediated mRNA decay (NMD). In addition to this, 5-LO protein expression was reduced on translational level in UPF1 knockdown cells, suggesting that UPF1 has a positive influence on 5-LO translation. Therefore, a mass spectrometry-based proteomics study was performed to identify compartment-specific protein expression changes upon UPF1 knockdown in differentiated and undifferentiated MM6 cells. The proteomics analysis revealed that the knockdown of UPF1 results in numerous protein changes in the microsomal fraction (~21%) but not in the cytosolic fraction (<1%). The results suggest that UPF1 is a critical gene expression regulator in a compartment-specific way. During differentiation by TGFβ and calcitriol, the majority of UPF1 regulated proteins were adjusted to normal level. This indicates that the translational regulation by UPF1 can potentially be cell differentiation-dependent.

  16. Differential skeletal muscle proteome of high- and low-active mice

    PubMed Central

    Dangott, Lawrence J.; Schmitt, Emily E.; Vellers, Heather L.; Lightfoot, J. Timothy

    2014-01-01

    Physical inactivity contributes to cardiovascular disease, type II diabetes, obesity, and some types of cancer. While the literature is clear that there is genetic regulation of physical activity with existing gene knockout data suggesting that skeletal muscle mechanisms contribute to the regulation of activity, actual differences in end-protein expression between high- and low-active mice have not been investigated. This study used two-dimensional differential gel electrophoresis coupled with mass spectrometry to evaluate the proteomic differences between high-active (C57L/J) and low-active (C3H/HeJ) mice in the soleus and extensor digitorum longus (EDL). Furthermore, vivo-morpholinos were used to transiently knockdown candidate proteins to confirm their involvement in physical activity regulation. Proteins with higher expression patterns generally fell into the calcium-regulating and Krebs (TCA) cycle pathways in the high-active mice (e.g., annexin A6, P = 0.0031; calsequestrin 1; P = 0.000025), while the overexpressed proteins in the low-active mice generally fell into cytoskeletal structure- and electron transport chain-related pathways (e.g., ATPase, P = 0.031; NADH dehydrogenase, P = 0.027). Transient knockdown of annexin A6 and calsequestrin 1 protein of high-active mice with vivo-morpholinos resulted in decreased physical activity levels (P = 0.001). These data suggest that high- and low-active mice have unique protein expression patterns and that each pattern contributes to the peripheral capability to be either high- or low-active, suggesting that different specific mechanisms regulate activity leading to the high- or low-activity status of the animal. PMID:24505100

  17. Differential skeletal muscle proteome of high- and low-active mice.

    PubMed

    Ferguson, David P; Dangott, Lawrence J; Schmitt, Emily E; Vellers, Heather L; Lightfoot, J Timothy

    2014-04-15

    Physical inactivity contributes to cardiovascular disease, type II diabetes, obesity, and some types of cancer. While the literature is clear that there is genetic regulation of physical activity with existing gene knockout data suggesting that skeletal muscle mechanisms contribute to the regulation of activity, actual differences in end-protein expression between high- and low-active mice have not been investigated. This study used two-dimensional differential gel electrophoresis coupled with mass spectrometry to evaluate the proteomic differences between high-active (C57L/J) and low-active (C3H/HeJ) mice in the soleus and extensor digitorum longus (EDL). Furthermore, vivo-morpholinos were used to transiently knockdown candidate proteins to confirm their involvement in physical activity regulation. Proteins with higher expression patterns generally fell into the calcium-regulating and Krebs (TCA) cycle pathways in the high-active mice (e.g., annexin A6, P = 0.0031; calsequestrin 1; P = 0.000025), while the overexpressed proteins in the low-active mice generally fell into cytoskeletal structure- and electron transport chain-related pathways (e.g., ATPase, P = 0.031; NADH dehydrogenase, P = 0.027). Transient knockdown of annexin A6 and calsequestrin 1 protein of high-active mice with vivo-morpholinos resulted in decreased physical activity levels (P = 0.001). These data suggest that high- and low-active mice have unique protein expression patterns and that each pattern contributes to the peripheral capability to be either high- or low-active, suggesting that different specific mechanisms regulate activity leading to the high- or low-activity status of the animal.

  18. Differential proteome association study of freeze-thaw damage in ram sperm.

    PubMed

    He, Yuxuan; Wang, Ke; Zhao, Xingxu; Zhang, Yong; Ma, Youji; Hu, Junjie

    2016-02-01

    In this study proteomics analysis was performed to investigate damage caused to ram sperm by the freeze-thaw process. Sperm motility, viability, reactive oxygen species (ROS) and adenosine triphosphate (ATP) content were measured to evaluate sperm quality. Compared with fresh groups, motility, viability and ATP content were all lower in freeze-thawed sperm (P < 0.001), and ROS content was higher (P < 0.001). Moreover, 25 differential protein spots were detected in two-dimensional gels using PDQuest 8.0 software and the corresponding proteins were identified using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF-TOF MS) coupled with searching of the NCBI protein sequence database. Among these proteins, hexokinase1 (HXK1), the enzyme that catalyzes the first step of glycolysis in the sperm glycolytic pathway, is known to be associated with sperm motility. Casein kinase II subunit alpha (CSNK2A2), a serine/threonine-selective protein kinase, is associated with sperm apoptosis. We used immunoblotting and immunofluorescence to analyze the expression and localization of these two proteins. HXK1 and CSNK2A2 expression levels in fresh sperm were significantly higher than that in freeze-thawed sperm (P < 0.001). HXK1 and CSNK2A2 were detected in the main part of the sperm flagellum, and the immunofluorescence signal from these proteins was weakened in the freeze-thawed group. Decreased expression of HXK1 and CSNK2A2 may be associated with decreased sperm motility and viability following freeze-thawing. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Diversity in TAF Proteomics: Consequences for Cellular Differentiation and Migration

    PubMed Central

    Kazantseva, Jekaterina; Palm, Kaia

    2014-01-01

    Development is a highly controlled process of cell proliferation and differentiation driven by mechanisms of dynamic gene regulation. Specific DNA binding factors for establishing cell- and tissue-specific transcriptional programs have been characterised in different cell and animal models. However, much less is known about the role of “core transcription machinery” during cell differentiation, given that general transcription factors and their spatiotemporally patterned activity govern different aspects of cell function. In this review, we focus on the role of TATA-box associated factor 4 (TAF4) and its functional isoforms generated by alternative splicing in controlling lineage-specific differentiation of normal mesenchymal stem cells and cancer stem cells. In the light of our recent findings, induction, control and maintenance of cell differentiation status implies diversification of the transcription initiation apparatus orchestrated by alternative splicing. PMID:25244017

  20. NSI and NSMT: usages of MS/MS fragment ion intensity for sensitive differential proteome detection and accurate protein fold change calculation in relative label-free proteome quantification.

    PubMed

    Wu, Qi; Zhao, Qun; Liang, Zhen; Qu, Yanyan; Zhang, Lihua; Zhang, Yukui

    2012-07-07

    Although widely applied in the label-free quantification of proteomics, spectral count (SC)-based abundance measurements suffer from the narrow dynamic range of attainable ratios, leading to the serious underestimation of true protein abundance fold changes, especially when studying biological samples that exhibit very large fold changes in protein expression. MS/MS fragment ion intensity, as an alternative to SC, has recently gained acceptance as the abundance feature of protein in label-free proteomic studies. Herein, we implemented two formats of MS/MS fragment ion intensity, Spectral Index (SI) and Summed MS/MS TIC (SMT), to alleviate this particular deficiency arising from SC. Both were in forms of replacing SC in the Normalized Spectral Abundance Factor (NSAF) formula, resulting in two algorithms, abbreviated as NSI and NSMT, respectively. The necessity of the normalization process was validated using a publicly available dataset. Furthermore, when applied to another well characterized benchmark dataset, both NSI and NSMT showed improved overall accuracy over NSAF for the relative quantification of proteomes. Hereinto, NSI enabled the sensitive detection of differentially expressed proteins, while NSMT ensured accurate calculation for protein abundance fold change. Therefore, the selective use of both algorithms might facilitate the screening and quantification of potential biomarkers on the proteome scale.

  1. Proteomic Analysis of Human Pluripotent Stem Cell Cardiomyogenesis Revealed Altered Expression of Metabolic Enzymes and PDLIM5 Isoforms.

    PubMed

    Konze, Sarah A; Werneburg, Sebastian; Oberbeck, Astrid; Olmer, Ruth; Kempf, Henning; Jara-Avaca, Monica; Pich, Andreas; Zweigerdt, Robert; Buettner, Falk F R

    2017-03-03

    Human pluripotent stem cells (hPSCs), both embryonic (hESCs) and induced (hiPSCs), can be differentiated into derivatives of the three germ layers and are promising tools in regenerative medicine. Cardiovascular diseases are the top-ranking cause of premature death worldwide, and cell replacement therapies based on in vitro differentiated cardiomyocytes might provide a promising perspective to cure patients in the future. The molecular processes during hPSC cardiomyogenesis are far from being fully understood, and we thus have focused here on characterizing the proteome along hESC in vitro differentiation into cardiomyocytes (CMs). Stable isotope labeling of amino acids in cell culture was applied to quantitatively assess the proteome throughout defined stages of hESC cardiomyogenesis. Genetically enriched, >90% pure CM populations were used for shotgun proteomics, leading to the identification and quantitative determination of several thousand proteins. Pathway analysis revealed alterations in energy metabolism during cardiomyogenesis. Enzymes of glycolysis were identified as up-regulated upon differentiation, whereas enzymes involved in oxidative phosphorylation were down-regulated in aggregates on day 20 of differentiation (<10% CMs) and reconstituted on day 35 in >90% pure CMs. A structural protein that attracted our attention was the PDZ and LIM domain containing protein 5 (PDLIM5), which was strongly up-regulated during cardiomyogenesis and for which we detected novel stage-specific isoforms. Notably, expression of the 53 kDa isoforms b and g (corresponding to transcript variants 2 and 7) of PDLIM5 occurred simultaneously to the onset of expression of the early cardiac transcription factor NKX2.5, known to play a key role in cardiac development.

  2. Analysis of differentially expressed proteins in Yersinia enterocolitica-infected HeLa cells.

    PubMed

    Alugubelly, Navatha; Hercik, Kamil; Kibler, Peter; Nanduri, Bindu; Edelmann, Mariola J

    2016-05-01

    Yersinia enterocolitica is a facultative intracellular pathogen and a causative agent of yersiniosis, which can be contracted by ingestion of contaminated food. Yersinia secretes virulence factors to subvert critical pathways in the host cell. In this study we utilized shotgun label-free proteomics to study differential protein expression in epithelial cells infected with Y.enterocolitica. We identified a total of 551 proteins, amongst which 42 were downregulated (including Prostaglandin E Synthase 3, POH-1 and Karyopherin alpha) and 22 were upregulated (including Rab1 and RhoA) in infected cells. We validated some of these results by western blot analysis of proteins extracted from Caco-2 and HeLa cells. The proteomic dataset was used to identify host canonical pathways and molecular functions modulated by this infection in the host cells. This study constitutes a proteome of Yersinia-infected cells and can support new discoveries in the area of host-pathogen interactions. We describe a proteome of Yersinia enterocolitica-infected HeLa cells, including a description of specific proteins differentially expressed upon infection, molecular functions as well as pathways altered during infection. This proteomic study can lead to a better understanding of Y. enterocolitica pathogenesis in human epithelial cells. Copyright © 2016. Published by Elsevier B.V.

  3. Species and tissues specific differentiation of processed animal proteins in aquafeeds using proteomics tools.

    PubMed

    Rasinger, J D; Marbaix, H; Dieu, M; Fumière, O; Mauro, S; Palmblad, M; Raes, M; Berntssen, M H G

    2016-09-16

    The rapidly growing aquaculture industry drives the search for sustainable protein sources in fish feed. In the European Union (EU) since 2013 non-ruminant processed animal proteins (PAP) are again permitted to be used in aquafeeds. To ensure that commercial fish feeds do not contain PAP from prohibited species, EU reference methods were established. However, due to the heterogeneous and complex nature of PAP complementary methods are required to guarantee the safe use of this fish feed ingredient. In addition, there is a need for tissue specific PAP detection to identify the sources (i.e. bovine carcass, blood, or meat) of illegal PAP use. In the present study, we investigated and compared different protein extraction, solubilisation and digestion protocols on different proteomics platforms for the detection and differentiation of prohibited PAP. In addition, we assessed if tissue specific PAP detection was feasible using proteomics tools. All work was performed independently in two different laboratories. We found that irrespective of sample preparation gel-based proteomics tools were inappropriate when working with PAP. Gel-free shotgun proteomics approaches in combination with direct spectral comparison were able to provide quality species and tissue specific data to complement and refine current methods of PAP detection and identification. To guarantee the safe use of processed animal protein (PAP) in aquafeeds efficient PAP detection and monitoring tools are required. The present study investigated and compared various proteomics workflows and shows that the application of shotgun proteomics in combination with direct comparison of spectral libraries provides for the desired species and tissue specific classification of this heat sterilized and pressure treated (≥133°C, at 3bar for 20min) protein feed ingredient. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Characterization of human reflex tear proteome reveals high expression of lacrimal proline-rich protein 4 (PRR4).

    PubMed

    Perumal, Natarajan; Funke, Sebastian; Wolters, Dominik; Pfeiffer, Norbert; Grus, Franz H

    2015-10-01

    In-depth studies on the proteome of reflex tears are still inadequate. Hence, further studies on this subject will unravel the key proteins which are conjectured to possess vital functions in the protection of the ocular surface. Therefore, this study investigated the differences in the expression levels in proteome of reflex compared to basal tears. Basal (n = 10) and reflex (n = 10) tear samples from healthy subjects were collected employing the capillary method, subsequently pooled and the proteomes were characterized employing 1DE combined with LC-ESI-MS/MS strategy for label-free quantitative (LFQ) analysis. The differentially expressed proteins were validated by 2DE combined with LC-ESI-MS/MS and targeted-MS approach called accurate inclusion mass screening (AIMS) strategies. The analysis of the reflex tear proteome demonstrated increased abundance in proline-rich protein 4 (PRR4) and zymogen granule protein 16 homolog B (ZG16B) for the first time. Other abundant lacrimal proteins, e.g. lactotransferrin and lysozyme remained constant. Predominantly, the lacrimal gland-specific PRR4 represents the major increased protein in reflex tears in an attempt to wash out irritants that come into contact with the eye. Conversely, decreased abundance in Ig alpha-1 chain C, polymeric immunoglobulin receptor, cystatin S/SN, clusterin and mammaglobin were observed. This study had further unraveled the intricate proteome regulation during reflex tearing, especially the potential role of PRR4, which may be the key player in the protection and maintenance of dynamic balance of the ocular surface. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Differential proteome analysis of the cell differentiation regulated by BCC, CRH, CXCR4, GnRH, GPCR, IL1 signaling pathways in Chinese fire-bellied newt limb regeneration.

    PubMed

    Geng, Xiaofang; Xu, Tiantian; Niu, Zhipeng; Zhou, Xiaochun; Zhao, Lijun; Xie, Zhaohui; Xue, Deming; Zhang, Fuchun; Xu, Cunshuan

    2014-01-01

    Following amputation, the newt has the remarkable ability to regenerate its limb, and this process involves dedifferentiation, proliferation and differentiation. To investigate the potential proteome during a dynamic network of Chinese fire-bellied newt limb regeneration (CNLR), two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mass spectrum (MS) were applied to examine changes in the proteome that occurred at 11 time points after amputation. Meanwhile, several proteins were selected to validate their expression levels by Western blot. The results revealed that 1476 proteins had significantly changed as compared to the control group. Gene Ontology annotation and protein network analysis by Ingenuity Pathway Analysis 9.0 (IPA) software suggested that the differentially expressed proteins were involved in 33 kinds of physiological activities including signal transduction, cell proliferation, cell differentiation, etc. Among these proteins, 407 proteins participated in cell differentiation with 212 proteins in the differentiation of skin cell, myocyte, neurocyte, chondrocyte and osteocyte, and 37 proteins participated in signaling pathways of BCC, CRH, CXCR4, GnRH, GPCR and IL1 which regulated cell differentiation and redifferentiation. On the other hand, the signal transduction activity and cell differentiation activity were analyzed by IPA based on the changes in the expression of these proteins. The results showed that BCC, CRH, CXCR4, GnRH, GPCR and IL1 signaling pathways played an important role in regulating the differentiation of skin cell, myocyte, neurocyte, chondrocyte and osteocyte during CNLR.

  6. Combined gene expression and proteomic analysis of EGF induced apoptosis in A431 cells suggests multiple pathways trigger apoptosis.

    PubMed

    Alanazi, Ibrahim; Ebrahimie, Esmaeil; Hoffmann, Peter; Adelson, David L

    2013-11-01

    A431 cells, derived from epidermoid carcinoma, overexpress the epidermal growth factor receptor (EGFR) and when treated with a high dose of EGF will undergo apoptosis. We exploited microarray and proteomics techniques and network prediction to study the regulatory mechanisms of EGF-induced apoptosis in A431 cells. We observed significant changes in gene expression in 162 genes, approximately evenly split between pro-apoptotic and anti-apoptotic genes and identified 30 proteins from the proteomic data that had either pro or anti-apoptotic annotation. Our correlation analysis of gene expression and proteome modeled a number of distinct sub-networks that are associated with the onset of apoptosis, allowing us to identify specific pathways and components. These include components of the interferon signalling pathway, and down stream components, including cytokines and suppressors of cytokine signalling. A central component of almost all gene expression sub-networks identified was TP53, which is mutated in A431 cells, and was down regulated. This down regulation of TP53 appeared to be correlated with proteomic sub-networks of cytoskeletal or cell adhesion components that might induce apoptosis by triggering cytochrome C release. Of the only three genes also differentially expressed as proteins, only serpinb1 had a known association with apoptosis. We confirmed that up regulation and cleavage of serpinb1 into L-DNAaseII was correlated with the induction of apoptosis. It is unlikely that a single pathway, but more likely a combination of pathways is needed to trigger EGF induced apoptosis in A431cells.

  7. Differential abundance of sarcoplasmic proteome explains animal effect on beef Longissimus lumborum color stability.

    PubMed

    Canto, Anna C V C S; Suman, Surendranath P; Nair, Mahesh N; Li, Shuting; Rentfrow, Gregg; Beach, Carol M; Silva, Teofilo J P; Wheeler, Tommy L; Shackelford, Steven D; Grayson, Adria; McKeith, Russell O; King, D Andy

    2015-04-01

    The sarcoplasmic proteome of beef Longissimus lumborum demonstrating animal-to-animal variation in color stability was examined to correlate proteome profile with color. Longissimus lumborum (36 h post-mortem) muscles were obtained from 73 beef carcasses, aged for 13 days, and fabricated to 2.5-cm steaks. One steak was allotted to retail display, and another was immediately vacuum packaged and frozen at -80°C. Aerobically packaged steaks were stored under display, and color was evaluated on days 0 and 11. The steaks were ranked based on redness and color stability on day 11, and ten color-stable and ten color-labile carcasses were identified. Sarcoplasmic proteome of frozen steaks from the selected carcasses was analyzed. Nine proteins were differentially abundant in color-stable and color-labile steaks. Three glycolytic enzymes (phosphoglucomutase-1, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase M2) were over-abundant in color-stable steaks and positively correlated (P<0.05) to redness and color stability. These results indicated that animal variations in proteome contribute to differences in beef color.

  8. Expression Profiling and Proteomic Analysis of JIN Chinese Herbal Formula in Lung Carcinoma H460 Xenografts

    PubMed Central

    Zheng, Luyu; Zhang, Weiyi; Jiang, Miao; Zhang, Huarong; Xiong, Fei; Yu, Yang; Chen, Meijuan; Zhou, Jing; Dai, Xiaoming; Jiang, Ming; Wang, Mingyan; Cheng, Ge; Duan, Jinao; Yu, Wei; Lin, Biaoyang; Fu, Haian; Zhang, Xu

    2013-01-01

    Many traditional Chinese medicine (TCM) formulae have been used in cancer therapy. The JIN formula is an ancient herbal formula recorded in the classic TCM book Jin Kui Yao Lue (Golden Chamber). The JIN formula significantly delayed the growth of subcutaneous human H460 xenografted tumors in vivo compared with the growth of mock controls. Gene array analysis of signal transduction in cancer showed that the JIN formula acted on multiple targets such as the mitogen-activated protein kinase, hedgehog, and Wnt signaling pathways. The coformula treatment of JIN and diamminedichloroplatinum (DDP) affected the stress/heat shock pathway. Proteomic analysis showed 36 and 84 differentially expressed proteins between the mock and DDP groups and between the mock and JIN groups, respectively. GoMiner analysis revealed that the differentially expressed proteins between the JIN and mock groups were enriched during cellular metabolic processes, and so forth. The ones between the DDP and mock groups were enriched during protein-DNA complex assembly, and so forth. Most downregulated proteins in the JIN group were heat shock proteins (HSPs) such as HSP90AA1 and HSPA1B, which could be used as markers to monitor responses to the JIN formula therapy. The mechanism of action of the JIN formula on HSP proteins warrants further investigation. PMID:24066008

  9. Differential Response and Priming Dose Effect on the Proteome of Human Fibroblast and Stem Cells Induced by Exposure to Low Doses of Ionizing Radiation.

    PubMed

    Hauptmann, Monika; Haghdoost, Siamak; Gomolka, Maria; Sarioglu, Hakan; Ueffing, Marius; Dietz, Anne; Kulka, Ulrike; Unger, Kristian; Babini, Gabriele; Harms-Ringdahl, Mats; Ottolenghi, Andrea; Hornhardt, Sabine

    2016-03-01

    It has been suggested that a mechanistic understanding of the cellular responses to low dose and dose rate may be valuable in reducing some of the uncertainties involved in current risk estimates for cancer- and non-cancer-related radiation effects that are inherited in the linear no-threshold hypothesis. In this study, the effects of low-dose radiation on the proteome in both human fibroblasts and stem cells were investigated. Particular emphasis was placed on examining: 1. the dose-response relationships for the differential expression of proteins in the low-dose range (40-140 mGy) of low-linear energy transfer (LET) radiation; and 2. the effect on differential expression of proteins of a priming dose given prior to a challenge dose (adaptive response effects). These studies were performed on cultured human fibroblasts (VH10) and human adipose-derived stem cells (ADSC). The results from the VH10 cell experiments demonstrated that low-doses of low-LET radiation induced unique patterns of differentially expressed proteins for each dose investigated. In addition, a low priming radiation dose significantly changed the protein expression induced by the subsequent challenge exposure. In the ADSC the number of differentially expressed proteins was markedly less compared to VH10 cells, indicating that ADSC differ in their intrinsic response to low doses of radiation. The proteomic results are further discussed in terms of possible pathways influenced by low-dose irradiation.

  10. Efficient isolation and proteomic analysis of cell plasma membrane proteins in gastric cancer reveal a novel differentiation and progression related cell surface marker, R-cadherin.

    PubMed

    Chen, Bo; Luo, Qi-Cong; Chen, Jian-Bo; Lin, Li-E; Luo, Ming-Xu; Ren, Hong-Yue; Chen, Pei-Qiong; Shi, Lian-Guo

    2016-09-01

    Cell plasma membrane proteins, playing a crucial role in cell malignant transformation and development, were the main targets of tumor detection and therapy. In this study, CyDye/biotin double-labeling proteomic approach was adopted to profile the membrane proteome of gastric cancer cell line BGC-823 and paired immortalized gastric epithelial cell GES-1. Real-time PCR, Western blotting, and immunohistochemical staining were used to validate the differential expression of a novel identified cell surface marker R-cadherin in gastric cancer cells and tissues. Clinicopathological study and survival analysis were performed to estimate its roles in tumor progression and outcome prediction. Real-time PCR and Western blotting showed that the expression level of R-cadherin in gastric cancer were significantly lower than non-cancerous epithelial cell and tissues. Clinicopathological study indicated that R-cadherin was dominantly expressed on cell surface of normal gastric epithelium, and its expression deletion in gastric cancer tissues was associated with tumor site, differentiation, lymph node metastasis, and pTNM (chi-square test, P < 0.05). Those patients with R-cadherin positive expression displayed better overall survivals than negative expression group (log-rank test, P = 0.000). Cox multivariate survival analysis revealed lacking the expression of R-cadherin was a main independent predictor for poor clinical outcome in gastric cancer (RR = 5.680, 95 % CI 2.250-14.341, P < 0.01). We have established a fundamental membrane proteome database for gastric cancer and identified R-cadherin as a tumor differentiation and progression-related cell surface marker of gastric cancer. Lacking the expression of R-cadherin indicates poor prognosis in patients with gastric cancer.

  11. The expression of tomato prosystemin gene in tobacco plants highly affects host proteomic repertoire.

    PubMed

    Rocco, Mariapina; Corrado, Giandomenico; Arena, Simona; D'Ambrosio, Chiara; Tortiglione, Claudia; Sellaroli, Stefano; Marra, Mauro; Rao, Rosa; Scaloni, Andrea

    2008-07-21

    Systemin, an octadecapeptide isolated from tomato, is a primary signal molecule involved in the local and systemic responses to pest attack, elicited by activation of a set of defence genes. It derives from processing of prosystemin, a prohormone of almost 200 amino acids. Prosystemin orthologues have been found in other Solanaceae species but not in tobacco, where are present hydroxyproline-rich peptides functionally but not structurally related to tomato systemin. Molecular events leading to the release of signalling peptides from protein precursors are unknown in plants; the occurrence of a family of signal molecules suggests that initiation of wound response may involve different processing mechanisms. It has been previously shown that the protein product from an engineered tomato prosystemin gene is processed in tobacco, thus suggesting that the components responsible for its post-translational modifications are present in this species. By analyzing analysing the proteome repertoire of transformed tobacco plant leaves with 2-DE, here we demonstrate that the constitutive expression of the tomato prosystemin gene highly affected host protein synthesis. In particular, engineered plants showed a number of differentially synthesized proteins that were identified by PMF MALDI-TOF and microLC-ESI-IT-MS/MS experiments as polypeptide species involved in protection from pathogens and oxidative stress, or in carbon/energy metabolism. Significant differences in over-produced proteins were observed with respect to previous data reported on systemin-engineered tomato plants. Our results strongly support the need of using proteomic approaches during systematic analysis of plant tissues to investigate the principle of substantial equivalence in transgenic plants expressing a transgene coding for a signalling molecule.

  12. Epigenetic and Proteomic Expression Changes Promoted by Eating Addictive-Like Behavior.

    PubMed

    Mancino, Samantha; Burokas, Aurelijus; Gutiérrez-Cuesta, Javier; Gutiérrez-Martos, Miriam; Martín-García, Elena; Pucci, Mariangela; Falconi, Anastasia; D'Addario, Claudio; Maccarrone, Mauro; Maldonado, Rafael

    2015-11-01

    An increasing perspective conceptualizes obesity and overeating as disorders related to addictive-like processes that could share common neurobiological mechanisms. In the present study, we aimed at validating an animal model of eating addictive-like behavior in mice, based on the DSM-5 substance use disorder criteria, using operant conditioning maintained by highly palatable chocolate-flavored pellets. For this purpose, we evaluated persistence of food-seeking during a period of non-availability of food, motivation for food, and perseverance of responding when the reward was associated with a punishment. This model has allowed identifying extreme subpopulations of mice related to addictive-like behavior. We investigated in these subpopulations the epigenetic and proteomic changes. A significant decrease in DNA methylation of CNR1 gene promoter was revealed in the prefrontal cortex of addict-like mice, which was associated with an upregulation of CB1 protein expression in the same brain area. The pharmacological blockade (rimonabant 3 mg/kg; i.p.) of CB1 receptor during the late training period reduced the percentage of mice that accomplished addiction criteria, which is in agreement with the reduced performance of CB1 knockout mice in this operant training. Proteomic studies have identified proteins differentially expressed in mice vulnerable or not to addictive-like behavior in the hippocampus, striatum, and prefrontal cortex. These changes included proteins involved in impulsivity-like behavior, synaptic plasticity, and cannabinoid signaling modulation, such as alpha-synuclein, phosphatase 1-alpha, doublecortin-like kinase 2, and diacylglycerol kinase zeta, and were validated by immunoblotting. This model provides an excellent tool to investigate the neurobiological substrate underlying the vulnerability to develop eating addictive-like behavior.

  13. Epigenetic and Proteomic Expression Changes Promoted by Eating Addictive-Like Behavior

    PubMed Central

    Mancino, Samantha; Burokas, Aurelijus; Gutiérrez-Cuesta, Javier; Gutiérrez-Martos, Miriam; Martín-García, Elena; Pucci, Mariangela; Falconi, Anastasia; D'Addario, Claudio; Maccarrone, Mauro; Maldonado, Rafael

    2015-01-01

    An increasing perspective conceptualizes obesity and overeating as disorders related to addictive-like processes that could share common neurobiological mechanisms. In the present study, we aimed at validating an animal model of eating addictive-like behavior in mice, based on the DSM-5 substance use disorder criteria, using operant conditioning maintained by highly palatable chocolate-flavored pellets. For this purpose, we evaluated persistence of food-seeking during a period of non-availability of food, motivation for food, and perseverance of responding when the reward was associated with a punishment. This model has allowed identifying extreme subpopulations of mice related to addictive-like behavior. We investigated in these subpopulations the epigenetic and proteomic changes. A significant decrease in DNA methylation of CNR1 gene promoter was revealed in the prefrontal cortex of addict-like mice, which was associated with an upregulation of CB1 protein expression in the same brain area. The pharmacological blockade (rimonabant 3 mg/kg; i.p.) of CB1 receptor during the late training period reduced the percentage of mice that accomplished addiction criteria, which is in agreement with the reduced performance of CB1 knockout mice in this operant training. Proteomic studies have identified proteins differentially expressed in mice vulnerable or not to addictive-like behavior in the hippocampus, striatum, and prefrontal cortex. These changes included proteins involved in impulsivity-like behavior, synaptic plasticity, and cannabinoid signaling modulation, such as alpha-synuclein, phosphatase 1-alpha, doublecortin-like kinase 2, and diacylglycerol kinase zeta, and were validated by immunoblotting. This model provides an excellent tool to investigate the neurobiological substrate underlying the vulnerability to develop eating addictive-like behavior. PMID:25944409

  14. Effect by acupuncture on hypothalamic expression of maternally separated rats: proteomic approach.

    PubMed

    Kim, Hak-Jae; Park, Hae Jeong; Hong, Mee Sook; Song, Jeong Yoon; Park, Hyun-Kyung; Jo, Dae Jean; Park, Sung Wook; HwanYun, Dong; Park, Hun-Kuk; Yang, Jong-Soo; Ban, Ju Yeon; Chung, Joo-Ho

    2010-02-01

    Early stressors can influence the development of biological and neurological systems. Maternal separation (social isolation) in early life may increase vulnerability to neurodegenerative diseases and neuropsychiatric disorders over the lifespan. To identify new proteins on acupuncture effects in maternally separated rats, an animal model for study of early environmental insults, proteomic approach on the expression of the hypothalamic proteins was performed. On post-natal day 14, rat pups were randomly divided into four groups: pups kept with their mothers for 7 days; pups kept with their mothers with acupuncture daily to HT8 (Sobu); maternally separated pups; maternally separated pups with acupuncture. The hypothalamic proteins were analysed by two-dimensional electrophoresis coupled with matrix assisted laser desorption/ionization time-of-flight mass spectrometer. The results showed that 27 spots were differentially and commonly expressed. Of 27 spots, 21 spots were identified while six spots were not, and 15 proteins were known proteins. In maternally separated group, the expressions of 14 proteins were down-regulated, compared to control group. In group of maternally separation with acupuncture, five proteins were down-regulated and nine were up-regulated, compared to the maternally separated group. Among nine proteins up-regulated by acupuncture treatment, we found four proteins (dihydropyrimidinase-like 2, dystrophin-related protein 2, tubulin, alpha 1a and syntaxin 1b) related to neurodevelopment. The result suggests that acupuncture to HT8 may affect neurodevelopment, and acupuncture may be a possible therapy for neurodevelopmental disorders.

  15. Differentiating Proteomic Biomarkers in Breast Cancer by Laser Capture Microdissection and MALDI MS

    PubMed Central

    Sanders, Melinda E.; Dias, Eduardo C.; Xu, Baogang J.; Mobley, James A.; Billheimer, Dean; Roder, Heinrich; Grigorieva, Julia; Dowsett, Mitchell; Arteaga, Carlos L.; Caprioli, Richard M.

    2009-01-01

    We assessed proteomic patterns in breast cancer using MALDI MS and laser capture microdissected cells. Protein and peptide expression in invasive mammary carcinoma versus normal mammary epithelium and estrogen-receptor positive versus estrogen-receptor negative tumors were compared. Biomarker candidates were identified by statistical analysis and classifiers were developed and validated in blinded test sets. Several of the m/z features used in the classifiers were identified by LC–MS/MS and two were confirmed by immunohistochemistry. PMID:18386930

  16. Proteomic profiling of the mesenteric lymph after hemorrhagic shock: Differential gel electrophoresis and mass spectrometry analysis

    PubMed Central

    2011-01-01

    Experiments show that upon traumatic injury the composition of mesenteric lymph changes such that it initiates an immune response that can ultimately result in multiple organ dysfunction syndrome (MODS). To identify candidate protein mediators of this process we carried out a quantitative proteomic study on mesenteric lymph from a well characterized rat shock model. We analyzed three animals using analytical 2D differential gel electrophoresis. Intra-animal variation for the majority of protein spots was minor. Functional clustering of proteins revealed changes arising from several global classes that give novel insight into fundamental mechanisms of MODS. Mass spectrometry based proteomic analysis of proteins in mesenteric lymph can effectively be used to identify candidate mediators and loss of protective agents in shock models. PMID:21906351

  17. Comparing calpain- and caspase-3-mediated degradation patterns in traumatic brain injury by differential proteome analysis

    PubMed Central

    Liu, Ming Cheng; Akle, Veronica; Zheng, Wenrong; Dave, Jitendra R.; Tortella, Frank C.; Hayes, Ronald L.; Wang, Kevin K. W.

    2005-01-01

    A major theme of TBI (traumatic brain injury) pathology is the over-activation of multiple proteases. We have previously shown that calpain-1 and -2, and caspase-3 simultaneously produced αII-spectrin BDPs (breakdown products) following TBI. In the present study, we attempted to identify a comprehensive set of protease substrates (degradome) for calpains and caspase-3. We further hypothesized that the TBI differential proteome is likely to overlap significantly with the calpain- and caspase-3-degradomes. Using a novel HTPI (high throughput immunoblotting) approach and 1000 monoclonal antibodies (PowerBlot™), we compared rat hippocampal lysates from 4 treatment groups: (i) naïve, (ii) TBI (48 h after controlled cortical impact), (iii) in vitro calpain-2 digestion and (iv) in vitro caspase-3 digestion. In total, we identified 54 and 38 proteins that were vulnerable to calpain-2 and caspase-3 proteolysis respectively. In addition, the expression of 48 proteins was down-regulated following TBI, whereas that of only 9 was up-regulated. Among the proteins down-regulated in TBI, 42 of them overlapped with the calpain-2 and/or caspase-3 degradomes, suggesting that they might be proteolytic targets after TBI. We further confirmed several novel TBI-linked proteolytic substrates, including βII-spectrin, striatin, synaptotagmin-1, synaptojanin-1 and NSF (N-ethylmaleimide-sensitive fusion protein) by traditional immunoblotting. In summary, we demonstrated that HTPI is a novel and powerful method for studying proteolytic pathways in vivo and in vitro. PMID:16351572

  18. Comparative Analysis of Salivary Gland Proteomes of Two Glossina Species that Exhibit Differential Hytrosavirus Pathologies

    PubMed Central

    Kariithi, Henry M.; İnce, İkbal Agah; Boeren, Sjef; Murungi, Edwin K.; Meki, Irene K.; Otieno, Everlyne A.; Nyanjom, Steven R. G.; van Oers, Monique M.; Vlak, Just M.; Abd-Alla, Adly M. M.

    2016-01-01

    Glossina pallidipes salivary gland hypertrophy virus (GpSGHV; family Hytrosaviridae) is a dsDNA virus exclusively pathogenic to tsetse flies (Diptera; Glossinidae). The 190 kb GpSGHV genome contains 160 open reading frames and encodes more than 60 confirmed proteins. The asymptomatic GpSGHV infection in flies can convert to symptomatic infection that is characterized by overt salivary gland hypertrophy (SGH). Flies with SGH show reduced general fitness and reproductive dysfunction. Although the occurrence of SGH is an exception rather than the rule, G. pallidipes is thought to be the most susceptible to expression of overt SGH symptoms compared to other Glossina species that are largely asymptomatic. Although Glossina salivary glands (SGs) play an essential role in GpSGHV transmission, the functions of the salivary components during the virus infection are poorly understood. In this study, we used mass spectrometry to study SG proteomes of G. pallidipes and G. m. morsitans, two Glossina model species that exhibit differential GpSGHV pathologies (high and low incidence of SGH, respectively). A total of 540 host proteins were identified, of which 23 and 9 proteins were significantly up- and down-regulated, respectively, in G. pallidipes compared to G. m. morsitans. Whereas 58 GpSGHV proteins were detected in G. pallidipes F1 progenies, only 5 viral proteins were detected in G. m. morsitans. Unlike in G. pallidipes, qPCR assay did not show any significant increase in virus titers in G. m. morsitans F1 progenies, confirming that G. m. morsitans is less susceptible to GpSGHV infection and replication compared to G. pallidipes. Based on our results, we speculate that in the case of G. pallidipes, GpSGHV employs a repertoire of host intracellular signaling pathways for successful infection. In the case of G. m. morsitans, antiviral responses appeared to be dominant. These results are useful for designing additional tools to investigate the Glossina-GpSGHV interactions

  19. Comparative Analysis of Salivary Gland Proteomes of Two Glossina Species that Exhibit Differential Hytrosavirus Pathologies.

    PubMed

    Kariithi, Henry M; İnce, İkbal Agah; Boeren, Sjef; Murungi, Edwin K; Meki, Irene K; Otieno, Everlyne A; Nyanjom, Steven R G; van Oers, Monique M; Vlak, Just M; Abd-Alla, Adly M M

    2016-01-01

    Glossina pallidipes salivary gland hypertrophy virus (GpSGHV; family Hytrosaviridae) is a dsDNA virus exclusively pathogenic to tsetse flies (Diptera; Glossinidae). The 190 kb GpSGHV genome contains 160 open reading frames and encodes more than 60 confirmed proteins. The asymptomatic GpSGHV infection in flies can convert to symptomatic infection that is characterized by overt salivary gland hypertrophy (SGH). Flies with SGH show reduced general fitness and reproductive dysfunction. Although the occurrence of SGH is an exception rather than the rule, G. pallidipes is thought to be the most susceptible to expression of overt SGH symptoms compared to other Glossina species that are largely asymptomatic. Although Glossina salivary glands (SGs) play an essential role in GpSGHV transmission, the functions of the salivary components during the virus infection are poorly understood. In this study, we used mass spectrometry to study SG proteomes of G. pallidipes and G. m. morsitans, two Glossina model species that exhibit differential GpSGHV pathologies (high and low incidence of SGH, respectively). A total of 540 host proteins were identified, of which 23 and 9 proteins were significantly up- and down-regulated, respectively, in G. pallidipes compared to G. m. morsitans. Whereas 58 GpSGHV proteins were detected in G. pallidipes F1 progenies, only 5 viral proteins were detected in G. m. morsitans. Unlike in G. pallidipes, qPCR assay did not show any significant increase in virus titers in G. m. morsitans F1 progenies, confirming that G. m. morsitans is less susceptible to GpSGHV infection and replication compared to G. pallidipes. Based on our results, we speculate that in the case of G. pallidipes, GpSGHV employs a repertoire of host intracellular signaling pathways for successful infection. In the case of G. m. morsitans, antiviral responses appeared to be dominant. These results are useful for designing additional tools to investigate the Glossina-GpSGHV interactions.

  20. Differential proteomics and functional research following gene therapy in a mouse model of Leber congenital amaurosis.

    PubMed

    Zheng, Qinxiang; Ren, Yueping; Tzekov, Radouil; Zhang, Yuanping; Chen, Bo; Hou, Jiangping; Zhao, Chunhui; Zhu, Jiali; Zhang, Ying; Dai, Xufeng; Ma, Shan; Li, Jia; Pang, Jijing; Qu, Jia; Li, Wensheng

    2012-01-01

    Leber congenital amaurosis (LCA) is one of the most severe forms of inherited retinal degeneration and can be caused by mutations in at least 15 different genes. To clarify the proteomic differences in LCA eyes, a cohort of retinal degeneration 12 (rd12) mice, an LCA2 model caused by a mutation in the RPE65 gene, were injected subretinally with an AAV vector (scAAV5-smCBA-hRPE65) in one eye, while the contralateral eye served as a control. Proteomics were compared between untreated rd12 and normal control retinas on P14 and P21, and among treated and untreated rd12 retinas and control retinas on P42. Gene therapy in rd12 mice restored retinal function in treated eyes, which was demonstrated by electroretinography (ERG). Proteomic analysis successfully identified 39 proteins expressed differently among the 3 groups. The expression of 3 proteins involved in regulation of apoptosis and neuroptotection (alpha A crystallin, heat shock protein 70 and peroxiredoxin 6) were investigated further. Immunofluorescence, Western blot and real-time PCR confirmed the quantitative changes in their expression. Furthermore, cell culture studies suggested that peroxiredoxin 6 could act in an antioxidant role in rd12 mice. Our findings support the feasibility of gene therapy in LCA2 patients and support a role for alpha A crystallin, heat shock protein 70 and peroxiredoxin 6 in the pathogenetic mechanisms involved in LCA2 disease process.

  1. In-depth proteomic analyses of exosomes isolated from expressed prostatic secretions in urine

    PubMed Central

    Sinha, Ankit; Nyalwidhe, Julius O.; Brooks, Jasmin; Semmes, O. John; Troyer, Dean A.; Lance, Raymond S.; Kislinger, Thomas; Drake, Richard R.

    2013-01-01

    Expressed prostatic secretions (EPS) are proximal fluids of the prostate that are increasingly being utilized as a clinical source for diagnostic and prognostic assays for prostate cancer (PCa). These fluids contain an abundant amount of microvesicles reflecting the secretory function of the prostate gland, and their protein composition remains poorly defined in relation to PCa. Using expressed prostatic secretions in urine (EPS-urine), exosome preparations were characterized by a shotgun proteomics procedure. In pooled EPS-urine exosome samples, ~900 proteins were detected. Many of these have not been previously observed in the soluble proteome of EPS generated by our labs or other related exosome proteomes. We performed systematic comparisons of our data against previously published, prostate-related proteomes and global annotation analyses to highlight functional processes within the proteome of EPS-urine derived exosomes. The acquired proteomic data has been deposited to the Tranche repository and will lay the foundation for more extensive investigations of PCa derived exosomes in the context of biomarker discovery and cancer biology. PMID:23533145

  2. Quantitative Proteomics Reveals GIMAP Family Proteins 1 and 4 to Be Differentially Regulated during Human T Helper Cell Differentiation *S⃞

    PubMed Central

    Filén, Jan-Jonas; Filén, Sanna; Moulder, Robert; Tuomela, Soile; Ahlfors, Helena; West, Anne; Kouvonen, Petri; Kantola, Suvi; Björkman, Mari; Katajamaa, Mikko; Rasool, Omid; Nyman, Tuula A.; Lahesmaa, Riitta

    2009-01-01

    T helper (Th) cells differentiate into functionally distinct effector cell subsets of which Th1 and Th2 cells are best characterized. Besides T cell receptor signaling, IL-12-induced STAT4 and T-bet- and IL-4-induced STAT6 and GATA3 signaling pathways are the major players regulating the Th1 and Th2 differentiation process, respectively. However, there are likely to be other yet unknown factors or pathways involved. In this study we used quantitative proteomics exploiting cleavable ICAT labeling and LC-MS/MS to identify IL-4-regulated proteins from the microsomal fractions of CD4+ cells extracted from umbilical cord blood. We were able to identify 557 proteins of which 304 were also quantified. This study resulted in the identification of the down-regulation of small GTPases GIMAP1 and GIMAP4 by IL-4 during Th2 differentiation. We also showed that both GIMAP1 and GIMAP4 genes are up-regulated by IL-12 and other Th1 differentiation-inducing cytokines in cells induced to differentiate toward Th1 lineage and down-regulated by IL-4 in cells induced to Th2. Our results indicate that the GIMAP (GTPase of the immunity-associated protein) family of proteins is differentially regulated during Th cell differentiation. PMID:18701445

  3. Utility of proteomics techniques for assessing protein expression

    USDA-ARS?s Scientific Manuscript database

    Proteomic technologies are currently used as an effective analytical tool for examining modifications in protein profiles for assessing the bio-safety of genetically modified (GM) crop organisms. Understanding the natural variation of soybean seed proteins is necessary to evaluate potential uninten...

  4. Utility of proteomics techniques for assessing protein expression

    USDA-ARS?s Scientific Manuscript database

    In recent years, proteomic technologies have been frequently used as an effective analytical tool for examining modifications of protein profiles for accessing the bio-safety of genetically modified crops (GMO). Understanding of natural variation of soybean seed proteins is critical for determining...

  5. Proteomic analysis of preovulatory follicular fluid reveals differentially abundant proteins in less fertile dairy cows.

    PubMed

    Zachut, Maya; Sood, Pankaj; Levin, Yishai; Moallem, Uzi

    2016-04-29

    The follicular fluid (FF) proteome can provide an indication of follicular quality. High-yielding dairy cows suffer from low fertility, which could be related to follicular function. However, the proteome of preovulatory follicles has never been described in cows. Our objectives were to: 1) define the bovine preovulatory FF proteome, and 2) examine differentially abundant proteins in FF of controls (CTL, n=10) and less fertile cows (LFC; failed to conceive following ≥6 inseminations, n=8). Follicles ≥7mm in diameter were aspirated in vivo, and estradiol (E2) and progesterone (P4) were examined. The FF from 10 preovulatory follicles (E2/P4>1) was analyzed; E2 was higher and follicle diameter tended to be larger in LFC. As aspirations were conducted at a fixed time, this suggests accelerated follicular growth in LFC. The 219 identified and quantified proteins consisted mainly of binding proteins, proteases, receptor ligands, enzymes and transporters. Differential abundance of 8 relevant proteins was found in LFC compared to CTL: SERPINA1, TIMP2, ITIH1, HSPG2, C8A, COL1A2, F2, and IL1RAP. These proteins could influence follicular function-e.g., decreased SERPINA1 may be related to accelerated follicular growth-and therefore, further examination of their roles in the etiology of LFC is warranted. High yielding dairy cows suffer from infertility that leads to major economic losses worldwide. In Israel, about 30% of dairy cows fail to conceive following ≥4 inseminations. The etiology of this low fertility is multifactorial and remains a serious challenge. Follicular fluid proteome can provide indication to follicular quality, yet the proteome of pre-ovulatory follicles has not been described in cows. This work examined the differential abundance of proteins in less fertile dairy cows compared to controls, and found 8 relevant novel proteins that could influence follicular function. The role of these proteins in the etiology of less fertile cows should be further

  6. Differential Gene Expression in Human Cerebrovascular Malformations

    PubMed Central

    Shenkar, Robert; Elliott, J. Paul; Diener, Katrina; Gault, Judith; Hu, Ling-Jia; Cohrs, Randall J.; Phang, Tzulip; Hunter, Lawrence; Breeze, Robert E.; Awad, Issam A.

    2009-01-01

    OBJECTIVE We sought to identify genes with differential expression in cerebral cavernous malformations (CCMs), arteriovenous malformations (AVMs), and control superficial temporal arteries (STAs) and to confirm differential expression of genes previously implicated in the pathobiology of these lesions. METHODS Total ribonucleic acid was isolated from four CCM, four AVM, and three STA surgical specimens and used to quantify lesion-specific messenger ribonucleic acid expression levels on human gene arrays. Data were analyzed with the use of two separate methodologies: gene discovery and confirmation analysis. RESULTS The gene discovery method identified 42 genes that were significantly up-regulated and 36 genes that were significantly down-regulated in CCMs as compared with AVMs and STAs (P = 0.006). Similarly, 48 genes were significantly up-regulated and 59 genes were significantly down-regulated in AVMs as compared with CCMs and STAs (P = 0.006). The confirmation analysis showed significant differential expression (P < 0.05) in 11 of 15 genes (angiogenesis factors, receptors, and structural proteins) that previously had been reported to be expressed differentially in CCMs and AVMs in immunohistochemical analysis. CONCLUSION We identify numerous genes that are differentially expressed in CCMs and AVMs and correlate expression with the immunohistochemistry of genes implicated in cerebrovascular malformations. In future efforts, we will aim to confirm candidate genes specifically related to the pathobiology of cerebrovascular malformations and determine their biological systems and mechanistic relevance. PMID:12535382

  7. Wounding, insect chewing and phloem sap feeding differentially alter the leaf proteome of potato, Solanum tuberosum L.

    PubMed Central

    2012-01-01

    Background Various factors shape the response of plants to herbivorous insects, including wounding patterns, specific chemical effectors and feeding habits of the attacking herbivore. Here we performed a comparative proteomic analysis of the plant's response to wounding and herbivory, using as a model potato plants (Solanum tuberosum L.) subjected to mechanical wounding, defoliation by the Colorado potato beetle Leptinotarsa decemlineata Say, or phloem sap feeding by the potato aphid Macrosiphum euphorbiae Thomas. Results Out of ~500 leaf proteins monitored by two-dimensional gel electrophoresis (2-DE), 31 were up- or downregulated by at least one stress treatment compared to healthy control plants. Of these proteins, 29 were regulated by beetle chewing, 8 by wounding and 8 by aphid feeding. Some proteins were up- or downregulated by two different treatments, while others showed diverging expression patterns in response to different treatments. A number of modulated proteins identified by mass spectrometry were typical defense proteins, including wound-inducible protease inhibitors and pathogenesis-related proteins. Proteins involved in photosynthesis were also modulated, notably by potato beetle feeding inducing a strong decrease of some photosystem I proteins. Quantitative RT PCR assays were performed with nucleotide primers for photosynthesis-related proteins to assess the impact of wounding and herbivory at the gene level. Whereas different, sometimes divergent, responses were observed at the proteome level in response to wounding and potato beetle feeding, downregulating effects were systematically observed for both treatments at the transcriptional level. Conclusions These observations illustrate the differential impacts of wounding and insect herbivory on defense- and photosynthesis-related components of the potato leaf proteome, likely associated with the perception of distinct physical and chemical cues in planta. PMID:23268880

  8. Proteome profiling of the growth phases of Leishmania pifanoi promastigotes in axenic culture reveals differential abundance of immunostimulatory proteins.

    PubMed

    Alcolea, Pedro J; Alonso, Ana; García-Tabares, Francisco; Mena, María del Carmen; Ciordia, Sergio; Larraga, Vicente

    2016-06-01

    Leishmaniasis is a term that encompasses a compendium of neglected tropical diseases caused by dimorphic and digenetic protozoan parasites from the genus Leishmania (Kinetoplastida: Trypanosomatidae). The clinical manifestations of neotropical cutaneous leishmaniasis (NCL) caused by Leishmania pifanoi and other species of the "Leishmania mexicana complex" mainly correspond to anergic diffuse cutaneous leishmaniasis (ADCL), which is the origin of considerable morbidity. Despite the outstanding advances in the characterization of the trypanosomatid genomes and proteomes, the biology of this species has been scarcely explored. However, the close relation of L. pifanoi to the sequenced species L. mexicana and others included in the "L. mexicana complex" allowed us to perform a two-dimension electrophoresis (2DE) approach to the promastigote proteome at the differential expression level. Protein identifications were performed by matrix-assisted laser desorption-ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF). This insight has revealed similarities and differences between L. pifanoi and other species responsible for cutaneous and visceral leishmaniasis. Interestingly, certain proteins that were previously described as immunostimulatory (elongation factor 1β, trypanothione peroxidase, heat shock protein 70, enolase, GDP-forming succinyl-CoA and aldehyde dehydrogenase) are more abundant in the final growth stages of promastigotes (late-logarithmic and/or stationary phase) in the case of L. pifanoi.

  9. Proteomic profiling and characterization of differential allergens in the nematodes Anisakis simplex sensu stricto and A. pegreffii.

    PubMed

    Arcos, Susana C; Ciordia, Sergio; Roberston, Lee; Zapico, Inés; Jiménez-Ruiz, Yolanda; Gonzalez-Muñoz, Miguel; Moneo, Ignacio; Carballeda-Sangiao, Noelia; Rodriguez-Mahillo, Ana; Albar, Juan P; Navas, Alfonso

    2014-06-01

    The parasite species complex Anisakis simplex sensu lato (Anisakis simplex sensu stricto; (A. simplex s.s.), A. pegreffii, A. simplex C) is the main cause of severe anisakiasis (allergy) worldwide and is now an important health matter. In this study, the relationship of this Anisakis species complex and their allergenic capacities is assessed by studying the differences between the two most frequent species (A. simplex s.s., A. pegreffii) and their hybrid haplotype by studying active L3 larvae parasiting Merluccius merluccius. They were compared by 2D gel electrophoresis and parallel Western blot (2DE gels were hybridized with pools of sera from Anisakis allergenic patients). Unambiguous spot differences were detected and protein assignation was made by MALDI-TOF/TOF analysis or de novo sequencing. Seventy-five gel spots were detected and the corresponding proteins were identified. Differentially expressed proteins for A. simplex s.s., A. pegreffii, and their hybrid are described and results are statistically supported. Twenty-eight different allergenic proteins are classified according to different families belonging to different biological functions. These proteins are described for the first time as antigenic and potentially new allergens in Anisakis. Comparative proteomic analyses of allergenic capacities are useful for diagnosis, epidemiological surveys, and clinical research. All MS data have been deposited in the ProteomeXchange with identifier PXD000662 (http://proteomecentral.proteomexchange.org/dataset/PXD000662). © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Assessment of the red cell proteome of young patients with unexplained hemolytic anemia by two-dimensional differential in-gel electrophoresis (DIGE).

    PubMed

    von Löhneysen, Katharina; Scott, Thomas M; Soldau, Katrin; Xu, Xiuling; Friedman, Jeffrey S

    2012-01-01

    Erythrocyte cytosolic protein expression profiles of children with unexplained hemolytic anemia were compared with profiles of close relatives and controls by two-dimensional differential in-gel electrophoresis (2D-DIGE). The severity of anemia in the patients varied from compensated (i.e., no medical intervention required) to chronic transfusion dependence. Common characteristics of all patients included chronic elevation of reticulocyte count and a negative workup for anemia focusing on hemoglobinopathies, morphologic abnormalities that would suggest a membrane defect, immune-mediated red cell destruction, and evaluation of the most common red cell enzyme defects, glucose-6-phosphate dehydrogenase and pyruvate kinase deficiency. Based upon this initial workup and presentation during infancy or early childhood, four patients classified as hereditary nonspherocytic hemolytic anemia (HNSHA) of unknown etiology were selected for proteomic analysis. DIGE analysis of red cell cytosolic proteins clearly discriminated each anemic patient from both familial and unrelated controls, revealing both patient-specific and shared patterns of differential protein expression. Changes in expression pattern shared among the four patients were identified in several protein classes including chaperons, cytoskeletal and proteasome proteins. Elevated expression in patient samples of some proteins correlated with high reticulocyte count, likely identifying a subset of proteins that are normally lost during erythroid maturation, including proteins involved in mitochondrial metabolism and protein synthesis. Proteins identified with patient-specific decreased expression included components of the glutathione synthetic pathway, antioxidant pathways, and proteins involved in signal transduction and nucleotide metabolism. Among the more than 200 proteins identified in this study are 21 proteins not previously described as part of the erythrocyte proteome. These results demonstrate the

  11. Investigating the Correspondence Between Transcriptomic and Proteomic Expression Profiles Using Coupled Cluster Models.

    SciTech Connect

    Rogers, Simon; Girolami, Mark; Kolch, Walter; Waters, Katrina M.; Liu, Tao; Thrall, Brian D.; Wiley, H. S.

    2008-12-01

    Modern transcriptomics and proteomics enable us to survey the expression of RNAs and proteins at large scales. While these data are usually generated and analysed separately, there is an increasing interest in comparing and co-analysing transcriptome and proteome expression data. A major open question is whether transcriptome and proteome expression is linked and how it is coordinated. Results: Here we have developed a probabilistic clustering model that permits analysis of the links between transcriptomic and proteomic profiles in a sensible and flexible manner. Our coupled mixture model defines a prior probability distribution over the component to which a protein profile should be assigned conditioned on which component the associated mRNA profile belongs to. By providing probabilistic assignments this approach sits between the two extremes of concatenating the data on the assumption that mRNA and protein clusters would have a one-to-one relationship, and independent clustering where the mRNA profile provides no information on the protein profile and vice-versa. We apply this approach to a large dataset of quantitative transcriptomic and proteomic expression data obtained from a human breast epithelial cell line (HMEC) stimulated by epidermal growth factor (EGF) over a series of timepoints corresponding to one cell cycle. The results reveal a complex relationship between transcriptome and proteome with most mRNA clusters linked to at least two protein clusters, and vice versa. A more detailed analysis incorporating information on gene function from the gene ontology database shows that a high correlation of mRNA and protein expression is limited to the components of some molecular machines, such as the ribosome, cell adhesion complexes and the TCP-1 chaperonin involved in protein folding. Conclusions: The dynamic regulation of the transcriptome and proteome in mammalian cells in response to an acute mitogenic stimulus appears largely independent with very little

  12. Differential effects of Zn exposure on male and female oysters (Crassostrea angulata) as revealed by label-free quantitative proteomics.

    PubMed

    Luo, Lianzhong; Zhang, Qinghong; Kong, Xue; Huang, Heqing; You, Weiwei; Ke, Caihuan

    2017-03-17

    Oysters accumulate Zn as an adaptation to Zn exposure; however, it is not known whether male and female oysters respond differently to Zn exposure. Here, proteomic and real-time polymerase chain reaction analyses were used to investigate differential responses of male and female oysters (Crassostrea angulata) to Zn exposure. After exposure to 50 µg.L(-1) or 500 µg.L(-1) Zn for 30 days, gonads of female oysters accumulated more Zn than those of males, and gonadal development was accelerated in females but was abnormal in males. Differentially expressed proteins after exposure to Zn were identified and shown to function in Zn transport, Ca transport, phosphate metabolism, energy metabolism, immune regulation, oxidative stress responses, gene expression regulation, and fat metabolism. Proteins with functions in Zn transportation and storage, and multi-functional proteins, such as hemicentin-1 and histidinol dehydrogenase, were expressed at significantly higher levels in the gonads of female than male oysters after Zn exposure. This article is protected by copyright. All rights reserved.

  13. Plumbagin elicits differential proteomic responses mainly involving cell cycle, apoptosis, autophagy, and epithelial-to-mesenchymal transition pathways in human prostate cancer PC-3 and DU145 cells.

    PubMed

    Qiu, Jia-Xuan; Zhou, Zhi-Wei; He, Zhi-Xu; Zhao, Ruan Jin; Zhang, Xueji; Yang, Lun; Zhou, Shu-Feng; Mao, Zong-Fu

    2015-01-01

    Plumbagin (PLB) has exhibited a potent anticancer effect in preclinical studies, but the molecular interactome remains elusive. This study aimed to compare the quantitative proteomic responses to PLB treatment in human prostate cancer PC-3 and DU145 cells using the approach of stable-isotope labeling by amino acids in cell culture (SILAC). The data were finally validated using Western blot assay. First, the bioinformatic analysis predicted that PLB could interact with 78 proteins that were involved in cell proliferation and apoptosis, immunity, and signal transduction. Our quantitative proteomic study using SILAC revealed that there were at least 1,225 and 267 proteins interacting with PLB and there were 341 and 107 signaling pathways and cellular functions potentially regulated by PLB in PC-3 and DU145 cells, respectively. These proteins and pathways played a critical role in the regulation of cell cycle, apoptosis, autophagy, epithelial to mesenchymal transition (EMT), and reactive oxygen species generation. The proteomic study showed substantial differences in response to PLB treatment between PC-3 and DU145 cells. PLB treatment significantly modulated the expression of critical proteins that regulate cell cycle, apoptosis, and EMT signaling pathways in PC-3 cells but not in DU145 cells. Consistently, our Western blotting analysis validated the bioinformatic and proteomic data and confirmed the modulating effects of PLB on important proteins that regulated cell cycle, apoptosis, autophagy, and EMT in PC-3 and DU145 cells. The data from the Western blot assay could not display significant differences between PC-3 and DU145 cells. These findings indicate that PLB elicits different proteomic responses in PC-3 and DU145 cells involving proteins and pathways that regulate cell cycle, apoptosis, autophagy, reactive oxygen species production, and antioxidation/oxidation homeostasis. This is the first systematic study with integrated computational, proteomic, and

  14. In vitro differentiation of bone marrow mesenchymal stem cells into endometrial epithelial cells in mouse: a proteomic analysis

    PubMed Central

    Cong, Qing; Li, Bin; Wang, Yisheng; Zhang, Wenbi; Cheng, Mingjun; Wu, Zhiyong; Zhang, Xiaoyan; Jiang, Wei; Xu, Congjian

    2014-01-01

    Objective: Mouse bone marrow mesenchymal stem cells (BMSCs) have been demonstrated to differentiate into female endometrial epithelial cells (EECs) in vivo. Our previous studies demonstrated that BMSCs can differentiate in the direction of EECs when co-cultured with endometrial stromal cells in vitro. Here, we obtain and analyse differential proteins and their relevant pathways in the process of BMSCs differentiating into EECs by isobaric tags for relative and absolute quantitation (iTRAQ) proteomic analysis. Methods: A 0.4-μm pore size indirect co-culture system was established with female mice endometrial stromal cells (EStCs) restricted in the upper Transwell chamber and BMSCs in the lower well plate. After indirect co-culture for several days, the BMSCs were revealed to progressively differentiate towards EECs in vitro. Then, four groups were divided according to different co-culture days with single culture groups of BMSCs as controls. Proteins were detected using iTRAQ based on 2DLC-ESI-MS/MS and data were analysed by bioinformatics. Results: A total number of 311 proteins were detected, of which 210 proteins were identified with relative quantitation. Among them, 107 proteins were differentially expressed with a 1.2-fold change as the benchmark, with 61 up-regulated and 46 down-regulated proteins. Differential proteins CK19 and CK8 were epithelial markers and upregulated. Stromal marker vimentin were downregulated. Top canonical pathways was “remodeling of epithelial adhesions junctions” and “actin cytoskeleton signaling”. Top networks was “cell-to-cell signaling and interaction, tissue development and cellular movement” regulated by ERK/MAPK and α-catenin. Conclusion: To the best of our knowledge, this is the first preliminary study of differential protein expression in the differentiation process of BMSCs into EECs in vitro. We further elucidated BMSCs differentiated in the direction of EECs. In addition, ERK/MAPK and α-catenin played

  15. Proteomic characterization of the effects of clofibrate on protein expression in rat liver.

    PubMed

    Léonard, Jean-François; Courcol, Martine; Mariet, Claire; Charbonnier, Aurore; Boitier, Eric; Duchesne, Marc; Parker, Fabienne; Genet, Bruno; Supatto, Françoise; Roberts, Ruth; Gautier, Jean-Charles

    2006-03-01

    Clofibrate is a peroxisome proliferator known to induce liver tumours in rats. A proteomics study was conducted to provide new insights into the molecular mechanisms of clofibrate-induced non-genotoxic hepatocarcinogenesis. Rats were treated with 250 mg/kg day clofibrate orally and sacrificed after 7 days. Proteins extracted from the liver were analysed by 2-DE using DIGE technology. The protein identification performed by MS showed that clofibrate induced up-regulation of 77 proteins and down-regulation of 27 proteins. The highest expression ratios corresponded to proteins involved in a series of biochemical pathways such as lipid metabolism, fatty acid metabolism, amino acid metabolism, protein metabolism, citric acid cycle, xenobiotic detoxification and oxidative stress. Proteins implicated in cell proliferation and apoptosis, such as prohibitin, 10-formyl tetrahydrofolate dehydrogenase, senescence marker protein-30, pyridoxine 5'-phosphate oxidase and vimentin, were also identified as being regulated. These results provide leads for further investigations into the molecular mechanisms of liver tumours induced by clofibrate. In addition, MS results showed that a series of regulated proteins were detected as several spots corresponding to different pI and/or M(r). Differential effects on those variants could result from specific PTM and could be a specific molecular signature of the clofibrate-induced protein expression modulation in rat liver.

  16. Triphenyltin degradation and proteomic response by an engineered Escherichia coli expressing cytochrome P450 enzyme.

    PubMed

    Yi, Wenying; Yang, Kunliang; Ye, Jinshao; Long, Yan; Ke, Jing; Ou, Huase

    2017-03-01

    Although triphenyltin (TPT) degradation pathway has been determined, information about the enzyme and protein networks involved was severely limited. To this end, a cytochrome P450 hydroxylase (CYP450) gene from Bacillus thuringiensis was cloned and expressed in Escherichia coli BL21 (DE3), namely E. coli pET32a-CYP450, whose dosage at 1gL(-1) could degrade 54.6% TPT at 1mgL(-1) within 6 d through attacking the carbon-tin bonds of TPT by CYP450. Sequence analysis verified that the CYP450 gene had a 1214bp open reading frame, encoding a protein with 404 amino acids. Proteomic analysis determined that 60 proteins were significantly differentially regulated expression in E. coli pET32a-CYP450 after TPT degradation. The up-regulated proteins enriched in a network related to transport, cell division, biosynthesis of amino acids and secondary metabolites, and microbial metabolism in diverse environments. The current findings demonstrated for the first time that P450 received electrons transferring from NADH could effectively cleave carbon-metal bonds.

  17. Proteomic analysis of protein expression in Lactobacillus plantarum in response to alkaline stress.

    PubMed

    Lee, KiBeom; Rho, Beom-Seop; Pi, KyungBae; Kim, Ho-Jin; Choi, Yun-Jaie

    2011-04-20

    Lactobacillus plantarum, a probiotic organism that plays an important role in the microbial fermentation of alkaline materials in fermenting foods, faces alkaline stress during the fermentation process. Here, we report the patterns of protein expression in L. plantarum subjected to transient (1h) alkaline stress at pH 7.7, 8.7 or 9.7. Thirty-three alkaline-responsive proteins were identified by two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS). Identification of proteins showing differential expression in response to alkaline stress revealed that the alkaline stress response of L. plantarum is a complex process. Some proteins appear to be induced, others repressed. These proteins could be clustered into nine groups based on their probable functions: energy metabolism, transport system, purine/pyrimidine metabolism, amino acid metabolism, proteolytic activity, transcription-translation, stress-related, general function, and unknown functions. These proteomic analyses are expected to prove useful in understanding the adaptive response of L. plantarum strains to alkaline stress and may facilitate future investigations into the genetic and physiological aspects of this response.

  18. Differential proteome analysis of bone marrow mesenchymal stem cells from adolescent idiopathic scoliosis patients.

    PubMed

    Zhuang, Qianyu; Li, Jing; Wu, Zhihong; Zhang, Jianguo; Sun, Wei; Li, Tao; Yan, Yujuan; Jiang, Ying; Zhao, Robert Chunhua; Qiu, Guixing

    2011-04-22

    Adolescent idiopathic scoliosis (AIS) is a complex three-dimensional deformity of the spine. The cause and pathogenesis of scoliosis and the accompanying generalized osteopenia remain unclear despite decades of extensive research. In this study, we utilized two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry (MS) to analyze the differential proteome of bone marrow mesenchymal stem cells (BM-MSCs) from AIS patients. In total, 41 significantly altered protein spots were detected, of which 34 spots were identified by MALDI-TOF/TOF analysis and found to represent 25 distinct gene products. Among these proteins, five related to bone growth and development, including pyruvate kinase M2, annexin A2, heat shock 27 kDa protein, γ-actin, and β-actin, were found to be dysregulated and therefore selected for further validation by Western blot analysis. At the protein level, our results supported the previous hypothesis that decreased osteogenic differentiation ability of MSCs is one of the mechanisms leading to osteopenia in AIS. In summary, we analyzed the differential BM-MSCs proteome of AIS patients for the first time, which may help to elucidate the underlying molecular mechanisms of bone loss in AIS and also increase understanding of the etiology and pathogenesis of AIS.

  19. Differentiating heart failure phenotypes using sex-specific transcriptomic and proteomic biomarker panels.

    PubMed

    Toma, Mustafa; Mak, George J; Chen, Virginia; Hollander, Zsuzsanna; Shannon, Casey P; Lam, Karen K Y; Ng, Raymond T; Tebbutt, Scott J; Wilson-McManus, Janet E; Ignaszewski, Andrew; Anderson, Todd; Dyck, Jason R B; Howlett, Jonathan; Ezekowitz, Justin; McManus, Bruce M; Oudit, Gavin Y

    2017-08-01

    Heart failure with preserved ejection fraction (HFpEF) accounts for 30-50% of patients with heart failure (HF). A major obstacle in HF management is the difficulty in differentiating between HFpEF and heart failure with reduced ejection fraction (HFrEF) using conventional clinical and laboratory investigations. The aim of this study is to develop robust transcriptomic and proteomic biomarker signatures that can differentiate HFpEF from HFrEF. A total of 210 HF patients were recruited in participating institutions from the Alberta HEART study. An expert clinical adjudicating panel differentiated between patients with HFpEF and HFrEF. The discovery cohort consisted of 61 patients, and the replication cohort consisted of 70 patients. Transcriptomic and proteomic data were analysed to find panels of differentiating HFpEF from HFrEF. In the discovery cohort, a 22-transcript panel was found to differentiate HFpEF from HFrEF in male patients with a cross-validation AUC of 0.74, as compared with 0.70 for N-terminal pro-B-type natriuretic peptide (NT-proBNP) in those same patients. An ensemble of the transcript panel and NT-pro-BNP yielded a cross-validation AUC of 0.80. This performance improvement was also observed in the replication cohort. An ensemble of the transcriptomic panel with NT-proBNP produced a replication AUC of 0.90, as compared with 0.74 for NT-proBNP alone and 0.73 for the transcriptomic panel. We have identified a male-specific transcriptomic biomarker panel that can differentiate between HFpEF and HFrEF. These biosignatures could be further replicated on other patients and potentially be developed into a blood test for better management of HF patients. © 2017 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of the European Society of Cardiology.

  20. Proteomic exploration of the impacts of pomegranate fruit juice on the global gene expression of prostate cancer cell.

    PubMed

    Lee, Song-Tay; Wu, Yi-Ling; Chien, Lan-Hsiang; Chen, Szu-Ting; Tzeng, Yu-Kai; Wu, Ting-Feng

    2012-11-01

    Prostate cancer has been known to be the second highest cause of death in cancer among men. Pomegranate is rich in polyphenols with the potent antioxidant activity and inhibits cell proliferation, invasion, and promotes apoptosis in various cancer cells. This study demonstrated that pomegranate fruit juice could effectively hinder the proliferation of human prostate cancer DU145 cell. The results of apoptotic analyses implicated that fruit juice might trigger the apoptosis in DU145 cells via death receptor signaling and mitochondrial damage pathway. In this study, we exploited 2DE-based proteomics to compare nine pairs of the proteome maps collected from untreated and treated DU145 cells to identify the differentially expressed proteins. Comparative proteomics indicated that 11 proteins were deregulated in affected DU145 cells with three upregulated and eight downregulated proteins. These dys-regulated proteins participated in cytoskeletal functions, antiapoptosis, proteasome activity, NF-κB signaling, cancer cell proliferation, invasion, and angiogenesis. Western immunoblotting were implemented to confirm the deregulated proteins and the downstream signaling proteins. The analytical results of this study help to provide insight into the molecular mechanism of inducing prostate cancer cell apoptosis by pomegranate fruit juice and to develop a novel mechanism-based chemopreventive strategy for prostate cancer.

  1. A proteomic study showing differential regulation of stress, redox regulation and peroxidase proteins by iron supply and the transcription factor FER.

    PubMed

    Brumbarova, Tzvetina; Matros, Andrea; Mock, Hans-Peter; Bauer, Petra

    2008-04-01

    Plants need to mobilize iron in the soil, and the basic helix-loop-helix transcription factor FER is a central regulator of iron acquisition in tomato roots. FER activity is controlled by iron supply. To analyse to what extent FER influences Fe-regulated protein expression, we investigated the root proteome of wild-type tomato, the fer mutant and a transgenic FER overexpression line under low-iron conditions versus sufficient and generous iron supply. The root proteomes were analysed by two-dimensional gel electrophoresis with three technical and three biological replicates. Statistical analysis identified 39 protein spots that were differentially regulated in selected pairwise comparisons of experimental conditions. Of these, 24 were correlated with expression clusters revealed by principal component analysis. The 39 protein spots were analysed by MALDI-TOF and nanoLC-MS/MS to deduce their possible functions. We investigated the functional representation in the identified expression clusters, and found that loss of FER function in iron-cultured plants mimicked an iron-deficiency status. The largest identified protein expression cluster was upregulated by iron deficiency and in the fer mutant. Two iron-regulated proteins required FER activity for induction by iron deficiency. Few proteins were suppressed by iron deficiency. The differentially expressed proteins belonged predominantly to the functional categories 'stress', 'redox regulation' and 'miscellaneous peroxidases'. Hence, we were able to identify distinct expression clusters of proteins with distinct functions.

  2. An integrated transcriptomics-guided genome-wide promoter analysis and next-generation proteomics approach to mine factor(s) regulating cellular differentiation

    PubMed Central

    Mandal, Kamal; Bader, Samuel L.; Kumar, Pankaj; Malakar, Dipankar; Campbell, David S.; Pradhan, Bhola Shankar; Sarkar, Rajesh K.; Wadhwa, Neerja; Sensharma, Souvik; Jain, Vaibhav; Moritz, Robert L.

    2017-01-01

    Abstract Differential next-generation-omics approaches aid in the visualization of biological processes and pave the way for divulging important events and/or interactions leading to a functional output at cellular or systems level. To this end, we undertook an integrated Nextgen transcriptomics and proteomics approach to divulge differential gene expression of infant and pubertal rat Sertoli cells (Sc).Unlike, pubertal Sc, infant Sc are immature and fail to support spermatogenesis. We found exclusive association of 14 and 19 transcription factor binding sites to infantile and pubertal states of Sc, respectively, using differential transcriptomics-guided genome-wide computational analysis of relevant promoters employing 220 Positional Weight Matrices from the TRANSFAC database. Proteomic SWATH-MS analysis provided extensive quantification of nuclear and cytoplasmic protein fractions revealing 1,670 proteins differentially located between the nucleus and cytoplasm of infant Sc and 890 proteins differentially located within those of pubertal Sc. Based on our multi-omics approach, the transcription factor YY1 was identified as one of the lead candidates regulating differentiation of Sc.YY1 was found to have abundant binding sites on promoters of genes upregulated during puberty. To determine its significance, we generated transgenic rats with Sc specific knockdown of YY1 that led to compromised spermatogenesis. PMID:28065881

  3. An integrated transcriptomics-guided genome-wide promoter analysis and next-generation proteomics approach to mine factor(s) regulating cellular differentiation.

    PubMed

    Mandal, Kamal; Bader, Samuel L; Kumar, Pankaj; Malakar, Dipankar; Campbell, David S; Pradhan, Bhola Shankar; Sarkar, Rajesh K; Wadhwa, Neerja; Sensharma, Souvik; Jain, Vaibhav; Moritz, Robert L; Majumdar, Subeer S

    2017-04-01

    Differential next-generation-omics approaches aid in the visualization of biological processes and pave the way for divulging important events and/or interactions leading to a functional output at cellular or systems level. To this end, we undertook an integrated Nextgen transcriptomics and proteomics approach to divulge differential gene expression of infant and pubertal rat Sertoli cells (Sc).Unlike, pubertal Sc, infant Sc are immature and fail to support spermatogenesis. We found exclusive association of 14 and 19 transcription factor binding sites to infantile and pubertal states of Sc, respectively, using differential transcriptomics-guided genome-wide computational analysis of relevant promoters employing 220 Positional Weight Matrices from the TRANSFAC database. Proteomic SWATH-MS analysis provided extensive quantification of nuclear and cytoplasmic protein fractions revealing 1,670 proteins differentially located between the nucleus and cytoplasm of infant Sc and 890 proteins differentially located within those of pubertal Sc. Based on our multi-omics approach, the transcription factor YY1 was identified as one of the lead candidates regulating differentiation of Sc.YY1 was found to have abundant binding sites on promoters of genes upregulated during puberty. To determine its significance, we generated transgenic rats with Sc specific knockdown of YY1 that led to compromised spermatogenesis. © The Author 2017. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

  4. Differential expression of proteins in response to molybdenum deficiency in winter wheat leaves under low-temperature stress

    USDA-ARS?s Scientific Manuscript database

    Molybdenum (Mo) is an essential micronutrient for plants. To obtain a better understanding of the molecular mechanisms of cold resistance enhanced by molybdenum application in winter wheat, we applied a proteomic approach to investigate the differential expression of proteins in response to molybden...

  5. Differential protein expression in Phalaenopsis under low temperature.

    PubMed

    Yuan, Xiu-Yun; Liang, Fang; Jiang, Su-Hua; Wan, Mo-Fei; Ma, Jie; Zhang, Xian-Yun; Cui, Bo

    2015-01-01

    A comparative proteomic analysis was carried out to explore the molecular mechanisms of responses to cold stress in Phalaenopsis after treated by low temperature (13/8 °C day/night) for 15 days. Differentially expressed proteins were examined using two-dimensional electrophoresis (2-DE) and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-TOF/MS). Among 85 differentially expressed proteins, 73 distinct proteins were identified. Comparative analysis revealed that the identified proteins mainly participate in photosynthesis, protein synthesis, folding and degradation, respiration, defense response, amino acid metabolism, energy pathway, cytoskeleton, transcription regulation, signal transduction, and seed storage protein, while the functional classification of the remaining four proteins was not determined. These data suggested that the proteins might work cooperatively to establish a new homeostasis under cold stress; 37 % of the identified cold-responsive proteins were associated with various aspects of chloroplast physiology, and 56 % of them were predicted to be located in the chloroplasts, implying that the cold stress tolerance of Phalaenopsis was achieved, at least partly, by regulation of chloroplast function. Moreover, the protein destination control, which was mediated by chaperones and proteases, plays an important role in tolerance to cold stress.

  6. Screening for Expressed Nonribosomal Peptide Synthetases and Polyketide Synthases Using LC-MS/MS-Based Proteomics

    PubMed Central

    Chen, Yunqiu; McClure, Ryan A.; Kelleher, Neil L.

    2016-01-01

    Liquid chromatography–mass spectrometry (LC-MS)-based proteomics is a powerful technique for the profiling of protein expression in cells in a high-throughput fashion. Herein we report a protocol using LC-MS/MS-based proteomics for the screening of enzymes involved in natural product biosynthesis, such as nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) from bacterial strains. Taking advantage of the large size of modular NRPSs and PKSs (often >200 kDa), size-based separation (SDS-PAGE) is employed prior to LC-MS/MS analysis. Based upon the protein identifications obtained through software search, we can accurately pinpoint the expressed NRPS and/or PKS gene clusters from a given strain and growth condition. The proteomics screening result can be used to guide the discovery of potentially new nonribosomal peptide and polyketide natural products. PMID:26831706

  7. High-throughput sperm differential proteomics suggests that epigenetic alterations contribute to failed assisted reproduction.

    PubMed

    Azpiazu, Rubén; Amaral, Alexandra; Castillo, Judit; Estanyol, Josep Maria; Guimerà, Marta; Ballescà, Josep Lluís; Balasch, Juan; Oliva, Rafael

    2014-06-01

    Are there quantitative alterations in the proteome of normozoospermic sperm samples that are able to complete IVF but whose female partner does not achieve pregnancy? Normozoospermic sperm samples with different IVF outcomes (pregnancy versus no pregnancy) differed in the levels of at least 66 proteins. The analysis of the proteome of sperm samples with distinct fertilization capacity using low-throughput proteomic techniques resulted in the detection of a few differential proteins. Current high-throughput mass spectrometry approaches allow the identification and quantification of a substantially higher number of proteins. This was a case-control study including 31 men with normozoospermic sperm and their partners who underwent IVF with successful fertilization recruited between 2007 and 2008. Normozoospermic sperm samples from 15 men whose female partners did not achieve pregnancy after IVF (no pregnancy) and 16 men from couples that did achieve pregnancy after IVF (pregnancy) were included in this study. To perform the differential proteomic experiments, 10 no pregnancy samples and 10 pregnancy samples were separately pooled and subsequently used for tandem mass tags (TMT) protein labelling, sodium dodecyl sulphate-polyacrylamide gel electrophoresis, liquid chromatography tandem mass spectrometry (LC-MS/MS) identification and peak intensity relative protein quantification. Bioinformatic analyses were performed using UniProt Knowledgebase, DAVID and Reactome. Individual samples (n = 5 no pregnancy samples; n = 6 pregnancy samples) and aliquots from the above TMT pools were used for western blotting. By using TMT labelling and LC-MS/MS, we have detected 31 proteins present at lower abundance (ratio no pregnancy/pregnancy < 0.67) and 35 at higher abundance (ratio no pregnancy/pregnancy > 1.5) in the no pregnancy group. Bioinformatic analyses showed that the proteins with differing abundance are involved in chromatin assembly and lipoprotein metabolism (P values < 0

  8. Protein synthesis rate is the predominant regulator of protein expression during differentiation

    PubMed Central

    Kristensen, Anders R; Gsponer, Joerg; Foster, Leonard J

    2013-01-01

    External perturbations, by forcing cells to adapt to a new environment, often elicit large-scale changes in gene expression resulting in an altered proteome that improves the cell's fitness in the new conditions. Steady-state levels of a proteome depend on transcription, the levels of transcripts, translation and protein degradation but system-level contribution that each of these processes make to the final protein expression change has yet to be explored. We therefore applied a systems biology approach to characterize the regulation of protein expression during cellular differentiation using quantitative proteomics. As a general rule, it seems that protein expression during cellular differentiation is largely controlled by changes in the relative synthesis rate, whereas the relative degradation rate of the majority of proteins stays constant. In these data, we also observe that the proteins in defined sub-structures of larger protein complexes tend to have highly correlated synthesis and degradation rates but that this does not necessarily extend to the holo-complex. Finally, we provide strong evidence that the generally poor correlation observed between transcript and protein levels can fully be explained once the protein synthesis and degradation rates are taken into account. PMID:24045637

  9. Proteomics unveil corticoid-induced S100A11 shuttling in keratinocyte differentiation

    SciTech Connect

    Dezitter, Xavier; Hammoudi, Fatma; Belverge, Nicolas; Deloulme, Jean-Christophe; Drobecq, Herve; Masselot, Bernadette; Formstecher, Pierre; Mendy, Denise; Idziorek, Thierry . E-mail: thierry.idziorek@lille.inserm.fr

    2007-08-31

    Unlike classical protein extraction techniques, proteomic mapping using a selective subcellular extraction kit revealed S100A11 as a new member of the S100 protein family modulated by glucocorticoids in keratinocytes. Glucocorticoids (GC)-induced S100A11 redistribution in the 'organelles and membranes' compartment. Microscopic examination indicated that glucocorticoids specifically routed cytoplasmic S100A11 toward perinuclear compartment. Calcium, a key component of skin terminal differentiation, directed S100A11 to the plasma membrane as previously reported. When calcium was added to glucocorticoids, minor change was observed at the proteomic level while confocal microscopy revealed a rapid and dramatic translocation of S100A11 toward plasma membrane. This effect was accompanied by strong nuclear condensation, loss of mitochondrial potential and DNA content, and increased high molecular weight S100A11 immunoreactivity, suggesting corticoids accelerate calcium-induced terminal differentiation. Finally, our results suggest GC-induced S100A11 relocalization could be a key step in both keratinocyte homeostasis and glucocorticoids side effects in human epidermis.

  10. Introducing Knowledge into Differential Expression Analysis

    PubMed Central

    Biecek, Przemysław; Tiuryn, Jerzy; Vingron, Martin

    2010-01-01

    Abstract Gene expression measurements allow determining sets of up- or down-regulated, or unchanged genes in a particular experimental condition. Additional biological knowledge can suggest examples of genes from one of these sets. For instance, known target genes of a transcriptional activator are expected, but are not certain to go down after this activator is knocked out. Available differential expression analysis tools do not take such imprecise examples into account. Here we put forward a novel partially supervised mixture modeling methodology for differential expression analysis. Our approach, guided by imprecise examples, clusters expression data into differentially expressed and unchanged genes. The partially supervised methodology is implemented by two methods: a newly introduced belief-based mixture modeling, and soft-label mixture modeling, a method proved efficient in other applications. We investigate on synthetic data the input example settings favorable for each method. In our tests, both belief-based and soft-label methods prove their advantage over semi-supervised mixture modeling in correcting for erroneous examples. We also compare them to alternative differential expression analysis approaches, showing that incorporation of knowledge yields better performance. We present a broad range of knowledge sources and data to which our partially supervised methodology can be applied. First, we determine targets of Ste12 based on yeast knockout data, guided by a Ste12 DNA-binding experiment. Second, we distinguish miR-1 from miR-124 targets in human by clustering expression data under transfection experiments of both microRNAs, using their computationally predicted targets as examples. Finally, we utilize literature knowledge to improve clustering of time-course expression profiles. PMID:20726790

  11. Copper response of Proteus hauseri based on proteomic and genetic expression and cell morphology analyses.

    PubMed

    Ng, I-Son; Zheng, Xuesong; Wang, Nan; Chen, Bor-Yann; Zhang, Xia; Lu, Yinghua

    2014-07-01

    The copper response of Proteus hauseri ZMd44 was determined using one-dimensional (1D) gel electrophoresis coupled with MALDI-TOF-TOF mass spectrometry for a similarity analysis of proteins isolated from P. hauseri ZMd44 cultured in CuSO4-bearing LB medium. Candidate proteins identified as a copper-transporting P-type ATPase (CTPP), phosphoenolpyruvate carboxykinase (PEPCK), flagellin (Fla), and outer membrane proteins (Omps) were the major copper-associated proteins in P. hauseri. In a comparative analysis of subcellular (i.e., periplasmic, intracellular, and inner membranes) and cellular debris, proteomics analysis revealed a distinct differential expression of proteins in P. hauseri with and without copper ion exposure. These findings were consistent with the transcription level dynamics determined using quantitative real-time PCR. Based on a genetic cluster analysis of copper-associated proteins from P. hauseri, Fla and one of the Omps showed greater diversity in their protein sequences compared to those of other Proteus species. Transmission electron microscopy (TEM) and the observed growth on LB agar plates showed that the swarming motility of cells was significantly suppressed and inhibited upon Cu(II) exposure. Thus, copper stress could have important therapeutic significance due to the loss of swarming motility capacity in P. hauseri, which causes urinary tract infections.

  12. Potential applications of global protein expression analysis (proteomics) in morbid obesity and bariatric surgery.

    PubMed

    Brandacher, Gerald; Golderer, Georg; Kienzl, Katrin; Werner, Ernst R; Margreiter, Raimund; Weiss, Helmut G

    2008-07-01

    Global protein expression analysis, known as proteomics, has emerged as a novel scientific technology currently successfully applied to several fields of medicine including cancer and transplantation. Thereby, a thorough exploration of the pathogenic mechanisms and a better understanding of the pathophysiology of diseases as well as identification of diagnostic biomarkers have been achieved. In this paper, we outline the basic principles and potential applications of this promising tool in bariatric surgery where proteomics might hold great potential for new insights into diagnostic and therapeutic decision making based on improved knowledge of metabolic regulations pre- and postsurgical interventions in morbidly obese patients.

  13. Transcriptome study of differential expression in schizophrenia

    PubMed Central

    Sanders, Alan R.; Göring, Harald H. H.; Duan, Jubao; Drigalenko, Eugene I.; Moy, Winton; Freda, Jessica; He, Deli; Shi, Jianxin; Gejman, Pablo V.

    2013-01-01

    Schizophrenia genome-wide association studies (GWAS) have identified common SNPs, rare copy number variants (CNVs) and a large polygenic contribution to illness risk, but biological mechanisms remain unclear. Bioinformatic analyses of significantly associated genetic variants point to a large role for regulatory variants. To identify gene expression abnormalities in schizophrenia, we generated whole-genome gene expression profiles using microarrays on lymphoblastoid cell lines (LCLs) from 413 cases and 446 controls. Regression analysis identified 95 transcripts differentially expressed by affection status at a genome-wide false discovery rate (FDR) of 0.05, while simultaneously controlling for confounding effects. These transcripts represented 89 genes with functions such as neurotransmission, gene regulation, cell cycle progression, differentiation, apoptosis, microRNA (miRNA) processing and immunity. This functional diversity is consistent with schizophrenia's likely significant pathophysiological heterogeneity. The overall enrichment of immune-related genes among those differentially expressed by affection status is consistent with hypothesized immune contributions to schizophrenia risk. The observed differential expression of extended major histocompatibility complex (xMHC) region histones (HIST1H2BD, HIST1H2BC, HIST1H2BH, HIST1H2BG and HIST1H4K) converges with the genetic evidence from GWAS, which find the xMHC to be the most significant susceptibility locus. Among the differentially expressed immune-related genes, B3GNT2 is implicated in autoimmune disorders previously tied to schizophrenia risk (rheumatoid arthritis and Graves’ disease), and DICER1 is pivotal in miRNA processing potentially linking to miRNA alterations in schizophrenia (e.g. MIR137, the second strongest GWAS finding). Our analysis provides novel candidate genes for further study to assess their potential contribution to schizophrenia. PMID:23904455

  14. IAA8 expression during vascular cell differentiation

    Treesearch

    Andrew T. Groover; Amy Pattishall; Alan M. Jones

    2003-01-01

    We report the characterization of a member of the auxin-induced IAA gene family from zinnia, designated zIAA8, which is expressed by mesophyll cells differentiating as tracheary elements in vitro. Transcription of zIAA8 is upregulated within 3 h after cell isolation in inductive medium,...

  15. [The application of proteomics in epididymis researches].

    PubMed

    Zou, Mei; Wang, Hai-Feng; Hu, Jian-Min

    2008-04-01

    Proteomics, a hot new research area studied by biologists in the post-genome era, leads an integrated study of the expression and function of proteins on a large scale. Epididymis is responsible for the sperm maturation and storage, and its gene expression is highly region-specific, so it maybe an ideal model for proteomics, especially for differential proteomics study. This paper gives a brief introduction of the application of proteomics in the epididymis and the differential proteins expressed in different epididymal regions. These epididymis-specific/selective proteins identified by proteomics technology may prove to be targets for contraception or may provide insight to the unique responsibilities of the epididymis in supporting male fertility.

  16. Proteomic Profiling Of Two-Dimensional Gel Electrophoresis Protein Expression Data

    NASA Astrophysics Data System (ADS)

    Ahmad, Norhaiza; Zhang, J.; Brown, P. J.; James, D. C.; Birch, J. R.; Racher, A. J.; Smales, C. M.

    2008-01-01

    We have undertaken two-dimensional gel electrophoresis (2-DE) proteomic profiling on a series of cell lines with different recombinant antibody production rates. Due to the nature of 2-DE proteomic investigations there will always be `process variability' factors in any data set collected in this way. Some of this variation will arise during sample preparation, gel running and staining, while further variation will arise from the gel analysis procedure. Therefore, in order to identify all significant changes in protein expression between biological samples when analysed by 2-DE, the system precision or `error', and how this correlates to protein abundance, must be known. Only then can the system be considered robust and investigators accurately and confidently report all observable statistically significant changes in protein expression. We introduce an expression variability test to identify protein spots whose expression correlates with increased antibody production. The results have highlighted a small number of candidate proteins for further investigation.

  17. Differential expression of hemolymph proteins between susceptible and insecticide-resistant Blattella germanica (Blattodea: Blattellidae).

    PubMed

    Zhang, F; Wang, X J; Huang, Y H; Zhao, Z G; Zhang, S S; Gong, X S; Xie, L; Kang, D M; Jing, X

    2014-08-01

    A proteomic approach combining two-dimensional polyacrylamide gel electrophoresis and tandem mass spectrometry was used to compare hemolymph expression profiles of a beta-cypermethrin-resistant Blattella germanica L. strain and a beta-cypermethrin-susceptible strain. Twenty-eight hemolymph proteins were differentially expressed in the resistant cockroach strain; 19 proteins were upregulated and 9 proteins were downregulated compared with the susceptible strain. Protein identification indicated that expression of putative cuticular protein, nitric oxide synthase, triosephosphate isomerase, alpha-amylase, ABC transporter, and Per a 3 allergen was elevated, and expression of arginine kinase and glycosidase was reduced. The differential expression of these proteins reflects the overall change in cellular structure and metabolism related to the resistance of pyrethroid insecticides.

  18. Protein expression in a transformed trabecular meshwork cell line: proteome analysis.

    PubMed

    Steely, H Thomas; Dillow, Glen W; Bian, Liangqian; Grundstad, Jason; Braun, Terry A; Casavant, Thomas L; McCartney, Mitchell D; Clark, Abbot F

    2006-04-18

    Characterization of the human trabecular meshwork (TM) proteome is hindered by the small mass of intact tissue and the slow growth of cultured cell strains. We have previously characterized a transformed TM cell strain (GTM3) that demonstrates many of the same protein expression and cell signaling systems of nontransformed cell strains. The aim of this study was to initiate a proteomic survey of GTM3 cells as the initial step toward characterization of the complete human TM proteome. GTM3 cells were cultured to confluence, harvested and solubilized in urea/Nonidet. The protein extract (600 mug) was focused in immobilized isoelectric focusing (IEF) strips, separated by 10% SDS PAGE, and visualized with colloidal Coomassie Blue. Spots of interest were excised, destained, and the contained proteins subjected to in-gel reduction, derivatization, and tryptic digestion. Tryptic peptides were extracted and analyzed by electrospray LC/MS/MS. Protein identification was made using the TurboSequest search algorithm and a recent version of the nonredundant human protein database downloaded from the National Center for Biotechnology Information (NCBI). Eighty-seven (87) primary proteins and 93 variants of these proteins were identified. A website was created (TM proteome) that combines data such as graphic spot location within the gel, peptide sequence, apparent and calculated pI, apparent and calculated mass, percentage of coverage, and protein informatic website links. Proteomic analysis of a transformed human TM cell line has been initiated combining preparative two-dimensional PAGE separation, LC/MS/MS analysis of major proteins, and bioinformatic cataloging of the data. Further investigation of data from the transformed cell strain will be used in a comparative fashion for spot identification of analytical proteomic gels of human TM tissue and cultured normal cells. These initial data will form the base from which the characterization of protein expression in the normal

  19. Differential Proteomic Analysis of Human Erythroblasts Undergoing Apoptosis Induced by Epo-Withdrawal

    PubMed Central

    Pellegrin, Stéphanie; Heesom, Kate J.; Satchwell, Timothy J.; Hawley, Bethan R.; Daniels, Geoff; van den Akker, Emile; Toye, Ashley M.

    2012-01-01

    The availability of Erythropoietin (Epo) is essential for the survival of erythroid progenitors. Here we study the effects of Epo removal on primary human erythroblasts grown from peripheral blood CD34+ cells. The erythroblasts died rapidly from apoptosis, even in the presence of SCF, and within 24 hours of Epo withdrawal 60% of the cells were Annexin V positive. Other classical hallmarks of apoptosis were also observed, including cytochrome c release into the cytosol, loss of mitochondrial membrane potential, Bax translocation to the mitochondria and caspase activation. We adopted a 2D DIGE approach to compare the proteomes of erythroblasts maintained for 12 hours in the presence or absence of Epo. Proteomic comparisons demonstrated significant and reproducible alterations in the abundance of proteins between the two growth conditions, with 18 and 31 proteins exhibiting altered abundance in presence or absence of Epo, respectively. We observed that Epo withdrawal induced the proteolysis of the multi-functional proteins Hsp90 alpha, Hsp90 beta, SET, 14-3-3 beta, 14-3-3 gamma, 14-3-3 epsilon, and RPSA, thereby targeting multiple signaling pathways and cellular processes simultaneously. We also observed that 14 proteins were differentially phosphorylated and confirmed the phosphorylation of the Hsp90 alpha and Hsp90 beta proteolytic fragments in apoptotic cells using Nano LC mass spectrometry. Our analysis of the global changes occurring in the proteome of primary human erythroblasts in response to Epo removal has increased the repertoire of proteins affected by Epo withdrawal and identified proteins whose aberrant regulation may contribute to ineffective erythropoiesis. PMID:22723854

  20. Differential proteomic analysis of human erythroblasts undergoing apoptosis induced by epo-withdrawal.

    PubMed

    Pellegrin, Stéphanie; Heesom, Kate J; Satchwell, Timothy J; Hawley, Bethan R; Daniels, Geoff; van den Akker, Emile; Toye, Ashley M

    2012-01-01

    The availability of Erythropoietin (Epo) is essential for the survival of erythroid progenitors. Here we study the effects of Epo removal on primary human erythroblasts grown from peripheral blood CD34(+) cells. The erythroblasts died rapidly from apoptosis, even in the presence of SCF, and within 24 hours of Epo withdrawal 60% of the cells were Annexin V positive. Other classical hallmarks of apoptosis were also observed, including cytochrome c release into the cytosol, loss of mitochondrial membrane potential, Bax translocation to the mitochondria and caspase activation. We adopted a 2D DIGE approach to compare the proteomes of erythroblasts maintained for 12 hours in the presence or absence of Epo. Proteomic comparisons demonstrated significant and reproducible alterations in the abundance of proteins between the two growth conditions, with 18 and 31 proteins exhibiting altered abundance in presence or absence of Epo, respectively. We observed that Epo withdrawal induced the proteolysis of the multi-functional proteins Hsp90 alpha, Hsp90 beta, SET, 14-3-3 beta, 14-3-3 gamma, 14-3-3 epsilon, and RPSA, thereby targeting multiple signaling pathways and cellular processes simultaneously. We also observed that 14 proteins were differentially phosphorylated and confirmed the phosphorylation of the Hsp90 alpha and Hsp90 beta proteolytic fragments in apoptotic cells using Nano LC mass spectrometry. Our analysis of the global changes occurring in the proteome of primary human erythroblasts in response to Epo removal has increased the repertoire of proteins affected by Epo withdrawal and identified proteins whose aberrant regulation may contribute to ineffective erythropoiesis.

  1. Label-Free Proteome Analysis of Plasma from Patients with Breast Cancer: Stage-Specific Protein Expression

    PubMed Central

    Lobo, Marina Duarte Pinto; Moreno, Frederico Bruno Mendes Batista; Souza, Gustavo Henrique Martins Ferreira; Verde, Sara Maria Moreira Lima; Moreira, Renato de Azevedo; Monteiro-Moreira, Ana Cristina de Oliveira

    2017-01-01

    Breast cancer is one of the most commonly diagnosed types of cancer among women. Breast cancer mortality rates remain high probably because its diagnosis is hampered by inaccurate detection methods. Since changes in protein expression as well as modifications in protein glycosylation have been frequently reported in cancer development, the aim of this work was to study the differential expression as well as modifications of glycosylation of proteins from plasma of women with breast cancer at different stages of disease (n = 30) compared to healthy women (n = 10). A proteomics approach was used that depleted albumin and IgG from plasma followed by glycoprotein enrichment using immobilized Moraceae lectin (frutalin)-affinity chromatography and data-independent label-free mass spectrometric analysis. Data are available via ProteomeXchange with identifier PXD003106. As result, 57,016 peptides and 4,175 proteins among all samples were identified. From this, 40 proteins present in unbound (PI—proteins that did not interact with lectin) and bound (PII—proteins that interacted with lectin) fractions were differentially expressed. High levels of apolipoprotein A-II were detected here that were elevated significantly in the early and advanced stages of the disease. Apolipoprotein C-III was detected in both fractions, and its level was increased slightly in the PI fraction of patients with early-stage breast cancer and expressed at higher levels in the PII fraction of patients with early and intermediate stages. Clusterin was present at higher levels in both fractions of patients with early and intermediate stages of breast cancer. Our findings reveal a correlation between alterations in protein glycosylation, lipid metabolism, and the progression of breast cancer. PMID:28210565

  2. Differential gene detection incorporating common expression patterns

    NASA Astrophysics Data System (ADS)

    Oba, Shigeyuki; Ishii, Shin

    2009-12-01

    In detection of differentially expressed (DE) genes between different groups of samples based on a high-throughput expression measurement system, we often use a classical statistical testing based on a simple assumption that the expression of a certain DE gene in one group is higher or lower in average than that in the other group. Based on this simple assumption, the theory of optimal discovery procedure (ODP) (Storey, 2005) provided an optimal thresholding function for DE gene detection. However, expression patterns of DE genes over samples may have such a structure that is not exactly consistent with group labels assigned to the samples. Appropriate treatment of such a structure can increase the detection ability. Namely, genes showing similar expression patterns to other biologically meaningful genes can be regarded as statistically more significant than those showing expression patterns independent of other genes, even if differences in mean expression levels are comparable. In this study, we propose a new statistical thresholding function based on a latent variable model incorporating expression patterns together with the ODP theory. The latent variable model assumes hidden common signals behind expression patterns over samples and the ODP theory is extended to involve the latent variables. When applied to several gene expression data matrices which include cluster structures or 'cancer outlier' structures, the newly-proposed thresholding functions showed prominently better detection performance of DE genes than the original ODP thresholding function did. We also demonstrate how the proposed methods behave through analyses of real breast cancer and lymphoma datasets.

  3. Label-Free Differential Proteomics and Quantification of Exoenzymes from Isolates of the Entomopathogenic Fungus Beauveria bassiana.

    PubMed

    Dionisio, Giuseppe; Kryger, Per; Steenberg, Tove

    2016-10-14

    Beauveria bassiana is an entomopathogenic fungus that grows both in vivo and in vitro. In vivo it can colonize live insect hosts, and tissue digestion occurs by secreted hydrolytic exoenzymes. It can also colonize dead insect tissue provided this is free from competing microorganisms. Depending on whether the host is alive or dead the expression (quality/quantity) of the exoenzymes may vary. We have grown several isolates of B. bassiana in shaking flasks for 120 h at 25 °C in order to evaluate the maximal exoenzyme production using two diet regimes. As sole carbon, nitrogen, and phosphate sources we used 1% shrimp chitin and either 0.5% w/v of dead intact American cockroach (Periplaneta americana) or their isolated cuticles. This is the first report of a differential proteomics of B. bassiana exoenzymes performed by label-free nano-LC MS/MS. Total proteolytic enzyme activity was mainly due to Pr1A or Pr1B depending on the isolate and the diet regime. The most differentially secreted enzymes were: the cuticle-degrading subtilisin Pr1A, GH13 alpha-glycosidase, glucan endo-1,3-beta-glucosidase, subtilisin-like proteinase Spm1, lipase 1, beta-1,3 exoglucanase, and endo-1,3-beta-glucosidase. Among the B. bassiana isolates analyzed, Bb 678 and Bb BG were the most active in Pr1A secretion.

  4. Label-Free Differential Proteomics and Quantification of Exoenzymes from Isolates of the Entomopathogenic Fungus Beauveria bassiana

    PubMed Central

    Dionisio, Giuseppe; Kryger, Per; Steenberg, Tove

    2016-01-01

    Beauveria bassiana is an entomopathogenic fungus that grows both in vivo and in vitro. In vivo it can colonize live insect hosts, and tissue digestion occurs by secreted hydrolytic exoenzymes. It can also colonize dead insect tissue provided this is free from competing microorganisms. Depending on whether the host is alive or dead the expression (quality/quantity) of the exoenzymes may vary. We have grown several isolates of B. bassiana in shaking flasks for 120 h at 25 °C in order to evaluate the maximal exoenzyme production using two diet regimes. As sole carbon, nitrogen, and phosphate sources we used 1% shrimp chitin and either 0.5% w/v of dead intact American cockroach (Periplaneta americana) or their isolated cuticles. This is the first report of a differential proteomics of B. bassiana exoenzymes performed by label-free nano-LC MS/MS. Total proteolytic enzyme activity was mainly due to Pr1A or Pr1B depending on the isolate and the diet regime. The most differentially secreted enzymes were: the cuticle-degrading subtilisin Pr1A, GH13 alpha-glycosidase, glucan endo-1,3-beta-glucosidase, subtilisin-like proteinase Spm1, lipase 1, beta-1,3 exoglucanase, and endo-1,3-beta-glucosidase. Among the B. bassiana isolates analyzed, Bb 678 and Bb BG were the most active in Pr1A secretion. PMID:27754403

  5. Proteomic Screening and Lasso Regression Reveal Differential Signaling in Insulin and Insulin-like Growth Factor I (IGF1) Pathways *

    PubMed Central

    Erdem, Cemal; Nagle, Alison M.; Casa, Angelo J.; Litzenburger, Beate C.; Wang, Yu-fen; Taylor, D. Lansing; Lee, Adrian V.; Lezon, Timothy R.

    2016-01-01

    Insulin and insulin-like growth factor I (IGF1) influence cancer risk and progression through poorly understood mechanisms. To better understand the roles of insulin and IGF1 signaling in breast cancer, we combined proteomic screening with computational network inference to uncover differences in IGF1 and insulin induced signaling. Using reverse phase protein array, we measured the levels of 134 proteins in 21 breast cancer cell lines stimulated with IGF1 or insulin for up to 48 h. We then constructed directed protein expression networks using three separate methods: (i) lasso regression, (ii) conventional matrix inversion, and (iii) entropy maximization. These networks, named here as the time translation models, were analyzed and the inferred interactions were ranked by differential magnitude to identify pathway differences. The two top candidates, chosen for experimental validation, were shown to regulate IGF1/insulin induced phosphorylation events. First, acetyl-CoA carboxylase (ACC) knock-down was shown to increase the level of mitogen-activated protein kinase (MAPK) phosphorylation. Second, stable knock-down of E-Cadherin increased the phospho-Akt protein levels. Both of the knock-down perturbations incurred phosphorylation responses stronger in IGF1 stimulated cells compared with insulin. Overall, the time-translation modeling coupled to wet-lab experiments has proven to be powerful in inferring differential interactions downstream of IGF1 and insulin signaling, in vitro. PMID:27364358

  6. Differential proteomic analysis of midguts from Nosema ceranae-infected honeybees reveals manipulation of key host functions.

    PubMed

    Vidau, Cyril; Panek, Johan; Texier, Catherine; Biron, David G; Belzunces, Luc P; Le Gall, Morgane; Broussard, Cédric; Delbac, Frédéric; El Alaoui, Hicham

    2014-09-01

    Many invasive pathogens effectively bypass the insect defenses to ensure the completion of their life cycle. Among those, an invasive microsporidian species, Nosema ceranae, can cause nosemosis in honeybees. N. ceranae was first described in the Asian honeybee Apis cerana and is suspected to be involved in Western honeybee (Apis mellifera) declines worldwide. The midgut of honeybees is the first barrier against N. ceranae attacks. To bring proteomics data on honeybee/N. ceranae crosstalk and more precisely to decipher the worker honeybee midgut response after an oral inoculation of N. ceranae (10days post-infection), we used 2D-DIGE (2-Dimensional Differential In-Gel Electrophoresis) combined with mass spectrometry. Forty-five protein spots produced by the infected worker honeybee group were shown to be differentially expressed when compared to the uninfected group; 14 were subsequently identified by mass spectrometry. N. ceranae mainly caused a modulation of proteins involved in three key host biological functions: (i) energy production, (ii) innate immunity (reactive oxygen stress) and (iii) protein regulation. The modulation of these host biological functions suggests that N. ceranae creates a zone of "metabolic habitat modification" in the honeybee midgut favoring its development by enhancing availability of nutrients and reducing the worker honeybee defense.

  7. Proteomic responses of European flounder to temperature and hypoxia as interacting stressors: Differential sensitivities of populations.

    PubMed

    Pédron, Nicolas; Artigaud, Sébastien; Infante, José-Luis Zambonino; Le Bayon, Nicolas; Charrier, Grégory; Pichereau, Vianney; Laroche, Jean

    2017-02-16

    In the context of global change, ectotherms are increasingly impacted by abiotic perturbations. Along the distribution area of a species, the populations at low latitudes are particularly exposed to temperature increase and hypoxic events. In this study, we have compared the proteomic responses in the liver of European flounder populations, by using 2-D electrophoresis. One southern peripheral population from Portugal vs two northern core populations from France, were reared in a common garden experiment. Most of the proteomic differences were observed between the two experimental conditions, a cold vs a warm and hypoxic conditions. Consistent differentiations between populations were observed in accumulation of proteins involved in the bioenergetics- and methionine-metabolisms, fatty acids transport, and amino-acid catabolism. The specific regulation of crucial enzymes like ATP-synthase and G6PDH, in the liver of the southern population, could be related to a possible local adaptation. This southern peripheral population is spatially distant from northern core populations and has experienced dissimilar ecological conditions; thus it may contain genotypes that confer resilience to climate changes.

  8. A Quantitative Proteomic Analysis of Hemogenic Endothelium Reveals Differential Regulation of Hematopoiesis by SOX17

    PubMed Central

    Clarke, Raedun L.; Robitaille, Aaron M.; Moon, Randall T.; Keller, Gordon

    2015-01-01

    Summary The in vitro derivation of hematopoietic stem cells (HSCs) from pluripotent stem cells (PSCs) is complicated by the existence of multiple overlapping embryonic blood cell programs called primitive, erythromyeloid progenitor (EMP), and definitive. As HSCs are only generated during the definitive stage of hematopoiesis, deciphering the regulatory pathways that control the emergence of this program and identifying markers that distinguish it from the other programs are essential. To identify definitive specific pathways and marker sets, we used label-free proteomics to determine the proteome of embryo-derived and mouse embryonic stem cell-derived VE-CADHERIN+CD45− definitive hematopoietic progenitors. With this approach, we identified Stat1 as a marker that distinguishes the definitive erythroid lineage from the primitive- and EMP-derived lineages. Additionally, we provide evidence that the generation of the Stat1+ definitive lineage is dependent on Sox17. These findings establish an approach for monitoring the emergence of definitive hematopoiesis in the PSC differentiation cultures. PMID:26267830

  9. Differential proteomic analysis of bovine conceptus fluid proteins in pregnancies generated by assisted reproductive technologies.

    PubMed

    Riding, George A; Hill, Jonathan R; Jones, Alun; Holland, Michael K; Josh, Peter F; Lehnert, Sigrid A

    2008-07-01

    Proteomic analysis of bovine conceptus fluid proteins during early pregnancy has the potential to expose protein species indicative of both the overall health of the fetal-maternal environment and fetal developmental status. In this study, we examined the differential abundance of bovine conceptus fluid proteins (5-50 kDa fraction) from naturally conceived, in vitro fertilisation (IVF) and somatic cell nuclear transfer (SCNT)-derived pregnancies at days 45 and 90 of gestation. In day 45 allantoic fluid (AllF) samples, an atypical cluster of low molecular weight ( approximately 14-16 kDa), low pI (between 3.0 and 4.5 pH units) protein species was increased in three of four IVF samples (30-100-fold increase in protein spot volumes compared to normal). These proteins were identified as paralogs of the bovine cathelicidin antimicrobial protein (CAMP) by MALDI-TOF MS peptide mass fingerprint and MALDI-TOF MS/MS peptide sequence analysis. Peptidoglycan recognition protein and serine (or cysteine) proteinase inhibitor clade B1, were also significantly increased in the corresponding IVF samples. In two of four SCNT AllF samples, a 2-10-fold increase in CAMP protein spot volumes were detected. No aberrant abundance levels of individual protein species were observed in amniotic fluid samples, or in day 90 IVF AllF samples. Identification of unique protein species present in the normal bovine AllF proteome at day 45 is also reported.

  10. Classical Galactosemia: Insight into Molecular Pathomechanisms by Differential Membrane Proteomics of Fibroblasts under Galactose Stress.

    PubMed

    Staubach, Simon; Müller, Stefan; Pekmez, Murat; Hanisch, Franz-Georg

    2017-02-03

    Classical galactosemia, a hereditary metabolic disease caused by the deficiency of galactose-1-phosphate uridyltransferase (GALT; EC 2.7.712), results in an impaired galactose metabolism and serious long-term developmental affection of the CNS and ovaries, potentially related in part to endogenous galactose-induced protein dysglycosylation. In search for galactose-induced changes in membrane raft proteomes of GALT-deficient cells, we performed differential analyses of lipid rafts from patient-derived (Q) and sex- and age-matched control fibroblasts (H) in the presence or absence of the stressor. Label-based proteomics revealed of the total 454 (female) or 678 (male) proteins a proportion of ∼12% in at least one of four relevant ratios as fold-changed. GALT(-) cell-specific effects in the absence of stressor revealed cell-model-dependent affection of biological processes related to protein targeting to the plasma membrane (female) or to cellular migration (male). However, a series of common galactose-induced effects were observed, among them the strongly increased ER-stress marker GRP78 and calreticulin involved in N-glycoprotein quality control. The membrane-anchored N-glycoprotein receptor CD109 was concertedly decreased under galactose-stress together with cadherin-13, GLIPR1, glypican-1, and semaphorin-7A. A series of proteins showed opposite fold-changes in the two cell models, whereas others fluctuated in only one of the two models.

  11. Reference proteome of highly purified human Th1 cells reveals strong effects on metabolism and protein ubiquitination upon differentiation.

    PubMed

    Pagani, Massimiliano; Rockstroh, Maxie; Schuster, Maj; Rossetti, Grazisa; Moro, Monica; Crosti, Mariacristina; Tomm, Janina M

    2015-11-01

    The differentiation of human CD4(+) T cells into T helper cell subtypes and regulatory T cells is crucial to the immune response. Among subtypes, Th1 cells are dominant, representing approximately 50% of all lymphocytes. Thus far, most global proteomic studies have used only partially purified T helper cell subpopulations and/or have employed artificial protocols for inducing specific T helper cell subtypes and/or used gel-based approaches. These studies have shed light on molecular details of certain aspects of the proteome; nevertheless a global analysis of high purity primary naïve and Th1 cells by LC-MS/MS is required to provide a reference dataset for proteome-based T cell subtype characterization. The utilization of highly purified Th1 cells for a global proteome assessment and the bioinformatic comparison to naïve cells reveals changes in cell metabolism and the ubiquitination pathway upon T cell differentiation. All MS data have been deposited in the ProteomeXchange with identifier PXD001066 (http://proteomecentral.proteomexchange.org/dataset/PXD001066). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Use of proteomic differential displays to assess functional discrepancies and adjustments of human bone marrow- and Wharton jelly-derived mesenchymal stem cells.

    PubMed

    Kuo, Hsing-Chun; Chiu, Chi-Chin; Chang, Wan-Ching; Sheen, Jiunn-Ming; Ou, Chia-Yu; Kuo, Ho-Chang; Chen, Rong-Fu; Hsu, Te-Yao; Chang, Jen-Chieh; Hsaio, Chang-Chun; Wang, Feng-Sheng; Huang, Chung-Cheng; Huang, Hsuan-Ying; Yang, Kuender D

    2011-03-04

    Mesenchymal stem cells (MSCs) from bone marrow are suitable for the reconstruction of connective tissues and even brain tissue but have limitations in terms of cell expansion and fully specific differentiation. In our current study, we have attempted to adjust and improve the cell expansion and differentiation properties of human MSCs from different tissues. MSCs from normal bone marrow and Wharton jelly were subjected to proteomic differential displays, followed by functional adjustments based on these displays. Bone marrow MSCs expressed more transgelin-2 and differentiated more rapidly into bone nodules but showed a slower growth rate. A knockdown of transgelin-2 expression by specific small interfering RNA (siRNA) significantly increased the growth rate of these cells, the G1/S phase cell cycle transition, and the interaction of cyclin D1 with cdk2. Wharton jelly MSCs expressed the chaperone protein HSP90β at higher levels and differentiated slowly toward an osteogenic lineage. However, the knockdown of HSP90β expression significantly increased bone nodule formation, inhibited cell growth, decreased the number of cells in the G1/S phase of the cell cycle, and decreased the interaction of cyclin D1 with cdk2 and of cyclin E with cdk2. These results were validated by the in vivo repair of segmental bone defects in a mouse model with severe combined immunodeficiency. We thus demonstrate an improvement in the cell expansion and tissue regeneration properties of human MSCs through specific adjustments.

  13. Gibberellin-Stimulation of Rhizome Elongation and Differential GA-Responsive Proteomic Changes in Two Grass Species

    PubMed Central

    Ma, Xiqing; Huang, Bingru

    2016-01-01

    Rapid and extensive rhizome development is a desirable trait for perennial grass growth and adaptation to environmental stresses. The objective of this study was to determine proteomic changes and associated metabolic pathways of gibberellin (GA) -regulation of rhizome elongation in two perennial grass species differing in rhizome development. Plants of a short-rhizome bunch-type tall fescue (TF; Festuca arundinacea; ‘BR’) and an extensive rhizomatous Kentucky bluegrass (KB; Poa pratensis; ‘Baron’) were treated with 10 μM GA3 in hydroponic culture in growth chambers. The average rhizome length in KB was significantly longer than that in TF regardless of GA3 treatment, and increased significantly with GA3 treatment, to a greater extent than that in TF. Comparative proteomic analysis using two-dimensional electrophoresis and mass spectrometry was performed to further investigate proteins and associated metabolic pathways imparting increased rhizome elongation by GA. A total of 37 and 38 differentially expressed proteins in response to GA3 treatment were identified in TF and KB plants, respectively, which were mainly involved in photosynthesis, energy and amino acid metabolism, protein synthesis, defense and cell development processes. Accelerated rhizome elongation in KB by GA could be mainly associated with the increased abundance of proteins involved in energy metabolism (glyceraldehyde-3-phosphate dehydrogenase, fructose-bisphosphate aldolase, and ATP synthase), amino acid metabolism (S-adenosylmethionine and adenosylhomocysteinase), protein synthesis (HSP90, elongation factor Tu and eukaryotic translation initiation factor 5A), cell-wall development (cell dividion cycle protein, alpha tubulin-2A and actin), and signal transduction (calreticulin). These proteins could be used as candidate proteins for further analysis of molecular mechanisms controlling rhizome growth. PMID:27446135

  14. Specific changes in the Arabidopsis proteome in response to bacterial challenge: differentiating basal and R-gene mediated resistance.

    PubMed

    Jones, Alexandra M E; Thomas, Vincent; Truman, Bill; Lilley, Kathryn; Mansfield, John; Grant, Murray

    2004-06-01

    Alterations in the proteome of Arabidopsis thaliana leaves during early responses to challenge by Pseudomonas syringae pv. tomato DC3000 (DC3000) were analysed using two-dimensional (2D) gel electrophoresis. Protein changes characteristic of the establishment of basal resistance and R-gene mediated resistance were examined by comparing responses to DC3000, a hrp mutant and DC3000 expressing avrRpm1 respectively. The abundance of selected transcripts was also analysed in GeneChip experiments. Here we present data from the soluble fraction of leaf protein, highlighting changes in two antioxidant enzyme groups; the glutathione S-transferases (GSTs F2, F6, F7 and F8) and peroxiredoxins (PrxA, B and IIE). Members of both enzyme groups showed signs of specific post-translational modifications, represented by multiple spots on gels. We suggest that oxidation of specific residues is responsible for some of the spot shifts. All forms of the GST proteins identified here increased following inoculation with bacteria. GSTF8 showed particularly dynamic responses to pathogen challenge, the corresponding transcript was significantly up-regulated by 2 h after inoculation, and the protein showed post-translational modifications specific to an incompatible interaction. Differential changes were observed with the peroxiredoxin proteins; PrxIIE and to a lesser extent PrxB, no change was observed with PrxA, but a truncated form PrxA-L was greatly reduced in abundance following bacterial challenges. Our data suggest that bacterial challenge generally induces Prxs and the antioxidants GSTs, however individual members of these families may be specifically modified dependent upon the virulence of the DC3000 strain and outcome of the interaction. Finally, proteomic and transcriptomic data derived from the same inoculation system are compared and the advantages offered by 2D gel analysis discussed in light of our results.

  15. Serum immune-related proteins are differentially expressed during hibernation in the American black bear.

    PubMed

    Chow, Brian A; Donahue, Seth W; Vaughan, Michael R; McConkey, Brendan; Vijayan, Mathilakath M

    2013-01-01

    Hibernation is an adaptation to conserve energy in the face of extreme environmental conditions and low food availability that has risen in several animal phyla. This phenomenon is characterized by reduced metabolic rate (∼25% of the active basal metabolic rate in hibernating bears) and energy demand, while other physiological adjustments are far from clear. The profiling of the serum proteome of the American black bear (Ursus americanus) may reveal specific proteins that are differentially modulated by hibernation, and provide insight into the remarkable physiological adaptations that characterize ursid hibernation. In this study, we used differential gel electrophoresis (DIGE) analysis, liquid chromatography coupled to tandem mass spectrometry, and subsequent MASCOT analysis of the mass spectra to identify candidate proteins that are differentially expressed during hibernation in captive black bears. Seventy serum proteins were identified as changing by ±1.5 fold or more, out of which 34 proteins increased expression during hibernation. The majority of identified proteins are involved in immune system processes. These included α2-macroglobulin, complement components C1s and C4, immunoglobulin μ and J chains, clusterin, haptoglobin, C4b binding protein, kininogen 1, α2-HS-glycoprotein, and apoplipoproteins A-I and A-IV. Differential expression of a subset of these proteins identified by proteomic analysis was also confirmed by immunodetection. We propose that the observed serum protein changes contribute to the maintenance of the hibernation phenotype and health, including increased capacities for bone maintenance and wound healing during hibernation in bears.

  16. Serum Immune-Related Proteins are Differentially Expressed during Hibernation in the American Black Bear

    PubMed Central

    Chow, Brian A.; Donahue, Seth W.; Vaughan, Michael R.; McConkey, Brendan; Vijayan, Mathilakath M.

    2013-01-01

    Hibernation is an adaptation to conserve energy in the face of extreme environmental conditions and low food availability that has risen in several animal phyla. This phenomenon is characterized by reduced metabolic rate (∼25% of the active basal metabolic rate in hibernating bears) and energy demand, while other physiological adjustments are far from clear. The profiling of the serum proteome of the American black bear (Ursus americanus) may reveal specific proteins that are differentially modulated by hibernation, and provide insight into the remarkable physiological adaptations that characterize ursid hibernation. In this study, we used differential gel electrophoresis (DIGE) analysis, liquid chromatography coupled to tandem mass spectrometry, and subsequent MASCOT analysis of the mass spectra to identify candidate proteins that are differentially expressed during hibernation in captive black bears. Seventy serum proteins were identified as changing by ±1.5 fold or more, out of which 34 proteins increased expression during hibernation. The majority of identified proteins are involved in immune system processes. These included α2-macroglobulin, complement components C1s and C4, immunoglobulin μ and J chains, clusterin, haptoglobin, C4b binding protein, kininogen 1, α2-HS-glycoprotein, and apoplipoproteins A-I and A-IV. Differential expression of a subset of these proteins identified by proteomic analysis was also confirmed by immunodetection. We propose that the observed serum protein changes contribute to the maintenance of the hibernation phenotype and health, including increased capacities for bone maintenance and wound healing during hibernation in bears. PMID:23825529

  17. Differential proteomic and genomic profiling of mouse striatal cell model of Huntington's disease and control; probable implications to the disease biology.

    PubMed

    Choudhury, Kamalika Roy; Das, Srijit; Bhattacharyya, Nitai P

    2016-01-30

    Huntington's disease (HD) is an autosomal dominant disorder of central nervous system caused by expansion of CAG repeats in exon1 of the huntingtin gene (Htt). Among various dysfunctions originated from the mutation in Htt gene, transcriptional deregulation has been considered to be one of the most important abnormalities. Large numbers of investigations identified altered expressions of genes in brains of HD patients and many models of HD. In this study we employed 2D SDS-PAGE/MALDI-MS coupled with 2D-DIGE and real-time PCR experiments of an array of genes focused to HD pathway to determine altered protein and gene expressions in STHdh(Q111)/Hdh(Q111) cells, a cell model of HD and compared with STHdh(Q7)/Hdh(Q7) cells, its wild type counterpart. We annotated 76 proteins from these cells and observed differential expressions of 31 proteins (by 2D-DIGE) involved in processes like unfolded protein binding, negative regulation of neuron apoptosis, response to superoxides etc. Our PCR array experiments identified altered expressions of 47 genes. Altogether significant alteration of 77 genes/proteins could be identified in this HD cell line with potential relevance to HD biology. In this study we intended to find out differential proteomic and genomic profiles in HD condition. We used the STHdh cells, a cellular model for HD and control. These are mouse striatal neuronal cell lines harboring 7 and 111 knock-in CAG repeats in their two alleles. The 111Q containing cell line (STHdh(Q111)/Hdh(Q111)) mimics diseased condition, whereas the 7Q containing ones (STHdh(Q7)/Hdh(Q7)), serves as the proper control cell line. Proteomic experiments were performed earlier to obtain differential expressions of proteins in R6/2 mice models, Hdh(Q) knock-in mice and in plasma and CSF from HD patients. However, no earlier report on proteomic alterations in these two HD cell lines and control was available in literature. It was, therefore, an important objective to find out differential

  18. Towards a Proteomic Catalogue and Differential Annotation of Salivary Gland Proteins in Blood Fed Malaria Vector Anopheles culicifacies by Mass Spectrometry.

    PubMed

    Rawal, Ritu; Vijay, Sonam; Kadian, Kavita; Singh, Jagbir; Pande, Veena; Sharma, Arun

    2016-01-01

    In order to understand the importance of functional proteins in mosquito behavior, following blood meal, a baseline proteomic dataset is essential for providing insights into the physiology of blood feeding. Therefore, in this study as first step, in solution and 1-D electrophoresis digestion approach combined with tandem mass spectrometry (nano LC-MS/MS) and computational bioinformatics for data mining was used to prepare a baseline proteomic catalogue of salivary gland proteins of sugar fed An. culicifacies mosquitoes. A total of 106 proteins were identified and analyzed by SEQUEST algorithm against mosquito protein database from Uniprot/NCBI. Importantly, D7r1, D7r2, D7r4, salivary apyrase, anti-platelet protein, calreticulin, antigen 5 family proteins were identified and grouped on the basis of biological and functional roles. Secondly, differential protein expression and annotations between salivary glands of sugar fed vs blood fed mosquitoes was analyzed using 2-Delectrophoresis combined with MALDI-TOF mass spectrometry. The alterations in the differential expression of total 38 proteins was observed out of which 29 proteins like beclin-1, phosphorylating proteins, heme oxygenase 1, ferritin, apoptotic proteins, coagulation and immunity like, serine proteases, serpins, c-type lectin and protein in regulation of blood feeding behavior were found to be up regulated while 9 proteins related to blood feeding, juvenile hormone epoxide hydrolase ii, odorant binding proteins and energy metabolic enzymes were found to be down regulated. To our knowledge, this study provides a first time baseline proteomic dataset and functional annotations of An. culicifacies salivary gland proteins that may be involved during the blood feeding. Identification of differential salivary proteins between sugar fed and blood fed mosquitoes and their plausible role may provide insights into the physiological processes associated with feeding behavior and sporozoite transmission during the

  19. Towards a Proteomic Catalogue and Differential Annotation of Salivary Gland Proteins in Blood Fed Malaria Vector Anopheles culicifacies by Mass Spectrometry

    PubMed Central

    Rawal, Ritu; Vijay, Sonam; Kadian, Kavita; Singh, Jagbir; Pande, Veena; Sharma, Arun

    2016-01-01

    In order to understand the importance of functional proteins in mosquito behavior, following blood meal, a baseline proteomic dataset is essential for providing insights into the physiology of blood feeding. Therefore, in this study as first step, in solution and 1-D electrophoresis digestion approach combined with tandem mass spectrometry (nano LC-MS/MS) and computational bioinformatics for data mining was used to prepare a baseline proteomic catalogue of salivary gland proteins of sugar fed An. culicifacies mosquitoes. A total of 106 proteins were identified and analyzed by SEQUEST algorithm against mosquito protein database from Uniprot/NCBI. Importantly, D7r1, D7r2, D7r4, salivary apyrase, anti-platelet protein, calreticulin, antigen 5 family proteins were identified and grouped on the basis of biological and functional roles. Secondly, differential protein expression and annotations between salivary glands of sugar fed vs blood fed mosquitoes was analyzed using 2-Delectrophoresis combined with MALDI-TOF mass spectrometry. The alterations in the differential expression of total 38 proteins was observed out of which 29 proteins like beclin-1, phosphorylating proteins, heme oxygenase 1, ferritin, apoptotic proteins, coagulation and immunity like, serine proteases, serpins, c-type lectin and protein in regulation of blood feeding behavior were found to be up regulated while 9 proteins related to blood feeding, juvenile hormone epoxide hydrolase ii, odorant binding proteins and energy metabolic enzymes were found to be down regulated. To our knowledge, this study provides a first time baseline proteomic dataset and functional annotations of An. culicifacies salivary gland proteins that may be involved during the blood feeding. Identification of differential salivary proteins between sugar fed and blood fed mosquitoes and their plausible role may provide insights into the physiological processes associated with feeding behavior and sporozoite transmission during the

  20. Comparative proteomic analysis of differential responses of Pinus massoniana and Taxus wallichiana var. mairei to simulated acid rain.

    PubMed

    Hu, Wen-Jun; Chen, Juan; Liu, Ting-Wu; Simon, Martin; Wang, Wen-Hua; Chen, Juan; Wu, Fei-Hua; Liu, Xiang; Shen, Zhi-Jun; Zheng, Hai-Lei

    2014-03-12

    Acid rain (AR), a serious environmental issue, severely affects plant growth and development. As the gymnosperms of conifer woody plants, Pinus massoniana (AR-sensitive) and Taxus wallichiana var. mairei (AR-resistant) are widely distributed in southern China. Under AR stress, significant necrosis and collapsed lesions were found in P. massoniana needles with remarkable yellowing and wilting tips, whereas T. wallichiana var. mairei did not exhibit chlorosis and visible damage. Due to the activation of a large number of stress-related genes and the synthesis of various functional proteins to counteract AR stress, it is important to study the differences in AR-tolerance mechanisms by comparative proteomic analysis of tolerant and sensitive species. This study revealed a total of 65 and 26 differentially expressed proteins that were identified in P. massoniana and T. wallichiana var. mairei, respectively. Among them, proteins involved in metabolism, photosynthesis, signal transduction and transcription were drastically down-regulated in P. massoniana, whereas most of the proteins participating in metabolism, cell structure, photosynthesis and transcription were increased in T. wallichiana var. mairei. These results suggest the distinct patterns of protein expression in the two woody species in response to AR, allowing a deeper understanding of diversity on AR tolerance in forest tree species.

  1. Comparative Proteomic Analysis of Differential Responses of Pinus massoniana and Taxus wallichiana var. mairei to Simulated Acid Rain

    PubMed Central

    Hu, Wen-Jun; Chen, Juan; Liu, Ting-Wu; Simon, Martin; Wang, Wen-Hua; Chen, Juan; Wu, Fei-Hua; Liu, Xiang; Shen, Zhi-Jun; Zheng, Hai-Lei

    2014-01-01

    Acid rain (AR), a serious environmental issue, severely affects plant growth and development. As the gymnosperms of conifer woody plants, Pinus massoniana (AR-sensitive) and Taxus wallichiana var. mairei (AR-resistant) are widely distributed in southern China. Under AR stress, significant necrosis and collapsed lesions were found in P. massoniana needles with remarkable yellowing and wilting tips, whereas T. wallichiana var. mairei did not exhibit chlorosis and visible damage. Due to the activation of a large number of stress-related genes and the synthesis of various functional proteins to counteract AR stress, it is important to study the differences in AR-tolerance mechanisms by comparative proteomic analysis of tolerant and sensitive species. This study revealed a total of 65 and 26 differentially expressed proteins that were identified in P. massoniana and T. wallichiana var. mairei, respectively. Among them, proteins involved in metabolism, photosynthesis, signal transduction and transcription were drastically down-regulated in P. massoniana, whereas most of the proteins participating in metabolism, cell structure, photosynthesis and transcription were increased in T. wallichiana var. mairei. These results suggest the distinct patterns of protein expression in the two woody species in response to AR, allowing a deeper understanding of diversity on AR tolerance in forest tree species. PMID:24625662

  2. Differential proteome analysis of a selected bacterial strain isolated from a high background radiation area in response to radium stress.

    PubMed

    Zakeri, Farideh; Sadeghizadeh, Majid; Kardan, Mohammad Reza; Shahbani Zahiri, Hossein; Ahmadian, Gholamreza; Masoumi, Fatemeh; Sharafi, Hakimeh; Rigi, Garshasb; Vali, Hojatollah; Akbari Noghabi, Kambiz

    2012-08-03

    The present study describes the response of a bacterial strain, isolated from a hot spring in an area with the highest levels of natural radiation, under radium ((226)Ra) stress. The bacterium has been characterized as a novel and efficient radium biosorbent and identified as a variant of Serratia marcescens by biochemical tests and molecular recognition. In order to gain insights into key cellular events that allow this strain to survive and undergo (226)Ra adaptation and biosorption, the strain was tested under two experimental conditions of 1000 and 6000 Bq (226)Ra stress. A proteomic approach involving two-dimensional polyacrylamide gel electrophoresis and mass spectrometry was used to identify the differentially expressed proteins under (226)Ra stress. Functional assessment of identified proteins with significantly altered expression levels revealed several mechanisms thought to be involved in (226)Ra adaptation and conferring resistant phenotype to the isolate, including general stress adaptation, anti-oxidative stress, protein and nucleic acid synthesis, energy metabolism, efflux and transport proteins. It suggests that this strain through evolution is particularly well adapted to the high background radiation environment and could represent an alternative source to remove (226)Ra from such areas as well as industrial radionuclide polluted wastewaters. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. BRCA1 deficiency in ovarian cancer is associated with alteration in expression of several key regulators of cell motility – A proteomics study

    PubMed Central

    Gau, David M; Lesnock, Jamie L; Hood, Brian L; Bhargava, Rohit; Sun, Mai; Darcy, Kathleen; Luthra, Soumya; Chandran, Uma; Conrads, Thomas P; Edwards, Robert P; Kelley, Joseph L; Krivak, Thomas C; Roy, Partha

    2015-01-01

    Functional loss of expression of breast cancer susceptibility gene 1(BRCA1) has been implicated in genomic instability and cancer progression. There is emerging evidence that BRCA1 gene product (BRCA1) also plays a role in cancer cell migration. We performed a quantitative proteomics study of EOC patient tumor tissues and identified changes in expression of several key regulators of actin cytoskeleton/cell adhesion and cell migration (CAPN1, 14-3-3, CAPG, PFN1, SPTBN1, CFN1) associated with loss of BRCA1 function. Gene expression analyses demonstrate that several of these proteomic hits are differentially expressed between early and advanced stage EOC thus suggesting clinical relevance of these proteins to disease progression. By immunohistochemistry of ovarian tumors with BRCA1+/+ and BRCA1null status, we further verified our proteomic-based finding of elevated PFN1 expression associated with BRCA1 deficiency. Finally, we established a causal link between PFN1 and BRCA1-induced changes in cell migration thus uncovering a novel mechanistic basis for BRCA1-dependent regulation of ovarian cancer cell migration. Overall, findings of this study open up multiple avenues by which BRCA1 can potentially regulate migration and metastatic phenotype of EOC cells. PMID:25927284

  4. BRCA1 deficiency in ovarian cancer is associated with alteration in expression of several key regulators of cell motility - A proteomics study.

    PubMed

    Gau, David M; Lesnock, Jamie L; Hood, Brian L; Bhargava, Rohit; Sun, Mai; Darcy, Kathleen; Luthra, Soumya; Chandran, Uma; Conrads, Thomas P; Edwards, Robert P; Kelley, Joseph L; Krivak, Thomas C; Roy, Partha

    2015-01-01

    Functional loss of expression of breast cancer susceptibility gene 1(BRCA1) has been implicated in genomic instability and cancer progression. There is emerging evidence that BRCA1 gene product (BRCA1) also plays a role in cancer cell migration. We performed a quantitative proteomics study of EOC patient tumor tissues and identified changes in expression of several key regulators of actin cytoskeleton/cell adhesion and cell migration (CAPN1, 14-3-3, CAPG, PFN1, SPTBN1, CFN1) associated with loss of BRCA1 function. Gene expression analyses demonstrate that several of these proteomic hits are differentially expressed between early and advanced stage EOC thus suggesting clinical relevance of these proteins to disease progression. By immunohistochemistry of ovarian tumors with BRCA1(+/+) and BRCA1(null) status, we further verified our proteomic-based finding of elevated PFN1 expression associated with BRCA1 deficiency. Finally, we established a causal link between PFN1 and BRCA1-induced changes in cell migration thus uncovering a novel mechanistic basis for BRCA1-dependent regulation of ovarian cancer cell migration. Overall, findings of this study open up multiple avenues by which BRCA1 can potentially regulate migration and metastatic phenotype of EOC cells.

  5. Proteomic Profiles in Acute Respiratory Distress Syndrome Differentiates Survivors from Non-Survivors

    PubMed Central

    Bhargava, Maneesh; Becker, Trisha L.; Viken, Kevin J.; Jagtap, Pratik D.; Dey, Sanjoy; Steinbach, Michael S.; Wu, Baolin; Kumar, Vipin; Bitterman, Peter B.; Ingbar, David H.; Wendt, Christine H.

    2014-01-01

    Acute Respiratory Distress Syndrome (ARDS) continues to have a high mortality. Currently, there are no biomarkers that provide reliable prognostic information to guide clinical management or stratify risk among clinical trial participants. The objective of this study was to probe the bronchoalveolar lavage fluid (BALF) proteome to identify proteins that differentiate survivors from non-survivors of ARDS. Patients were divided into early-phase (1 to 7 days) and late-phase (8 to 35 days) groups based on time after initiation of mechanical ventilation for ARDS (Day 1). Isobaric tags for absolute and relative quantitation (iTRAQ) with LC MS/MS was performed on pooled BALF enriched for medium and low abundance proteins from early-phase survivors (n = 7), early-phase non-survivors (n = 8), and late-phase survivors (n = 7). Of the 724 proteins identified at a global false discovery rate of 1%, quantitative information was available for 499. In early-phase ARDS, proteins more abundant in survivors mapped to ontologies indicating a coordinated compensatory response to injury and stress. These included coagulation and fibrinolysis; immune system activation; and cation and iron homeostasis. Proteins more abundant in early-phase non-survivors participate in carbohydrate catabolism and collagen synthesis, with no activation of compensatory responses. The compensatory immune activation and ion homeostatic response seen in early-phase survivors transitioned to cell migration and actin filament based processes in late-phase survivors, revealing dynamic changes in the BALF proteome as the lung heals. Early phase proteins differentiating survivors from non-survivors are candidate biomarkers for predicting survival in ARDS. PMID:25290099

  6. Utility of proteomics techniques for assessing protein expression.

    PubMed

    Natarajan, Savithiry S; Xu, Chenping; Cregan, Perry; Caperna, Thomas J; Garrett, Wesley M; Luthria, Devanand

    2009-08-01

    Proteomic technologies are currently used as an effective analytical tool for examining modifications in protein profiles. Understanding the natural variation of soybean seed proteins is necessary to evaluate potential unintended (collateral) effects due to transgenic modifications in genetically modified (GMO) soybeans. We used two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry, and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) to separate, identify and quantify the different classes of soybean seed proteins. Sixteen soybean genotypes, including four wild and twelve cultivated genotypes, belonging to four different subgroups were used as models for protein profile evaluation. Significant variations of allergen and anti-nutritional protein profiles were observed between two different groups, cultivated and wild soybean genotypes. However, only minor variations in protein profiles were observed within the soybean samples from the same group (cultivated or wild). These results may be useful to scientists needing to compare GMO and non-GMO soybeans once additional data are generated on additional soybean varieties and the same varieties grown at different geographical locations.

  7. Differential proteomics reveals novel insights into Nosema-honey bee interactions.

    PubMed

    Kurze, Christoph; Dosselli, Ryan; Grassl, Julia; Le Conte, Yves; Kryger, Per; Baer, Boris; Moritz, Robin F A

    2016-12-01

    Host manipulation is a common strategy by parasites to reduce host defense responses, enhance development, host exploitation, reproduction and, ultimately, transmission success. As these parasitic modifications can reduce host fitness, increased selection pressure may result in reciprocal adaptations of the host. Whereas the majority of studies on host manipulation have explored resistance against parasites (i.e. ability to prevent or limit an infection), data describing tolerance mechanisms (i.e. ability to limit harm of an infection) are scarce. By comparing differential protein abundance, we provide evidence of host-parasite interactions in the midgut proteomes of N. ceranae-infected and uninfected honey bees from both Nosema-tolerant and Nosema-sensitive lineages. We identified 16 proteins out of 661 protein spots that were differentially abundant between experimental groups. In general, infections of Nosema resulted in an up-regulation of the bee's energy metabolism. Additionally, we identified 8 proteins that were differentially abundant between tolerant and sensitive honey bees regardless of the Nosema infection. Those proteins were linked to metabolism, response to oxidative stress and apoptosis. In addition to bee proteins, we also identified 3 Nosema ceranae proteins. Interestingly, abundance of two of these Nosema proteins were significantly higher in infected Nosema-sensitive honeybees relative to the infected Nosema-tolerant lineage. This may provide a novel candidate for studying the molecular interplay between N. ceranae and its honey bee host in more detail.

  8. Differential antennal proteome comparison of adult honeybee drone, worker and queen (Apis mellifera L.).

    PubMed

    Fang, Yu; Song, Feifei; Zhang, Lan; Aleku, Dereje Woltedji; Han, Bin; Feng, Mao; Li, Jianke

    2012-01-04

    To understand the olfactory mechanism of honeybee antennae in detecting specific volatile compounds in the atmosphere, antennal proteome differences of drone, worker and queen were compared using 2-DE, mass spectrometry and bioinformatics. Therefore, 107 proteins were altered their expressions in the antennae of drone, worker and queen bees. There were 54, 21 and 32 up-regulated proteins in the antennae of drone, worker and queen, respectively. Proteins upregulated in the drone antennae were involved in fatty acid metabolism, antioxidation, carbohydrate metabolism and energy production, protein folding and cytoskeleton. Proteins upregulated in the antennae of worker and queen bees were related to carbohydrate metabolism and energy production while molecular transporters were upregulated in the queen antennae. Our results explain the role played by the antennae of drone is to aid in perceiving the queen sexual pheromones, in the worker antennae to assist for food search and social communication and in the queen antennae to help pheromone communication with the worker and the drone during the mating flight. This first proteomic study significantly extends our understanding of honeybee olfactory activities and the possible mechanisms played by the antennae in response to various environmental, social, biological and biochemical signals.

  9. 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.

  10. Difference gel electrophoresis (DiGE) identifies differentially expressed proteins in endoscopically-collected pancreatic fluid

    PubMed Central

    Paulo, Joao A.; Lee, Linda S.; Banks, Peter A.; Steen, Hanno; Conwell, Darwin L.

    2012-01-01

    Alterations in the pancreatic fluid proteome of individuals with chronic pancreatitis may offer insights into the development and progression of the disease. The endoscopic pancreas function test (ePFT) can safely collect large volumes of pancreatic fluid that are potentially amenable to proteomic analyses using difference gel electrophoresis (DiGE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Pancreatic fluid was collected endoscopically using the ePFT method following secretin stimulation from three individuals with severe chronic pancreatitis and three chronic abdominal pain controls. The fluid was processed to minimize protein degradation and the protein profiles of each cohort, as determined by DiGE and LC-MS/MS, were compared. This DiGE-LC-MS/MS analysis reveals proteins that are differentially expressed in chronic pancreatitis compared to chronic abdominal pain controls. Proteins with higher abundance in pancreatic fluid from chronic pancreatitis individuals include: actin, desmoplankin, alpha-1-antitrypsin, SNC73, and serotransferrin. Those of relatively lower abundance include carboxypeptidase B, lipase, alpha-1-antichymotrypsin, alpha-2-macroglobulin, Arp2/3 subunit 4, glyceraldehyde-3-phosphate dehydrogenase, and protein disulfide isomerase. Endoscopic collection (ePFT) in tandem with DiGE-LC-MS/MS is a suitable approach for pancreatic fluid proteome analysis, however, further optimization of our protocol, as outlined herein, may improve proteome coverage in future analyses. PMID:21792986

  11. A Bayesian algorithm for detecting differentially expressed proteins and its application in breast cancer research

    NASA Astrophysics Data System (ADS)

    Santra, Tapesh; Delatola, Eleni Ioanna

    2016-07-01

    Presence of considerable noise and missing data points make analysis of mass-spectrometry (MS) based proteomic data a challenging task. The missing values in MS data are caused by the inability of MS machines to reliably detect proteins whose abundances fall below the detection limit. We developed a Bayesian algorithm that exploits this knowledge and uses missing data points as a complementary source of information to the observed protein intensities in order to find differentially expressed proteins by analysing MS based proteomic data. We compared its accuracy with many other methods using several simulated datasets. It consistently outperformed other methods. We then used it to analyse proteomic screens of a breast cancer (BC) patient cohort. It revealed large differences between the proteomic landscapes of triple negative and Luminal A, which are the most and least aggressive types of BC. Unexpectedly, majority of these differences could be attributed to the direct transcriptional activity of only seven transcription factors some of which are known to be inactive in triple negative BC. We also identified two new proteins which significantly correlated with the survival of BC patients, and therefore may have potential diagnostic/prognostic values.

  12. Bayesian modeling of differential gene expression.

    PubMed

    Lewin, Alex; Richardson, Sylvia; Marshall, Clare; Glazier, Anne; Aitman, Tim

    2006-03-01

    We present a Bayesian hierarchical model for detecting differentially expressing genes that includes simultaneous estimation of array effects, and show how to use the output for choosing lists of genes for further investigation. We give empirical evidence that expression-level dependent array effects are needed, and explore different nonlinear functions as part of our model-based approach to normalization. The model includes gene-specific variances but imposes some necessary shrinkage through a hierarchical structure. Model criticism via posterior predictive checks is discussed. Modeling the array effects (normalization) simultaneously with differential expression gives fewer false positive results. To choose a list of genes, we propose to combine various criteria (for instance, fold change and overall expression) into a single indicator variable for each gene. The posterior distribution of these variables is used to pick the list of genes, thereby taking into account uncertainty in parameter estimates. In an application to mouse knockout data, Gene Ontology annotations over- and underrepresented among the genes on the chosen list are consistent with biological expectations.

  13. Global analysis of the Brucella melitensis proteome: Identification of proteins expressed in laboratory-grown culture.

    PubMed

    Wagner, Mary Ann; Eschenbrenner, Michel; Horn, Troy A; Kraycer, Jo Ann; Mujer, Cesar V; Hagius, Sue; Elzer, Philip; DelVecchio, Vito G

    2002-08-01

    Brucella melitensis is a facultative intracellular bacterial pathogen that causes brucellosis, a zoonotic disease primarily infecting sheep and goats, characterized by undulant fever, arthritic pain and other neurological disorders in humans. A comprehensive proteomic study of strain 16M was conducted to identify and characterize the proteins expressed in laboratory-grown culture. Using overlapping narrow range immobilized pH gradient strips for two-dimensional gel electrophoresis, 883 protein spots were detected between pH 3.5 and 11. The average isoelectric point and molecular weight values of the detected spots were 5.22 and 46.5 kDa, respectively. Of the 883 observed protein spots, 440 have been identified by matrix-assisted laser desorption/ionization-mass spectrometry. These proteins represent 187 discrete open reading frames (ORFs) or 6% of the predicted 3197 ORFs contained in the genome. The corresponding ORFs of the identified proteins are distributed evenly between each of the two circular B. melitensis chromosomes, indicating that both replicons are functionally active. The presented proteome map lists those protein spots identified to date in this study. This map may serve as a baseline reference for future proteomic studies aimed at the definition of biochemical pathways associated with stress responses, host specificity, pathogenicity and virulence. It will also assist in characterization of global proteomic effects in gene-knockout mutants. Ultimately, it may aid in our overall understanding of the cell biology of B. melitensis, an important bacterial pathogen.

  14. Combining Click Chemistry-Based Proteomics With Dox-Inducible Gene Expression.

    PubMed

    Gebert, J; Schnölzer, M; Warnken, U; Kopitz, J

    2017-01-01

    Inactivating mutations in single genes can trigger, prevent, promote, or alleviate diseases. Identifying such disease-related genes is a main pillar of medical research. Since proteins play a crucial role in mediating these effects, their impact on the diseased cells' proteome including posttranslational modifications has to be elucidated for a detailed understanding of the role of these genes in the disease process. In complex disorders, like cancer, several genes contribute to the disease process, thereby hampering the assignment of a proteomic change to the corresponding causative gene. To enable comprehensive screening for the impact of inactivation of a gene, e.g., loss of a tumor suppressor in cancer, on the cellular proteome, we present a strategy based on combination of three technologies that is recombinase-mediated cassette exchange, click chemistry, and mass spectrometry. The methodology is exemplified by the analysis of the proteomic changes induced by the loss of a tumor suppressor gene in colorectal cancer cells. To demonstrate the applicability to screen for posttranslational modification changes, we also describe the analysis of protein glycosylation changes caused by the tumor suppressor inactivation. In principle, this strategy can be applied to analyze the effects of any gene of interest on protein expression as well as posttranslational modification by glycosylation. Moreover adaptation of the strategy to an appropriate cell culture model has the potential for application on a broad range of diseases where the disease-promoting mutations have been identified. © 2017 Elsevier Inc. All rights reserved.

  15. Unifying expression scale for peptide hydrophobicity in proteomic reversed phase high-pressure liquid chromatography experiments.

    PubMed

    Grigoryan, Marine; Shamshurin, Dmitry; Spicer, Victor; Krokhin, Oleg V

    2013-11-19

    As an initial step in our efforts to unify the expression of peptide retention times in proteomic liquid chromatography-mass spectrometry (LC-MS) experiments, we aligned the chromatographic properties of a number of peptide retention standards against a collection of peptides commonly observed in proteomic experiments. The standard peptide mixtures and tryptic digests of samples of different origins were separated under the identical chromatographic condition most commonly employed in proteomics: 100 Å C18 sorbent with 0.1% formic acid as an ion-pairing modifier. Following our original approach (Krokhin, O. V.; Spicer, V. Anal. Chem. 2009, 81, 9522-9530) the retention characteristics of these standards and collection of tryptic peptides were mapped into hydrophobicity index (HI) or acetonitrile percentage units. This scale allows for direct visualization of the chromatographic outcome of LC-MS acquisitions, monitors the performance of the gradient LC system, and simplifies method development and interlaboratory data alignment. Wide adoption of this approach would significantly aid understanding the basic principles of gradient peptide RP-HPLC and solidify our collective efforts in acquiring confident peptide retention libraries, a key component in the development of targeted proteomic approaches.

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

    PubMed

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

    2016-03-01

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

  17. Differential expression of genes and proteins associated with wool follicle cycling.

    PubMed

    Liu, Nan; Li, Hegang; Liu, Kaidong; Yu, Juanjuan; Cheng, Ming; De, Wei; Liu, Jifeng; Shi, Shuyan; He, Yanghua; Zhao, Jinshan

    2014-08-01

    Sheep are valuable resources for the wool industry. Wool growth of Aohan fine wool sheep has cycled during different seasons in 1 year. Therefore, identifying genes that control wool growth cycling might lead to ways for improving the quality and yield of fine wool. In this study, we employed Agilent sheep gene expression microarray and proteomic technology to compare the gene expression patterns of the body side skins at August and December time points in Aohan fine wool sheep (a Chinese indigenous breed). Microarray study revealed that 2,223 transcripts were differentially expressed, including 1,162 up-regulated and 1,061 down-regulated transcripts, comparing body side skin at the August time point to the December one (A/D) in Aohan fine wool sheep. Then seven differentially expressed genes were selected to validated the reliability of the gene chip data. The majority of the genes possibly related to follicle development and wool growth could be assigned into the categories including regulation of receptor binding, extracellular region, protein binding and extracellular space. Proteomic study revealed that 84 protein spots showed significant differences in expression levels. Of the 84, 63 protein spots were upregulated and 21 were downregulated in A/D. Finally, 55 protein points were determined through MALDI-TOF/MS analyses. Furthermore, the regulation mechanism of hair follicle might resemble that of fetation.

  18. dbDEPC 2.0: updated database of differentially expressed proteins in human cancers

    PubMed Central

    He, Ying; Zhang, Menghuan; Ju, Yuanhu; Yu, Zhonghao; Lv, Daqing; Sun, Han; Yuan, Weilan; He, Fei; Zhang, Jianshe; Li, Hong; Li, Jing; Wang-Sattler, Rui; Li, Yixue; Zhang, Guoqing; Xie, Lu

    2012-01-01

    A large amount of differentially expressed proteins (DEPs) have been identified in various cancer proteomics experiments, curation and annotation of these proteins are important in deciphering their roles in oncogenesis and tumor progression, and may further help to discover potential protein biomarkers for clinical applications. In 2009, we published the first database of DEPs in human cancers (dbDEPCs). In this updated version of 2011, dbDEPC 2.0 has more than doubly expanded to over 4000 protein entries, curated from 331 experiments across 20 types of human cancers. This resource allows researchers to search whether their interested proteins have been reported changing in certain cancers, to compare their own proteomic discovery with previous studies, to picture selected protein expression heatmap across multiple cancers and to relate protein expression changes with aberrance in other genetic level. New important developments include addition of experiment design information, advanced filter tools for customer-specified analysis and a network analysis tool. We expect dbDEPC 2.0 to be a much more powerful tool than it was in its first release and can serve as reference to both proteomics and cancer researchers. dbDEPC 2.0 is available at http://lifecenter.sgst.cn/dbdepc/index.do. PMID:22096234

  19. Application of highly sensitive saturation labeling to the analysis of differential protein expression in infected ticks from limited samples

    PubMed Central

    2010-01-01

    Background Ticks are vectors of pathogens that affect human and animal health worldwide. Proteomics and genomics studies of infected ticks are required to understand tick-pathogen interactions and identify potential vaccine antigens to control pathogen transmission. One of the limitations for proteomics research in ticks is the amount of protein that can be obtained from these organisms. In the work reported here, individual naturally-infected and uninfected Rhipicephalus spp. ticks were processed using a method that permits simultaneous extraction of DNA, RNA and proteins. This approach allowed using DNA to determine pathogen infection, protein for proteomics studies and RNA to characterize mRNA levels for some of the differentially expressed proteins. Differential protein expression in response to natural infection with different pathogens was characterized by two-dimensional (2-D) differential in gel electrophoresis (DIGE) saturation labeling in combination with mass spectrometry analysis. To our knowledge, this is the first report of the application of DIGE saturation labeling to study tick proteins. Results Questing and feeding Rhipicephalus spp. adult ticks were collected in 27 farms located in different Sicilian regions. From 300 collected ticks, only 16 were found to be infected: R. sanguineus with Rickettsia conorii and Ehrlichia canis; R. bursa with Theileria annulata; and R. turanicus with Anaplasma ovis. The proteomic analysis conducted from a limited amount of proteins allowed the identification of host, pathogen and tick proteins differentially expressed as a consequence of infection. Conclusion These results showed that DIGE saturation labeling is a powerful technology for proteomics studies in small number of ticks and provided new information about the effect of pathogen infection in ticks. PMID:20704695

  20. Genetic Basis of Differential Heat Resistance between Two Species of Congeneric Freshwater Snails: Insights from Quantitative Proteomics and Base Substitution Rate Analysis.

    PubMed

    Mu, Huawei; Sun, Jin; Fang, Ling; Luan, Tiangang; Williams, Gray A; Cheung, Siu Gin; Wong, Chris K C; Qiu, Jian-Wen

    2015-10-02

    We compared the heat tolerance, proteomic responses to heat stress, and adaptive sequence divergence in the invasive snail Pomacea canaliculata and its noninvasive congener Pomacea diffusa. The LT50 of P. canaliculata was significantly higher than that of P. diffusa. More than 3350 proteins were identified from the hepatopancreas of the snails exposed to acute and chronic thermal stress using iTRAQ-coupled mass spectrometry. Acute exposure (3 h exposure at 37 °C with 25 °C as control) resulted in similar numbers (27 in P. canaliculata and 23 in P. diffusa) of differentially expressed proteins in the two species. Chronic exposure (3 weeks of exposure at 35 °C with 25 °C as control) caused differential expression of more proteins (58 in P. canaliculata and 118 in P. diffusa), with many of them related to restoration of damaged molecules, ubiquitinating dysfunctional molecules, and utilization of energy reserves in both species; but only in P. diffusa was there a shift from carbohydrate to lipid catabolism. Analysis of orthologous genes encoding the differentially expressed proteins revealed two genes having clear evidence of positive selection (Ka/Ks > 1) and seven candidates for more detailed analysis of positive selection (Ka/Ks between 0.5 and 1). These nine genes are related to energy metabolism, cellular oxidative homeostasis, signaling, and binding processes. Overall, the proteomic and base substitution rate analyses indicate genetic basis of differential resistance to heat stress between the two species, and such differences could affect their further range expansion in a warming climate.

  1. Preliminary identification of differentially expressed tear proteins in keratoconus

    PubMed Central

    Wasinger, Valerie C.; Pye, David C.; Willcox, Mark D. P.

    2013-01-01

    Purpose To examine the proteins differentially expressed in the tear film of people with keratoconus and normal subjects. Methods Unstimulated tears from people with keratoconus (KC) and controls (C) were collected using a capillary tube. Tear proteins from people with KC and controls were partitioned using a novel in-solution electrophoresis, Microflow 10 (ProteomeSep), and analyzed using linear ion trap quadrupole fourier transform mass spectrometry. Spectral counting was used to quantify the individual tear proteins. Results Elevated levels of cathepsin B (threefold) were evident in the tears of people with KC. Polymeric immunoglobulin receptor (ninefold), fibrinogen alpha chain (eightfold), cystatin S (twofold), and cystatin SN (twofold) were reduced in tears from people with KC. Keratin type-1 cytoskeletal-14 and keratin type-2 cytoskeletal-5 were present only in the tears of people with KC. Conclusions The protein changes in tears, that is, the decrease in protease inhibitors and increase in proteases, found in the present and other previously published studies reflect the pathological events involved in KC corneas. Further investigations into tear proteins may help elucidate the underlying molecular mechanisms of KC, which could result in better treatment options. PMID:24194634

  2. Differential gene expression during multistage carcinogenesis

    SciTech Connect

    Bowden, G.T. ); Krieg, P. )

    1991-06-01

    The use of the mouse skin multistage model of carcinogenesis has aided our understanding of critical target genes in chemical carcinogenesis. The mutagenic activation of the Harvey-ras proto-oncogene has been found to be an early event associated with the initiation of mouse skin tumors by the polycyclic aromatic hydrocarbon 7,12 dimethylbenz(a)anthracene and the pure initiator ethyl carbamate (urethane). In contrast to chemical initiation of mouse skin tumors, ionizing radiation-initiated malignant skin tumors have been shown to possess distinct non-ras transforming gene(s). Differential screening of cDNA libraries made from chemically initiated malignant skin tumors has been used to identify a number of cellular gene transcripts that are overexpressed during mouse skin tumor progression. These differentially expressed genes include {beta}-actin, ubiquitin, a hyperproliferative keratin (K6), a gene whose product is a member of a fatty acid or lipid-binding protein family, and a gene called transin or stromelysin. The overexpression of the stromelysin gene, which encodes a metalloproteinase that degrades proteins in the basement membrane, is hypothesized to play a functional role in malignant tumor cell invasion and metastasis. The authors believe that the cloning, identification, and characterization of gene sequences that are differentially expressed during tumor progression could lead to the discovery of gene products that either play functional roles in skin tumor progression or in the maintenance of various progressive tumor phenotypes.

  3. Comparative Proteomics and Expression Analysis of Five Genes in Epicauta chinensis Larvae from the First to Fifth Instar

    PubMed Central

    Li, Qiurong; Wang, Dun; Lv, Shumin; Zhang, Yalin

    2014-01-01

    Blister beetle is an important insect model for both medicinal and pure research. Previous research has mainly focused on its biology and biochemistry, but very little data is yet available in the molecular biology. This study uses differential proteomics technology to analyze the soluble proteins extracted from each of the 5 instars larvae of Epicauta chinensis. 42 of the differentially-expressed proteins were identified successfully by MALDI-TOF/TOF-MS. Some of these proteins’ function and their expression profiles are analyzed. Our analysis revealed dynamics regulation of the following proteins: Axin-like protein pry-1 (APR-1), dihydrolipoyl dehydrogenase (DLD), vitellogenin (Vg) and lysozyme C (Lmz-S). APR-1 negatively regulates the Wnt signaling pathway. Its overexpression could result in embryo, leg, eye and ovary ectopica or malformation. DLD catalyzes the pyruvate into acetyl-CoA, the latter is the starting material of juvenile hormone (JH) and ipsdienol biosynthesis through the MVA pathway in insects. While Vg synthesis can be regulated by JH and stimulated by food factors. So DLD may affect the synthesis of JH, ipsdienol and Vg indirectly. The activity of lysozyme is an indicator of the immunity. Nutrition/food should be taken into account for its potential role during the development of larva in the future. Among the five genes and their corresponding proteins’ expression, only hsc70 gene showed a good correspondence with the protein level. This reflects the fluctuating relationship between mRNA and protein levels. PMID:24586908

  4. Effects of simulated microgravity on the expression of presynaptic proteins distorting the GABA/glutamate equilibrium--A proteomics approach.

    PubMed

    Wang, Yun; Iqbal, Javed; Liu, Yahui; Su, Rui; Lu, Song; Peng, Guang; Zhang, Yongqian; Qing, Hong; Deng, Yulin

    2015-11-01

    Microgravity may cause cognition-related changes in the animal nervous system due to the resulting uneven flow of fluids in the body. These changes may restrict the long-term stay of humans in space for various purposes. In this study, a rat tail suspension model (30°) was used to explore the effects of 21 days of prolonged simulated microgravity (SM) on the expression of proteins involved in cognitive functions in the rat hippocampus. SM decreased the content of γ-aminobutyric acid (GABA) and increased the content of glutamate (Glu) in the rat hippocampus. A comparative (18)O-labeled quantitative proteomics strategy was applied to detect the differential expression of synaptic proteins under SM. Fifty-three proteins were found to be differentially expressed under SM. Microgravity induces difficulty in the formation of the SNARE complex due to the down-regulation of vesicle-associated membrane protein 3(VAMP3) and syntaxin-1A. Synaptic vesicle recycling may also be affected due to the dysregulation of syntaxin-binding protein 5 (tomosyn), rab3A and its effector rim2. Both processes are disturbed, indicating that presynaptic proteins mediate a GABA/Glu imbalance under SM. These findings provide clues for understanding the mechanism of the GABA/Glu equilibrium in the hippocampus induced by microgravity in space and represent steps toward safe space travel. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Differential protein expression in the midgut of Culex quinquefasciatus mosquitoes induced by the insecticide temephos.

    PubMed

    Games, P D; Alves, S N; Katz, B B; Tomich, J M; Serrão, J E

    2016-09-01

    Mosquitoes are vectors for pathogens of malaria, lymphatic filariasis, dengue, chikungunya, yellow fever and Japanese encephalitis. Culex quinquefasciatus Say, 1823 (Diptera: Culicidae) is a known vector of lymphatic filariasis. Its control in Brazil has been managed using the organophosphate temephos. Studies examining the proteins of Cx. quinquefasciatus that are differentially expressed in response to temephos further understanding of the modes of action of the insecticide and may potentially identify resistance factors in the mosquito. In the present study, a comparative proteomic analysis, using 2-dimensional electrophoresis coupled with matrix-assisted laser desorption/ionization (MALDI) time of flight (TOF)/TOF mass spectrometry, and bioinformatics analyses were performed to identify midgut proteins in Cx. quinquefasciatus larvae that were differentially expressed in response to exposure to temephos relative to those in untreated controls. A total of 91 protein spots were differentially expressed; 40 were upregulated and 51 were downregulated by temephos. A total of 22 proteins, predominantly upregulated, were identified as known to play a role in the immune response, whereas the downregulated proteins were involved in energy and protein catabolism. This is the first proteome study of the midgut of Cx. quinquefasciatus and it provides insights into the molecular mechanisms of insecticide-induced responses in the mosquito. © 2016 The Royal Entomological Society.

  6. Sulfur isotopic and proteomic profiles of sulfate reducers grown under differential steady-states

    NASA Astrophysics Data System (ADS)

    Leavitt, W.; Venceslau, S.; Waldbauer, J.; Smith, D. A.; Boidi, F. J.; Bradley, A. S.

    2016-12-01

    Microbial sulfate reducers (MSR) drive the Earth's biogeochemical sulfur cycle. At the heart of this energy metabolism is a cascade of redox transformations coupling organic carbon and/or hydrogen oxidation to the dissimilatory reduction of sulfate to sulfide. The product sulfide is depleted in the heavier isotopes of sulfur, relative to the reactant sulfate, consistent with a normal kinetic isotope effect. However, the magnitude of the net fractionation during MSR can range over a range of 70 permil, consistent with a multi-step set of reactions. This range in MSR fractionation has been shown to mainly depend on: i) the cell-specific sulfate reduction rate (csSRR), and ii) the ambient sulfate concentration. However, the fractionation under identical conditions differs among strains (Bradley et al. 2016. Geobio), and so must also be mediated by strain-specific processes, such as the nature and quantity of individual proteins involved in sulfate reduction, electron transport, and growth. In recent work we have examined the influence of electron donor, electron acceptor, and co-limitation under controlled steady-state culture conditions in order better inform models of MSR isotope fractionation, and the physiological and isotopic response to differential environmental forcings (e.g. Leavitt et al. (2013) PNAS). Recent models of the fractionation response to MSR rate (c.f. Bradley 2016; Wing & Halevy, 2016) make specific predictions for the responses of the cellular metabolome and proteome. Here we compare the steady-state S-isotopic fractionation and proteome of `fast' versus `slow' grown D. vulgaris, using replicate chemostats under electron donor limitation. We observe clear and statistically robust changes in some key central MSR and C-metabolism enzymes, though a host of the critical energy-transfer enzymes show no statistically discernable change. We discuss these results in light of recent theoretical advances and their relevance to modern and ancient

  7. A Proteomic Approach to Investigating Gene Cluster Expression and Secondary Metabolite Functionality in Aspergillus fumigatus

    PubMed Central

    Owens, Rebecca A.; Hammel, Stephen; Sheridan, Kevin J.; Jones, Gary W.; Doyle, Sean

    2014-01-01

    A combined proteomics and metabolomics approach was utilised to advance the identification and characterisation of secondary metabolites in Aspergillus fumigatus. Here, implementation of a shotgun proteomic strategy led to the identification of non-redundant mycelial proteins (n = 414) from A. fumigatus including proteins typically under-represented in 2-D proteome maps: proteins with multiple transmembrane regions, hydrophobic proteins and proteins with extremes of molecular mass and pI. Indirect identification of secondary metabolite cluster expression was also achieved, with proteins (n = 18) from LaeA-regulated clusters detected, including GliT encoded within the gliotoxin biosynthetic cluster. Biochemical analysis then revealed that gliotoxin significantly attenuates H2O2-induced oxidative stress in A. fumigatus (p>0.0001), confirming observations from proteomics data. A complementary 2-D/LC-MS/MS approach further elucidated significantly increased abundance (p<0.05) of proliferating cell nuclear antigen (PCNA), NADH-quinone oxidoreductase and the gliotoxin oxidoreductase GliT, along with significantly attenuated abundance (p<0.05) of a heat shock protein, an oxidative stress protein and an autolysis-associated chitinase, when gliotoxin and H2O2 were present, compared to H2O2 alone. Moreover, gliotoxin exposure significantly reduced the abundance of selected proteins (p<0.05) involved in de novo purine biosynthesis. Significantly elevated abundance (p<0.05) of a key enzyme, xanthine-guanine phosphoribosyl transferase Xpt1, utilised in purine salvage, was observed in the presence of H2O2 and gliotoxin. This work provides new insights into the A. fumigatus proteome and experimental strategies, plus mechanistic data pertaining to gliotoxin functionality in the organism. PMID:25198175

  8. Proteomic mapping of differentially vulnerable pre-synaptic populations identifies regulators of neuronal stability in vivo.

    PubMed

    Llavero Hurtado, Maica; Fuller, Heidi R; Wong, Andrew M S; Eaton, Samantha L; Gillingwater, Thomas H; Pennetta, Giuseppa; Cooper, Jonathan D; Wishart, Thomas M

    2017-09-29

    Synapses are an early pathological target in many neurodegenerative diseases ranging from well-known adult onset conditions such as Alzheimer and Parkinson disease to neurodegenerative conditions of childhood such as spinal muscular atrophy (SMA) and neuronal ceroid lipofuscinosis (NCLs). However, the reasons why synapses are particularly vulnerable to such a broad range of neurodegeneration inducing stimuli remains unknown. To identify molecular modulators of synaptic stability and degeneration, we have used the Cln3 (-/-) mouse model of a juvenile form of NCL. We profiled and compared the molecular composition of anatomically-distinct, differentially-affected pre-synaptic populations from the Cln3 (-/-) mouse brain using proteomics followed by bioinformatic analyses. Identified protein candidates were then tested using a Drosophila CLN3 model to study their ability to modify the CLN3-neurodegenerative phenotype in vivo. We identified differential perturbations in a range of molecular cascades correlating with synaptic vulnerability, including valine catabolism and rho signalling pathways. Genetic and pharmacological targeting of key 'hub' proteins in such pathways was sufficient to modulate phenotypic presentation in a Drosophila CLN3 model. We propose that such a workflow provides a target rich method for the identification of novel disease regulators which could be applicable to the study of other conditions where appropriate models exist.

  9. Differential proteins of the optic ganglion in octopus vulgaris under methanol stress revealed using proteomics.

    PubMed

    Huang, Lin; Huang, Qing-Yu; Chen, Hai-Bin; Huang, Fu-Sheng; Huang, He-Qing

    2011-10-01

    An analytical approach using the two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) technique separated the proteome from the optic ganglia of Octopus vulgaris (OVOG). Approximately 600 protein spots were detected from the extraction when applying 150 μg protein to a 2D-PAGE gel in the pH range 5.0-8.0. Compared to the control, significant changes of 18 protein spots were observed in OVOG under the stress of native seawater containing 2% methanol for 72 h. Among these spots, we found that eight were down-regulated and ten were up-regulated in the gels, which were further identified using both peptide mass fingerprinting and database searches. Significant proteins such as glyceraldehyde-3-phosphate dehydrogenase, alpha subunit of succinyl-CoA synthetase, alcohol dehydrogenase, and long-chain specific acyl-CoA dehydrogenase were up-regulated proteins, whereas putative ABC transporter was a down -regulated protein. These differential proteins at the level of subcellular localization were further classified using LOCtree software with a hierarchical system of support vector machines. We found that most of the differential proteins in the gel could be identified as mitochondrial proteins, suggesting that these protective or marker proteins might help to prevent methanol poisoning via the mitochondria in the optical ganglia. The results indicated that both beta-tubulin and beta-actin were potential biomarkers as up-regulated proteins for monitoring methanol toxicosis associated with fish foods such as octopus and shark.

  10. Differential lysine acetylation profiles of Erwinia amylovora strains revealed by proteomics.

    PubMed

    Wu, Xia; Vellaichamy, Adaikkalam; Wang, Dongping; Zamdborg, Leonid; Kelleher, Neil L; Huber, Steven C; Zhao, Youfu

    2013-02-21

    Protein lysine acetylation (LysAc) has recently been demonstrated to be widespread in E. coli and Salmonella, and to broadly regulate bacterial physiology and metabolism. However, LysAc in plant pathogenic bacteria is largely unknown. Here we first report the lysine acetylome of Erwinia amylovora, an enterobacterium causing serious fire blight disease of apples and pears. Immunoblots using generic anti-lysine acetylation antibodies demonstrated that growth conditions strongly affected the LysAc profiles in E. amylovora. Differential LysAc profiles were also observed for two E. amylovora strains, known to have differential virulence in plants, indicating translational modification of proteins may be important in determining virulence of bacterial strains. Proteomic analysis of LysAc in two E. amylovora strains identified 141 LysAc sites in 96 proteins that function in a wide range of biological pathways. Consistent with previous reports, 44% of the proteins are involved in metabolic processes, including central metabolism, lipopolysaccharide, nucleotide and amino acid metabolism. Interestingly, for the first time, several proteins involved in E. amylovora virulence, including exopolysaccharide amylovoran biosynthesis- and type III secretion-associated proteins, were found to be lysine acetylated, suggesting that LysAc may play a major role in bacterial virulence. Comparative analysis of LysAc sites in E. amylovora and E. coli further revealed the sequence and structural commonality for LysAc in the two organisms. Collectively, these results reinforce the notion that LysAc of proteins is widespread in bacterial metabolism and virulence.

  11. Teaching expression proteomics: From the wet-lab to the laptop.

    PubMed

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

    2009-09-01

    Expression proteomics has become, in recent years, a key genome-wide expression approach in fundamental and applied life sciences. This postgenomic technology aims the quantitative analysis of all the proteins or protein forms (the so-called proteome) of a given organism in a given environmental and genetic context. It is a challenge to provide effective training in this area due to its demanding laboratory procedures and laborious computational data analysis. However, the effective training of undergraduates and postgraduates in this field is highly recommended to prepare them for the challenges of postgenomic research and of medical, industrial and other economical activities. Since 2004, the area of Biological Sciences at the Department of Chemical and Biological Engineering of Instituto Superior Técnico (IST) has been teaching Expression Proteomics to undergraduate and postgraduate students in three formats: 1) as modules of curricular units (CU), in particular of Functional Genomics and Bioinformatics (FGB), offered as a mandatory CU to IST Biological Engineering or Biotechnology Master courses students, or as an elective CU to other MSc courses with a biological component and to the MSc in Information Systems and Computer Engineering; the topic is also part of the PhD program in Biotechnology; 2) as mentored coaching, in which IST students integrate ongoing research programs at the Biological Sciences Research Group of IBB at IST; and 3) as intensive thematic courses open to the external community. In this article, educational programs and teaching methodologies and tools that we have been using are outlined, from the wet-lab to the laptop. The current role of quantitative proteomics in biological research, with emphasis on microbial stress response and on biomedical and biotechnological applications, is addressed, as a case-study, anchored on our group research activities.

  12. Differential Proteomic Analysis by iTRAQ Reveals the Mechanism of Pyropia haitanensis Responding to High Temperature Stress.

    PubMed

    Shi, Jianzhi; Chen, Yuting; Xu, Yan; Ji, Dehua; Chen, Changsheng; Xie, Chaotian

    2017-03-17

    Global warming increases sea temperature and leads to high temperature stress, which affects the yield and quality of Pyropia haitanensis. To understand the molecular mechanisms underlying high temperature stress in a high temperature tolerance strain Z-61, the iTRAQ technique was employed to reveal the global proteomic response of Z-61 under different durations of high temperature stress. We identified 151 differentially expressed proteins and classified them into 11 functional categories. The 4 major categories of these are protein synthesis and degradation, photosynthesis, defense response, and energy and carbohydrate metabolism. These findings indicated that photosynthesis, protein synthesis, and secondary metabolism are inhibited by heat to limit damage to a repairable level. As time progresses, misfolded proteins and ROS accumulate and lead to the up-regulation of molecular chaperones, proteases, and antioxidant systems. Furthermore, to cope with cells injured by heat, PCD works to remove them. Additionally, sulfur assimilation and cytoskeletons play essential roles in maintaining cellular and redox homeostasis. These processes are based on signal transduction in the phosphoinositide pathway and multiple ways to supply energy. Conclusively, Z-61 establishes a new steady-state balance of metabolic processes and survives under higher temperature stress.

  13. Differential Proteomic Analysis by iTRAQ Reveals the Mechanism of Pyropia haitanensis Responding to High Temperature Stress

    PubMed Central

    Shi, Jianzhi; Chen, Yuting; Xu, Yan; Ji, Dehua; Chen, Changsheng; Xie, Chaotian

    2017-01-01

    Global warming increases sea temperature and leads to high temperature stress, which affects the yield and quality of Pyropia haitanensis. To understand the molecular mechanisms underlying high temperature stress in a high temperature tolerance strain Z-61, the iTRAQ technique was employed to reveal the global proteomic response of Z-61 under different durations of high temperature stress. We identified 151 differentially expressed proteins and classified them into 11 functional categories. The 4 major categories of these are protein synthesis and degradation, photosynthesis, defense response, and energy and carbohydrate metabolism. These findings indicated that photosynthesis, protein synthesis, and secondary metabolism are inhibited by heat to limit damage to a repairable level. As time progresses, misfolded proteins and ROS accumulate and lead to the up-regulation of molecular chaperones, proteases, and antioxidant systems. Furthermore, to cope with cells injured by heat, PCD works to remove them. Additionally, sulfur assimilation and cytoskeletons play essential roles in maintaining cellular and redox homeostasis. These processes are based on signal transduction in the phosphoinositide pathway and multiple ways to supply energy. Conclusively, Z-61 establishes a new steady-state balance of metabolic processes and survives under higher temperature stress. PMID:28303955

  14. A peptide identification-free, genome sequence-independent shotgun proteomics workflow for strain-level bacterial differentiation

    PubMed Central

    Shao, Wenguang; Zhang, Min; Lam, Henry; Lau, Stanley C. K.

    2015-01-01

    Shotgun proteomics is an emerging tool for bacterial identification and differentiation. However, the identification of the mass spectra of peptides to genome-derived peptide sequences remains a key issue that limits the use of shotgun proteomics to bacteria with genome sequences available. In this proof-of-concept study, we report a novel bacterial fingerprinting method that enjoys the resolving power and accuracy of mass spectrometry without the burden of peptide identification (i.e. genome sequence-independent). This method uses a similarity-clustering algorithm to search for mass spectra that are derived from the same peptide and merge them into a unique consensus spectrum as the basis to generate proteomic fingerprints of bacterial isolates. In comparison to a traditional peptide identification-based shotgun proteomics workflow and a PCR-based DNA fingerprinting method targeting the repetitive extragenic palindromes elements in bacterial genomes, the novel method generated fingerprints that were richer in information and more discriminative in differentiating E. coli isolates by their animal sources. The novel method is readily deployable to any cultivable bacteria, and may be used for several fields of study such as environmental microbiology, applied microbiology, and clinical microbiology. PMID:26395646

  15. A peptide identification-free, genome sequence-independent shotgun proteomics workflow for strain-level bacterial differentiation.

    PubMed

    Shao, Wenguang; Zhang, Min; Lam, Henry; Lau, Stanley C K

    2015-09-23

    Shotgun proteomics is an emerging tool for bacterial identification and differentiation. However, the identification of the mass spectra of peptides to genome-derived peptide sequences remains a key issue that limits the use of shotgun proteomics to bacteria with genome sequences available. In this proof-of-concept study, we report a novel bacterial fingerprinting method that enjoys the resolving power and accuracy of mass spectrometry without the burden of peptide identification (i.e. genome sequence-independent). This method uses a similarity-clustering algorithm to search for mass spectra that are derived from the same peptide and merge them into a unique consensus spectrum as the basis to generate proteomic fingerprints of bacterial isolates. In comparison to a traditional peptide identification-based shotgun proteomics workflow and a PCR-based DNA fingerprinting method targeting the repetitive extragenic palindromes elements in bacterial genomes, the novel method generated fingerprints that were richer in information and more discriminative in differentiating E. coli isolates by their animal sources. The novel method is readily deployable to any cultivable bacteria, and may be used for several fields of study such as environmental microbiology, applied microbiology, and clinical microbiology.

  16. Shoot differentiation from protocorm callus cultures of Vanilla planifolia (Orchidaceae): proteomic and metabolic responses at early stage

    PubMed Central

    2010-01-01

    Background Vanilla planifolia is an important Orchid commercially cultivated for the production of natural vanilla flavour. Vanilla plants are conventionally propagated by stem cuttings and thus causing injury to the mother plants. Regeneration and in vitro mass multiplication are proposed as an alternative to minimize damage to mother plants. Because mass production of V. planifolia through indirect shoot differentiation from callus culture is rare and may be a successful use of in vitro techniques for producing somaclonal variants, we have established a novel protocol for the regeneration of vanilla plants and investigated the initial biochemical and molecular mechanisms that trigger shoot organogenesis from embryogenic/organogenic callus. Results For embryogenic callus induction, seeds obtained from 7-month-old green pods of V. planifolia were inoculated on MS basal medium (BM) containing TDZ (0.5 mg l-1). Germination of unorganized mass callus such as protocorm -like structure (PLS) arising from each seed has been observed. The primary embryogenic calli have been formed after transferring on BM containing IAA (0.5 mg l-1) and TDZ (0.5 mg l-1). These calli were maintained by subculturing on BM containing IAA (0.5 mg l-1) and TDZ (0.3 mg l-1) during 6 months and formed embryogenic/organogenic calli. Histological analysis showed that shoot organogenesis was induced between 15 and 20 days after embryogenic/organogenic calli were transferred onto MS basal medium with NAA (0.5 mg l-1). By associating proteomics and metabolomics analyses, the biochemical and molecular markers responsible for shoot induction have been studied in 15-day-old calli at the stage where no differentiating part was visible on organogenic calli. Two-dimensional electrophoresis followed by matrix-assisted laser desorption ionization time-of-flight-tandem mass spectrometry (MALDI-TOF-TOF-MS) analysis revealed that 15 protein spots are significantly expressed (P < 0.05) at earlier stages of shoot

  17. Quantitative proteomics of Xenopus laevis embryos: expression kinetics of nearly 4000 proteins during early development

    NASA Astrophysics Data System (ADS)

    Sun, Liangliang; Bertke, Michelle M.; Champion, Matthew M.; Zhu, Guijie; Huber, Paul W.; Dovichi, Norman J.

    2014-03-01

    While there is a rich literature on transcription dynamics during the development of many organisms, protein data is limited. We used iTRAQ isotopic labeling and mass spectrometry to generate the largest developmental proteomic dataset for any animal. Expression dynamics of nearly 4,000 proteins of Xenopus laevis was generated from fertilized egg to neurula embryo. Expression clusters into groups. The cluster profiles accurately reflect the major events that mark changes in gene expression patterns during early Xenopus development. We observed decline in the expression of ten DNA replication factors after the midblastula transition (MBT), including a marked decline of the licensing factor XCdc6. Ectopic expression of XCdc6 leads to apoptosis; temporal changes in this protein are critical for proper development. Measurement of expression in single embryos provided no evidence for significant protein heterogeneity between embryos at the same stage of development.

  18. Profiling of antioxidative enzyme expression induced by various food components using targeted proteome analysis.

    PubMed

    Bartenbacher, Sven; Östreicher, Christiane; Pischetsrieder, Monika

    2017-09-01

    A method was developed for targeted proteome analysis of the expression profile of a set of antioxidative enzymes in rat macrophages and applied to screen the antioxidative potential of several food components/foods. Expression profiles of heme oxygenase 1, peroxiredoxin 1, thioredoxin reductase 1, glutathione reductase, glutathione-S transferase P1, and superoxide dismutase 1 were analyzed by nanoLC-MS/MS in selected scheduled reaction monitoring (sSRM) mode monitoring two to three peptides per protein and three transitions per peptide. Relative quantification was performed by metabolic labeling. The validated method was used to profile the activity of capsaicin, carnosol, diallyl trisulfide, maslinic acid, quercetin, sulforaphane, cinnamaldehyde and coffee extract to modulate the expression levels of antioxidative enzymes. Carnosol and sulforaphane most effectively induced protein expression, leading to upregulation of at least five out of the six antioxidative enzymes by a maximum factor of 22.80 ± 6.71 (heme oxygenase 1 by carnosol). Heme oxygenase 1 was most susceptible to nutritive modulation, whereas glutathione reductase expression rates were hardly affected. Targeted mass proteome analysis allows comprehensive evaluation of antioxidative effects by food ingredients. Simultaneous expression analysis of a set of proteins provided valuable insights how various enzymes were differently affected by food components. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Cancer outlier differential gene expression detection.

    PubMed

    Wu, Baolin

    2007-07-01

    We study statistical methods to detect cancer genes that are over- or down-expressed in some but not all samples in a disease group. This has proven useful in cancer studies where oncogenes are activated only in a small subset of samples. We propose the outlier robust t-statistic (ORT), which is intuitively motivated from the t-statistic, the most commonly used differential gene expression detection method. Using real and simulation studies, we compare the ORT to the recently proposed cancer outlier profile analysis (Tomlins and others, 2005) and the outlier sum statistic of Tibshirani and Hastie (2006). The proposed method often has more detection power and smaller false discovery rates. Supplementary information can be found at http://www.biostat.umn.edu/~baolin/research/ort.html.

  20. A high efficiency cloning and expression system for proteomic analysis.

    PubMed

    Ding, Xuan Z; Paulsen, Ian T; Bhattacharjee, Apurba K; Nikolich, Mikeljon P; Myers, Gary; Hoover, David L

    2006-07-01

    The recent description of the complete genomes of the two most pathogenic species of Brucella opens the way for genome-based analysis of the antigenicity of their proteins. In the present report, we describe a bench-level high-efficiency cloning and expression system (HECES) that allow expression of large numbers of Brucella proteins based on genomic sequence information. Purified proteins are produced with high efficiency in a microarray format conducive to analysis of their sero-reactivity against serum from immunized animals. This method is applicable at either small or large scale of protein processing. While it does not require robotics, the format is amenable to robotic implementation for all aspects of the process and subsequent analysis of protein characteristics. This method will allow selection of new reagents for diagnosis of brucellosis and development of vaccine against Brucella, an important zoonotic disease and biothreat agent.

  1. Proteomic analysis of increased Parkin expression and its interactants provides evidence for a role in modulation of mitochondrial function.

    PubMed

    Davison, Eleanor J; Pennington, Kyla; Hung, Chao-Chun; Peng, Jianhe; Rafiq, Rumana; Ostareck-Lederer, Antje; Ostareck, Dirk H; Ardley, Helen C; Banks, Rosamonde E; Robinson, Philip A

    2009-09-01

    Parkin is an ubiquitin-protein ligase (E3), mutations of which cause juvenile onset - autosomal recessive Parkinson's disease, and result in reduced enzymic activity. In contrast, increased levels are protective against mitochondrial dysfunction and neurodegeneration, the mechanism of which is largely unknown. In this study, 2-DE and MS proteomic techniques were utilised to investigate the effects of increased Parkin levels on protein expression in whole cell lysates using in an inducible Parkin expression system in HEK293 cells, and also to isolate potential interactants of Parkin using tandem affinity purification and MS. Nine proteins were significantly differentially expressed (+/-2-fold change; p<0.05) using 2-DE analysis. MS revealed the identity of these proteins to be ACAT2, HNRNPK, HSPD1, PGK1, PRDX6, VCL, VIM, TPI1, and IMPDH2. The first seven of these were reduced in expression. Western blot analysis confirmed the reduction in one of these proteins (HNRNPK), and that its levels were dependent on 26S proteasomal activity. Tandem affinity purification/MS revealed 14 potential interactants of Parkin; CKB, DBT, HSPD1, HSPA9, LRPPRC, NDUFS2, PRDX6, SLC25A5, TPI1, UCHL1, UQCRC1, VCL, YWHAZ, YWHAE. Nine of these are directly involved in mitochondrial energy metabolism and glycolysis; four were also identified in the 2-DE study (HSP60, PRDX6, TPI1, and VCL). This study provides further evidence for a role for Parkin in regulating mitochondrial activity within cells.

  2. Differential proteomic analysis of virus-enriched fractions obtained from plasma pools of patients with dengue fever or severe dengue.

    PubMed

    Fragnoud, Romain; Flamand, Marie; Reynier, Frederic; Buchy, Philippe; Duong, Vasna; Pachot, Alexandre; Paranhos-Baccala, Glaucia; Bedin, Frederic

    2015-11-14

    Dengue is the most widespread mosquito-borne viral disease of public health concern. In some patients, endothelial cell and platelet dysfunction lead to life-threatening hemorrhagic dengue fever or dengue shock syndrome. Prognostication of disease severity is urgently required to improve patient management. The pathogenesis of severe dengue has not been fully elucidated, and the role of host proteins associated with viral particles has received little exploration. The proteomes of virion-enriched fractions purified from plasma pools of patients with dengue fever or severe dengue were compared. Virions were purified by ultracentrifugation combined with a water-insoluble polyelectrolyte-based technique. Following in-gel hydrolysis, peptides were analyzed by nano-liquid chromatography coupled to ion trap mass spectrometry and identified using data libraries. Both dengue fever and severe dengue viral-enriched fractions contained identifiable viral envelope proteins and host cellular proteins. Canonical pathway analysis revealed the identified host proteins are mainly involved in the coagulation cascade, complement pathway or acute phase response signaling pathway. Some host proteins were over- or under-represented in plasma from patients with severe dengue compared to patients with dengue fever. ELISAs were used to validate differential expression of a selection of identified host proteins in individual plasma samples of patients with dengue fever compared to patients with severe dengue. Among 22 host proteins tested, two could differentiate between dengue fever and severe dengue in two independent cohorts (olfactomedin-4: area under the curve (AUC), 0.958; and platelet factor-4: AUC, 0.836). A novel technique of virion-enrichment from plasma has allowed to identify two host proteins that have prognostic value for classifying patients with acute dengue who are more likely to develop a severe dengue. The impact of these host proteins on pathogenicity and disease outcome

  3. Differentially correlated genes in co-expression networks control phenotype transitions.

    PubMed

    Thomas, Lina D; Vyshenska, Dariia; Shulzhenko, Natalia; Yambartsev, Anatoly; Morgun, Andrey

    2016-01-01

    Co-expression networks are a tool widely used for analysis of "Big Data" in biology that can range from transcriptomes to proteomes, metabolomes and more recently even microbiomes. Several methods were proposed to answer biological questions interrogating these networks. Differential co-expression analysis is a recent approach that measures how gene interactions change when a biological system transitions from one state to another. Although the importance of differentially co-expressed genes to identify dysregulated pathways has been noted, their role in gene regulation is not well studied. Herein we investigated differentially co-expressed genes in a relatively simple mono-causal process (B lymphocyte deficiency) and in a complex multi-causal system (cervical cancer). Co-expression networks of B cell deficiency (Control and BcKO) were reconstructed using Pearson correlation coefficient for two mus musculus datasets: B10.A strain (12 normal, 12 BcKO) and BALB/c strain (10 normal, 10 BcKO). Co-expression networks of cervical cancer (normal and cancer) were reconstructed using local partial correlation method for five datasets (total of 64 normal, 148 cancer). Differentially correlated pairs were identified along with the location of their genes in BcKO and in cancer networks. Minimum Shortest Path and Bi-partite Betweenness Centrality where statistically evaluated for differentially co-expressed genes in corresponding networks.    Results: We show that in B cell deficiency the differentially co-expressed genes are highly enriched with immunoglobulin genes (causal genes). In cancer we found that differentially co-expressed genes act as "bottlenecks" rather than causal drivers with most flows that come from the key driver genes to the peripheral genes passing through differentially co-expressed genes. Using in vitro knockdown experiments for two out of 14 differentially co-expressed genes found in cervical cancer (FGFR2 and CACYBP), we showed that they play

  4. Differentially correlated genes in co-expression networks control phenotype transitions

    PubMed Central

    Thomas, Lina D.; Vyshenska, Dariia; Shulzhenko, Natalia; Yambartsev, Anatoly; Morgun, Andrey

    2016-01-01

    Background: Co-expression networks are a tool widely used for analysis of “Big Data” in biology that can range from transcriptomes to proteomes, metabolomes and more recently even microbiomes. Several methods were proposed to answer biological questions interrogating these networks. Differential co-expression analysis is a recent approach that measures how gene interactions change when a biological system transitions from one state to another. Although the importance of differentially co-expressed genes to identify dysregulated pathways has been noted, their role in gene regulation is not well studied. Herein we investigated differentially co-expressed genes in a relatively simple mono-causal process (B lymphocyte deficiency) and in a complex multi-causal system (cervical cancer). Methods: Co-expression networks of B cell deficiency (Control and BcKO) were reconstructed using Pearson correlation coefficient for two mus musculus datasets: B10.A strain (12 normal, 12 BcKO) and BALB/c strain (10 normal, 10 BcKO). Co-expression networks of cervical cancer (normal and cancer) were reconstructed using local partial correlation method for five datasets (total of 64 normal, 148 cancer). Differentially correlated pairs were identified along with the location of their genes in BcKO and in cancer networks. Minimum Shortest Path and Bi-partite Betweenness Centrality where statistically evaluated for differentially co-expressed genes in corresponding networks.    Results: We show that in B cell deficiency the differentially co-expressed genes are highly enriched with immunoglobulin genes (causal genes). In cancer we found that differentially co-expressed genes act as “bottlenecks” rather than causal drivers with most flows that come from the key driver genes to the peripheral genes passing through differentially co-expressed genes. Using in vitro knockdown experiments for two out of 14 differentially co-expressed genes found in cervical cancer (FGFR2 and CACYBP), we

  5. A proteomic chronology of gene expression through the cell cycle in human myeloid leukemia cells

    PubMed Central

    Ly, Tony; Ahmad, Yasmeen; Shlien, Adam; Soroka, Dominique; Mills, Allie; Emanuele, Michael J; Stratton, Michael R; Lamond, Angus I

    2014-01-01

    Technological advances have enabled the analysis of cellular protein and RNA levels with unprecedented depth and sensitivity, allowing for an unbiased re-evaluation of gene regulation during fundamental biological processes. Here, we have chronicled the dynamics of protein and mRNA expression levels across a minimally perturbed cell cycle in human myeloid leukemia cells using centrifugal elutriation combined with mass spectrometry-based proteomics and RNA-Seq, avoiding artificial synchronization procedures. We identify myeloid-specific gene expression and variations in protein abundance, isoform expression and phosphorylation at different cell cycle stages. We dissect the relationship between protein and mRNA levels for both bulk gene expression and for over ∼6000 genes individually across the cell cycle, revealing complex, gene-specific patterns. This data set, one of the deepest surveys to date of gene expression in human cells, is presented in an online, searchable database, the Encyclopedia of Proteome Dynamics (http://www.peptracker.com/epd/). DOI: http://dx.doi.org/10.7554/eLife.01630.001 PMID:24596151

  6. A proteomic chronology of gene expression through the cell cycle in human myeloid leukemia cells.

    PubMed

    Ly, Tony; Ahmad, Yasmeen; Shlien, Adam; Soroka, Dominique; Mills, Allie; Emanuele, Michael J; Stratton, Michael R; Lamond, Angus I

    2014-01-01

    Technological advances have enabled the analysis of cellular protein and RNA levels with unprecedented depth and sensitivity, allowing for an unbiased re-evaluation of gene regulation during fundamental biological processes. Here, we have chronicled the dynamics of protein and mRNA expression levels across a minimally perturbed cell cycle in human myeloid leukemia cells using centrifugal elutriation combined with mass spectrometry-based proteomics and RNA-Seq, avoiding artificial synchronization procedures. We identify myeloid-specific gene expression and variations in protein abundance, isoform expression and phosphorylation at different cell cycle stages. We dissect the relationship between protein and mRNA levels for both bulk gene expression and for over ∼6000 genes individually across the cell cycle, revealing complex, gene-specific patterns. This data set, one of the deepest surveys to date of gene expression in human cells, is presented in an online, searchable database, the Encyclopedia of Proteome Dynamics (http://www.peptracker.com/epd/). DOI: http://dx.doi.org/10.7554/eLife.01630.001.

  7. INDEED: Integrated differential expression and differential network analysis of omic data for biomarker discovery.

    PubMed

    Zuo, Yiming; Cui, Yi; Di Poto, Cristina; Varghese, Rency S; Yu, Guoqiang; Li, Ruijiang; Ressom, Habtom W

    2016-12-01

    Differential expression (DE) analysis is commonly used to identify biomarker candidates that have significant changes in their expression levels between distinct biological groups. One drawback of DE analysis is that it only considers the changes on single biomolecule level. Recently, differential network (DN) analysis has become popular due to its capability to measure the changes on biomolecular pair level. In DN analysis, network is typically built based on correlation and biomarker candidates are selected by investigating the network topology. However, correlation tends to generate over-complicated networks and the selection of biomarker candidates purely based on network topology ignores the changes on single biomolecule level. In this paper, we propose a novel approach, INDEED, that builds sparse differential network based on partial correlation and integrates DE and DN analyses for biomarker discovery. We applied this approach on real proteomic and glycomic data generated by liquid chromatography coupled with mass spectrometry for hepatocellular carcinoma (HCC) biomarker discovery study. For each omic data, we used one dataset to select biomarker candidates, built a disease classifier and evaluated the performance of the classifier on an independent dataset. The biomarker candidates, selected by INDEED, were more reproducible across independent datasets, and led to a higher classification accuracy in predicting HCC cases and cirrhotic controls compared with those selected by separate DE and DN analyses. INDEED also identified some candidates previously reported to be relevant to HCC, such as intercellular adhesion molecule 2 (ICAM2) and c4b-binding protein alpha chain (C4BPA), which were missed by both DE and DN analyses. In addition, we applied INDEED for survival time prediction based on transcriptomic data acquired by analysis of samples from breast cancer patients. We selected biomarker candidates and built a regression model for survival time prediction

  8. Proteomic approach for identifying gonad differential proteins in the oyster (Crassostrea angulata) following food-chain contamination with HgCl2.

    PubMed

    Zhang, Qing-Hong; Huang, Lin; Zhang, Yong; Ke, Cai-Huan; Huang, He-Qing

    2013-12-06

    Hg discharged into the environmental waters can generally be bioaccumulated, transformed and transmited by living organisms, thus resulting in the formation of Hg-toxicity food chains. The pathway and toxicology of food chain contaminated with environmental Hg are rarely revealed by proteomics. Here, we showed that differential proteomics had the potential to understand reproduction toxicity mechanism in marine molluscs through the Hg-contaminated food chain. Hg bioaccumulation was found in every link of the HgCl2-Chlorella vulgaris-oyster-mice food chain. Morphological observations identified the lesions in both the oyster gonad and the mice ovary. Differential proteomics was used to study the mechanisms of Hg toxicity in the oyster gonad and to find some biomarkers of Hg contamination in food chain. Using 2-DE and MALDI-TOF/TOF MS, we identified 13 differential protein spots, of which six were up-regulated, six were down-regulated, while one was undecided. A portion of these differential proteins was further confirmed using real-time PCR and western blotting methods. Their major functions involved binding, protein translocation, catalysis, regulation of energy metabolism, reproductive functioning and structural molecular activity. Among these proteins, 14-3-3 protein, GTP binding protein, arginine kinase (AK) and 71kDa heat shock connate protein (HSCP 71) are considered to be suitable biomarkers of environmental Hg contamination. Furthermore, we established the gene correspondence, responding to Hg reproductive toxicity, between mouse and oyster, and then used real-time PCR to analyze mRNA differential expression of the corresponding genes in mice. The results indicated that the mechanism of Hg reproductive toxicity in mouse was similar to that in oyster. We suggest that the proteomics would be further developed in application research of food safety including toxicological mechanism. It is well known that mercury (Hg) is one of the best toxic metal elements in

  9. Clinical implications from proteomic studies in neurodegenerative diseases: lessons from mitochondrial proteins.

    PubMed

    Butterfield, D Allan; Palmieri, Erika M; Castegna, Alessandra

    2016-01-01

    Mitochondria play a key role in eukaryotic cells, being mediators of energy, biosynthetic and regulatory requirements of these cells. Emerging proteomics techniques have allowed scientists to obtain the differentially expressed proteome or the proteomic redox status in mitochondria. This has unmasked the diversity of proteins with respect to subcellular location, expression and interactions. Mitochondria have become a research 'hot spot' in subcellular proteomics, leading to identification of candidate clinical targets in neurodegenerative diseases in which mitochondria are known to play pathological roles. The extensive efforts to rapidly obtain differentially expressed proteomes and unravel the redox proteomic status in mitochondria have yielded clinical insights into the neuropathological mechanisms of disease, identification of disease early stage and evaluation of disease progression. Although current technical limitations hamper full exploitation of the mitochondrial proteome in neurosciences, future advances are predicted to provide identification of specific therapeutic targets for neurodegenerative disorders.

  10. Clinical implications from proteomic studies in neurodegenerative diseases: lessons from mitochondrial proteins

    PubMed Central

    Butterfield, D. Allan; Palmieri, Erika M.; Castegna, Alessandra

    2016-01-01

    Mitochondria play a key role in eukaryotic cells, being mediators of energy, biosynthetic and regulatory requirements of these cells. Emerging proteomics techniques have allowed scientists to obtain the differentially expressed proteome or the proteomic redox status in mitochondria. This has unmasked the diversity of proteins with respect to subcellular location, expression and interactions. Mitochondria have become a research ‘hot spot’ in subcellular proteomics, leading to identification of candidate clinical targets in neurodegenerative diseases in which mitochondria are known to play pathological roles. The extensive efforts to rapidly obtain differentially expressed proteomes and unravel the redox proteomic status in mitochondria have yielded clinical insights into the neuropathological mechanisms of disease, identification of disease early stage and evaluation of disease progression. Although current technical limitations hamper full exploitation of the mitochondrial proteome in neurosciences, future advances are predicted to provide identification of specific therapeutic targets for neurodegenerative disorders. PMID:26837425

  11. In-depth LC-MS/MS analysis of the chicken ovarian cancer proteome reveals conserved and novel differentially regulated proteins in humans

    PubMed Central

    Nepomuceno, Angelito I.; Shao, Huanjie; Jing, Kai; Ma, Yibao; Petitte, James N.; Idowu, Michael O.; Muddiman, David C.; Fang, Xianjun

    2017-01-01

    Ovarian cancer (OVC) remains the most lethal gynecological malignancy in the world due to the combined lack of early-stage diagnostics and effective therapeutic strategies. The development and application of advanced proteomics technology and new experimental models has created unique opportunities for translational studies. In this study, we investigated the ovarian cancer proteome of the chicken, an emerging experimental model of OVC that develops ovarian tumors spontaneously. Matched plasma, ovary, and oviduct tissue biospecimens derived from healthy, early-stage OVC, and late-stage OVC birds were quantitatively characterized by label-free proteomics. Over 2600 proteins were identified in this study, 348 of which were differentially expressed by more than twofold (p≤0.05) in early- and late-stage ovarian tumor tissue specimens relative to healthy ovarian tissues. Several of the 348 proteins are known to be differentially regulated in human cancers including B2M, CLDN3, EPCAM, PIGR, S100A6, S100A9, S100A11, and TPD52. Of particular interest was ovostatin 2 (OVOS2), a novel 165-kDa protease inhibitor found to be strongly upregulated in chicken ovarian tumors (p=0.0005) and matched plasma (p=0.003). Indeed, RT-quantitative PCR and Western blot analysis demonstrated that OVOS2 mRNA and protein were also upregulated in multiple human OVC cell lines compared to normal ovarian epithelia (NOE) cells and immunohistochemical staining confirmed overexpression of OVOS2 in primary human ovarian cancers relative to non-cancerous tissues. Collectively, these data provide the first evidence for involvement of OVOS2 in the pathogenesis of both chicken and human ovarian cancer. PMID:26159569

  12. Differential expression of renal proteins in a rodent model of Meckel syndrome.

    PubMed

    Mason, Stephen B; Lai, Xianyin; Ringham, Heather N; Bacallao, Robert L; Harris, Peter C; Witzmann, Frank A; Gattone, Vincent H

    2011-01-01

    Meckel syndrome (MKS) is a fatal autosomal recessive condition with prominent renal cystic pathology. Renal protein misexpression was evaluated in the Wpk rat model of human MKS3 gene disease to identify biomarkers for the staging of renal cystic progression. Misexpressed proteins were compared between early and late stages of MKS renal cystic disease using proteomic analysis (two-dimensional gel electrophoresis with LC-MS/MS identification) followed by Western blot analysis. A proteomic analysis identified 76 proteins with statistically different, normalized abundance in at least one group. Subsequently, Western blot was used to confirm differential expression in several of these and polycystic kidney disease (PKD)-associated proteins. Galectin-1 and vimentin were identified as overexpressed proteins, which have been previously found in the jck mouse model of nephronophthisis 9. Ciliopathic PKD proteins, polycystins 1 & 2, and fibrocystin were also differentially expressed in Wpk kidney. In the Wpk rat, misexpressed proteins were identified that were also implicated in other forms of cystic disease. Numerous proteins were either over- or underexpressed in late-stage disease. Differences in protein expression may serve as biomarkers of cystic disease and its progression. Copyright © 2010 S. Karger AG, Basel.

  13. High-specificity quantification method for almond-by-products, based on differential proteomic analysis.

    PubMed

    Zhang, Shiwei; Wang, Shifeng; Huang, Jingmin; Lai, Xintian; Du, Yegang; Liu, Xiaoqing; Li, Bifang; Feng, Ronghu; Yang, Guowu

    2016-03-01

    A highly specific competitive enzyme-linked immunosorbent assay (ELISA) protocol has been developed to identify and classify almond products based on differential proteomic analysis. We applied two-dimensional electrophoresis to compare the differences between almond and apricot kernels to search for almond-specific proteins. The amino acid of apricot Pru-1 was sequenced and aligned to almond Pru-1. One peptide, RQGRQQGRQQQEEGR, which exists in almond but not in apricot, was used as hapten to prepare monoclonal antibody against almond Pru-1. An optimized ELISA method was established using this antibody. The assay did not exhibit cross-reactivity with the tested apricot kernels and other edible plant seeds. The limit of detection (LOD) was 2.5-100μg/g based on different food samples. The recoveries of fortified samples at levels of twofold and eightfold LOD ranged from 82% to 96%. The coefficients of variation were less than 13.0%. Using 7M urea as extracting solution, the heat-treated protein loss ratios were 2%, 5% and 15% under pasteurization (65°C for 30min), baking (150°C for 30min) and autoclaved sterilization (120°C for 15min), respectively. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Differential Proteomics of Urinary Exovesicles from Classical Galactosemic Patients Reveals Subclinical Kidney Insufficiency.

    PubMed

    Staubach, Simon; Pekmez, Murat; Hanisch, Franz-Georg

    2016-06-03

    Classical galactosemia is caused by a nearly complete deficiency of galactose-1-phosphate uridyltransferase (GALT; EC 2.7.712), resulting in a severely impaired galactose metabolism with galactose-1-phosphate and galactitol accumulation. Even on a galactose-restricted diet, patients develop serious long-term complications of the central nervous system and ovaries that may result from chronic cell-toxic effects exerted by endogenous galactose. To address the question of whether disease-associated cellular perturbations could affect the kidney function of the patients, we performed differential proteomics of detergent-resistant membranes from urinary exovesicles. Galactosemic samples (showing drastic shifts from high-mannose to complex-type N-glycosylation on exosomal N-glycoproteins) and healthy, sex-matched controls were analyzed in quadruplex iTRAQ experiments performed in biological and technical replicates. Particularly in the female patient group, the most striking finding was a drastic increase of abundant serum (glyco)proteins, like albumin, leucine-rich α-2-glycoprotein, fetuin, immunoglobulins, prostaglandin H2 d-isomerase, and α-1-microglobulin protein (AMBP), pointing to a subclinical failure of kidney filter function in galactosemic patients and resulting in a heavy overload of exosomal membranes with adsorbed serum (glyco)proteins. Several of these proteins are connected to TBMN and IgAN, proteinuria, and renal damage. The impairment of renal protein filtration was also indicated by increased protein contents derived from extracellular matrices and lysosomes.

  15. Detection of Structural and Metabolic Changes in Traumatically Injured Hippocampus by Quantitative Differential Proteomics

    PubMed Central

    Wu, Ping; Zhao, Yingxin; Haidacher, Sigmund J.; Wang, Enyin; Parsley, Margaret O.; Gao, Junling; Sadygov, Rovshan G.; Starkey, Jonathan M.; Luxon, Bruce A.; Spratt, Heidi; DeWitt, Douglas S.; Prough, Donald S.

    2013-01-01

    Abstract Traumatic brain injury (TBI) is a complex and common problem resulting in the loss of cognitive function. In order to build a comprehensive knowledge base of the proteins that underlie these cognitive deficits, we employed unbiased quantitative mass spectrometry, proteomics, and bioinformatics to identify and quantify dysregulated proteins in the CA3 subregion of the hippocampus in the fluid percussion model of TBI in rats. Using stable isotope 18O-water differential labeling and multidimensional tandem liquid chromatography (LC)-MS/MS with high stringency statistical analyses and filtering, we identified and quantified 1002 common proteins, with 124 increased and 76 decreased. The Ingenuity Pathway Analysis (IPA) bioinformatics tool identified that TBI had profound effects on downregulating global energy metabolism, including glycolysis, the Krebs cycle, and oxidative phosphorylation, as well as cellular structure and function. Widespread upregulation of actin-related cytoskeletal dynamics was also found. IPA indicated a common integrative signaling node, calcineurin B1 (CANB1, CaNBα, or PPP3R1), which was downregulated by TBI. Western blotting confirmed that the calcineurin regulatory subunit, CANB1, and its catalytic binding partner PP2BA, were decreased without changes in other calcineurin subunits. CANB1 plays a critical role in downregulated networks of calcium signaling and homeostasis through calmodulin and calmodulin-dependent kinase II to highly interconnected structural networks dominated by tubulins. This large-scale knowledge base lays the foundation for the identification of novel therapeutic targets for cognitive rescue in TBI. PMID:22757692

  16. Differential abundance of sarcoplasmic proteome explains animal effect on beef Longissimus lumborum color stability

    USDA-ARS?s Scientific Manuscript database

    The sarcoplasmic proteome of beef Longissimus lumborum demonstrating animal-to-animal variation in color stability was examined to correlate proteome profile with color. Longissimus lumborum (36 h post-mortem) muscles were obtained from 73 beef carcasses, aged for 13 days, and fabricated to 2.5-cm s...

  17. High-Throughput Cloning and Expression Library Creation for Functional Proteomics

    PubMed Central

    Festa, Fernanda; Steel, Jason; Bian, Xiaofang; Labaer, Joshua

    2013-01-01

    The study of protein function usually requires the use of a cloned version of the gene for protein expression and functional assays. This strategy is particular important when the information available regarding function is limited. The functional characterization of the thousands of newly identified proteins revealed by genomics requires faster methods than traditional single gene experiments, creating the need for fast, flexible and reliable cloning systems. These collections of open reading frame (ORF) clones can be coupled with high-throughput proteomics platforms, such as protein microarrays and cell-based assays, to answer biological questions. In this tutorial we provide the background for DNA cloning, discuss the major high-throughput cloning systems (Gateway® Technology, Flexi® Vector Systems, and Creator™ DNA Cloning System) and compare them side-by-side. We also report an example of high-throughput cloning study and its application in functional proteomics. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP12). Details can be found at http://www.proteomicstutorials.org. PMID:23457047

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

    PubMed Central

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

    2008-01-01

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

  19. Primer: Shotgun Proteomics in Neuroscience

    PubMed Central

    Liao, Lujian; McClatchy, Daniel B.; Yates, John R.

    2009-01-01

    Mass spectrometry-based proteomics is increasingly used to address basic and clinical questions in biomedical research through studies of differential protein expression, protein-protein interactions, and post-translational modifications. The complex structural and functional organization of the human brain warrants the application of high-throughput, systematic approaches to understand the functional alterations under normal physiological conditions and the perturbations of neurological diseases. This primer focuses on shotgun proteomics based tandem mass spectrometry for the identification of proteins in a complex mixture. It describes the basic concepts of protein differential expression analysis and post-translational modification analysis and discusses several strategies to improve the coverage of the proteome. PMID:19607789

  20. The Effect of Pericellular Oxygen Levels on Proteomic Profile and Lipogenesis in 3T3-L1 Differentiated Preadipocytes Cultured on Gas-Permeable Cultureware

    PubMed Central

    Weiszenstein, Martin; Pavlikova, Nela; Elkalaf, Moustafa; Halada, Petr; Seda, Ondrej; Trnka, Jan; Kovar, Jan; Polak, Jan

    2016-01-01

    Pericellular oxygen concentration represents an important factor in the regulation of cell functions, including cell differentiation, growth and mitochondrial energy metabolism. Hypoxia in adipose tissue has been associated with altered adipokine secretion profile and suggested as a possible factor in the development of type 2 diabetes. In vitro experiments provide an indispensable tool in metabolic research, however, physical laws of gas diffusion make prolonged exposure of adherent cells to desired pericellular O2 concentrations questionable. The aim of this study was to investigate the direct effect of various O2 levels (1%, 4% and 20% O2) on the proteomic profile and triglyceride accumulation in 3T3-L1 differentiated preadipocytes using gas-permeable cultureware. Following differentiation of cells under desired pericellular O2 concentrations, cell lysates were subjected to two-dimensional gel electrophoresis and protein visualization using Coomassie blue staining. Spots showing differential expression under hypoxia were analyzed using matrix-assisted laser desorption/ionization mass spectrometry. All identified proteins were subjected to pathway analysis. We observed that protein expression of 26 spots was reproducibly affected by 4% and 1% O2 (17 upregulated and 9 downregulated). Pathway analysis showed that mitochondrial energy metabolism and triglyceride synthesis were significantly upregulated by hypoxia. In conclusion, this study demonstrated the direct effects of pericellular O2 levels on adipocyte energy metabolism and triglyceride synthesis, probably mediated through the reversed tricarboxylic acid cycle flux. PMID:27023342

  1. The Effect of Pericellular Oxygen Levels on Proteomic Profile and Lipogenesis in 3T3-L1 Differentiated Preadipocytes Cultured on Gas-Permeable Cultureware.

    PubMed

    Weiszenstein, Martin; Pavlikova, Nela; Elkalaf, Moustafa; Halada, Petr; Seda, Ondrej; Trnka, Jan; Kovar, Jan; Polak, Jan

    2016-01-01

    Pericellular oxygen concentration represents an important factor in the regulation of cell functions, including cell differentiation, growth and mitochondrial energy metabolism. Hypoxia in adipose tissue has been associated with altered adipokine secretion profile and suggested as a possible factor in the development of type 2 diabetes. In vitro experiments provide an indispensable tool in metabolic research, however, physical laws of gas diffusion make prolonged exposure of adherent cells to desired pericellular O2 concentrations questionable. The aim of this study was to investigate the direct effect of various O2 levels (1%, 4% and 20% O2) on the proteomic profile and triglyceride accumulation in 3T3-L1 differentiated preadipocytes using gas-permeable cultureware. Following differentiation of cells under desired pericellular O2 concentrations, cell lysates were subjected to two-dimensional gel electrophoresis and protein visualization using Coomassie blue staining. Spots showing differential expression under hypoxia were analyzed using matrix-assisted laser desorption/ionization mass spectrometry. All identified proteins were subjected to pathway analysis. We observed that protein expression of 26 spots was reproducibly affected by 4% and 1% O2 (17 upregulated and 9 downregulated). Pathway analysis showed that mitochondrial energy metabolism and triglyceride synthesis were significantly upregulated by hypoxia. In conclusion, this study demonstrated the direct effects of pericellular O2 levels on adipocyte energy metabolism and triglyceride synthesis, probably mediated through the reversed tricarboxylic acid cycle flux.

  2. Lupus anti-ribosomal P autoantibody proteomes express convergent biclonal signatures.

    PubMed

    Al Kindi, M A; Colella, A D; Beroukas, D; Chataway, T K; Gordon, T P

    2016-04-01

    Lupus-specific anti-ribosomal P (anti-Rib-P) autoantibodies have been implicated in the pathogenesis of neurological complications in systemic lupus erythematosus (SLE). The aim of the present study was to determine variable (V)-region signatures of secreted autoantibody proteomes specific for the Rib-P heterocomplex and investigate the molecular basis of the reported cross-reactivity with Sm autoantigen. Anti-Rib-P immunoglobulins (IgGs) were purified from six anti-Rib-P-positive sera by elution from enzyme-linked immunosorbent assay (ELISA) plates coated with either native Rib-P proteins or an 11-amino acid peptide (11-C peptide) representing the conserved COOH-terminal P epitope. Rib-P- and 11-C peptide-specific IgGs were analysed for heavy (H) and light (L) chain clonality and V-region expression using an electrophoretic and de-novo and database-driven mass spectrometric sequencing workflow. Purified anti-Rib-P and anti-SmD IgGs were tested for cross-reactivity on ELISA and their proteome data sets analysed for shared clonotypes. Anti-Rib-P autoantibody proteomes were IgG1 kappa-restricted and comprised two public clonotypes defined by unique H/L chain pairings. The major clonotypic population was specific for the common COOH-terminal epitope, while the second shared the same pairing signature as a recently reported anti-SmD clonotype, accounting for two-way immunoassay cross-reactivity between these lupus autoantibodies. Sequence convergence of anti-Rib-P proteomes suggests common molecular pathways of autoantibody production and identifies stereotyped clonal populations that are thought to play a pathogenic role in neuropsychiatric lupus. Shared clonotypic structures for anti-Rib-P and anti-Sm responses suggest a common B cell clonal origin for subsets of these lupus-specific autoantibodies.

  3. Application of the SILAC (stable isotope labelling with amino acids in cell culture) technique in quantitative comparisons for tissue proteome expression.

    PubMed

    Xu, Yuhuan; Liang, Shufang; Shen, Guobo; Xu, Xuejiao; Liu, Qingping; Xu, Zhizhong; Gong, Fengming; Tang, Minghai; Wei, Yuquan

    2009-07-06

    Stable isotope labelling has recently become a popular tool for the quantitative profiling of the proteome, especially the emergence and development of the SILAC (stable isotope labelling with amino acids in cell culture) technique. Here we have expanded the application of SILAC to comparison of the relative protein expression levels between two different states of tissues based on cultured cells with [2H]leucine labelling as an internal standard in mass spectra. The SILAC ratio of tissue proteins versus labelled cells was determined by the calculation of peak intensity of the pair of labelled and unlabelled peptide fragment ions from the mass spectra, and the relative expression level of proteins in two groups of tissues was estimated by calculating the ratio of their SILAC ratio. To validate our [2H]leucine-based differential proteome analysis for tissues, we successfully compared two known proteins, one up-regulated vimentin and one down-regulated enoyl-CoA hydratase in human renal cancerous tissues versus human normal kidney tissues, which was previously confirmed by other groups using conventional two-dimensional PAGE analysis. Furthermore, we identified a previously unknown down-regulated protein, COX4I1 (cytochrome c oxidase subunit 4 isoform 1), in renal carcinoma tissues by this [2H]leucine-based quantitative proteomics method, which was also validated by immunohistochemistry and Western-blot analysis. In conclusion, the application of the [2H]leucine-based quantitative technique can be effectively expanded to comparison of the expression levels for the tissue proteome at different states, which would help us to identify new candidate biomarkers for tumours.

  4. 2D-DIGE-based proteome expression changes in leaves of rice seedlings exposed to low-level gamma radiation at Iitate village, Fukushima

    PubMed Central

    Hayashi, Gohei; Moro, Carlo F; Rohila, Jai Singh; Shibato, Junko; Kubo, Akihiro; Imanaka, Tetsuji; Kimura, Shinzo; Ozawa, Shoji; Fukutani, Satoshi; Endo, Satoru; Ichikawa, Katsuki; Agrawal, Ganesh Kumar; Shioda, Seiji; Hori, Motohide; Fukumoto, Manabu; Rakwal, Randeep

    2015-01-01

    The present study continues our previous research on investigating the biological effects of low-level gamma radiation in rice at the heavily contaminated Iitate village in Fukushima, by extending the experiments to unraveling the leaf proteome. 14-days-old plants of Japonica rice (Oryza sativa L. cv. Nipponbare) were subjected to gamma radiation level of upto 4 µSv/h, for 72 h. Following exposure, leaf samples were taken from the around 190 µSv/3 d exposed seedling and total proteins were extracted. The gamma irradiated leaf and control leaf (harvested at the start of the experiment) protein lysates were used in a 2-D differential gel electrophoresis (2D-DIGE) experiment using CyDye labeling in order to asses which spots were differentially represented, a novelty of the study. 2D-DIGE analysis revealed 91 spots with significantly different expression between samples (60 positive, 31 negative). MALDI-TOF and TOF/TOF mass spectrometry analyses revealed those as comprising of 59 different proteins (50 up-accumulated, 9 down-accumulated). The identified proteins were subdivided into 10 categories, according to their biological function, which indicated that the majority of the differentially expressed proteins consisted of the general (non-energy) metabolism and stress response categories. Proteome-wide data point to some effects of low-level gamma radiation exposure on the metabolism of rice leaves. PMID:26451896

  5. 2D-DIGE-based proteome expression changes in leaves of rice seedlings exposed to low-level gamma radiation at Iitate village, Fukushima.

    PubMed

    Hayashi, Gohei; Moro, Carlo F; Rohila, Jai Singh; Shibato, Junko; Kubo, Akihiro; Imanaka, Tetsuji; Kimura, Shinzo; Ozawa, Shoji; Fukutani, Satoshi; Endo, Satoru; Ichikawa, Katsuki; Agrawal, Ganesh Kumar; Shioda, Seiji; Hori, Motohide; Fukumoto, Manabu; Rakwal, Randeep

    2015-01-01

    The present study continues our previous research on investigating the biological effects of low-level gamma radiation in rice at the heavily contaminated Iitate village in Fukushima, by extending the experiments to unraveling the leaf proteome. 14-days-old plants of Japonica rice (Oryza sativa L. cv. Nipponbare) were subjected to gamma radiation level of upto 4 µSv/h, for 72 h. Following exposure, leaf samples were taken from the around 190 µSv/3 d exposed seedling and total proteins were extracted. The gamma irradiated leaf and control leaf (harvested at the start of the experiment) protein lysates were used in a 2-D differential gel electrophoresis (2D-DIGE) experiment using CyDye labeling in order to asses which spots were differentially represented, a novelty of the study. 2D-DIGE analysis revealed 91 spots with significantly different expression between samples (60 positive, 31 negative). MALDI-TOF and TOF/TOF mass spectrometry analyses revealed those as comprising of 59 different proteins (50 up-accumulated, 9 down-accumulated). The identified proteins were subdivided into 10 categories, according to their biological function, which indicated that the majority of the differentially expressed proteins consisted of the general (non-energy) metabolism and stress response categories. Proteome-wide data point to some effects of low-level gamma radiation exposure on the metabolism of rice leaves.

  6. Differential gene expression in ripening banana fruit.

    PubMed

    Clendennen, S K; May, G D

    1997-10-01

    During banana (Musa acuminata L.) fruit ripening ethylene production triggers a developmental cascade that is accompanied by a massive conversion of starch to sugars, an associated burst of respiratory activity, and an increase in protein synthesis. Differential screening of cDNA libraries representing banana pulp at ripening stages 1 and 3 has led to the isolation of 11 nonredundant groups of differentially expressed mRNAs. Identification of these transcripts by partial sequence analysis indicates that two of the mRNAs encode proteins involved in carbohydrate metabolism, whereas others encode proteins thought to be associated with pathogenesis, senescence, or stress responses in plants. Their relative abundance in the pulp and tissue-specific distribution in greenhouse-grown banana plants were determined by northern-blot analyses. The relative abundance of transcripts encoding starch synthase, granule-bound starch synthase, chitinase, lectin, and a type-2 metallothionein decreased in pulp during ripening. Transcripts encoding endochitinase, beta-1,3-glucanase, a thaumatin-like protein, ascorbate peroxidase, metallothionein, and a putative senescence-related protein increased early in ripening. The elucidation of the molecular events associated with banana ripening will facilitate a better understanding and control of these processes, and will allow us to attain our long-term goal of producing candidate oral vaccines in transgenic banana plants.

  7. Collagen gene expression during limb cartilage differentiation

    PubMed Central

    1986-01-01

    As limb mesenchymal cells differentiate into chondrocytes, they initiate the synthesis of type II collagen and cease synthesizing type I collagen. Changes in the cytoplasmic levels of type I and type II collagen mRNAs during the course of limb chondrogenesis in vivo and in vitro were examined using cloned cDNA probes. A striking increase in cytoplasmic type II collagen mRNA occurs coincident with the crucial condensation stage of chondrogenesis in vitro, in which prechondrogenic mesenchymal cells become closely juxtaposed before depositing a cartilage matrix. Thereafter, a continuous and progressive increase in the accumulation of cytoplasmic type II collagen mRNA occurs which parallels the progressive accumulation of cartilage matrix by cells. The onset of overt chondrogenesis, however, does not involve activation of the transcription of the type II collagen gene. Low levels of type II collagen mRNA are present in the cytoplasm of prechondrogenic mesenchymal cells at the earliest stages of limb development, well before the accumulation of detectable levels of type II collagen. Type I collagen gene expression during chondrogenesis is regulated, at least in part, at the translational level. Type I collagen mRNAs are present in the cytoplasm of differentiated chondrocytes, which have ceased synthesizing detectable amounts of type I collagen. PMID:3754261

  8. Analysis of root proteome unravels differential molecular responses during compatible and incompatible interaction between chickpea (Cicer arietinum L.) and Fusarium oxysporum f. sp. ciceri Race1 (Foc1).

    PubMed

    Chatterjee, Moniya; Gupta, Sumanti; Bhar, Anirban; Chakraborti, Dipankar; Basu, Debabrata; Das, Sampa

    2014-11-03

    Vascular wilt caused by Fusarium oxysporum f. sp. ciceri Race 1 (Foc1) is a serious disease of chickpea (Cicer arietinum L.) accounting for approximately 10-15% annual crop loss. The fungus invades the plant via roots, colonizes the xylem vessels and prevents the upward translocation of water and nutrients, finally resulting in wilting of the entire plant. Although comparative transcriptomic profiling have highlighted some important signaling molecules, but proteomic studies involving chickpea-Foc1 are limited. The present study focuses on comparative root proteomics of susceptible (JG62) and resistant (WR315) chickpea genotypes infected with Foc1, to understand the mechanistic basis of susceptibility and/or resistance. The differential and unique proteins of both genotypes were identified at 48 h, 72 h, and 96 h post Foc1 inoculation. 2D PAGE analyses followed by MALDI-TOF MS and MS/MS identified 100 differentially (>1.5 fold<, p<0.05) or uniquely expressed proteins. These proteins were further categorized into 10 functional classes and grouped into GO (gene ontology) categories. Network analyses of identified proteins revealed intra and inter relationship of these proteins with their neighbors as well as their association with different defense signaling pathways. qRT-PCR analyses were performed to correlate the mRNA and protein levels of some proteins of representative classes. The differential and unique proteins identified indicate their involvement in early defense signaling of the host. Comparative analyses of expression profiles of obtained proteins suggest that albeit some common components participate in early defense signaling in both susceptible and resistant genotypes, but their roles and regulation differ in case of compatible and/or incompatible interactions. Thus, functional characterization of identified PR proteins (PR1, BGL2, TLP), Trypsin protease inhibitor, ABA responsive protein, cysteine protease, protein disulphide isomerase, ripening

  9. Natural Genetic Variation Differentially Affects the Proteome and Transcriptome in Caenorhabditis elegans.

    PubMed

    Kamkina, Polina; Snoek, L Basten; Grossmann, Jonas; Volkers, Rita J M; Sterken, Mark G; Daube, Michael; Roschitzki, Bernd; Fortes, Claudia; Schlapbach, Ralph; Roth, Alexander; von Mering, Christian; Hengartner, Michael O; Schrimpf, Sabine P; Kammenga, Jan E

    2016-05-01

    Natural genetic variation is the raw material of evolution and influences disease development and progression. An important question is how this genetic variation translates into variation in protein abundance. To analyze the effects of the genetic background on gene and protein expression in the nematode Caenorhabditis elegans, we quantitatively compared the two genetically highly divergent wild-type strains N2 and CB4856. Gene expression was analyzed by microarray assays, and proteins were quantified using stable isotope labeling by amino acids in cell culture. Among all transcribed genes, we found 1,532 genes to be differentially transcribed between the two wild types. Of the total 3,238 quantified proteins, 129 proteins were significantly differentially expressed between N2 and CB4856. The differentially expressed proteins were enriched for genes that function in insulin-signaling and stress-response pathways, underlining strong divergence of these pathways in nematodes. The protein abundance of the two wild-type strains correlates more strongly than protein abundance versus transcript abundance within each wild type. Our findings indicate that in C. elegans only a fraction of the changes in protein abundance can be explained by the changes in mRNA abundance. These findings corroborate with the observations made across species.

  10. Deep immune profiling by mass cytometry links human T and NK cell differentiation and cytotoxic molecule expression patterns.

    PubMed

    Bengsch, Bertram; Ohtani, Takuya; Herati, Ramin Sedaghat; Bovenschen, Niels; Chang, Kyong-Mi; Wherry, E John

    2017-03-19

    The elimination of infected or tumor cells by direct lysis is a key T and NK cell effector function. T and NK cells can kill target cells by coordinated secretion of cytotoxic granules containing one or both pore-forming proteins, perforin and granulysin and combinations of granzyme (Gzm) family effector proteases (in humans: Gzm A, B, K, M and H). Understanding the pattern of expression of cytotoxic molecules and the relationship to different states of T and NK cells may have direct relevance for immune responses in autoimmunity, infectious disease and cancer. Approaches capable of simultaneously evaluating expression of multiple cytotoxic molecules with detailed information on T and NK differentiation state, however, remain limited. Here, we established a high dimensional mass cytometry approach to comprehensively interrogate single cell proteomic expression of cytotoxic programs and lymphocyte differentiation. This assay identified a coordinated expression pattern of cytotoxic molecules linked to CD8 T cell differentiation stages. Coordinated high expression of perforin, granulysin, Gzm A, Gzm B and Gzm M was associated with markers of late effector memory differentiation and expression of chemokine receptor CX3CR1. However, classical gating and dimensionality reduction approaches also identified other discordant patterns of cytotoxic molecule expression in CD8 T cells, including reduced perforin, but high Gzm A, Gzm K and Gzm M expression. When applied to non-CD8 T cells, this assay identified different patterns of cytotoxic molecule co-expression by CD56(hi) versus CD56(dim) defined NK cell developmental stages; in CD4 T cells, low expression of cytotoxic molecules was found mainly in TH1 phenotype cells, but not in Tregs or T follicular helper cells (TFH). Thus, this comprehensive, single cell, proteomic assessment of cytotoxic protein co-expression patterns demonstrates specialized cytotoxic programs in T cells and NK cells linked to their differentiation

  11. Proteomics of Plant Pathogenic Fungi

    PubMed Central

    González-Fernández, Raquel; Prats, Elena; Jorrín-Novo, Jesús V.

    2010-01-01

    Plant pathogenic fungi cause important yield losses in crops. In order to develop efficient and environmental friendly crop protection strategies, molecular studies of the fungal biological cycle, virulence factors, and interaction with its host are necessary. For that reason, several approaches have been performed using both classical genetic, cell biology, and biochemistry and the modern, holistic, and high-throughput, omic techniques. This work briefly overviews the tools available for studying Plant Pathogenic Fungi and is amply focused on MS-based Proteomics analysis, based on original papers published up to December 2009. At a methodological level, different steps in a proteomic workflow experiment are discussed. Separate sections are devoted to fungal descriptive (intracellular, subcellular, extracellular) and differential expression proteomics and interactomics. From the work published we can conclude that Proteomics, in combination with other techniques, constitutes a powerful tool for providing important information about pathogenicity and virulence factors, thus opening up new possibilities for crop disease diagnosis and crop protection. PMID:20589070

  12. Microfluidic chips for protein differential expression profiling.

    PubMed

    Armenta, Jenny M; Dawoud, Abdulilah A; Lazar, Iulia M

    2009-04-01

    Biomarker discovery and screening using novel proteomic technologies is an area that is attracting increased attention in the biomedical community. Early detection of abnormal physiological conditions will be highly beneficial for diagnosing various diseases and increasing survivability rates. Clearly, progress in this area will depend on the development of fast, reliable, and highly sensitive and specific sample bioanalysis methods. Microfluidics has emerged as a technology that could become essential in proteomics research as it enables the integration of all sample preparation, separation, and detection steps, with the added benefit of enhanced sample throughput. The combination of these advantages with the sensitivity and capability of MS detection to deliver precise structural information makes microfluidics-MS a very competitive technology for biomarker discovery. The integration of LC microchip devices with MS detection, and specifically their applicability to biomarker screening applications in MCF-7 breast cancer cellular extracts is reported in this manuscript. Loading approximately 0.1-1 microg of crude protein extract tryptic digest on the chip has typically resulted in the reliable identification of approximately 40-100 proteins. The potential of an LC-ESI-MS chip for comparative proteomic analysis of isotopically labeled MCF-7 breast cancer cell extracts is explored for the first time.

  13. Integrated Analysis of Transcriptomic and Proteomic Datasets Reveals Information on Protein Expressivity and Factors Affecting Translational Efficiency.

    PubMed

    Wang, Jiangxin; Wu, Gang; Chen, Lei; Zhang, Weiwen

    2016-01-01

    Integrated analysis of large-scale transcriptomic and proteomic data can provide important insights into the metabolic mechanisms underlying complex biological systems. In this chapter, we present methods to address two aspects of issues related to integrated transcriptomic and proteomic analysis. First, due to the fact that proteomic datasets are often incomplete, and integrated analysis of partial proteomic data may introduce significant bias. To address these issues, we describe a zero-inflated Poisson (ZIP)-based model to uncover the complicated relationships between protein abundances and mRNA expression levels, and then apply them to predict protein abundance for the proteins not experimentally detected. The ZIP model takes into consideration the undetected proteins by assuming that there is a probability mass at zero representing expressed proteins that were undetected owing to technical limitations. The model validity is demonstrated using biological information of operons, regulons, and pathways. Second, weak correlation between transcriptomic and proteomic datasets is often due to biological factors affecting translational processes. To quantify the effects of these factors, we describe a multiple regression-based statistical framework to quantitatively examine the effects of various translational efficiency-related sequence features on mRNA-protein correlation. Using the datasets from sulfate-reducing bacteria Desulfovibrio vulgaris, the analysis shows that translation-related sequence features can contribute up to 15.2-26.2% of the total variation of the correlation between transcriptomic and proteomic datasets, and also reveals the relative importance of various features in translation process.

  14. Proteomic analysis of human hepatoma cells expressing methionine adenosyltransferase I/III☆

    PubMed Central

    Schröder, Paul C.; Fernández-Irigoyen, Joaquín; Bigaud, Emilie; Serna, Antonio; Renández-Alcoceba, Rubén; Lu, Shelly C.; Mato, José M.; Prieto, Jesús; Corrales, Fernando J.

    2015-01-01

    Methionine adenosyltransferase I/III (MATI/III) synthesizes S-adenosylmethionine (SAM) in quiescent hepatocytes. Its activity is compromised in most liver diseases including liver cancer. Since SAM is a driver of hepatocytes fate we have studied the effect of re-expressing MAT1A in hepatoma Huh7 cells using proteomics. MAT1A expression leads to SAM levels close to those found in quiescent hepatocytes and induced apoptosis. Normalization of intracellular SAM induced alteration of 128 proteins identified by 2D-DIGE and gel-free methods, accounting for deregulation of central cellular functions including apoptosis, cell proliferation and survival. Human Dead-box protein 3 (DDX3X), a RNA helicase regulating RNA splicing, export, transcription and translation was down-regulated upon MAT1A expression. Our data support the regulation of DDX3X levels by SAM in a concentration and time dependent manner. Consistently, DDX3X arises as a primary target of SAM and a principal intermediate of its antitumoral effect. Based on the parallelism between SAM and DDX3X along the progression of liver disorders, and the results reported here, it is tempting to suggest that reduced SAM in the liver may lead to DDX3X up-regulation contributing to the pathogenic process and that replenishment of SAM might prove to have beneficial effects, at least in part by reducing DDX3X levels. This article is part of a Special Issue entitled: Proteomics: The clinical link. PMID:22270009

  15. 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.

  16. Analysis of differentially expressed proteins in Yersinia enterocolitica-infected HeLa cells

    PubMed Central

    Alugubelly, Navatha; Hercik, Kamil; Kibler, Peter; Nanduri, Bindu; Edelmann, Mariola J.

    2017-01-01

    Yersinia enterocolitica is a facultative intracellular pathogen and a causative agent of yersiniosis, which can be contracted by ingestion of contaminated food. Yersinia secretes virulence factors to subvert critical pathways in the host cell. In this study we utilized shotgun label-free proteomics to study differential protein expression in epithelial cells infected with Yersinia enterocolitica. We identified a total of 551 proteins, amongst which 42 were downregulated (e.g. Prostaglandin E Synthase 3, POH-1 and Karyopherin alpha) and 22 were upregulated (e.g. Rab1c and RhoA) in infected cells. We validated some of these results by western blot analysis of proteins extracted from Caco-2 and HeLa cells. The proteomic dataset was used to identify host canonical pathways and molecular functions modulated by this infection in the host cells. This study constitutes a proteome of Yersinia-infected cells and can support new discoveries in the area of host-pathogen interactions. PMID:26854600

  17. Differential protein expression in the estuarine copepod Eurytemora affinis after diuron and alkylphenol exposures.

    PubMed

    Boulangé-Lecomte, Céline; Rocher, Béatrice; Cailleaud, Kévin; Cosette, Pascal; Legrand, Eléna; Devreker, David; Budzinski, Hélène; Souissi, Sami; Forget-Leray, Joëlle

    2016-07-01

    Proteomics was used in the calanoid copepod Eurytemora affinis for screening of protein expression modifications induced by organic contaminants. The copepods were exposed in a continuous flow-through system for 86 h to environmentally relevant concentrations of contaminants representative of the pollution in the Seine Estuary (Haute-Normandie, France; diuron, 500 ng L(-1) ; alkylphenol mixture, 1000 ng L(-1) ). Proteome analysis of whole-body copepod extracts by 2-dimensional gel electrophoresis revealed that the contaminants induced modifications in protein expression, with the highest quantitative variations occurring after diuron exposure. Specifically, 88 and 41 proteins were differentially expressed after diuron and alkylphenol treatments, respectively. After mass spectrometry analysis, 51 (diuron exposure) and 15 (alkylphenol exposure) proteins were identified. The identified proteins were potentially related to energy metabolism, cell growth, nervous signal conductivity, excitotoxicity, oxidative stress response, and antioxidant defense. The data suggest a massive general disturbance of physiological functions of E. affinis after diuron exposure, whereas alkylphenols induced an alteration of a few targeted physiological functions. The protein expression signatures identified after contaminant exposure deserve further investigation in terms of the development of novel potential biomarkers for water quality assessment. Environ Toxicol Chem 2016;35:1860-1871. © 2015 SETAC. © 2015 SETAC.

  18. Differential diagnosis of azoospermia with proteomic biomarkers ECM1 and TEX101 quantified in seminal plasma.

    PubMed

    Drabovich, Andrei P; Dimitromanolakis, Apostolos; Saraon, Punit; Soosaipillai, Antoninus; Batruch, Ihor; Mullen, Brendan; Jarvi, Keith; Diamandis, Eleftherios P

    2013-11-20

    Male fertility problems range from diminished production of sperm, or oligozoospermia, to nonmeasurable levels of sperm in semen, or azoospermia, which is diagnosed in nearly 2% of men in the general population. Testicular biopsy is the only definitive diagnostic method to distinguish between obstructive (OA) and nonobstructive (NOA) azoospermia and to identify the NOA subtypes of hypospermatogenesis, maturation arrest and Sertoli cell-only syndrome. We measured by selected reaction monitoring assay 18 biomarker candidates in 119 seminal plasma samples from men with normal spermatogenesis and azoospermia, and identified two proteins, epididymis-expressed ECM1 and testis-expressed TEX101, which differentiated OA and NOA with high specificities and sensitivities. The performance of ECM1 was confirmed by enzyme-linked immunosorbent assay. On the basis of a cutoff level of 2.3 μg/ml derived from the current data, we could distinguish OA from normal spermatogenesis with 100% specificity, and OA from NOA with 73% specificity, at 100% sensitivity. Immunohistochemistry and an immunoenrichment mass spectrometry-based assay revealed the differential expression of TEX101 in distinct NOA subtypes. TEX101 semen concentrations differentiated Sertoli cell-only syndrome from the other categories of NOA. As a result, we propose a simple two-biomarker decision tree for the differential diagnosis of OA and NOA and, in addition, for the differentiation of NOA subtypes. Clinical assays for ECM1 and TEX101 have the potential to replace most of the diagnostic testicular biopsies and facilitate the prediction of outcome of sperm retrieval procedures, thus increasing the reliability and success of assisted reproduction techniques.

  19. The workflow for quantitative proteome analysis of chloroplast development and differentiation, chloroplast mutants, and protein interactions by spectral counting.

    PubMed

    Friso, Giulia; Olinares, Paul Dominic B; van Wijk, Klaas J

    2011-01-01

    This chapter outlines a quantitative proteomics workflow using a label-free spectral counting technique. The workflow has been tested on different aspects of chloroplast biology in maize and Arabidopsis, including chloroplast mutant analysis, cell-type specific chloroplast differentiation, and the proplastid-to-chloroplast transition. The workflow involves one-dimensional SDS-PAGE of the proteomes of leaves or chloroplast subfractions, tryptic digestions, online LC-MS/MS using a mass spectrometer with high mass accuracy and duty cycle, followed by semiautomatic data processing. The bioinformatics analysis can effectively select best gene models and deals with quantification of closely related proteins; the workflow avoids overidentification of proteins and results in more accurate protein quantification. The final output includes pairwise comparative quantitative analysis, as well as hierarchical clustering for discovery of temporal and spatial patterns of protein accumulation. A brief discussion about potential pitfalls, as well as the advantages and disadvantages of spectral counting, is provided.

  20. Differential protein expression in metallothionein protection from depleted uranium-induced nephrotoxicity

    PubMed Central

    Hao, Yuhui; Huang, Jiawei; Liu, Cong; Li, Hong; Liu, Jing; Zeng, Yiping; Yang, Zhangyou; Li, Rong

    2016-01-01

    The purpose of this study was to investigate the underlying mechanism of metallothionein (MT) protection from depleted uranium (DU) using a proteomics approach to search for a DU toxicity-differential protein. MT−/− and MT+/+ mice were administrated with a single dose of DU (10 mg/kg, i.p.) or equal volume of saline. After 4 days, protein changes in kidney tissues were evaluated using a proteomics approach. A total of 13 differentially expressed proteins were identified using two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The validating results showed that the expression of aminoacylase-3 (ACY-3) and the mitochondrial ethylmalonic encephalopathy 1 (ETHE1) decreased significantly after DU exposure; in addition, the reduction in MT−/− mice was more significant than that in MT+/+ mice. The results also showed that exogenous ETHE1 or ACY-3 could increase the survival rate of human embryonic kidney 293 (HEK293) cells after DU exposure. A specific siRNA of ETHE1 significantly increased cell apoptosis rates after DU exposure, whereas exogenous ETHE1 significantly decreased cell apoptosis rates. In summary, ACY-3 and ETHE1 might involve in protection roles of MT. ETHE1 could be a new sensitive molecular target of DU-induced cell apoptosis. PMID:27966587

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

    PubMed Central

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

    2014-01-01

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

  2. Differential Proteomic Analysis of Platelets Suggested Possible Signal Cascades Network in Platelets Treated with Salvianolic Acid B

    PubMed Central

    Ma, Chao; Yao, Yan; Yue, Qing-Xi; Zhou, Xin-Wen; Yang, Peng-Yuan; Wu, Wan-Ying; Guan, Shu-Hong; Jiang, Bao-Hong; Yang, Min; Liu, Xuan; Guo, De-An

    2011-01-01

    Background Salvianolic acid B (SB) is an active component isolated from Danshen, a traditional Chinese medicine widely used for the treatment of cardiovascular disorders. Previous study suggested that SB might inhibit adhesion as well as aggregation of platelets by a mechanism involving the integrin α2β1. But, the signal cascades in platelets after SB binding are still not clear. Methodology/Principal Findings In the present study, a differential proteomic analysis (two-dimensional electrophoresis) was conducted to check the protein expression profiles of rat platelets with or without treatment of SB. Proteins altered in level after SB exposure were identified by MALDI-TOF MS/MS. Treatment of SB caused regulation of 20 proteins such as heat shock-related 70 kDa protein 2 (hsp70), LIM domain protein CLP-36, copine I, peroxiredoxin-2, coronin-1 B and cytoplasmic dynein intermediate chain 2C. The regulation of SB on protein levels was confirmed by Western blotting. The signal cascades network induced by SB after its binding with integrin α2β1 was predicted. To certify the predicted network, binding affinity of SB to integrin α2β1 was checked in vitro and ex vivo in platelets. Furthermore, the effects of SB on protein levels of hsp70, coronin-1B and intracellular levels of Ca(2+) and reactive oxygen species (ROS) were checked with or without pre-treatment of platelets using antibody against integrin α2β1. Electron microscopy study confirmed that SB affected cytoskeleton structure of platelets. Conclusions/Significance Integrin α2β1 might be one of the direct target proteins of SB in platelets. The signal cascades network of SB after binding with integrin α2β1 might include regulation of intracellular Ca(2+) level, cytoskeleton-related proteins such as coronin-1B and cytoskeleton structure of platelets. PMID:21379382

  3. Differential proteomic analysis of grapevine leaves by iTRAQ reveals responses to heat stress and subsequent recovery

    PubMed Central

    2014-01-01

    Background High temperature is a major environmental factor limiting grape yield and affecting berry quality. Thermotolerance includes the direct response to heat stress and the ability to recover from heat stress. To better understand the mechanism of the thermotolerance of Vitis, we combined a physiological analysis with iTRAQ-based proteomics of Vitis vinifera cv Cabernet Sauvignon, subjected to 43°C for 6 h, and then followed by recovery at 25/18°C. Results High temperature increased the concentrations of TBARS and inhibited electronic transport in photosynthesis apparatus, indicating that grape leaves were damaged by heat stress. However, these physiological changes rapidly returned to control levels during the subsequent recovery phase from heat stress. One hundred and seventy-four proteins were differentially expressed under heat stress and/or during the recovery phase, in comparison to unstressed controls, respectively. Stress and recovery conditions shared 42 proteins, while 113 and 103 proteins were respectively identified under heat stress and recovery conditions alone. Based on MapMan ontology, functional categories for these dysregulated proteins included mainly photosynthesis (about 20%), proteins (13%), and stress (8%). The subcellular localization using TargetP showed most proteins were located in the chloroplasts (34%), secretory pathways (8%) and mitochondrion (3%). Conclusion On the basis of these findings, we proposed that some proteins related to electron transport chain of photosynthesis, antioxidant enzymes, HSPs and other stress response proteins, and glycolysis may play key roles in enhancing grapevine adaptation to and recovery capacity from heat stress. These results provide a better understanding of the proteins involved in, and mechanisms of thermotolerance in grapevines. PMID:24774513

  4. Proteomic analysis of cAMP-mediated signaling during differentiation of 3 T3-L1 preadipocytes.

    PubMed

    Borkowski, Kamil; Wrzesinski, Krzysztow; Rogowska-Wrzesinska, Adelina; Audouze, Karine; Bakke, Jesse; Petersen, Rasmus Koefoed; Haj, Fawaz G; Madsen, Lise; Kristiansen, Karsten

    2014-12-01

    Initiation of adipocyte differentiation is promoted by the synergistic action of insulin/insulin-like growth factor, glucocorticoids, and agents activating cAMP-dependent signaling. The action of cAMP is mediated via PKA and Epac, where at least part of the PKA function relates to strong repression of Rho kinase activity, whereas Epac counteracts the reduction in insulin/insulin-like growth factor signaling associated with complete repression of Rho kinase activity. However, detailed knowledge of the Epac-dependent branch and the interplay with PKA is still limited. In the present study, we present a comprehensive evaluation of Epac-mediated processes and their interplay with PKA during the initiation of 3 T3-L1 preadipocyte differentiation using a combination of proteomics, molecular approaches, and bioinformatics. Proteomic analyses revealed 7 proteins specifically regulated in response to Epac activation, 4 in response to PKA activation, and 11 in response to the combined activation of Epac and PKA during the initial phase of differentiation. Network analyses indicated that the identified proteins are involved in pathways of importance for glucose metabolism, inositol metabolism, and calcium-dependent signaling thereby adding a novel facet to our understanding of cAMP-mediated potentiation of adipocyte differentiation.

  5. Differential proteome analysis of serum proteins associated with the development of type 2 diabetes mellitus in the KK-A(y) mouse model using the iTRAQ technique.

    PubMed

    Takahashi, Eri; Okumura, Akinori; Unoki-Kubota, Hiroyuki; Hirano, Hisashi; Kasuga, Masato; Kaburagi, Yasushi

    2013-06-12

    To identify candidate serum molecules associated with the progression of type 2 diabetes mellitus (T2DM), we carried out differential proteomic analysis using the KK-A(y) mouse, an animal model of T2DM with obesity. We employed an iTRAQ-based quantitative proteomic approach to analyze the proteomic changes in the sera collected from a pair of 4-week-old KK-A(y) versus C57BL/6 mice. Among the 227 proteins identified, a total of 45 proteins were differentially expressed in KK-A(y) versus C57BL/6 mice. We comparatively analyzed a series of the sera collected at 4 and 12weeks of age from KK-A(y) and C57BL/6 mice for the target protein using multiple reaction monitoring analysis, and identified 8 differentially expressed proteins between the sera of these mice at both time points. Among them, serine (or cysteine) peptidase inhibitor, clade A, member 3K (SERPINA3K) levels were elevated significantly in the sera of KK-A(y) mice compared to C57BL/6 mice. An in vitro assay revealed that the human homologue SERPINA3 increased the transendothelial permeability of retinal microvascular endothelial cells, which may be involved in the pathogenesis of diabetes and/or diabetic retinopathy. With the identified proteins, our proteomics study could provide valuable clues for a better understanding of the underlying mechanisms associated with T2DM. In this paper, we investigated the serum proteome of KK-A(y) mice in a pre-diabetic state compared to that of wild type controls in an attempt to uncover early diagnostic markers of diabetes that are maintained through a diabetic phenotype. We used iTRAQ-based two-dimensional LC-MS/MS serum profiling, and identified several differentially expressed proteins at the pre-diabetic stage. The differential expression was confirmed by multiple reaction monitoring assay, which is fast gaining ground as a sensitive, specific, and cost-effective methodology for relative quantification of the candidate proteins. Using these techniques, we have

  6. Regional Differences of Proteins Expressing in Adipose Depots Isolated from Cows, Steers and Bulls as Identified by a Proteomic Approach

    PubMed Central

    Cho, Jin Hyoung; Jeong, Jin Young; Lee, Ra Ham; Park, Mi Na; Kim, Seok-Ho; Park, Seon-Min; Shin, Jae-Cheon; Jeon, Young-Joo; Shim, Jung-Hyun; Choi, Nag-Jin; Seo, Kang Seok; Cho, Young Sik; Kim, MinSeok S.; Ko, Sungho; Seo, Jae-Min; Lee, Seung-Youp; Chae, Jung-Il; Lee, Hyun-Jeong

    2016-01-01

    Adipose tissue in the loin muscle area of beef cattle as a marbling factor is directly associated with beef quality. To elucidate whether properties of proteins involved in depot specific adipose tissue were sex-dependent, we analyzed protein expression of intramuscular adipose tissue (IMAT) and omental adipose tissue (OMAT) from Hanwoo cows, steers, and bulls of Korean native beef cattle by liquid chromatography-tandem mass spectrometry (LC-MS/MS)–based proteomic analysis, quantitative polymerase chain reaction (PCR) and western blot analysis. Two different adipose depots (i.e. intramuscular and omental) were collected from cows (n = 7), steers (n = 7), or bulls (n = 7). LC-MS/MS revealed a total of 55 and 35 proteins in IMAT and OMAT, respectively. Of the 55 proteins identified, 44, 40, and 42 proteins were confirmed to be differentially expressed in IMAT of cows, steers, and bulls, respectively. In OMAT of cows, steers, and bulls, 33, 33, and 22 were confirmed to be differentially expressed, respectively. Tropomyosin (TPM) 1, TPM 2, and TPM3 were subjected to verification by quantitative PCR and western blot analysis in IMAT and OMAT of Hanwoo cows, steers, and bulls as key factors closely associated with muscle development. Both mRNA levels and protein levels of TPM1, TPM2, and TPM3 in IMAT were lower in bulls compared to in cows or steers suggesting that they were positively correlated with marbling score and quality grade. Our results may aid the regulation of marbling development and improvement of meat quality grades in beef cattle. PMID:27165017

  7. Regional Differences of Proteins Expressing in Adipose Depots Isolated from Cows, Steers and Bulls as Identified by a Proteomic Approach.

    PubMed

    Cho, Jin Hyoung; Jeong, Jin Young; Lee, Ra Ham; Park, Mi Na; Kim, Seok-Ho; Park, Seon-Min; Shin, Jae-Cheon; Jeon, Young-Joo; Shim, Jung-Hyun; Choi, Nag-Jin; Seo, Kang Seok; Cho, Young Sik; Kim, MinSeok S; Ko, Sungho; Seo, Jae-Min; Lee, Seung-Youp; Chae, Jung-Il; Lee, Hyun-Jeong

    2016-08-01

    Adipose tissue in the loin muscle area of beef cattle as a marbling factor is directly associated with beef quality. To elucidate whether properties of proteins involved in depot specific adipose tissue were sex-dependent, we analyzed protein expression of intramuscular adipose tissue (IMAT) and omental adipose tissue (OMAT) from Hanwoo cows, steers, and bulls of Korean native beef cattle by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic analysis, quantitative polymerase chain reaction (PCR) and western blot analysis. Two different adipose depots (i.e. intramuscular and omental) were collected from cows (n = 7), steers (n = 7), or bulls (n = 7). LC-MS/MS revealed a total of 55 and 35 proteins in IMAT and OMAT, respectively. Of the 55 proteins identified, 44, 40, and 42 proteins were confirmed to be differentially expressed in IMAT of cows, steers, and bulls, respectively. In OMAT of cows, steers, and bulls, 33, 33, and 22 were confirmed to be differentially expressed, respectively. Tropomyosin (TPM) 1, TPM 2, and TPM3 were subjected to verification by quantitative PCR and western blot analysis in IMAT and OMAT of Hanwoo cows, steers, and bulls as key factors closely associated with muscle development. Both mRNA levels and protein levels of TPM1, TPM2, and TPM3 in IMAT were lower in bulls compared to in cows or steers suggesting that they were positively correlated with marbling score and quality grade. Our results may aid the regulation of marbling development and improvement of meat quality grades in beef cattle.

  8. Proteomic Analysis of Acetaminophen-Induced Changes in Mitochondrial Protein Expression Using Spectral Counting

    PubMed Central

    Stamper, Brendan D.; Mohar, Isaac; Kavanagh, Terrance J.; Nelson, Sidney D.

    2011-01-01

    Comparative proteomic analysis following treatment with acetaminophen (APAP) was performed on two different models of APAP-mediated hepatocellular injury in order to both identify common targets for adduct formation and track drug-induced changes in protein expression. Male C57BL/6 mice were used as a model for APAP-mediated liver injury in vivo and TAMH cells were used as a model for APAP-mediated cytotoxicity in vitro. SEQUEST was unable to identify the precise location of sites of adduction following treatment with APAP in either system. However, semiquantitative analysis of the proteomic datasets using spectral counting revealed a downregulation of P450 isoforms associated with APAP bioactivation, and an upregulation of proteins related to the electron transport chain by APAP compared to control. Both mechanisms are likely compensatory in nature as decreased P450 expression is likely to attenuate toxicity associated with N-acetyl-p-quinoneimine (NAPQI) formation, whereas APAP-induced electron transport chain component upregulation may be an attempt to promote cellular bioenergetics. PMID:21329376

  9. Adaptation to beta-myrcene catabolism in Pseudomonas sp. M1: an expression proteomics analysis.

    PubMed

    Santos, Pedro M; Sá-Correia, Isabel

    2009-11-01

    Beta-myrcene, a monoterpene widely used as a fragrance and flavoring additive, also possesses analgesic, anti-mutagenic, and tyrosinase inhibitory properties. In order to get insights into the molecular mechanisms underlying the ability of Pseudomonas sp. M1 to catabolize beta-myrcene, an expression proteomics approach was used in this study. Results indicate that the catabolic enzyme machinery for beta-myrcene utilization (MyrB, MyrC, and MyrD and other uncharacterized proteins) is strongly induced when beta-myrcene is present in the growth medium. Since an M1 mutant, lacking a functional 2-methylisocitrate dehydratase, is not able to grow in mineral medium with beta-myrcene or propionic acid as the sole C-source, and also based on the expression proteomic analysis carried out in this study, it is suggested that the beta-myrcene catabolic intermediate propionyl-CoA is channeled into the central metabolism via the 2-methylcitrate cycle. Results also suggest that the major alteration occurring in the central carbon metabolism of cells growing in beta-myrcene-containing media is related with the redistribution of the metabolic fluxes leading to increased oxaloacetate production. Other up-regulated proteins are believed to prevent protein misfolding and aggregation or to play important structural roles, contributing to the adaptive alteration of cell wall and membrane organization and integrity, which are essential features to allow the bacterium to cope with the highly lipophilic beta-myrcene as C-source.

  10. Proteome analysis of tobacco leaves reveals dynamic changes in protein expression among different cultivation areas.

    PubMed

    Lu, L M; Ye, K Y; Tang, Z X; Liu, L; Chen, Y; Luo, J; Huang, Y B

    2015-11-30

    The leaves of tobacco plants were used to analyze differences in protein content of tobacco grown in the four main flue-cured tobacco-producing areas of Sichuan Province, China. An improved protein extraction method, isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis two-dimensional gel electrophoretic separation, was used to extract and separate total protein from tobacco leaves. Proteomic maps with relatively high resolution and repeatability were produced. At isoelectric points 4 to 7 and molecular weight ranging from 20-100 kDa, we detected 1032, 1030, 1019, and 1011 clearly visible protein spots in tobacco leaves from the four study areas. Proteome comparison between these protein spots showed that 119 spots with a greater than 2-fold change in expression quantity contributed to the variation in expression. Of which, 115 were successfully identified and annotated. According to the annotation results, these proteins participate in photosynthesis, energy metabolism, mineral nutrition, terpene metabolism, defensive reaction, and other physiological and biochemical processes. This study preliminarily explains the effects of ecological conditions on the physiological metabolism of tobacco leaves and how such effects directly or indirectly contribute to tobacco leaf quality.

  11. Differential quantitative proteomics of Porphyromonas gingivalis by linear ion trap mass spectrometry: Non-label methods comparison, q-values and LOWESS curve fitting

    NASA Astrophysics Data System (ADS)

    Xia, Qiangwei; Wang, Tiansong; Park, Yoonsuk; Lamont, Richard J.; Hackett, Murray

    2007-01-01

    Differential analysis of whole cell proteomes by mass spectrometry has largely been applied using various forms of stable isotope labeling. While metabolic stable isotope labeling has been the method of choice, it is often not possible to apply such an approach. Four different label free ways of calculating expression ratios in a classic "two-state" experiment are compared: signal intensity at the peptide level, signal intensity at the protein level, spectral counting at the peptide level, and spectral counting at the protein level. The quantitative data were mined from a dataset of 1245 qualitatively identified proteins, about 56% of the protein encoding open reading frames from Porphyromonas gingivalis, a Gram-negative intracellular pathogen being studied under extracellular and intracellular conditions. Two different control populations were compared against P. gingivalis internalized within a model human target cell line. The q-value statistic, a measure of false discovery rate previously applied to transcription microarrays, was applied to proteomics data. For spectral counting, the most logically consistent estimate of random error came from applying the locally weighted scatter plot smoothing procedure (LOWESS) to the most extreme ratios generated from a control technical replicate, thus setting upper and lower bounds for the region of experimentally observed random error.

  12. Differential proteomic responses in hepatopancreas and adductor muscles of the green-lipped mussel Perna viridis to stresses induced by cadmium and hydrogen peroxide.

    PubMed

    Leung, Priscilla T Y; Wang, Yu; Mak, Sarah S T; Ng, W C; Leung, Kenneth M Y

    2011-09-01

    This study aimed to reveal the proteomic responses in the hepatopancreas and adductor muscle of a common biomonitor, Perna viridis after 14-day exposure to two model chemicals, cadmium (Cd; a toxic metal) and hydrogen peroxide (H(2)O(2); a pro-oxidant), using two-dimensional gel electrophoresis coupled with multivariate statistical analyses. Unique sets of tissue-specific protein expression signatures were revealed corresponding to the two treatment groups. In the hepatopancreas, 15 and 2 spots responded to Cd and H(2)O(2) treatments respectively. 6 and 7 spots were differentially expressed in the adductor muscle for Cd and H(2)O(2) treatments, respectively. 15 differentially expressed spots were successfully identified by MALDI-TOF/TOF MS analysis. These proteins are involved in glycolysis, amino acid metabolism, energy homeostasis, oxidative stress response, redox homeostasis and protein folding, heat-shock response, and muscle contraction modulation. This is the first time, to have demonstrated that Cd exposure not only leads to substantial oxidative stress but also results in endoplasmic reticulum stress in hepatopancreas of the mussel. Such notable stress responses may be attributable to high Cd accumulation in this tissue. Our results suggested that investigations on these stress-associated protein changes could be used as a new and complementary approach in pollution monitoring by this popular biomonitor species. Copyright © 2011 Elsevier B.V. All rights reserved.

  13. Proteomic Analysis of Tissue from α1,3-galactosyltransferase Knockout Mice Reveals That a Wide Variety of Proteins and Protein Fragments Change Expression Level

    PubMed Central

    Thorlacius-Ussing, Louise; Ludvigsen, Maja; Kirkeby, Svend

    2013-01-01

    A barrier in a pig-to-man xenotransplantation is that the Galα1-3Galβ1-4GlcNAc-R carbohydrate (α-Gal epitope) expressed on pig endothelial cells reacts with naturally occurring antibodies in the recipient’s blood leading to rejection. Deletion of the α1,3-galactosyltransferase gene prevents the synthesis of the α-Gal epitope. Therefore, knockout models of the α1,3-galactosyltransferase gene are widely used to study xenotransplantation. We have performed proteomic studies on liver and pancreas tissues from wild type and α1,3-galactosyltransferase gene knockout mice. The tissues were analyzed by two-dimensional polyacrylamide gel electrophoresis and liquid chromatography - tandem mass spectrometry. The analyses revealed that a wide variety of proteins and protein fragments are differentially expressed suggesting that knockout of the α1,3-galactosyltransferase gene affects the expression of several other genes. PMID:24244699

  14. Heterologous Expression and Purification Systems for Structural Proteomics of Mammalian Membrane Proteins

    PubMed Central

    2002-01-01

    Membrane proteins (MPs) are responsible for the interface between the exterior and the interior of the cell. These proteins are implicated in numerous diseases, such as cancer, cystic fibrosis, epilepsy, hyperinsulinism, heart failure, hypertension and Alzheimer's disease. However, studies on these disorders are hampered by a lack of structural information about the proteins involved. Structural analysis requires large quantities of pure and active proteins. The majority of medically and pharmaceutically relevant MPs are present in tissues at very low concentration, which makes heterologous expression in large-scale production-adapted cells a prerequisite for structural studies. Obtaining mammalian MP structural data depends on the development of methods that allow the production of large quantities of MPs. This review focuses on the different heterologous expression systems, and the purification strategies, used to produce large amounts of pure mammalian MPs for structural proteomics. PMID:18629259

  15. A Targeted Proteomics Approach for Profiling Murine Cytochrome P450 Expression

    PubMed Central

    Hersman, Elisabeth M.

    2014-01-01

    The cytochrome P450 (P450) superfamily of enzymes plays a prominent role in drug metabolism. Although mice are a widely used preclinical model in pharmacology, the expression of murine P450 enzymes at the protein level has yet to be fully defined. Twenty-seven proteins belonging to P450 subfamilies 1A, 2A, 2B, 2C, 2D, 2E, 2F, 2J, 2U, 3A, 4A, 4B, 4F, and 4V were readily detectable in Balb/c mouse tissue using a global mass spectrometry–based proteomics approach. Subsequently, a targeted mass spectrometry–based assay was developed to simultaneously quantify these enzymes in ranges of femtomoles of P450 per microgram of total protein concentration range. This screen was applied to mouse liver microsomes and tissue lysates of kidney, lung, intestine, heart, and brain isolated from mixed-sex fetuses; male and female mice that were 3–4 weeks, 9–10 weeks, and 8–10 months of age; and pregnant mice. CYP1A2 was consistently more abundant in male mouse liver microsomes compared with age-matched females. Hepatic expression of CYP2B9 was more abundant in 3- to 4-week-old male and female mice than in mice of other ages; in addition, CYP2B9 was the only enzyme that was detectable at higher levels in pregnant mouse liver microsomes compared with age-matched females. Interestingly, sexually dimorphic expression of CYP2B9, 2D26, 2E1, and 4B1 was observed in kidney only. The targeted proteomics assay described here can be broadly used as a tool for investigating the expression patterns of P450 enzymes in mice. PMID:24594750

  16. Differential cysteine labeling and global label-free proteomics reveals an altered metabolic state in skeletal muscle aging.

    PubMed

    McDonagh, Brian; Sakellariou, Giorgos K; Smith, Neil T; Brownridge, Philip; Jackson, Malcolm J

    2014-11-07

    The molecular mechanisms underlying skeletal muscle aging and associated sarcopenia have been linked to an altered oxidative status of redox-sensitive proteins. Reactive oxygen and reactive nitrogen species (ROS/RNS) generated by contracting skeletal muscle are necessary for optimal protein function, signaling, and adaptation. To investigate the redox proteome of aging gastrocnemius muscles from adult and old male mice, we developed a label-free quantitative proteomic approach that includes a differential cysteine labeling step. The approach allows simultaneous identification of up- and downregulated proteins between samples in addition to the identification and relative quantification of the reversible oxidation state of susceptible redox cysteine residues. Results from muscles of adult and old mice indicate significant changes in the content of chaperone, glucose metabolism, and cytoskeletal regulatory proteins, including Protein DJ-1, cAMP-dependent protein kinase type II, 78 kDa glucose regulated protein, and a reduction in the number of redox-responsive proteins identified in muscle of old mice. Results demonstrate skeletal muscle aging causes a reduction in redox-sensitive proteins involved in the generation of precursor metabolites and energy metabolism, indicating a loss in the flexibility of the redox energy response. Data is available via ProteomeXchange with identifier PXD001054.

  17. Differential Cysteine Labeling and Global Label-Free Proteomics Reveals an Altered Metabolic State in Skeletal Muscle Aging

    PubMed Central

    2014-01-01

    The molecular mechanisms underlying skeletal muscle aging and associated sarcopenia have been linked to an altered oxidative status of redox-sensitive proteins. Reactive oxygen and reactive nitrogen species (ROS/RNS) generated by contracting skeletal muscle are necessary for optimal protein function, signaling, and adaptation. To investigate the redox proteome of aging gastrocnemius muscles from adult and old male mice, we developed a label-free quantitative proteomic approach that includes a differential cysteine labeling step. The approach allows simultaneous identification of up- and downregulated proteins between samples in addition to the identification and relative quantification of the reversible oxidation state of susceptible redox cysteine residues. Results from muscles of adult and old mice indicate significant changes in the content of chaperone, glucose metabolism, and cytoskeletal regulatory proteins, including Protein DJ-1, cAMP-dependent protein kinase type II, 78 kDa glucose regulated protein, and a reduction in the number of redox-responsive proteins identified in muscle of old mice. Results demonstrate skeletal muscle aging causes a reduction in redox-sensitive proteins involved in the generation of precursor metabolites and energy metabolism, indicating a loss in the flexibility of the redox energy response. Data is available via ProteomeXchange with identifier PXD001054. PMID:25181601

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

    PubMed

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

    2015-03-01

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

  19. Differential proteome and cellular adhesion analyses of the probiotic bacterium Lactobacillus acidophilus NCFM grown on raffinose - an emerging prebiotic.

    PubMed

    Celebioglu, Hasan Ufuk; Ejby, Morten; Majumder, Avishek; Købler, Carsten; Goh, Yong Jun; Thorsen, Kristian; Schmidt, Bjarne; O'Flaherty, Sarah; Abou Hachem, Maher; Lahtinen, Sampo J; Jacobsen, Susanne; Klaenhammer, Todd R; Brix, Susanne; Mølhave, Kristian; Svensson, Birte

    2016-05-01

    Whole cell and surface proteomes were analyzed together with adhesive properties of the probiotic bacterium Lactobacillus acidophilus NCFM (NCFM) grown on the emerging prebiotic raffinose, exemplifying a synbiotic. Adhesion of NCFM to mucin and intestinal HT-29 cells increased three-fold after culture with raffinose versus glucose, as also visualized by scanning electron microscopy. Comparative proteomics using 2D-DIGE showed 43 unique proteins to change in relative abundance in whole cell lysates from NCFM grown on raffinose compared to glucose. Furthermore, 14 unique proteins in 18 spots of the surface subproteome underwent changes identified by differential 2DE, including elongation factor G, thermostable pullulanase, and phosphate starvation inducible stress-related protein increasing in a range of +2.1 - +4.7 fold. By contrast five known moonlighting proteins decreased in relative abundance by up to -2.4 fold. Enzymes involved in raffinose catabolism were elevated in the whole cell proteome; α-galactosidase (+13.9 fold); sucrose phosphorylase (+5.4 fold) together with metabolic enzymes from the Leloir pathway for galactose utilization and the glycolysis; β-galactosidase (+5.7 fold); galactose (+2.9/+3.1 fold) and fructose (+2.8 fold) kinases. The insights at the molecular and cellular levels contributed to the understanding of the interplay of a synbiotic composed of NCFM and raffinose with the host. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Differential effects of growth hormone versus insulin-like growth factor-I on the mouse plasma proteome.

    PubMed

    Ding, Juan; List, Edward O; Bower, Brian D; Kopchick, John J

    2011-10-01

    The GH/IGF-I axis has both pre- and postpubertal metabolic effects. However, the differential effects of GH and/or IGF-I on animal physiology or the plasma proteome are still being unraveled. In this report, we analyzed several physiological effects along with the plasma proteome after treatment of mice with recombinant bovine GH or recombinant human IGF-I. GH and IGF-I showed similar effects in increasing body length, body weight, lean and fluid masses, and organ weights including muscle, kidney, and spleen. However, GH significantly increased serum total cholesterol, whereas IGF-I had no effect on it. Both acute and longer-term effects on the plasma proteome were determined. Proteins found to be significantly changed by recombinant bovine GH and/or recombinant human IGF-I injections were identified by mass spectrometry (MS) and MS/MS. The identities of these proteins were further confirmed by Western blotting analysis. Isoforms of apolipoprotein A4, apolipoprotein E, serum amyloid protein A-1, clusterin, transthyretin, and several albumin fragments were found to be differentially regulated by GH vs. IGF-I in mouse plasma. Thus, we have identified several plasma protein biomarkers that respond specifically and differentially to GH or IGF-I and may represent new physiological targets of these hormones. These findings may lead to better understanding of the independent biological effects of GH vs. IGF-I. In addition, these novel biomarkers may be useful for the development of tests to detect illicit use of GH or IGF-I.

  1. Differential Effects of Growth Hormone Versus Insulin-Like Growth Factor-I on the Mouse Plasma Proteome

    PubMed Central

    Ding, Juan; List, Edward O.; Bower, Brian D.

    2011-01-01

    The GH/IGF-I axis has both pre- and postpubertal metabolic effects. However, the differential effects of GH and/or IGF-I on animal physiology or the plasma proteome are still being unraveled. In this report, we analyzed several physiological effects along with the plasma proteome after treatment of mice with recombinant bovine GH or recombinant human IGF-I. GH and IGF-I showed similar effects in increasing body length, body weight, lean and fluid masses, and organ weights including muscle, kidney, and spleen. However, GH significantly increased serum total cholesterol, whereas IGF-I had no effect on it. Both acute and longer-term effects on the plasma proteome were determined. Proteins found to be significantly changed by recombinant bovine GH and/or recombinant human IGF-I injections were identified by mass spectrometry (MS) and MS/MS. The identities of these proteins were further confirmed by Western blotting analysis. Isoforms of apolipoprotein A4, apolipoprotein E, serum amyloid protein A-1, clusterin, transthyretin, and several albumin fragments were found to be differentially regulated by GH vs. IGF-I in mouse plasma. Thus, we have identified several plasma protein biomarkers that respond specifically and differentially to GH or IGF-I and may represent new physiological targets of these hormones. These findings may lead to better understanding of the independent biological effects of GH vs. IGF-I. In addition, these novel biomarkers may be useful for the development of tests to detect illicit use of GH or IGF-I. PMID:21791560

  2. Differential proteome analysis of mature and germinated embryos of Araucaria angustifolia.

    PubMed

    Balbuena, Tiago S; Jo, Leonardo; Pieruzzi, Fernanda P; Dias, Leonardo L C; Silveira, Vanildo; Santa-Catarina, Claudete; Junqueira, Magno; Thelen, Jay J; Shevchenko, Andrej; Floh, Eny I S

    2011-04-01

    Araucaria angustifolia is an endangered Brazilian native conifer tree. The aim of the present work was to identify differentially expressed proteins between mature and germinated embryos of A. angustifolia, using one and two dimensional gel electrophoresis approaches followed by protein identification by tandem mass spectrometry. The identities of 32 differentially expressed protein spots from two dimensional gel maps were successfully determined, including proteins and enzymes involved in storage mobilization such as the vicilin-like storage protein and proteases. A label free approach, based on spectral counts, resulted in detection of 10 and 14 mature and germinated enriched proteins, respectively. Identified proteins were mainly related to energetic metabolism pathways, translational processes, oxidative stress regulation and cellular signaling. The integrated use of both strategies permitted a comprehensive protein expression overview of changes in germinated embryos in relation to matures, providing insights into the this process in a recalcitrant seed species. Applications of the data generated on the monitoring and control of in vitro somatic embryos were discussed.

  3. Differential expression and modification of proteins during ontogenesis in Malus domestica.

    PubMed

    Cao, Xin; Gao, Yan; Wang, Yi; Li, Chun M; Zhao, Yong B; Han, Zhen H; Zhang, Xin Z

    2011-12-01

    Many morphological and physiological changes have been widely reported during ontogeny in higher plants. In order for the better understanding of the proteomic differences between ontogenetic phases, protein compositions between leaves of juvenile, adult vegetative and reproductive phases were compared in an apple (Malus domestica Borkh., Jonathan × Golden Delicious) seedling. Totally, 122 differentially expressed or modified protein spots were separated by DIGE. Of the 122 protein spots, 44, 17 and 29 were abundant in the leaf samples from the juvenile, adult vegetative and reproductive phases, respectively, two spots showed a lower level in the adult vegetative tissue, while the amount of protein increased in 21 spots during ontogeny and declined in nine spots. One hundred and fifteen spots were successfully picked and 95 spots were identified by MALDI-TOF-TOF high-resolution tandem mass spectrometry. Twenty-three juvenile phase abundant or down-regulated spots were photosynthesis-associated proteins, implying a juvenile phase-related photosynthesis enhancement. The expression of 10 enzymes and coenzymes involved in protein synthesis and catabolism was elevated in the adult reproductive phase or up-regulated during ontogeny, contributing a phase change-related activation in protein metabolism. Six proteins generated 30 differential gel spots via post-translational modifications. The differential expression of NADP-dependent D-sorbitol-6-phosphate dehydrogenase was confirmed by Western blotting in six seedlings derived from two hybrid populations. The results of semi-quantitative PCR indicate that some but not all of these proteomic changes were transcriptionally regulated. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    PubMed Central

    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. PMID:26867742

  5. 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.

  6. Differential expression, distinct localization and opposite effect on Golgi structure and cell differentiation by a novel splice variant of human PRMT5.

    PubMed

    Sohail, Muhammad; Zhang, Manli; Litchfield, David; Wang, Lisheng; Kung, Sam; Xie, Jiuyong

    2015-10-01

    Alternative splicing contributes greatly to the proteomic diversity of metazoans. Protein arginine methyltransferase 5 (PRMT5) methylates arginines of Golgi components and other factors exerting diverse effects on cell growth/differentiation, but the underlying molecular basis for its subcellular distribution and diverse roles has not been fully understood. Here we show the detailed properties of an evolutionarily emerged splice variant of human PRMT5 (PRMT5S) that is distinct from the original isoform (PRMT5L). The isoforms are differentially expressed among mammalian cells and tissues. The PRMT5S is distributed all over the cell but PRMT5L mainly colocalizes with Giantin, a Golgi marker. PRMT5 knockdown led to an enlarged Giantin pattern, which was prevented by the expression of either isoform. Rescuing PRMT5S also increased the percentage of cells with an interphase Giantin pattern compacted at one end of the nucleus, consistent with its cell cycle-arresting effect, while rescuing PRMT5L increased that of the mitotic Giantin patterns of dynamically fragmented structures. Moreover, the isoforms are differentially expressed during neuronal or dendritic cell differentiation, and their ectopic expression showed an opposite effect on dendritic cell differentiation. Furthermore, besides their differential regulation of gene expression, both isoforms also similarly regulate over a thousand genes particularly those involved in apoptosis and differentiation. Taking these properties together, we propose that their differential expression and subcellular localization contribute to spatial and temporal regulation of arginine methylation and gene expression to exert different effects. The novel PRMT5S likely contributes to the observed diverse effects of PRMT5 in cells. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. 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

  8. 2D DIGE Does Not Reveal all: A Scotopic Report Suggests Differential Expression of a Single "Calponin Family Member" Protein for Tetany of Sphincters!

    PubMed

    Chaudhury, Arun

    2015-01-01

    Using 2D differential gel electrophoresis (DIGE) and mass spectrometry (MS), a recent report by Rattan and Ali (2015) compared proteome expression between tonically contracted sphincteric smooth muscles of the internal anal sphincter (IAS), in comparison to the adjacent rectum [rectal smooth muscles (RSM)] that contracts in a phasic fashion. The study showed the differential expression of a single 23 kDa protein SM22, which was 1.87 fold, overexpressed in RSM in comparison to IAS. Earlier studies have shown differences in expression of different proteins like Rho-associated protein kinase II, myosin light chain kinase, myosin phosphatase, and protein kinase C between IAS and RSM. The currently employed methods, despite its high-throughput potential, failed to identify these well-characterized differences between phasic and tonic muscles. This calls into question the fidelity and validatory potential of the otherwise powerful technology of 2D DIGE/MS. These discrepancies, when redressed in future studies, will evolve this recent report as an important baseline study of "sphincter proteome." Proteomics techniques are currently underutilized in examining pathophysiology of hypertensive/hypotensive disorders involving gastrointestinal sphincters, including achalasia, gastroesophageal reflux disease (GERD), spastic pylorus, seen during diabetes or chronic chemotherapy, intestinal pseudo-obstruction, and recto-anal incontinence. Global proteome mapping may provide instant snapshot of the complete repertoire of differential proteins, thus expediting to identify the molecular pathology of gastrointestinal motility disorders currently labeled "idiopathic" and facilitating practice of precision medicine.

  9. Global features of gene expression on the proteome and transcriptome levels in S. coelicolor during germination.

    PubMed

    Strakova, Eva; Bobek, Jan; Zikova, Alice; Vohradsky, Jiri

    2013-01-01

    Streptomycetes have been studied mostly as producers of secondary metabolites, while the transition from dormant spores to an exponentially growing culture has largely been ignored. Here, we focus on a comparative analysis of fluorescently and radioactively labeled proteome and microarray acquired transcriptome expressed during the germination of Streptomyces coelicolor. The time-dynamics is considered, starting from dormant spores through 5.5 hours of growth with 13 time points. Time series of the gene expressions were analyzed using correlation, principal components analysis and an analysis of coding genes utilization. Principal component analysis was used to identify principal kinetic trends in gene expression and the corresponding genes driving S. coelicolor germination. In contrast with the correlation analysis, global trends in the gene/protein expression reflected by the first principal components showed that the prominent patterns in both the protein and the mRNA domains are surprisingly well correlated. Analysis of the number of expressed genes identified functional groups activated during different time intervals of the germination.