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Sample records for differential expression proteomics

  1. 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.

  2. Proteomic analysis of the differentially expressed proteins by airborne nanoparticles.

    PubMed

    Park, Seul Ki; Jeon, Yu Mi; Son, Bu Soon; Youn, Hyung Sun; Lee, Mi Young

    2011-07-01

    Airborne nanoparticles with thermodynamic diameters less than 56 nm (PM(0.056)) were collected using a Moudi cascade impactor, and the differentially expressed proteins upon exposure to the airborne nanoparticles were identified in human bronchial epithelial cells. More than 600 protein spots were detected on the two-dimensional gel, and the identified 13 of these proteins showed notable changes. Nine were up-regulated and four were down-regulated following treatment with the airborne nanoparticles. Notably, malignant transformation-associated multiple forms of keratins, epigenetic regulation-related MBD1-containing chromatin associated factor 2, epithelial malignancy-related vimentin and exocytosis-related annexin A2 were changed upon exposure to airborne nanoparticle PM(0.056). PMID:21491466

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

    PubMed

    Xu, Jia; Wang, Lily; Li, Jing

    2014-12-01

    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. PMID:25327611

  4. 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.

  5. 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

  6. 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

    2013-04-01

    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. PMID:23464874

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

    PubMed

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

    2015-01-01

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

  8. 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

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

    PubMed

    Wang, Lei; Xu, Wenrong; Cao, Lei; 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. 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

    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

  11. 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

    2016-06-01

    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. PMID:23968773

  13. Proteomic analysis of differentially expressed proteins in Penaeus monodon hemocytes after Vibrio harveyi infection

    PubMed Central

    2010-01-01

    Background Viral and bacterial diseases can cause mass mortalities in commercial shrimp aquaculture. In contrast to studies on the antiviral response, the responses of shrimps to bacterial infections by high throughput techniques have been reported only at the transcriptional level and not at the translational level. In this study, a proteomic analysis of shrimp hemocytes to identify differentially expressed proteins in response to a luminous bacterium Vibrio harveyi was evaluated for its feasibility and is reported for the first time. Results The two-dimensional gel electrophoresis (2-DE) patterns of the hemocyte proteins from the unchallenged and V. harveyi challenged shrimp, Penaeus monodon, at 24 and 48 h post infection were compared. From this, 27 differentially expressed protein spots, and a further 12 weakly to non-differentially regulated control spots, were selected for further analyses by the LC-ESI-MS/MS. The 21 differentially expressed proteins that could be identified by homologous annotation were comprised of proteins that are directly involved in the host defense responses, such as hemocyanin, prophenoloxidase, serine proteinase-like protein, heat shock protein 90 and alpha-2-macroglobulin, and those involved in signal transduction, such as the14-3-3 protein epsilon and calmodulin. Western blot analysis confirmed the up-regulation of hemocyanin expression upon bacterial infection. The expression of the selected proteins which were the representatives of the down-regulated proteins (the 14-3-3 protein epsilon and alpha-2-macroglobulin) and of the up-regulated proteins (hemocyanin) was further assessed at the transcription level using real-time RT-PCR. Conclusions This work suggests the usefulness of a proteomic approach to the study of shrimp immunity and revealed hemocyte proteins whose expression were up regulated upon V. harveyi infection such as hemocyanin, arginine kinase and down regulated such as alpha-2-macroglobulin, calmodulin and 14

  14. Proteomic identification of differentially expressed proteins in sea cucumber Apostichopus japonicus coelomocytes after Vibrio splendidus infection.

    PubMed

    Zhang, Peng; Li, Chenghua; Li, Ye; Zhang, Pengjuan; Shao, Yina; Jin, Chunhua; Li, Taiwu

    2014-06-01

    Skin ulceration syndrome (SUS) was the main limitation in the development of Apostichopus japonicus culture industries. To better understand how Vibrio splendidus modulates SUS outbreak, the immune response of A. japonicus coelomocytes after the pathogen challenge were investigated through comparative proteomics approach, and differentially expressed proteins were screened and characterized in the present study. A total of 40 protein spots representing 30 entries were identified at 24, 72 and 96 h post-infection. Of these proteins, 32 were up-regulated and 8 were down-regulated in the V. splendidus challenged samples compared to those of control. These differentially expressed proteins were mainly classified into four categories by GO analysis, in which approximate 33% of proteins showed to be related to immunity response. The mRNA expression levels of 6 differentially expressed proteins were further validated by qRT-PCR. Similar protein-mRNA-level expression patterns were detected in genes of phospholipase (spot 4), G protein (spot 20), annexin (spot 30) and filamin (spot 31). Whilst the levels of ficolin (spot 12) and calumenin (spot 14) transcripts were not corresponded with those of their translation products. These data provide a new insight to understand the molecular immune mechanism of sea cucumber responsive towards pathogen infection. PMID:24468075

  15. 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

  16. Proteomic analysis of differentially expressed proteins induced by salicylic acid in suspension-cultured ginseng cells

    PubMed Central

    Sun, Jiaman; Fu, Junfan; Zhou, Rujun

    2013-01-01

    In this study, optimized 2-DE sample preparation methodologies were established for suspension-cultured ginseng cells. Three commonly used protein extraction methods (Trichloroacetic acid-acetone, urea/thiourea and phenol extraction method) were evaluated for proteomic analysis of suspension cultures of ginseng. A comparative analysis of suspension-cultured ginseng cells proteome induced by salicylic acid (SA) was reported. The results demonstrated that phenol extraction method was the best method based on protein extraction efficiency and the good quality of 2-DE patterns for suspension-cultured ginseng cells. Fifteen differentially expressed proteins induced by salicylic acid in suspension-cultured ginseng cells were identified by MALDI-TOF-MS. These identified proteins were involved in defense and stress response, energy metabolism, signal transduction/transcription, protein synthesis and metabolism, and photosynthesis. Chaperonin 60, related to defense responses, was more abundant in suspension-cultured ginseng cells after application of SA. Vacuolar ATPase subunit B was newly induced in SA treatment. PMID:24600313

  17. Bioinformatics analysis of differentially expressed proteins in prostate cancer based on proteomics data

    PubMed Central

    Chen, Chen; Zhang, Li-Guo; Liu, Jian; Han, Hui; Chen, Ning; Yao, An-Liang; Kang, Shao-San; Gao, Wei-Xing; Shen, Hong; Zhang, Long-Jun; Li, Ya-Peng; Cao, Feng-Hong; Li, Zhi-Guo

    2016-01-01

    We mined the literature for proteomics data to examine the occurrence and metastasis of prostate cancer (PCa) through a bioinformatics analysis. We divided the differentially expressed proteins (DEPs) into two groups: the group consisting of PCa and benign tissues (P&b) and the group presenting both high and low PCa metastatic tendencies (H&L). In the P&b group, we found 320 DEPs, 20 of which were reported more than three times, and DES was the most commonly reported. Among these DEPs, the expression levels of FGG, GSN, SERPINC1, TPM1, and TUBB4B have not yet been correlated with PCa. In the H&L group, we identified 353 DEPs, 13 of which were reported more than three times. Among these DEPs, MDH2 and MYH9 have not yet been correlated with PCa metastasis. We further confirmed that DES was differentially expressed between 30 cancer and 30 benign tissues. In addition, DEPs associated with protein transport, regulation of actin cytoskeleton, and the extracellular matrix (ECM)–receptor interaction pathway were prevalent in the H&L group and have not yet been studied in detail in this context. Proteins related to homeostasis, the wound-healing response, focal adhesions, and the complement and coagulation pathways were overrepresented in both groups. Our findings suggest that the repeatedly reported DEPs in the two groups may function as potential biomarkers for detecting PCa and predicting its aggressiveness. Furthermore, the implicated biological processes and signaling pathways may help elucidate the molecular mechanisms of PCa carcinogenesis and metastasis and provide new targets for clinical treatment. PMID:27051295

  18. Proteomic Identification of Differentially Expressed Proteins between Male and Female Plants in Pistacia chinensis

    PubMed Central

    Shi, Jiang; Wang, Xiaoyan; Wang, Wei

    2013-01-01

    Pistacia chinensis is a strict dioecious plant with male and female flowers in individuals. In China, P. chinensis is widely planted for biodiesel oil due to high oil content in seeds. In practice it requires to grow more female plants for biodiesel production. At present, there are still no reliable methods for sex determination during the long juvenile stage of this species. In order to develop protein molecular markers for sex determination in P. chinensis, proteomic approach was used to identify differentially expressed proteins between male and female plants. Vegetative organs (leaf and stem) rather than reproductive organs/tissues were used for protein extraction so as to develop protein markers which can be used in siblings before flowering. Protein was extracted using a phenol-based protocol. By using two-dimensional electrophoresis, a total of 10 protein spots were found to be differentially expressed in leaf and stem between both sexes, of which 7 were successfully identified by mass spectrometry and matched to 6 functional proteins such as NB-ARC domain containing protein, light harvesting chlorophyll a/b binding protein, asorbate peroxidase (APX), eukaryotic translation initiation factor 5A2, temperature-induced lipocalin (TIL) and phosphoglycerate kinase (PGK). The sex-related difference displayed in a tissue-specific way, especially in stem. PGK existed in high abundance in stem phloem in the female, but was almost not detected in the male; APX and two TIL species were highly abundant in the stem of male plants, while their abundance was much lower in female plants. Moreover, these abundance differences were further confirmed in individual plants. Hence, it is assumed that APX, PGK and TIL might be promising candidates to serve as protein molecular markers for sex determination in P. chinensis. Our results form the basis for a further understanding of the biochemical mechanisms of sex determination in P. chinensis. PMID:23691188

  19. Proteomic identification of differentially expressed proteins between male and female plants in Pistacia chinensis.

    PubMed

    Xiong, Erhui; Wu, Xiaolin; Shi, Jiang; Wang, Xiaoyan; Wang, Wei

    2013-01-01

    Pistacia chinensis is a strict dioecious plant with male and female flowers in individuals. In China, P. chinensis is widely planted for biodiesel oil due to high oil content in seeds. In practice it requires to grow more female plants for biodiesel production. At present, there are still no reliable methods for sex determination during the long juvenile stage of this species. In order to develop protein molecular markers for sex determination in P. chinensis, proteomic approach was used to identify differentially expressed proteins between male and female plants. Vegetative organs (leaf and stem) rather than reproductive organs/tissues were used for protein extraction so as to develop protein markers which can be used in siblings before flowering. Protein was extracted using a phenol-based protocol. By using two-dimensional electrophoresis, a total of 10 protein spots were found to be differentially expressed in leaf and stem between both sexes, of which 7 were successfully identified by mass spectrometry and matched to 6 functional proteins such as NB-ARC domain containing protein, light harvesting chlorophyll a/b binding protein, asorbate peroxidase (APX), eukaryotic translation initiation factor 5A2, temperature-induced lipocalin (TIL) and phosphoglycerate kinase (PGK). The sex-related difference displayed in a tissue-specific way, especially in stem. PGK existed in high abundance in stem phloem in the female, but was almost not detected in the male; APX and two TIL species were highly abundant in the stem of male plants, while their abundance was much lower in female plants. Moreover, these abundance differences were further confirmed in individual plants. Hence, it is assumed that APX, PGK and TIL might be promising candidates to serve as protein molecular markers for sex determination in P. chinensis. Our results form the basis for a further understanding of the biochemical mechanisms of sex determination in P. chinensis. PMID:23691188

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  1. The cassava (Manihot esculenta Crantz) root proteome: protein identification and differential expression.

    PubMed

    Sheffield, Jeanne; Taylor, Nigel; Fauquet, Claude; Chen, Sixue

    2006-03-01

    Using high-resolution 2-DE, we resolved proteins extracted from fibrous and tuberous root tissues of 3-month-old cassava plants. Gel image analysis revealed an average of 1467 electrophoretically resolved spots on the fibrous gels and 1595 spots on the tuberous gels in pH 3-10 range. Protein spots from both sets of gels were digested with trypsin. The digests were subjected to nanoelectrospray quadrupole TOF tandem mass analysis. Currently, we have obtained 299 protein identifications for 292 gel spots corresponding to 237 proteins. The proteins span various functional categories from energy, primary and secondary metabolism, disease and defense, destination and storage, transport, signal transduction, protein synthesis, cell structure, and transcription to cell growth and division. Gel image analysis has shown unique, as well as up- and down-regulated proteins, present in the tuberous and the fibrous tissues. Quantitative and qualitative analysis of the cassava root proteome is an important step towards further characterization of differentially expressed proteins and the elucidation of the mechanisms underlying the development and biological functions of the two types of roots. PMID:16421938

  2. 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. PMID:19662628

  3. Differential proteomic expression of human placenta and fetal development following e-waste lead and cadmium exposure in utero.

    PubMed

    Xu, Long; Ge, Jingjing; Huo, Xia; Zhang, Yuling; Lau, Andy T Y; Xu, Xijin

    2016-04-15

    Prenatal exposure to lead (Pb) and cadmium (Cd) has been associated with a series of physiological problems resulting in fetal growth restriction. We aimed to investigate the effects of Pb and Cd exposure on placental function and the potential mechanisms involved in fetal development. Placental specimens and questionnaires were collected from an e-waste area and a reference area in China. Two-dimensional electrophoresis combined with MALDI-TOF-MS/MS and molecular network relationship were performed to analyze differentially expressed proteins using a compositing sample pool. Compared with the reference group, the exposed group exhibited significantly higher levels of placental Pb and Cd (p<0.01), shorter body length and higher gestational age (p<0.01). After bivariate adjustment in a linear regression model, decreases of 205.05g in weight and 0.44cm in body length were associated with a 10ng/g wt increase in placental Cd. Pb showed a negative trend but lacked statistical significance. Proteomic analysis showed 32 differentially-expressed proteins and were predominantly involved in protein translocation, cytoskeletal structure, and energy metabolism. Fumarate hydratase was down-regulated in the exposed placenta tissues and validated by ELISA. Alterations in placental proteome suggest that imbalances in placental mitochondria respiration might be a vital pathway targeting fetal growth restriction induced by exposure to Cd. PMID:26895036

  4. 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

  5. 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

  6. Microsomal membrane proteome of low grade diffuse astrocytomas: Differentially expressed proteins and candidate surveillance biomarkers

    PubMed Central

    Polisetty, Ravindra Varma; Gautam, Poonam; Gupta, Manoj Kumar; Sharma, Rakesh; Gowda, Harsha; Renu, Durairaj; Shivakumar, Bhadravathi Marigowda; Lakshmikantha, Akhila; Mariswamappa, Kiran; Ankathi, Praveen; Purohit, Aniruddh K.; Uppin, Megha S.; Sundaram, Challa; Sirdeshmukh, Ravi

    2016-01-01

    Diffuse astrocytoma (DA; WHO grade II) is a low-grade, primary brain neoplasm with high potential of recurrence as higher grade malignant form. We have analyzed differentially expressed membrane proteins from these tumors, using high-resolution mass spectrometry. A total of 2803 proteins were identified, 340 of them differentially expressed with minimum of 2 fold change and based on ≥2 unique peptides. Bioinformatics analysis of this dataset also revealed important molecular networks and pathways relevant to tumorigenesis, mTOR signaling pathway being a major pathway identified. Comparison of 340 differentially expressed proteins with the transcript data from Grade II diffuse astrocytomas reported earlier, revealed about 190 of the proteins correlate in their trends in expression. Considering progressive and recurrent nature of these tumors, we have mapped the differentially expressed proteins for their secretory potential, integrated the resulting list with similar list of proteins from anaplastic astrocytoma (WHO Grade III) tumors and provide a panel of proteins along with their proteotypic peptides, as a resource that would be useful for investigation as circulatory plasma markers for post-treatment surveillance of DA patients. PMID:27246909

  7. Microsomal membrane proteome of low grade diffuse astrocytomas: Differentially expressed proteins and candidate surveillance biomarkers.

    PubMed

    Polisetty, Ravindra Varma; Gautam, Poonam; Gupta, Manoj Kumar; Sharma, Rakesh; Gowda, Harsha; Renu, Durairaj; Shivakumar, Bhadravathi Marigowda; Lakshmikantha, Akhila; Mariswamappa, Kiran; Ankathi, Praveen; Purohit, Aniruddh K; Uppin, Megha S; Sundaram, Challa; Sirdeshmukh, Ravi

    2016-01-01

    Diffuse astrocytoma (DA; WHO grade II) is a low-grade, primary brain neoplasm with high potential of recurrence as higher grade malignant form. We have analyzed differentially expressed membrane proteins from these tumors, using high-resolution mass spectrometry. A total of 2803 proteins were identified, 340 of them differentially expressed with minimum of 2 fold change and based on ≥2 unique peptides. Bioinformatics analysis of this dataset also revealed important molecular networks and pathways relevant to tumorigenesis, mTOR signaling pathway being a major pathway identified. Comparison of 340 differentially expressed proteins with the transcript data from Grade II diffuse astrocytomas reported earlier, revealed about 190 of the proteins correlate in their trends in expression. Considering progressive and recurrent nature of these tumors, we have mapped the differentially expressed proteins for their secretory potential, integrated the resulting list with similar list of proteins from anaplastic astrocytoma (WHO Grade III) tumors and provide a panel of proteins along with their proteotypic peptides, as a resource that would be useful for investigation as circulatory plasma markers for post-treatment surveillance of DA patients. PMID:27246909

  8. Proteomic Analysis of Human Brain Microvascular Endothelial Cells Reveals Differential Protein Expression in Response to Enterovirus 71 Infection

    PubMed Central

    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. PMID:25821824

  9. Differential Expression of Durum Wheat Gluten Proteome under Water Stress during Grain Filling.

    PubMed

    Giuliani, Marcella Michela; Palermo, Carmen; De Santis, Michele Andrea; Mentana, Annalisa; Pompa, Marianna; Giuzio, Luigia; Masci, Stefania; Centonze, Diego; Flagella, Zina

    2015-07-29

    Environmental stress during grain filling may affect wheat protein composition, thus influencing its final quality. A proteomic approach was used to evaluate changes in storage protein composition under water stress of two Italian durum wheat (Triticum turgidum ssp. durum) cultivars, Ciccio and Svevo. The high-molecular-weight glutenin region increased progressively in both cultivars and under two water regimens. The L48-35 region, corresponding to low-molecular-weight (LMW) glutenin subunits, increased slightly during grain development and decreased under water stress in both cultivars. In particular, an s-type LMW related to superior technological quality was down-expressed in the early-mid period in Svevo and in the mid-late period in Ciccio. Finally, the L<35 region, corresponding to gliadin-like proteins, decreased slightly during grain development and increased under stress in both cultivars. Several α-gliadins, associated with immunological potential, increased their expression under water stress, especially in Svevo in the early-mid stage of grain filling. PMID:26138860

  10. 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

  11. Differential proteome and gene expression reveal response to carbon ion irradiation in pubertal mice testes.

    PubMed

    Li, Hongyan; He, Yuxuan; Zhang, Hong; Miao, Guoying

    2014-03-21

    Heavy ion radiation, a high linear energy transfer (LET) radiation, has been shown to have adverse effects on reproduction in male mice. The aim of this study was to profile and investigate the differentially expressed proteins in pubertal male mice testes following carbon ion radiation (CIR). Male mice underwent whole-body irradiation with CIR (1 and 4 Gy), and MALDI-TOF/TOF analysis was used to investigate the alteration in protein expression in 2-DE (two-dimensional gel electrophoresis) gels of testes caused by irradiation after 14 days. 8 differentially expressed proteins 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. Our results indicated 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. PMID:24440814

  12. Identification of differentially expressed proteins from primary versus metastatic pancreatic cancer cells using subcellular proteomics.

    PubMed

    McKinney, Kimberly Q; Lee, Jin-Gyun; Sindram, David; Russo, Mark W; Han, David K; Bonkovsky, Herbert L; Hwang, Sun-Il

    2012-01-01

    Pancreatic cancer is an aggressive disease with nearly equal yearly rates of diagnosis and death. Current therapies have failed to improve outcomes due to rapid disease progression and late stage at presentation. Recently, pathways involved in progression and metastasis have been elucidated; however, new knowledge has not generated more effective therapies. We report on the use of subcellular fractionation and liquid chromatography (LC)-mass spectrometry to identify 3,907 proteins in four pancreatic cancer cell lines, 540 of which are unique to primary cancer cells, and 487 unique to cells derived from metastatic sites. Statistical analysis identified 134 proteins significantly differentially expressed between the two populations. The subcellular localization of these proteins was determined and expression levels for four targets were validated using western blot techniques. These identified proteins can be further investigated to determine their roles in progression and metastasis and may serve as therapeutic targets in the development of more effective treatments for pancreatic cancer. PMID:22990105

  13. 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.

  14. 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. PMID:27303420

  15. 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

  16. The differentially-expressed proteome in Zn/Cd hyperaccumulator Arabis paniculata Franch. in response to Zn and Cd.

    PubMed

    Zeng, Xiao-Wen; Qiu, Rong-Liang; Ying, Rong-Rong; Tang, Ye-Tao; Tang, Lu; Fang, Xiao-Hang

    2011-01-01

    The Zn/Cd hyperaccumulator Arabis paniculata is able to tolerate high level of Zn and Cd. To clarify the molecular basis of Zn and Cd tolerance, proteomic approaches were applied to identify proteins involved in Zn and Cd stress response in A. paniculata. Plants were exposed to both low and high Zn or Cd levels for 10 d. Proteins of leaves in each treatment were separated by 2-DE (two-dimensional electrophoresis). Nineteen differentially-expressed proteins upon Zn treatments and 18 proteins upon Cd treatments were observed. Seventeen out of 19 of Zn-responsive proteins and 16 out of 18 of Cd-responsive proteins were identified using MALDI-TOF/TOF-MS (matrix-assisted laser desorption/ionization time of flight mass spectrometry). The most of identified proteins were known to function in energy metabolism, xenobiotic/antioxidant defense, cellular metabolism, protein metabolism, suggesting the responses of A. paniculata to Zn and Cd share similar pathway to certain extend. However, the different metal defense was also revealed between Zn and Cd treatment in A. paniculata. These results indicated that A. paniculata against to Zn stress mainly by enhancement of energy metabolism including auxin biosynthesis and protein metabolism to maintain plant growth and correct misfolded proteins. In the case of Cd, plants adopted antioxidative/xenobiotic defense and cellular metabolism to keep cellular redox homeostasis and metal-transportation under Cd stress. PMID:21074242

  17. Proteomics beyond large-scale protein expression analysis.

    PubMed

    Boersema, Paul J; Kahraman, Abdullah; Picotti, Paola

    2015-08-01

    Proteomics is commonly referred to as the application of high-throughput approaches to protein expression analysis. Typical results of proteomics studies are inventories of the protein content of a sample or lists of differentially expressed proteins across multiple conditions. Recently, however, an explosion of novel proteomics workflows has significantly expanded proteomics beyond the analysis of protein expression. Targeted proteomics methods, for example, enable the analysis of the fine dynamics of protein systems, such as a specific pathway or a network of interacting proteins, and the determination of protein complex stoichiometries. Structural proteomics tools allow extraction of restraints for structural modeling and identification of structurally altered proteins on a proteome-wide scale. Other variations of the proteomic workflow can be applied to the large-scale analysis of protein activity, location, degradation and turnover. These exciting developments provide new tools for multi-level 'omics' analysis and for the modeling of biological networks in the context of systems biology studies. PMID:25636126

  18. 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

  19. Tissue Proteomics Reveals Differential and Compartment-Specific Expression of the Homologs Transgelin and Transgelin-2 in Lung Adenocarcinoma and Its Stroma

    PubMed Central

    Rho, Jung-hyun; Roehrl, Michael H. A.; Wang, Julia Y.

    2009-01-01

    Discovery of tissue-specific biomarkers for human cancer is crucial for early diagnosis and molecular understanding of the disease. To overcome the limitations posed by the large dynamic concentration range and compositional complexity of tissue biomacromolecules, we applied heparin affinity fractionation for proteomic enrichment. Comparing the proteomes of five paired samples of normal lung and pulmonary adenocarcinoma tissue by 2-D difference gel electrophoresis, 14 spots were found to be differentially expressed. From these candidate spots, three proteins overexpressed in cancer were identified by mass spectrometry as transgelin (TAGLN, SM22-α, WS3-10), transgelin-2 (TAGLN2), and cyclophilin A (PPIA). Quantitative RT-PCR indicated that both TAGLN2 and PPIA were upregulated at transcriptional level. Differential protein expression levels were validated by Western blot analysis using an independent set of 10 paired lung adenocarcinoma samples. Using immunohistochemistry on human tissue sections, we discovered that overexpression of TAGLN was strictly localized to the tumor-induced reactive myofibroblastic stromal tissue compartment, whereas overexpression of TAGLN2 was exclusively localized to the neoplastic glandular compartment. Thus, the highly homologous protein pair TAGLN and TAGLN2 displayed mutually exclusive, compartment-specific cell type expression regulation in tumor stroma vs. neoplastic epithelial cells. Our data further suggest that TGLN may be a marker of active stromal remodeling in the vicinity of invasive carcinomas. It may shed light on mechanisms of tumor-stroma interaction and could be useful for early diagnosis, treatment guidance, and treatment response monitoring. PMID:19848416

  20. RegStatGel: proteomic software for identifying differentially expressed proteins based on 2D gel images

    PubMed Central

    Li, Feng; Seillier-Moiseiwitsch, Françoise

    2011-01-01

    Image analysis of two-dimensional gel electrophoresis is a key step in proteomic workflow for identifying proteins that change under different experimental conditions. Since there are usually large amount of proteins and variations shown in the gel images, the use of software for analysis of 2D gel images is inevitable. We developed open-source software with graphical user interface for differential analysis of 2D gel images. The user-friendly software, RegStatGel, contains fully automated as well as interactive procedures. It was developed and has been tested under Matlab 7.01. Availability The database is available for free at http://www.mediafire.com/FengLi/2DGelsoftware PMID:21904427

  1. 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.

  2. A Pipeline for Differential Proteomics in Unsequenced Species.

    PubMed

    Yılmaz, Şule; Victor, Bjorn; Hulstaert, Niels; Vandermarliere, Elien; Barsnes, Harald; Degroeve, Sven; Gupta, Surya; Sticker, Adriaan; Gabriël, Sarah; Dorny, Pierre; Palmblad, Magnus; Martens, Lennart

    2016-06-01

    Shotgun proteomics experiments often take the form of a differential analysis, where two or more samples are compared against each other. The objective is to identify proteins that are either unique to a specific sample or a set of samples (qualitative differential proteomics), or that are significantly differentially expressed in one or more samples (quantitative differential proteomics). However, the success depends on the availability of a reliable protein sequence database for each sample. To perform such an analysis in the absence of a database, we here propose a novel, generic pipeline comprising an adapted spectral similarity score derived from database search algorithms that compares samples at the spectrum level to detect unique spectra. We applied our pipeline to compare two parasitic tapeworms: Taenia solium and Taenia hydatigena, of which only the former poses a threat to humans. Furthermore, because the genome of T. solium recently became available, we were able to prove the effectiveness and reliability of our pipeline a posteriori. PMID:27089233

  3. Proteomic analysis of differentially expressed proteins in the lymphoid organ of Vibrio harveyi-infected Penaeus monodon.

    PubMed

    Chaikeeratisak, Vorrapon; Somboonwiwat, Kunlaya; Wang, Hao-Ching; Lo, Chu Fang; Tassanakajon, Anchalee

    2012-05-01

    The protein expression profiles of the lymphoid organ, taken from mock and systemic Vibrio harveyi-infected Penaeus monodon at 6 and 48 h post infection, were revealed. The considerable changes in the expression level of several proteins were observed between the mock and V. harveyi-infected shrimps. From 30 analyzed protein spots with 27 differentially expressed, 21 were known proteins with the most common of these being cytoskeleton proteins (33%) which were all down-regulated upon systemic bacterial infection. Other six proteins including four proteins that are involved in the shrimp immunity (alpha-2-macroglobulin, transglutaminase, heat shock protein 1 and hemocyanin subunit Y), and two proteins that are involved in metabolism (triosephosphate isomerase) and cell signaling (14-3-3 like protein), displayed significantly decreased expression levels. There was, however, an increase in the expression level of the ATP synthase beta subunit, a protein involved in energy balance. Transcription levels of ATP synthase beta subunit and 14-3-3 like protein were up- and down-regulated, respectively, in accord with the observed protein expression levels, but the alpha-2-macroglobulin transcript levels were significantly increased in contrast to the decreased protein expression levels. Interestingly, partial gene silencing of ATP synthase beta subunit revealed a high cumulative mortality of the knockdown shrimps (73.3%) and a dramatic reduction of the total hemocyte numbers in the survival shrimps. These altered proteins are likely to play essential roles in shrimp defense against the pathogenic bacterium V. harveyi. PMID:22302389

  4. Comparative proteomics reveals highly and differentially expressed proteins in field-collected and laboratory-cultured blooming cells of the diatom Skeletonema costatum.

    PubMed

    Zhang, Hao; Wang, Da-Zhi; Xie, Zhang-Xian; Zhang, Shu-Fei; Wang, Ming-Hua; Lin, Lin

    2015-10-01

    Diatoms are a major phytoplankton group causing extensive blooms in the ocean. However, little is known about the intracellular biological processes occurring during the blooming period. This study compared the protein profiles of field-collected and laboratory-cultured blooming cells of Skeletonema costatum, and identified highly and differentially expressed proteins using the shotgun proteomic approach. A total of 1372 proteins were confidently identified with two or more peptides. Among them, 222 and 311 proteins were unique to the laboratory and field samples respectively. Proteins involved in photosynthesis, translation, nucleosome assembly, carbohydrate and energy metabolism dominated the protein profiles in both samples. However, different features of specific proteins were also found: proteins participated in light harvesting, photosynthetic pigment biosynthesis, photoprotection, cell division and redox homeostasis were highly detected in the field sample, whereas proteins involved in translation, amino acid and protein metabolic processes, and nitrogen and carbon assimilation presented high detection rates in the laboratory sample. ATP synthase cf1 subunit beta and light harvest complex protein were the most abundant protein in the laboratory and field samples respectively. These results indicated that S. costatum had evolved adaptive mechanisms to the changing environment, and integrating field and laboratory proteomic data should provide comprehensive understanding of bloom mechanisms. PMID:26014042

  5. 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

  6. 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

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

    2015-09-01

    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

  7. Differential expression of proteins in naïve and IL-2 stimulated primary human NK cells identified by global proteomic analysis

    PubMed Central

    Ma, Di; Felder, Mildred; Scarlett, Cameron O.; Patankar, Manish S.; Li, Lingjun

    2015-01-01

    Natural killer (NK) cells efficiently cytolyse tumors and virally infected cells. Despite the important role that interleukin (IL)-2 plays in stimulating the proliferation of NK cells and increasing NK cell activity, little is known about the alterations in the global NK cell proteome following IL-2 activation. To compare the proteomes of naïve and IL-2-activated primary NK cells and identify key cellular pathways involved in IL-2 signaling, we isolated proteins from naïve and IL-2-activated NK cells from healthy donors, the proteins were trypsinized and the resulting peptides were analyzed by 2D LC ESI-MS/MS followed by label-free quantification. In total, more than 2000 proteins were identified from naïve and IL-2-activated NK cells where 383 proteins were found to be differentially expressed following IL-2 activation. Functional annotation of IL-2 regulated proteins revealed potential targets for future investigation of IL-2 signaling in human primary NK cells. A pathway analysis was performed and revealed several pathways that were not previously known to be involved in IL-2 response, including ubiquitin proteasome pathway, integrin signaling pathway, platelet derived growth factor (PDGF) signaling pathway, epidermal growth factor receptor (EGFR) signaling pathway and Wnt signaling pathway. PMID:23806757

  8. 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.

    2006-01-02

    While genomic approaches have been applied to the detection and identification of individual bacteria within microbial communities, analogous proteomics approaches have been effectively precluded due to the inherent complexity. An in silico assessment of peptides derived from artificial simple and complex communities was performed to evaluate the effect of proteome complexity on species detection. Detection and validation of predicted peptides initially identified as distinctive within the simple community was experimentally performed using a mass spectrometry-based proteomics approach. 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 assessment performed in silico of peptide distinctiveness for a complex community of 25 microorganisms was also conducted. The experimental data for a simple community, and the in silico data for a complex community revealed that it is feasible to predict, observe, and quantify distinctive peptides from one organism in the presence of at least a 100-fold greater abundance of another, thus yielding putative markers for the identification of a bacterium of interest. This work represents a first step towards quantitative proteomic characterization of complex microbial communities.

  9. Erratum to “Proteomic analysis of differential protein expression in Acidithiobacillus ferrooxidans cultivated in high potassium concentration” [Microbiol. Res. 168 (7) (2013) 455–460].

    PubMed

    Ouyang, Jianping; Guo, Wenbin; Li, Bo; Gu, Li; Zhang, Huijun; Xinhua Chen, Huijun

    2016-01-01

    Acidithiobacillus ferrooxidans is a chemolithoautotrophic acidophile that oxidizes ferrous iron or sulfur compounds to obtain energy in the presence of various ions. To investigate the potassium ion response of A. ferrooxidans, we conducted a proteomics analysis. We identified eight proteins that were differentially expressed in the presence of high potassium concentration, including four up-regulated and four down-regulated proteins. Transcription levels of the genes encoding differential expressed proteins were subsequently analyzed by Northern blot in the presence of high potassium concentration. Among the up-regulated proteins, GDP-mannose 4,6-dehydratase, ribose 5-phosphate isomerase A and ribose-phosphate pyrophosphokinase were known to be implicated in the synthesis of glycocalyx, suggesting that the formation of glycocalyx might be involved in the A. ferrooxidans response to high potassium concentration. Thickening of the glycocalyx layer was also observed in cells cultivated under high potassium concentration via transmission electronic microscopy (TEM) analysis. Among the down-regulated proteins, ATP synthase F1 delta subunit and ATP synthase F1 beta subunit were two important components of ATP synthase. ATP synthase (P-ATPase) is directly linked to the transport of potassium into the cell, thus Acidithiobacillus ferrooxidans might just reduce the quantity of ATP synthase to offset the high potassium level in the culture medium. Therefore, the results obtained here provide some new clues to improve our understanding of the response of A. ferrooxidans to high potassium concentration. PMID:27062771

  10. Gene-Expression Novelty in Allopolyploid Cotton: A Proteomic Perspective

    PubMed Central

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

    2015-01-01

    Allopolyploidization is accompanied by changes in gene expression that are thought to contribute to phenotypic diversification. Here we describe global changes in the single-celled cotton fiber proteome of two natural allopolyploid species (Gossypium hirsutum and G. barbadense) and living models of their diploid parents using two different proteomic approaches. In total, 1323 two-dimensional gel electrophoresis spots and 1652 identified proteins by isobaric tags for relative and absolute quantitation were quantitatively profiled during fiber elongation. Between allopolyploids and their diploid A- and D-genome progenitors, amounts of differential expression ranged from 4.4 to 12.8%. Over 80% of the allopolyploid proteome was additively expressed with respect to progenitor diploids. Interestingly, the fiber proteome of G. hirsutum resembles the parental A-genome more closely, where long, spinable fiber first evolved, than does the fiber proteome of G. barbadense. More protein expression patterns were A-dominant than D-dominant in G. hirsutum, but in G. barbadense, the direction of expression-level dominance switched from the D-genome to the A-genome during fiber development. Comparison of developmental changes between the two allopolyploid species revealed a high level of proteomic differentiation despite their shared ancestry, relatively recent evolutionary divergence, and similar gross morphology. These results suggest that the two allopolyploid species have achieved superficially similar modern fiber phenotypes through different evolutionary routes at the proteome level. We also detected homeolog-specific expression for 1001 proteins and present a novel approach to infer the relationship between homeolog-specific and duplicate expression patterns. Our study provides a proteomic perspective on understanding evolutionary consequences of allopolyploidization, showing how protein expression has been altered by polyploidization and subsequently has diversified among

  11. 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

  12. Proteomic identification of differentially expressed genes in neural stem cells and neurons differentiated from embryonic stem cells of cynomolgus monkey (Macaca fascicularis) in vitro.

    PubMed

    Akama, Kuniko; Horikoshi, Tomoe; Nakayama, Takashi; Otsu, Masahiro; Imaizumi, Noriaki; Nakamura, Megumi; Toda, Tosifusa; Inuma, Michiko; Hirano, Hisashi; Kondo, Yasushi; Suzuki, Yutaka; Inoue, Nobuo

    2011-02-01

    Understanding neurogenesis is valuable for the treatment of nervous system disorders. However, there is currently limited information about the molecular events associated with the transition from primate ES cells to neural cells. We therefore sought to identify the proteins involved in neurogenesis, from Macaca fascicularis ES cells (CMK6 cell line) to neural stem (NS) cells to neurons using two-dimensional gel electrophoresis (2-DE), peptide mass fingerprinting (PMF), and liquid chromatography-tandem mass spectrometry (LC-MS-MS). During the differentiation of highly homogeneous ES cells to NS cells, we identified 17 proteins with increased expression, including fatty acid binding protein 7 (FABP7), collapsin response mediator protein 2 (CRMP2), and cellular retinoic acid binding protein 1 (CRABP1), and seven proteins with decreased expression. In the differentiation of NS cells to neurons, we identified three proteins with increased expression, including CRMP2, and 10 proteins with decreased expression. Of these proteins, FABP7 is a marker of NS cells, CRMP2 is involved in axon guidance, and CRABP1 is thought to regulate retinoic acid access to its nuclear receptors. Western blot analysis confirmed the upregulation of FABP7 and CRABP1 in NS cells, and the upregulation of CRMP2 in NS cells and neurons. RT-PCR results showed that CRMP2 and FABP7 mRNAs were also upregulated in NS cells, while CRABP1 mRNA was unchanged. These results provide insight into the molecular basis of monkey neural differentiation. PMID:21047566

  13. 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

  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. 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. PMID:26528309

  16. Proteome and Differential Expression Analysis of Membrane and Cytosolic Proteins from Mycobacterium avium subsp. paratuberculosis Strains K-10 and 187.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Little is known of the protein expression in Mycobacterium avium subspecies paratuberculosis (MAP) and how this contributes to pathogenesis. In the present study, proteins from both outer membranes and cytosol were prepared from two strains of MAP; a laboratory-adapted strain K-10 and a recent isola...

  17. 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.

  18. Proteomic analysis and differential expression in protein extracted from chicken with a varying growth rate and water-holding capacity.

    PubMed

    Phongpa-Ngan, Phodchanee; Grider, Arthur; Mulligan, Jake H; Aggrey, Samuel E; Wicker, Louise

    2011-12-28

    Chickens from a randomly bred genetic line were segregated into high and low growth rates and high and low water-holding capacities (WHCs). The objective of this study was to identify protein markers associated with slow and fast growth rates and low and high WHCs from water-soluble protein (WSP) and crude myofibrillar protein (CMP) extracts of chicken breast muscle. Proteins were fractionated using two-dimensional electrophoresis, and a total of 22 protein spots were selected, excised, and analyzed by in-gel tryptic digestion and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Proteins expressed in extracts from slow and fast growth rates and low and high WHCs included metabolic enzymes, such as creatine kinase, pyruvate kinase, triosephosphate isomerase, and ubiqitin; housekeeping proteins, such as heat shock protein; contractile proteins, such as myosin heavy chain; actin; and also MHC isoforms and actin isoforms. The mass spectra of 20 protein spots significantly matched (protein score >83; P < 0.05) an online database. In CMP, there were unique proteins that were present only in the fast-growth population: gi|118099530 , gi|20664362 , gi|71895043 , gi|114794125 , gi|297343122 , and gi|71895043 . This information identified protein markers associated with growth rate and water holding capacity. Some of those protein markers could be added to the chicken database. PMID:22010637

  19. Proteomics for Protein Expression Profiling in Neuroscience*

    PubMed Central

    Freeman, Willard M.; Hemby, Scott E.

    2013-01-01

    As the technology of proteomics moves from a theoretical approach to a practical reality, neuroscientists will have to determine the most appropriate applications for this technology. Neuroscientists will have to surmount difficulties particular to their research, such as limited sample amounts, heterogeneous cellular compositions in samples, and the fact that many proteins of interest are rare, hydrophobic proteins. This review examines protein isolation and protein fractionation and separation using two-dimensional electrophoresis (2-DE) and mass spectrometry proteomic methods. Methods for quantifying relative protein expression between samples (e.g., 2-DIGE, and ICAT) are also described. The coverage of the proteome, ability to detect membrane proteins, resource requirements, and quantitative reliability of different approaches is also discussed. Although there are many challenges in proteomic neuroscience, this field promises many rewards in the future. PMID:15176464

  20. Improving the TFold test for differential shotgun proteomics

    PubMed Central

    Carvalho, Paulo C.; Yates, John R.; Barbosa, Valmir C.

    2012-01-01

    Summary: We present an updated version of the TFold software for pinpointing differentially expressed proteins in shotgun proteomics experiments. Given an FDR bound, the updated approach uses a theoretical FDR estimator to maximize the number of identifications that satisfy both a fold-change cutoff that varies with the t-test P-value as a power law and a stringency criterion that aims to detect lowly abundant proteins. The new version has yielded significant improvements in sensitivity over the previous one. Availability: Freely available for academic use at http://pcarvalho.com/patternlab. Contact: paulo@pcarvalho.com PMID:22539673

  1. Proteome and Differential Expression Analysis of Membrane and Cytosolic Proteins from Mycobacterium avium subsp. paratuberculosis Strains K-10 and 187▿†

    PubMed Central

    Radosevich, Thomas J.; Reinhardt, Timothy A.; Lippolis, John D.; Bannantine, John P.; Stabel, Judith R.

    2007-01-01

    Little is known of protein expression in Mycobacterium avium subsp. paratuberculosis and how this contributes to pathogenesis. In the present study, proteins from both membranes and cytosol were prepared from two strains of M. avium subsp. paratuberculosis, i.e., laboratory-adapted strain K-10 and a recent isolate, strain 187, obtained from a cow exhibiting clinical signs of Johne's disease. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cytosol and membrane proteins from K-10 and 187 showed marked differences in protein expression. Relative levels of protein expression from both M. avium subsp. paratuberculosis strains were measured by using amine-reactive isobaric tagging reagents (iTRAQ) and tandem mass spectroscopy. Protein identification and relative expression data were obtained for 874 membrane and cytosolic proteins from the M. avium subsp. paratuberculosis proteome. These data showed a number of significant differences in protein expression between strain K-10 and clinical isolate 187. Examples of proteins expressed at higher levels in clinical isolate 187 compared to strain K-10 are AtpC, RpoA, and several proteins involved in fatty acid biosynthesis. In contrast, proteins such as AhpC and several proteins involved in nitrogen metabolism were expressed at higher levels in strain K-10 compared to strain 187. These data may provide insights into the proteins whose expression is important in natural infection but are modified once M. avium subsp. paratuberculosis is adapted to laboratory cultivation. Results from these studies will provide tools for developing a better understanding of M. avium subsp. paratuberculosis infection in the host and offer potential as diagnostic reagents and vaccine candidates. PMID:17142399

  2. 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

  3. Differential proteome analysis of replicative senescence in rat embryo fibroblasts.

    PubMed

    Benvenuti, Silvia; Cramer, Rainer; Quinn, Christopher C; Bruce, Jim; Zvelebil, Marketa; Corless, Steven; Bond, Jacquelyn; Yang, Alice; Hockfield, Susan; Burlingame, Alma L; Waterfield, Michael D; Jat, Parmjit S

    2002-04-01

    Normal somatic cells undergo a finite number of divisions and then cease dividing whereas cancer cells are able to proliferate indefinitely. To identify the underlying mechanisms that limit the mitotic potential, a two-dimensional differential proteome analysis of replicative senescence in serially passaged rat embryo fibroblasts was undertaken. Triplicate independent two-dimensional gels containing over 1200 spots each were run, curated, and analyzed. This revealed 49 spots whose expression was altered more than 2-fold. Of these, 42 spots yielded positive protein identification by mass spectrometry comprising a variety of cytoskeletal, heat shock, and metabolic proteins, as well as proteins involved in trafficking, differentiation, and protein synthesis, turnover, and modification. These included gelsolin, a candidate tumor suppressor for breast cancer, and alpha-glucosidase II, a member of the family of glucosidases that includes klotho; a defect in klotho expression in mice results in a syndrome that resembles human aging. Changes in expression of TUC-1, -2, -4, and -4 beta, members of the TUC family critical for neuronal differentiation, were also identified. Some of the identified changes were also shown to occur in two other models of senescence, premature senescence of REF52 cells and replicative senescence of mouse embryo fibroblasts. The majority of these candidate proteins were unrecognized previously in replicative senescence. They are now implicated in a new role. PMID:12096110

  4. 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.

  5. Cellular Proteome Dynamics during Differentiation of Human Primary Myoblasts.

    PubMed

    Le Bihan, Marie-Catherine; Barrio-Hernandez, Inigo; Mortensen, Tenna Pavia; Henningsen, Jeanette; Jensen, Søren Skov; Bigot, Anne; Blagoev, Blagoy; Butler-Browne, Gillian; Kratchmarova, Irina

    2015-08-01

    Muscle stem cells, or satellite cells, play an important role in the maintenance and repair of muscle tissue and have the capacity to proliferate and differentiate in response to physiological or environmental changes. Although they have been extensively studied, the key regulatory steps and the complex temporal protein dynamics accompanying the differentiation of primary human muscle cells remain poorly understood. Here, we demonstrate the advantages of applying a MS-based quantitative approach, stable isotope labeling by amino acids in cell culture (SILAC), for studying human myogenesis in vitro and characterize the fine-tuned changes in protein expression underlying the dramatic phenotypic conversion of primary mononucleated human muscle cells during in vitro differentiation to form multinucleated myotubes. Using an exclusively optimized triple encoding SILAC procedure, we generated dynamic expression profiles during the course of myogenic differentiation and quantified 2240 proteins, 243 of which were regulated. These changes in protein expression occurred in sequential waves and underlined vast reprogramming in key processes governing cell fate decisions, i.e., cell cycle withdrawal, RNA metabolism, cell adhesion, proteolysis, and cytoskeletal organization. In silico transcription factor target analysis demonstrated that the observed dynamic changes in the proteome could be attributed to a cascade of transcriptional events involving key myogenic regulatory factors as well as additional regulators not yet known to act on muscle differentiation. In addition, we created of a dynamic map of the developing myofibril, providing valuable insights into the formation and maturation of the contractile apparatus in vitro. Finally, our SILAC-based quantitative approach offered the possibility to follow the expression profiles of several muscle disease-associated proteins simultaneously and therefore could be a valuable resource for future studies investigating

  6. Identification of Thalidomide-Specific Transcriptomics and Proteomics Signatures during Differentiation of Human Embryonic Stem Cells

    PubMed Central

    Meganathan, Kesavan; Jagtap, Smita; Wagh, Vilas; Winkler, Johannes; Gaspar, John Antonydas; Hildebrand, Diana; Trusch, Maria; Lehmann, Karola; Hescheler, Jürgen; Schlüter, Hartmut; Sachinidis, Agapios

    2012-01-01

    Embryonic development can be partially recapitulated in vitro by differentiating human embryonic stem cells (hESCs). Thalidomide is a developmental toxicant in vivo and acts in a species-dependent manner. Besides its therapeutic value, thalidomide also serves as a prototypical model to study teratogenecity. Although many in vivo and in vitro platforms have demonstrated its toxicity, only a few test systems accurately reflect human physiology. We used global gene expression and proteomics profiling (two dimensional electrophoresis (2DE) coupled with Tandem Mass spectrometry) to demonstrate hESC differentiation and thalidomide embryotoxicity/teratogenecity with clinically relevant dose(s). Proteome analysis showed loss of POU5F1 regulatory proteins PKM2 and RBM14 and an over expression of proteins involved in neuronal development (such as PAK2, PAFAH1B2 and PAFAH1B3) after 14 days of differentiation. The genomic and proteomic expression pattern demonstrated differential expression of limb, heart and embryonic development related transcription factors and biological processes. Moreover, this study uncovered novel possible mechanisms, such as the inhibition of RANBP1, that participate in the nucleocytoplasmic trafficking of proteins and inhibition of glutathione transferases (GSTA1, GSTA2), that protect the cell from secondary oxidative stress. As a proof of principle, we demonstrated that a combination of transcriptomics and proteomics, along with consistent differentiation of hESCs, enabled the detection of canonical and novel teratogenic intracellular mechanisms of thalidomide. PMID:22952932

  7. Analyzing marginal cases in differential shotgun proteomics

    PubMed Central

    Carvalho, Paulo C.; Fischer, Juliana S. G.; Perales, Jonas; Yates, John R.; Barbosa, Valmir C.; Bareinboim, Elias

    2011-01-01

    Summary: We present an approach to statistically pinpoint differentially expressed proteins that have quantitation values near the quantitation threshold and are not identified in all replicates (marginal cases). Our method uses a Bayesian strategy to combine parametric statistics with an empirical distribution built from the reproducibility quality of the technical replicates. Availability:The software is freely available for academic use at http://pcarvalho.com/patternlab. Contact: paulo@pcarvalho.com Supplementary information: Supplementary data are available at Bioinformatics online. PMID:21075743

  8. Proteomic identification of glutamine synthetase as a differential marker for oligodendrogliomas and astrocytomas

    PubMed Central

    Zhuang, Zhengping; Qi, Meng; Li, Jie; Okamoto, Hiroaki; Xu, David S.; Iyer, Rajiv R.; Lu, Jie; Yang, Chunzhang; Weil, Robert J.; Vortmeyer, Alexander; Lonser, Russell R.

    2016-01-01

    Object Astrocytomas and oligodendrogliomas are primary CNS tumors that remain a challenge to differentiate histologically because of their morphological variability and because there is a lack of reliable differential diagnostic markers. To identify proteins that are differentially expressed between astrocytomas and oligodendrogliomas, the authors analyzed the proteomic expression patterns and identified uniquely expressed proteins in these neoplasms. Methods Proteomes of astrocytomas and oligodendrogliomas were analyzed using 2D gel electrophoresis and subsequent computerized gel analysis to detect differentially expressed proteins. The proteins were identified using high-performance liquid chromatography accompanied by tandem mass spectrometry. To determine the role of the differentially expressed proteins in astrocytes, undifferentiated glial cell cultures were treated with dibutyryl–cyclic adenosine monophosphate (cAMP). Results Two-dimensional gel electrophoresis revealed that glutamine synthetase was differentially expressed in astrocytomas and oligodendrogliomas. Western blot and immunohistochemical analyses confirmed the increased expression of glutamine synthetase in astrocytomas compared with oligodendrogliomas. Whereas glutamine synthetase expression was demonstrated across all grades of astrocytomas (Grade II–IV [15 tumors]) and oligoastrocytomas (4 tumors), it was expressed in only 1 oligodendroglioma (6% [16 tumors]). Treatment of undifferentiated glial cell cultures with dibutyryl-cAMP resulted in astrocyte differentiation that was associated with increased levels of glial fibrillary acidic protein and glutamine synthetase. Conclusions These data indicate that glutamine synthetase expression can be used to distinguish astrocytic from oligodendroglial tumors and may play a role in the pathogenesis of astrocytomas. PMID:21682567

  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. PMID:17001089

  10. Proteomic Profiles of Mesenchymal Stem Cells Induced by a Liver Differentiation Protocol

    PubMed Central

    Leelawat, Kawin; Narong, Siriluck; Chaijan, Suthidarak; Sa-ngiamsuntorn, Khanit; Disthabanchong, Sinee; Wongkajornsilp, Adisak; Hongeng, Suradej

    2010-01-01

    The replacement of disease hepatocytes and the stimulation of endogenous or exogenous regeneration by human mesenchymal stem cells (MSCs) are promising candidates for liver-directed cell therapy. In this study, we isolated MSCs from adult bone marrow by plastic adhesion and induced differentiation with a liver differentiation protocol. Western blot analyses were used to assess the expression of liver-specific markers. Next, MSC-specific proteins were analyzed with two-dimensional (2D) gel electrophoresis and peptide mass fingerprinting matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF)-mass spectrometry (MS). To confirm the results from the proteomic study, semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses were performed. We demonstrated that MSCs treated with the liver differentiation protocol expressed significantly more albumin, CK19 and CK20, than did undifferentiated cells. In addition the results of proteomic study demonstrated increases expression of FEM1B, PSMC2 and disulfide-isomerase A3 in MSCs treated with the liver differentiation protocol. These results from proteomic profiling will not only provide insight into the global responses of MSCs to hepatocyte differentiation, but will also lead to in-depth studies on the mechanisms of proteomic changes in MSCs. PMID:21614181

  11. Free Radical Scavenging Activity: Antiproliferative and Proteomics Analyses of the Differential Expression of Apoptotic Proteins in MCF-7 Cells Treated with Acetone Leaf Extract of Diospyros lycioides (Ebenaceae)

    PubMed Central

    Pilane, M. C.; Bagla, V. P.; Mokgotho, M. P.; Mbazima, V.; Matsebatlela, T. M.; Ncube, I.; Mampuru, L.

    2015-01-01

    Breast cancer is the most common cancer in South Africa. The acetone leaf extract of Diospyros lycioides was evaluated qualitatively and quantitatively for its antioxidant potential using DPPH assay and nitric oxide radical scavenging effect, while the viability of MCF-7 cells was evaluated using the MTT. MCF-7 treated cells were stained with Hoechst 335258 dye and annexin-V-FITC to be evaluated for apoptotic effect of the extract, while mRNA expression levels of apoptotic genes were assessed by quantitative real-time PCR and deferential protein expression levels using 2D gel electrophoresis and mass spectrometry. Results revealed presence of antioxidant constituents in the extract. Extract was shown to be cytotoxic in a concentration- and time-dependent manner. Cytotoxicity was demonstrated to be due to apoptosis, with 70% of the extract-treated cells being annexin-V-positive/PI negative at 48 hours. The extract was also shown to upregulate the expression of p53 gene with concomitant downregulation of the Bcl-2 antiapoptotic gene while differentially expressed proteins were identified as enolase, pyruvate kinase, and glyceraldehyde-3-phosphate. The extract in this study was shown to induce apoptosis at an early stage which makes it an ideal source that can be explored for compounds that may be used in the treatment and management of cancer. PMID:26457109

  12. 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

  13. 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

  14. Differential proteomic analysis of respiratory failure in peripheral blood mononuclear cells using iTRAQ technology

    PubMed Central

    SUN, GUOPING; CAO, CUIHUI; CHEN, WENBIAO; ZHANG, YANG; DAI, YONG

    2016-01-01

    Respiratory failure (RF) is a state in which the respiratory system fails by its gas exchange functions. Failure of the lung, which is caused by all types of lung diseases, leads to hypoxaemia with type I respiratory failure. Failure of the pump leads to hypercapnia or type II respiratory failure. Using isobaric tags for relative and absolute quantification (iTRAQ) technology to identify and quantify the total proteins in peripheral blood mononuclear cells (PBMCs) of RF patients and identify the differentially expressed proteome. The present study analyzed the total proteins in the PBMCs of RF patients and healthy controls using the eight-plex iTRAQ added with strong cation-exchange chromatography and liquid chromatography coupled with tandem mass spectrometry. The differentially expressed proteins were identified by MASCOT. A total of 4,795 differentially expressed proteins were identified, and 403 proteins were upregulated and 421 were downregulated. Among them, 4 proteins were significantly differentially expressed, which were upregulated KIAA1520 protein and γ fibrinogen type B (AA at 202) and downregulated chain A, crystal structure of recombinant human platelet factor 4 and myosin regulatory light polypeptide 9. iTRAQ technology is suitable for identifying and quantifying the proteome in the PBMCs of RF patients. The differentially expressed proteins of RF patients have been identified in the present study, and further research of the molecular mechanism of the differentially expressed proteins is required to clarify the pathogenesis and identify novel biomarkers of RF. PMID:27123249

  15. 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

  16. 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. PMID:27064846

  17. Differential Expression of Prognostic Proteomic Markers in Primary Tumour, Venous Tumour Thrombus and Metastatic Renal Cell Cancer Tissue and Correlation with Patient Outcome

    PubMed Central

    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

  18. 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

  19. [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. PMID:27220253

  20. [Transcriptomics and proteomics in studies of induced differentiation of leukemia cells].

    PubMed

    Novikova, S E; Zgoda, V G

    2015-01-01

    Induced differentiation of leukemia cells is in the focus of basic and applied biomedical studies medicine and biology for more than 30 years. During this period specific regulatory molecules involved in the maturation process have been identified by biochemical and molecular biological methods. Recent developments of high-throughput transcriptomic and proteomic techniques made it possible to analyze large sets of mRNA and proteins; this resulted in identification of functionally important signal transduction pathways and networks of molecular interactions, and thus extent existing knowledge on the molecular mechanisms of induced differentiation. Despite significant advances in mechanisms of induced differentiation, many problems related to the molecular mechanism of cell maturation, a phenomenon of therapeutic resistance of leukemic cells need better understanding and thus require further detailed study. Transcriptomics and proteomics methods provide a suitable methodological platform for the implementation of such studies. This review highlights the use of transcriptomic and proteomic methods in studies aimed at various aspects of the induced differentiation. Special attention is paid to the employment of the systems approach for investigation of various aspects of cell maturation. The use of the systems approach in studies of induced differentiation is an important step for the transition from the formal data accumulation on expression of mRNA and proteins towards creating models of biological processes in silico. PMID:26539862

  1. Differential Proteomics Analysis of Bacillus amyloliquefaciens and Its Genome-Shuffled Mutant for Improving Surfactin Production

    PubMed Central

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

    2014-01-01

    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. PMID:25365175

  2. The proteome of the differentiating mesencephalic progenitor cell line CSM14.1 in vitro.

    PubMed

    Weiss, B; Haas, S; Lessner, G; Mikkat, S; Kreutzer, M; Glocker, M O; Wree, A; Schmitt, O

    2014-01-01

    The treatment of Parkinson's disease by transplantation of dopaminergic (DA) neurons from human embryonic mesencephalic tissue is a promising approach. However, the origin of these cells causes major problems: availability and standardization of the graft. Therefore, the generation of unlimited numbers of DA neurons from various types of stem or progenitor cells has been brought into focus. A source for DA neurons might be conditionally immortalized progenitor cells. The temperature-sensitive immortalized cell line CSM14.1 derived from the mesencephalon of an embryonic rat has been used successfully for transplantation experiments. This cell line was analyzed by unbiased stereology of cell type specific marker proteins and 2D-gel electrophoresis followed by mass spectrometry to characterize the differentially expressed proteome. Undifferentiated CSM14.1 cells only expressed the stem cell marker nestin, whereas differentiated cells expressed GFAP or NeuN and tyrosine hydroxylase. An increase of the latter cells during differentiation could be shown. By using proteomics an explanation on the protein level was found for the observed changes in cell morphology during differentiation, when CSM14.1 cells possessed the morphology of multipolar neurons. The results obtained in this study confirm the suitability of CSM14.1 cells as an in vitro model for the study of neuronal and dopaminergic differentiation in rats. PMID:24592386

  3. Differential proteomics analysis of mononuclear cells in cerebrospinal fluid of Parkinson’s disease

    PubMed Central

    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. PMID:26823915

  4. Differential alkylation-based redox proteomics – Lessons learnt

    PubMed Central

    Wojdyla, Katarzyna; Rogowska-Wrzesinska, Adelina

    2015-01-01

    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. PMID:26282677

  5. 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. PMID:26282677

  6. Identification of Differentiation-Related Proteins in Gastric Adenocarcinoma Tissues by Proteomics.

    PubMed

    Zhou, Xin; Yao, Kun; Zhang, Lang; Zhang, Ying; Han, Yin; Liu, Hui-Ling; Liu, Xiang-Wen; Su, Gang; Yuan, Wen-Zhen; Wei, Xiao-Dong; Guan, Quan-Lin; Zhu, Bing-Dong

    2016-10-01

    There is a significant correlation between the degree of tumor differentiation and the survival of patients with gastric cancers. In this report, we compared proteomic differences between poorly differentiated gastric adenocarcinoma tissues and well-differentiated gastric adenocarcinoma tissues in order to identify differentiation-related proteins that may be closely correlated with differentiation of gastric cancer pathogenesis. We identified 7 proteins, of which calreticulin precursor, tapasinERP57 heterodimer, pyruvate kinase isozymes M1/M2 isoform M2, class Pi glutathione S-transferase, and chain A crystal structure of human enolase 1 were upregulated in poorly differentiated gastric adenocarcinoma compared with well-differentiated gastric adenocarcinoma, while myosin-11 isoform SM2A and actin alpha cardiac were downregulated. Two of them, pyruvate kinase isozymes M1/M2 isoform M2 and enolase 1 are enzymes involved in glycolytic pathway. The upregulation of pyruvate kinase isozymes M1/M2 isoform M2 and enolase 1 in poorly differentiated gastric adenocarcinoma was confirmed by Western blotting and immunohistochemistry. Furthermore, we observed 107 cases with gastric adenocarcinoma and found that the high expression of pyruvate kinase isozymes M1/M2 isoform M2 and enolase 1 correlates with tumor size (P = .0001 and P = .0017, respectively), depth of invasion (P = .0024 and P = .0261, respectively), and poor prognosis of patients. In conclusion, with this proteomic analysis, pyruvate kinase isozymes M1/M2 isoform M2 and enolase 1 were identified upregulated in poorly differentiated gastric adenocarcinoma comparing with well-differentiated gastric adenocarcinoma. The expression level of pyruvate kinase isozymes M1/M2 isoform M2 and enolase 1 was significantly correlated with overall survival. Some of them would be differentiation-related cancer biomarkers and are associated with tumor metastasis, invasion, and prognosis. PMID:27624754

  7. Constraints imposed by nonfunctional protein-protein interactions on gene expression and proteome size

    NASA Astrophysics Data System (ADS)

    Zhang, Jingshan; Maslov, Sergei; Shakhnovich, Eugene

    2009-03-01

    Crowded intracellular environments present a challenge for proteins to form functional specific complexes while reducing nonfunctional interactions with promiscuous nonfunctional partners. Here we show how nonfunctional interactions limit the proteome diversity and the average concentration of co-expressed and co-localized proteins. We use yeast compartments to verify our hypothesis that the yeast proteome has evolved to operate closely to the upper limit of its size, while keeping individual protein concentrations sufficiently low to reduce nonfunctional interactions. These findings have implication for conceptual understanding of intracellular compartmentalization, multicellularity, and differentiation.

  8. 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

  9. Metabolome-Proteome Differentiation Coupled to Microbial Divergence

    SciTech Connect

    Wilmes, P; Bowen, Benjamin P.; Thomas, Brian; Mueller, Ryan; Denef, Vincent; Verberkmoes, Nathan C; Hettich, Robert {Bob} L; Northen, Trent R.; Banfield, Jillian F.

    2010-01-01

    Tandem high-throughput proteomics and metabolomics were employed to functionally characterize natural microbial biofilm communities. Distinct molecular signatures exist for each analyzed sample. Deconvolution of the high-resolution molecular data demonstrates that identified proteins and detected metabolites exhibit organism-specific correlation patterns. These patterns are reflective of the functional differentiation of two bacterial species that share the same genus and that co-occur in the sampled microbial communities. Our analyses indicate that the two species have similar niche breadths and are not in strong competition with one another.

  10. Differential proteomic profiles from distinct Toxoplasma gondii strains revealed by 2D-difference gel electrophoresis.

    PubMed

    Zhou, Huaiyu; Zhao, Qunli; Das Singla, Lachhman; Min, Juan; He, Shenyi; Cong, Hua; Li, Ying; Su, Chunlei

    2013-04-01

    Toxoplasma gondii is an obligate intracellular protozoan that infects mammals and birds. Human infection during pregnancy may cause severe damage to the fetus. Reactivation of latent infection in immunocompromised patients can cause life-threatening encephalitis. T. gondii strains are highly diverse but only a few lineages (Type I, II and III) are widely spread. In mouse model, Type I strains are highly virulent, whereas Type II and III strains are intermediately or non virulent. It is not clear how much quantitative difference exists in proteomic profiles among these distinct T. gondii lineages. In the present study, the proteomic profiles of T. gondii tachyzoites from these lineages were investigated by two dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS) technologies. A total of 2321 protein spots were detected. Overall, the GT1 strain of Type I lineage and the strain PTG of Type II lineage have highly similar proteomic profiles and both are different from that of the CTG strain of Type III lineage. Eighty-four protein spots were differentially expressed by greater than 1.5-fold in relative abundance and 10 of them were identified to 7 T. gondii proteins in existing database. Investigation of the quantitative differences in proteomics among distinct T. gondii strains should facilitate our understanding of difference in biological processes and pathogenesis of distinct T. gondii genotypes, which will provide basic information to determine treatment regimen for different manifestation of toxoplasmosis. PMID:23340323

  11. Proteomic Analysis of MG132-Treated Germinating Pollen Reveals Expression Signatures Associated with Proteasome Inhibition

    PubMed Central

    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. PMID:25265451

  12. Differential Proteomic Analysis of Noncardia Gastric Cancer from Individuals of Northern Brazil

    PubMed Central

    Leal, Mariana Ferreira; Chung, Janete; Calcagno, Danielle Queiroz; Assumpção, Paulo Pimentel; Demachki, Samia; da Silva, Ismael Dale Cotrim Guerreiro; Chammas, Roger; Burbano, Rommel Rodríguez; de Arruda Cardoso Smith, Marília

    2012-01-01

    Gastric cancer is the second leading cause of cancer-related death worldwide. The identification of new cancer biomarkers is necessary to reduce the mortality rates through the development of new screening assays and early diagnosis, as well as new target therapies. In this study, we performed a proteomic analysis of noncardia gastric neoplasias of individuals from Northern Brazil. The proteins were analyzed by two-dimensional electrophoresis and mass spectrometry. For the identification of differentially expressed proteins, we used statistical tests with bootstrapping resampling to control the type I error in the multiple comparison analyses. We identified 111 proteins involved in gastric carcinogenesis. The computational analysis revealed several proteins involved in the energy production processes and reinforced the Warburg effect in gastric cancer. ENO1 and HSPB1 expression were further evaluated. ENO1 was selected due to its role in aerobic glycolysis that may contribute to the Warburg effect. Although we observed two up-regulated spots of ENO1 in the proteomic analysis, the mean expression of ENO1 was reduced in gastric tumors by western blot. However, mean ENO1 expression seems to increase in more invasive tumors. This lack of correlation between proteomic and western blot analyses may be due to the presence of other ENO1 spots that present a slightly reduced expression, but with a high impact in the mean protein expression. In neoplasias, HSPB1 is induced by cellular stress to protect cells against apoptosis. In the present study, HSPB1 presented an elevated protein and mRNA expression in a subset of gastric cancer samples. However, no association was observed between HSPB1 expression and clinicopathological characteristics. Here, we identified several possible biomarkers of gastric cancer in individuals from Northern Brazil. These biomarkers may be useful for the assessment of prognosis and stratification for therapy if validated in larger clinical study

  13. 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

  14. 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. PMID:26261351

  15. LC–MS Based Detection of Differential Protein Expression

    PubMed Central

    Tuli, Leepika; Ressom, Habtom W.

    2010-01-01

    While several techniques are available in proteomics, LC-MS based analysis of complex protein/peptide mixtures has turned out to be a mainstream analytical technique for quantitative proteomics. Significant technical advances at both sample preparation/separation and mass spectrometry levels have revolutionized comprehensive proteome analysis. Moreover, automation and robotics for sample handling process permit multiple sampling with high throughput. For LC-MS based quantitative proteomics, sample preparation turns out to be critical step, as it can significantly influence sensitivity of downstream analysis. Several sample preparation strategies exist, including depletion of high abundant proteins or enrichment steps that facilitate protein quantification but with a compromise of focusing on a smaller subset of a proteome. While several experimental strategies have emerged, certain limitations such as physiochemical properties of a peptide/protein, protein turnover in a sample, analytical platform used for sample analysis and data processing, still imply challenges to quantitative proteomics. Other aspects that make analysis of a proteome a challenging task include dynamic nature of a proteome, need for efficient and fast analysis of protein due to its constant modifications inside a cell, concentration range of proteins that exceed dynamic range of a single analytical method, and absence of appropriate bioinformatics tools for analysis of large volume and high dimensional data. This paper gives an overview of various LC-MS methods currently used in quantitative proteomics and their potential for detecting differential protein expression. Fundamental steps such as sample preparation, LC separation, mass spectrometry, quantitative assessment and protein identification are discussed. For quantitative assessment of protein expression, both label and label free approaches are evaluated for their set of merits and demerits. While most of these methods edge on providing

  16. 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

  17. Differential proteomic response of Sydney rock oysters (Saccostrea glomerata) to prolonged environmental stress.

    PubMed

    Melwani, A R; Thompson, E L; Raftos, D A

    2016-04-01

    Exposure to prolonged environmental stress can have impacts on the cellular homeostasis of aquatic organisms. The current study employed two-dimensional electrophoresis (2-DE) to test whether exposure to impaired water quality conditions in the Sydney Harbour estuary has significantly altered the proteomes of the resident Sydney rock oyster (Saccostrea glomerata). Adult S. glomerata were sampled from four bays in the estuary. Each bay consisted of a "high-impact" site adjacent to point sources of chemical contamination (e.g., storm drains/canals or legacy hotspots) and a "low-impact" site located ∼5km away from point sources. A mixture of environmental stressors differed significantly between high- and low-impact sites. Specifically, PAHs, PCBs, tributyltin, Pb, and Zn were significantly elevated in oyster tissues from high-impact sites, together with depleted dissolved oxygen and low pH in the water column. A 2-DE proteomics analysis subsequently identified 238 protein spots across 24 2-DE gels, of which 27-50 spots differed significantly in relative intensity between high- and low-impact sites per bay. Twenty-five percent of the differential spots were identified in more than one bay. The identities of 80 protein spots were determined by mass spectrometry. HSP 70, PPIB, and radixin were the three most highly expressed differential proteins. Despite the largely unique proteomes evident in each bay, functional annotations revealed that half of the differentially expressed proteins fell into just two subcellular functional categories-energy metabolism and the cytoskeleton. These findings provide a framework to further investigate adaptation of cellular mechanisms to prolonged stress in S. glomerata. PMID:26844780

  18. 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.

  19. Differentially delayed root proteome responses to salt stress in sugar cane varieties.

    PubMed

    Pacheco, Cinthya Mirella; Pestana-Calsa, Maria Clara; Gozzo, Fabio Cesar; Mansur Custodio Nogueira, Rejane Jurema; Menossi, Marcelo; Calsa, Tercilio

    2013-12-01

    Soil salinity is a limiting factor to sugar cane crop development, although in general plants present variable mechanisms of tolerance to salinity stress. The molecular basis underlying these mechanisms can be inferred by using proteomic analysis. Thus, the objective of this work was to identify differentially expressed proteins in sugar cane plants submitted to salinity stress. For that, a greenhouse experiment was established with four sugar cane varieties and two salt conditions, 0 mM (control) and 200 mM NaCl. Physiological and proteomics analyses were performed after 2 and 72 h of stress induction by salt. Distinct physiological responses to salinity stress were observed in the varieties and linked to tolerance mechanisms. In proteomic analysis, the roots soluble protein fraction was extracted, quantified, and analyzed through bidimensional electrophoresis. Gel images analyses were done computationally, where in each contrast only one variable was considered (salinity condition or variety). Differential spots were excised, digested by trypsin, and identified via mass spectrometry. The tolerant variety RB867515 showed the highest accumulation of proteins involved in growth, development, carbohydrate and energy metabolism, reactive oxygen species metabolization, protein protection, and membrane stabilization after 2 h of stress. On the other hand, the presence of these proteins in the sensitive variety was verified only in stress treatment after 72 h. These data indicate that these stress responses pathways play a role in the tolerance to salinity in sugar cane, and their effectiveness for phenotypical tolerance depends on early stress detection and activation of the coding genes expression. PMID:24251627

  20. Proteomic Cornerstones of Hematopoietic Stem Cell Differentiation: Distinct Signatures of Multipotent Progenitors and Myeloid Committed Cells*

    PubMed Central

    Klimmeck, Daniel; Hansson, Jenny; Raffel, Simon; Vakhrushev, Sergey Y.; Trumpp, Andreas; Krijgsveld, Jeroen

    2012-01-01

    Regenerative tissues such as the skin epidermis, the intestinal mucosa or the hematopoietic system are organized in a hierarchical manner with stem cells building the top of this hierarchy. Somatic stem cells harbor the highest self-renewal activity and generate a series of multipotent progenitors which differentiate into lineage committed progenitors and subsequently mature cells. In this report, we applied an in-depth quantitative proteomic approach to analyze and compare the full proteomes of ex vivo isolated and FACS-sorted populations highly enriched for either multipotent hematopoietic stem/progenitor cells (HSPCs, LinnegSca-1+c-Kit+) or myeloid committed precursors (LinnegSca-1−c-Kit+). By employing stable isotope dimethyl labeling and high-resolution mass spectrometry, more than 5000 proteins were quantified. From biological triplicate experiments subjected to rigorous statistical evaluation, 893 proteins were found differentially expressed between multipotent and myeloid committed cells. The differential protein content in these cell populations points to a distinct structural organization of the cytoskeleton including remodeling activity. In addition, we found a marked difference in the expression of metabolic enzymes, including a clear shift of specific protein isoforms of the glycolytic pathway. Proteins involved in translation showed a collective higher expression in myeloid progenitors, indicating an increased translational activity. Strikingly, the data uncover a unique signature related to immune defense mechanisms, centering on the RIG-I and type-1 interferon response systems, which are installed in multipotent progenitors but not evident in myeloid committed cells. This suggests that specific, and so far unrecognized, mechanisms protect these immature cells before they mature. In conclusion, this study indicates that the transition of hematopoietic stem/progenitors toward myeloid commitment is accompanied by a profound change in processing of

  1. 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. PMID:21810490

  2. Proteomics-based confirmation of protein expression and correction of annotation errors in the Brucella abortus genome

    PubMed Central

    2010-01-01

    Background Brucellosis is a major bacterial zoonosis affecting domestic livestock and wild mammals, as well as humans around the globe. While conducting proteomics studies to better understand Brucella abortus virulence, we consolidated the proteomic data collected and compared it to publically available genomic data. Results The proteomic data was compiled from several independent comparative studies of Brucella abortus that used either outer membrane blebs, cytosols, or whole bacteria grown in media, as well as intracellular bacteria recovered at different times following macrophage infection. We identified a total of 621 bacterial proteins that were differentially expressed in a condition-specific manner. For 305 of these proteins we provide the first experimental evidence of their expression. Using a custom-built protein sequence database, we uncovered 7 annotation errors. We provide experimental evidence of expression of 5 genes that were originally annotated as non-expressed pseudogenes, as well as start site annotation errors for 2 other genes. Conclusions An essential element for ensuring correct functional studies is the correspondence between reported genome sequences and subsequent proteomics studies. In this study, we have used proteomics evidence to confirm expression of multiple proteins previously considered to be putative, as well as correct annotation errors in the genome of Brucella abortus strain 2308. PMID:20462421

  3. 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

  4. 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

  5. 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.

  6. Susceptibility to COPD: Differential Proteomic Profiling after Acute Smoking

    PubMed Central

    Franciosi, Lorenza; Postma, Dirkje S.; van den Berge, Maarten; Govorukhina, Natalia; Horvatovich, Peter L.; Fusetti, Fabrizia; Poolman, Bert; Lodewijk, Monique E.; Timens, Wim; Bischoff, Rainer; ten Hacken, Nick H. T.

    2014-01-01

    Cigarette smoking is the main risk factor for COPD (Chronic Obstructive Pulmonary Disease), yet only a subset of smokers develops COPD. Family members of patients with severe early-onset COPD have an increased risk to develop COPD and are therefore defined as “susceptible individuals”. Here we perform unbiased analyses of proteomic profiles to assess how “susceptible individuals” differ from age-matched “non-susceptible individuals” in response to cigarette smoking. Epithelial lining fluid (ELF) was collected at baseline and 24 hours after smoking 3 cigarettes in young individuals susceptible or non-susceptible to develop COPD and older subjects with established COPD. Controls at baseline were older healthy smoking and non-smoking individuals. Five samples per group were pooled and analysed by stable isotope labelling (iTRAQ) in duplicate. Six proteins were selected and validated by ELISA or immunohistochemistry. After smoking, 23 proteins increased or decreased in young susceptible individuals, 7 in young non-susceptible individuals, and 13 in COPD in the first experiment; 23 proteins increased or decreased in young susceptible individuals, 32 in young non-susceptible individuals, and 11 in COPD in the second experiment. SerpinB3 and Uteroglobin decreased after acute smoke exposure in young non-susceptible individuals exclusively, whereas Peroxiredoxin I, S100A9, S100A8, ALDH3A1 (Aldehyde dehydrogenase 3A1) decreased both in young susceptible and non-susceptible individuals, changes being significantly different between groups for Uteroglobin with iTRAQ and for Serpin B3 with iTRAQ and ELISA measures. Peroxiredoxin I, SerpinB3 and ALDH3A1 increased in COPD patients after smoking. We conclude that smoking induces a differential protein response in ELF of susceptible and non-susceptible young individuals, which differs from patients with established COPD. This is the first study applying unbiased proteomic profiling to unravel the underlying mechanisms

  7. 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

  8. Differential proteomic profile of spermatogenic and Sertoli cells from peri-pubertal testes of three different bovine breeds

    PubMed Central

    Tripathi, Utkarsh K.; Aslam, Muhammad K. M.; Pandey, Shashank; Nayak, Samiksha; Chhillar, Shivani; Srinivasan, A.; Mohanty, T. K.; Kadam, Prashant H.; Chauhan, M. S.; Yadav, Savita; Kumaresan, Arumugam

    2014-01-01

    Sub-fertility is one of the most common problems observed in crossbred males, but the etiology remains unknown in most of the cases. Although proteomic differences in the spermatozoa and seminal plasma between breeds have been investigated, the possible differences at the sperm precursor cells and supporting/nourishing cells have not been studied. The present study reports the differential proteomic profile of spermatogenic and Sertoli cells in crossbred and purebred bulls. Testis was removed by unilateral castration of 12 peri-pubertal bulls (10 months age), four each from crossbred (Holstein Friesian × Tharparkar), exotic purebred [Holstein Friesian (HF)] and indigenous purebred [Tharparkar (TP)] bulls. Spermatogenic and Sertoli cells were isolated and subjected to proteomic analysis. Protein extracts from the Sertoli and spermatogenic cells of each breed were analyzed with 2-dimensional difference gel electrophoresis (2D-DIGE) and analyzed with Decyder™ software. Compared to HF, 26 protein spots were over expressed and 14 protein spots were under expressed in spermatogenic cells of crossbred bulls. Similarly, 7 protein spots were over expressed and 15 protein spots were under expressed in the spermatogenic cells of TP bulls compared to that of crossbred bulls. Out of 12 selected protein spots identified through mass spectrometry, Phosphatidyl ethanolamine binding protein was found to be over expressed in the spermatogenic cells of crossbred bulls compared to TP bulls. The protein, gamma actin was found to be over expressed in the Sertoli cells of HF bulls, whereas Speedy Protein-A was found to be over expressed in Sertoli cells of crossbred bulls. It may be concluded that certain proteomic level differences exist in sperm precursor cells and nourishing cells between breeds, which might be associated with differences in the fertility among these breeds. PMID:25364731

  9. Proteomics Based Identification of Cell Migration Related Proteins in HBV Expressing HepG2 Cells

    PubMed Central

    Feng, Huixing; Li, Xi; Chan, Vincent; Chen, Wei Ning

    2014-01-01

    Proteomics study was performed to investigate the specific protein expression profiles of HepG2 cells transfected with mutant HBV compared with wildtype HBV genome, aiming to identify the specific functions of SH3 binding domain (proline rich region) located in HBx. In addition to the cell movement and kinetics changes due to the expression of HBV genome we have observed previously, here we further targeted to explore the specific changes of cellular proteins and potential intracellular protein interactions, which might provide more information of the potential cellular mechanism of the differentiated cell movements. Specific changes of a number of proteins were shown in global protein profiling in HepG2 cells expressing wildtype HBV, including cell migration related proteins, and interestingly the changes were found recovered by SH3 binding domain mutated HBV. The distinctive expressions of proteins were validated by Western blot analysis. PMID:24763314

  10. 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

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

    PubMed

    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

  12. 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

    2016-01-01

    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. PMID:27525926

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

    PubMed Central

    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

  14. 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…

  15. Proteomic Identification of Novel Differentiation Plasma Protein Markers in Hypobaric Hypoxia-Induced Rat Model

    PubMed Central

    Ahmad, Mohammad Faiz; Sharma, Manish; Garg, Iti; Bhargava, Kalpana

    2014-01-01

    Background Hypobaric hypoxia causes complex changes in the expression of genes, including stress related genes and corresponding proteins that are necessary to maintain homeostasis. Whereas most prior studies focused on single proteins, newer methods allowing the simultaneous study of many proteins could lead to a better understanding of complex and dynamic changes that occur during the hypobaric hypoxia. Methods In this study we investigated the temporal plasma protein alterations of rat induced by hypobaric hypoxia at a simulated altitude of 7620 m (25,000 ft, 282 mm Hg) in a hypobaric chamber. Total plasma proteins collected at different time points (0, 6, 12 and 24 h), separated by two-dimensional electrophoresis (2-DE) and identified using matrix assisted laser desorption ionization time of flight (MALDI-TOF/TOF). Biological processes that were enriched in the plasma proteins during hypobaric hypoxia were identified using Gene Ontology (GO) analysis. According to their properties and obvious alterations during hypobaric hypoxia, changes of plasma concentrations of Ttr, Prdx-2, Gpx -3, Apo A-I, Hp, Apo-E, Fetub and Nme were selected to be validated by Western blot analysis. Results Bioinformatics analysis of 25 differentially expressed proteins showed that 23 had corresponding candidates in the database. The expression patterns of the eight selected proteins observed by Western blot were in agreement with 2-DE results, thus confirming the reliability of the proteomic analysis. Most of the proteins identified are related to cellular defense mechanisms involving anti-inflammatory and antioxidant activity. Their presence reflects the consequence of serial cascades initiated by hypobaric hypoxia. Conclusion/Significance This study provides information about the plasma proteome changes induced in response to hypobaric hypoxia and thus identification of the candidate proteins which can act as novel biomarkers. PMID:24842778

  16. 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. PMID:25173675

  17. Investigation of proteomic biomarkers in in vivo hepatotoxicity study of rat liver: toxicity differentiation in hepatotoxicants.

    PubMed

    Yamamoto, Toshinori; Kikkawa, Rie; Yamada, Hiroshi; Horii, Ikuo

    2006-02-01

    We investigated the overall protein expression profiles in the in vivo hepatotoxicity of rats induced by four well-recognized hepatotoxicants. Acetaminophen (APAP), amiodarone (AMD), tetracycline (TC) and carbon tetrachloride (CTC) were administered to male rats by gavages and the liver at 24 hr post-dosing was applied to the proteomic experiment. Blood biochemistry and histopathology were examined to identify specific changes related to the compounds given. Protein expression in the liver was investigated by 2-dimensional gel electrophoresis (2DE), and spots showing a significantly different expression in treated versus control group were excised from gels and identified by Q-Tof mass spectrometer. They were well characterized based on their functions related to the mechanisms of toxicity of the compounds. Among them, we focused on the 8 proteins that were affected by all 4 compounds examined. Proteins related to oxidative stress response such as carbonic anhydrase III (CA3) and 60kDa heat shock protein (HSP60), and energy metabolism such as adenylate kinase 4 (AK4) were found. Moreover, hierarchical clustering analysis using 2D-gel spots information revealed the possibility to differentiate the groups based on their toxicity levels such as severity of liver damage. These results suggested that assessing the effects of hepatotoxicants on protein expression is worth trying to screen candidate compounds at the developmental stage of drugs. PMID:16538043

  18. 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. PMID:24136547

  19. Stage-specific proteomic expression patterns of the human filarial parasite Brugia malayi and its endosymbiont Wolbachia

    PubMed Central

    Bennuru, Sasisekhar; Meng, Zhaojing; Ribeiro, José M. C.; Semnani, Roshanak Tolouei; Ghedin, Elodie; Chan, King; Lucas, David A.; Veenstra, Timothy D.; Nutman, Thomas B.

    2011-01-01

    Global proteomic analyses of pathogens have thus far been limited to unicellular organisms (e.g., protozoa and bacteria). Proteomic analyses of most eukaryotic pathogens (e.g., helminths) have been restricted to specific organs, specific stages, or secretomes. We report here a large-scale proteomic characterization of almost all the major mammalian stages of Brugia malayi, a causative agent of lymphatic filariasis, resulting in the identification of more than 62% of the products predicted from the Bm draft genome. The analysis also yielded much of the proteome of Wolbachia, the obligate endosymbiont of Bm that also expressed proteins in a stage-specific manner. Of the 11,610 predicted Bm gene products, 7,103 were definitively identified from adult male, adult female, blood-borne and uterine microfilariae, and infective L3 larvae. Among the 4,956 gene products (42.5%) inferred from the genome as “hypothetical,” the present study was able to confirm 2,336 (47.1%) as bona fide proteins. Analysis of protein families and domains coupled with stage-specific expression highlight the important pathways that benefit the parasite during its development in the host. Gene set enrichment analysis identified extracellular matrix proteins and those with immunologic effects as enriched in the microfilarial and L3 stages. Parasite sex- and stage-specific protein expression identified those pathways related to parasite differentiation and demonstrates stage-specific expression by the Bm endosymbiont Wolbachia as well. PMID:21606368

  20. Stage-specific proteomic expression patterns of the human filarial parasite Brugia malayi and its endosymbiont Wolbachia.

    PubMed

    Bennuru, Sasisekhar; Meng, Zhaojing; Ribeiro, José M C; Semnani, Roshanak Tolouei; Ghedin, Elodie; Chan, King; Lucas, David A; Veenstra, Timothy D; Nutman, Thomas B

    2011-06-01

    Global proteomic analyses of pathogens have thus far been limited to unicellular organisms (e.g., protozoa and bacteria). Proteomic analyses of most eukaryotic pathogens (e.g., helminths) have been restricted to specific organs, specific stages, or secretomes. We report here a large-scale proteomic characterization of almost all the major mammalian stages of Brugia malayi, a causative agent of lymphatic filariasis, resulting in the identification of more than 62% of the products predicted from the Bm draft genome. The analysis also yielded much of the proteome of Wolbachia, the obligate endosymbiont of Bm that also expressed proteins in a stage-specific manner. Of the 11,610 predicted Bm gene products, 7,103 were definitively identified from adult male, adult female, blood-borne and uterine microfilariae, and infective L3 larvae. Among the 4,956 gene products (42.5%) inferred from the genome as "hypothetical," the present study was able to confirm 2,336 (47.1%) as bona fide proteins. Analysis of protein families and domains coupled with stage-specific expression highlight the important pathways that benefit the parasite during its development in the host. Gene set enrichment analysis identified extracellular matrix proteins and those with immunologic effects as enriched in the microfilarial and L3 stages. Parasite sex- and stage-specific protein expression identified those pathways related to parasite differentiation and demonstrates stage-specific expression by the Bm endosymbiont Wolbachia as well. PMID:21606368

  1. Expression profiles in surgically-induced carotid stenosis: a combined transcriptomic and proteomic investigation

    PubMed Central

    Forte, A; Finicelli, M; De Luca, P; Quarto, C; Onorati, F; Santè, P; Renzulli, A; Galderisi, U; Berrino, L; De Feo, M; Rossi, F; Cotrufo, M; Cascino, A; Cipollaro, M

    2008-01-01

    Vascular injury aimed at stenosis removal induces local reactions often leading to restenosis. The aim of this study was a concerted transcriptomic-proteomics analysis of molecular variations in a model of rat carotid arteriotomy, to dissect the molecular pathways triggered by vascular surgical injury and to identify new potential anti-restenosis targets. RNA and proteins extracted from inbred Wistar Kyoro (WKY) rat carotids harvested 4 hrs, 48 hrs and 7 days after arteriotomy were analysed by Affymetrix rat microarrays and by bidi-mensional electrophoresis followed by liquid chromatography and tandem mass spectrometry, using as reference the RNA and the proteins extracted from uninjured rat carotids. Results were classified according to their biological function, and the most significant Kyoro Encyclopedia of Genes and Genomes (KEGG) pathways were identified. A total of 1163 mRNAs were differentially regulated in arteriotomy-injured carotids 4 hrs, 48 hrs and 7 days after injury (P < 0.0001, fold-change ≥2), while 48 spots exhibited significant changes after carotid arteriotomy (P < 0.05, fold-change ≥2). Among them, 16 spots were successfully identified and resulted to correspond to a set of 19 proteins. mRNAs were mainly involved in signal transduction, oxidative stress/inflammation and remodelling, including many new potential targets for limitation of surgically induced (re)stenosis (e.g. Arginase I, Kruppel like factors). Proteome analysis confirmed and extended the microrarray data, revealing time-dependent post-translational modifications of Hsp27, haptoglobin and contrapsin-like protease inhibitor 6, and the differential expression of proteins mainly involved in contractility. Transcriptomic and proteomic methods revealed functional categories with different preferences, related to the experimental sensitivity and to mechanisms of regulation. The comparative analysis revealed correlation between transcriptional and translational expression for 47% of

  2. 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

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

    PubMed

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

    2016-01-01

    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

  4. 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. PMID:26241871

  5. 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.

  6. 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. PMID:23977166

  7. 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

  8. Two Oyster Species That Show Differential Susceptibility to Virus Infection Also Show Differential Proteomic Responses to Generic dsRNA.

    PubMed

    Masood, Muhammad; Raftos, David A; Nair, Sham V

    2016-06-01

    Viral diseases are a significant cause of mortality and morbidity in oysters, resulting in significant economic losses. We investigated the proteomic responses of these two species of oysters to generic double-stranded RNAs (poly I:C and poly A:U). Analysis of proteomic data using isobaric tags for relative and absolute quantitaion (iTRAQ) indicated that there were significant differences in the proteomic responses of the two oyster species resulting from this treatment. Gene ontology analysis showed that several biological processes, cellular components, and molecular function were unique to the different data sets. For example, a number of proteins implicated in the TLR signaling pathway were associated with the Saccostrea glomerata data set but were absent in the Crassostra gigas data set. These results suggest that the differences in the proteomic responses to dsRNA may underpin the biological differences in viral susceptibility. Molecular targets previously shown to be expressed in C. gigas in response to OsHV1 infections were not present in our proteomic data sets, although they were present in the RNA extracted from the very same tissues. Taken together, our data indicate that there are substantial disparities between transcriptomic and proteomic responses to dsRNA challenge, and a comprehensive account of the oysters' biological responses to these treatments must take into account that disparity. PMID:27072892

  9. Differential proteomics of the synovial membrane between bilateral and unilateral knee osteoarthritis in surgery‑induced rabbit models.

    PubMed

    Liu, Weilin; He, Jian; Lin, Ruhui; Liang, Jie; Luo, Qinglu

    2016-09-01

    The present study investigated the differential proteomics of synovial membranes between bilateral and unilateral anterior cruciate ligament transection (ACLT) in rabbits with knee osteoarthritis (KOA), in order to elucidate the pathological biomarkers of different degrees of KOA. A total of 6 New Zealand rabbits were randomly divided into groups A and B (three rabbits per group). The two groups were subjected to bilateral and unilateral ACLT, respectively. A total of 6 weeks following surgery, proteins were extracted from the knee joint synovial membranes of KOA rabbits and were separated by two‑dimensional polyacrylamide gel electrophoresis. The differentially expressed proteins in the OA synovial membranes were selected for further analysis by linear ion trap‑Fourier transform ion cyclotron resonance mass spectrometry. Ten protein spots were identified to be different between the synovial membranes of the bilateral and unilateral KOA rabbits. Protein disulfide‑isomerase and creatine kinase M‑type were identified in the unilateral KOA rabbit synovial membranes. Serum albumin (three spots), lumican, α‑2‑HS‑glycoprotein and three uncharacterized proteins were identified in the synovial membranes of the bilateral KOA rabbits. The differential proteomic expression demonstrated the different biomarkers associated with bilateral and unilateral KOA, and indicated that spontaneous and secondary KOA require diverse methods of treatment; thus the underlying mechanism of KOA requires further investigation. PMID:27430254

  10. Proteomic analysis in pterygium; upregulated protein expression of ALDH3A1, PDIA3, and PRDX2

    PubMed Central

    Kim, Sun Woong; Lee, Jonghoon; Lee, Boram

    2014-01-01

    Purpose To identify differentially expressed proteins in the pterygium compared to healthy conjunctiva using a proteomic analysis. Methods Pterygial and healthy conjunctival tissues were obtained from 24 patients undergoing pterygium excision. Total proteins of the pterygia and healthy conjunctiva were analyzed with one-dimensional electrophoresis, and protein bands of interest were excised and subjected to liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-MS/MS) using Thermo’s Finnigan ProteomeX workstation LTQ linear ion trap MS/MS. Using bioinformatics, differentially expressed proteins were classified, and three proteins closely involved in the response to oxidative stress were selected for further validation. Differential expression of these proteins was confirmed with western blot and immunohistochemistry. Results A web-based gene ontology program, DAVID, was used to classify 230 proteins that were differentially expressed in pterygial tissues. Among these genes, we chose three proteins, aldehyde dehydrogenase, dimeric NADP-preferring (ALDH3A1), protein disulfide-isomerase A3 (PDIA3), and peroxiredoxin-2 (PRDX2), that were significantly upregulated in pterygium and further increased in recurrent pterygium. Immunohistochemistry and western blot analysis confirmed that these three proteins were mainly detected in the basal epithelial layer, and their expression was significantly increased in the pterygium compared to normal conjunctiva. Conclusions This study reported increased expression of ALDH3A1, PDIA3, and PRDX2 in pterygia using a proteomic approach. These proteins are presumed to have a protective role against oxidative stress-induced apoptosis. This result is consistent with the hypothesis that oxidative stress is a significant factor in the pathogenesis of pterygia. PMID:25221425

  11. 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. PMID:24297927

  12. 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

  13. Bile proteomics for differentiation of malignant from benign biliary strictures: a pilot study

    PubMed Central

    Navaneethan, Udayakumar; Lourdusamy, Vennisvasanth; GK Venkatesh, Preethi; Willard, Belinda; Sanaka, Madhusudhan R; Parsi, Mansour A

    2015-01-01

    Background: Determining the etiology of biliary strictures is challenging, and the sensitivities of the current tests to diagnose them are low. Protein biomarkers in bile, in combination with other tests, may improve sensitivity in diagnosing biliary strictures. Objective: To analyse the differential abundance of proteins in benign and malignant biliary strictures through proteomic analysis of bile. Methods: In this prospective, cross-sectional study, bile was aspirated in 24 patients undergoing endoscopic retrograde cholangiopancreatography (ERCP) including six patients with primary sclerosing cholangitis (PSC), three with cholangiocarcinoma (CCA), ten with pancreatic cancer, and five with benign biliary conditions. Liquid chromatography/mass spectrometry was used to examine the bile for differential abundance of protein biomarkers. The relative abundance of various proteins was compared in the malignant vs. benign groups and in CCA vs. PSC. Results: The majority of the proteins identified in bile were similar to those of the plasma (plasma proteins) and certain proteins were differentially expressed among the different groups (CCA, pancreatic cancer, PSC or benign). A total of 18 proteins were identified as being more abundant in the malignant group (CCA and pancreatic cancer) than in the benign strictures group, including myeloperoxidase, complement C3, inter-alpha-trypsin inhibitor heavy chain H4, apolipoprotein B-100, and kininogen-1 isoform 2. A total of 30 proteins were identified to be less abundant in the malignant group than in the benign group, including trefoil factor 2, superoxide dismutase [Cu-Zn], kallikrein-1, carboxypeptidase B and trefoil factor 1. Conclusions: Protein biomarkers in bile may differentiate malignant from benign biliary strictures. Larger studies are warranted to validate these observations. PMID:25304323

  14. Proteomic Analysis of the Retina: Removal of RPE Alters Outer Segment Assembly and Retinal Protein Expression

    PubMed Central

    Wang, XiaoFei; Nookala, Suba; Narayanan, Chidambarathanu; Giorgianni, Francesco; Beranova-Giorgianni, Sarka; McCollum, Gary; Gerling, Ivan; Penn, John S.; Jablonski, Monica M.

    2008-01-01

    The mechanisms that regulate the complex physiologic task of photoreceptor outer segment assembly remain an enigma. One limiting factor in revealing the mechanism(s) by which this process is modulated is that not all of the role players that participate in this process are known. The purpose of this study was to determine some of the retinal proteins that likely play a critical role in regulating photoreceptor outer segment assembly. To do so, we analyzed and compared the proteome map of tadpole Xenopus laevis retinal pigment epithelium (RPE)-supported retinas containing organized outer segments with that of RPE-deprived retinas containing disorganized outer segments. Solubilized proteins were labeled with CyDye fluors followed by multiplexed two-dimensional separation. The intensity of protein spots and comparison of proteome maps was performed using DeCyder software. Identification of differentially regulated proteins was determined using nanoLC-ESI-MS/MS analysis. We found a total of 27 protein spots, 21 of which were unique proteins, which were differentially expressed in retinas with disorganized outer segments. We predict that in the absence of the RPE, oxidative stress initiates an unfolded protein response. Subsequently, downregulation of several candidate Müller glial cell proteins may explain the inability of photoreceptors to properly fold their outer segment membranes. In this study we have used identification and bioinformatics assessment of proteins that are differentially expressed in retinas with disorganized outer segments as a first step in determining probable key molecules involved in regulating photoreceptor outer segment assembly. PMID:18803304

  15. Dynamic Proteomic Analysis of Pancreatic Mesenchyme Reveals Novel Factors That Enhance Human Embryonic Stem Cell to Pancreatic Cell Differentiation

    PubMed Central

    Russ, Holger A.; Landsman, Limor; Moss, Christopher L.; Higdon, Roger; Greer, Renee L.; Kaihara, Kelly; Salamon, Randy; Kolker, Eugene; Hebrok, Matthias

    2016-01-01

    Current approaches in human embryonic stem cell (hESC) to pancreatic beta cell differentiation have largely been based on knowledge gained from developmental studies of the epithelial pancreas, while the potential roles of other supporting tissue compartments have not been fully explored. One such tissue is the pancreatic mesenchyme that supports epithelial organogenesis throughout embryogenesis. We hypothesized that detailed characterization of the pancreatic mesenchyme might result in the identification of novel factors not used in current differentiation protocols. Supplementing existing hESC differentiation conditions with such factors might create a more comprehensive simulation of normal development in cell culture. To validate our hypothesis, we took advantage of a novel transgenic mouse model to isolate the pancreatic mesenchyme at distinct embryonic and postnatal stages for subsequent proteomic analysis. Refined sample preparation and analysis conditions across four embryonic and prenatal time points resulted in the identification of 21,498 peptides with high-confidence mapping to 1,502 proteins. Expression analysis of pancreata confirmed the presence of three potentially important factors in cell differentiation: Galectin-1 (LGALS1), Neuroplastin (NPTN), and the Laminin α-2 subunit (LAMA2). Two of the three factors (LGALS1 and LAMA2) increased expression of pancreatic progenitor transcript levels in a published hESC to beta cell differentiation protocol. In addition, LAMA2 partially blocks cell culture induced beta cell dedifferentiation. Summarily, we provide evidence that proteomic analysis of supporting tissues such as the pancreatic mesenchyme allows for the identification of potentially important factors guiding hESC to pancreas differentiation. PMID:26681951

  16. Differentially proteomic analysis of the Chinese shrimp at WSSV latent and acute infection stages by iTRAQ approach.

    PubMed

    Li, Shihao; Li, Fuhua; Sun, Zheng; Zhang, Xiaojun; Xiang, Jianhai

    2016-07-01

    As the direct executors of biological function, the expression level of proteins will reveal the molecular mechanisms regulating WSSV acute infection more directly. In the present study, the iTRAQ approach was applied to identifying differentially expressed proteins in Chinese shrimp during WSSV latent infection and acute infection. A total of 4051 unique peptides corresponding to 1286 proteins were identified. 118 unique proteins showed differential up-regulation and 122 proteins were down-regulated in shrimp during WSSV acute infection compared with those in WSSV latent infection stage. A number of proteins related to actin-myosin cytoskeleton process, including myosin, actin, tubulin, clathrin, and tropomyosin were found up-regulated in shrimp at WSSV AI stage, indicating that the phagocytosis process was involved in WSSV AI stage. The apoptosis process in shrimp during WSSV AI seemed to be inhibited because some proteins suppressive on apoptosis were up-regulated, such as ALG-2 interacting protein x, Hsp90, 14-3-3-like protein, peroxiredoxin 5, peroxiredoxin 6 and adenine nucleotide translocase 2. Association analysis between the proteomic data and the previous transcriptome data was performed. Quantitative real-time PCR and western blot were carried out to verify the reliability of the proteomics data. The present study provided a comprehensive view of molecular mechanisms regulating WSSV acute infection at the protein level. PMID:27192146

  17. 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. PMID:26046300

  18. In silico Proteome-wide Amino aCid and Elemental Composition (PACE) Analysis of Expression Proteomics Data Provides A Fingerprint of Dominant Metabolic Processes

    PubMed Central

    Good, David M.; Mamdoh, Anwer; Budamgunta, Harshavardhan; Zubarev, Roman A.

    2013-01-01

    Proteome-wide Amino aCid and Elemental composition (PACE) analysis is a novel and informative way of interrogating the proteome. The PACE approach consists of in silico decomposition of proteins detected and quantified in a proteomics experiment into 20 amino acids and five elements (C, H, N, O and S), with protein abundances converted to relative abundances of amino acids and elements. The method is robust and very sensitive; it provides statistically reliable differentiation between very similar proteomes. In addition, PACE provides novel insights into proteome-wide metabolic processes, occurring, e.g., during cell starvation. For instance, both Escherichia coli and Synechocystis down-regulate sulfur-rich proteins upon sulfur deprivation, but E. coli preferentially down-regulates cysteine-rich proteins while Synechocystis mainly down-regulates methionine-rich proteins. Due to its relative simplicity, flexibility, generality and wide applicability, PACE analysis has the potential of becoming a standard analytical tool in proteomics. PMID:23917074

  19. Application of Differential Proteomic Analysis to Authenticate Ophiocordyceps sinensis.

    PubMed

    Zhang, Shiwei; Lai, Xintian; Li, Bifang; Wu, Cong; Wang, Shifeng; Chen, Xuejian; Huang, Jingmin; Yang, Guowu

    2016-03-01

    Ophiocordyceps sinensis (Berk.) Sacc. is one of the most well-known fungi in traditional Chinese medicine and is attracting attention because of its nutritious and medicinal properties. The present study aimed to produce a proteomic map to identify common O. sinensis proteins. The caterpillar body and stroma of O. sinensis collected from five locations and four fungal specimens of similar appearance were examined by two-dimensional electrophoresis (2-DE). Five proteins were identified using MALDI-TOF--TOF/MS, and the 2-DE identification pattern was provided. OCS_04585 and β-lactamase domain-containing protein, the two abundant and characteristic proteins, were separated and purified using liquid-phase isoelectric focusing. The products were high-quality materials that can be used for future protein-function studies and immunoassay development. PMID:26660081

  20. Proteomics Based Identification of Proteins with Deregulated Expression in B Cell Lymphomas.

    PubMed

    Wu, Rui; Nijland, Marcel; Rutgers, Bea; Veenstra, Rianne; Langendonk, Myra; van der Meeren, Lotte E; Kluin, Philip M; Li, Guanwu; Diepstra, Arjan; Chiu, Jen-Fu; van den Berg, Anke; Visser, Lydia

    2016-01-01

    Follicular lymphoma and diffuse large B cell lymphomas comprise the main entities of adult B cell malignancies. Although multiple disease driving gene aberrations have been identified by gene expression and genomic studies, only a few studies focused at the protein level. We applied 2 dimensional gel electrophoresis to compare seven GC B cell non Hodgkin lymphoma (NHL) cell lines with a lymphoblastoid cell line (LCL). An average of 130 spots were at least two folds different in intensity between NHL cell lines and the LCL. We selected approximately 38 protein spots per NHL cell line and linked them to 145 unique spots based on the location in the gel. 34 spots that were found altered in at least three NHL cell lines when compared to LCL, were submitted for LC-MS/MS. This resulted in 28 unique proteins, a substantial proportion of these proteins were involved in cell motility and cell metabolism. Loss of expression of B2M, and gain of expression of PRDX1 and PPIA was confirmed in the cell lines and primary lymphoma tissue. Moreover, inhibition of PPIA with cyclosporine A blocked cell growth of the cell lines, the effect size was associated with the PPIA expression levels. In conclusion, we identified multiple differentially expressed proteins by 2-D proteomics, and showed that some of these proteins might play a role in the pathogenesis of NHL. PMID:26752561

  1. 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

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

    PubMed

    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

  3. 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. PMID:24030930

  4. DIFFERENTIAL PROTEOME ANALYSIS OF THE MALE AND FEMALE ANTENNAE FROM Holotrichia parallela.

    PubMed

    Zhang, Ju-Hong; Wang, Shang; Yang, Shuang; Yi, Jiankun; Liu, Yan; Xi, Jing-Hui

    2016-08-01

    To understand the olfactory mechanisms of Holotrichia parallela antennae in detecting volatile compounds in the environment, protein profiles of H. parallela antennae were analyzed using two-dimensional electrophoresis followed by mass spectrometry and bioinformatics analyses. Approximately 1,100 protein spots in silver staining gel were detected. Quantitative image analysis revealed that in total 47 protein spots showed significant changes in different genders of adult antennae. Thirty-five differentially expressed proteins were identified by Matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/TOF) tandem mass spectrometer, among which 65.7% are involved in carbohydrate and energy metabolism, antioxidant system, transport, and amino acid/nucleotide metabolism. Some proteins identified here have not been reported previously in insect antennae. Identified male-biased proteins included odorant-binding protein 4, pheromone-binding protein-related protein 2, odorant-binding protein 14, prophenoloxidase-I, acyl-CoA dehydrogenase, aldo-keto reductase-like, carbamoyl phosphate synthetase, etc. whereas some proteins are female biased, such as antennae-rich cytochrome P450, aldehyde dehydrogenase, and putative glutamine synthetase. Alterations in the levels of some proteins were further confirmed by real time polymerase chain reaction (RT-PCR). The proteomic resources displayed here are valuable for the discovery of proteins from H. parallela antennae. PMID:27396371

  5. 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. PMID:26617253

  6. 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

  7. Proteomic analysis for the impact of hypercholesterolemia on expressions of hepatic drug transporters and metabolizing enzymes.

    PubMed

    Liu, Yan; Pu, Qiang-Hong; Wu, Ming-Jun; Yu, Chao

    2016-10-01

    1. Our objective is to investigate the alterations of hepatic drug transporters and metabolizing enzymes in hypercholesterolemia. Male Sprague-Dawley rats were fed high-cholesterol chows for 8 weeks to induce hypercholesterolemia. Protein levels of hepatic drug transporters and metabolizing enzymes were analyzed by iTRAQ labeling coupled with LC TRIPLE-TOF. 2. Total 239 differentially expressed proteins were identified using proteomic analysis. Among those, protein levels of hepatic drug transporters (MRP2, ABCD3, OAT2, SLC25A12, SCL38A3, SLC2A2 and SLC25A5) and metabolizing enzymes (CYP2B3, CYP2C7, CYP2C11, CYP2C13, CYP4A2 and UGT2B) were markedly reduced, but the levels of CYP2C6 and CYP2E1 were increased in hypercholesterolemia group compared to control. Decreased expressions of drug transporters MRP2 and OAT2 were further confirmed by real time quantitative PCR (RT-qPCR) and western blot. 3. Ingenuity pathway analysis revealed that these differentially expressed proteins were regulated by various signaling pathways including nuclear receptors and inflammatory cytokines. One of the nuclear receptor candidates, liver X receptor alpha (LXRα), was further validated by RT-qPCR and western blot. Additionally, LXRα agonist T0901317 rescued the reduced expressions of MRP2 and OAT2 in HepG2 cells in hypercholesterolemic serum treatment. 4. Our present results indicated that hypercholesterolemia affected the expressions of various drug transporters and metabolizing enzymes in liver via nuclear receptors pathway. Especially, decreased function of LXRα contributes to the reduced expressions of MRP2 and OAT2. PMID:26887802

  8. Heat-shock response in Arabidopsis thaliana explored by multiplexed quantitative proteomics using differential metabolic labeling.

    PubMed

    Palmblad, Magnus; Mills, Davinia J; Bindschedler, Laurence V

    2008-02-01

    We have developed a general method for multiplexed quantitative proteomics using differential metabolic stable isotope labeling and mass spectrometry. The method was successfully used to study the dynamics of heat-shock response in Arabidopsis thaliana. A number of known heat-shock proteins were confirmed, and some proteins not previously associated with heat shock were discovered. The method is applicable in stable isotope labeling and allows for high degrees of multiplexing. PMID:18189342

  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. PMID:26934482

  10. 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. PMID:25944409

  11. Bayesian identification of protein differential expression in multi-group isobaric labelled mass spectrometry data.

    PubMed

    Jow, Howsun; Boys, Richard J; Wilkinson, Darren J

    2014-10-01

    In this paper we develop a Bayesian statistical inference approach to the unified analysis of isobaric labelled MS/MS proteomic data across multiple experiments. An explicit probabilistic model of the log-intensity of the isobaric labels' reporter ions across multiple pre-defined groups and experiments is developed. This is then used to develop a full Bayesian statistical methodology for the identification of differentially expressed proteins, with respect to a control group, across multiple groups and experiments. This methodology is implemented and then evaluated on simulated data and on two model experimental datasets (for which the differentially expressed proteins are known) that use a TMT labelling protocol. PMID:25153608

  12. Upc2p-associated differential protein expression in Candida albicans.

    PubMed

    Hoehamer, Christopher F; Cummings, Edwin D; Hilliard, George M; Morschhäuser, Joachim; David Rogers, Phillip

    2009-10-01

    The gain-of-function mutation G648D in UPC2 causes ERG11 up-regulation and increased fluconazole resistance in Candida albicans. In this study, we performed 2-DE and PMF to identify proteomic alterations in an ERG11-overexpressing fluconazole-resistant C. albicans clinical isolate compared with its fluconazole-susceptible parent strain. We identified 23 differentially expressed proteins, and among them, seven became differentially expressed in a C. albicans wild-type strain after the introduction of a UPC2 allele carrying this mutation. These Upc2p-regulated proteins may contribute to fluconazole resistance in C. albicans. PMID:19750515

  13. Large-scale differential proteome analysis in Plasmodium falciparum under drug treatment.

    PubMed

    Prieto, Judith Helena; Fischer, Elisabeth; Koncarevic, Sasa; Yates, John; Becker, Katja

    2015-01-01

    Here, we establish a methodology for large-scale quantitative proteomics using SIL (stable isotope labeling) to examine protein expression changes in trophozoite stages of the malaria parasite Plasmodium falciparum following drug treatment. For this purpose, exposure to (13)C6 (15)N1-isoleucine was optimized in order to obtain 99% atomic enrichment. Proteome fractionation with anion exchange chromatography was used to reduce sample complexity and increase quantitative coverage of protein expression. Tryptic peptides of subfractions were subjected to SCX/RP separation, measured by LC-MS/MS, and quantified using the software tool Census. In drug-treated parasites, we identified a total number of 1,253 proteins, thus increasing the overall number of proteins so far identified in the trophozoite stage by 30% in the previous literature. A relative quantification was obtained for more than 800 proteins. About 5% of proteins showed a clear up- or downregulation upon drug treatment. PMID:25388121

  14. Mature adipocyte proteome reveals differentially altered protein abundances between lean, overweight and morbidly obese human subjects.

    PubMed

    Benabdelkamel, Hicham; Masood, Afshan; Almidani, Ghaith M; Alsadhan, Abdulmajeed A; Bassas, Abdulelah F; Duncan, Mark W; Alfadda, Assim A

    2015-02-01

    Overweight (OW) and obese individuals are considered to be graded parts of the scale having increasing weight as a common feature. They may not, however, be part of the same continuum and may differ metabolically. In this study we applied an untargeted proteomic approach to compare protein abundances in mature adipocytes derived from the subcutaneous adipose tissue of overweight and morbidly obese female subjects to those of lean age matched controls. Mature adipocytes were isolated from liposuction samples of abdominal subcutaneous adipose tissue collected from both lean (L; n = 7, 23.3 ± 0.4 kg/m(2); mean BMI ± SD), overweight (OW; n = 8, 27.9 ± 0.6 kg/m(2); mean BMI ± SD) and morbidly obese (MOB; n = 7, 44.8 ± 3.8 kg/m(2); mean BMI ± SD) individuals. Total protein extracts were then compared by two-dimensional difference in gel electrophoresis (2D DIGE). One hundred and ten differentially expressed protein spots (i.e., fitting the statistical criteria ANOVA test, p < 0.05; fold-change ≥1.5) were detected, and of these, 89 were identified by MALDI-TOF mass spectrometry. Of these, 66 protein spots were common to both groups whereas 23 were unique to the MOB group. Significant differences were evident in the abundances of key proteins involved in glucose and lipid metabolism, energy regulation, cytoskeletal structure and redox control signaling pathways. Differences in the abundance of some chaperones were also evident. The differentially abundant proteins were investigated using Ingenuity Pathway Analysis (IPA) to establish their associations with known biological functions. The network identified in the OW group with the highest score relates to-: cell-to-cell signaling and interaction; in contrast, in the MOB group the major interacting pathways are associated with lipid metabolism, small molecule biochemistry and cancer. The differences in abundance of the differentially regulated proteins were validated by

  15. Inferring differentiation pathways from gene expression

    PubMed Central

    Costa, Ivan G.; Roepcke, Stefan; Hafemeister, Christoph; Schliep, Alexander

    2008-01-01

    Motivation: The regulation of proliferation and differentiation of embryonic and adult stem cells into mature cells is central to developmental biology. Gene expression measured in distinguishable developmental stages helps to elucidate underlying molecular processes. In previous work we showed that functional gene modules, which act distinctly in the course of development, can be represented by a mixture of trees. In general, the similarities in the gene expression programs of cell populations reflect the similarities in the differentiation path. Results: We propose a novel model for gene expression profiles and an unsupervised learning method to estimate developmental similarity and infer differentiation pathways. We assess the performance of our model on simulated data and compare it with favorable results to related methods. We also infer differentiation pathways and predict functional modules in gene expression data of lymphoid development. Conclusions: We demonstrate for the first time how, in principal, the incorporation of structural knowledge about the dependence structure helps to reveal differentiation pathways and potentially relevant functional gene modules from microarray datasets. Our method applies in any area of developmental biology where it is possible to obtain cells of distinguishable differentiation stages. Availability: The implementation of our method (GPL license), data and additional results are available at http://algorithmics.molgen.mpg.de/Supplements/InfDif/ Contact: filho@molgen.mpg.de, schliep@molgen.mpg.de Supplementary information: Supplementary data is available at Bioinformatics online. PMID:18586709

  16. Differential Proteomics and Functional Research following Gene Therapy in a Mouse Model of Leber Congenital Amaurosis

    PubMed Central

    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. PMID:22953002

  17. 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. PMID:22953002

  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. Utility of proteomics techniques for assessing protein expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  20. Utility of proteomics techniques for assessing protein expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  1. 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

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

    PubMed

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

    2016-01-01

    Valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA) are both HDAC inhibitors (HDACi). Previous studies indicated that both inhibitors show therapeutic effects on acute myeloid leukaemia (AML), while the differential impacts of the two different HDACi on AML treatment still remains elusive. In this study, using 3-plex SILAC based quantitative proteomics technique, anti-acetyllysine antibody based affinity enrichment, high resolution LC-MS/MS and intensive bioinformatic analysis, the quantitative proteome and acetylome in SAHA and VPA treated AML HL60 cells were extensively studied. In total, 5,775 proteins and 1,124 lysine acetylation sites were successfully obtained in response to VAP and SAHA treatment. It is found that VPA and SAHA treatment differently induced proteome and acetylome profiling in AML HL60 cells. This study revealed the differential impacts of VPA and SAHA on proteome/acetylome in AML cells, deepening our understanding of HDAC inhibitor mediated AML therapeutics. PMID:26822725

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

    PubMed Central

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

    2016-01-01

    Valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA) are both HDAC inhibitors (HDACi). Previous studies indicated that both inhibitors show therapeutic effects on acute myeloid leukaemia (AML), while the differential impacts of the two different HDACi on AML treatment still remains elusive. In this study, using 3-plex SILAC based quantitative proteomics technique, anti-acetyllysine antibody based affinity enrichment, high resolution LC-MS/MS and intensive bioinformatic analysis, the quantitative proteome and acetylome in SAHA and VPA treated AML HL60 cells were extensively studied. In total, 5,775 proteins and 1,124 lysine acetylation sites were successfully obtained in response to VAP and SAHA treatment. It is found that VPA and SAHA treatment differently induced proteome and acetylome profiling in AML HL60 cells. This study revealed the differential impacts of VPA and SAHA on proteome/acetylome in AML cells, deepening our understanding of HDAC inhibitor mediated AML therapeutics. PMID:26822725

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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. 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. PMID:26992294

  6. 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. PMID:21356255

  7. Genomic and proteomic analyses reveal multiple homologs of genes encoding enzymes of the methanol:coenzyme M methyltransferase system that are differentially expressed in methanol- and acetate-grown Methanosarcina thermophila.

    PubMed

    Ding, Yan-Huai R; Zhang, Shi-Ping; Tomb, Jean-Francois; Ferry, James G

    2002-09-24

    Each of the genomic sequences of Methanosarcina acetivorans, Methanosarcina mazei, and Methanosarcina thermophila revealed two homologs of mtaA, three homologs of mtaB, and three homologs of mtaC encoding enzymes specific for methanogenesis from methanol. Two-dimensional gel electrophoretic analyses of polypeptides from M. thermophila established that methanol induces the expression of mtaA-1, mtaB-1, mtaB-2, mtaB-3, mtaC-1, mtaC-2, and mtaC-3 whereas mtaB-3 and mtaC-3 are constitutively expressed in acetate-grown cells. The gene product of one of three mttC homologs, encoding trimethylamine-specific methyltransferase I, was detected in methanol- but not acetate-grown M. thermophila. A postulated role for the multiple homologs is discussed. PMID:12393212

  8. 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

  9. 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 Central

    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

  10. Differential expression of proteins in monozygotic twins with discordance of infantile esotropic phenotypes

    PubMed Central

    Bai, Haiqing; Yan, Zhiyong; Ma, Yuna; Li, Hui

    2011-01-01

    Purpose To identify strabismus-related proteins, we performed proteome analysis in monozygotic twins with discordance of congenital esotropic phenotypes and in normal children. Methods Surface-enhanced laser desorption/ ionization time-of-flight mass spectrometry (SELDI-TOF-MS) technology was used to detect changes in protein expression in a pair of twins with discordant esotropic phenotypes (twin A is orthotropic and twin B is esotropic). In addition, two non-twin esotropic children and two orthotropic children of the same age were chosen. The differentially expressed proteome obtained was validated in twelve non-twin esotropic children and eighteen orthotropic children and compared to the protein database. Results We detected four differentially expressed proteins in monozygotic twins with discordance of congenital esotropic phenotypes. The corresponding molecular weights were 4,146 Da, 4,801 Da, 7,786 Da, and 5,859 Da, respectively. Among these 4 proteins, the first three proteins were down-regulated and the last was upregulated. The initial characterization of these detected proteins via protein library revealed that their characteristics were similar to those of the glucagon precursor, pituitary adenylate cyclase-activating polypeptide (PACAP), camp-dependent protein kinase inhibitor α, and anti-metastasis gene (antigen), respectively. Conclusions There were differentially expressed proteins between monozygotic twins with discordance of congenital esotropic phenotypes and normal children. These differentially expressed proteins were mainly down-regulated in the strabismus patients and may be involved in the occurrence and development of congenital esotropia. PMID:21738391

  11. Differential placental gene expression in severe preeclampsia.

    PubMed

    Sitras, V; Paulssen, R H; Grønaas, H; Leirvik, J; Hanssen, T A; Vårtun, A; Acharya, G

    2009-05-01

    We investigated the global placental gene expression profile in severe preeclampsia. Twenty-one women were randomly selected from 50 participants with uncomplicated pregnancies to match 21 patients with severe preeclampsia. A 30K Human Genome Survey Microarray v.2.0 (Applied Biosystems) was used to evaluate the gene expression profile. After RNA isolation, five preeclamptic placentas were excluded due to poor RNA quality. The series composed of 37 hybridizations in a one-channel detection system of chemiluminescence emitted by the microarrays. An empirical Bayes analysis was applied to find differentially expressed genes. In preeclamptic placentas 213 genes were significantly (fold-change>or=2 and pdifferentially expressed genes were associated with Alzheimer disease, angiogenesis, Notch-, TGFbeta- and VEGF-signalling pathways. Sixteen genes best discriminated preeclamptic from normal placentas. Comparison between early- (<34 weeks) and late-onset preeclampsia showed 168 differentially expressed genes with oxidative stress, inflammation, and endothelin signalling pathways mainly involved in early-onset disease. Validation of the microarray results was performed by RT-PCR, quantitative urine hCG measurement and placental histopathologic examination. In summary, placental gene expression is altered in preeclampsia and we provide a comprehensive list of the differentially expressed genes. Placental gene expression is different between early- and late-onset preeclampsia, suggesting differences in pathophysiology. PMID:19249095

  12. 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

  13. Proteomic analysis of MCF-7 cell lines expressing the zinc-finger or the proline-rich domain of retinoblastoma-interacting-zinc-finger protein.

    PubMed

    Chambery, Angela; Farina, Annarita; Di Maro, Antimo; Rossi, Mariangela; Abbondanza, Ciro; Moncharmont, Bruno; Malorni, Livia; Cacace, Giuseppina; Pocsfalvi, Gabriella; Malorni, Antonio; Parente, Augusto

    2006-05-01

    To identify a growth-promoting activity related to retinoblastoma-interacting-zinc-finger (RIZ) protein, differential protein expression of MCF-7 cell lines expressing the zinc-finger or the proline-rich domain of RIZ protein was analyzed by a robust bottom-up mass-spectrometry proteomic approach. Spots corresponding to qualitative and quantitative differences in protein expression have been selected and identified. Some of these proteins have been previously reported as being associated with different types of carcinomas or involved in cell proliferation and differentiation. Knowledge of specific differentially expressed proteins by MCF-7-derived cell lines expressing RIZ different domains will provide the basis for identifying a growth-promoting activity related to RIZ gene products. PMID:16674107

  14. Comprehensive analyses of prostate gene expression: convergence of expressed sequence tag databases, transcript profiling and proteomics.

    PubMed

    Nelson, P S; Han, D; Rochon, Y; Corthals, G L; Lin, B; Monson, A; Nguyen, V; Franza, B R; Plymate, S R; Aebersold, R; Hood, L

    2000-05-01

    Several methods have been developed for the comprehensive analysis of gene expression in complex biological systems. Generally these procedures assess either a portion of the cellular transcriptome or a portion of the cellular proteome. Each approach has distinct conceptual and methodological advantages and disadvantages. We have investigated the application of both methods to characterize the gene expression pathway mediated by androgens and the androgen receptor in prostate cancer cells. This pathway is of critical importance for the development and progression of prostate cancer. Of clinical importance, modulation of androgens remains the mainstay of treatment for patients with advanced disease. To facilitate global gene expression studies we have first sought to define the prostate transcriptome by assembling and annotating prostate-derived expressed sequence tags (ESTs). A total of 55000 prostate ESTs were assembled into a set of 15953 clusters putatively representing 15953 distinct transcripts. These clusters were used to construct cDNA microarrays suitable for examining the androgen-response pathway at the level of transcription. The expression of 20 genes was found to be induced by androgens. This cohort included known androgen-regulated genes such as prostate-specific antigen (PSA) and several novel complementary DNAs (cDNAs). Protein expression profiles of androgen-stimulated prostate cancer cells were generated by two-dimensional electrophoresis (2-DE). Mass spectrometric analysis of androgen-regulated proteins in these cells identified the metastasis-suppressor gene NDKA/nm23, a finding that may explain a marked reduction in metastatic potential when these cells express a functional androgen receptor pathway. PMID:10870968

  15. Proteome changes during bone mesenchymal stem cell differentiation into photoreceptor-like cells in vitro

    PubMed Central

    Hong, Yu; Xu, Guo-Xing

    2011-01-01

    Human bone marrow stem cell (BMSC) may be directed to differentiate into multiple cell types, including adipocyte, chondrocyte, osteocyte and photoreceptor, among others. At present, little is known about the features of the BMSC and the protein control mechanism underlying their differentiation into photoreceptor-like cells. In the present study, BMSCs are induced to differentiate into photoreceptor-like cells in an in vitro model simulating the in vivo microenvironment. Up to 32 proteins are identified and differentially expressed through two-dimensional difference gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to establish a differential protein database for photoreceptor-like cells from BMSC-induced differentiation. Western blot analysis further confirms the expression of some of the identified proteins. The present study proposes the total protein expression and possible molecular mechanism during the differentiation of BMSCs into photoreceptor cells. PMID:22553704

  16. Differential Protein Expression in Honeybee (Apis mellifera L.) Larvae: Underlying Caste Differentiation

    PubMed Central

    Li, Jianke; Wu, Jing; Begna Rundassa, Desalegn; Song, Feifei; Zheng, Aijuan; Fang, Yu

    2010-01-01

    Honeybee (Apis mellifera) exhibits divisions in both morphology and reproduction. The queen is larger in size and fully developed sexually, while the worker bees are smaller in size and nearly infertile. To better understand the specific time and underlying molecular mechanisms of caste differentiation, the proteomic profiles of larvae intended to grow into queen and worker castes were compared at 72 and 120 hours using two dimensional electrophoresis (2-DE), network, enrichment and quantitative PCR analysis. There were significant differences in protein expression between the two larvae castes at 72 and 120 hours, suggesting the queen and the worker larvae have already decided their fate before 72 hours. Specifically, at 72 hours, queen intended larvae over-expressed transketolase, aldehyde reductase, and enolase proteins which are involved in carbohydrate metabolism and energy production, imaginal disc growth factor 4 which is a developmental related protein, long-chain-fatty-acid CoA ligase and proteasome subunit alpha type 5 which metabolize fatty and amino acids, while worker intended larvae over-expressed ATP synthase beta subunit, aldehyde dehydrogenase, thioredoxin peroxidase 1 and peroxiredoxin 2540, lethal (2) 37 and 14-3-3 protein epsilon, fatty acid binding protein, and translational controlled tumor protein. This differential protein expression between the two caste intended larvae was more pronounced at 120 hours, with particular significant differences in proteins associated with carbohydrate metabolism and energy production. Functional enrichment analysis suggests that carbohydrate metabolism and energy production and anti-oxidation proteins play major roles in the formation of caste divergence. The constructed network and validated gene expression identified target proteins for further functional study. This new finding is in contrast to the existing notion that 72 hour old larvae has bipotential and can develop into either queen or worker based on

  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. PMID:25182623

  18. Ion Current-Based Proteomic Profiling for Understanding the Inhibitory Effect of Tumor Necrosis Factor Alpha on Myogenic Differentiation.

    PubMed

    Tu, Chengjian; Bu, Yahao; Vujcic, Marija; Shen, Shichen; Li, Jun; Qu, Miao; Hangauer, David; Clements, James L; Qu, Jun

    2016-09-01

    Despite a demonstrated role for TNF-α in promoting muscle wasting and cachexia, the associated molecular mechanisms and signaling pathways of myoblast differentiation dysregulated by TNF-α remain poorly understood. This study presents well-controlled proteomic profiling as a means to investigate the mechanisms of TNF-α-regulated myogenic differentiation. Primary human muscle precursor cells (MPCs) cultured in growth medium (GM), differentiation medium (DM) to induce myogenic differentiation, and DM with 20 ng/mL of TNF-α (n = 5/group) were comparatively analyzed by an ion current-based quantitative platform consisting of reproducible sample preparation/on-pellet digestion, a long-column nano-LC separation, and ion current-based differential analysis. The inhibition of myogenic differentiation by TNF-α was confirmed by reduced formation of multinucleated myotubes and the recovered expression of altered myogenic proteins such as MYOD and myogenin during myogenic differentiation. Functional analysis and validation by immunoassay analysis suggested that the cooperation of NF-κB and STAT proteins is responsible for dysregulated differentiation in MPCs by TNF-α treatment. Increased MHC class I components such as HLA-A, HLA-B, HLA-C, and beta-2-microglobulin were also observed in cultures in DM treated with TNF-α. Interestingly, inhibition of the cholesterol biosynthesis pathway during myogenic differentiation induced by serum starvation was not recovered by TNF-α treatment, which combined with previous reports, implies that this process may be an early event of myogenesis. This finding could lay the foundation for the potential use of statins in modulating myogenesis through cholesterol, for example, in stem cell-based myocardial infarction treatment, where differentiation of myoblasts and stem cells into force-generating mature muscle cells is a key step to the therapeutic capacity. In conclusion, the landscapes of altered transcription regulators, metabolic

  19. Proteomic analysis reveals the differential histone programs between male germline cells and vegetative cells in Lilium davidii.

    PubMed

    Yang, Hao; Yang, Ning; Wang, Tai

    2016-03-01

    In flowering plants, male germline fate is determined after asymmetric division of the haploid microspore. Daughter cells have distinct fates: the generative cell (GC) undergoes further mitosis to generate sperm cells (SCs), and the vegetative cell (VC) terminally differentiates. However, our understanding of the mechanisms underlying germline development remains limited. Histone variants and modifications define chromatin states, and contribute to establishing and maintaining cell identities by affecting gene expression. Here, we constructed a lily protein database, then extracted and detailed histone entries into a comprehensive lily histone database. We isolated large amounts of nuclei from VCs, GCs and SCs from lily, and profiled histone variants of all five histone families in all three cell types using proteomics approaches. We revealed 92 identities representing 32 histone variants: six for H1, 11 for H2A, eight for H2B, five for H3 and two for H4. Nine variants, including five H1, two H2B, one H3 and one H4 variant, specifically accumulated in GCs and SCs. We also detected H3 modification patterns in the three cell types. GCs and SCs had almost identical histone profiles and similar H3 modification patterns, which were significantly different from those of VCs. Our study also revealed the presence of multiple isoforms, and differential expression patterns between isoforms of a variant. The results suggest that differential histone programs between the germline and companion VCs may be established following the asymmetric division, and are important for identity establishment and differentiation of the male germline as well as the VC. PMID:26846354

  20. Comparative Proteomic Analysis of Extracellular Vesicles Isolated by Acoustic Trapping or Differential Centrifugation.

    PubMed

    Rezeli, Melinda; Gidlöf, Olof; Evander, Mikael; Bryl-Górecka, Paulina; Sathanoori, Ramasri; Gilje, Patrik; Pawłowski, Krzysztof; Horvatovich, Péter; Erlinge, David; Marko-Varga, György; Laurell, Thomas

    2016-09-01

    Extracellular vesicles (ECVs), including microparticles and exosomes, are submicrometer membrane vesicles released by diverse cell types upon activation or stress. Circulating ECVs are potential reservoirs of disease biomarkers, and the complexity of these vesicles is significantly lower compared to their source, blood plasma, which makes ECV-based biomarker studies more promising. Proteomic profiling of ECVs is important not only to discover new diagnostic or prognostic markers but also to understand their roles in biological function. In the current study, we investigated the protein composition of plasma-derived ECVs isolated by acoustic seed trapping. Additionally, the protein composition of ECVs isolated with acoustic trapping was compared to that isolated with a conventional differential centrifugation protocol. Finally, the proteome of ECVs originating from ST-elevation myocardial infarction patients was compared with that of healthy controls using label-free LC-MS quantification. The acoustic trapping platform allows rapid and automated preparation of ECVs from small sample volumes, which are therefore well-suited for biobank repositories. We found that the protein composition of trapped ECVs is very similar to that isolated by the conventional differential centrifugation method. PMID:27487081

  1. 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.

  2. 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.

  3. 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. PMID:23825529

  4. 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

  5. Changes in the proteomic profile during differentiation and maturation of human monocyte-derived dendritic cells stimulated with granulocyte macrophage colony stimulating factor/interleukin-4 and lipopolysaccharide.

    PubMed

    Pereira, Sandra Rodrigues; Faça, Vitor Marcel; Gomes, Glauce Gaspar; Chammas, Roger; Fontes, Aparecida Maria; Covas, Dimas Tadeu; Greene, Lewis Joel

    2005-04-01

    Dendritic cells (DCs) are highly specialized antigen-presenting cells that play an essential role in the immune response. We used the proteomic approach based on two-dimensional gel electrophoresis and mass spectrometry to identify the protein changes that occur during differentiation of DCs from monocytes (Mo) stimulated with granulocyte macrophage colony stimulating factor/interleukin-4 (GM-CSF/IL-4) and during the maturation of immature DCs stimulated with lipopolysaccharide. Sixty-three differentially expressed proteins (+/- two-fold) were unambiguously identified with sequence coverage greater than 20%. They corresponded to only 36 different proteins, because 11 were present as 38 electrophoretic forms. Some proteins such as tropomyosin 4 and heat shock protein 71 presented differentially expressed electrophoretic forms, suggesting that many of the changes in protein expression that accompany differentiation and maturation of DCs occur in post-translationally modified proteins. The largest differences in expression were observed for actin (21-fold in Mo), Rho GDP-dissociation inhibitor 2 (20-fold in Mo), vimentin (eight-fold in immature DCs), lymphocyte-specific protein 1 (12-fold in mature DCs) and thioredoxin (14-fold in mature DCs). Several proteins are directly related to functional and morphological characteristics of DCs, such as cytoskeletal proteins (cytoskeleton rearrangement) and chaperones (antigen processing and presentation), but other proteins have not been assigned specific functions in DCs. Only a few proteins identified here were the same as those reported in proteomic studies of DCs, which used different stimuli to produce the cells (GM-CSF/IL-4 and tumor necrosis factor-alpha). These data suggest that the DC protein profile depends on the stimuli used for differentiation and especially for maturation. PMID:15800872

  6. Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and Quantitative Comparison of the Membrane Proteomes of Self-renewing and Differentiating Human Embryonic Stem Cells*S⃞

    PubMed Central

    Prokhorova, Tatyana A.; Rigbolt, Kristoffer T. G.; Johansen, Pia T.; Henningsen, Jeanette; Kratchmarova, Irina; Kassem, Moustapha; Blagoev, Blagoy

    2009-01-01

    Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful quantitative proteomics platform for comprehensive characterization of complex biological systems. However, the potential of SILAC-based approaches has not been fully utilized in human embryonic stem cell (hESC) research mainly because of the complex nature of hESC culture conditions. Here we describe complete SILAC labeling of hESCs with fully preserved pluripotency, self-renewal capabilities, and overall proteome status that was quantitatively analyzed to a depth of 1556 proteins and 527 phosphorylation events. SILAC-labeled hESCs appear to be perfectly suitable for functional studies, and we exploited a SILAC-based proteomics strategy for discovery of hESC-specific surface markers. We determined and quantitatively compared the membrane proteomes of the self-renewing versus differentiating cells of two distinct human embryonic stem cell lines. Of the 811 identified membrane proteins, six displayed significantly higher expression levels in the undifferentiated state compared with differentiating cells. This group includes the established marker CD133/Prominin-1 as well as novel candidates for hESC surface markers: Glypican-4, Neuroligin-4, ErbB2, receptor-type tyrosine-protein phosphatase ζ (PTPRZ), and Glycoprotein M6B. Our study also revealed 17 potential markers of hESC differentiation as their corresponding protein expression levels displayed a dramatic increase in differentiated embryonic stem cell populations. PMID:19151416

  7. Differential proteomic analysis of STAT6 knockout mice reveals new regulatory function in liver lipid homeostasis.

    PubMed

    Iff, Joël; Wang, Wei; Sajic, Tatjana; Oudry, Nathalie; Gueneau, Estelle; Hopfgartner, Gérard; Varesio, Emmanuel; Szanto, Ildiko

    2009-10-01

    Increased inflammatory signaling is a key feature of metabolic disorders. In this context, the role of increased pro-inflammatory signals has been extensively studied. By contrast, no efforts have been dedicated to study the contrasting scenario: the attenuation of anti-inflammatory signals and their role in metabolic homeostasis. IL-4 and IL-13 are anti-inflammatory cytokines signaling through the Signal Transducer and Activator of Transcription 6 (STAT6). Our study was aimed at evaluating the lack of STAT6 signaling on liver homeostasis. To this end we analyzed the liver proteome of wild type and STAT6 knock-out mice using 2D nanoscale LC-MS/MS with iTRAQ labeling technique. The coordinated changes in proteins identified by this quantitative proteome analysis indicated disturbed lipid homeostasis and a state of hepatocellular stress. Most significantly, the expression of the liver fatty acid binding protein (FABP1) was increased in the knock-out mice. In line with the elevated FABP1 expression we found latent liver lipid accumulation in the STAT6-deficient mice which was further aggravated when mice were challenged by a high fat diet. In conclusion, our study revealed a so far uncharacterized role for STAT6 in regulating liver lipid homeostasis and demonstrates the importance of anti-inflammatory signaling in the defense against the development of liver steatosis. PMID:19663508

  8. Multiplexed, Proteome-Wide Protein Expression Profiling: Yeast Deubiquitylating Enzyme Knockout Strains

    PubMed Central

    Isasa, Marta; Rose, Christopher M.; Elsasser, Suzanne; Navarrete-Perea, José; Paulo, Joao A.; Finley, Daniel J.; Gygi, Steven P.

    2016-01-01

    Characterizing a protein’s function often requires a description of the cellular state in its absence. Multiplexing in mass spectrometry-based proteomics has now achieved the ability to globally measure protein expression levels in yeast from 10 cell states simultaneously. We applied this approach to quantify expression differences in wild type and nine deubiquitylating enzyme (DUB) knockout strains with the goal of creating “information networks” that might provide deeper, mechanistic insights into a protein’s biological role. In total, more than 3700 proteins were quantified with high reproducibility across three biological replicates (30 samples in all). DUB mutants demonstrated different proteomics profiles, consistent with distinct roles for each family member. These included differences in total ubiquitin levels and specific chain linkages. Moreover, specific expression changes suggested novel functions for several DUB family members. For instance, the ubp3Δ mutant showed large expression changes for members of the cytochrome C oxidase complex, consistent with a role for Ubp3 in mitochondrial regulation. Several DUBs also showed broad expression changes for phosphate transporters as well as other components of the inorganic phosphate signaling pathway, suggesting a role for these DUBs in regulating phosphate metabolism. These data highlight the potential of multiplexed proteome-wide analyses for biological investigation and provide a framework for further study of the DUB family. Our methods are readily applicable to the entire collection of yeast deletion mutants and may help facilitate systematic analysis of yeast and other organisms. PMID:26503604

  9. 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

  10. 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.

  11. A Bayesian algorithm for detecting differentially expressed proteins and its application in breast cancer research.

    PubMed

    Santra, Tapesh; Delatola, Eleni Ioanna

    2016-01-01

    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. PMID:27444576

  12. Difference gel electrophoresis identifies differentially expressed proteins in endoscopically collected pancreatic fluid.

    PubMed

    Paulo, Joao A; Lee, Linda S; Banks, Peter A; Steen, Hanno; Conwell, Darwin L

    2011-08-01

    Alterations in the pancreatic fluid proteome of individuals with chronic pancreatitis (CP) may offer insights into the development and progression of the disease. The endoscopic pancreatic 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 CP and three chronic abdominal pain (CAP) 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 CP compared with CAP controls. Proteins with higher abundance in pancreatic fluid from CP individuals include: actin, desmoplankin, α-1-antitrypsin, SNC73, and serotransferrin. Those of relatively lower abundance include carboxypeptidase B, lipase, α-1-antichymotrypsin, α-2-macroglobulin, actin-related protein (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

  13. A Bayesian algorithm for detecting differentially expressed proteins and its application in breast cancer research

    PubMed Central

    Santra, Tapesh; Delatola, Eleni Ioanna

    2016-01-01

    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. PMID:27444576

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

    PubMed

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

    2015-08-11

    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

  15. Differential expression of cysteine desulfurases in soybean

    PubMed Central

    2011-01-01

    Background Iron-sulfur [Fe-S] clusters are prosthetic groups required to sustain fundamental life processes including electron transfer, metabolic reactions, sensing, signaling, gene regulation and stabilization of protein structures. In plants, the biogenesis of Fe-S protein is compartmentalized and adapted to specific needs of the cell. Many environmental factors affect plant development and limit productivity and geographical distribution. The impact of these limiting factors is particularly relevant for major crops, such as soybean, which has worldwide economic importance. Results Here we analyze the transcriptional profile of the soybean cysteine desulfurases NFS1, NFS2 and ISD11 genes, involved in the biogenesis of [Fe-S] clusters, by quantitative RT-PCR. NFS1, ISD11 and NFS2 encoding two mitochondrial and one plastid located proteins, respectively, are duplicated and showed distinct transcript levels considering tissue and stress response. NFS1 and ISD11 are highly expressed in roots, whereas NFS2 showed no differential expression in tissues. Cold-treated plants showed a decrease in NFS2 and ISD11 transcript levels in roots, and an increased expression of NFS1 and ISD11 genes in leaves. Plants treated with salicylic acid exhibited increased NFS1 transcript levels in roots but lower levels in leaves. In silico analysis of promoter regions indicated the presence of different cis-elements in cysteine desulfurase genes, in good agreement with differential expression of each locus. Our data also showed that increasing of transcript levels of mitochondrial genes, NFS1/ISD11, are associated with higher activities of aldehyde oxidase and xanthine dehydrogenase, two cytosolic Fe-S proteins. Conclusions Our results suggest a relationship between gene expression pattern, biochemical effects, and transcription factor binding sites in promoter regions of cysteine desulfurase genes. Moreover, data show proportionality between NFS1 and ISD11 genes expression. PMID:22099069

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

    PubMed

    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

  17. 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

  18. 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. PMID:15276439

  19. 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. PMID:24847760

  20. 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

  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. High molecular mass proteomics analyses of left ventricle from rats subjected to differential swimming training

    PubMed Central

    2012-01-01

    Background Regular exercises are commonly described as an important factor in health improvement, being directly related to contractile force development in cardiac cells. In order to evaluate the links between swimming exercise intensity and cardiac adaptation by using high molecular mass proteomics, isogenic Wistar rats were divided into four groups: one control (CG) and three training groups (TG’s), with low, moderate and high intensity of exercises. In order to evaluate the links between swimming exercise intensity and cardiac adaptation by using high molecular mass proteomics, isogenic Wistar rats were divided into four groups: one control (CG) and three training groups (TG’s), with low, moderate and high intensity of exercises. Results Findings here reported demonstrated clear morphologic alterations, significant cellular injury and increased energy supplies at high exercise intensities. α-MyHC, as well proteins associated with mitochondrial oxidative metabolism were shown to be improved. α-MyHC expression increase 1.2 fold in high intensity training group when compared with control group. α-MyHC was also evaluated by real-time PCR showing a clear expression correlation with protein synthesis data increase in 8.48 fold in high intensity training group. Other myofibrillar protein, troponin , appear only in high intensity group, corroborating the cellular injury data. High molecular masses proteins such as MRS2 and NADH dehydrogenase, involved in metabolic pathways also demonstrate increase expression, respectily 1.5 and 1.3 fold, in response to high intensity exercise. Conclusions High intensity exercise demonstrated an increase expression in some high molecular masses myofibrilar proteins, α-MyHC and troponin. Furthermore this intensity also lead a significant increase of other high molecular masses proteins such as MRS2 and NADH dehydrogenase in comparison to low and moderate intensities. However, high intensity exercise also represented a

  3. 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

  4. Proteomic Analysis of Terminalia chebula Extract-Dependent Changes in Human Lymphoblastic T Cell Protein Expression

    PubMed Central

    Das, Nando Dulal; Jung, Kyoung Hwa; Park, Ji Hyun; Choi, Mi Ran; Lee, Hyung Tae; Kim, Moo Sung; Lee, Sang Rin

    2012-01-01

    Abstract Terminalia chebula is a native plant from southern Asia to southwestern China that is used in traditional medicine for the treatment of malignant tumors and diabetes. This plant also has antibacterial and immunomodulatory properties. The present study assessed T. chebula extract-dependent protein expression changes in Jurkat cells. Matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry and Ingenuity Pathways Analysis (IPA) were performed to assess protein expression and networks, respectively. A comparative proteomic profile was determined in T. chebula extract (50 μg/mL)-treated and control cells; the expressions of β-tubulin, ring finger and CHY zinc finger domain containing 1, and insulin-like growth factor 1 receptor kinase were significantly down-regulated in T. chebula extract-treated Jurkat cells. Moreover, the molecular basis for the T. chebula extract-dependent protein expression changes in Jurkat cells was determined by IPA. Treatment with the T. chebula extract significantly inhibited nuclear factor-κB activity and affected the proteomic profile of Jurkat cells. The molecular network signatures and functional proteomics obtained in this study may facilitate the evaluation of potential antitumor therapeutic targets and elucidate the molecular mechanism of T. chebula extract-dependent effects in Jurkat cells. PMID:22471968

  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. PMID:27072633

  6. Comprehensive identification of substrates for F-box proteins by differential proteomics analysis.

    PubMed

    Yumimoto, Kanae; Matsumoto, Masaki; Oyamada, Koji; Moroishi, Toshiro; Nakayama, Keiichi I

    2012-06-01

    Although elucidation of enzyme-substrate relations is fundamental to the advancement of biology, universal approaches to the identification of substrates for a given enzyme have not been established. It is especially difficult to identify substrates for ubiquitin ligases, given that most such substrates are immediately ubiquitylated and degraded as a result of their association with the enzyme. We here describe the development of a new approach, DiPIUS (differential proteomics-based identification of ubiquitylation substrates), to the discovery of substrates for ubiquitin ligases. We applied DiPIUS to Fbxw7α, Skp2, and Fbxl5, three of the most well-characterized F-box proteins, and identified candidate substrates including previously known targets. DiPIUS is thus a powerful tool for unbiased and comprehensive screening for substrates of ubiquitin ligases. PMID:22524983

  7. Proteomics-Based Identification of Differentially Abundant Proteins from Human Keratinocytes Exposed to Arsenic Trioxide

    PubMed Central

    Udensi, Udensi K; Tackett, Alan J; Byrum, Stephanie; Avaritt, Nathan L; Sengupta, Deepanwita; Moreland, Linley W; Tchounwou, Paul B; Isokpehi, Raphael D

    2014-01-01

    Introduction Arsenic is a widely distributed environmental toxicant that can cause multi-tissue pathologies. Proteomic assays allow for the identification of biological processes modulated by arsenic in diverse tissue types. Method The altered abundance of proteins from HaCaT human keratinocyte cell line exposed to arsenic was quantified using a label-free LC-MS/MS mass spectrometry workflow. Selected proteomics results were validated using western blot and RT-PCR. A functional annotation analytics strategy that included visual analytical integration of heterogeneous data sets was developed to elucidate functional categories. The annotations integrated were mainly tissue localization, biological process and gene family. Result The abundance of 173 proteins was altered in keratinocytes exposed to arsenic; in which 96 proteins had increased abundance while 77 proteins had decreased abundance. These proteins were also classified into 69 Gene Ontology biological process terms. The increased abundance of transferrin receptor protein (TFRC) was validated and also annotated to participate in response to hypoxia. A total of 33 proteins (11 increased abundance and 22 decreased abundance) were associated with 18 metabolic process terms. The Glutamate--cysteine ligase catalytic subunit (GCLC), the only protein annotated with the term sulfur amino acid metabolism process, had increased abundance while succinate dehydrogenase [ubiquinone] iron-sulfur subunit, mitochondrial precursor (SDHB), a tumor suppressor, had decreased abundance. Conclusion A list of 173 differentially abundant proteins in response to arsenic trioxide was grouped using three major functional annotations covering tissue localization, biological process and protein families. A possible explanation for hyperpigmentation pathologies observed in arsenic toxicity is that arsenic exposure leads to increased iron uptake in the normally hypoxic human skin. The proteins mapped to metabolic process terms and

  8. Proteomic profiles in acute respiratory distress syndrome differentiates survivors from non-survivors.

    PubMed

    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

  9. 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

    2012-01-01

    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. PMID:21982827

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

    PubMed Central

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

    2016-01-01

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

  11. Differential secretome analysis of Pseudomonas syringae pv tomato using gel-free MS proteomics

    PubMed Central

    Schumacher, Jörg; Waite, Christopher J.; Bennett, Mark H.; Perez, Marcos F.; Shethi, Kishwar; Buck, Martin

    2014-01-01

    The plant pathogen Pseudomonas syringae pv.tomato (DC3000) causes virulence by delivering effector proteins into host plant cells through its type three secretion system (T3SS). In response to the plant environment DC3000 expresses hypersensitive response and pathogenicity genes (hrp). Pathogenesis depends on the ability of the pathogen to manipulate the plant metabolism and to inhibit plant immunity, which depends to a large degree on the plant's capacity to recognize both pathogen and microbial determinants (PAMP/MAMP-triggered immunity). We have developed and employed MS-based shotgun and targeted proteomics to (i) elucidate the extracellular and secretome composition of DC3000 and (ii) evaluate temporal features of the assembly of the T3SS and the secretion process together with its dependence of pH. The proteomic screen, under hrp inducing in vitro conditions, of extracellular and cytoplasmatic fractions indicated the segregated presence of not only T3SS implicated proteins such as HopP1, HrpK1, HrpA1 and AvrPto1, but also of proteins not usually associated with the T3SS or with pathogenicity. Using multiple reaction monitoring MS (MRM-MS) to quantify HrpA1 and AvrPto1, we found that HrpA1 is rapidly expressed, at a strict pH-dependent rate and is post-translationally processed extracellularly. These features appear to not interfere with rapid AvrPto1 expression and secretion but may suggest some temporal post-translational regulatory mechanism of the T3SS assembly. The high specificity and sensitivity of the MRM-MS approach should provide a powerful tool to measure secretion and translocation in infected tissues. PMID:25071788

  12. Comparative proteomics and expression analysis of five genes in Epicauta chinensis larvae from the first to fifth instar.

    PubMed

    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

  13. 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. PMID:26359799

  14. 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

  15. Differential proteomics analysis of proteins from human diabetic and age-related cataractous lenses

    PubMed Central

    Zhu, Jing; Shao, Jun; Yao, Yong; Chu, Zhao Dong; Yu, Qian Qian; Zhao, Wei; Lin, Qing; Zhang, Zi Yin

    2013-01-01

    Backgound: To investigate the differential lens proteomics between diabetic cataract, age-related cataract, and natural subjects. Materials and Methods: Two-dimensional electrophoresis (2-DE), mass spectrometry (MS), and enzyme-linked immunosorbent assay (ELISA) were employed. Total soluble proteins in lenses of type I diabetic cataract, age-related cataract (nondiabetic) patients, and normal control were extracted and subjected to 2-DE. The differential protein spots were recovered, digested with trypsin, and further applied to MALDI-TOF-MS. ELISA analysis was used to determine the levels of differential proteins in lenses of three groups. Results: 2-DE analysis reflected that lens proteins of normal control, diabetic, and age-related cataract subjects were in the section of pH 5-9 and the relative molecular weights were 14-97 kDa, while relative molecular weight of more abundant crystallines was localized at 20-31 kDa. five differential protein spots were detected and identified using MALDI-TOF-MS, including beta-crystallin A3, alpha-crystallin B chain, chain A of crystal structure of truncated human beta-B1-crystallin, beta-crystallin B1, and an interesting unnamed protein product highly similar to alpha-crystallin B chain, respectively. ELISA analysis revealed that lenses of diabetic cataract patients should contain significantly more concentrations of beta-crystallin A3, alpha-crystallin B chain, and beta-crystallin B1 than those of age-related cataract patients and normal control. Conclusion: This study clearly reflected the differential proteins of diabetic cataract, age-related cataract lenses compared with natural subjects, and it is helpful for the further research on the principles and mechanisms of different types of cataract. PMID:24520233

  16. 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.

  17. A proteomic approach to investigating gene cluster expression and secondary metabolite functionality in Aspergillus fumigatus.

    PubMed

    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

  18. 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

  19. Differential lysine acetylation profiles of Erwinia amylovora strains revealed by proteomics

    PubMed Central

    Wu, Xia; Vellaichamy, Adaikkalam; Wang, Dongping; Zamdborg, Leonid; Kelleher, Neil L.; Huber, Steven C.; Zhao, Youfu

    2015-01-01

    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. PMID:23234799

  20. 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. PMID:23234799

  1. 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

  2. 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

  3. 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

    2015-01-01

    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

  4. Differential proteomic profiling reveals regulatory proteins and novel links between primary metabolism and spinosad production in Saccharopolyspora spinosa

    PubMed Central

    2014-01-01

    Background Saccharopolyspora spinosa is an important producer of antibiotic spinosad with clarified biosynthesis pathway but its complex regulation networks associated with primary metabolism and secondary metabolites production almost have never been concerned or studied before. The proteomic analysis of a novel Saccharopolyspora spinosa CCTCC M206084 was performed and aimed to provide a global profile of regulatory proteins. Results Two-dimensional-liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified 1090, 1166, 701, and 509 proteins from four phases respectively, i.e., the logarithmic growth phase (T1), early stationary phase (T2), late stationary phase (T3), and decline phase (T4). Among the identified proteins, 1579 were unique to the S. spinosa proteome, including almost all the enzymes for spinosad biosynthesis. Trends in protein expression over the various time phases were deduced from using the modified protein abundance index (PAI), revealed the importance of stress pathway proteins and other global regulatory network proteins during spinosad biosynthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis followed by one-dimensional LC-MS/MS identification revealed similar trend of protein expression from four phases with the results of semi-quantification by PAI. qRT-PCR analysis revealed that 6 different expressed genes showed a positive correlation between changes at translational and transcriptional expression level. Expression of three proteins that likely promote spinosad biosynthesis, namely, 5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase (MHSM), glutamine synthetase (GS) and cyclic nucleotide-binding domain-containing protein (CNDP) was validated by western blot, which confirmed the results of proteomic analysis. Conclusions This study is the first systematic analysis of the S. spinosa proteome during fermentation and its valuable proteomic data of regulatory proteins may be used to enhance

  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. 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. PMID:9342866

  7. Comparative proteomic analysis of tobacco expressing cyanobacterial flavodoxin and its wild type under drought stress.

    PubMed

    Gharechahi, Javad; Hajirezaei, Mohammad-Reza; Salekdeh, Ghasem Hosseini

    2015-03-01

    Tobacco plants expressing cyanobacterial flavodoxin (Fld) show enhanced tolerance to a wide range of abiotic stresses including drought, temperature and UV. The mechanisms of adaptation to stress conditions under Fld expression are largely unknown. Here, we applied comparative proteomic analysis to uncover the changes in the proteome profile of Fld-expressing plants in response to drought stress. Using high-resolution two-dimensional gel electrophoresis, we were able to detect 930 protein spots and compare their abundance. We found changes up to 1.5 fold for 52 spots under drought in transgenic and/or wild type plants. Using combined MALDI-TOF/TOF and ESI-Q/TOF analysis 39 (24 in wild type, 11 in transgenic, and 4 in both) drought-responsive proteins (DRPs) could be identified. The majority of DRPs are known to be involved in photosynthesis, carbohydrate and energy metabolism, amino acid and protein synthesis and processing, and oxidative stress responses. Among candidate DRPs, the abundance of remurin, ferredoxin-NADP reductase, chloroplast manganese stabilizing protein-II, phosphoglycerate mutase, and glutathione S-transferase decreased in drought stressed Fld-tobacco while S-formylglutathione hydrolase and pyridoxine biosynthesis protein abundance increased. In wild type plants, drought caused a reduction of proteins related to carbohydrate metabolism. These results suggest that the stress tolerance conferred by Fld expression is strongly related to control mechanisms regarding carbohydrate and energy metabolism as well as oxidative stress responses. PMID:25506766

  8. 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

  9. Differential proteomic analysis of outer membrane enriched extracts of Bacteroides fragilis grown under bile salts stress.

    PubMed

    Boente, Renata F; Pauer, Heidi; Silva, Deborah N S; Filho, Joaquim Santos; Sandim, Vanessa; Antunes, Luis Caetano M; Ferreira, Rosana Barreto Rocha; Zingali, Russolina B; Domingues, Regina M C P; Lobo, Leandro A

    2016-06-01

    Bacteroides fragilis is the most commonly isolated anaerobic bacteria from infectious processes. Several virulence traits contribute to the pathogenic nature of this bacterium, including the ability to tolerate the high concentrations of bile found in the gastrointestinal tract (GIT). The activity of bile salts is similar to detergents and may lead to membrane permeabilization and cell death. Modulation of outer membrane proteins (OMPs) is considered a crucial event to bile salts resistance. The primary objective of the current work was to identify B. fragilis proteins associated with the stress induced by high concentration of bile salts. The outer membrane of B. fragilis strain 638R was isolated after growth either in the presence of 2% conjugated bile salts or without bile salts. The membrane fractions were separated on SDS-PAGE and analyzed by ESI-Q/TOF tandem mass spectrometry. A total of 37 proteins were identified; among them nine were found to be expressed exclusively in the absence of bile salts whereas eight proteins were expressed only in the presence of bile salts. These proteins are related to cellular functions such as transport through membrane, nutrient uptake, and protein-protein interactions. This study demonstrates the alteration of OMPs composition in B. fragilis during bile salts stress resistance and adaptation to environmental changes. Proteomics of OMPs was also shown to be a useful approach in the identification of new targets for functional analyses. PMID:26948242

  10. Quantitative Proteomic Analysis of the Orbital Frontal Cortex in Rats Following Extended Exposure to Caffeine Reveals Extensive Changes to Protein Expression: Implications for Neurological Disease.

    PubMed

    Franklin, Jane L; Mirzaei, Mehdi; Wearne, Travis A; Homewood, Judi; Goodchild, Ann K; Haynes, Paul A; Cornish, Jennifer L

    2016-05-01

    Caffeine is a plant-derived psychostimulant and a common additive found in a wide range of foods and pharmaceuticals. The orbitofrontal cortex (OFC) is rapidly activated by flavours, integrates gustatory and olfactory information, and plays a critical role in decision-making, with dysfunction contributing to psychopathologies and neurodegenerative conditions. This study investigated whether long-term consumption of caffeine causes changes to behavior and protein expression in the OFC. Male adult Sprague-Dawley rats (n = 8 per group) were treated for 26 days with either water or a 0.6 g/L caffeine solution. Locomotor behavior was measured on the first and last day of treatment, then again after 9 days treatment free following exposure to a mild stressor. When tested drug free, caffeine-treated animals were hyperactive compared to controls. Two hours following final behavioral testing, brains were rapidly removed and prepared for proteomic analysis of the OFC. Label free shotgun proteomics found 157 proteins differentially expressed in the caffeine-drinking rats compared to control. Major proteomic effects were seen for cell-to-cell communication, cytoskeletal regulation, and mitochondrial function. Similar changes have been observed in neurological disorders including Alzheimer's disease, Parkinson's disease, and schizophrenia. PMID:26941107

  11. Comparative proteomic analysis of proteins expression changes in the mammary tissue of cows infected with Escherichia coli mastitis

    PubMed Central

    Zhao, Xiao-wei; Huang, Dong-wei; Cheng, Guang-long; Zhao, Hui-ling

    2015-01-01

    Cows infected with Escherichia (E.) coli usually experience severe clinical symptoms, including damage to mammary tissues, reduced milk yield, and altered milk composition. In order to investigate the host response to E. coli infection and discover novel markers for mastitis treatment, mammary tissue samples were collected from healthy cows and bovines with naturally occurring severe E. coli mastitis. Changes of mammary tissue proteins were examined using two-dimensional gel electrophoresis and label-free proteomic approaches. A total of 95 differentially expressed proteins were identified. Of these, 56 proteins were categorized according to molecular function, cellular component, and biological processes. The most frequent biological processes influenced by the proteins were response to stress, transport, and establishment of localization. Furthermore, a network analysis of the proteins with altered expression in mammary tissues demonstrated that these factors are predominantly involved with binding and structural molecule activities. Vimentin and α-enolase were central "functional hubs" in the network. Based on results from the present study, disease-induced alterations of protein expression in mammary glands and potential markers for the effective treatment of E. coli mastitis were identified. These data have also helped elucidate defense mechanisms that protect the mammary glands and promote the pathogenesis of E. coli mastitis. PMID:25549220

  12. 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. PMID:26677818

  13. 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

  14. Quantitative Proteomics Reveals a Role for Epigenetic Reprogramming During Human Monocyte Differentiation*

    PubMed Central

    Nicholas, Dequina; Tang, Hui; Zhang, Qiongyi; Rudra, Jai; Xu, Feng; Langridge, William; Zhang, Kangling

    2015-01-01

    The differentiation of monocytes into macrophages and dendritic cells involves mechanisms for activation of the innate immune system in response to inflammatory stimuli, such as pathogen infection and environmental cues. Epigenetic reprogramming is thought to play an important role during monocyte differentiation. Complementary to cell surface markers, the characterization of monocytic cell lineages by mass spectrometry based protein/histone expression profiling opens a new avenue for studying immune cell differentiation. Here, we report the application of mass spectrometry and bioinformatics to identify changes in human monocytes during their differentiation into macrophages and dendritic cells. Our data show that linker histone H1 proteins are significantly down-regulated during monocyte differentiation. Although highly enriched H3K9-methyl/S10-phos/K14-acetyl tri-modification forms of histone H3 were identified in monocytes and macrophages, they were dramatically reduced in dendritic cells. In contrast, histone H4 K16 acetylation was found to be markedly higher in dendritic cells than in monocytes and macrophages. We also found that global hyperacetylation generated by the nonspecific histone deacetylase HDAC inhibitor Apicidin induces monocyte differentiation. Together, our data suggest that specific regulation of inter- and intra-histone modifications including H3 K9 methylation, H3 S10 phosphorylation, H3 K14 acetylation, and H4 K16 acetylation must occur in concert with chromatin remodeling by linker histones for cell cycle progression and differentiation of human myeloid cells into macrophages and dendritic cells. PMID:25316709

  15. Quantitative proteomics reveals a role for epigenetic reprogramming during human monocyte differentiation.

    PubMed

    Nicholas, Dequina; Tang, Hui; Zhang, Qiongyi; Rudra, Jai; Xu, Feng; Langridge, William; Zhang, Kangling

    2015-01-01

    The differentiation of monocytes into macrophages and dendritic cells involves mechanisms for activation of the innate immune system in response to inflammatory stimuli, such as pathogen infection and environmental cues. Epigenetic reprogramming is thought to play an important role during monocyte differentiation. Complementary to cell surface markers, the characterization of monocytic cell lineages by mass spectrometry based protein/histone expression profiling opens a new avenue for studying immune cell differentiation. Here, we report the application of mass spectrometry and bioinformatics to identify changes in human monocytes during their differentiation into macrophages and dendritic cells. Our data show that linker histone H1 proteins are significantly down-regulated during monocyte differentiation. Although highly enriched H3K9-methyl/S10-phos/K14-acetyl tri-modification forms of histone H3 were identified in monocytes and macrophages, they were dramatically reduced in dendritic cells. In contrast, histone H4 K16 acetylation was found to be markedly higher in dendritic cells than in monocytes and macrophages. We also found that global hyperacetylation generated by the nonspecific histone deacetylase HDAC inhibitor Apicidin induces monocyte differentiation. Together, our data suggest that specific regulation of inter- and intra-histone modifications including H3 K9 methylation, H3 S10 phosphorylation, H3 K14 acetylation, and H4 K16 acetylation must occur in concert with chromatin remodeling by linker histones for cell cycle progression and differentiation of human myeloid cells into macrophages and dendritic cells. PMID:25316709

  16. 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. PMID:26646815

  17. 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. PMID:27023342

  18. 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

  19. Structural and Metabolic Transitions of C4 Leaf Development and Differentiation Defined by Microscopy and Quantitative Proteomics in Maize[W

    PubMed Central

    Majeran, Wojciech; Friso, Giulia; Ponnala, Lalit; Connolly, Brian; Huang, Mingshu; Reidel, Edwin; Zhang, Cankui; Asakura, Yukari; Bhuiyan, Nazmul H.; Sun, Qi; Turgeon, Robert; van Wijk, Klaas J.

    2010-01-01

    C4 grasses, such as maize (Zea mays), have high photosynthetic efficiency through combined biochemical and structural adaptations. C4 photosynthesis is established along the developmental axis of the leaf blade, leading from an undifferentiated leaf base just above the ligule into highly specialized mesophyll cells (MCs) and bundle sheath cells (BSCs) at the tip. To resolve the kinetics of maize leaf development and C4 differentiation and to obtain a systems-level understanding of maize leaf formation, the accumulation profiles of proteomes of the leaf and the isolated BSCs with their vascular bundle along the developmental gradient were determined using large-scale mass spectrometry. This was complemented by extensive qualitative and quantitative microscopy analysis of structural features (e.g., Kranz anatomy, plasmodesmata, cell wall, and organelles). More than 4300 proteins were identified and functionally annotated. Developmental protein accumulation profiles and hierarchical cluster analysis then determined the kinetics of organelle biogenesis, formation of cellular structures, metabolism, and coexpression patterns. Two main expression clusters were observed, each divided in subclusters, suggesting that a limited number of developmental regulatory networks organize concerted protein accumulation along the leaf gradient. The coexpression with BSC and MC markers provided strong candidates for further analysis of C4 specialization, in particular transporters and biogenesis factors. Based on the integrated information, we describe five developmental transitions that provide a conceptual and practical template for further analysis. An online protein expression viewer is provided through the Plant Proteome Database. PMID:21081695

  20. 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. PMID:26471588

  1. Proteome dynamics during contractile and metabolic differentiation of bovine foetal muscle.

    PubMed

    Chaze, T; Meunier, B; Chambon, C; Jurie, C; Picard, B

    2009-07-01

    Contractile and metabolic properties of bovine muscles play an important role in meat sensorial quality, particularly tenderness. Earlier studies based on Myosin heavy chain isoforms analyses and measurements of glycolytic and oxidative enzyme activities have demonstrated that the third trimester of foetal life in bovine is characterized by contractile and metabolic differentiation. In order to complete this data and to obtain a precise view of this phase and its regulation, we performed a proteomic analysis of Semitendinosus muscle from Charolais foetuses analysed at three stages of the third trimester of gestation (180, 210 and 260 days). The results complete the knowledge of important changes in the profiles of proteins from metabolic and contractile pathways. They provide new insights about proteins such as Aldehyde dehydrogenase family, Enolase, Dihydrolipoyl dehydrogenase, Troponin T or Myosin light chains isoforms. These data have agronomical applications not only for the management of beef sensorial quality but also in medical context, as bovine myogenesis appears very similar to human one. PMID:22444818

  2. 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

  3. Differential Proteomics of Urinary Exovesicles from Classical Galactosemic Patients Reveals Subclinical Kidney Insufficiency.

    PubMed

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

    2016-06-01

    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. PMID:27103203

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  5. 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. PMID:26944343

  6. 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

  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. PMID:27165017

  8. 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

  9. 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

    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

  10. 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