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Sample records for dynamic proteomic analysis

  1. Proteomic analysis.

    Science.gov (United States)

    Cosette, Pascal; Jouenne, Thierry

    2014-01-01

    Proteome provides highly valuable information on the amount, modifications, and subcellular localization of polypeptides. Accordingly, geneticists, molecular biologists, and biochemists have logically applied these new tools to respond to different lines of biological questions (inventory of proteins, impact of a mutation, dynamics of protein regulation under a given exposure, …). However, even if the results obtained are very informative, this approach needs an excellent experimental design which ensures robustness and thus yields reproducibility. The present chapter gives appropriate methods for assessing the proteome of Pseudomonas aeruginosa by using a two-dimensional gel electrophoresis approach. Protocols for crude protein extraction, protein separation by using immobilized pH gradients, and protein identification by Liquid Chromatography coupled with tandem Mass Spectrometry (LC-MS/MS) are given.

  2. Dynamic proteome analysis of Cyanothece sp. ATCC 51142 under constant light

    Energy Technology Data Exchange (ETDEWEB)

    Aryal, Uma K.; Stockel, Jana; Welsh, Eric A.; Gritsenko, Marina A.; Nicora, Carrie D.; Koppenaal, David W.; Smith, Richard D.; Pakrasi, Himadri B.; Jacobs, Jon M.

    2012-02-03

    Understanding the dynamic nature of protein abundances provides insights into protein turnover not readily apparent from conventional, static mass spectrometry measurements. This level of data is particularly informative when surveying protein abundances in biological systems subjected to large perturbations or alterations in environment such as cyanobacteria. Our current analysis expands upon conventional proteomic approaches in cyanobacteria by measuring dynamic changes of the proteome using a 13C15N-L-leucine metabolic labeling in Cyanothece ATCC51142. Metabolically labeled Cyanothece ATCC51142 cells grown under nitrogen sufficient conditions in continuous light were monitored longitudinally for isotope incorporation over a 48 h period, revealing 422 proteins with dynamic changes in abundances. In particular, proteins involved in carbon fixation, pentose phosphate pathway, cellular protection, redox regulation, protein folding, assembly and degradation showed higher levels of isotope incorporation suggesting that these biochemical pathways are important for growth under non-diazotrophic conditions. Calculation of relative isotope abundances (RIA) values allowed to measure actual active protein synthesis over time for different biochemical pathways under non-diazotrophic conditions. Overall results demonstrated the utility of 'non-steady state' pulsed metabolic labeling for systems-wide dynamic quantification of the proteome in Cyanothece ATCC51142 that can also be applied to other cyanobacteria.

  3. Dynamic proteome analysis of Cyanothece sp. ATCC 51142 under constant light.

    Science.gov (United States)

    Aryal, Uma K; Stöckel, Jana; Welsh, Eric A; Gritsenko, Marina A; Nicora, Carrie D; Koppenaal, David W; Smith, Richard D; Pakrasi, Himadri B; Jacobs, Jon M

    2012-02-03

    Understanding the dynamic nature of protein abundances provides insights into protein turnover not readily apparent from conventional, static mass spectrometry measurements. This level of data is particularly informative when surveying protein abundances in biological systems subjected to large perturbations or alterations in environment such as cyanobacteria. Our current analysis expands upon conventional proteomic approaches in cyanobacteria by measuring dynamic changes of the proteome using a (13)C(15)N-l-leucine metabolic labeling in Cyanothece ATCC51142. Metabolically labeled Cyanothece ATCC51142 cells grown under nitrogen-sufficient conditions in continuous light were monitored longitudinally for isotope incorporation over a 48 h period, revealing 414 proteins with dynamic changes in abundances. In particular, proteins involved in carbon fixation, pentose phosphate pathway, cellular protection, redox regulation, protein folding, assembly, and degradation showed higher levels of isotope incorporation, suggesting that these biochemical pathways are important for growth under continuous light. Calculation of relative isotope abundances (RIA) values allowed the measurement of actual active protein synthesis over time for different biochemical pathways under high light exposure. Overall results demonstrated the utility of "non-steady state" pulsed metabolic labeling for systems-wide dynamic quantification of the proteome in Cyanothece ATCC51142 that can also be applied to other cyanobacteria.

  4. Dynamic Proteomic Insights of Seed Germination

    Institute of Scientific and Technical Information of China (English)

    Marc Galland; Romain Huguet; Erwann Arc; Gwendal Cueff; Dominique Job; Lo(i)c Rajjou

    2012-01-01

    Proteome analysis,which involves the identification and characterization of expressed proteins,is a powerful tool for determining the biological roles and functions of individual proteins.Furthermore,by providing a systematic and without any a priori mean for large-scale identification of cellular proteins,proteomics is expected to accelerate discoveries in complex processes such as plant development.Our research activity is mainly focused on the "Functional proteomics" approach in the field of seed biology.We are developing a proteome analysis of the model plant,Arabidopsis thaliana,in order to investigate seed development,dormancy,germination and longevity and identify related changes in the seed proteome.Combined approaches associating classical 2D gel-based proteome and dynamic radiolabeled proteome disclosed data regarding protein turnover and protein stability (http://www.seed-proteome.com).The selective translation of mRNAs emerges as an important mechanism regulating molecular functions involved in the control of seed germination.

  5. Proteomics analysis reveals a dynamic diurnal pattern of photosynthesis-related pathways in maize leaves.

    Science.gov (United States)

    Feng, Dan; Wang, Yanwei; Lu, Tiegang; Zhang, Zhiguo; Han, Xiao

    2017-01-01

    Plant leaves exhibit differentiated patterns of photosynthesis rates under diurnal light regulation. Maize leaves show a single-peak pattern without photoinhibition at midday when the light intensity is maximized. This mechanism contributes to highly efficient photosynthesis in maize leaves. To understand the molecular basis of this process, an isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomics analysis was performed to reveal the dynamic pattern of proteins related to photosynthetic reactions. Steady, single-peak and double-peak protein expression patterns were discovered in maize leaves, and antenna proteins in these leaves displayed a steady pattern. In contrast, the photosystem, carbon fixation and citrate pathways were highly controlled by diurnal light intensity. Most enzymes in the limiting steps of these pathways were major sites of regulation. Thus, maize leaves optimize photosynthesis and carbon fixation outside of light harvesting to adapt to the changes in diurnal light intensity at the protein level.

  6. Quantitative analysis of proteome and lipidome dynamics reveals functional regulation of global lipid metabolism

    DEFF Research Database (Denmark)

    Casanovas, Albert; Sprenger, Richard R; Tarasov, Kirill

    2015-01-01

    architecture and processes during physiological adaptations in yeast. Our results reveal that activation of cardiolipin synthesis and remodeling supports mitochondrial biogenesis in the transition from fermentative to respiratory metabolism, that down-regulation of de novo sterol synthesis machinery prompts......Elucidating how and to what extent lipid metabolism is remodeled under changing conditions is essential for understanding cellular physiology. Here, we analyzed proteome and lipidome dynamics to investigate how regulation of lipid metabolism at the global scale supports remodeling of cellular...

  7. Proteomic analysis of arginine methylation sites in human cells reveals dynamic regulation during transcriptional arrest

    DEFF Research Database (Denmark)

    Sylvestersen, Kathrine B; Horn, Heiko; Jungmichel, Stephanie

    2014-01-01

    contain regulated functions on their own. Collectively, we present a site-specific MMA dataset in human cells and demonstrate for the first time that MMA is a dynamic post-translational modification regulated during transcriptional arrest by a hitherto uncharacterized arginine demethylase......., transcription, and chromatin remodeling are predominantly found modified with MMA. Despite this, MMA sites prominently are located outside RNA-binding domains as compared to the proteome-wide distribution of arginine residues. Quantification of arginine methylation in cells treated with Actinomycin D uncovers......The covalent attachment of methyl groups to the side-chain of arginine residues is known to play essential roles in regulation of transcription, protein function and RNA metabolism. The specific N-methylation of arginine residues is catalyzed by a small family of gene products known as protein...

  8. Quantitative analysis of proteome and lipidome dynamics reveals functional regulation of global lipid metabolism.

    Science.gov (United States)

    Casanovas, Albert; Sprenger, Richard R; Tarasov, Kirill; Ruckerbauer, David E; Hannibal-Bach, Hans Kristian; Zanghellini, Jürgen; Jensen, Ole N; Ejsing, Christer S

    2015-03-19

    Elucidating how and to what extent lipid metabolism is remodeled under changing conditions is essential for understanding cellular physiology. Here, we analyzed proteome and lipidome dynamics to investigate how regulation of lipid metabolism at the global scale supports remodeling of cellular architecture and processes during physiological adaptations in yeast. Our results reveal that activation of cardiolipin synthesis and remodeling supports mitochondrial biogenesis in the transition from fermentative to respiratory metabolism, that down-regulation of de novo sterol synthesis machinery prompts differential turnover of lipid droplet-associated triacylglycerols and sterol esters during respiratory growth, that sphingolipid metabolism is regulated in a previously unrecognized growth stage-specific manner, and that endogenous synthesis of unsaturated fatty acids constitutes an in vivo upstream activator of peroxisomal biogenesis, via the heterodimeric Oaf1/Pip2 transcription factor. Our work demonstrates the pivotal role of lipid metabolism in adaptive processes and provides a resource to investigate its regulation at the cellular level.

  9. Mass Spectrometry-Based Proteomics for the Analysis of Chromatin Structure and Dynamics

    Directory of Open Access Journals (Sweden)

    Monica Soldi

    2013-03-01

    Full Text Available Chromatin is a highly structured nucleoprotein complex made of histone proteins and DNA that controls nearly all DNA-dependent processes. Chromatin plasticity is regulated by different associated proteins, post-translational modifications on histones (hPTMs and DNA methylation, which act in a concerted manner to enforce a specific “chromatin landscape”, with a regulatory effect on gene expression. Mass Spectrometry (MS has emerged as a powerful analytical strategy to detect histone PTMs, revealing interplays between neighbouring PTMs and enabling screens for their readers in a comprehensive and quantitative fashion. Here we provide an overview of the recent achievements of state-of-the-art mass spectrometry-based proteomics for the detailed qualitative and quantitative characterization of histone post-translational modifications, histone variants, and global interactomes at specific chromatin regions. This synopsis emphasizes how the advances in high resolution MS, from “Bottom Up” to “Top Down” analysis, together with the uptake of quantitative proteomics methods by chromatin biologists, have made MS a well-established method in the epigenetics field, enabling the acquisition of original information, highly complementary to that offered by more conventional, antibody-based, assays.

  10. Protein turnover analysis in Salmonella Typhimurium during infection by dynamic SILAC, Topograph, and quantitative proteomics.

    Science.gov (United States)

    Wang, Zhe; Han, Qiang-Qiang; Zhou, Mao-Tian; Chen, Xi; Guo, Lin

    2016-07-01

    Protein turnover affects protein abundance and phenotypes. Comprehensive investigation of protein turnover dynamics has the potential to provide substantial information about gene expression. Here we report a large-scale protein turnover study in Salmonella Typhimurium during infection by quantitative proteomics. Murine macrophage-like RAW 264.7 cells were infected with SILAC labeled Salmonella. Bacterial cells were extracted after 0, 30, 60, 120, and 240 min. Mass spectrometry analyses yielded information about Salmonella protein turnover dynamics and a software program named Topograph was used for the calculation of protein half lives. The half lives of 311 proteins from intracellular Salmonella were obtained. For bacteria cultured in control medium (DMEM), the half lives for 870 proteins were obtained. The calculated median of protein half lives was 69.13 and 99.30 min for the infection group and the DMEM group, respectively, indicating an elevated protein turnover at the initial stage of infection. Gene ontology analyses revealed that a number of protein functional groups were significantly regulated by infection, including proteins involved in ribosome, periplasmic space, cellular amino acid metabolic process, ion binding, and catalytic activity. The half lives of proteins involved in purine metabolism pathway were found to be significantly shortened during infection.

  11. Proteome analysis of tobacco leaves reveals dynamic changes in protein expression among different cultivation areas.

    Science.gov (United States)

    Lu, L M; Ye, K Y; Tang, Z X; Liu, L; Chen, Y; Luo, J; Huang, Y B

    2015-11-30

    The leaves of tobacco plants were used to analyze differences in protein content of tobacco grown in the four main flue-cured tobacco-producing areas of Sichuan Province, China. An improved protein extraction method, isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis two-dimensional gel electrophoretic separation, was used to extract and separate total protein from tobacco leaves. Proteomic maps with relatively high resolution and repeatability were produced. At isoelectric points 4 to 7 and molecular weight ranging from 20-100 kDa, we detected 1032, 1030, 1019, and 1011 clearly visible protein spots in tobacco leaves from the four study areas. Proteome comparison between these protein spots showed that 119 spots with a greater than 2-fold change in expression quantity contributed to the variation in expression. Of which, 115 were successfully identified and annotated. According to the annotation results, these proteins participate in photosynthesis, energy metabolism, mineral nutrition, terpene metabolism, defensive reaction, and other physiological and biochemical processes. This study preliminarily explains the effects of ecological conditions on the physiological metabolism of tobacco leaves and how such effects directly or indirectly contribute to tobacco leaf quality.

  12. Proteomics and the dynamic plasma membrane

    DEFF Research Database (Denmark)

    Sprenger, Richard R; Jensen, Ole Nørregaard

    2010-01-01

    plasma membrane is of particular interest, by not only serving as a barrier between the "cell interior" and the external environment, but moreover by organizing and clustering essential components to enable dynamic responses to internal and external stimuli. Defining and characterizing the dynamic plasma...... membrane proteome is crucial for understanding fundamental biological processes, disease mechanisms and for finding drug targets. Protein identification, characterization of dynamic PTMs and protein-ligand interactions, and determination of transient changes in protein expression and composition are among...... the challenges in functional proteomic studies of the plasma membrane. We review the recent progress in MS-based plasma membrane proteomics by presenting key examples from eukaryotic systems, including mammals, yeast and plants. We highlight the importance of enrichment and quantification technologies required...

  13. Bioinformatics Resources for In Silico Proteome Analysis

    Directory of Open Access Journals (Sweden)

    Pruess Manuela

    2003-01-01

    Full Text Available In the growing field of proteomics, tools for the in silico analysis of proteins and even of whole proteomes are of crucial importance to make best use of the accumulating amount of data. To utilise this data for healthcare and drug development, first the characteristics of proteomes of entire species—mainly the human—have to be understood, before secondly differentiation between individuals can be surveyed. Specialised databases about nucleic acid sequences, protein sequences, protein tertiary structure, genome analysis, and proteome analysis represent useful resources for analysis, characterisation, and classification of protein sequences. Different from most proteomics tools focusing on similarity searches, structure analysis and prediction, detection of specific regions, alignments, data mining, 2D PAGE analysis, or protein modelling, respectively, comprehensive databases like the proteome analysis database benefit from the information stored in different databases and make use of different protein analysis tools to provide computational analysis of whole proteomes.

  14. Proteomic Analysis of Chinese Hamster Ovary Cells

    DEFF Research Database (Denmark)

    Baycin-Hizal, Deniz; Tabb, David L.; Chaerkady, Raghothama;

    2012-01-01

    To complement the recent genomic sequencing of Chinese hamster ovary (CHO) cells, proteomic analysis was performed on CHO cells including the cellular proteome, secretome, and glycoproteome using tandem mass spectrometry (MS/MS) of multiple fractions obtained from gel electrophoresis, multidimens......To complement the recent genomic sequencing of Chinese hamster ovary (CHO) cells, proteomic analysis was performed on CHO cells including the cellular proteome, secretome, and glycoproteome using tandem mass spectrometry (MS/MS) of multiple fractions obtained from gel electrophoresis...

  15. Analysis of dynamic changes in the proteome of a Bcl-XL overexpressing Chinese hamster ovary cell culture during exponential and stationary phases.

    Science.gov (United States)

    Carlage, Tyler; Kshirsagar, Rashmi; Zang, Li; Janakiraman, Vijay; Hincapie, Marina; Lyubarskaya, Yelena; Weiskopf, Andy; Hancock, William S

    2012-01-01

    Mammalian cell cultures used for biopharmaceutical production undergo various dynamic biological changes over time, including the transition of cells from an exponential growth phase to a stationary phase during cell culture. To better understand the dynamic aspects of cell culture, a quantitative proteomics approach was used to identify dynamic trends in protein expression over the course of a Chinese hamster ovary (CHO) cell culture for the production of a recombinant monoclonal antibody and overexpressing the antiapoptotic gene Bcl-xl. Samples were analyzed using a method incorporating iTRAQ labeling, two-dimensional LC/MS, and linear regression calculations to identify significant dynamic trends in protein abundance. Using this approach, 59 proteins were identified with significant temporal changes in expression. Pathway analysis tools were used to identify a putative network of proteins associated with cell growth and apoptosis. Among the differentially expressed proteins were molecular chaperones and isomerases, such as GRP78 and PDI, and reported cell growth markers MCM2 and MCM5. In addition, two proteins with growth-regulating properties, transglutaminase-2 and clusterin, were identified. These proteins are associated with tumor proliferation and apoptosis and were observed to be expressed at relatively high levels during stationary phase, which was confirmed by western blotting. The proteomic methodology described here provides a dynamic view of protein expression throughout a CHO fed-batch cell culture, which may be useful for further elucidating the biological processes driving mammalian cell culture performance. Copyright © 2012 American Institute of Chemical Engineers (AIChE).

  16. Dynamic proteomic analysis reveals a switch between central carbon metabolism and alcoholic fermentation in rice filling grains.

    Science.gov (United States)

    Xu, Sheng Bao; Li, Tang; Deng, Zhu Yun; Chong, Kang; Xue, Yongbiao; Wang, Tai

    2008-10-01

    Accumulation of reserve materials in filling grains involves the coordination of different metabolic and cellular processes, and understanding the molecular mechanisms underlying the interconnections remains a major challenge for proteomics. Rice (Oryza sativa) is an excellent model for studying grain filling because of its importance as a staple food and the available genome sequence database. Our observations showed that embryo differentiation and endosperm cellularization in developing rice seeds were completed approximately 6 d after flowering (DAF); thereafter, the immature seeds mainly underwent cell enlargement and reached the size of mature seeds at 12 DAF. Grain filling began at 6 DAF and lasted until 20 DAF. Dynamic proteomic analyses revealed 396 protein spots differentially expressed throughout eight sequential developmental stages from 6 to 20 DAF and determined 345 identities. These proteins were involved in different cellular and metabolic processes with a prominently functional skew toward metabolism (45%) and protein synthesis/destination (20%). Expression analyses of protein groups associated with different functional categories/subcategories showed that substantially up-regulated proteins were involved in starch synthesis and alcoholic fermentation, whereas the down-regulated proteins in the process were involved in central carbon metabolism and most of the other functional categories/subcategories such as cell growth/division, protein synthesis, proteolysis, and signal transduction. The coordinated changes were consistent with the transition from cell growth and differentiation to starch synthesis and clearly indicated that a switch from central carbon metabolism to alcoholic fermentation may be important for starch synthesis and accumulation in the developmental process.

  17. A dynamic range compression and three-dimensional peptide fractionation analysis platform expands proteome coverage and the diagnostic potential of whole saliva.

    Science.gov (United States)

    Bandhakavi, Sricharan; Stone, Matthew D; Onsongo, Getiria; Van Riper, Susan K; Griffin, Timothy J

    2009-12-01

    Comprehensive identification of proteins in whole human saliva is critical for appreciating its full diagnostic potential. However, this is challenged by the large dynamic range of protein abundance within the fluid. To address this problem, we used an analysis platform that coupled hexapeptide libraries for dynamic range compression (DRC) with three-dimensional (3D) peptide fractionation. Our approach identified 2340 proteins in whole saliva and represents the largest saliva proteomic dataset generated using a single analysis platform. Three-dimensional peptide fractionation involving sequential steps of preparative isoelectric focusing (IEF), strong cation exchange, and capillary reversed-phase liquid chromatography was essential for maximizing gains from DRC. Compared to saliva not treated with hexapeptide libraries, DRC substantially increased identified proteins across physicochemical and functional categories. Approximately 20% of total salivary proteins are also seen in plasma, and proteins in both fluids show comparable functional diversity and disease-linkage. However, for a subset of diseases, saliva has higher apparent diagnostic potential. These results expand the potential for whole saliva in health monitoring/diagnostics and provide a general platform for improving proteomic coverage of complex biological samples.

  18. Analysis of mass spectrometry data in proteomics

    DEFF Research Database (Denmark)

    Matthiesen, Rune; Jensen, Ole N

    2008-01-01

    that in turn allow protein identification, annotation of secondary modifications, and determination of the absolute or relative abundance of individual proteins. Advances in mass spectrometry-driven proteomics rely on robust bioinformatics tools that enable large-scale data analysis. This chapter describes......The systematic study of proteins and protein networks, that is, proteomics, calls for qualitative and quantitative analysis of proteins and peptides. Mass spectrometry (MS) is a key analytical technology in current proteomics and modern mass spectrometers generate large amounts of high-quality data...... some of the basic concepts and current approaches to the analysis of MS and MS/MS data in proteomics....

  19. Dynamic Proteomics: In Vivo Proteome-Wide Measurement of Protein Kinetics Using Metabolic Labeling.

    Science.gov (United States)

    Holmes, W E; Angel, T E; Li, K W; Hellerstein, M K

    2015-01-01

    Control of biosynthetic and catabolic rates of polymers, including proteins, stands at the center of phenotype, physiologic adaptation, and disease pathogenesis. Advances in stable isotope-labeling concepts and mass spectrometric instrumentation now allow accurate in vivo measurement of protein synthesis and turnover rates, both for targeted proteins and for unbiased screening across the proteome. We describe here the underlying principles and operational protocols for measuring protein dynamics, focusing on metabolic labeling with (2)H2O (heavy water) combined with tandem mass spectrometric analysis of mass isotopomer abundances in trypsin-generated peptides. The core principles of combinatorial analysis (mass isotopomer distribution analysis or MIDA) are reviewed in detail, including practical advantages, limitations, and technical procedures to ensure optimal kinetic results. Technical factors include heavy water labeling protocols, optimal duration of labeling, clean up and simplification of sample matrices, accurate quantitation of mass isotopomer abundances in peptides, criteria for adequacy of mass spectrometric abundance measurements, and calculation algorithms. Some applications are described, including the noninvasive "virtual biopsy" strategy for measuring molecular flux rates in tissues through measurements in body fluids. In addition, application of heavy water labeling to measure flux lipidomics is noted. In summary, the combination of stable isotope labeling, particularly from (2)H2O, with tandem mass spectrometric analysis of mass isotopomer abundances in peptides, provides a powerful approach for characterizing the dynamics of proteins across the global proteome. Many applications in research and clinical medicine have been achieved and many others can be envisioned.

  20. Bioinformatic analysis of proteomics data.

    Science.gov (United States)

    Schmidt, Andreas; Forne, Ignasi; Imhof, Axel

    2014-01-01

    Most biochemical reactions in a cell are regulated by highly specialized proteins, which are the prime mediators of the cellular phenotype. Therefore the identification, quantitation and characterization of all proteins in a cell are of utmost importance to understand the molecular processes that mediate cellular physiology. With the advent of robust and reliable mass spectrometers that are able to analyze complex protein mixtures within a reasonable timeframe, the systematic analysis of all proteins in a cell becomes feasible. Besides the ongoing improvements of analytical hardware, standardized methods to analyze and study all proteins have to be developed that allow the generation of testable new hypothesis based on the enormous pre-existing amount of biological information. Here we discuss current strategies on how to gather, filter and analyze proteomic data sates using available software packages.

  1. Proteomic Analysis of Chinese Hamster Ovary Cells

    Science.gov (United States)

    Baycin-Hizal, Deniz; Tabb, David L.; Chaerkady, Raghothama; Chen, Lily; Lewis, Nathan E.; Nagarajan, Harish; Sarkaria, Vishaldeep; Kumar, Amit; Wolozny, Daniel; Colao, Joe; Jacobson, Elena; Tian, Yuan; O'Meally, Robert N.; Krag, Sharon S.; Cole, Robert N.; Palsson, Bernhard O.; Zhang, Hui; Betenbaugh, Michael

    2013-01-01

    In order to complement the recent genomic sequencing of Chinese hamster ovary (CHO) cells, proteomic analysis was performed on CHO including the cellular proteome, secretome, and glycoproteome using tandem mass spectrometry (MS/MS) of multiple fractions obtained from gel electrophoresis, multi-dimensional liquid chromatography, and solid phase extraction of glycopeptides (SPEG). From the 120 different mass spectrometry analyses generating 682,097 MS/MS spectra, 93,548 unique peptide sequences were identified with at most a 0.02 false discovery rate (FDR). A total of 6164 grouped proteins were identified from both glycoproteome and proteome analysis, representing an 8-fold increase in the number of proteins currently identified in the CHO proteome. Furthermore, this is the first proteomic study done using CHO genome exclusively which provides for more accurate identification of proteins. From this analysis, the CHO codon frequency was determined and found to be distinct from humans, which will facilitate expression of human proteins in CHO cells. Analysis of the combined proteomic and mRNA data sets indicated the enrichment of a number of pathways including protein processing and apoptosis but depletion of proteins involved in steroid hormone and glycosphingolipid metabolism. 504 of the detected proteins included N-acetylation modifications and 1292 different proteins were observed to be N-glycosylated. This first large-scale proteomic analysis will enhance the knowledge base about CHO capabilities for recombinant expression and provide information useful in cell engineering efforts aimed at modifying CHO cellular functions. PMID:22971049

  2. Network-based analysis of proteomic profiles

    KAUST Repository

    Wong, Limsoon

    2016-01-26

    Mass spectrometry (MS)-based proteomics is a widely used and powerful tool for profiling systems-wide protein expression changes. It can be applied for various purposes, e.g. biomarker discovery in diseases and study of drug responses. Although RNA-based high-throughput methods have been useful in providing glimpses into the underlying molecular processes, the evidences they provide are indirect. Furthermore, RNA and corresponding protein levels have been known to have poor correlation. On the other hand, MS-based proteomics tend to have consistency issues (poor reproducibility and inter-sample agreement) and coverage issues (inability to detect the entire proteome) that need to be urgently addressed. In this talk, I will discuss how these issues can be addressed by proteomic profile analysis techniques that use biological networks (especially protein complexes) as the biological context. In particular, I will describe several techniques that we have been developing for network-based analysis of proteomics profile. And I will present evidence that these techniques are useful in identifying proteomics-profile analysis results that are more consistent, more reproducible, and more biologically coherent, and that these techniques allow expansion of the detected proteome to uncover and/or discover novel proteins.

  3. Pulsatile dynamics in the yeast proteome.

    Science.gov (United States)

    Dalal, Chiraj K; Cai, Long; Lin, Yihan; Rahbar, Kasra; Elowitz, Michael B

    2014-09-22

    The activation of transcription factors in response to environmental conditions is fundamental to cellular regulation. Recent work has revealed that some transcription factors are activated in stochastic pulses of nuclear localization, rather than at a constant level, even in a constant environment [1-12]. In such cases, signals control the mean activity of the transcription factor by modulating the frequency, duration, or amplitude of these pulses. Although specific pulsatile transcription factors have been identified in diverse cell types, it has remained unclear how prevalent pulsing is within the cell, how variable pulsing behaviors are between genes, and whether pulsing is specific to transcriptional regulators or is employed more broadly. To address these issues, we performed a proteome-wide movie-based screen to systematically identify localization-based pulsing behaviors in Saccharomyces cerevisiae. The screen examined all genes in a previously developed fluorescent protein fusion library of 4,159 strains [13] in multiple media conditions. This approach revealed stochastic pulsing in ten proteins, all transcription factors. In each case, pulse dynamics were heterogeneous and unsynchronized among cells in clonal populations. Pulsing is the only dynamic localization behavior that we observed, and it tends to occur in pairs of paralogous and redundant proteins. Taken together, these results suggest that pulsatile dynamics play a pervasive role in yeast and may be similarly prevalent in other eukaryotic species.

  4. Toward a standardized urine proteome analysis methodology.

    Science.gov (United States)

    Court, Magali; Selevsek, Nathalie; Matondo, Mariette; Allory, Yves; Garin, Jerome; Masselon, Christophe D; Domon, Bruno

    2011-03-01

    Urine is an easily accessible bodily fluid particularly suited for the routine clinical analysis of disease biomarkers. Actually, the urinary proteome is more diverse than anticipated a decade ago. Hence, significant analytical and practical issues of urine proteomics such as sample collection and preparation have emerged, in particular for large-scale studies. We have undertaken a systematic study to define standardized and integrated analytical protocols for a biomarker development pipeline, employing two LC-MS analytical platforms, namely accurate mass and time tags and selected reaction monitoring, for the discovery and verification phase, respectively. Urine samples collected from hospital patients were processed using four different protocols, which were evaluated and compared on both analytical platforms. Addition of internal standards at various stages of sample processing allowed the estimation of protein extraction yields and the absolute quantification of selected urinary proteins. Reproducibility of the entire process and dynamic range of quantification were also evaluated. Organic solvent precipitation followed by in-solution digestion provided the best performances and was thus selected as the standard method common to the discovery and verification phases. Finally, we applied this protocol for platforms' cross-validation and obtained excellent consistency between urinary protein concentration estimates by both analytical methods performed in parallel in two laboratories. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Quantitative Map of Proteome Dynamics during Neuronal Differentiation

    NARCIS (Netherlands)

    Frese, Christian K; Mikhaylova, Marina; Stucchi, Riccardo; Gautier, Violette; Liu, Qingyang; Mohammed, Shabaz; Heck, Albert J R; Altelaar, A F Maarten; Hoogenraad, Casper C

    2017-01-01

    Neuronal differentiation is a multistep process that shapes and re-shapes neurons by progressing through several typical stages, including axon outgrowth, dendritogenesis, and synapse formation. To systematically profile proteome dynamics throughout neuronal differentiation, we took cultured rat hip

  6. Prefractionation techniques in proteome analysis.

    Science.gov (United States)

    Righetti, Pier Giorgio; Castagna, Annalisa; Herbert, Ben; Reymond, Frederic; Rossier, Joël S

    2003-08-01

    The present review deals with a number of prefractionation protocols in preparation for two-dimensional map analysis, both in the fields of chromatography and in the field of electrophoresis. In the first case, Fountoulaki's groups has reported just about any chromatographic procedure useful as a prefractionation step, including affinity, ion-exchange, and reversed-phase resins. As a result of the various enrichment steps, several hundred new species, previously undetected in unfractionated samples, could be revealed for the first time. Electrophoretic prefractionation protocols include all those electrokinetic methodologies which are performed in free solution, essentially all relying on isoelectric focusing steps. The devices here reviewed include multichamber apparatus, such as the multicompartment electrolyzer with Immobiline membranes, Off-Gel electrophoresis in a multicup device and the Rotofor, an instrument also based on a multichamber system but exploiting the conventional technique of carrier-ampholyte-focusing. Other instruments of interest are the Octopus, a continuous-flow device for isoelectric focusing in a upward flowing liquid curtain, and the Gradiflow, where different pI cuts are obtained by a multistep passage through two compartments buffered at different pH values. It is felt that this panoply of methods could offer a strong step forward in "mining below the tip of the iceberg" for detecting the "unseen proteome".

  7. Inspection, visualisation and analysis of quantitative proteomics data

    OpenAIRE

    Gatto, Laurent

    2016-01-01

    Material Quantitative Proteomics and Data Analysis Course. 4 - 5 April 2016, Queen Hotel, Chester, UK Table D - Inspection, visualisation and analysis of quantitative proteomics data, Laurent Gatto (University of Cambridge)

  8. Directed proteomic analysis of the human nucleolus

    DEFF Research Database (Denmark)

    Andersen, Jens S; Lyon, Carol E; Fox, Archa H

    2002-01-01

    of their structure and function remain uncharacterized. RESULTS: We report a proteomic analysis of human nucleoli. Using a combination of mass spectrometry (MS) and sequence database searches, including online analysis of the draft human genome sequence, 271 proteins were identified. Over 30% of the nucleolar...

  9. iTRAQ-based analysis of developmental dynamics in the soybean leaf proteome reveals pathways associated with leaf photosynthetic rate.

    Science.gov (United States)

    Qin, Jun; Zhang, Jianan; Liu, Duan; Yin, Changcheng; Wang, Fengmin; Chen, Pengyin; Chen, Hao; Ma, Jinbing; Zhang, Bo; Xu, Jin; Zhang, Mengchen

    2016-08-01

    Photosynthetic rate which acts as a vital limiting factor largely affects the potential of soybean production, especially during the senescence phase. However, the physiological and molecular mechanisms that underlying the change of photosynthetic rate during the developmental process of soybean leaves remain unclear. In this study, we compared the protein dynamics during the developmental process of leaves between the soybean cultivar Hobbit and the high-photosynthetic rate cultivar JD 17 using the iTRAQ (isobaric tags for relative and absolute quantification) method. A total number of 1269 proteins were detected in the leaves of these two cultivars at three different developmental stages. These proteins were classified into nine expression patterns depending on the expression levels at different developmental stages, and the proteins in each pattern were also further classified into three large groups and 20 small groups depending on the protein functions. Only 3.05-6.53 % of the detected proteins presented a differential expression pattern between these two cultivars. Enrichment factor analysis indicated that proteins involved in photosynthesis composed an important category. The expressions of photosynthesis-related proteins were also further confirmed by western blotting. Together, our results suggested that the reduction in photosynthetic rate as well as chloroplast activity and composition during the developmental process was a highly regulated and complex process which involved a serial of proteins that function as potential candidates to be targeted by biotechnological approaches for the improvement of photosynthetic rate and production.

  10. Quantitative proteomics and bioinformatic analysis provide new insight into the dynamic response of porcine intestine to Salmonella Typhimurium.

    Directory of Open Access Journals (Sweden)

    Melania eCollado-Romero

    2015-09-01

    Full Text Available The enteropathogen Salmonella Typhimurium (S. Typhimurium is the most commonly nontyphoideal serotype isolated in pig worldwide. Currently, one of the main sources of human infection is by consumption of pork meat. Therefore, prevention and control of salmonellosis in pigs is crucial for minimizing risks to public health. The aim of the present study was to use isobaric tags for relative and absolute quantification (iTRAQ to explore differences in the response to Salmonella in two segment of the porcine gut (ileum and colon along a time course of 1, 2 and 6 days post infection (dpi with S. Typhimurium. A total of 298 proteins were identified in the infected ileum samples of which, 112 displayed significant expression differences due to Salmonella infection. In colon, 184 proteins were detected in the infected samples of which 46 resulted differentially expressed with respect to the controls. The higher number of changes in protein expression was quantified in ileum at 2 dpi. Further biological interpretation of proteomics data using bioinformatics tools demonstrated that the expression changes in colon were found in proteins involved in cell death and survival, tissue morphology or molecular transport at the early stages and tissue regeneration at 6 dpi. In ileum, however, changes in protein expression were mainly related to immunological and infection diseases, inflammatory response or connective tissue disorders at 1 and 2 dpi. iTRAQ has proved to be a proteomic robust approach allowing us to identify ileum as the earliest response focus upon S. Typhimurium in the porcine gut. In addition, new functions involved in the response to bacteria such as eIF2 signalling, free radical scavengers or antimicrobial peptides expression have been identified. Finally, the impairment at of the enterohepatic circulation of bile acids and lipid metabolism by means the under regulation of FABP6 protein and FXR/RXR and LXR/RXR signalling pathway in ileum has

  11. Proteomic analysis of zebrafish caudal fin regeneration.

    Science.gov (United States)

    Saxena, Sandeep; Singh, Sachin K; Lakshmi, Mula G Meena; Meghah, Vuppalapaty; Bhatti, Bhawna; Swamy, Cherukuvada V Brahmendra; Sundaram, Curam S; Idris, Mohammed M

    2012-06-01

    The epimorphic regeneration of zebrafish caudal fin is rapid and complete. We have analyzed the biomechanism of zebrafish caudal fin regeneration at various time points based on differential proteomics approaches. The spectrum of proteome changes caused by regeneration were analyzed among controls (0 h) and 1, 12, 24, 48, and 72 h postamputation involving quantitative differential proteomics analysis based on two-dimensional gel electrophoresis matrix-assisted laser desorption/ionization and differential in-gel electrophoresis Orbitrap analysis. A total of 96 proteins were found differentially regulated between the control nonregenerating and regenerating tissues of different time points for having at least 1.5-fold changes. 90 proteins were identified as differentially regulated for regeneration based on differential in-gel electrophoresis analysis between the control and regenerating tissues. 35 proteins were characterized for its expression in all of the five regenerating time points against the control samples. The proteins identified and associated with regeneration were found to be directly allied with various molecular, biological, and cellular functions. Based on network pathway analysis, the identified proteome data set for regeneration was majorly associated in maintaining cellular structure and architecture. Also the proteins were found associated for the cytoskeleton remodeling pathway and cellular immune defense mechanism. The major proteins that were found differentially regulated during zebrafish caudal fin regeneration includes keratin and its 10 isoforms, cofilin 2, annexin a1, skeletal α1 actin, and structural proteins. Annexin A1 was found to be exclusively undergoing phosphorylation during regeneration. The obtained differential proteome and the direct association of the various proteins might lead to a new understanding of the regeneration mechanism.

  12. Proteome analysis of adenovirus using mass spectrometry.

    Science.gov (United States)

    Lind, Sara Bergström; Artemenko, Konstantin A; Pettersson, Ulf

    2014-01-01

    Analysis of proteins and their posttranslational modifications is important for understanding different biological events. For analysis of viral proteomes, an optimal protocol includes production of a highly purified virus that can be investigated with a high-resolving analytical method. In this Methods in Molecular Biology paper we describe a working strategy for how structural proteins in the Adenovirus particle can be studied using liquid chromatography-high-resolving mass spectrometry. This method provides information on the chemical composition of the virus particle. Further, knowledge about amino acids carrying modifications that could be essential for any part of the virus life cycle is collected. We describe in detail alternatives available for preparation of virus for proteome analysis as well as choice of mass spectrometric instrumentation suitable for this kind of analysis.

  13. Shotgun proteomic analysis of mulberry dwarf phytoplasma

    Directory of Open Access Journals (Sweden)

    Zheng Chengchao

    2010-04-01

    Full Text Available Abstract Background Mulberry dwarf (MD, which is caused by phytoplasma, is one of the most serious infectious diseases of mulberry. Phytoplasmas have been associated with diseases in several hundred plant species. The inability to culture phytoplasmas in vitro has hindered their characterization at the molecular level. Though the complete genomes of two phytoplasmas have been published, little information has been obtained about the proteome of phytoplasma. Therefore, the proteomic information of phytoplasmas would be useful to elucidate the functional mechanisms of phytoplasma in many biological processes. Results MD phytoplasmas, which belong to the 16SrI-B subgroup based on the 16S DNA analysis, were purified from infected tissues using a combination of differential centrifugation and density gradient centrifugation. The expressed proteome of phytoplasma was surveyed by one-dimensional SDS-PAGE and nanocapillary liquid chromatography-tandem mass spectrometry. A total of 209 phytoplasma proteins were unambiguously assigned, including the proteins with the functions of amino acid biosynthesis, cell envelope, cellular processes, energy metabolism, nucleosides and nucleotide metabolism, replication, transcription, translation, transport and binding as well as the proteins with other functions. In addition to these known function proteins, 63 proteins were annotated as hypothetical or conserved hypothetical proteins. Conclusions Taken together, a total of 209 phytoplasma proteins have been experimentally verified, representing the most extensive survey of any phytoplasma proteome to date. This study provided a valuable dataset of phytoplasma proteins, and a better understanding of the energy metabolism and virulence mechanisms of MD phytoplasma.

  14. Proteomic Analysis of Hair Follicles

    Science.gov (United States)

    Ishioka, Noriaki; Terada, Masahiro; Yamada, Shin; Seki, Masaya; Takahashi, Rika; Majima, Hideyuki J.; Higashibata, Akira; Mukai, Chiaki

    2013-02-01

    Hair root cells actively divide in a hair follicle, and they sensitively reflect physical conditions. By analyzing the human hair, we can know stress levels on the human body and metabolic conditions caused by microgravity environment and cosmic radiation. The Japan Aerospace Exploration Agency (JAXA) has initiated a human research study to investigate the effects of long-term space flight on gene expression and mineral metabolism by analyzing hair samples of astronauts who stayed in the International Space Station (ISS) for 6 months. During long-term flights, the physiological effects on astronauts include muscle atrophy and bone calcium loss. Furthermore, radiation and psychological effects are important issue to consider. Therefore, an understanding of the effects of the space environment is important for developing countermeasures against the effects experienced by astronauts. In this experiment, we identify functionally important target proteins that integrate transcriptome, mineral metabolism and proteome profiles from human hair. To compare the protein expression data with the gene expression data from hair roots, we developed the protein processing method. We extracted the protein from five strands of hair using ISOGEN reagents. Then, these extracted proteins were analyzed by LC-MS/MS. These collected profiles will give us useful physiological information to examine the effect of space flight.

  15. Proteome analysis of grape skins during ripening.

    Science.gov (United States)

    Deytieux, Christelle; Geny, Laurence; Lapaillerie, Delphine; Claverol, Stéphane; Bonneu, Marc; Donèche, Bernard

    2007-01-01

    The characterization of proteins isolated from skin tissue is apparently an essential parameter for understanding grape ripening as this tissue contains the key compounds for wine quality. It has been particularly difficult to extract proteins from skins for analysis by two-dimensional electrophoresis gels and, therefore, a protocol for this purpose has been adapted. The focus was on the evolution of the proteome profile of grape skin during maturation. Proteome maps obtained at three stages of ripening were compared to assess the extent to which protein distribution differs in grape skin during ripening. The comparative analysis shows that numerous soluble skin proteins evolve during ripening and reveal specific distributions at different stages. Proteins involved in photosynthesis, carbohydrate metabolisms, and stress response are identified as being over-expressed at the beginning of colour-change. The end of colour-change is characterized by the over-expression of proteins involved in anthocyanin synthesis and, at harvest, the dominant proteins are involved in defence mechanisms. In particular, increases in the abundance of different chitinase and beta-1,3-glucanase isoforms were found as the berry ripens. This observation can be correlated with the increase of the activities of both of these enzymes during skin ripening. The differences observed in proteome maps clearly show that significant metabolic changes occur in grape skin during this crucial phase of ripening. This comparative analysis provides more detailed characterization of the fruit ripening process.

  16. Proteomics

    DEFF Research Database (Denmark)

    Tølbøll, Trine Højgaard; Danscher, Anne Mette; Andersen, Pia Haubro;

    2012-01-01

    different proteins were identified, with 146 proteins available for identification in C, 279 proteins in D and 269 proteins in L. A functional annotation of the identified proteins was obtained using the on-line Blast2GO tool. Three hundred and sixteen of the identified proteins could be subsequently...... grouped manually to one or more of five major functional groups related to metabolism, cell structure, immunity, apoptosis and angiogenesis. These were chosen to represent basic cell functions and biological processes potentially involved in the pathogenesis of CHD. The LC–MS/MS-based proteomic analysis...

  17. Proteomic analysis of SETD6 interacting proteins.

    Science.gov (United States)

    Cohn, Ofir; Chen, Ayelet; Feldman, Michal; Levy, Dan

    2016-03-01

    SETD6 (SET-domain-containing protein 6) is a mono-methyltransferase that has been shown to methylate RelA and H2AZ. Using a proteomic approach we recently identified several new SETD6 substrates. To identify novel SETD6 interacting proteins, SETD6 was immunoprecipitated (IP) from Human erythromyeloblastoid leukemia K562 cells. SETD6 binding proteins were subjected to mass-spectrometry analysis resulting in 115 new SETD6 binding candidates. STRING database was used to map the SETD6 interactome network. Network enrichment analysis of biological processes with Gene Ontology (GO) database, identified three major groups; metabolic processes, muscle contraction and protein folding.

  18. Quantitative and qualitative proteome characteristics extracted from in-depth integrated genomics and proteomics analysis.

    Science.gov (United States)

    Low, Teck Yew; van Heesch, Sebastiaan; van den Toorn, Henk; Giansanti, Piero; Cristobal, Alba; Toonen, Pim; Schafer, Sebastian; Hübner, Norbert; van Breukelen, Bas; Mohammed, Shabaz; Cuppen, Edwin; Heck, Albert J R; Guryev, Victor

    2013-12-12

    Quantitative and qualitative protein characteristics are regulated at genomic, transcriptomic, and posttranscriptional levels. Here, we integrated in-depth transcriptome and proteome analyses of liver tissues from two rat strains to unravel the interactions within and between these layers. We obtained peptide evidence for 26,463 rat liver proteins. We validated 1,195 gene predictions, 83 splice events, 126 proteins with nonsynonymous variants, and 20 isoforms with nonsynonymous RNA editing. Quantitative RNA sequencing and proteomics data correlate highly between strains but poorly among each other, indicating extensive nongenetic regulation. Our multilevel analysis identified a genomic variant in the promoter of the most differentially expressed gene Cyp17a1, a previously reported top hit in genome-wide association studies for human hypertension, as a potential contributor to the hypertension phenotype in SHR rats. These results demonstrate the power of and need for integrative analysis for understanding genetic control of molecular dynamics and phenotypic diversity in a system-wide manner. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  19. Quantitative and Qualitative Proteome Characteristics Extracted from In-Depth Integrated Genomics and Proteomics Analysis

    Directory of Open Access Journals (Sweden)

    Teck Yew Low

    2013-12-01

    Full Text Available Quantitative and qualitative protein characteristics are regulated at genomic, transcriptomic, and posttranscriptional levels. Here, we integrated in-depth transcriptome and proteome analyses of liver tissues from two rat strains to unravel the interactions within and between these layers. We obtained peptide evidence for 26,463 rat liver proteins. We validated 1,195 gene predictions, 83 splice events, 126 proteins with nonsynonymous variants, and 20 isoforms with nonsynonymous RNA editing. Quantitative RNA sequencing and proteomics data correlate highly between strains but poorly among each other, indicating extensive nongenetic regulation. Our multilevel analysis identified a genomic variant in the promoter of the most differentially expressed gene Cyp17a1, a previously reported top hit in genome-wide association studies for human hypertension, as a potential contributor to the hypertension phenotype in SHR rats. These results demonstrate the power of and need for integrative analysis for understanding genetic control of molecular dynamics and phenotypic diversity in a system-wide manner.

  20. Proteomic Analysis of Bovine Nucleolus

    Institute of Scientific and Technical Information of China (English)

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

    2010-01-01

    Nucleolus is the most prominent subnuclear structure, which performs a wide variety of functions in the eu-karyotic cellular processes. In order to understand the structural and functional role of the nucleoli in bovine cells,we analyzed the proteomie composition of the bovine nueleoli. The nucleoli were isolated from Madin Darby bo-vine kidney cells and subjected to proteomie analysis by LC-MS/MS after fractionation by SDS-PAGE and strongcation exchange chromatography. Analysis of the data using the Mascot database search and the GPM databasesearch identified 311 proteins in the bovine nucleoli, which contained 22 proteins previously not identified in theproteomic analysis of human nucleoli. Analysis of the identified proteins using the GoMiner software suggestedthat the bovine nueleoli contained proteins involved in ribosomal biogenesis, cell cycle control, transcriptional,translational and post-translational regulation, transport, and structural organization.

  1. Analysis of Peanut Leaf Proteome

    DEFF Research Database (Denmark)

    Ramesh, R.; Suravajhala, Prashanth; Pechan, T.

    2010-01-01

    Peanut (Arachis hypogaea) is one of the most important sources of plant protein. Current selection of genotypes requires molecular characterization of available populations. Peanut genome database has several EST cDNAs which can be used to analyze gene expression. Analysis of proteins is a direct...

  2. Quantitative Proteomic Approaches for Analysis of Protein S-Nitrosylation.

    Science.gov (United States)

    Qu, Zhe; Greenlief, C Michael; Gu, Zezong

    2016-01-01

    S-Nitrosylation is a redox-based post-translational modification of a protein in response to nitric oxide (NO) signaling, and it participates in a variety of processes in diverse biological systems. The significance of this type of protein modification in health and diseases is increasingly recognized. In the central nervous system, aberrant S-nitrosylation, due to excessive NO production, is known to cause protein misfolding, mitochondrial dysfunction, transcriptional dysregulation, and neuronal death. This leads to an altered physiological state and consequently contributes to pathogenesis of neurodegenerative disorders. To date, much effort has been made to understand the mechanisms underlying protein S-nitrosylation, and several approaches have been developed to unveil S-nitrosylated proteins from different organisms. Interest in determining the dynamic changes of protein S-nitrosylation under different physiological and pathophysiological conditions has underscored the need for the development of quantitative proteomic approaches. Currently, both gel-based and gel-free mass spectrometry-based quantitative methods are widely used, and they each have advantages and disadvantages but may also be used together to produce complementary data. This review evaluates current available quantitative proteomic techniques for the analysis of protein S-nitrosylation and highlights recent advances, with emphasis on applications in neurodegenerative diseases. An important goal is to provide a comprehensive guide of feasible quantitative proteomic methodologies for examining protein S-nitrosylation in research to yield insights into disease mechanisms, diagnostic biomarkers, and drug discovery.

  3. Quantitative Map of Proteome Dynamics during Neuronal Differentiation

    Directory of Open Access Journals (Sweden)

    Christian K. Frese

    2017-02-01

    Full Text Available Neuronal differentiation is a multistep process that shapes and re-shapes neurons by progressing through several typical stages, including axon outgrowth, dendritogenesis, and synapse formation. To systematically profile proteome dynamics throughout neuronal differentiation, we took cultured rat hippocampal neurons at different developmental stages and monitored changes in protein abundance using a combination of stable isotope labeling and high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS. Almost one third of all 4,500 proteins quantified underwent a more than 2-fold expression change during neuronal differentiation, indicating extensive remodeling of the neuron proteome. To highlight the strength of our resource, we studied the neural-cell-adhesion molecule 1 (NCAM1 and found that it stimulates dendritic arbor development by promoting actin filament growth at the dendritic growth cone. We anticipate that our quantitative map of neuronal proteome dynamics is a rich resource for further analyses of the many identified proteins in various neurodevelopmental processes.

  4. Proteome analysis in the assessment of ageing.

    Science.gov (United States)

    Nkuipou-Kenfack, Esther; Koeck, Thomas; Mischak, Harald; Pich, Andreas; Schanstra, Joost P; Zürbig, Petra; Schumacher, Björn

    2014-11-01

    Based on demographic trends, the societies in many developed countries are facing an increasing number and proportion of people over the age of 65. The raise in elderly populations along with improved health-care will be concomitant with an increased prevalence of ageing-associated chronic conditions like cardiovascular, renal, and respiratory diseases, arthritis, dementia, and diabetes mellitus. This is expected to pose unprecedented challenges both for individuals and societies and their health care systems. An ultimate goal of ageing research is therefore the understanding of physiological ageing and the achievement of 'healthy' ageing by decreasing age-related pathologies. However, on a molecular level, ageing is a complex multi-mechanistic process whose contributing factors may vary individually, partly overlap with pathological alterations, and are often poorly understood. Proteome analysis potentially allows modelling of these multifactorial processes. This review summarises recent proteomic research on age-related changes identified in animal models and human studies. We combined this information with pathway analysis to identify molecular mechanisms associated with ageing. We identified some molecular pathways that are affected in most or even all organs and others that are organ-specific. However, appropriately powered studies are needed to confirm these findings based in in silico evaluation. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. 2 D gel based analysis of biological variability of the human plasma proteome

    DEFF Research Database (Denmark)

    Rentsch, Maria Louise; Jessen, Flemming

    Human blood plasma is a valuable specimen for the biomarker discovery process, since it is easily accessible and contains proteins that are synthesised, secreted or lost from cells and tissue. In this way, changes in plasma proteome reflect the current state of the organism. The analysis of plasma...... by one-week interval. Blood samples were drawn before the meal intake and five times during 24 hours for proteome analysis. Plasma was fractionated by use of IgY-12 spin column depleting the 12 highly abundant proteins and further processed for two-dimensional gel electrophoresis. The plasma proteome...... proteome is yet challenging due to the huge dynamic range of protein abundance. When evaluating a potential biomarker, stable basal level of the protein is needed before it can be considered a functional biomarker. However, basal level differences of plasma proteins are naturally occurring between...

  6. Structural dynamics flexibility informs function and evolution at a proteome scale

    Science.gov (United States)

    Nevin Gerek, Zeynep; Kumar, Sudhir; Banu Ozkan, Sefika

    2013-01-01

    Protein structures are dynamic entities with a myriad of atomic fluctuations, side-chain rotations, and collective domain movements. Although the importance of these dynamics to proper functioning of proteins is emerging in the studies of many protein families, there is a lack of broad evidence for the critical role of protein dynamics in shaping the biological functions of a substantial fraction of residues for a large number of proteins in the human proteome. Here, we propose a novel dynamic flexibility index (dfi) to quantify the dynamic properties of individual residues in any protein and use it to assess the importance of protein dynamics in 100 human proteins. Our analyses involving functionally critical positions, disease-associated and putatively neutral population variations, and the rate of interspecific substitutions per residue produce concordant patterns at a proteome scale. They establish that the preservation of dynamic properties of residues in a protein structure is critical for maintaining the protein/biological function. Therefore, structural dynamics needs to become a major component of the analysis of protein function and evolution. Such analyses will be facilitated by the dfi, which will also enable the integrative use of structural dynamics with evolutionary conservation in genomic medicine as well as functional genomics investigations. PMID:23745135

  7. Proteome Dynamics during Plastid Differentiation in Rice1[W

    Science.gov (United States)

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

    2007-01-01

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

  8. Comparative Proteomic Analysis of the Fiber Elongating Process in Cotton

    Institute of Scientific and Technical Information of China (English)

    LIU Jin-yuan; YANG Yi-wei; BIAN Shao-min

    2008-01-01

    @@ A comparative proteomic analysis was performed to explore the mechanism of cell elongation in developing cotton fibers.The temporal changes of global proteomes at five representative development stages (5~25 days post-anthesis [DPA]) were examined using 2-D electrophoresis.

  9. Dynamic proteomic profiling of a unicellular cyanobacterium Cyanothece ATCC51142 across light-dark diurnal cycles

    Energy Technology Data Exchange (ETDEWEB)

    Aryal, Uma K.; Stockel, Jana; Krovvidi, Ravi K.; Gritsenko, Marina A.; Monroe, Matthew E.; Moore, Ronald J.; Koppenaal, David W.; Smith, Richard D.; Pakrasi, Himadri B.; Jacobs, Jon M.

    2011-12-01

    Unicellular cyanobacteria of the genus Cyanothece are recognized for their ability to execute nitrogen (N2)-fixation in the dark and photosynthesis in the light. Systems-wide dynamic proteomic profiling with mass spectrometry (MS) analysis reveals fundamental insights into the control and regulation of these functions. To expand upon the current knowledge of protein expression patterns in Cyanothece ATCC51142, we performed quantitative proteomic analysis using partial ("unsaturated") metabolic labeling and high mass accuracy LC-MS analysis. This dynamic proteomic profiling identified 721 actively synthesized proteins with significant temporal changes in expression throughout the light-dark cycles, of which 425 proteins matched with previously characterized cycling transcripts. The remaining 296 proteins contained a cluster of proteins uniquely involved in DNA replication and repair, protein degradation, tRNA synthesis and modification, transport and binding, and regulatory functions. Analysis of protein functions revealed that the expression of nitrogenase in the dark is mediated by higher respiration and glycogen metabolism. We have also shown that Cyanothece ATCC51142 utilizes alternative pathways for carbon (C) and nitrogen (N) acquisition, particularly, aspartic acid and glutamate as substrates of C and N, respectively. Utilization of phosphoketolase (PHK) pathway for the conversion of xylulose-5P to pyruvate and acetyl-P likely constitutes an alternative strategy to compensate higher ATP and NADPH demand. In conclusion, this study provides a deeper insight into how Cyanothece ATCC51142 modulates cellular functions to accommodate photosynthesis and N2-fixation within the single cell.

  10. Representative proteomes: a stable, scalable and unbiased proteome set for sequence analysis and functional annotation.

    Directory of Open Access Journals (Sweden)

    Chuming Chen

    Full Text Available The accelerating growth in the number of protein sequences taxes both the computational and manual resources needed to analyze them. One approach to dealing with this problem is to minimize the number of proteins subjected to such analysis in a way that minimizes loss of information. To this end we have developed a set of Representative Proteomes (RPs, each selected from a Representative Proteome Group (RPG containing similar proteomes calculated based on co-membership in UniRef50 clusters. A Representative Proteome is the proteome that can best represent all the proteomes in its group in terms of the majority of the sequence space and information. RPs at 75%, 55%, 35% and 15% co-membership threshold (CMT are provided to allow users to decrease or increase the granularity of the sequence space based on their requirements. We find that a CMT of 55% (RP55 most closely follows standard taxonomic classifications. Further analysis of this set reveals that sequence space is reduced by more than 80% relative to UniProtKB, while retaining both sequence diversity (over 95% of InterPro domains and annotation information (93% of experimentally characterized proteins. All sets can be browsed and are available for sequence similarity searches and download at http://www.proteininformationresource.org/rps, while the set of 637 RPs determined using a 55% CMT are also available for text searches. Potential applications include sequence similarity searches, protein classification and targeted protein annotation and characterization.

  11. Identification of Metal Reductases using Proteomic Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Lipton, Mary

    2006-06-01

    Central to the NABIR goal to develop the scientific basis for in situ remediation of radioactive contaminants is the fundamental understanding of microorganisms with dissimilatory metal reducing activity. In order to effectively exploit these bacteria, it is necessary to know which enzymes and pathways are involved. Additionally, it would be advantageous to understand the similarities and differences of these pathways across different bacteria for effective deployment in bioremediation, as well as to identify new microbes capable of such activities. Most approaches to identify these enzymes or enzyme complexes rely on biochemical purification to homogeneity with subsequent Nterminal sequencing of digested peptides. However, loss of activity before achieving purity often necessitates repetition of the entire process. Newly developed proteomics capabilities at PNNL allow for the identification of many proteins from a single sample through mass spectrometry analysis.

  12. Proteomic analysis of mature Lagenaria siceraria seed.

    Science.gov (United States)

    Kumari, Neha; Tajmul, Md; Yadav, Savita

    2015-04-01

    Lagenaria siceraria (bottle gourd) class belongs to Magnoliopsida family curcurbitaceae that is a traditionally used medicinal plant. Fruit of this plant are widely used as a therapeutic vegetable in various diseases, all over the Asia and Africa. Various parts of this plant like fruit, seed, leaf and root are used as alternative medicine. In the present study, primarily, we have focused on proteomic analysis of L. siceraria seed using phenol extraction method for protein isolation. Twenty-four colloidal coomassie blue stained protein spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) after resolving on two-dimensional gel electrophoresis. Out of 24 identified protein spots, four were grouped as unidentified proteins which clearly suggest that less work has been done in the direction of plant seed proteomics. These proteins have been found to implicate in various functions such as biosynthesis of plant cell wall polysaccharides and glycoproteins, serine/threonine kinase activity, plant disease resistance and transferase activity against insects by means of insecticidal and larval growth inhibitory, anti-HIV, antihelmintic and antimicrobial properties. By Blast2GO annotation analysis, amongst the identified proteins of L. siceraria, molecular function for majority of proteins has indispensable role in catalytic activity, few in binding activity and antioxidant activity; it is mostly distributed in cell, organelle, membrane and macromolecular complex. Most of them involved in biological process such as metabolic process, cellular process, response to stimulus, single organism process, signalling, biological recognition, cellular component organization or biogenesis and localization.

  13. Preliminary analysis of cerebrospinal fluid proteome in patients with neurocysticercosis

    Institute of Scientific and Technical Information of China (English)

    TIAN Xiao-jun; LI Jing-yi; HUANG Yong; XUE Yan-ping

    2009-01-01

    Background Neurocysticercosis is the infection of the nervous system by the larvae of Taenia solium (T. solium). Despite continuous effort, the experimental diagnosis of neurocysticercosis remains unresolved. Since the cerebrospinal fluid (CSF) contacts with the brain, dynamic information about pathological processes of the brain is likely to be reflected in CSF. Therefore, CSF may serve as a rich source of putative biomarkers related to neurocysticercosis. Comparative proteomic analysis of CSF of neurocysticercosis patients and control subjects may find differentially expressed proteins. Methods Two-dimensional difference in gel electrophoresis (2D-DIGE) was used to investigate differentially expressed proteins in CSF of patients with neurocysticercosis by comparing the protein profile of CSF from neurocysticercosis patients with that from control subjects. The differentially expressed spots/proteins were recognized with matrix-assisted laser desorption/ionization-time of flight-time of flight (MALDI-TOF-TOF) mass spectrometry. Results Forty-four enzyme digested peptides were obtained from 4 neurocysticercotic patients. Twenty-three were identified through search of the NCBI protein database with Mascot software, showing 19 up-expressed and 4 down-expressed. Of these proteins, 26S proteosome related to ATP- and ubiquitin-dependent degradation of proteins and lipocalin type prostaglandin D synthase involved in PGD2-synthesis and extracellular transporter activities were up-expressed, while transferrin related to iron metabolism within the brain was down-expressed. Conclusions This study established the proteomic profile of pooled CSF from 4 patients with neurocysticercosis, suggesting the potential value of proteomic analysis for the study of candidate biomarkers involved in the diagnosis or pathogenesis of neurocysticercosis.

  14. Proteomics

    DEFF Research Database (Denmark)

    Dam, Svend; Stougaard, Jens

    2014-01-01

    proteomics data. Two characteristics of legumes are the high seed protein level and the nitrogen fixing symbiosis. Thus, the majority of the proteomics studies in Lotus have been performed on seed/pod and nodule/root tissues in order to create proteome reference maps and to enable comparative analyses within...... Lotus tissues or toward similar tissues from other legume species. More recently, N-glycan structures and compositions have been determined from mature Lotus seeds using glycomics and glycoproteomics, and finally, phosphoproteomics has been employed...... and annotated Lotus japonicus (Lotus) genome has been essential for obtaining high-quality protein identifications from proteomics studies. Furthermore, additional genomics and transcriptomics studies from several Lotus species/ecotypes support putative gene structures and these can be further supported using...

  15. High-density lipoprotein proteome dynamics in human endotoxemia

    Directory of Open Access Journals (Sweden)

    Stroes Erik SG

    2011-06-01

    Full Text Available Abstract Background A large variety of proteins involved in inflammation, coagulation, lipid-oxidation and lipid metabolism have been associated with high-density lipoprotein (HDL and it is anticipated that changes in the HDL proteome have implications for the multiple functions of HDL. Here, SELDI-TOF mass spectrometry (MS was used to study the dynamic changes of HDL protein composition in a human experimental low-dose endotoxemia model. Ten healthy men with low HDL cholesterol (0.7+/-0.1 mmol/L and 10 men with high HDL cholesterol levels (1.9+/-0.4 mmol/L were challenged with endotoxin (LPS intravenously (1 ng/kg bodyweight. We previously showed that subjects with low HDL cholesterol are more susceptible to an inflammatory challenge. The current study tested the hypothesis that this discrepancy may be related to differences in the HDL proteome. Results Plasma drawn at 7 time-points over a 24 hour time period after LPS challenge was used for direct capture of HDL using antibodies against apolipoprotein A-I followed by subsequent SELDI-TOF MS profiling. Upon LPS administration, profound changes in 21 markers (adjusted p-value Conclusions This study shows that the semi-quantitative differences in the HDL proteome as assessed by SELDI-TOF MS cannot explain why subjects with low HDL cholesterol are more susceptible to a challenge with LPS than those with high HDL cholesterol. Instead the results indicate that hierarchical clustering could be useful to predict HDL functionality in acute phase responses towards LPS.

  16. PROTEINCHALLENGE: Crowd sourcing in proteomics analysis and software development

    DEFF Research Database (Denmark)

    Martin, Sarah F.; Falkenberg, Heiner; Dyrlund, Thomas Franck

    2013-01-01

    , including arguments for community-wide open source software development and “big data” compatible solutions for the future. For the meantime, we have laid out ten top tips for data processing. With these at hand, a first large-scale proteomics analysis hopefully becomes less daunting to navigate.......However there is clearly a real need for robust tools, standard operating procedures and general acceptance of best practises. Thus we submit to the proteomics community a call for a community-wide open set of proteomics analysis challenges—PROTEINCHALLENGE—that directly target and compare data analysis workflows......In large-scale proteomics studies there is a temptation, after months of experimental work, to plug resulting data into a convenient—if poorly implemented—set of tools, which may neither do the data justice nor help answer the scientific question. In this paper we have captured key concerns...

  17. Characterisation of the circulating acellular proteome of healthy sheep using LC-MS/MS-based proteomics analysis of serum

    OpenAIRE

    Chemonges, Saul; Gupta, Rajesh; Mills, Paul C.; Steven R. Kopp; Sadowski, Pawel

    2017-01-01

    Background Unlike humans, there is currently no publicly available reference mass spectrometry-based circulating acellular proteome data for sheep, limiting the analysis and interpretation of a range of physiological changes and disease states. The objective of this study was to develop a robust and comprehensive method to characterise the circulating acellular proteome in ovine serum. Methods Serum samples from healthy sheep were subjected to shotgun proteomic analysis using nano liquid chro...

  18. Proteome-wide analysis and diel proteomic profiling of the cyanobacterium Arthrospira platensis PCC 8005.

    Directory of Open Access Journals (Sweden)

    Sabine Matallana-Surget

    Full Text Available The filamentous cyanobacterium Arthrospira platensis has a long history of use as a food supply and it has been used by the European Space Agency in the MELiSSA project, an artificial microecosystem which supports life during long-term manned space missions. This study assesses progress in the field of cyanobacterial shotgun proteomics and light/dark diurnal cycles by focusing on Arthrospira platensis. Several fractionation workflows including gel-free and gel-based protein/peptide fractionation procedures were used and combined with LC-MS/MS analysis, enabling the overall identification of 1306 proteins, which represents 21% coverage of the theoretical proteome. A total of 30 proteins were found to be significantly differentially regulated under light/dark growth transition. Interestingly, most of the proteins showing differential abundance were related to photosynthesis, the Calvin cycle and translation processes. A novel aspect and major achievement of this work is the successful improvement of the cyanobacterial proteome coverage using a 3D LC-MS/MS approach, based on an immobilized metal affinity chromatography, a suitable tool that enabled us to eliminate the most abundant protein, the allophycocyanin. We also demonstrated that cell growth follows a light/dark cycle in A. platensis. This preliminary proteomic study has highlighted new characteristics of the Arthrospira platensis proteome in terms of diurnal regulation.

  19. Proteome-wide analysis and diel proteomic profiling of the cyanobacterium Arthrospira platensis PCC 8005.

    Science.gov (United States)

    Matallana-Surget, Sabine; Derock, Jérémy; Leroy, Baptiste; Badri, Hanène; Deschoenmaeker, Frédéric; Wattiez, Ruddy

    2014-01-01

    The filamentous cyanobacterium Arthrospira platensis has a long history of use as a food supply and it has been used by the European Space Agency in the MELiSSA project, an artificial microecosystem which supports life during long-term manned space missions. This study assesses progress in the field of cyanobacterial shotgun proteomics and light/dark diurnal cycles by focusing on Arthrospira platensis. Several fractionation workflows including gel-free and gel-based protein/peptide fractionation procedures were used and combined with LC-MS/MS analysis, enabling the overall identification of 1306 proteins, which represents 21% coverage of the theoretical proteome. A total of 30 proteins were found to be significantly differentially regulated under light/dark growth transition. Interestingly, most of the proteins showing differential abundance were related to photosynthesis, the Calvin cycle and translation processes. A novel aspect and major achievement of this work is the successful improvement of the cyanobacterial proteome coverage using a 3D LC-MS/MS approach, based on an immobilized metal affinity chromatography, a suitable tool that enabled us to eliminate the most abundant protein, the allophycocyanin. We also demonstrated that cell growth follows a light/dark cycle in A. platensis. This preliminary proteomic study has highlighted new characteristics of the Arthrospira platensis proteome in terms of diurnal regulation.

  20. Connecting Genomic Alterations to Cancer Biology with Proteomics: The NCI Clinical Proteomic Tumor Analysis Consortium

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-10-03

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

  1. Network Tools for the Analysis of Proteomic Data.

    Science.gov (United States)

    Chisanga, David; Keerthikumar, Shivakumar; Mathivanan, Suresh; Chilamkurti, Naveen

    2017-01-01

    Recent advancements in high-throughput technologies such as mass spectrometry have led to an increase in the rate at which data is generated and accumulated. As a result, standard statistical methods no longer suffice as a way of analyzing such gigantic amounts of data. Network analysis, the evaluation of how nodes relate to one another, has over the years become an integral tool for analyzing high throughput proteomic data as they provide a structure that helps reduce the complexity of the underlying data.Computational tools, including pathway databases and network building tools, have therefore been developed to store, analyze, interpret, and learn from proteomics data. These tools enable the visualization of proteins as networks of signaling, regulatory, and biochemical interactions. In this chapter, we provide an overview of networks and network theory fundamentals for the analysis of proteomics data. We further provide an overview of interaction databases and network tools which are frequently used for analyzing proteomics data.

  2. Proteomic analysis of post-translational modifications

    DEFF Research Database (Denmark)

    Mann, Matthias; Jensen, Ole N

    2003-01-01

    Post-translational modifications modulate the activity of most eukaryote proteins. Analysis of these modifications presents formidable challenges but their determination generates indispensable insight into biological function. Strategies developed to characterize individual proteins are now...... mass spectrometric peptide sequencing and analysis technologies hold tremendous potential. Finally, stable isotope labeling strategies in combination with mass spectrometry have been applied successfully to study the dynamics of modifications....

  3. Dynamic proteomic profiling of a unicellular cyanobacterium Cyanothece ATCC51142 across light-dark diurnal cycles

    Directory of Open Access Journals (Sweden)

    Aryal Uma K

    2011-12-01

    Full Text Available Abstract Background Unicellular cyanobacteria of the genus Cyanothece are recognized for their ability to execute nitrogen (N2-fixation in the dark and photosynthesis in the light. An understanding of these mechanistic processes in an integrated systems context should provide insights into how Cyanothece might be optimized for specialized environments and/or industrial purposes. Systems-wide dynamic proteomic profiling with mass spectrometry (MS analysis should reveal fundamental insights into the control and regulation of these functions. Results To expand upon the current knowledge of protein expression patterns in Cyanothece ATCC51142, we performed quantitative proteomic analysis using partial ("unsaturated" metabolic labeling and high mass accuracy LC-MS analysis. This dynamic proteomic profiling identified 721 actively synthesized proteins with significant temporal changes in expression throughout the light-dark cycles, of which 425 proteins matched with previously characterized cycling transcripts. The remaining 296 proteins contained a cluster of proteins uniquely involved in DNA replication and repair, protein degradation, tRNA synthesis and modification, transport and binding, and regulatory functions. Functional classification of labeled proteins suggested that proteins involved in respiration and glycogen metabolism showed increased expression in the dark cycle together with nitrogenase, suggesting that N2-fixation is mediated by higher respiration and glycogen metabolism. Results indicated that Cyanothece ATCC51142 might utilize alternative pathways for carbon (C and nitrogen (N acquisition, particularly, aspartic acid and glutamate as substrates of C and N, respectively. Utilization of phosphoketolase (PHK pathway for the conversion of xylulose-5P to pyruvate and acetyl-P likely constitutes an alternative strategy to compensate higher ATP and NADPH demand. Conclusion This study provides a deeper systems level insight into how

  4. A proteomic analysis of human bile

    DEFF Research Database (Denmark)

    Kristiansen, Troels Zakarias; Bunkenborg, Jakob; Gronborg, Mads

    2004-01-01

    We have carried out a comprehensive characterization of human bile to define the bile proteome. Our approach involved fractionation of bile by one-dimensional gel electrophoresis and lectin affinity chromatography followed by liquid chromatography tandem mass spectrometry. Overall, we identified ...

  5. Preprocessing and Analysis of LC-MS-Based Proteomic Data.

    Science.gov (United States)

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

    2016-01-01

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

  6. Dynamics of nuclear matrix proteome during embryonic development in Drosophila melanogaster

    Indian Academy of Sciences (India)

    Parul Varma; Rakesh K Mishra

    2011-08-01

    Embryonic development is a complex and dynamic process that involves spatiotemporal expression of genes in a highly coordinated manner. Multiple levels of nuclear architecture maintain the fidelity of gene expression programme. One of the components of nuclear architecture, which is believed to play an important role in regulation of gene expression, is the nuclear matrix (NuMat). Many studies over the past few years have tried to analyse the components of this non-chromatin scaffolding of the nucleus and have provided evidences of its structural and functional complexity. However, the relationship of NuMat with the process of embryonic development still remains poorly understood. Here, we report a comparative analysis of the NuMat proteomes of early and late stage Drosophila melanogaster embryos and show that 65% of the NuMat proteome is dynamic during development. Our study establishes links between the dynamics of nuclear architecture and embryonic development and provides tools to further understand the process such as cellular differentiation in the context of higher-order nuclear organization.

  7. Proteome Profile and Quantitative Proteomic Analysis of Buffalo (Bubalusbubalis) Follicular Fluid during Follicle Development.

    Science.gov (United States)

    Fu, Qiang; Huang, Yulin; Wang, Zhiqiang; Chen, Fumei; Huang, Delun; Lu, Yangqing; Liang, Xianwei; Zhang, Ming

    2016-04-29

    Follicular fluid (FF) accumulates in the antrum of the ovarian follicle and provides the microenvironment for oocyte development. FF plays an important role in follicle growth and oocyte maturation. The FF provides a unique window to investigate the processes occurring during buffalo follicular development. The observed low quality of buffalo oocytes may arise from the poor follicular microenvironment. Investigating proteins found in buffalo FF (BFF) should provide insight into follicular development processes and provide further understanding of intra-follicular maturation and oocytes quality. Here, a proteomic-based approach was used to analyze the proteome of BFF. SDS-PAGE separation combined with mass spectrometry was used to generate the proteomic dataset. In total, 363 proteins were identified and classified by Gene Ontology terms. The proteins were assigned to 153 pathways, including signaling pathways. To evaluate difference in proteins expressed between BFF with different follicle size (small, 8 mm), a quantitative proteomic analysis based on multi-dimensional liquid chromatography pre-fractionation tandem Orbitrap mass spectrometry identification was performed. Eleven differentially expressed proteins (six downregulated and five upregulated in large BFF) were identified and assigned to a variety of functional processes, including serine protease inhibition, oxidation protection and the complement cascade system. Three differentially expressed proteins, Vimentin, Peroxiredoxin-1 and SERPIND1, were verified by Western blotting, consistent with the quantitative proteomics results. Our datasets offers new information about proteins present in BFF and should facilitate the development of new biomarkers. These differentially expressed proteins illuminate the size-dependent protein changes in follicle microenvironment.

  8. Global dynamics of the Escherichia coli proteome and phosphoproteome during growth in minimal medium.

    Science.gov (United States)

    Soares, Nelson C; Spät, Philipp; Krug, Karsten; Macek, Boris

    2013-06-07

    Recent phosphoproteomics studies have generated relatively large data sets of bacterial proteins phosphorylated on serine, threonine, and tyrosine, implicating this type of phosphorylation in the regulation of vital processes of a bacterial cell; however, most phosphoproteomics studies in bacteria were so far qualitative. Here we applied stable isotope labeling by amino acids in cell culture (SILAC) to perform a quantitative analysis of proteome and phosphoproteome dynamics of Escherichia coli during five distinct phases of growth in the minimal medium. Combining two triple-SILAC experiments, we detected a total of 2118 proteins and quantified relative dynamics of 1984 proteins in all measured phases of growth, including 570 proteins associated with cell wall and membrane. In the phosphoproteomic experiment, we detected 150 Ser/Thr/Tyr phosphorylation events, of which 108 were localized to a specific amino acid residue and 76 were quantified in all phases of growth. Clustering analysis of SILAC ratios revealed distinct sets of coregulated proteins for each analyzed phase of growth and overrepresentation of membrane proteins in transition between exponential and stationary phases. The proteomics data indicated that proteins related to stress response typically associated with the stationary phase, including RpoS-dependent proteins, had increasing levels already during earlier phases of growth. Application of SILAC enabled us to measure median occupancies of phosphorylation sites, which were generally low (growth.

  9. A proteomic strategy for global analysis of plant protein complexes.

    Science.gov (United States)

    Aryal, Uma K; Xiong, Yi; McBride, Zachary; Kihara, Daisuke; Xie, Jun; Hall, Mark C; Szymanski, Daniel B

    2014-10-01

    Global analyses of protein complex assembly, composition, and location are needed to fully understand how cells coordinate diverse metabolic, mechanical, and developmental activities. The most common methods for proteome-wide analysis of protein complexes rely on affinity purification-mass spectrometry or yeast two-hybrid approaches. These methods are time consuming and are not suitable for many plant species that are refractory to transformation or genome-wide cloning of open reading frames. Here, we describe the proof of concept for a method allowing simultaneous global analysis of endogenous protein complexes that begins with intact leaves and combines chromatographic separation of extracts from subcellular fractions with quantitative label-free protein abundance profiling by liquid chromatography-coupled mass spectrometry. Applying this approach to the crude cytosolic fraction of Arabidopsis thaliana leaves using size exclusion chromatography, we identified hundreds of cytosolic proteins that appeared to exist as components of stable protein complexes. The reliability of the method was validated by protein immunoblot analysis and comparisons with published size exclusion chromatography data and the masses of known complexes. The method can be implemented with appropriate instrumentation, is applicable to any biological system, and has the potential to be further developed to characterize the composition of protein complexes and measure the dynamics of protein complex localization and assembly under different conditions.

  10. Proteomic analysis of an unsequenced plant--Mangifera indica.

    Science.gov (United States)

    Renuse, Santosh; Harsha, H C; Kumar, Praveen; Acharya, Pradip Kumar; Sharma, Jyoti; Goel, Renu; Kumar, Ghantasala S Sameer; Raju, Rajesh; Prasad, T S Keshava; Slotta, Tracey; Pandey, Akhilesh

    2012-10-22

    Mangifera indica (Mango) is an important fruit crop in tropical countries with India being the leading producer in the world. Substantial research efforts are being devoted to produce fruit that have desirable characteristics including those that pertain to taste, hardiness and resistance to pests. Characterization of the genome and proteome of mango would help in the improvement of cultivars. As the mango genome has not yet been sequenced, we employed a mass spectrometry-based approach followed by database searches of mango-derived ESTs and proteins along with proteins from six other closely related plant species to characterize its proteome. In addition to this, de novo sequencing followed by homology-based protein identification was also carried out. The LC-MS/MS analysis of the mango leaf proteome was performed using an accurate mass quadrupole time-of-flight mass spectrometer. This integrative approach enabled the identification of 1001 peptides that matched to 538 proteins. To our knowledge, this study is the first high-throughput analysis of mango leaf proteome and could pave the way for further genomic, transcriptomic and proteomic studies.

  11. Comprehensive proteomic analysis of human pancreatic juice

    DEFF Research Database (Denmark)

    Grønborg, Mads; Bunkenborg, Jakob; Kristiansen, Troels Zakarias

    2004-01-01

    ). In addition, we identified a number of proteins that have not been previously described in pancreatic juice (e.g., tumor rejection antigen (pg96) and azurocidin). Interestingly, a novel protein that is 85% identical to HIP/PAP was identified, which we have designated as PAP-2. The proteins identified...... in this study could be directly assessed for their potential as biomarkers for pancreatic cancer by quantitative proteomics methods or immunoassays....

  12. A proteomic analysis of human hemodialysis fluid

    DEFF Research Database (Denmark)

    Molina, Henrik; Bunkenborg, Jakob; Reddy, G Hanumanthu;

    2005-01-01

    The vascular compartment is an easily accessible compartment that provides an opportunity to measure analytes for diagnostic, prognostic, or therapeutic indications. Both serum and plasma have been analyzed extensively by proteomic approaches in an effort to catalog all proteins and polypeptides......., including cytokines, were only present as predicted transcripts in data bases and thus represent novel proteins. The proteins identified in this study could serve as biomarkers in serum using more sensitive methods such as ELISA-specific antibodies....

  13. Redefining the Breast Cancer Exosome Proteome by Tandem Mass Tag Quantitative Proteomics and Multivariate Cluster Analysis.

    Science.gov (United States)

    Clark, David J; Fondrie, William E; Liao, Zhongping; Hanson, Phyllis I; Fulton, Amy; Mao, Li; Yang, Austin J

    2015-10-20

    Exosomes are microvesicles of endocytic origin constitutively released by multiple cell types into the extracellular environment. With evidence that exosomes can be detected in the blood of patients with various malignancies, the development of a platform that uses exosomes as a diagnostic tool has been proposed. However, it has been difficult to truly define the exosome proteome due to the challenge of discerning contaminant proteins that may be identified via mass spectrometry using various exosome enrichment strategies. To better define the exosome proteome in breast cancer, we incorporated a combination of Tandem-Mass-Tag (TMT) quantitative proteomics approach and Support Vector Machine (SVM) cluster analysis of three conditioned media derived fractions corresponding to a 10 000g cellular debris pellet, a 100 000g crude exosome pellet, and an Optiprep enriched exosome pellet. The quantitative analysis identified 2 179 proteins in all three fractions, with known exosomal cargo proteins displaying at least a 2-fold enrichment in the exosome fraction based on the TMT protein ratios. Employing SVM cluster analysis allowed for the classification 251 proteins as "true" exosomal cargo proteins. This study provides a robust and vigorous framework for the future development of using exosomes as a potential multiprotein marker phenotyping tool that could be useful in breast cancer diagnosis and monitoring disease progression.

  14. Proteome Analysis of Rheumatoid Arthritis Gut Mucosa.

    Science.gov (United States)

    Bennike, Tue Bjerg; Ellingsen, Torkell; Glerup, Henning; Bonderup, Ole Kristian; Carlsen, Thomas Gelsing; Meyer, Michael Kruse; Bøgsted, Martin; Christiansen, Gunna; Birkelund, Svend; Andersen, Vibeke; Stensballe, Allan

    2017-01-06

    Rheumatoid arthritis (RA) is an inflammatory joint disease leading to cartilage damage and ultimately impaired joint function. To gain new insight into the systemic immune manifestations of RA, we characterized the colon mucosa proteome from 11 RA-patients and 10 healthy controls. The biopsies were extracted by colonoscopy and analyzed by label-free quantitative proteomics, enabling the quantitation of 5366 proteins. The abundance of dihydrofolate reductase (DHFR) was statistically significantly increased in RA-patient biopsies compared with controls and correlated with the administered dosage of methotrexate (MTX), the most frequently prescribed immunosuppressive drug for RA. Additionally, our data suggest that treatment with Leflunomide, a common alternative to MTX, increases DHFR. The findings were supported by immunohistochemistry with confocal microscopy, which furthermore demonstrated that DHFR was located in the cytosol of the intestinal epithelial and interstitial cells. Finally, we identified 223 citrullinated peptides from 121 proteins. Three of the peptides were unique to RA. The list of citrullinated proteins was enriched in extracellular and membrane proteins and included known targets of anticitrullinated protein antibodies (ACPAs). Our findings support that the colon mucosa could trigger the production of ACPAs, which could contribute to the onset of RA. The MS data have been deposited to ProteomeXchange with identifiers PXD001608 and PXD003082.

  15. Interaction Analysis through Proteomic Phage Display

    Directory of Open Access Journals (Sweden)

    Gustav N. Sundell

    2014-01-01

    Full Text Available Phage display is a powerful technique for profiling specificities of peptide binding domains. The method is suited for the identification of high-affinity ligands with inhibitor potential when using highly diverse combinatorial peptide phage libraries. Such experiments further provide consensus motifs for genome-wide scanning of ligands of potential biological relevance. A complementary but considerably less explored approach is to display expression products of genomic DNA, cDNA, open reading frames (ORFs, or oligonucleotide libraries designed to encode defined regions of a target proteome on phage particles. One of the main applications of such proteomic libraries has been the elucidation of antibody epitopes. This review is focused on the use of proteomic phage display to uncover protein-protein interactions of potential relevance for cellular function. The method is particularly suited for the discovery of interactions between peptide binding domains and their targets. We discuss the largely unexplored potential of this method in the discovery of domain-motif interactions of potential biological relevance.

  16. Proteomic analysis of normal murine brain parts.

    Science.gov (United States)

    Taraslia, Vasiliki K; Kouskoukis, Alexandros; Anagnostopoulos, Athanasios K; Stravopodis, Dimitrios J; Margaritis, Lukas H; Tsangaris, George Th

    2013-01-01

    Murine brain is an excellent tool for studying protein expression and brain function in mammals. Although mice are an extensively used model to recapitulate various pathological conditions, the proteome of the normal mouse brain has not been yet reported. In the present study, we identified the total proteins of different parts of the brain of CB7BL/6 mice, a widely used strain, by applying proteomic methodologies. The adult mouse brain was dissected anatomically into the following regions: frontal cortex, olfactory bulb, hippocampus, midbrain, cerebellum, hypothalamus and medulla. Total protein extracts of these regions were separated by two-dimensional gel electrophoresis and analyzed by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry, following in-gel digestion with trypsin. Protein identification was carried out by peptide mass fingerprint. Thus, 515 different single-gene products were identified in total, 54 expressed specifically in the olfactory bulb, 62 in the hippocampus, 36 in the frontal cortex, five in the cerebellum, nine in the midbrain, eight in the hypothamamus and 10 in the medulla. The majority of the proteins were enzymes, structural proteins and transporters. Moreover, the distribution of these molecules appears to exhibit direct correlation with the function of the brain regions where they were expressed. This study leads to the complete characterization of the normal mouse brain proteome as well as the protein expression profile of the different brain regions. These results will aid in addressing unmet scientific needs regarding physiological and pathological brain functions.

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

    Science.gov (United States)

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

    2015-01-01

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

  18. Dynamics of chloroplast proteome in salt-stressed mangrove Kandelia candel (L.) Druce.

    Science.gov (United States)

    Wang, Lingxia; Liang, Wenyu; Xing, Jianhong; Tan, Fanglin; Chen, Yiyong; Huang, Li; Cheng, Chi-Lien; Chen, Wei

    2013-11-01

    Kandelia candel is being established as a model xylophyte for ecoadaptation due to its salt tolerance. To adapt to high salinity, the photosynthesis apparatus must function efficiently under these conditions. Proteomic analysis of chloroplasts isolated from plants under different degrees of salt stress was performed to quantify the changes of individual proteins and to gain a global view of the total chloroplast protein dynamics. Among the 1030 proteins quantified (unique peptide ≥ 1), 76 showed a more than 1.5-fold change in abundance, of which 36 are involved in the light-dependent reactions and 12 in the Calvin cycle. The dynamic change of these proteins indicates that light-dependent reactions are maintained by up-regulating the levels of component proteins at both moderate and high salinity, and the Calvin cycle remained functional at moderate salinity but showed a decline at high salinity. In addition to proteins related to photosynthesis, some known abiotic-stress proteins and plastoglobuli were up-regulated in salt-stressed plants. Plastoglobuli might contribute to maintaining membrane integrity and fluidity. In conclusion, this extensive proteomic investigation on intact chloroplasts of the salt-tolerant xylophyte under salt stress provides some important novel information on adaptative mechanisms involving photosynthesis in responses to salt stress in K. candel.

  19. Noninvasive diagnosis of chronic kidney diseases using urinary proteome analysis

    DEFF Research Database (Denmark)

    Siwy, Justyna; Zürbig, Petra; Argiles, Angel

    2016-01-01

    BACKGROUND: In spite of its invasive nature and risks, kidney biopsy is currently required for precise diagnosis of many chronic kidney diseases (CKDs). Here, we explored the hypothesis that analysis of the urinary proteome can discriminate different types of CKD irrespective of the underlying...... mechanism of disease. METHODS: We used data from the proteome analyses of 1180 urine samples from patients with different types of CKD, generated by capillary electrophoresis coupled to mass spectrometry. A set of 706 samples served as the discovery cohort, and 474 samples were used for independent...

  20. Proteomic analysis of tissue samples in translational breast cancer research

    DEFF Research Database (Denmark)

    Gromov, Pavel; Moreira, José; Gromova, Irina

    2014-01-01

    , and both prognosis and prediction of outcome of chemotherapy. The purpose of this review is to critically appraise what has been achieved to date using proteomic technologies and to bring forward novel strategies - based on the analysis of clinically relevant samples - that promise to accelerate......In the last decade, many proteomic technologies have been applied, with varying success, to the study of tissue samples of breast carcinoma for protein expression profiling in order to discover protein biomarkers/signatures suitable for: characterization and subtyping of tumors; early diagnosis...

  1. PIQMIe: A web server for semi-quantitative proteomics data management and analysis

    NARCIS (Netherlands)

    A. Kuzniar (Arnold); R. Kanaar (Roland)

    2014-01-01

    textabstractWe present the Proteomics Identifications and Quantitations Data Management and Integration Service or PIQMIe that aids in reliable and scalable data management, analysis and visualization of semi-quantitative mass spectrometry based proteomics experiments. PIQMIe readily integrates pept

  2. Differential proteomic analysis reveals novel links between primary metabolism and antibiotic production in Amycolatopsis balhimycina

    DEFF Research Database (Denmark)

    Gallo, G.; Renzone, G.; Alduina, R.

    2010-01-01

    A differential proteomic analysis, based on 2-DE and MS procedures, was performed on Amycolatopsis balhimycina DSM5908, the actinomycete producing the vancomycin-like antibiotic balhimycin. A comparison of proteomic profiles before and during balhimycin production characterized differentially...

  3. PROTEINCHALLENGE: Crowd sourcing in proteomics analysis and software development

    DEFF Research Database (Denmark)

    Martin, Sarah F.; Falkenberg, Heiner; Dyrlund, Thomas Franck;

    2013-01-01

    , including arguments for community-wide open source software development and “big data” compatible solutions for the future. For the meantime, we have laid out ten top tips for data processing. With these at hand, a first large-scale proteomics analysis hopefully becomes less daunting to navigate...

  4. Comparative proteomic analysis of human pancreatic juice : Methodological study

    NARCIS (Netherlands)

    Zhou, Lu; Lu, ZhaoHui; Yang, AiMing; Deng, RuiXue; Mai, CanRong; Sang, XinTing; Faber, Klaas Nico; Lu, XingHua

    Pancreatic cancer is the most lethal of all the common malignancies. Markers for early detection of this disease are urgently needed. Here, we optimized and applied a proteome analysis of human pancreatic juice to identify biomarkers for pancreatic cancer. Pancreatic juice samples, devoid of blood

  5. Proteomic analysis of the Arabidopsis nucleolus suggests novel nucleolar functions

    DEFF Research Database (Denmark)

    Pendle, Alison F; Clark, Gillian P; Boon, Reinier

    2005-01-01

    The eukaryotic nucleolus is involved in ribosome biogenesis and a wide range of other RNA metabolism and cellular functions. An important step in the functional analysis of the nucleolus is to determine the complement of proteins of this nuclear compartment. Here, we describe the first proteomic ...

  6. Proteomic analysis of Arabidopsis seed germination and priming

    NARCIS (Netherlands)

    Gallardo, K.; Job, C.; Groot, S.P.C.; Puype, M.; Demol, H.; Vandekerckhove, J.; Job, D.

    2001-01-01

    To better understand seed germination, a complex developmental process, we developed a proteome analysis of the model plant Arabidopsis for which complete genome sequence is now available. Among about 1,300 total seed proteins resolved in two-dimensional gels, changes in the abundance (up- and down-

  7. Comparative proteomic analysis of human pancreatic juice : Methodological study

    NARCIS (Netherlands)

    Zhou, Lu; Lu, ZhaoHui; Yang, AiMing; Deng, RuiXue; Mai, CanRong; Sang, XinTing; Faber, Klaas Nico; Lu, XingHua

    2007-01-01

    Pancreatic cancer is the most lethal of all the common malignancies. Markers for early detection of this disease are urgently needed. Here, we optimized and applied a proteome analysis of human pancreatic juice to identify biomarkers for pancreatic cancer. Pancreatic juice samples, devoid of blood o

  8. Comparative proteomic analysis of human pancreatic juice : Methodological study

    NARCIS (Netherlands)

    Zhou, Lu; Lu, ZhaoHui; Yang, AiMing; Deng, RuiXue; Mai, CanRong; Sang, XinTing; Faber, Klaas Nico; Lu, XingHua

    2007-01-01

    Pancreatic cancer is the most lethal of all the common malignancies. Markers for early detection of this disease are urgently needed. Here, we optimized and applied a proteome analysis of human pancreatic juice to identify biomarkers for pancreatic cancer. Pancreatic juice samples, devoid of blood o

  9. Comparative proteomic analysis of human pancreatic juice: Methodological study

    NARCIS (Netherlands)

    Zhou, Lu; Lu, Z.H.; Yang, A.M.; Deng, R.X.; Mai, C.R.; Sang, X.T.; Faber, Klaas Nico; Lu, X.H.

    2007-01-01

    Pancreatic cancer is the most lethal of all the common malignancies. Markers for early detection of this disease are urgently needed. Here, we optimized and applied a proteome analysis of human pancreatic juice to identify biomarkers for pancreatic cancer. Pancreatic juice samples, devoid of blood o

  10. PROTEINCHALLENGE: crowd sourcing in proteomics analysis and software development.

    Science.gov (United States)

    Martin, Sarah F; Falkenberg, Heiner; Dyrlund, Thomas F; Khoudoli, Guennadi A; Mageean, Craig J; Linding, Rune

    2013-08-02

    In large-scale proteomics studies there is a temptation, after months of experimental work, to plug resulting data into a convenient-if poorly implemented-set of tools, which may neither do the data justice nor help answer the scientific question. In this paper we have captured key concerns, including arguments for community-wide open source software development and "big data" compatible solutions for the future. For the meantime, we have laid out ten top tips for data processing. With these at hand, a first large-scale proteomics analysis hopefully becomes less daunting to navigate. However there is clearly a real need for robust tools, standard operating procedures and general acceptance of best practises. Thus we submit to the proteomics community a call for a community-wide open set of proteomics analysis challenges--PROTEINCHALLENGE--that directly target and compare data analysis workflows, with the aim of setting a community-driven gold standard for data handling, reporting and sharing. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. Proteomic analysis of hippocampus in the rat

    Institute of Scientific and Technical Information of China (English)

    ZHANG Bo; WANG Ren-zhi; LIAN Zhi-gang; YAO Yong

    2004-01-01

    Objective To analyze the protein expression in the rat hippocampus by the proteomic approach.Methods Proteins from hippocampal tissue homogenates of the rat were separated by two-dimensional gel electrophoresis(2-DE),and stained with colloidal Coomassie blue to produce a high-resolution map of the rat hippocampus proteome.Selected proteins from this map were digested with trypsin,and the resulting tryptic peptides were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS).The mass spectrometric data were used to identify the proteins through searches of the NCBI protein sequence database.Results 37 prominent proteins with various functional characteristics were identified.The identified brain protein classes covered metabolism enzymes,cytoskeleton proteins,heat shock proteins,antioxidant proteins,signalling proteins,proteasome-related proteins,neuron-specific proteins and glial-associated proteins.Furthermore,3 hypothetical proteins,unknown proteins so far only proposed from their nucleic acid structure,were identified.Conclusion This study provides the first unbiased characterization of proteins of the rat hippocampus and will be used for future studies of differential protein expression in rat models of neurological disorders.

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

    Science.gov (United States)

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

    2014-06-13

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

  13. Proteome research in food science.

    Science.gov (United States)

    Pischetsrieder, Monika; Baeuerlein, Rainer

    2009-09-01

    The proteome is the totality of proteins present in a biological sample. In contrast to the static genome, the proteome is highly dynamic, influenced by the genome and many external factors, such as the state of development, tissue type, metabolic state, and various interactions. Thus, the proteome reflects very closely the biological (and chemical) processes occurring in a system. For proteome analysis, gel based and shotgun methods are most widely applied. Because of the potential to generate a systematic view of protein composition and biological as well as chemical interactions, the application of proteome analysis in food science is steadily growing. This tutorial review introduces several fields in food science, where proteomics has been successfully applied: analysis of food composition, safety assessment of genetically modified food, the search for marker proteins for food authentication, identification of food allergens, systematic analysis of the physiological activity of food, analysis of the effects of processing on food proteins and the improvement of food quality.

  14. HiQuant: Rapid Postquantification Analysis of Large-Scale MS-Generated Proteomics Data.

    Science.gov (United States)

    Bryan, Kenneth; Jarboui, Mohamed-Ali; Raso, Cinzia; Bernal-Llinares, Manuel; McCann, Brendan; Rauch, Jens; Boldt, Karsten; Lynn, David J

    2016-06-01

    Recent advances in mass-spectrometry-based proteomics are now facilitating ambitious large-scale investigations of the spatial and temporal dynamics of the proteome; however, the increasing size and complexity of these data sets is overwhelming current downstream computational methods, specifically those that support the postquantification analysis pipeline. Here we present HiQuant, a novel application that enables the design and execution of a postquantification workflow, including common data-processing steps, such as assay normalization and grouping, and experimental replicate quality control and statistical analysis. HiQuant also enables the interpretation of results generated from large-scale data sets by supporting interactive heatmap analysis and also the direct export to Cytoscape and Gephi, two leading network analysis platforms. HiQuant may be run via a user-friendly graphical interface and also supports complete one-touch automation via a command-line mode. We evaluate HiQuant's performance by analyzing a large-scale, complex interactome mapping data set and demonstrate a 200-fold improvement in the execution time over current methods. We also demonstrate HiQuant's general utility by analyzing proteome-wide quantification data generated from both a large-scale public tyrosine kinase siRNA knock-down study and an in-house investigation into the temporal dynamics of the KSR1 and KSR2 interactomes. Download HiQuant, sample data sets, and supporting documentation at http://hiquant.primesdb.eu .

  15. Proteomics Analysis of the Causative Agent of Typhoid Fever

    Energy Technology Data Exchange (ETDEWEB)

    Ansong, Charles; Yoon, Hyunjin; Norbeck, Angela D.; Gustin, Jean K.; McDermott, Jason E.; Mottaz, Heather M.; Rue, Joanne; Adkins, Joshua N.; Heffron, Fred; Smith, Richard D.

    2008-02-01

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

  16. Aging synaptic mitochondria exhibit dynamic proteomic changes while maintaining bioenergetic function.

    Science.gov (United States)

    Stauch, Kelly L; Purnell, Phillip R; Fox, Howard S

    2014-04-01

    Aging correlates with a progressive impairment of mitochondrial homeostasis and is an influential factor for several forms of neurodegeneration. However, the mechanisms underlying age-related alterations in synaptosomal mitochondria, a neuronal mitochondria population highly susceptible to insults and critical for brain function, remain incompletely understood. Therefore this study investigates the synaptic mitochondrial proteomic and bioenergetic alterations that occur with age. The utilization of a state of the art quantitative proteomics approach allowed for the comparison of protein expression levels in synaptic mitochondria isolated from 5 (mature), 12 (old), and 24 (aged) month old mice. During the process of aging we find that dynamic proteomic alterations occur in synaptic mitochondria. Despite direct (mitochondrial DNA deletions) and indirect (increased antioxidant protein levels) signs of mitochondrial damage in the aged mice, there was an overall maintenance of mitochondrial function. Therefore the synaptic mitochondrial proteomic changes that occur with aging correlate with preservation of synaptic mitochondrial function.

  17. Dynamics of the proteome in human and farm animal milk

    NARCIS (Netherlands)

    Zhang, L.

    2015-01-01

    Abstract

    The milk proteome changes due to many factors, such as lactation, individual, health status, processing, and species differences. The objective of the work described in this thesis was to increase our

  18. Dynamics of the proteome in human and farm animal milk

    NARCIS (Netherlands)

    Zhang, L.

    2015-01-01

    Abstract

    The milk proteome changes due to many factors, such as lactation, individual, health status, processing, and species differences. The objective of the work described in this thesis was to increase our understan

  19. Analysis of the soluble cell wall proteome of gymnosperms.

    Science.gov (United States)

    Uzal, Esther Novo; Gómez-Ros, Laura V; Hernández, Jose A; Pedreño, María A; Cuello, Juan; Ros Barceló, Alfonso

    2009-05-15

    We analyzed the cell wall proteome of lignifying suspension cell cultures (SCCs) from four gymnosperms that differ in evolution degree. This analysis showed the presence of "peptide sequence tags" (PSTs) corresponding to glucan endo-1,3-beta-D-glucosidase, xyloglucan-endotrans-glucosylase/hydrolase, chitinases, thaumatin-like proteins and proteins involved in lignin/lignan biosynthesis, such as dirigent-like proteins and peroxidases. Surprisingly, and given the abundance of peroxidases in the cell wall proteome of these gymnosperms, PSTs corresponding to peroxidases were only detected in tryptic fragments of the cell wall proteome of Cycas revoluta. The current lack of knowledge regarding C. revoluta peroxidases led us to purify, characterize and partially sequence the peroxidases responsible for lignin biosynthesis in this species. This yielded three peroxidase-enriched fractions: CrPrx 1, CrPrx 2 and CrPrx 3. Analyses of tryptic peptides of CrPrx 2 (32kDa) and CrPrx 3 (26kDa) suggest that CrPrx 3 arises from CrPrx 2 by protein truncation, and that CrPrx 3 apparently constitutes a post-translational modification of CrPrx 2. That CrPrx 2 and CrPrx 3 are apparently the same enzyme was also deduced from the similarity between the k(cat) shown by both peroxidases for the three monolignols. These results emphasize the analogies between the cell wall proteome of gymnosperms and angiosperms, the complexity of the peroxidase proteome, and the difficulties involved in establishing fine structure-function relationships.

  20. Differential proteome analysis of chikungunya virus infection on host cells.

    Directory of Open Access Journals (Sweden)

    Christina Li-Ping Thio

    Full Text Available BACKGROUND: Chikungunya virus (CHIKV is an emerging mosquito-borne alphavirus that has caused multiple unprecedented and re-emerging outbreaks in both tropical and temperate countries. Despite ongoing research efforts, the underlying factors involved in facilitating CHIKV replication during early infection remains ill-characterized. The present study serves to identify host proteins modulated in response to early CHIKV infection using a proteomics approach. METHODOLOGY AND PRINCIPAL FINDINGS: The whole cell proteome profiles of CHIKV-infected and mock control WRL-68 cells were compared and analyzed using two-dimensional gel electrophoresis (2-DGE. Fifty-three spots were found to be differentially modulated and 50 were successfully identified by MALDI-TOF/TOF. Eight were significantly up-regulated and 42 were down-regulated. The mRNA expressions of 15 genes were also found to correlate with the corresponding protein expression. STRING network analysis identified several biological processes to be affected, including mRNA processing, translation, energy production and cellular metabolism, ubiquitin-proteasome pathway (UPP and cell cycle regulation. CONCLUSION/SIGNIFICANCE: This study constitutes a first attempt to investigate alteration of the host cellular proteome during early CHIKV infection. Our proteomics data showed that during early infection, CHIKV affected the expression of proteins that are involved in mRNA processing, host metabolic machinery, UPP, and cyclin-dependent kinase 1 (CDK1 regulation (in favour of virus survival, replication and transmission. While results from this study complement the proteomics results obtained from previous late host response studies, functional characterization of these proteins is warranted to reinforce our understanding of their roles during early CHIKV infection in humans.

  1. Quest for novel cardiovascular biomarkers by proteomic analysis.

    Science.gov (United States)

    Vivanco, Fernando; Martín-Ventura, Jose L; Duran, Mari Carmen; Barderas, Maria G; Blanco-Colio, Luis; Dardé, Verónica M; Mas, Sebastián; Meilhac, Olivier; Michel, Jean B; Tuñón, Jose; Egido, Jesús

    2005-01-01

    Atherosclerosis, and the resulting coronary heart disease and stroke, is the most common cause of death in developed countries. Atherosclerosis is an inflammatory process that results in the development of complex lesions or plaques that protrude into the arterial lumen. Plaque rupture and thrombosis result in the acute clinical complications of myocardial infarction (MI) and stroke. Although certain risk factors (dyslipidemias, diabetes, hypertension) and humoral markers of plaque vulnerability (C-reactive protein, interleukin-6, 10 and 18, CD40L) have been identified, a highly sensitive and specific biomarker or protein profile, which could provide information on the stability/vulnerability of atherosclerotic lesions, remains to be identified. In this review, we report several proteomic approaches which have been applied to circulating or resident cells, atherosclerotic plaques or plasma, in the search for new proteins that could be used as cardiovascular biomarkers. First, an example using a differential proteomic approach (2-DE and MS) comparing the secretome from control mammary arteries and atherosclerotic plaques is displayed. Among the different proteins identified, we showed that low levels of HSP-27 could be a potential marker of atherosclerosis. Second, we have revised several studies performed in cells involved in the pathogenesis of atherosclerosis (foam cells and smooth muscle cells). Another approach consists of performing proteomic analysis on circulating cells or plasma, which will provide a global view of the whole body response to atherosclerotic aggression. Circulating cells can bear information reflecting directly an inflammatory or pro-coagulant state related to the pathology. As an illustration, we report that circulating monocytes and plasma in patients with acute coronary syndromes has disclosed that mature Cathepsin D is increased both in the plasma and monocytes of these patients. Finally, the problems of applying proteomic approach

  2. Data from quantitative label free proteomics analysis of rat spleen

    Directory of Open Access Journals (Sweden)

    Khadar Dudekula

    2016-09-01

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

  3. Data from quantitative label free proteomics analysis of rat spleen.

    Science.gov (United States)

    Dudekula, Khadar; Le Bihan, Thierry

    2016-09-01

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

  4. Proteomic analysis of acetylation in thermophilic Geobacillus kaustophilus.

    Science.gov (United States)

    Lee, Dong-Woo; Kim, Dooil; Lee, Yong-Jik; Kim, Jung-Ae; Choi, Ji Young; Kang, Sunghyun; Pan, Jae-Gu

    2013-08-01

    Recent analysis of prokaryotic N(ε)-lysine-acetylated proteins highlights the posttranslational regulation of a broad spectrum of cellular proteins. However, the exact role of acetylation remains unclear due to a lack of acetylated proteome data in prokaryotes. Here, we present the N(ε)-lysine-acetylated proteome of gram-positive thermophilic Geobacillus kaustophilus. Affinity enrichment using acetyl-lysine-specific antibodies followed by LC-MS/MS analysis revealed 253 acetylated peptides representing 114 proteins. These acetylated proteins include not only common orthologs from mesophilic Bacillus counterparts, but also unique G. kaustophilus proteins, indicating that lysine acetylation is pronounced in thermophilic bacteria. These data complement current knowledge of the bacterial acetylproteome and provide an expanded platform for better understanding of the function of acetylation in cellular metabolism.

  5. Statistics in experimental design, preprocessing, and analysis of proteomics data.

    Science.gov (United States)

    Jung, Klaus

    2011-01-01

    High-throughput experiments in proteomics, such as 2-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS), yield usually high-dimensional data sets of expression values for hundreds or thousands of proteins which are, however, observed on only a relatively small number of biological samples. Statistical methods for the planning and analysis of experiments are important to avoid false conclusions and to receive tenable results. In this chapter, the most frequent experimental designs for proteomics experiments are illustrated. In particular, focus is put on studies for the detection of differentially regulated proteins. Furthermore, issues of sample size planning, statistical analysis of expression levels as well as methods for data preprocessing are covered.

  6. High pH reversed-phase chromatography with fraction concatenation as an alternative to strong-cation exchange chromatography for two-dimensional proteomic analysis

    OpenAIRE

    Yang, Feng; Shen, Yufeng; Camp, David G.; Smith, Richard D.

    2012-01-01

    Orthogonal high-resolution separations are critical for attaining improved analytical dynamic range and protein coverage in proteomic measurements. High pH reversed-phase liquid chromatography (RPLC) followed by fraction concatenation affords better peptide analysis than conventional strong-cation exchange (SCX) chromatography applied for the two-dimensional proteomic analysis. For example, concatenated high pH reversed-phase liquid chromatography increased identification for peptides (1.8-fo...

  7. Impact of replicate types on proteomic expression analysis.

    Science.gov (United States)

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

    2005-01-01

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

  8. Human myelin proteome and comparative analysis with mouse myelin

    OpenAIRE

    Ishii, Akihiro; Dutta, Ranjan; Wark, Greg M.; Hwang, Sun-Il; Han, David K.; Trapp, Bruce D.; Pfeiffer, Steven E.; Bansal, Rashmi

    2009-01-01

    Myelin, formed by oligodendrocytes (OLs) in the CNS, is critical for axonal functions, and its damage leads to debilitating neurological disorders such as multiple sclerosis. Understanding the molecular mechanisms of myelination and the pathogenesis of human myelin disease has been limited partly by the relative lack of identification and functional characterization of the repertoire of human myelin proteins. Here, we present a large-scale analysis of the myelin proteome, using the shotgun ap...

  9. SWATH-MS data of Drosophila melanogaster proteome dynamics during embryogenesis.

    Science.gov (United States)

    Fabre, Bertrand; Korona, Dagmara; Nightingale, Daniel J H; Russell, Steven; Lilley, Kathryn S

    2016-12-01

    Embryogenesis is one of the most important processes in the life of an animal. During this dynamic process, progressive cell division and cellular differentiation are accompanied by significant changes in protein expression at the level of the proteome. However, very few studies to date have described the dynamics of the proteome during the early development of an embryo in any organism. In this dataset, we monitor changes in protein expression across a timecourse of more than 20 h of Drosophila melanogaster embryonic development. Mass-spectrometry data were produced using a SWATH acquisition mode on a Sciex Triple-TOF 6600. A spectral library built in-house was used to analyse these data and more than 1950 proteins were quantified at each embryonic timepoint. The files presented here are a permanent digital map and can be reanalysed to test against new hypotheses. The data have been deposited with the ProteomeXchange Consortium with the dataset identifier PRIDE: PXD0031078.

  10. PARIS (Proteomic Analysis and Resources Indexation System) VERSION 2.0 User's Guide

    OpenAIRE

    Wang, Juhui

    2015-01-01

    International audience; PARIS (Proteomic Analysis and Resources Indexation System) is a suit of software to manage and analyse the data from 2D electrophoresis based proteomic analysis. It stores information about experiments and analysis procedure, allows the user to search and navigate in genomic and proteomic data, supports visual verification and validation of the analysis results, and provides tools for cross multi-experiment and multi-experimenter data validation and exploration.

  11. A Combined Metabolomic and Proteomic Analysis of Gestational Diabetes Mellitus.

    Science.gov (United States)

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

    2015-12-16

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

  12. A Combined Metabolomic and Proteomic Analysis of Gestational Diabetes Mellitus

    Directory of Open Access Journals (Sweden)

    Joanna Hajduk

    2015-12-01

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

  13. Plastid Proteomic Analysis in Tomato Fruit Development.

    Directory of Open Access Journals (Sweden)

    Miho Suzuki

    Full Text Available To better understand the mechanism of plastid differentiation from chloroplast to chromoplast, we examined proteome and plastid changes over four distinct developmental stages of 'Micro-Tom' fruit. Additionally, to discover more about the relationship between fruit color and plastid differentiation, we also analyzed and compared 'Micro-Tom' results with those from two other varieties, 'Black' and 'White Beauty'. We confirmed that proteins related to photosynthesis remain through the orange maturity stage of 'Micro-Tom', and also learned that thylakoids no longer exist at this stage. These results suggest that at a minimum there are changes in plastid morphology occurring before all related proteins change. We also compared 'Micro-Tom' fruits with 'Black' and 'White Beauty' using two-dimensional gel electrophoresis. We found a decrease of CHRC (plastid-lipid-associated protein and HrBP1 (harpin binding protein-1 in the 'Black' and 'White Beauty' varieties. CHRC is involved in carotenoid accumulation and stabilization. HrBP1 in Arabidopsis has a sequence similar to proteins in the PAP/fibrillin family. These proteins have characteristics and functions similar to lipocalin, an example of which is the transport of hydrophobic molecules. We detected spots of TIL (temperature-induced lipocalin in 2D-PAGE results, however the number of spots and their isoelectric points differed between 'Micro-Tom' and 'Black'/'White Beauty'. Lipocalin has various functions including those related to environmental stress response, apoptosis induction, membrane formation and fixation, regulation of immune response, cell growth, and metabolism adjustment. Lipocalin related proteins such as TIL and HrBP1 could be related to the accumulation of carotenoids, fruit color and the differentiation of chromoplast.

  14. Plastid Proteomic Analysis in Tomato Fruit Development.

    Science.gov (United States)

    Suzuki, Miho; Takahashi, Sachiko; Kondo, Takanori; Dohra, Hideo; Ito, Yumihiko; Kiriiwa, Yoshikazu; Hayashi, Marina; Kamiya, Shiori; Kato, Masaya; Fujiwara, Masayuki; Fukao, Yoichiro; Kobayashi, Megumi; Nagata, Noriko; Motohashi, Reiko

    2015-01-01

    To better understand the mechanism of plastid differentiation from chloroplast to chromoplast, we examined proteome and plastid changes over four distinct developmental stages of 'Micro-Tom' fruit. Additionally, to discover more about the relationship between fruit color and plastid differentiation, we also analyzed and compared 'Micro-Tom' results with those from two other varieties, 'Black' and 'White Beauty'. We confirmed that proteins related to photosynthesis remain through the orange maturity stage of 'Micro-Tom', and also learned that thylakoids no longer exist at this stage. These results suggest that at a minimum there are changes in plastid morphology occurring before all related proteins change. We also compared 'Micro-Tom' fruits with 'Black' and 'White Beauty' using two-dimensional gel electrophoresis. We found a decrease of CHRC (plastid-lipid-associated protein) and HrBP1 (harpin binding protein-1) in the 'Black' and 'White Beauty' varieties. CHRC is involved in carotenoid accumulation and stabilization. HrBP1 in Arabidopsis has a sequence similar to proteins in the PAP/fibrillin family. These proteins have characteristics and functions similar to lipocalin, an example of which is the transport of hydrophobic molecules. We detected spots of TIL (temperature-induced lipocalin) in 2D-PAGE results, however the number of spots and their isoelectric points differed between 'Micro-Tom' and 'Black'/'White Beauty'. Lipocalin has various functions including those related to environmental stress response, apoptosis induction, membrane formation and fixation, regulation of immune response, cell growth, and metabolism adjustment. Lipocalin related proteins such as TIL and HrBP1 could be related to the accumulation of carotenoids, fruit color and the differentiation of chromoplast.

  15. Dynamic changes of histone H3 marks during Caenorhabditis elegans lifecycle revealed by middle-down proteomics

    DEFF Research Database (Denmark)

    Sidoli, Simone; Vandamme, Julien; Elisabetta Salcini, Anna;

    2016-01-01

    We applied a middle-down proteomics strategy for large scale protein analysis during in vivo development of Caenorhabditis elegans. We characterized post-translational modifications (PTMs) on histone H3 N-terminal tails at eight time points during the C. elegans lifecycle, including embryo, larval......-occurring PTMs. We measured temporally distinct combinatorial PTM profiles during C. elegans development. We show that the doubly modified form H3K23me3K27me3, which is rare or non-existent in mammals, is the most abundant PTM in all stages of C. elegans lifecycle. The abundance of H3K23me3 increased during...... that is transmitted during dauer formation. Collectively, our data describe the dynamics of histone H3 combinatorial code during C. elegans lifecycle and demonstrate the feasibility of using middle-down proteomics to study in vivo development of multicellular organisms. This article is protected by copyright. All...

  16. Proteomics-driven analysis of ovine whey colostrum.

    Directory of Open Access Journals (Sweden)

    Domenica Scumaci

    Full Text Available The aim of this study was to shed light in to the complexity of the ovine colostrum proteome, with a specific focus on the low abundance proteins. The ovine colostrum is characterized by a few dominating proteins, as the immunoglobulins, but it also contains less represented protein species, equally important for the correct development of neonates. Ovine colostrum, collected immediately after lambing, was separated by 1D SDS-PAGE. Proteins bands were digested with trypsin and the resulting peptides were analyzed by LC-MS/MS. On the basis of the Swiss-Prot database, a total of 343 unique proteins were identified. To our knowledge, this study represents the most comprehensive analysis of ovine colostrum proteome.

  17. Proteomic analysis of HIV-T cell interaction: an update.

    Directory of Open Access Journals (Sweden)

    Dave eSpeijer

    2012-07-01

    Full Text Available This mini-review summarizes techniques applied in, and results obtained with, proteomic studies of HIV-1 T cell interaction. Our group previously reported on the use of two-dimensional differential gel electrophoresis (2D-DIGE coupled to MALDI-TOF peptide mass fingerprint analysis, to study T cell responses upon HIV-1 infection. Only one in three differentially expressed proteins could be identified using this experimental setup. Here we report on our latest efforts to test models generated by this data set and extend its analysis by using novel bioinformatic algorithms. The 2D-DIGE results are compared with other studies including a pilot study using one-dimensional peptide separation coupled to MSE, a novel mass spectrometric approach. It can be concluded that although the latter method detects fewer proteins, it is much faster and less labor intensive. Last but not least, recent developments and remaining challenges in the field of proteomic studies of HIV-1 infection and proteomics in general are discussed.

  18. Proteomic analysis of formalin-fixed prostate cancer tissue.

    Science.gov (United States)

    Hood, Brian L; Darfler, Marlene M; Guiel, Thomas G; Furusato, Bungo; Lucas, David A; Ringeisen, Bradley R; Sesterhenn, Isabell A; Conrads, Thomas P; Veenstra, Timothy D; Krizman, David B

    2005-11-01

    Proteomic analysis of formalin-fixed paraffin-embedded (FFPE) tissue would enable retrospective biomarker investigations of this vast archive of pathologically characterized clinical samples that exist worldwide. These FFPE tissues are, however, refractory to proteomic investigations utilizing many state of the art methodologies largely due to the high level of covalently cross-linked proteins arising from formalin fixation. A novel tissue microdissection technique has been developed and combined with a method to extract soluble peptides directly from FFPE tissue for mass spectral analysis of prostate cancer (PCa) and benign prostate hyperplasia (BPH). Hundreds of proteins from PCa and BPH tissue were identified, including several known PCa markers such as prostate-specific antigen, prostatic acid phosphatase, and macrophage inhibitory cytokine-1. Quantitative proteomic profiling utilizing stable isotope labeling confirmed similar expression levels of prostate-specific antigen and prostatic acid phosphatase in BPH and PCa cells, whereas the expression of macrophage inhibitory cytokine-1 was found to be greater in PCa as compared with BPH cells.

  19. Nascent chromatin capture proteomics determines chromatin dynamics during DNA replication and identifies unknown fork components

    DEFF Research Database (Denmark)

    Alabert, Constance; Bukowski-Wills, Jimi-Carlo; Lee, Sung-Po

    2014-01-01

    To maintain genome function and stability, DNA sequence and its organization into chromatin must be duplicated during cell division. Understanding how entire chromosomes are copied remains a major challenge. Here, we use nascent chromatin capture (NCC) to profile chromatin proteome dynamics durin...

  20. Transcriptome and Proteome Dynamics of the Cellular Response of Shewanella oneidensis to Chromium Stress

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, D.K.

    2005-04-18

    The overall goal of this DOE NABIR project is to characterize the molecular basis and regulation of hexavalent chromium [Cr(VI)] stress response and reduction by Shewanella oneidensis strain MR-1. Temporal genomic profiling and mass spectrometry-based proteomic analysis were employed to characterize the dynamic molecular response of S. oneidensis MR-1 to both acute and chronic Cr(VI) exposure. The acute stress response of aerobic, mid-exponential phase cells shocked to a final concentration of 1 mM potassium chromate (K2CrO4) was examined at post-exposure time intervals of 5, 30, 60, and 90 min relative to untreated cells. The transcriptome of mid-exponential cultures was also analyzed 30 min after shock doses of 0.3, 0.5, or 1 mM K{sub 2}CrO{sub 4}. The tonB1-exbB1-exbD1 genes comprising the TonB1 iron transport system were some of the most highly induced coding sequences (CDSs) after 90 min (up to {approx}240 fold), followed by other genes involved in heme transport, sulfate transport, and sulfur assimilation pathways. In addition, transcript levels for CDSs with annotated functions in DNA repair (dinP, recX, recA, recN) and detoxification processes (so3585, so3586) were substantially increased in Cr(VI)-exposed cells compared to untreated cells. By contrast, genes predicted to encode hydrogenases (HydA, HydB), oxidoreductases (SO0902-03-04, SO1911), iron-sulfur cluster binding proteins (SO4404), decaheme cytochrome c proteins (MtrA, OmcA, OmcB), and a number of LysR or TetR family transcriptional regulators were some of the most highly repressed CDSs following the 90-min shock period. Transcriptome profiles generated from MR-1 cells adapted to 0.3 mM Cr(VI) differed significantly from those characterizing cells exposed to acute Cr(VI) stress without adaptation. Parallel proteomic characterization of soluble protein and membrane protein fractions extracted from Cr(VI)-shocked and Cr(VI)-adapted MR-1 cells was performed using multidimensional HPLC-ESI-MS/MS (both

  1. Integrative analysis of the mitochondrial proteome in yeast.

    Directory of Open Access Journals (Sweden)

    Holger Prokisch

    2004-06-01

    Full Text Available In this study yeast mitochondria were used as a model system to apply, evaluate, and integrate different genomic approaches to define the proteins of an organelle. Liquid chromatography mass spectrometry applied to purified mitochondria identified 546 proteins. By expression analysis and comparison to other proteome studies, we demonstrate that the proteomic approach identifies primarily highly abundant proteins. By expanding our evaluation to other types of genomic approaches, including systematic deletion phenotype screening, expression profiling, subcellular localization studies, protein interaction analyses, and computational predictions, we show that an integration of approaches moves beyond the limitations of any single approach. We report the success of each approach by benchmarking it against a reference set of known mitochondrial proteins, and predict approximately 700 proteins associated with the mitochondrial organelle from the integration of 22 datasets. We show that a combination of complementary approaches like deletion phenotype screening and mass spectrometry can identify over 75% of the known mitochondrial proteome. These findings have implications for choosing optimal genome-wide approaches for the study of other cellular systems, including organelles and pathways in various species. Furthermore, our systematic identification of genes involved in mitochondrial function and biogenesis in yeast expands the candidate genes available for mapping Mendelian and complex mitochondrial disorders in humans.

  2. Proteomic analysis of purified Newcastle disease virus particles

    Directory of Open Access Journals (Sweden)

    Ren Xiangpeng

    2012-05-01

    Full Text Available Abstract Background Newcastle disease virus (NDV is an enveloped RNA virus, bearing severe economic losses to the poultry industry worldwide. Previous virion proteomic studies have shown that enveloped viruses carry multiple host cellular proteins both internally and externally during their life cycle. To address whether it also occurred during NDV infection, we performed a comprehensive proteomic analysis of highly purified NDV La Sota strain particles. Results In addition to five viral structural proteins, we detected thirty cellular proteins associated with purified NDV La Sota particles. The identified cellular proteins comprised several functional categories, including cytoskeleton proteins, annexins, molecular chaperones, chromatin modifying proteins, enzymes-binding proteins, calcium-binding proteins and signal transduction-associated proteins. Among these, three host proteins have not been previously reported in virions of other virus families, including two signal transduction-associated proteins (syntenin and Ras small GTPase and one tumor-associated protein (tumor protein D52. The presence of five selected cellular proteins (i.e., β-actin, tubulin, annexin A2, heat shock protein Hsp90 and ezrin associated with the purified NDV particles was validated by Western blot or immunogold labeling assays. Conclusions The current study presented the first standard proteomic profile of NDV. The results demonstrated the incorporation of cellular proteins in NDV particles, which provides valuable information for elucidating viral infection and pathogenesis.

  3. Proteomic analysis of purified coronavirus infectious bronchitis virus particles

    Directory of Open Access Journals (Sweden)

    Shu Dingming

    2010-06-01

    Full Text Available Abstract Background Infectious bronchitis virus (IBV is the coronavirus of domestic chickens causing major economic losses to the poultry industry. Because of the complexity of the IBV life cycle and the small number of viral structural proteins, important virus-host relationships likely remain to be discovered. Toward this goal, we performed two-dimensional gel electrophoresis fractionation coupled to mass spectrometry identification approaches to perform a comprehensive proteomic analysis of purified IBV particles. Results Apart from the virus-encoded structural proteins, we detected 60 host proteins in the purified virions which can be grouped into several functional categories including intracellular trafficking proteins (20%, molecular chaperone (18%, macromolcular biosynthesis proteins (17%, cytoskeletal proteins (15%, signal transport proteins (15%, protein degradation (8%, chromosome associated proteins (2%, ribosomal proteins (2%, and other function proteins (3%. Interestingly, 21 of the total host proteins have not been reported to be present in virions of other virus families, such as major vault protein, TENP protein, ovalbumin, and scavenger receptor protein. Following identification of the host proteins by proteomic methods, the presence of 4 proteins in the purified IBV preparation was verified by western blotting and immunogold labeling detection. Conclusions The results present the first standard proteomic profile of IBV and may facilitate the understanding of the pathogenic mechanisms.

  4. Organellar proteomics: analysis of pancreatic zymogen granule membranes.

    Science.gov (United States)

    Chen, Xuequn; Walker, Angela K; Strahler, John R; Simon, Eric S; Tomanicek-Volk, Sarah L; Nelson, Bradley B; Hurley, Mary C; Ernst, Stephen A; Williams, John A; Andrews, Philip C

    2006-02-01

    The zymogen granule (ZG) is the specialized organelle in pancreatic acinar cells for digestive enzyme storage and regulated secretion and has been a model for studying secretory granule functions. In an initial effort to comprehensively understand the functions of this organelle, we conducted a proteomic study to identify proteins from highly purified ZG membranes. By combining two-dimensional gel electrophoresis and two-dimensional LC with tandem mass spectrometry, 101 proteins were identified from purified ZG membranes including 28 known ZG proteins and 73 previously unknown proteins, including SNAP29, Rab27B, Rab11A, Rab6, Rap1, and myosin Vc. Moreover several hypothetical proteins were identified that represent potential novel proteins. The ZG localization of nine of these proteins was further confirmed by immunocytochemistry. To distinguish intrinsic membrane proteins from soluble and peripheral membrane proteins, a quantitative proteomic strategy was used to measure the enrichment of intrinsic membrane proteins through the purification process. The iTRAQ ratios correlated well with known or Transmembrane Hidden Markov Model-predicted soluble or membrane proteins. By combining subcellular fractionation with high resolution separation and comprehensive identification of proteins, we have begun to elucidate zymogen granule functions through proteomic and subsequent functional analysis of its membrane components.

  5. Proteomic analysis of post translational modifications in cyanobacteria.

    Science.gov (United States)

    Xiong, Qian; Chen, Zhuo; Ge, Feng

    2016-02-16

    Cyanobacteria are a diverse group of Gram-negative bacteria and the only prokaryotes capable of oxygenic photosynthesis. Recently, cyanobacteria have attracted great interest due to their crucial roles in global carbon and nitrogen cycles and their ability to produce clean and renewable biofuels. To survive in various environmental conditions, cyanobacteria have developed a complex signal transduction network to sense environmental signals and implement adaptive changes. The post-translational modifications (PTMs) systems play important regulatory roles in the signaling networks of cyanobacteria. The systematic investigation of PTMs could contribute to the comprehensive description of protein species and to elucidate potential biological roles of each protein species in cyanobacteria. Although the proteomic studies of PTMs carried out in cyanobacteria were limited, these data have provided clues to elucidate their sophisticated sensing mechanisms that contribute to their evolutionary and ecological success. This review aims to summarize the current status of PTM studies and recent publications regarding PTM proteomics in cyanobacteria, and discuss the novel developments and applications for the analysis of PTMs in cyanobacteria. Challenges, opportunities and future perspectives in the proteomics studies of PTMs in cyanobacteria are also discussed.

  6. A Systematic Analysis of a Deep Mouse Epididymal Sperm Proteome

    Energy Technology Data Exchange (ETDEWEB)

    Chauvin, Theodore; Xie, Fang; Liu, Tao; Nicora, Carrie D.; Yang, Feng; Camp, David G.; Smith, Richard D.; Roberts, Kenneth P.

    2012-12-21

    Spermatozoa are highly specialized cells that, when mature, are capable of navigating the female reproductive tract and fertilizing an oocyte. The sperm cell is thought to be largely quiescent in terms of transcriptional and translational activity. As a result, once it has left the male reproductive tract, the sperm cell is essentially operating with a static population of proteins. It is therefore theoretically possible to understand the protein networks contained in a sperm cell and to deduce its cellular function capabilities. To this end we have performed a proteomic analysis of mouse sperm isolated from the cauda epididymis and have confidently identified 2,850 proteins, which is the most comprehensive sperm proteome for any species reported to date. These proteins comprise many complete cellular pathways, including those for energy production via glycolysis, β-oxidation and oxidative phosphorylation, protein folding and transport, and cell signaling systems. This proteome should prove a useful tool for assembly and testing of protein networks important for sperm function.

  7. Comprehensive Proteomic Analysis of Human Milk-derived Extracellular Vesicles Unveils a Novel Functional Proteome Distinct from Other Milk Components.

    Science.gov (United States)

    van Herwijnen, Martijn J C; Zonneveld, Marijke I; Goerdayal, Soenita; Nolte-'t Hoen, Esther N M; Garssen, Johan; Stahl, Bernd; Maarten Altelaar, A F; Redegeld, Frank A; Wauben, Marca H M

    2016-11-01

    Breast milk contains several macromolecular components with distinctive functions, whereby milk fat globules and casein micelles mainly provide nutrition to the newborn, and whey contains molecules that can stimulate the newborn's developing immune system and gastrointestinal tract. Although extracellular vesicles (EV) have been identified in breast milk, their physiological function and composition has not been addressed in detail. EV are submicron sized vehicles released by cells for intercellular communication via selectively incorporated lipids, nucleic acids, and proteins. Because of the difficulty in separating EV from other milk components, an in-depth analysis of the proteome of human milk-derived EV is lacking. In this study, an extensive LC-MS/MS proteomic analysis was performed of EV that had been purified from breast milk of seven individual donors using a recently established, optimized density-gradient-based EV isolation protocol. A total of 1963 proteins were identified in milk-derived EV, including EV-associated proteins like CD9, Annexin A5, and Flotillin-1, with a remarkable overlap between the different donors. Interestingly, 198 of the identified proteins are not present in the human EV database Vesiclepedia, indicating that milk-derived EV harbor proteins not yet identified in EV of different origin. Similarly, the proteome of milk-derived EV was compared with that of other milk components. For this, data from 38 published milk proteomic studies were combined in order to construct the total milk proteome, which consists of 2698 unique proteins. Remarkably, 633 proteins identified in milk-derived EV have not yet been identified in human milk to date. Interestingly, these novel proteins include proteins involved in regulation of cell growth and controlling inflammatory signaling pathways, suggesting that milk-derived EVs could support the newborn's developing gastrointestinal tract and immune system. Overall, this study provides an expansion of

  8. Recent insights into plant-virus interactions through proteomic analysis.

    Science.gov (United States)

    Di Carli, Mariasole; Benvenuto, Eugenio; Donini, Marcello

    2012-10-05

    Plant viruses represent a major threat for a wide range of host species causing severe losses in agricultural practices. The full comprehension of mechanisms underlying events of virus-host plant interaction is crucial to devise novel plant resistance strategies. Until now, functional genomics studies in plant-virus interaction have been limited mainly on transcriptomic analysis. Only recently are proteomic approaches starting to provide important contributions to this area of research. Classical two-dimensional electrophoresis (2-DE) coupled to mass spectrometry (MS) is still the most widely used platform in plant proteome analysis, although in the last years the application of quantitative "second generation" proteomic techniques (such as differential in gel electrophoresis, DIGE, and gel-free protein separation methods) are emerging as more powerful analytical approaches. Apparently simple, plant-virus interactions reveal a really complex pathophysiological context, in which resistance, defense and susceptibility, and direct virus-induced reactions interplay to trigger expression responses of hundreds of genes. Given that, this review is specifically focused on comparative proteome-based studies on pathogenesis of several viral genera, including some of the most important and widespread plant viruses of the genus Tobamovirus, Sobemovirus, Cucumovirus and Potyvirus. In all, this overview reveals a widespread repression of proteins associated with the photosynthetic apparatus, while energy metabolism/protein synthesis and turnover are typically up-regulated, indicating a major redirection of cell metabolism. Other common features include the modulation of metabolisms concerning sugars, cell wall, and reactive oxigen species as well as pathogenesis-related (PR) proteins. The fine-tuning between plant development and antiviral defense mechanisms determines new patterns of regulation of common metabolic pathways. By offering a 360-degree view of protein modulation

  9. Temporal proteomic analysis reveals continuous impairment of intestinal development in neonatal piglets with intrauterine growth restriction.

    Science.gov (United States)

    Wang, Xiaoqiu; Wu, Weizong; Lin, Gang; Li, Defa; Wu, Guoyao; Wang, Junjun

    2010-02-01

    Efficiency of nutrient utilization is reduced in neonates with intrauterine growth restriction (IUGR) compared with those with a normal birth weight (NBW). However, the underlying mechanisms are largely unknown. In this study, we applied temporal proteomic approach, coupled with histological and biochemical analyses, to study dynamic changes of the proteome in the small intestinal mucosa of IUGR piglets during the nursing period (Days 1, 7 and 21). We identified 56 differentially expressed protein spots between IUGR and NBW piglets. These proteins participate in key biological processes, including (1) absorption, digestion and transport of nutrients; (2) cell structure and motility; (3) glucose and energy metabolism; (4) lipid metabolism; (5) amino acid metabolism; (6) mineral and vitamin metabolism; (7) cellular redox homeostasis; (8) stress response; and (9) apoptosis. The results of our temporal proteomics analysis reveal continuous impairment of intestinal development in neonatal piglets with IUGR. The findings have important implications for understanding metabolic defects in the small intestine of IUGR neonates and are expected to provide new strategies to improve their survival and growth.

  10. Proteomic analysis of GPI-anchored membrane proteins

    DEFF Research Database (Denmark)

    Jung, Hye Ryung; Jensen, Ole Nørregaard

    2006-01-01

    Glycosyl-phosphatidyl-inositol-anchored proteins (GPI-APs) represent a subset of post-translationally modified proteins that are tethered to the outer leaflet of the plasma membrane via a C-terminal GPI anchor. GPI-APs are found in a variety of eukaryote species, from pathogenic microorganisms...... to humans. GPI-APs confer important cellular functions as receptors, enzymes and scaffolding molecules. Specific enzymes and detergent extraction methods combined with separation technologies and mass spectrometry permit proteomic analysis of GPI-APs from plasma membrane preparations to reveal cell...

  11. Proteomic analysis of GPI-anchored membrane proteins

    DEFF Research Database (Denmark)

    Jung, Hye Ryung; Jensen, Ole Nørregaard

    2006-01-01

    to humans. GPI-APs confer important cellular functions as receptors, enzymes and scaffolding molecules. Specific enzymes and detergent extraction methods combined with separation technologies and mass spectrometry permit proteomic analysis of GPI-APs from plasma membrane preparations to reveal cell......Glycosyl-phosphatidyl-inositol-anchored proteins (GPI-APs) represent a subset of post-translationally modified proteins that are tethered to the outer leaflet of the plasma membrane via a C-terminal GPI anchor. GPI-APs are found in a variety of eukaryote species, from pathogenic microorganisms...

  12. Statistics for proteomics: experimental design and 2-DE differential analysis.

    Science.gov (United States)

    Chich, Jean-François; David, Olivier; Villers, Fanny; Schaeffer, Brigitte; Lutomski, Didier; Huet, Sylvie

    2007-04-15

    Proteomics relies on the separation of complex protein mixtures using bidimensional electrophoresis. This approach is largely used to detect the expression variations of proteins prepared from two or more samples. Recently, attention was drawn on the reliability of the results published in literature. Among the critical points identified were experimental design, differential analysis and the problem of missing data, all problems where statistics can be of help. Using examples and terms understandable by biologists, we describe how a collaboration between biologists and statisticians can improve reliability of results and confidence in conclusions.

  13. Proteomic Analysis Identifies Outcome-Predictive Clusters in Patients with Peripheral T-Cell Lymphoma, Not otherwise specified

    DEFF Research Database (Denmark)

    Ludvigsen, Maja; Pedersen, Martin Bjerregård; Poulsen, T.S.

    2014-01-01

    Proteomic Analysis Identifies Outcome-Predictive Clusters in Patients with Peripheral T-Cell Lymphoma, Not otherwise specified......Proteomic Analysis Identifies Outcome-Predictive Clusters in Patients with Peripheral T-Cell Lymphoma, Not otherwise specified...

  14. Transcriptome and proteome analysis of Eucalyptus infected with Calonectria pseudoreteaudii.

    Science.gov (United States)

    Chen, Quanzhu; Guo, Wenshuo; Feng, Lizhen; Ye, Xiaozhen; Xie, Wanfeng; Huang, Xiuping; Liu, Jinyan

    2015-02-06

    Cylindrocladium leaf blight is one of the most severe diseases in Eucalyptus plantations and nurseries. There are Eucalyptus cultivars with resistance to the disease. However, little is known about the defense mechanism of resistant cultivars. Here, we investigated the transcriptome and proteome of Eucalyptus leaves (E. urophylla×E. tereticornis M1), infected or not with Calonectria pseudoreteaudii. A total of 8585 differentially expressed genes (|log2 ratio| ≥1, FDR ≤0.001) at 12 and 24hours post-inoculation were detected using RNA-seq. Transcriptional changes for five genes were further confirmed by qRT-PCR. A total of 3680 proteins at the two time points were identified using iTRAQ technique.The combined transcriptome and proteome analysis revealed that the shikimate/phenylpropanoid pathway, terpenoid biosynthesis, signalling pathway (jasmonic acid and sugar) were activated. The data also showed that some proteins (WRKY33 and PR proteins) which have been reported to involve in plant defense response were up-regulated. However, photosynthesis, nucleic acid metabolism and protein metabolism were impaired by the infection of C. pseudoreteaudii. This work will facilitate the identification of defense related genes and provide insights into Eucalyptus defense responses to Cylindrocladium leaf blight. In this study, a total of 130 proteins and genes involved in the shikimate/phenylpropanoid pathway, terpenoid biosynthesis, signalling pathway, cell transport, carbohydrate and energy metabolism, nucleic acid metabolism and protein metabolism in Eucalyptus leaves after infected with C. pseudoreteaudii were identified. This is the first report of a comprehensive transcriptomic and proteomic analysis of Eucalyptus in response to Calonectria sp. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Extending the boundaries of MS-based proteomics: towards comprehensive analysis of the proteome and the HLA ligandome

    NARCIS (Netherlands)

    Marino, F.

    2016-01-01

    The work, described in this thesis, has been aimed at the improvement of proteomic workflows; from the sample preparation, to the optimization of state-of-art chromatographical separations and alternative MS fragmentation techniques. An important focus of this work is represented by the analysis of

  16. Identification and proteomic analysis of osteoblast-derived exosomes.

    Science.gov (United States)

    Ge, Min; Ke, Ronghu; Cai, Tianyi; Yang, Junyi; Mu, Xiongzheng

    2015-11-01

    Exosomes are nanometer-sized vesicles with the function of intercellular communication, and they are released by various cell types. To reveal the knowledge about the exosomes from osteoblast, and explore the potential functions of osteogenesis, we isolated microvesicles from supernatants of mouse Mc3t3 by ultracentrifugation, characterized exosomes by electron microscopy and immunoblotting and presented the protein profile by proteomic analysis. The result demonstrated that microvesicles were between 30 and 100 nm in diameter, round shape with cup-like concavity and expressed exosomal marker tumor susceptibility gene (TSG) 101 and flotillin (Flot) 1. We identified a total number of 1069 proteins among which 786 proteins overlap with ExoCarta database. Gene Oncology analysis indicated that exosomes mostly derived from plasma membrane and mainly involved in protein localization and intracellular signaling. The Ingenuity Pathway Analysis showed pathways are mostly involved in exosome biogenesis, formation, uptake and osteogenesis. Among the pathways, eukaryotic initiation factor 2 pathways played an important role in osteogenesis. Our study identified osteoblast-derived exosomes, unveiled the content of them, presented potential osteogenesis-related proteins and pathways and provided a rich proteomics data resource that will be valuable for further studies of the functions of individual proteins in bone diseases.

  17. Comparative Proteomic Analysis of Arabidopsis Mature Pollen and Germinated Pollen

    Institute of Scientific and Technical Information of China (English)

    Junjie Zou; Lianfen Song; Wenzheng Zhang; Yi Wang; Songlin Ruan; Wei-Hua Wu

    2009-01-01

    Proteomic analysis was applied to generating the map of Arabidopsis mature pollen proteins and analyzing the differentially expressed proteins that are potentially involved in the regulation of Arabidopsis pollen germination. By applying 2-D electrophoresis and silver staining, we resolved 499 and 494 protein spots from protein samples extracted from pollen grains and pollen tubes, respectively. Using the matrix-assisted laser desorption ionization time-of-flight mass spectrometry method, we identified 189 distinct proteins from 213 protein spots expressed in mature pollen or pollen tubes, and 75 new identified proteins that had not been reported before in research into the Arabidopsis pollen proteome. Comparative analysis revealed that 40 protein spots exhibit reproducible significant changes between mature pollen and pollen tubes. And 21 proteins from 17 downregulated and six upregulated protein spots were identified. Functional category analysis indicated that these differentially expressed proteins mainly involved in signaling, cellular structure, transport, defense/stress responses, transcription, metabolism, and energy production. The patterns of changes at protein level suggested the important roles for energy metabolism-related proteins in pollen tube growth, accompanied by the activation of the stress response pathway and modifications to the cell wall.

  18. A data processing pipeline for mammalian proteome dynamics studies using stable isotope metabolic labeling.

    Science.gov (United States)

    Guan, Shenheng; Price, John C; Prusiner, Stanley B; Ghaemmaghami, Sina; Burlingame, Alma L

    2011-12-01

    In a recent study, in vivo metabolic labeling using (15)N traced the rate of label incorporation among more than 1700 proteins simultaneously and enabled the determination of individual protein turnover rate constants over a dynamic range of three orders of magnitude (Price, J. C., Guan, S., Burlingame, A., Prusiner, S. B., and Ghaemmaghami, S. (2010) Analysis of proteome dynamics in the mouse brain. Proc. Natl. Acad. Sci. U. S. A. 107, 14508-14513). These studies of protein dynamics provide a deeper understanding of healthy development and well-being of complex organisms, as well as the possible causes and progression of disease. In addition to a fully labeled food source and appropriate mass spectrometry platform, an essential and enabling component of such large scale investigations is a robust data processing and analysis pipeline, which is capable of the reduction of large sets of liquid chromatography tandem MS raw data files into the desired protein turnover rate constants. The data processing pipeline described in this contribution is comprised of a suite of software modules required for the workflow that fulfills such requirements. This software platform includes established software tools such as a mass spectrometry database search engine together with several additional, novel data processing modules specifically developed for (15)N metabolic labeling. These fulfill the following functions: (1) cross-extraction of (15)N-containing ion intensities from raw data files at varying biosynthetic incorporation times, (2) computation of peptide (15)N isotopic incorporation distributions, and (3) aggregation of relative isotope abundance curves for multiple peptides into single protein curves. In addition, processing parameter optimization and noise reduction procedures were found to be necessary in the processing modules in order to reduce propagation of errors in the long chain of the processing steps of the entire workflow.

  19. A comparative proteome analysis of hippocampal tissue from schizophrenic and Alzheimer's disease individuals.

    Science.gov (United States)

    Edgar, P F; Schonberger, S J; Dean, B; Faull, R L; Kydd, R; Cooper, G J

    1999-03-01

    The proteins expressed by a genome have been termed the proteome. Comparative proteome analysis of brain tissue offers a novel means to identify biologically significant gene products that underlie psychopathology. In this study we collected post mortem hippocampal tissue from the brains of seven schizophrenic, seven Alzheimer's disease (AD) and seven control individuals. Hippocampal proteomes were visualised by two-dimensional gel electrophoresis of homogenised tissue. A mean of 549 (s.d. 35) proteins were successfully matched between each disease group and the control group. In comparison with the control hippocampal proteome, eight proteins in the schizophrenic hippocampal proteome were found to be decreased and eight increased in concentration, whereas, in the AD hippocampal proteome, 35 proteins were decreased and 73 were increased in concentration (Pdiazepam binding inhibitor (DBI) by N-terminal sequence analysis. DBI can regulate the action of the GABA(A) receptor. Protein changes involved 6% of the assessed AD hippocampal proteome, whereas, in schizophrenia protein changes involved less than 1% of the assessed hippocampal proteome. We conclude that schizophrenia has a subtle neuropathological presentation and comparative proteome analysis is a viable means by which to investigate diseases of the brain at the molecular level.

  20. Comprehensive proteomic analysis of the human spliceosome

    Science.gov (United States)

    Zhou, Zhaolan; Licklider, Lawrence J.; Gygi, Steven P.; Reed, Robin

    2002-09-01

    The precise excision of introns from pre-messenger RNA is performed by the spliceosome, a macromolecular machine containing five small nuclear RNAs and numerous proteins. Much has been learned about the protein components of the spliceosome from analysis of individual purified small nuclear ribonucleoproteins and salt-stable spliceosome `core' particles. However, the complete set of proteins that constitutes intact functional spliceosomes has yet to be identified. Here we use maltose-binding protein affinity chromatography to isolate spliceosomes in highly purified and functional form. Using nanoscale microcapillary liquid chromatography tandem mass spectrometry, we identify ~145 distinct spliceosomal proteins, making the spliceosome the most complex cellular machine so far characterized. Our spliceosomes comprise all previously known splicing factors and 58 newly identified components. The spliceosome contains at least 30 proteins with known or putative roles in gene expression steps other than splicing. This complexity may be required not only for splicing multi-intronic metazoan pre-messenger RNAs, but also for mediating the extensive coupling between splicing and other steps in gene expression.

  1. Proteome analysis of muscadine grape leaves

    Directory of Open Access Journals (Sweden)

    Sheikh M Basha

    2009-04-01

    Full Text Available Sheikh M Basha1, Ramesh Katam1, Hemanth Vasanthaiah1, Frank Matta21Center for Viticulture and Small Fruit Research, Florida A and M University, Tallahassee, FL, USA; 2Plant and Soil Science Department, Mississippi State University, Mississippi State, MS, USAAbstract: Muscadine grapes are native to the southeastern United States and are used for making wine and consumed as fresh fruit. Grape berries, as ‘sink organs,’ rely on the use of available carbohydrate resources produced by photosynthesis to support their development and composition. A high throughput two-dimensional gel electrophoresis (2-DE was conducted on muscadine (Vitis rotundifolia grape leaf proteins to document complexity in their composition and to determine protein identity and function for enhancing photosynthetic efficiency of muscadine grape. 2-DE resolved muscadine leaf proteins into >258 polypeptides with pIs between 3.5 and 8.0 and molecular weight between 12,000 to 15,0000 Daltons. The consistently expressed proteins were excised and subjected to sequencing. Homology search of protein sequences showed 84% identity with Viridi plantae database. Identity of some of these proteins included RuBisCO, glutamine synthetase, pathogenesis-related protein, glyoxisomal malate dehydrogenase, ribonucleoprotein, chloroplast precursor, oxygen evolving enhancer protein. Comparative analysis of 10 muscadine cultivars showed quantitative differences in expression of 39 polypeptides among these genotypes. The results suggested that the polypeptide composition of muscadine grape leaf is complex, and polypeptide number and amount vary widely among muscadine genotypes, and these variations may be responsible for differences in their physiology, berry and stress tolerance characteristics.Keywords: grapevine, leaves, muscadine, proteins, sequencing, 2-DE

  2. Comparative proteome analysis of saccular intracranial aneurysms with iTRAQ quantitative proteomics.

    Science.gov (United States)

    Wang, Jia; Yu, Lanbing; Huang, Xiahe; Wang, Yingchun; Zhao, Jizong

    2016-01-01

    To screen differentially expressed proteins of saccular intracranial aneurysms and superficial temporal artery by the proteomics analysis using isobaric tags for relative and absolute quantification (iTRAQ) combined with reverse phase high-performance liquid chromatography. Collecting 17 samples from intracranial aneurysm patients undergoing aneurysmectomy as experiment group and 17 matched STA as control group. After quantification and enzymolysis of the protein, the iTRAQ were used to label the peptides of the 2 groups respectively. Then, the mixture of the peptides was fractioned by RP-HPLC and analyzed by LC-MS/MS to identify the differential expression proteins. A total of 1699 proteins were identified from the ProteinPilot 4.5 software (AB SCIEX) using the Paragon database search algorithm. Comparing with STA, 54 proteins were significantly up-regulated (115:1142.0-fold). Furthermore, Integrin β3, Secreted frizzled-related protein 2 were significantly up-regulated (2.3 fold and 2.1 fold, respectively), whereas MyosinIIb, Alpha-actinin-1, Laminin β2, and Carboxypeptidase A3 were down-regulated (3.01 fold, 2.1 fold, 2.07 fold, and 2.01 fold, respectively) in sIAs. GO Ontology analysis showed that most differential proteins expressed in cytoskeletal; up-regulated proteins in sIAs play an important role in inflammatory reaction, enzymatic hydrolysis, cell adhesion and invasion, and cellular immune reaction; down-regulated proteins in sIAs involved in cytoskeletal protein, enzyme, and structural protein. ITGB3, ACTN1 and MYL2 play a role in aneurysm formation via focal adhesion pathway. The results of Western-blot assay were consistent with the proteomic changes of those 6 proteins. The differentially expressed proteins in sIAs that showed aneurysm formation are related to cytoskeleton abnormal and extracellular matrix changes. The iTRAQ technology provides scientific foundation for the further study to explore the pathogenic mechanism of sIAs. Copyright © 2015

  3. Prediction of acute coronary syndromes by urinary proteome analysis

    Science.gov (United States)

    Htun, Nay M.; Magliano, Dianna J.; Zhang, Zhen-Yu; Lyons, Jasmine; Petit, Thibault; Nkuipou-Kenfack, Esther; Ramirez-Torres, Adela; von zur Muhlen, Constantin; Maahs, David; Schanstra, Joost P.; Pontillo, Claudia; Pejchinovski, Martin; Snell-Bergeon, Janet K.; Delles, Christian; Mischak, Harald; Staessen, Jan A.; Shaw, Jonathan E.

    2017-01-01

    Identification of individuals who are at risk of suffering from acute coronary syndromes (ACS) may allow to introduce preventative measures. We aimed to identify ACS-related urinary peptides, that combined as a pattern can be used as prognostic biomarker. Proteomic data of 252 individuals enrolled in four prospective studies from Australia, Europe and North America were analyzed. 126 of these had suffered from ACS within a period of up to 5 years post urine sampling (cases). Proteomic analysis of 84 cases and 84 matched controls resulted in the discovery of 75 ACS-related urinary peptides. Combining these to a peptide pattern, we established a prognostic biomarker named Acute Coronary Syndrome Predictor 75 (ACSP75). ACSP75 demonstrated reasonable prognostic discrimination (c-statistic = 0.664), which was similar to Framingham risk scoring (c-statistics = 0.644) in a validation cohort of 42 cases and 42 controls. However, generating by a composite algorithm named Acute Coronary Syndrome Composite Predictor (ACSCP), combining the biomarker pattern ACSP75 with the previously established urinary proteomic biomarker CAD238 characterizing coronary artery disease as the underlying aetiology, and age as a risk factor, further improved discrimination (c-statistic = 0.751) resulting in an added prognostic value over Framingham risk scoring expressed by an integrated discrimination improvement of 0.273 ± 0.048 (P < 0.0001) and net reclassification improvement of 0.405 ± 0.113 (P = 0.0007). In conclusion, we demonstrate that urinary peptide biomarkers have the potential to predict future ACS events in asymptomatic patients. Further large scale studies are warranted to determine the role of urinary biomarkers in clinical practice. PMID:28273075

  4. Proteomic Analysis of Vitreous Humor in Retinal Vein Occlusion

    Science.gov (United States)

    Reich, Michael; Dacheva, Ivanka; Nobl, Matthias; Siwy, Justyna; Schanstra, Joost P.; Mullen, William; Koch, Frank H. J.; Kopitz, Jürgen; Kretz, Florian T. A.; Auffarth, Gerd U.; Koss, Michael J.

    2016-01-01

    Purpose To analyze the protein profile of human vitreous of untreated patients with retinal vein occlusion (RVO). Methods Sixty-eight vitreous humor (VH) samples (44 from patients with treatment naïve RVO, 24 controls with idiopathic floaters) were analyzed in this clinical-experimental study using capillary electrophoresis coupled to mass spectrometer and tandem mass spectrometry. To define potential candidate protein markers of RVO, proteomic analysis was performed on RVO patients (n = 30) and compared with controls (n = 16). To determine validity of potential biomarker candidates in RVO, receiver operating characteristic (ROC) was performed by using proteome data of independent RVO (n = 14) and control samples (n = 8). Results Ninety-four different proteins (736 tryptic peptides) could be identified. Sixteen proteins were found to be significant when comparing RVO and control samples (P = 1.43E-05 to 4.48E-02). Five proteins (Clusterin, Complement C3, Ig lambda-like polypeptide 5 (IGLL5), Opticin and Vitronectin), remained significant after using correction for multiple testing. These five proteins were also detected significant when comparing subgroups of RVO (central RVO, hemi-central RVO, branch RVO) to controls. Using independent samples ROC-Area under the curve was determined proving the validity of the results: Clusterin 0.884, Complement C3 0.955, IGLL5 1.000, Opticin 0.741, Vitronectin 0.786. In addition, validation through ELISA measurements was performed. Conclusion The results of the study reveal that the proteomic composition of VH differed significantly between the patients with RVO and the controls. The proteins identified may serve as potential biomarkers for pathogenesis induced by RVO. PMID:27362861

  5. Proteomic Analysis of Vitreous Humor in Retinal Vein Occlusion.

    Directory of Open Access Journals (Sweden)

    Michael Reich

    Full Text Available To analyze the protein profile of human vitreous of untreated patients with retinal vein occlusion (RVO.Sixty-eight vitreous humor (VH samples (44 from patients with treatment naïve RVO, 24 controls with idiopathic floaters were analyzed in this clinical-experimental study using capillary electrophoresis coupled to mass spectrometer and tandem mass spectrometry. To define potential candidate protein markers of RVO, proteomic analysis was performed on RVO patients (n = 30 and compared with controls (n = 16. To determine validity of potential biomarker candidates in RVO, receiver operating characteristic (ROC was performed by using proteome data of independent RVO (n = 14 and control samples (n = 8.Ninety-four different proteins (736 tryptic peptides could be identified. Sixteen proteins were found to be significant when comparing RVO and control samples (P = 1.43E-05 to 4.48E-02. Five proteins (Clusterin, Complement C3, Ig lambda-like polypeptide 5 (IGLL5, Opticin and Vitronectin, remained significant after using correction for multiple testing. These five proteins were also detected significant when comparing subgroups of RVO (central RVO, hemi-central RVO, branch RVO to controls. Using independent samples ROC-Area under the curve was determined proving the validity of the results: Clusterin 0.884, Complement C3 0.955, IGLL5 1.000, Opticin 0.741, Vitronectin 0.786. In addition, validation through ELISA measurements was performed.The results of the study reveal that the proteomic composition of VH differed significantly between the patients with RVO and the controls. The proteins identified may serve as potential biomarkers for pathogenesis induced by RVO.

  6. Shotgun proteomic analysis of porcine colostrum and mature milk.

    Science.gov (United States)

    Ogawa, Shohei; Tsukahara, Takamitsu; Nishibayashi, Ryoichiro; Nakatani, Masako; Okutani, Mie; Nakanishi, Nobuo; Ushida, Kazunari; Inoue, Ryo

    2014-04-01

    The epitheliochorial nature of the porcine placenta prevents the transfer of maternal immunity. Therefore, ingestion of the colostrum immediately after birth is crucial for neonatal piglets to acquire passive immunity from the sow. We performed a shotgun proteomic analysis of porcine milk to reveal in detail the protein composition of porcine milk. On the basis of the Swiss-Prot database, 113 and 118 proteins were identified in the porcine colostrum and mature milk, respectively, and 50 of these proteins were common to both samples. Some immune-related proteins, including interleukin-18 (IL-18), were unique to the colostrum. The IL-18 concentration in the colostrum and mature milk of four sows was measured to validate the proteomic analysis, and IL-18 was only detected in the colostrum (191.0 ± 53.9 pg/mL) and not in mature milk. In addition, some proteins involved in primary defense, such as azurocidin, which has never been detected in any other mammal's milk, were also identified in the colostrum.

  7. Layer-by-Layer Proteomic Analysis of Mytilus galloprovincialis Shell.

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    Peng Gao

    Full Text Available Bivalve shell is a biomineralized tissue with various layers/microstructures and excellent mechanical properties. Shell matrix proteins (SMPs pervade and envelop the mineral crystals and play essential roles in biomineralization. Despite that Mytilus is an economically important bivalve, only few proteomic studies have been performed for the shell, and current knowledge of the SMP set responsible for different shell layers of Mytilus remains largely patchy. In this study, we observed that Mytilus galloprovincialis shell contained three layers, including nacre, fibrous prism, and myostracum that is involved in shell-muscle attachment. A parallel proteomic analysis was performed for these three layers. By combining LC-MS/MS analysis with Mytilus EST database interrogations, a whole set of 113 proteins was identified, and the distribution of these proteins in different shell layers followed a mosaic pattern. For each layer, about a half of identified proteins are unique and the others are shared by two or all of three layers. This is the first description of the protein set exclusive to nacre, myostracum, and fibrous prism in Mytilus shell. Moreover, most of identified proteins in the present study are novel SMPs, which greatly extended biomineralization-related protein data of Mytilus. These results are useful, on one hand, for understanding the roles of SMPs in the deposition of different shell layers. On the other hand, the identified protein set of myostracum provides candidates for further exploring the mechanism of adductor muscle-shell attachment.

  8. Layer-by-Layer Proteomic Analysis of Mytilus galloprovincialis Shell

    Science.gov (United States)

    Wang, Xin-xing; Bao, Lin-fei; Fan, Mei-hua; Li, Xiao-min; Wu, Chang-wen; Xia, Shu-wei

    2015-01-01

    Bivalve shell is a biomineralized tissue with various layers/microstructures and excellent mechanical properties. Shell matrix proteins (SMPs) pervade and envelop the mineral crystals and play essential roles in biomineralization. Despite that Mytilus is an economically important bivalve, only few proteomic studies have been performed for the shell, and current knowledge of the SMP set responsible for different shell layers of Mytilus remains largely patchy. In this study, we observed that Mytilus galloprovincialis shell contained three layers, including nacre, fibrous prism, and myostracum that is involved in shell-muscle attachment. A parallel proteomic analysis was performed for these three layers. By combining LC-MS/MS analysis with Mytilus EST database interrogations, a whole set of 113 proteins was identified, and the distribution of these proteins in different shell layers followed a mosaic pattern. For each layer, about a half of identified proteins are unique and the others are shared by two or all of three layers. This is the first description of the protein set exclusive to nacre, myostracum, and fibrous prism in Mytilus shell. Moreover, most of identified proteins in the present study are novel SMPs, which greatly extended biomineralization-related protein data of Mytilus. These results are useful, on one hand, for understanding the roles of SMPs in the deposition of different shell layers. On the other hand, the identified protein set of myostracum provides candidates for further exploring the mechanism of adductor muscle-shell attachment. PMID:26218932

  9. Layer-by-Layer Proteomic Analysis of Mytilus galloprovincialis Shell.

    Science.gov (United States)

    Gao, Peng; Liao, Zhi; Wang, Xin-Xing; Bao, Lin-Fei; Fan, Mei-Hua; Li, Xiao-Min; Wu, Chang-Wen; Xia, Shu-Wei

    2015-01-01

    Bivalve shell is a biomineralized tissue with various layers/microstructures and excellent mechanical properties. Shell matrix proteins (SMPs) pervade and envelop the mineral crystals and play essential roles in biomineralization. Despite that Mytilus is an economically important bivalve, only few proteomic studies have been performed for the shell, and current knowledge of the SMP set responsible for different shell layers of Mytilus remains largely patchy. In this study, we observed that Mytilus galloprovincialis shell contained three layers, including nacre, fibrous prism, and myostracum that is involved in shell-muscle attachment. A parallel proteomic analysis was performed for these three layers. By combining LC-MS/MS analysis with Mytilus EST database interrogations, a whole set of 113 proteins was identified, and the distribution of these proteins in different shell layers followed a mosaic pattern. For each layer, about a half of identified proteins are unique and the others are shared by two or all of three layers. This is the first description of the protein set exclusive to nacre, myostracum, and fibrous prism in Mytilus shell. Moreover, most of identified proteins in the present study are novel SMPs, which greatly extended biomineralization-related protein data of Mytilus. These results are useful, on one hand, for understanding the roles of SMPs in the deposition of different shell layers. On the other hand, the identified protein set of myostracum provides candidates for further exploring the mechanism of adductor muscle-shell attachment.

  10. Proteomic analysis of fetal programming-related obesity markers.

    Science.gov (United States)

    Lee, Ji Hye; Yoo, Jae Young; You, Young-Ah; Kwon, Woo-Sung; Lee, Sang Mi; Pang, Myung-Geol; Kim, Young Ju

    2015-08-01

    The objectives of this study were to analyze fetal programming in rat brain using proteomic analysis and to identify fetal programming-related obesity markers. Sprague-Dawley rats were divided into four feeding groups: (i) the Ad Libitum (AdLib)/AdLib group was given a normal diet during pregnancy and the lactation period; (ii) the AdLib/maternal food restriction group (FR) was subjected to 50% FR during the lactation period; (iii) the FR/AdLib group was subjected to 50% FR during pregnancy; and (iv) the FR/FR group was subjected to 50% FR during pregnancy and the lactation period. Offspring from each group were sacrificed at 3 weeks of age and whole brains were dissected. To obtain a maximum number of protein markers related to obesity, 2DE and Pathway Studio bioinformatics analysis were performed. The identities of the markers among the selected and candidate proteins were confirmed by Western blotting and immunohistochemistry. Proteomic and bioinformatics analyses revealed that expression of ubiquitin carboxy-terminal hydrolase L1 (UCHL1) and Secernin 1 (SCRN1) were significantly different in the FR/AdLib group compared with the AdLib/AdLib group for both male and female offspring. These findings suggest that UCHL1 and SCRN1 may be used as fetal programming-related obesity markers.

  11. Differential Proteomic Analysis of Carbon Ion Radiation in Sheep Sperm

    Institute of Scientific and Technical Information of China (English)

    HE Yu-xuan; LI Hong-yan; ZHANG Yong; HE Jian-hua; ZHANG Hong; ZHAO Xing-xu

    2013-01-01

    This study is first to investigate proteomic changes in sheep sperm induced by carbon ion radiation using two-dimensional electrophoresis (2-DE) analysis in the project of breeding a new variety of sheep. Differential expression proteins were detected using the PDQuest 8.0 software after staining with Coomassie blue. Valid spots were then analyzed through liquid chromatography tandem mass spectrometry (LC-MS/MS). Among the 480 total protein spots displayed in 2-D gels, 6 specific protein spots were observed in sperm gels. A search against protein sequences in the National Center for Biotechnology Information databases (NCBI) indicated that differentially expressed proteins correspond to two proteins, identified to be enolase and transcription factor AP-2-alpha (TFAP-2α). The two proteins were up-regulated in the irradiated sperm. To the best of our knowledge, this study is the first to identify proteomic changes induced by carbon ion radiation in sheep sperm. The analysis of differential expression protein may be useful in identifying new breeding markers in sheep reproduction and in clarifying the mechanisms involved in irradiation or space breeding.

  12. Toward improving the proteomic analysis of formalin-fixed, paraffin-embedded tissue.

    Science.gov (United States)

    Fowler, Carol B; O'Leary, Timothy J; Mason, Jeffrey T

    2013-08-01

    Archival formalin-fixed, paraffin-embedded (FFPE) tissue and their associated diagnostic records represent an invaluable source of retrospective proteomic information on diseases for which the clinical outcome and response to treatment are known. However, analysis of archival FFPE tissues by high-throughput proteomic methods has been hindered by the adverse effects of formaldehyde fixation and subsequent tissue histology. This review examines recent methodological advances for extracting proteins from FFPE tissue suitable for proteomic analysis. These methods, based largely upon heat-induced antigen retrieval techniques borrowed from immunohistochemistry, allow at least a qualitative analysis of the proteome of FFPE archival tissues. The authors also discuss recent advances in the proteomic analysis of FFPE tissue; including liquid-chromatography tandem mass spectrometry, reverse phase protein microarrays and imaging mass spectrometry.

  13. Maximum entropy reconstructions of dynamic signaling networks from quantitative proteomics data.

    Science.gov (United States)

    Locasale, Jason W; Wolf-Yadlin, Alejandro

    2009-08-26

    Advances in mass spectrometry among other technologies have allowed for quantitative, reproducible, proteome-wide measurements of levels of phosphorylation as signals propagate through complex networks in response to external stimuli under different conditions. However, computational approaches to infer elements of the signaling network strictly from the quantitative aspects of proteomics data are not well established. We considered a method using the principle of maximum entropy to infer a network of interacting phosphotyrosine sites from pairwise correlations in a mass spectrometry data set and derive a phosphorylation-dependent interaction network solely from quantitative proteomics data. We first investigated the applicability of this approach by using a simulation of a model biochemical signaling network whose dynamics are governed by a large set of coupled differential equations. We found that in a simulated signaling system, the method detects interactions with significant accuracy. We then analyzed a growth factor mediated signaling network in a human mammary epithelial cell line that we inferred from mass spectrometry data and observe a biologically interpretable, small-world structure of signaling nodes, as well as a catalog of predictions regarding the interactions among previously uncharacterized phosphotyrosine sites. For example, the calculation places a recently identified tumor suppressor pathway through ARHGEF7 and Scribble, in the context of growth factor signaling. Our findings suggest that maximum entropy derived network models are an important tool for interpreting quantitative proteomics data.

  14. Characterisation of the circulating acellular proteome of healthy sheep using LC-MS/MS-based proteomics analysis of serum.

    Science.gov (United States)

    Chemonges, Saul; Gupta, Rajesh; Mills, Paul C; Kopp, Steven R; Sadowski, Pawel

    2016-01-01

    Unlike humans, there is currently no publicly available reference mass spectrometry-based circulating acellular proteome data for sheep, limiting the analysis and interpretation of a range of physiological changes and disease states. The objective of this study was to develop a robust and comprehensive method to characterise the circulating acellular proteome in ovine serum. Serum samples from healthy sheep were subjected to shotgun proteomic analysis using nano liquid chromatography nano electrospray ionisation tandem mass spectrometry (nanoLC-nanoESI-MS/MS) on a quadrupole time-of-flight instrument (TripleTOF® 5600+, SCIEX). Proteins were identified using ProteinPilot™ (SCIEX) and Mascot (Matrix Science) software based on a minimum of two unmodified highly scoring unique peptides per protein at a false discovery rate (FDR) of 1% software by searching a subset of the Universal Protein Resource Knowledgebase (UniProtKB) database (http://www.uniprot.org). PeptideShaker (CompOmics, VIB-UGent) searches were used to validate protein identifications from ProteinPilot™ and Mascot. ProteinPilot™ and Mascot identified 245 and 379 protein groups (IDs), respectively, and PeptideShaker validated 133 protein IDs from the entire dataset. Since Mascot software is considered the industry standard and identified the most proteins, these were analysed using the Protein ANalysis THrough Evolutionary Relationships (PANTHER) classification tool revealing the association of 349 genes with 127 protein pathway hits. These data are available via ProteomeXchange with identifier PXD004989. These results demonstrated for the first time the feasibility of characterising the ovine circulating acellular proteome using nanoLC-nanoESI-MS/MS. This peptide spectral data contributes to a protein library that can be used to identify a wide range of proteins in ovine serum.

  15. Proteomic analysis of the shistosome tegument and its surface membranes

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    Simon Braschi

    2006-10-01

    Full Text Available The tegument surface of the adult schistosome, bounded by a normal plasma membrane overlain by a secreted membranocalyx, holds the key to understanding how schistosomes evade host immune responses. Recent advances in mass spectrometry (MS, and the sequencing of the Schistosoma mansoni transcriptome/genome, have facilitated schistosome proteomics. We detached the tegument from the worm body and enriched its surface membranes by differential extraction, before subjecting the preparation to liquid chromatography-based proteomics to identify its constituents. The most exposed proteins on live worms were labelled with impearmeant biotinylation reagents, and we also developed methods to isolate the membranocalyx for analysis. We identified transporters for sugars, amino acids, inorganic ions and water, which confirm the importance of the tegument plasma membrane in nutrient acquisition and solute balance. Enzymes, including phosphohydrolases, esterases and carbonic anhydrase were located with their catalytic domains external to the plasma membrane, while five tetraspanins, annexin and dysferlin were implicated in membrane architecture. In contrast, few parasite proteins could be assigned to the membranocalyx but mouse immune response proteins, including three immunoglobulins and two complement factors, were detected, plus host membrane proteins such as CD44, integrin and a complement regulatory protein, testifying to the acquisitive properties of the secreted bilayer.

  16. Comparative proteomic analysis of indica and japonica rice varieties

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    Yanhua Yang

    2014-12-01

    Full Text Available Indica and japonica are two main subspecies of Asian cultivated rice (Oryza sativa L. that differ clearly in morphological and agronomic traits, in physiological and biochemical characteristics and in their genomic structure. However, the proteins and genes responsible for these differences remain poorly characterized. In this study, proteomic tools, including two-dimensional electrophoresis and mass spectrometry, were used to globally identify proteins that differed between two sequenced rice varieties (93-11 and Nipponbare. In all, 47 proteins that differed significantly between 93-11 and Nipponbare were identified using mass spectrometry and database searches. Interestingly, seven proteins were expressed only in Nipponbare and one protein was expressed specifically in 93-11; these differences were confirmed by quantitative real-time PCR and proteomic analysis of other indica and japonica rice varieties. This is the first report to successfully demonstrate differences in the protein composition of indica and japonica rice varieties and to identify candidate proteins and genes for future investigation of their roles in the differentiation of indica and japonica rice.

  17. Comparative proteome analysis of human epithelial ovarian cancer

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    Gagné Jean-Philippe

    2007-09-01

    Full Text Available Abstract Background Epithelial ovarian cancer is a devastating disease associated with low survival prognosis mainly because of the lack of early detection markers and the asymptomatic nature of the cancer until late stage. Using two complementary proteomics approaches, a differential protein expression profile was carried out between low and highly transformed epithelial ovarian cancer cell lines which realistically mimic the phenotypic changes observed during evolution of a tumour metastasis. This investigation was aimed at a better understanding of the molecular mechanisms underlying differentiation, proliferation and neoplastic progression of ovarian cancer. Results The quantitative profiling of epithelial ovarian cancer model cell lines TOV-81D and TOV-112D generated using iTRAQ analysis and two-dimensional electrophoresis coupled to liquid chromatography tandem mass spectrometry revealed some proteins with altered expression levels. Several of these proteins have been the object of interest in cancer research but others were unrecognized as differentially expressed in a context of ovarian cancer. Among these, series of proteins involved in transcriptional activity, cellular metabolism, cell adhesion or motility and cytoskeleton organization were identified, suggesting their possible role in the emergence of oncogenic pathways leading to aggressive cellular behavior. Conclusion The differential protein expression profile generated by the two proteomics approaches combined to complementary characterizations studies will open the way to more exhaustive and systematic representation of the disease and will provide valuable information that may be helpful to uncover the molecular mechanisms related to epithelial ovarian cancer.

  18. Predictive sequence analysis of the Candidatus Liberibacter asiaticus proteome.

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    Qian Cong

    Full Text Available Candidatus Liberibacter asiaticus (Ca. L. asiaticus is a parasitic gram-negative bacterium that is closely associated with Huanglongbing (HLB, a worldwide citrus disease. Given the difficulty in culturing the bacterium and thus in its experimental characterization, computational analyses of the whole Ca. L. asiaticus proteome can provide much needed insights into the mechanisms of the disease and guide the development of treatment strategies. In this study, we applied state-of-the-art sequence analysis tools to every Ca. L. asiaticus protein. Our results are available as a public website at http://prodata.swmed.edu/liberibacter_asiaticus/. In particular, we manually curated the results to predict the subcellular localization, spatial structure and function of all Ca. L. asiaticus proteins (http://prodata.swmed.edu/liberibacter_asiaticus/curated/. This extensive information should facilitate the study of Ca. L. asiaticus proteome function and its relationship to disease. Pilot studies based on the information from our website have revealed several potential virulence factors, discussed herein.

  19. Proteomic analysis of proton beam irradiated human melanoma cells.

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    Sylwia Kedracka-Krok

    Full Text Available Proton beam irradiation is a form of advanced radiotherapy providing superior distributions of a low LET radiation dose relative to that of photon therapy for the treatment of cancer. Even though this clinical treatment has been developing for several decades, the proton radiobiology critical to the optimization of proton radiotherapy is far from being understood. Proteomic changes were analyzed in human melanoma cells treated with a sublethal dose (3 Gy of proton beam irradiation. The results were compared with untreated cells. Two-dimensional electrophoresis was performed with mass spectrometry to identify the proteins. At the dose of 3 Gy a minimal slowdown in proliferation rate was seen, as well as some DNA damage. After allowing time for damage repair, the proteomic analysis was performed. In total 17 protein levels were found to significantly (more than 1.5 times change: 4 downregulated and 13 upregulated. Functionally, they represent four categories: (i DNA repair and RNA regulation (VCP, MVP, STRAP, FAB-2, Lamine A/C, GAPDH, (ii cell survival and stress response (STRAP, MCM7, Annexin 7, MVP, Caprin-1, PDCD6, VCP, HSP70, (iii cell metabolism (TIM, GAPDH, VCP, and (iv cytoskeleton and motility (Moesin, Actinin 4, FAB-2, Vimentin, Annexin 7, Lamine A/C, Lamine B. A substantial decrease (2.3 x was seen in the level of vimentin, a marker of epithelial to mesenchymal transition and the metastatic properties of melanoma.

  20. Proteomic Analysis of Pachytene Spermatocytes of Sterile Hybrid Male Mice.

    Science.gov (United States)

    Wang, Lu; Guo, Yueshuai; Liu, Wenjing; Zhao, Weidong; Song, Gendi; Zhou, Tao; Huang, Hefeng; Guo, Xuejiang; Sun, Fei

    2016-09-01

    Incompatibilities in interspecific hybrids, such as reduced hybrid fertility and lethality, are common features resulting from reproductive isolation that lead to speciation. Subspecies crosses of house mice produce offspring in which one sex is infertile or absent, yet the molecular mechanisms of hybrid sterility are poorly understood. In this study, we observed extensive asynapsis of chromosomes and disturbance of the sex body in pachytene spermatocytes of sterile F1 males (PWK/Ph female × C57BL/6J male). We report the high-confidence identification of 4005 proteins in the pachytene spermatocytes of fertile F1 males (PWK/Ph male × C57BL/6J female) and sterile F1 males (PWK/Ph female × C57BL/6J male), of which 215 were upregulated and 381 were downregulated. Bioinformatics analysis of the proteome led to the identification of 43 and 59 proteins known to be essential for male meiosis and spermatogenesis in mice, respectively. Characterization of the proteome of pachytene spermatocytes associated with hybrid male sterility provides an inventory of proteins that is useful for understanding meiosis and the mechanisms of hybrid male infertility.

  1. Proteomic analysis of the carotenogenic yeast Xanthophyllomyces dendrorhous

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    Baeza Marcelo

    2011-06-01

    Full Text Available Abstract Background The yeast Xanthophyllomyces dendrorhous is used for the microbiological production of the antioxidant carotenoid astaxanthin. In this study, we established an optimal protocol for protein extraction and performed the first proteomic analysis of the strain ATCC 24230. Protein profiles before and during the induction of carotenogenesis were determined by two-dimensional polyacrylamide gel electrophoresis and proteins were identified by mass spectrometry. Results Among the approximately 600 observed protein spots, 131 non-redundant proteins were identified. Proteomic analyses allowed us to identify 50 differentially expressed proteins that fall into several classes with distinct expression patterns. These analyses demonstrated that enzymes related to acetyl-CoA synthesis were more abundant prior to carotenogenesis. Later, redox- and stress-related proteins were up-regulated during the induction of carotenogenesis. For the carotenoid biosynthetic enzymes mevalonate kinase and phytoene/squalene synthase, we observed higher abundance during induction and/or accumulation of carotenoids. In addition, classical antioxidant enzymes, such as catalase, glutathione peroxidase and the cytosolic superoxide dismutases, were not identified. Conclusions Our results provide an overview of potentially important carotenogenesis-related proteins, among which are proteins involved in carbohydrate and lipid biosynthetic pathways as well as several redox- and stress-related proteins. In addition, these results might indicate that X. dendrorhous accumulates astaxanthin under aerobic conditions to scavenge the reactive oxygen species (ROS generated during metabolism.

  2. In-depth analysis of the adipocyte proteome by mass spectrometry and bioinformatics

    DEFF Research Database (Denmark)

    Adachi, Jun; Kumar, Chanchal; Zhang, Yanling;

    2007-01-01

    , mitochondria, membrane, and cytosol of 3T3-L1 adipocytes. We identified 3,287 proteins while essentially eliminating false positives, making this one of the largest high confidence proteomes reported to date. Comprehensive bioinformatics analysis revealed that the adipocyte proteome, despite its specialized...

  3. A two-dimensional protein map of Pleurotus ostreatus microsomes-proteome dynamics.

    Science.gov (United States)

    Petráčková, Denisa; Halada, Petr; Bezoušková, Silvia; Křesinová, Zdena; Svobodová, Kateřina

    2016-01-01

    Recent studies documented that several processes in filamentous fungi are connected with microsomal enzyme activities. In this work, microsomal subproteomes of Pleurotus ostreatus were analyzed by two-dimensional (2-D) polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry analysis. To assess proteome dynamics, microsomal proteins were isolated from fungal cultures after 7 and 12 days of cultivation. Additionally, 10 mg/L of 17α-ethinylestradiol (EE2) was treated with the cultures during 2 days. Despite the EE2 degradation by the fungus reached 97 and 76.3 % in 7- and 12-day-old cultures, respectively, only a minor effect on the composition of microsomal proteins was observed. The changes in protein maps related to ageing prevailed over those induced by EE2. Epoxide hydrolase, known to metabolize EE2, was detected in 12-day-old cultures only which suggests differences in EE2 degradation pathways utilized by fungal cultures of different age. The majority (32 %) of identified microsomal proteins were parts of mitochondrial energy metabolism.

  4. Pathway analysis of kidney cancer using proteomics and metabolic profiling

    Directory of Open Access Journals (Sweden)

    Fiehn Oliver

    2006-11-01

    Full Text Available Abstract Background Renal cell carcinoma (RCC is the sixth leading cause of cancer death and is responsible for 11,000 deaths per year in the US. Approximately one-third of patients present with disease which is already metastatic and for which there is currently no adequate treatment, and no biofluid screening tests exist for RCC. In this study, we have undertaken a comprehensive proteomic analysis and subsequently a pathway and network approach to identify biological processes involved in clear cell RCC (ccRCC. We have used these data to investigate urinary markers of RCC which could be applied to high-risk patients, or to those being followed for recurrence, for early diagnosis and treatment, thereby substantially reducing mortality of this disease. Results Using 2-dimensional electrophoresis and mass spectrometric analysis, we identified 31 proteins which were differentially expressed with a high degree of significance in ccRCC as compared to adjacent non-malignant tissue, and we confirmed some of these by immunoblotting, immunohistochemistry, and comparison to published transcriptomic data. When evaluated by several pathway and biological process analysis programs, these proteins are demonstrated to be involved with a high degree of confidence (p values Conclusion Extensive pathway and network analysis allowed for the discovery of highly significant pathways from a set of clear cell RCC samples. Knowledge of activation of these processes will lead to novel assays identifying their proteomic and/or metabolomic signatures in biofluids of patient at high risk for this disease; we provide pilot data for such a urinary bioassay. Furthermore, we demonstrate how the knowledge of networks, processes, and pathways altered in kidney cancer may be used to influence the choice of optimal therapy.

  5. Quantitative proteome and transcriptome analysis of the archaeon Thermoplasma acidophilum cultured under aerobic and anaerobic conditions.

    Science.gov (United States)

    Sun, Na; Pan, Cuiping; Nickell, Stephan; Mann, Matthias; Baumeister, Wolfgang; Nagy, István

    2010-09-03

    A comparative proteome and transcriptome analysis of Thermoplasma acidophilum cultured under aerobic and anaerobic conditions has been performed. One-thousand twenty-five proteins were identified covering 88% of the cytosolic proteome. Using a label-free quantitation method, we found that approximately one-quarter of the identified proteome (263 proteins) were significantly induced (>2 fold) under anaerobic conditions. Thirty-nine macromolecular complexes were identified, of which 28 were quantified and 15 were regulated under anaerobiosis. In parallel, a whole genome cDNA microarray analysis was performed showing that the expression levels of 445 genes were influenced by the absence of oxygen. Interestingly, more than 40% of the membrane protein-encoding genes (145 out of 335 ORFs) were up- or down-regulated at the mRNA level. Many of these proteins are functionally associated with extracellular protein or peptide degradation or ion and amino acid transport. Comparison of the transcriptome and proteome showed only a weak positive correlation between mRNA and protein expression changes, which is indicative of extensive post-transcriptional regulatory mechanisms in T. acidophilum. Integration of transcriptomics and proteomics data generated hypotheses for physiological adaptations of the cells to anaerobiosis, and the quantitative proteomics data together with quantitative analysis of protein complexes provide a platform for correlation of MS-based proteomics studies with cryo-electron tomography-based visual proteomics approaches.

  6. Proteomic analysis reveals key proteins and phosphoproteins upon seed germination of wheat (Triticum aestivum L.

    Directory of Open Access Journals (Sweden)

    Kun eDong

    2015-11-01

    Full Text Available Wheat (Triticum aestivum L. is one of the oldest cultivated crops and the second most important food crop in the world. Seed germination is the key developmental process in plant growth and development, and poor germination directly affects plant growth and subsequent grain yield. In this study, we performed the first dynamic proteome analysis of wheat seed germination using a two-dimensional differential gel electrophoresis (2D-DIGE-based proteomic approach. A total of 166 differentially expressed protein (DEP spots representing 73 unique proteins were identified, which are mainly involved in storage, stress/defense/detoxification, carbohydrate metabolism, photosynthesis, cell metabolism, and transcription/translation/ transposition. The identified DEPs and their dynamic expression profiles generally correspond to three distinct seed germination phases after imbibition: storage degradation, physiological processes/morphogenesis, and photosynthesis. Some key DEPs involved in storage substance degradation and plant defense mechanisms, such as globulin 3, sucrose synthase type I, serpin, beta-amylase, and plastid ADP-glucose pyrophosphorylase (AGPase small subunit, were found to be phosphorylated during seed germination. Particularly, the phosphorylation site Ser355 was found to be located in the enzyme active region of beta-amylase, which promotes substrate binding. Phosphorylated modification of several proteins could promote storage substance degradation and environmental stress defense during seed germination. The central metabolic pathways involved in wheat seed germination are proposed herein, providing new insights into the molecular mechanisms of cereal seed germination.

  7. Identification and proteomic analysis of osteoblast-derived exosomes

    Energy Technology Data Exchange (ETDEWEB)

    Ge, Min; Ke, Ronghu; Cai, Tianyi; Yang, Junyi; Mu, Xiongzheng, E-mail: cranio@vip.163.com

    2015-11-06

    Exosomes are nanometer-sized vesicles with the function of intercellular communication, and they are released by various cell types. To reveal the knowledge about the exosomes from osteoblast, and explore the potential functions of osteogenesis, we isolated microvesicles from supernatants of mouse Mc3t3 by ultracentrifugation, characterized exosomes by electron microscopy and immunoblotting and presented the protein profile by proteomic analysis. The result demonstrated that microvesicles were between 30 and 100 nm in diameter, round shape with cup-like concavity and expressed exosomal marker tumor susceptibility gene (TSG) 101 and flotillin (Flot) 1. We identified a total number of 1069 proteins among which 786 proteins overlap with ExoCarta database. Gene Oncology analysis indicated that exosomes mostly derived from plasma membrane and mainly involved in protein localization and intracellular signaling. The Ingenuity Pathway Analysis showed pathways are mostly involved in exosome biogenesis, formation, uptake and osteogenesis. Among the pathways, eukaryotic initiation factor 2 pathways played an important role in osteogenesis. Our study identified osteoblast-derived exosomes, unveiled the content of them, presented potential osteogenesis-related proteins and pathways and provided a rich proteomics data resource that will be valuable for further studies of the functions of individual proteins in bone diseases. - Highlights: • We for the first time identified exosomes from mouse osteoblast. • Osteoblasts-derived exosomes contain osteoblast peculiar proteins. • Proteins from osteoblasts-derived exosomes are intently involved in EIF2 pathway. • EIF2α from the EIF2 pathway plays an important role in osteogenesis.

  8. Proteome analysis of early lineage specification in bovine embryos.

    Science.gov (United States)

    Demant, Myriam; Deutsch, Daniela R; Fröhlich, Thomas; Wolf, Eckhard; Arnold, Georg J

    2015-02-01

    During mammalian embryo development, the zygote undergoes embryonic cleavage in the oviduct and reaches the uterus at the morula stage, when compaction and early lineage specification take place. To increase knowledge about the associated changes of the embryonic protein repertoire, we performed a comprehensive proteomic analysis of in vitro produced bovine morulae and blastocysts (six biological replicates), using an iTRAQ-based approach. A total of 560 proteins were identified of which 502 were quantified. The abundance of 140 proteins was significantly different between morulae and blastocysts, among them nucleophosmin (NPM1), eukaryotic translation initiation factor 5A-1 (EIF5A), receptor of activated protein kinase C 1 (GNB2L1/RACK1), and annexin A6 (ANXA6) with increased, and glutathione S-transferase mu 3 (GSTM3), peroxiredoxin 2 (PRDX2), and aldo-keto reductase family 1 member B1 (AKR1B1) with decreased abundance in blastocysts. Seventy-three percent of abundance altered proteins increased, reflecting an increase of translation activity in this period. This is further supported by an increase in the abundance of proteins involved in the translation machinery and the synthesis of ATP. Additionally, a complementary 2D saturation DIGE analysis led to the detection of protein isoforms, e.g. of GSTM3 and PRDX2, relevant for this period of mammalian development, and exemplarily verified the results of the iTRAQ approach. In summary, our systematic differential proteome analysis of bovine morulae and blastocysts revealed new molecular correlates of early lineage specification and differentiation events during bovine embryogenesis.

  9. SWATH-MS data of Drosophila melanogaster proteome dynamics during embryogenesis

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    Bertrand Fabre

    2016-12-01

    Full Text Available Embryogenesis is one of the most important processes in the life of an animal. During this dynamic process, progressive cell division and cellular differentiation are accompanied by significant changes in protein expression at the level of the proteome. However, very few studies to date have described the dynamics of the proteome during the early development of an embryo in any organism. In this dataset, we monitor changes in protein expression across a timecourse of more than 20 h of Drosophila melanogaster embryonic development. Mass-spectrometry data were produced using a SWATH acquisition mode on a Sciex Triple-TOF 6600. A spectral library built in-house was used to analyse these data and more than 1950 proteins were quantified at each embryonic timepoint. The files presented here are a permanent digital map and can be reanalysed to test against new hypotheses. The data have been deposited with the ProteomeXchange Consortium with the dataset identifier PRIDE: PXD0031078.

  10. Integration of Proteomics and Transcriptomics Data Sets for the Analysis of a Lymphoma B-Cell Line in the Context of the Chromosome-Centric Human Proteome Project.

    Science.gov (United States)

    Díez, Paula; Droste, Conrad; Dégano, Rosa M; González-Muñoz, María; Ibarrola, Nieves; Pérez-Andrés, Martín; Garin-Muga, Alba; Segura, Víctor; Marko-Varga, Gyorgy; LaBaer, Joshua; Orfao, Alberto; Corrales, Fernando J; De Las Rivas, Javier; Fuentes, Manuel

    2015-09-04

    A comprehensive study of the molecular active landscape of human cells can be undertaken to integrate two different but complementary perspectives: transcriptomics, and proteomics. After the genome era, proteomics has emerged as a powerful tool to simultaneously identify and characterize the compendium of thousands of different proteins active in a cell. Thus, the Chromosome-centric Human Proteome Project (C-HPP) is promoting a full characterization of the human proteome combining high-throughput proteomics with the data derived from genome-wide expression profiling of protein-coding genes. Here we present a full proteomic profiling of a human lymphoma B-cell line (Ramos) performed using a nanoUPLC-LTQ-Orbitrap Velos proteomic platform, combined to an in-depth transcriptomic profiling of the same cell type. Data are available via ProteomeXchange with identifier PXD001933. Integration of the proteomic and transcriptomic data sets revealed a 94% overlap in the proteins identified by both -omics approaches. Moreover, functional enrichment analysis of the proteomic profiles showed an enrichment of several functions directly related to the biological and morphological characteristics of B-cells. In turn, about 30% of all protein-coding genes present in the whole human genome were identified as being expressed by the Ramos cells (stable average of 30% genes along all the chromosomes), revealing the size of the protein expression-set present in one specific human cell type. Additionally, the identification of missing proteins in our data sets has been reported, highlighting the power of the approach. Also, a comparison between neXtProt and UniProt database searches has been performed. In summary, our transcriptomic and proteomic experimental profiling provided a high coverage report of the expressed proteome from a human lymphoma B-cell type with a clear insight into the biological processes that characterized these cells. In this way, we demonstrated the usefulness of

  11. Proteomic analysis of cellular response induced by boron neutron capture reaction in human squamous cell carcinoma SAS cells.

    Science.gov (United States)

    Sato, Akira; Itoh, Tasuku; Imamichi, Shoji; Kikuhara, Sota; Fujimori, Hiroaki; Hirai, Takahisa; Saito, Soichiro; Sakurai, Yoshinori; Tanaka, Hiroki; Nakamura, Hiroyuki; Suzuki, Minoru; Murakami, Yasufumi; Baiseitov, Diaz; Berikkhanova, Kulzhan; Zhumadilov, Zhaxybay; Imahori, Yoshio; Itami, Jun; Ono, Koji; Masunaga, Shinichiro; Masutani, Mitsuko

    2015-12-01

    To understand the mechanism of cell death induced by boron neutron capture reaction (BNCR), we performed proteome analyses of human squamous tumor SAS cells after BNCR. Cells were irradiated with thermal neutron beam at KUR after incubation under boronophenylalanine (BPA)(+) and BPA(-) conditions. BNCR mainly induced typical apoptosis in SAS cells 24h post-irradiation. Proteomic analysis in SAS cells suggested that proteins functioning in endoplasmic reticulum, DNA repair, and RNA processing showed dynamic changes at early phase after BNCR and could be involved in the regulation of cellular response to BNCR. We found that the BNCR induces fragments of endoplasmic reticulum-localized lymphoid-restricted protein (LRMP). The fragmentation of LRMP was also observed in the rat tumor graft model 20 hours after BNCT treatment carried out at the National Nuclear Center of the Republic of Kazakhstan. These data suggest that dynamic changes of LRMP could be involved during cellular response to BNCR.

  12. Functional proteomic analysis of Ankaferd® Blood Stopper

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    Duygu Özel Demiralp

    2010-06-01

    Full Text Available Objective: Ankaferd® Blood Stopper (ABS comprises a standardized mixture of the plants Thymus vulgaris, Glycyrrhiza glabra, Vitis vinifera, Alpinia officinarum, and Urtica dioica. The basic mechanism of action for ABS is the formation of an encapsulated protein network that provides focal points for vital erythrocyte aggregation. ABS–induced protein network formation with blood cells, particularly erythrocytes, covers the primary and secondary hemostatic system without disturbing individual coagulation factors. Materials and Methods: To understand the effect mechanisms of ABS on hemostasis, a proteomic analysis using 2D gel electrophoresis and mass spectrometer was performed. Results: Proteins of plant origin in Ankaferd® were NADP-dependent-malic enzyme, ribulose bisphosphate-carboxylase-large chain, maturase K, ATP synthase subunit-beta, ATP synthase subunit-alpha, chalcone-flavanone isomerase-1, chalcone-flavanone isomerase-2, and actin-depolymerizing factor. Furthermore, functional proteomic studies revealed that proteins resembling human peptides have been detected within Ankaferd®, including ATP synthase, mucin-16 (CD164 sialomucin-like 2 protein, coiled-coil domain containing 141 hypothetical protein LOC283638 isoform 1, hypothetical protein LOC283638 isoform 2, dynactin 5, complex I intermediate-associated protein 30, mitochondrial, NADH dehydrogenase (ubiquinone 1 alpha subcomplex, TP synthase, H+ transporting, mitochondrial actin binding 1 isoform, LIM domain and actin binding 1 isoform a, LIM domain and actin binding 1 isoform b, spectrin alpha non erythrocytic 1, prolactin releasing hormone receptor, utrophin, tet oncogene family member 2 isoform b, protein phosphatase 1 regulatory subunit 12A, NIMA (never in mitosis gene a-related kinase, ATP-binding cassette protein C12, Homo sapiens malic enzyme 1, mitochondrial NADP(+-dependent malic enzyme 3, ME2 protein, nuclear factor 1 B-type, abhydrolase domain-containing protein 12B, E

  13. Cellular Proteome Dynamics during Differentiation of Human Primary Myoblasts

    DEFF Research Database (Denmark)

    Le Bihan, Marie-Catherine; Barrio, Inigo; Mortensen, Tenna Pavia;

    2015-01-01

    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...... 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...

  14. An LC-IMS-MS Platform Providing Increased Dynamic Range for High-Throughput Proteomic Studies

    Energy Technology Data Exchange (ETDEWEB)

    Baker, Erin Shammel; Livesay, Eric A.; Orton, Daniel J.; Moore, Ronald J.; Danielson, William F.; Prior, David C.; Ibrahim, Yehia M.; Lamarche, Brian L.; Mayampurath, Anoop M.; Schepmoes, Athena A.; Hopkins, Derek F.; Tang, Keqi; Smith, Richard D.; Belov, Mikhail E.

    2010-02-05

    A high-throughput approach and platform using 15 minute reversed-phase capillary liquid chromatography (RPLC) separations in conjunction with ion mobility spectrometry-mass spectrometry (IMS-MS) measurements was evaluated for the rapid analysis of complex proteomics samples. To test the separation quality of the short LC gradient, a sample was prepared by spiking twenty reference peptides at varying concentrations from 1 ng/mL to 10 µg/mL into a tryptic digest of mouse blood plasma and analyzed with both a LC-Linear Ion Trap Fourier Transform (FT) MS and LC-IMS-TOF MS. The LC-FT MS detected thirteen out of the twenty spiked peptides that had concentrations ≥100 ng/mL. In contrast, the drift time selected mass spectra from the LC-IMS-TOF MS analyses yielded identifications for nineteen of the twenty peptides with all spiking level present. The greater dynamic range of the LC-IMS-TOF MS system could be attributed to two factors. First, the LC-IMS-TOF MS system enabled drift time separation of the low concentration spiked peptides from the high concentration mouse peptide matrix components, reducing signal interference and background, and allowing species to be resolved that would otherwise be obscured by other components. Second, the automatic gain control (AGC) in the linear ion trap of the hybrid FT MS instrument limits the number of ions that are accumulated to reduce space charge effects, but in turn limits the achievable dynamic range compared to the TOF detector.

  15. Recovery of Chinese hamster ovary host cell proteins for proteomic analysis.

    Science.gov (United States)

    Valente, Kristin N; Schaefer, Amy K; Kempton, Hannah R; Lenhoff, Abraham M; Lee, Kelvin H

    2014-01-01

    Identification and characterization of Chinese hamster ovary (CHO) host cell protein (HCP) impurities by proteomic techniques can aid bioprocess design and lead to more efficient development and improved biopharmaceutical manufacturing operations. Recovery of extracellular CHO HCP for proteomic analysis is particularly challenging due to the relatively low protein concentration and complex composition of media. In this article, we report the development of optimized protocols that improve proteome capture for CHO HCP. Eleven precipitation protocols were screened for protein recovery and optimized for a subset of precipitants by a design of experiments (DOE) approach. Because total protein recovery does not fully replicate a proteomics experiment, or detect non-protein agents that may interfere with proteomic methods, a subset of precipitation conditions were compared by two-dimensional electrophoresis and liquid chromatography coupled with mass spectrometry, with optimized recovery shown to differ between the two proteomic methods. This work demonstrates broadly applicable methods that can be applied as initial steps to optimize sample preparation of any sample type for proteomic analysis, and presents optimized precipitation protocols for extracellular CHO HCP recovery, which can vary appreciably between gel-based and shotgun proteomic methods.

  16. Proteomic analysis of indium embryotoxicity in cultured postimplantation rat embryos.

    Science.gov (United States)

    Usami, Makoto; Nakajima, Mikio; Mitsunaga, Katsuyoshi; Miyajima, Atsuko; Sunouchi, Momoko; Doi, Osamu

    2009-12-01

    Indium embryotoxicity was investigated by proteomic analysis with two-dimensional electrophoresis of rat embryos cultured from day 10.5 of gestation for 24h in the presence of 50 microM indium trichloride. In the embryo proper, indium increased quantity of several protein spots including those identified as serum albumin, phosphorylated cofilin 1, phosphorylated destrin and tyrosyl-tRNA synthetase. The increased serum albumin, derived from the culture medium composed of rat serum, may decrease the toxicity of indium. The increase of phosphorylated cofilin 1 might be involved in dysmorphogenicity of indium through perturbation of actin functions. In the yolk sac membrane, indium induced quantitative and qualitative changes in the protein spots. Proteins from appeared spots included stress proteins, and those from decreased or disappeared spots included serum proteins, glycolytic pathway enzymes and cytoskeletal proteins, indicating yolk sac dysfunction. Thus, several candidate proteins that might be involved in indium embryotoxicity were identified.

  17. Optimized protein extraction methods for proteomic analysis of Rhizoctonia solani.

    Science.gov (United States)

    Lakshman, Dilip K; Natarajan, Savithiry S; Lakshman, Sukla; Garrett, Wesley M; Dhar, Arun K

    2008-01-01

    Rhizoctonia solani (Teleomorph: Thanatephorus cucumeris, T. praticola) is a basidiomycetous fungus and a major cause of root diseases of economically important plants. Various isolates of this fungus are also beneficially associated with orchids, may serve as biocontrol agents or remain as saprophytes with roles in decaying and recycling of soil organic matter. R. solani displays several hyphal anastomosis groups (AG) with distinct host and pathogenic specializations. Even though there are reports on the physiological and histological basis of Rhizoctonia-host interactions, very little is known about the molecular biology and control of gene expression early during infection by this pathogen. Proteamic technologies are powerful tools for examining alterations in protein profiles. To aid studies on its biology and host pathogen interactions, a two-dimensional (2-D) gel-based global proteomic study has been initiated. To develop an optimized protein extraction protocol for R. solani, we compared two previously reported protein extraction protocols for 2-D gel analysis of R. solani (AG-4) isolate Rs23. Both TCA-acetone precipitation and phosphate solubilization before TCA-acetone precipitation worked well for R. solani protein extraction, although selective enrichment of some proteins was noted with either method. About 450 spots could be detected with the densitiometric tracing of Coomassie blue-stained 2-D PAGE gels covering pH 4-7 and 6.5-205 kDa. Selected protein spots were subjected to mass spectrometric analysis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Eleven protein spots were positively identified based on peptide mass fingerprinting match with fungal proteins in public databases with the Mascot search engine. These results testify to the suitability of the two optimized protein extraction protocols for 2-D proteomic studies of R. solani.

  18. Analysis of the SUMO2 Proteome during HSV-1 Infection.

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    Elizabeth Sloan

    2015-07-01

    Full Text Available Covalent linkage to members of the small ubiquitin-like (SUMO family of proteins is an important mechanism by which the functions of many cellular proteins are regulated. Sumoylation has roles in the control of protein stability, activity and localization, and is involved in the regulation of transcription, gene expression, chromatin structure, nuclear transport and RNA metabolism. Sumoylation is also linked, both positively and negatively, with the replication of many different viruses both in terms of modification of viral proteins and modulation of sumoylated cellular proteins that influence the efficiency of infection. One prominent example of the latter is the widespread reduction in the levels of cellular sumoylated species induced by herpes simplex virus type 1 (HSV-1 ubiquitin ligase ICP0. This activity correlates with relief from intrinsic immunity antiviral defence mechanisms. Previous work has shown that ICP0 is selective in substrate choice, with some sumoylated proteins such the promyelocytic leukemia protein PML being extremely sensitive, while RanGAP is completely resistant. Here we present a comprehensive proteomic analysis of changes in the cellular SUMO2 proteome during HSV-1 infection. Amongst the 877 potentially sumoylated species detected, we identified 124 whose abundance was decreased by a factor of 3 or more by the virus, several of which were validated by western blot and expression analysis. We found many previously undescribed substrates of ICP0 whose degradation occurs by a range of mechanisms, influenced or not by sumoylation and/or the SUMO2 interaction motif within ICP0. Many of these proteins are known or are predicted to be involved in the regulation of transcription, chromatin assembly or modification. These results present novel insights into mechanisms and host cell proteins that might influence the efficiency of HSV-1 infection.

  19. Identification of novel amelogenin-binding proteins by proteomics analysis.

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    Takao Fukuda

    Full Text Available Emdogain (enamel matrix derivative, EMD is well recognized in periodontology. It is used in periodontal surgery to regenerate cementum, periodontal ligament, and alveolar bone. However, the precise molecular mechanisms underlying periodontal regeneration are still unclear. In this study, we investigated the proteins bound to amelogenin, which are suggested to play a pivotal role in promoting periodontal tissue regeneration. To identify new molecules that interact with amelogenin and are involved in osteoblast activation, we employed coupling affinity chromatography with proteomic analysis in fractionated SaOS-2 osteoblastic cell lysate. In SaOS-2 cells, many of the amelogenin-interacting proteins in the cytoplasm were mainly cytoskeletal proteins and several chaperone molecules of heat shock protein 70 (HSP70 family. On the other hand, the proteomic profiles of amelogenin-interacting proteins in the membrane fraction of the cell extracts were quite different from those of the cytosolic-fraction. They were mainly endoplasmic reticulum (ER-associated proteins, with lesser quantities of mitochondrial proteins and nucleoprotein. Among the identified amelogenin-interacting proteins, we validated the biological interaction of amelogenin with glucose-regulated protein 78 (Grp78/Bip, which was identified in both cytosolic and membrane-enriched fractions. Confocal co-localization experiment strongly suggested that Grp78/Bip could be an amelogenin receptor candidate. Further biological evaluations were examined by Grp78/Bip knockdown analysis with and without amelogenin. Within the limits of the present study, the interaction of amelogenin with Grp78/Bip contributed to cell proliferation, rather than correlate with the osteogenic differentiation in SaOS-2 cells. Although the biological significance of other interactions are not yet explored, these findings suggest that the differential effects of amelogenin-derived osteoblast activation could be of

  20. Reproducible Analysis of Post-Translational Modifications in Proteomes--Application to Human Mutations.

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    Alex S Holehouse

    Full Text Available Protein post-translational modifications (PTMs are an important aspect of protein regulation. The number of PTMs discovered within the human proteome, and other proteomes, has been rapidly expanding in recent years. As a consequence of the rate in which new PTMs are identified, analysis done in one year may result in different conclusions when repeated in subsequent years. Among the various functional questions pertaining to PTMs, one important relationship to address is the interplay between modifications and mutations. Specifically, because the linear sequence surrounding a modification site often determines molecular recognition, it is hypothesized that mutations near sites of PTMs may be more likely to result in a detrimental effect on protein function, resulting in the development of disease.We wrote an application programming interface (API to make analysis of ProteomeScout, a comprehensive database of PTMs and protein information, easy and reproducible. We used this API to analyze the relationship between PTMs and human mutations associated with disease (based on the 'Clinical Significance' annotation from dbSNP. Proteins containing pathogenic mutations demonstrated a significant study bias which was controlled for by analyzing only well-studied proteins, based on their having at least one pathogenic mutation. We found that pathogenic mutations are significantly more likely to lie within eight amino acids of a phosphoserine, phosphotyrosine or ubiquitination site when compared to mutations in general, based on a Fisher's Exact test. Despite the skew of pathogenic mutations occurring on positively charged arginines, we could not account for this relationship based only on residue type. Finally, we hypothesize a potential mechanism for a pathogenic mutation on RAF1, based on its proximity to a phosphorylation site, which represents a subtle regulation difference that may explain why its biochemical effect has failed to be uncovered

  1. Quantitative and qualitative proteome characteristics extracted from in-depth integrated genomics and proteomics analysis

    NARCIS (Netherlands)

    Low, T.Y.; van Heesch, S.; van den Toorn, H.; Giansanti, P.; Cristobal, A.; Toonen, P.; Schafer, S.; Hubner, N.; van Breukelen, B.; Mohammed, S.; Cuppen, E.; Heck, A.J.R.; Guryev, V.

    2013-01-01

    Quantitative and qualitative protein characteristics are regulated at genomic, transcriptomic, and posttranscriptional levels. Here, we integrated in-depth transcriptome and proteome analyses of liver tissues from two rat strains to unravel the interactions within and between these layers. We obtain

  2. Comparative Proteomic Analysis of Rice at Filling Stage under Chilling Stress

    Institute of Scientific and Technical Information of China (English)

    Ying Wang; Weitong Cui; Shihua Shen

    2012-01-01

    Using comparative proteomic analysis,we carried out an investigation aiming at a better understanding of the molecular mechanisms of chilling stress adaptation in rice (O.sativa L.ssp.indica cultivar 93-11) at early filling stage.The rice plants at early filling stage were exposed to 12℃ for 6 h,12 h,24 h,48 h.Analysis of the dynamic proteome profile of the uppermost internodes showed that 99 protein spots were differentially expressed during this process.Among them,45 protein spots were found to be up-regulated and 54 protein spots were down-regulated in responding to chilling stress.81 protein spots were successfully identified through MALDITOF MS and database searching.Those identified proteins were the products of 65 genes,and could be sorted into 8 functional categories.Among them,secondary metabolism related proteins such as S-adenosylmethionine synthetase were up-regulated.That lignification-related proteins were up-regulated by chilling stress indicated the uppermost internodes supporting ear would be enhanced to resist the chilling stress.

  3. Recent developments of nanoparticle-based enrichment methods for mass spectrometric analysis in proteomics

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    In proteome research, rapid and effective separation strategies are essential for successful protein identification due to the broad dynamic range of proteins in biological samples. Some important proteins are often expressed in ultra low abundance, thus making the pre-concentration procedure before mass spectrometric analysis prerequisite. The main purpose of enrichment is to isolate target molecules from complex mixtures to reduce sample complexity and facilitate the subsequent analyzing steps. The introduction of nanoparticles into this field has accelerated the development of enrichment methods. In this review, we mainly focus on recent developments of using different nanomaterials for pre-concentration of low-abundance peptides/ proteins, including those containing post-translational modifications, such as phosphorylation and glycosylation, prior to mass spectrometric analysis.

  4. Proteomic Approaches for Site-specific O-GlcNAcylation Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Sheng; Yang, Feng; Camp, David G.; Rodland, Karin D.; Qian, Weijun; Liu, Tao; Smith, Richard D.

    2014-10-01

    O-GlcNAcylation is a dynamic protein post-translational modification of serine or threonine residues by an O-linked monosaccharide N-acetylglucosamine (O-GlcNAc). O-GlcNAcylation was discovered three decades ago, and it has been shown to contribute to various disease states, such as metabolic diseases, cancer and neurological diseases. Yet it remains technically difficult to characterize comprehensively and quantitatively, due to its exceptionally low abundance and extremely labile nature under conventional tandem mass spectrometry conditions. Herein, we review the recent efforts for tackling these challenges in developing proteomic approaches for site-specific O-GlcNAcylation analysis, such as specific enrichment of O-GlcNAc peptides/proteins, unambiguous site-determination of O-GlcNAc modification, and quantitative analysis of O-GlcNAcylation.

  5. Primary metabolism in Lactobacillus sakei food isolates by proteomic analysis

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    Champomier-Vergès Marie-Christine

    2010-04-01

    Full Text Available Abstract Background Lactobacillus sakei is an important food-associated lactic acid bacterium commonly used as starter culture for industrial meat fermentation, and with great potential as a biopreservative in meat and fish products. Understanding the metabolic mechanisms underlying the growth performance of a strain to be used for food fermentations is important for obtaining high-quality and safe products. Proteomic analysis was used to study the primary metabolism in ten food isolates after growth on glucose and ribose, the main sugars available for L. sakei in meat and fish. Results Proteins, the expression of which varied depending on the carbon source were identified, such as a ribokinase and a D-ribose pyranase directly involved in ribose catabolism, and enzymes involved in the phosphoketolase and glycolytic pathways. Expression of enzymes involved in pyruvate and glycerol/glycerolipid metabolism were also affected by the change of carbon source. Interestingly, a commercial starter culture and a protective culture strain down-regulated the glycolytic pathway more efficiently than the rest of the strains when grown on ribose. The overall two-dimensional gel electrophoresis (2-DE protein expression pattern was similar for the different strains, though distinct differences were seen between the two subspecies (sakei and carnosus, and a variation of about 20% in the number of spots in the 2-DE gels was observed between strains. A strain isolated from fermented fish showed a higher expression of stress related proteins growing on both carbon sources. Conclusions It is obvious from the data obtained in this study that the proteomic approach efficiently identifies differentially expressed proteins caused by the change of carbon source. Despite the basic similarity in the strains metabolic routes when they ferment glucose and ribose, there were also interesting differences. From the application point of view, an understanding of regulatory

  6. Proteomic analysis of blastema formation in regenerating axolotl limbs

    Directory of Open Access Journals (Sweden)

    Nye Holly LD

    2009-11-01

    neural and epidermal factors. Our findings indicate the general value of quantitative proteomic analysis in understanding the regeneration of complex structures.

  7. Discrimination analysis of mass spectrometry proteomics for ovarian cancer detection

    Institute of Scientific and Technical Information of China (English)

    Yan-jun HONG; Xiao-dan WANG; David SHEN; Su ZENG

    2008-01-01

    Aim:A discrimination analysis has been explored for the probabilistic classifica-tion of healthy versus ovarian cancer serum samples using proteomics data from mass spectrometry (MS).Methods:The method employs data normalization,clustering,and a linear discriminant analysis on surface-enhanced laser desorp-tion ionization (SELDI) time-of-flight MS data.The probabilistic classification method computes the optimal linear discriminant using the complex human blood serum SELDI spectra.Cross-validation and training/testing data-split experi-ments are conducted to verify the optimal discriminant and demonstrate the accu-racy and robustness of the method.Results:The cluster discrimination method achieves excellent performance.The sensitivity,specificity,and positive predic-tive values are above 97% on ovarian cancer.The protein fraction peaks,which significantly contribute to the classification,can be available from the analysis process.Conclusion:The discrimination analysis helps the molecular identities of differentially expressed proteins and peptides between the healthy and ovarian patients.

  8. Large-scale proteome comparative analysis of developing rhizomes of the ancient vascular plant Equisetum hyemale.

    Directory of Open Access Journals (Sweden)

    Tiago Santana Balbuena

    2012-06-01

    Full Text Available Equisetum hyemale is a widespread vascular plant species, whose reproduction is mainly dependent on the growth and development of the rhizomes. Due to its key evolutionary position, the identification of factors that could be involved in the existence of the rhizomatous trait may contribute to a better understanding of the role of this underground organ for the successful propagation of this and other plant species. In the present work, we characterized the proteome of E. hyemale rhizomes using a GeLC-MS spectral-counting proteomics strategy. A total of 1,911 and 1,860 non-redundant proteins were identified in the rhizomes apical tip and elongation zone, respectively. Rhizome- characteristic proteins were determined by comparisons of the developing rhizome tissues to developing roots. A total of 87 proteins were found to be up-regulated in both E. hyemale rhizome tissues in relation to developing roots. Hierarchical clustering indicated a vast dynamic range in the expression of the 87 characteristic proteins and revealed, based on the expression profile, the existence of 9 major protein groups. Gene ontology analyses suggested an over-representation of the terms involved in macromolecular and protein biosynthetic processes, gene expression and nucleotide and protein binding functions. Spatial differences analysis between the rhizome apical tip and the elongation zone revealed that only eight proteins were up-regulated in the apical tip including RNA-binding proteins and an acyl carrier protein, as well as a KH-domain protein and a T-complex subunit; while only seven proteins were up-regulated in the elongation zone including phosphomannomutase, galactomannan galactosyltransferase, endoglucanase 10 and 25 and mannose-1-phosphate guanyltransferase subunits alpha and beta. This is the first large scale characterization of the proteome of a plant rhizome. Implications of the findings were discussed in relation to other underground organs and related

  9. Quantitative proteome analysis using isotope-coded affinity tags and mass spectrometry.

    Science.gov (United States)

    Shiio, Yuzuru; Aebersold, Ruedi

    2006-01-01

    A main objective of proteomics research is to systematically identify and quantify proteins in a given proteome (cells, subcellular fractions, protein complexes, tissues or body fluids). Protein labeling with isotope-coded affinity tags (ICAT) followed by tandem mass spectrometry allows sequence identification and accurate quantification of proteins in complex mixtures, and has been applied to the analysis of global protein expression changes, protein changes in subcellular fractions, components of protein complexes, protein secretion and body fluids. This protocol describes protein-sample labeling with ICAT reagents, chromatographic fractionation of the ICAT-labeled tryptic peptides, and protein identification and quantification using tandem mass spectrometry. The method is suitable for both large-scale analysis of complex samples including whole proteomes and small-scale analysis of subproteomes, and allows quantitative analysis of proteins, including those that are difficult to analyze by gel-based proteomics technology.

  10. Proteomics analysis reveals diabetic kidney as a ketogenic organ in type 2 diabetes

    National Research Council Canada - National Science Library

    Dongjuan Zhang; Hang Yang; Xiaomu Kong; Kang Wang; Xuan Mao; Xianzhong Yan; Yuan Wang; Siqi Liu; Xiaoyan Zhang; Jing Li; Lihong Chen; Jing Wu; Mingfen Wei; Jichun Yang; Youfei Guan

    2011-01-01

    .... Proteomic analysis revealed that 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase 2 (HMGCS2), the key enzyme in ketogenesis, was increased fourfold in the kidneys of type 2 diabetic db/db mice...

  11. Stressor-induced proteome alterations in zebrafish: A meta-analysis of response patterns

    Energy Technology Data Exchange (ETDEWEB)

    Groh, Ksenia J., E-mail: ksenia.groh@eawag.ch [Eawag, Swiss Federal Institute of Aquatic Science and Technology, 8600 Dübendorf (Switzerland); ETH Zürich, Swiss Federal Institute of Technology, Department of Chemistry and Applied Biosciences, 8093 Zürich (Switzerland); Suter, Marc J.-F. [Eawag, Swiss Federal Institute of Aquatic Science and Technology, 8600 Dübendorf (Switzerland); ETH Zürich, Swiss Federal Institute of Technology, Department of Environmental Systems Science, 8092 Zürich (Switzerland)

    2015-02-15

    Highlights: • We compared reported proteome changes induced by various stressors in zebrafish. • Several proteins groups frequently responding to diverse stressors were identified. • These included energy metabolism enzymes, heat shock and cytoskeletal proteins. • Insufficient proteome coverage impedes identification of more specific responses. • Further research needs for proteomics in ecotoxicology are discussed. - Abstract: Proteomics approaches are being increasingly applied in ecotoxicology on the premise that the identification of specific protein expression changes in response to a particular chemical would allow elucidation of the underlying molecular pathways leading to an adverse effect. This in turn is expected to promote the development of focused testing strategies for specific groups of toxicants. Although both gel-based and gel-free global characterization techniques provide limited proteome coverage, the conclusions regarding the cellular processes affected are still being drawn based on the few changes detected. To investigate how specific the detected responses are, we analyzed a set of studies that characterized proteome alterations induced by various physiological, chemical and biological stressors in zebrafish, a popular model organism. Our analysis highlights several proteins and protein groups, including heat shock and oxidative stress defense proteins, energy metabolism enzymes and cytoskeletal proteins, to be most frequently identified as responding to diverse stressors. In contrast, other potentially more specifically responding protein groups are detected much less frequently. Thus, zebrafish proteome responses to stress reported by different studies appear to depend mostly on the level of stress rather than on the specific stressor itself. This suggests that the most broadly used current proteomics technologies do not provide sufficient proteome coverage to allow in-depth investigation of specific mechanisms of toxicant action

  12. [Bibliometric analysis of bacterial quantitative proteomics in English literatures].

    Science.gov (United States)

    Zhang, Xin; She, Danyang; Liu, Youning; Wang, Rui; Di, Xiuzhen; Liang, Beibei; Wang, Yue

    2014-07-01

    To analyze the worldwide advances on bacterial quantitative proteomics over the past fifteen years with bibliometric approach. Literature retrieval was conducted throughout the databases of Pubmed, Embase and Science citation index (SCI), using "bacterium" and "quantitative proteomics" as the key words. The deadline is July 2013. We sorted and analyzed these articles with Endnote X6 from the aspects of published year, the first author, name of journal, published institution, cited frequency and publication type. 932 English articles were included in our research after deleting the duplicates. The first article on bacterial quantitative proteomics was reported in 1999. The maximal publications were 163 related articles in 2012. Up till July 2013, authors from more than 23 countries and regions have published articles in this field. China ranks the fourth. The main publication type is original articles. The most frequently cited article is entitled with "Absolute quantification of proteins by LCMSE: a virtue of parallel MS acquisition" by Silva JC, Gorenstein MV, Li GZ, et al in Mol Cell Proteomics 2006. The most productive author is Smith RD from Biological Sciences Division, Pac. Northwest National Laboratory. The top journal publishing bacterial quantitative proteomics is Proteomics. More and more researchers pay attention to quantitative proteomics which will be widely used in bacteriology.

  13. Proteomic analysis of RCL2 paraffin-embedded tissues.

    Science.gov (United States)

    Bellet, V; Boissière, F; Bibeau, F; Desmetz, C; Berthe, M L; Rochaix, P; Maudelonde, T; Mangè, A; Solassol, J

    2008-10-01

    Histopathological diagnosis in most of the world's hospitals is based upon formalin-fixed and paraffin-embedded (FFPE) tissues. Although this standard fixation and embedding procedure keeps the tissue in excellent form for morphological and immunohistological analysis, FFPE is inappropriate for nucleic acids and protein studies. We investigated the potential value of RCL2, a new non-toxic fixative, for sparing proteins preserved in paraffin-embedded tissues. Normal colonic mucosa tissue was fixed in RCL2 prior to paraffin embedding (RCL2P), and then processed for quality and quantity of protein conservation, as compared to frozen and FFPE tissues using complementary proteomic analysis approaches. Using 4 different protein extraction protocols, RCL2P tissue consistently showed the highest protein yield. Similar protein patterns were observed with RCL2P and frozen tissues using mono and bi-dimensional electrophoresis. Moreover, membrane, cytoplasmic and nuclear proteins, as well as phosphorylated proteins, were successfully detected using western-blot. Furthermore, protein patterns observed by mass spectrometry analysis after laser-captured microdissection were found to be identical for frozen and RCL2-fixed tissues. At last, immunohistochemistry using various antibodies showed comparable results between both tissue storage methods. We concluded that RCL2 has great potential for performing both morphological and molecular analyses on the same archival paraffin-embedded tissue sample, and can be a new method for investigating protein biomarkers.

  14. Comparative Proteome Analysis of Human Lung Squamous Carcinoma Tissue

    Institute of Scientific and Technical Information of China (English)

    LI Cui; TANG Can'e; DUAN Chaojun; YI Hong; XIAO Zhiqiang; CHEN Zhuchu

    2006-01-01

    Objective: To establish the two-dimensional electrophoresis profiles with high resolution and reproducibility from human lung squamous carcinoma tissue and paired normal tumor-adjacent bronchial epithelial tissue, and to identify differential expression tumor-associated proteins by using proteome analysis. Methods: Comparative proteome analysis with 20 human lung squamous carcinoma tissues and the paired normal bronchial epithelial tissues adjacent to tumors was carried out. The total proteins of human lung squamous carcinoma tissue and paired normal tumor-adjacent bronchial epithelial tissue were separated by means of immobilized pH gradient-based two-dimensional gel electrophoresis (2-DE) and silver staining. The differential expression proteins were analyzed and then identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). Results: (1) Well-resolved, reproducible 2-DE patterns of human lung squamous carcinoma and adjacent normal bronchial epithelial tissues were obtained. For tumor tissue, average spots of 3 gels were 1567±46, and 1436±54 spots were matched with an average matching rate of 91.6%. For control, average spots of 3 gels were 1349±58, and 1228±35 spots were matched with an average matching rate of 91.03%. The average position deviation of matched spots was 0.924±0.128 mm in IEF direction, and 1.022±0.205 mm in SDS-PAGE direction; (2)A total of 1178±56 spots were matched between the electrophoretic maps of 20 human lung squamous carcinoma tissues and paired normal tumor-adjacent bronchial epithelial tissues. Seventy-six differentially expressed proteins were screened; (3) Sixty-eight differential proteins were identified by PMF, some proteins were the products of oncogenes, and others involved in the regulation of cell cycle and signal transduction;(4) In order to validate the reliability of the identified results, the expression of 3 proteins mdm2, c-jun and EGFR, which was correlated with lung

  15. Proteomic and protein interaction network analysis of human T lymphocytes during cell-cycle entry

    Science.gov (United States)

    Orr, Stephen J; Boutz, Daniel R; Wang, Rong; Chronis, Constantinos; Lea, Nicholas C; Thayaparan, Thivyan; Hamilton, Emma; Milewicz, Hanna; Blanc, Eric; Mufti, Ghulam J; Marcotte, Edward M; Thomas, N Shaun B

    2012-01-01

    Regulating the transition of cells such as T lymphocytes from quiescence (G0) into an activated, proliferating state involves initiation of cellular programs resulting in entry into the cell cycle (proliferation), the growth cycle (blastogenesis, cell size) and effector (functional) activation. We show the first proteomic analysis of protein interaction networks activated during entry into the first cell cycle from G0. We also provide proof of principle that blastogenesis and proliferation programs are separable in primary human T cells. We employed a proteomic profiling method to identify large-scale changes in chromatin/nuclear matrix-bound and unbound proteins in human T lymphocytes during the transition from G0 into the first cell cycle and mapped them to form functionally annotated, dynamic protein interaction networks. Inhibiting the induction of two proteins involved in two of the most significantly upregulated cellular processes, ribosome biogenesis (eIF6) and hnRNA splicing (SF3B2/SF3B4), showed, respectively, that human T cells can enter the cell cycle without growing in size, or increase in size without entering the cell cycle. PMID:22415777

  16. Toward characterizing seed vigor in alfalfa through proteomic analysis of germination and priming.

    Science.gov (United States)

    Yacoubi, Rafika; Job, Claudette; Belghazi, Maya; Chaibi, Wided; Job, Dominique

    2011-09-02

    Alfalfa, the most widely grown leguminous crop in the world, is generally exposed to severe salinity stress in Tunisia, notably affecting its germination performance. Toward a better understanding of alfalfa seed vigor, we have used proteomics to characterize protein changes occurring during germination and osmopriming, a pretreatment that accelerates germination and improves seedling uniformity particularly under stress conditions. The data revealed that germination was accompanied by dynamic changes of 79 proteins, which are mainly involved in protein metabolism, cell structure, metabolism, and defense. Comparative proteomic analysis also revealed 63 proteins specific to osmopriming, 65 proteins preferentially varying during germination, and 14 proteins common to both conditions. Thus, the present study unveiled the unexpected finding that osmopriming cannot simply be considered as an advance of germination-related processes but involves other mechanisms improving germination such as the mounting of defense mechanisms enabling osmoprimed seeds to surmount environmental stresses potentially occurring during germination. The present results therefore provide novel avenues toward understanding the mechanisms of invigoration of low vigor seeds by priming treatments that are widely used both in commercial applications and in developing countries (on farm seed priming) to better control crop yields.

  17. Proteome-Wide Analysis and Diel Proteomic Profiling of the Cyanobacterium Arthrospira platensis PCC 8005

    OpenAIRE

    2014-01-01

    The filamentous cyanobacterium Arthrospira platensis has a long history of use as a food supply and it has been used by the European Space Agency in the MELiSSA project, an artificial microecosystem which supports life during long-term manned space missions. This study assesses progress in the field of cyanobacterial shotgun proteomics and light/dark diurnal cycles by focusing on Arthrospira platensis. Several fractionation workflows including gel-free and gel-based protein/peptide fractionat...

  18. Expanding proteomics into the analysis of chiral drugs.

    Science.gov (United States)

    Sui, Jianjun; Zhang, Jianhua; Ching, Chi Bun; Chen, Wei Ning

    2009-06-01

    The chiralities of chiral drugs have been investigated extensively with the purpose of enlightening the role of chirality in drug action. Proteomics, though in its infancy, has recently emerged as the foremost technology in drug development research, possessing the advantage of providing more useful information about an organism than genomics, as it directly addresses the level of genome products and their interactions. In this review, we will discuss the background of chiral drug investigation from which contemporary drug chirality research has emerged, the techniques involved in proteomics technology, the application of proteomics in this exciting area, and the perspectives in future applications of this field.

  19. Insights into immune responses in oral cancer through proteomic analysis of saliva and salivary extracellular vesicles

    Science.gov (United States)

    Winck, Flavia V.; Prado Ribeiro, Ana Carolina; Ramos Domingues, Romênia; Ling, Liu Yi; Riaño-Pachón, Diego Mauricio; Rivera, César; Brandão, Thaís Bianca; Gouvea, Adriele Ferreira; Santos-Silva, Alan Roger; Coletta, Ricardo D.; Paes Leme, Adriana F.

    2015-01-01

    The development and progression of oral cavity squamous cell carcinoma (OSCC) involves complex cellular mechanisms that contribute to the low five-year survival rate of approximately 20% among diagnosed patients. However, the biological processes essential to tumor progression are not completely understood. Therefore, detecting alterations in the salivary proteome may assist in elucidating the cellular mechanisms modulated in OSCC and improve the clinical prognosis of the disease. The proteome of whole saliva and salivary extracellular vesicles (EVs) from patients with OSCC and healthy individuals were analyzed by LC-MS/MS and label-free protein quantification. Proteome data analysis was performed using statistical, machine learning and feature selection methods with additional functional annotation. Biological processes related to immune responses, peptidase inhibitor activity, iron coordination and protease binding were overrepresented in the group of differentially expressed proteins. Proteins related to the inflammatory system, transport of metals and cellular growth and proliferation were identified in the proteome of salivary EVs. The proteomics data were robust and could classify OSCC with 90% accuracy. The saliva proteome analysis revealed that immune processes are related to the presence of OSCC and indicate that proteomics data can contribute to determining OSCC prognosis. PMID:26538482

  20. Proteomic analysis of minute amount of colonic biopsies by enteroscopy sampling

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xing [Department of Analytical Chemistry and CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (China); Xu, Yanli [Fuyang People’s Hospital (China); Meng, Qian [Department of Analytical Chemistry and CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (China); Zheng, Qingqing [Digestive Endoscopic Center, Shanghai Jiaotong University Affiliated Sixth People’s Hospital (China); Wu, Jianhong [Department of Analytical Chemistry and CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (China); Wang, Chen; Jia, Weiping [Shanghai Key Laboratory of Diabetes Mellitus, Department of Endocrinology and Metabolism, Shanghai Diabetes Institute, Shanghai Clinical Center for Diabetes, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital (China); Figeys, Daniel [Department of Biochemistry, Microbiology and Immunology, and Department of Chemistry and Biomolecular Sciences, University of Ottawa (Canada); Chang, Ying, E-mail: emulan@163.com [Digestive Endoscopic Center, Shanghai Jiaotong University Affiliated Sixth People’s Hospital (China); Zhou, Hu, E-mail: zhouhu@simm.ac.cn [Department of Analytical Chemistry and CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (China)

    2016-08-05

    Colorectal cancer (CRC) is one of the most common types of malignant tumor worldwide. Currently, although many researchers have been devoting themselves in CRC studies, the process of locating biomarkers for CRC early diagnosis and prognostic is still very slow. Using a centrifugal proteomic reactor-based proteomic analysis of minute amount of colonic biopsies by enteroscopy sampling, 2620 protein groups were quantified between cancer mucosa and adjacent normal colorectal mucosa. Of which, 403 protein groups were differentially expressed with statistic significance between cancer and normal tissues, including 195 up-regulated and 208 down-regulated proteins in cancer tissues. Three proteins (SOD3, PRELP and NGAL) were selected for further Western blot validation. And the resulting Western blot experimental results were consistent with the quantitative proteomic data. SOD3 and PRELP are down-regulated in CRC mucosa comparing to adjacent normal tissue, while NGAL is up-regulated in CRC mucosa. In conclusion, the centrifugal proteomic reactor-based label-free quantitative proteomic approach provides a highly sensitive and powerful tool for analyzing minute protein sample from tiny colorectal biopsies, which may facilitate CRC biomarkers discovery for diagnoses and prognoses. -- Highlights: •Minute amount of colonic biopsies by endoscopy is suitable for proteomic analysis. •Centrifugal proteomic reactor can be used for processing tiny clinic biopsy sample. •SOD3 and PRELP are down-regulated in CRC, while NGAL is up-regulated in CRC.

  1. Current Proteomic Methods to Investigate the Dynamics of Histone Turnover in the Central Nervous System

    Science.gov (United States)

    Farrelly, L.A.; Dill, B.D.; Molina, H.; Birtwistle, M.R.; Maze, I.

    2016-01-01

    Characterizing the dynamic behavior of nucleosomes in the central nervous system is vital to our understanding of brain-specific chromatin-templated processes and their roles in transcriptional plasticity. Histone turnover—the complete loss of old, and replacement by new, nucleosomal histones—is one such phenomenon that has recently been shown to be critical for cell-type-specific transcription in brain, synaptic plasticity, and cognition. Such revelations that histones, long believed to static proteins in postmitotic cells, are highly dynamic in neurons were only possible owing to significant advances in analytical chemistry-based techniques, which now provide a platform for investigations of histone dynamics in both healthy and diseased tissues. Here, we discuss both past and present proteomic methods (eg, mass spectrometry, human “bomb pulse labeling”) for investigating histone turnover in brain with the hope that such information may stimulate future investigations of both adaptive and aberrant forms of “neuroepigenetic” plasticity. PMID:27423867

  2. Evaluation of sample extraction methods for proteomics analysis of green algae Chlorella vulgaris.

    Science.gov (United States)

    Gao, Yan; Lim, Teck Kwang; Lin, Qingsong; Li, Sam Fong Yau

    2016-05-01

    Many protein extraction methods have been developed for plant proteome analysis but information is limited on the optimal protein extraction method from algae species. This study evaluated four protein extraction methods, i.e. direct lysis buffer method, TCA-acetone method, phenol method, and phenol/TCA-acetone method, using green algae Chlorella vulgaris for proteome analysis. The data presented showed that phenol/TCA-acetone method was superior to the other three tested methods with regards to shotgun proteomics. Proteins identified using shotgun proteomics were validated using sequential window acquisition of all theoretical fragment-ion spectra (SWATH) technique. Additionally, SWATH provides protein quantitation information from different methods and protein abundance using different protein extraction methods was evaluated. These results highlight the importance of green algae protein extraction method for subsequent MS analysis and identification.

  3. Functional complexity of the axonal growth cone: a proteomic analysis.

    Directory of Open Access Journals (Sweden)

    Adriana Estrada-Bernal

    Full Text Available The growth cone, the tip of the emerging neurite, plays a crucial role in establishing the wiring of the developing nervous system. We performed an extensive proteomic analysis of axonal growth cones isolated from the brains of fetal Sprague-Dawley rats. Approximately 2000 proteins were identified at ≥ 99% confidence level. Using informatics, including functional annotation cluster and KEGG pathway analysis, we found great diversity of proteins involved in axonal pathfinding, cytoskeletal remodeling, vesicular traffic and carbohydrate metabolism, as expected. We also found a large and complex array of proteins involved in translation, protein folding, posttranslational processing, and proteasome/ubiquitination-dependent degradation. Immunofluorescence studies performed on hippocampal neurons in culture confirmed the presence in the axonal growth cone of proteins representative of these processes. These analyses also provide evidence for rough endoplasmic reticulum and reveal a reticular structure equipped with Golgi-like functions in the axonal growth cone. Furthermore, Western blot revealed the growth cone enrichment, relative to fetal brain homogenate, of some of the proteins involved in protein synthesis, folding and catabolism. Our study provides a resource for further research and amplifies the relatively recently developed concept that the axonal growth cone is equipped with proteins capable of performing a highly diverse range of functions.

  4. Proteomic analysis of peach fruit moth larvae treated with phosphine.

    Science.gov (United States)

    Liu, Tao; Li, Li; Li, Baishu; Zhang, Fanhua; Wang, Yuejin

    2012-01-01

    Phosphine has been used worldwide for the control of stored-product insects for many years. However, the molecular mechanism of its toxicity is not clearly understood. In the current study, larvae of the peach fruit moth were fumigated with phosphine. Proteomic analysis was then performed to identify the regulated proteins. Our results confirmed the phosphine toxicity on the peach fruit moth. The median lethal time LT50 was 38.5 h at 330 ppm at 25 degrees C. During fumigation, the respiration of the peach fruit moth was extremely inhibited. Of the 26 regulated proteins, 16 were identified by MALDI-TOF mass spectrometry after a 24 h treatment. The proteins were classified as related to metabolism (25 %), anti-oxidation (6 %), signal transduction (38 %), or defense (19 %). The rest (13 %) were unclassified. Phosphine regulation of ATP and glutathione contents, as well as of ATP synthase and glutathione S-transferase 2 activities were confirmed by enzyme activity analysis. These results demonstrate that complex transcriptional regulations underlie phosphine fumigation. New theories on the mechanism of phosphine toxicity may also be established based on these results.

  5. Proteomic analysis of honeybee worker (Apis mellifera hypopharyngeal gland development

    Directory of Open Access Journals (Sweden)

    Li Jianke

    2009-12-01

    Full Text Available Abstract Background Hypopharyngeal glands (HG of honeybee workers play an important role in honeybee nutrition and caste differentiation. Previous research mainly focused on age-dependent morphological, physiological, biochemical and genomic characters of the HG. Here proteomics and biochemical network analysis were used to follow protein changes during the HG development. Results A total of 87, 76, 85, 74, 71, and 55 proteins were unambiguously identified on day 1, 3, 6, 12, 15 and 20, respectively. These proteins were major royal jelly proteins (MRJPs, metabolism of carbohydrates, lipids and proteins, cytoskeleton, development regulation, antioxidant, molecule transporter, regulation of transcription/translation, proteins with folding functions. The most interesting is that MRJP's that have been detected in the HG of the newly emerged worker bees. The MRJP's expression is at peak level from 6-12 days, was validated by western blot analysis of MRJP1, 2 and 3. Moreover, 35 key node proteins were found in the biochemical networks of the HG. Conclusions HG secretes RJ at peak level within 6-12 days, but the worker bee can secrete royal jelly (RJ since birth, which is a new finding. Several key node proteins play an important role in the biochemical networks of the developing HG. This provides us some target proteins when genetically manipulating honeybees.

  6. Analysis of the biofilm proteome of Xylella fastidiosa

    Directory of Open Access Journals (Sweden)

    Labate Carlos A

    2011-09-01

    Full Text Available Abstract Background Xylella fastidiosa is limited to the xylem of the plant host and the foregut of insect vectors (sharpshooters. The mechanism of pathogenicity of this bacterium differs from other plant pathogens, since it does not present typical genes that confer specific interactions between plant and pathogens (avr and/or hrp. The bacterium is injected directly into the xylem vessels where it adheres and colonizes. The whole process leads to the formation of biofilms, which are considered the main mechanism of pathogenicity. Cells in biofilms are metabolically and phenotypically different from their planktonic condition. The mature biofilm stage (phase of higher cell density presents high virulence and resistance to toxic substances such as antibiotics and detergents. Here we performed proteomic analysis of proteins expressed exclusively in the mature biofilm of X. fastidiosa strain 9a5c, in comparison to planktonic growth condition. Results We found a total of 456 proteins expressed in the biofilm condition, which correspond to approximately 10% of total protein in the genome. The biofilm showed 37% (or 144 proteins different protein than we found in the planktonic growth condition. The large difference in protein pattern in the biofilm condition may be responsible for the physiological changes of the cells in the biofilm of X. fastidiosa. Mass spectrometry was used to identify these proteins, while real-time quantitative polymerase chain reaction monitored expression of genes encoding them. Most of proteins expressed in the mature biofilm growth were associated with metabolism, adhesion, pathogenicity and stress conditions. Even though the biofilm cells in this work were not submitted to any stress condition, some stress related proteins were expressed only in the biofilm condition, suggesting that the biofilm cells would constitutively express proteins in different adverse environments. Conclusions We observed overexpression of proteins

  7. Integrated proteomic and genomic analysis of colorectal cancer

    Science.gov (United States)

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

  8. Alteration of protein levels during influenza virus H1N1 infection in host cells: a proteomic survey of host and virus reveals differential dynamics.

    Directory of Open Access Journals (Sweden)

    Susann Kummer

    Full Text Available We studied the dynamics of the proteome of influenza virus A/PR/8/34 (H1N1 infected Madin-Darby canine kidney cells up to 12 hours post infection by mass spectrometry based quantitative proteomics using the approach of stable isotope labeling by amino acids in cell culture (SILAC. We identified 1311 cell proteins and, apart from the proton channel M2, all major virus proteins. Based on their abundance two groups of virus proteins could be distinguished being in line with the function of the proteins in genesis and formation of new virions. Further, the data indicate a correlation between the amount of proteins synthesized and their previously determined copy number inside the viral particle. We employed bioinformatic approaches such as functional clustering, gene ontology, and pathway (KEGG enrichment tests to uncover co-regulated cellular protein sets, assigned the individual subsets to their biological function, and determined their interrelation within the progression of viral infection. For the first time we are able to describe dynamic changes of the cellular and, of note, the viral proteome in a time dependent manner simultaneously. Through cluster analysis, time dependent patterns of protein abundances revealed highly dynamic up- and/or down-regulation processes. Taken together our study provides strong evidence that virus infection has a major impact on the cell status at the protein level.

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

    Directory of Open Access Journals (Sweden)

    Rader Robert

    2007-06-01

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

  10. Transcriptome and proteome dynamics in larvae of the barnacle Balanus Amphitrite from the Red Sea

    KAUST Repository

    Chandramouli, Kondethimmanahalli

    2015-12-15

    Background The barnacle Balanus amphitrite is widely distributed in marine shallow and tidal waters, and has significant economic and ecological importance. Nauplii, the first larval stage of most crustaceans, are extremely abundant in the marine zooplankton. However, a lack of genome information has hindered elucidation of the molecular mechanisms of development, settlement and survival strategies in extreme marine environments. We sequenced and constructed the genome dataset for nauplii to obtain comprehensive larval genetic information. We also investigated iTRAQ-based protein expression patterns to reveal the molecular basis of nauplii development, and to gain information on larval survival strategies in the Red Sea marine environment. Results A nauplii larval transcript dataset, containing 92,117 predicted open reading frames (ORFs), was constructed and used as a reference for the proteome analysis. Genes related to translation, oxidative phosphorylation and cytoskeletal development were highly abundant. We observed remarkable plasticity in the proteome of Red Sea larvae. The proteins associated with development, stress responses and osmoregulation showed the most significant differences between the two larval populations studied. The synergistic overexpression of heat shock and osmoregulatory proteins may facilitate larval survival in intertidal habitats or in extreme environments. Conclusions We presented, for the first time, comprehensive transcriptome and proteome datasets for Red Sea nauplii. The datasets provide a foundation for future investigations focused on the survival mechanisms of other crustaceans in extreme marine environments.

  11. Proteome of human colon cancer stem cells: A comparative analysis

    Institute of Scientific and Technical Information of China (English)

    Jian Zou; Xiao-Feng Yu; Zhi-Jun Bao; Jie Dong

    2011-01-01

    AIM: To isolate and identify the biological characteristics of human colon cancer stem cells (SW1116 cells) and further study their proteome. METHODS: SW1116 cells were isolated and cultured with a serum-free medium (SFM). Sphere formation was assayed to observe the formation of colon cancer stem cell spheres. SW1116 cells were inoculated into a serum-containing medium for observing their differentiation characteristics. Proliferation curve and cross-resistance of SW1116 cells to different drugs were detected by MTT. Percentage of SP cells in SW1116 cells was detected with Hoechst33342 staining. Telomerase activity in SW1116cells was checked by polymerase chain reaction (PCR)-enzyme linked immunosorbent assay. Expressions of stem cell relevant genes and proteins were detected by reverse transcription-PCR and Western blot, respectively. Total protein was isolated from SW1116 cells by two-dimensional gel electrophoresis (2-DE) and differentially expressed proteins were identified by tandem mass spectrometry (MALDI-TOF/TOF). RESULTS: The isolated SW1116 cells presented as spheroid and suspension growths in SFM with a strong self-renewal, proliferation, differentiation and drug-resistance ability. The percentage of SP cells in SW1116 cells was 38.9%. The SW1116 cells co-expressed the CD133 and CD29 proteins. The telomerase activity in SW1116 cells was increased. The expressions of different stem cell relevant genes and proteins were detected. The proteomic analysis showed that the 26 protein spots were differently expressed in SW1116 cells and 10 protein spots were identified as ubiquitin fusiondegradation 1-like protein, nuclear chloride channel protein, tubulin b, Raichu404X, stratifin, F-actin capping protein a-1 subunit, eukaryotic translation elongation factor 1 delta isoform 2, hypothetical protein, glyceraldehyde-3-phosphate dehydrogenase and guanine nucleotide binding protein b polypeptide 2-like 1, respectively. CONCLUSION: SW1116 cells are biologically

  12. MUMAL: Multivariate analysis in shotgun proteomics using machine learning techniques

    Directory of Open Access Journals (Sweden)

    Cerqueira Fabio R

    2012-10-01

    Full Text Available Abstract Background The shotgun strategy (liquid chromatography coupled with tandem mass spectrometry is widely applied for identification of proteins in complex mixtures. This method gives rise to thousands of spectra in a single run, which are interpreted by computational tools. Such tools normally use a protein database from which peptide sequences are extracted for matching with experimentally derived mass spectral data. After the database search, the correctness of obtained peptide-spectrum matches (PSMs needs to be evaluated also by algorithms, as a manual curation of these huge datasets would be impractical. The target-decoy database strategy is largely used to perform spectrum evaluation. Nonetheless, this method has been applied without considering sensitivity, i.e., only error estimation is taken into account. A recently proposed method termed MUDE treats the target-decoy analysis as an optimization problem, where sensitivity is maximized. This method demonstrates a significant increase in the retrieved number of PSMs for a fixed error rate. However, the MUDE model is constructed in such a way that linear decision boundaries are established to separate correct from incorrect PSMs. Besides, the described heuristic for solving the optimization problem has to be executed many times to achieve a significant augmentation in sensitivity. Results Here, we propose a new method, termed MUMAL, for PSM assessment that is based on machine learning techniques. Our method can establish nonlinear decision boundaries, leading to a higher chance to retrieve more true positives. Furthermore, we need few iterations to achieve high sensitivities, strikingly shortening the running time of the whole process. Experiments show that our method achieves a considerably higher number of PSMs compared with standard tools such as MUDE, PeptideProphet, and typical target-decoy approaches. Conclusion Our approach not only enhances the computational performance, and

  13. Chromatin Dynamics During DNA Replication and Uncharacterized Replication Factors determined by Nascent Chromatin Capture (NCC) Proteomics

    Science.gov (United States)

    Alabert, Constance; Bukowski-Wills, Jimi-Carlo; Lee, Sung-Bau; Kustatscher, Georg; Nakamura, Kyosuke; de Lima Alves, Flavia; Menard, Patrice; Mejlvang, Jakob; Rappsilber, Juri; Groth, Anja

    2014-01-01

    SUMMARY To maintain genome function and stability, DNA sequence and its organization into chromatin must be duplicated during cell division. Understanding how entire chromosomes are copied remains a major challenge. Here, we use Nascent Chromatin Capture (NCC) to profile chromatin proteome dynamics during replication in human cells. NCC relies on biotin-dUTP labelling of replicating DNA, affinity-purification and quantitative proteomics. Comparing nascent chromatin with mature post-replicative chromatin, we provide association dynamics for 3995 proteins. The replication machinery and 485 chromatin factors like CAF-1, DNMT1, SUV39h1 are enriched in nascent chromatin, whereas 170 factors including histone H1, DNMT3, MBD1-3 and PRC1 show delayed association. This correlates with H4K5K12diAc removal and H3K9me1 accumulation, while H3K27me3 and H3K9me3 remain unchanged. Finally, we combine NCC enrichment with experimentally derived chromatin probabilities to predict a function in nascent chromatin for 93 uncharacterized proteins and identify FAM111A as a replication factor required for PCNA loading. Together, this provides an extensive resource to understand genome and epigenome maintenance. PMID:24561620

  14. Cell-specific proteomic analysis in Caenorhabditis elegans.

    Science.gov (United States)

    Yuet, Kai P; Doma, Meenakshi K; Ngo, John T; Sweredoski, Michael J; Graham, Robert L J; Moradian, Annie; Hess, Sonja; Schuman, Erin M; Sternberg, Paul W; Tirrell, David A

    2015-03-03

    Proteomic analysis of rare cells in heterogeneous environments presents difficult challenges. Systematic methods are needed to enrich, identify, and quantify proteins expressed in specific cells in complex biological systems including multicellular plants and animals. Here, we have engineered a Caenorhabditis elegans phenylalanyl-tRNA synthetase capable of tagging proteins with the reactive noncanonical amino acid p-azido-L-phenylalanine. We achieved spatiotemporal selectivity in the labeling of C. elegans proteins by controlling expression of the mutant synthetase using cell-selective (body wall muscles, intestinal epithelial cells, neurons, and pharyngeal muscle) or state-selective (heat-shock) promoters in several transgenic lines. Tagged proteins are distinguished from the rest of the protein pool through bioorthogonal conjugation of the azide side chain to probes that permit visualization and isolation of labeled proteins. By coupling our methodology with stable-isotope labeling of amino acids in cell culture (SILAC), we successfully profiled proteins expressed in pharyngeal muscle cells, and in the process, identified proteins not previously known to be expressed in these cells. Our results show that tagging proteins with spatiotemporal selectivity can be achieved in C. elegans and illustrate a convenient and effective approach for unbiased discovery of proteins expressed in targeted subsets of cells.

  15. Proteome Analysis of Rice Root Proteins Regulated by Gibberellin

    Institute of Scientific and Technical Information of China (English)

    Setsuko Komatsu; Hirosato Konishi

    2005-01-01

    To gain an enhanced understanding of the mechanism by which gibberellins (GAs) regulate the growth and development of plants, it is necessary to identify proteins regulated by GA. Proteome analysis techniques have been applied as a direct,effective, and reliable tool in differential protein expressions. In previous studies,sixteen proteins showed differences in accumulation levels as a result of treatment with GA3, uniconazole, or abscisic acid (ABA), and/or the differences between the GA-deficient semi-dwarf mutant, Tan-ginbozu, and normal cultivars. Among these proteins, aldolase increased in roots treated with GA3, was present at low levels in Tan-ginbozu roots, and decreased in roots treated with uniconazole or ABA. In a root elongation assay, the growth of aldolase-antisense transgenic rice was half of that of vector control transgenic rice. These results indicate that increases in aldolase activity stimulate the glycolytic pathway and may play an important role in the GA-induced growth of roots. In this review, we discuss the relationship among GA, aldolase, and root growth.

  16. Proteomic analysis of the copper resistance of Streptococcus pneumoniae.

    Science.gov (United States)

    Guo, Zhong; Han, Junlong; Yang, Xiao-Yan; Cao, Kun; He, Ke; Du, Gaofei; Zeng, Guandi; Zhang, Liang; Yu, Guangchuang; Sun, Zhenghua; He, Qing-Yu; Sun, Xuesong

    2015-03-01

    Streptococcus pneumoniae is a Gram-positive bacterial pathogen causing a variety of diseases, including otitis media, bacteraemia and meningitis. Although copper is an essential trace metal for bacterial growth, high intracellular levels of free-copper are toxic. Copper resistance has emerged as an important virulence determinant of microbial pathogens. In this study, we determined the minimum inhibition concentration of copper for the growth inhibition of S. pneumoniae. Two-dimensional-electrophoresis coupled with mass spectrometry was applied to identify proteins involved in copper resistance of S. pneumoniae. In total, forty-four proteins with more than 1.5-fold alteration in expression (p < 0.05) were identified. Quantitative reverse transcription PCR was used to confirm the proteomic results. Bioinformatics analysis showed that the differentially expressed proteins were mainly involved in the cell wall biosynthesis, protein biosynthesis, purine biosynthesis, pyrimidine biosynthesis, primary metabolic process, and the nitrogen compound metabolic process. Many up-regulated proteins in response to the copper treatment directly or indirectly participated in the cell wall biosynthesis, indicating that the cell wall is a critical determinant in copper resistance of S. pneumoniae.

  17. Proteomic analysis of early seed development in Pinus massoniana L.

    Science.gov (United States)

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

    2012-05-01

    Understanding seed development is important for large-scale propagation and germplasm conservation for the Masson pine. We undertook a proteomic analysis of Masson pine seeds during the early stages of embryogenesis. Two-dimensional difference gel electrophoresis (2D DIGE) was used to quantify the differences in protein expression during early seed development. Using electrospray ionization mass spectrometry/mass spectrometry, we identified proteins from 43 gel spots that had been excised from preparative "pick" gels. Proteins involved in carbon metabolism were identified and were predominantly expressed at higher levels during the cleavage polyembryony and columnar embryo stages. Functional annotation of one seed protein revealed it involvement in programmed cell death and translation of selective mRNAs, which may play an important role in subordinate embryo elimination and suspensor degeneration in polyembryonic seed gymnosperms. Other identified proteins were associated with protein folding, nitrogen metabolism, disease/defense response, and protein storage, synthesis and stabilization. The comprehensive protein expression profiles generated by this study will provide new insights into the complex developmental process of seed development in Masson pine.

  18. Proteomic analysis of zebrafish embryos exposed to simulated-microgravity

    Science.gov (United States)

    Hang, Xiaoming; Ma, Wenwen; Wang, Wei; Liu, Cong; Sun, Yeqing

    Microgravity can induce a serial of physiological and pathological changes in human body, such as cardiovascular functional disorder, bone loss, muscular atrophy and impaired immune system function, etc. In this research, we focus on the influence of microgravity to vertebrate embryo development. As a powerful model for studying vertebrate development, zebrafish embryos at 8 hpf (hour past fertilization) and 24 hpf were placed into a NASA developed bioreac-tor (RCCS) to simulate microgravity for 64 and 48 hours, respectively. The same number of control embryos from the same parents were placed in a tissue culture dish at the same temper-ature of 28° C. Each experiment was repeated 3 times and analyzed by two-dimensional (2-D) gel electrophoresis. Image analysis of silver stained 2-D gels revealed that 64 from total 292 protein spots showed quantitative and qualitative variations that were significantly (Pprotein spots with significant expression alteration (Pproteins, 3 down-regulated proteins were identified as bectin 2, centrosomal protein of 135kDa and tropomyosin 4, while the up-regulated protein was identified as creatine kinase muscle B. Other protein spots showed significant expression alteration will be identified successively and the corresponding genes expression will also be measured by Q-PCR method at different development stages. The data presented in this study illustrate that zebrafish embryo can be significantly induced by microgravity on the expression of proteins involved in bone and muscle formation. Key Words: Danio rerio; Simulated-microgravity; Proteomics

  19. Proteomic Analysis the Seed Development of Jatropha curcas

    Institute of Scientific and Technical Information of China (English)

    Hui Liu; Setsuko Komatsu; Qing Yang; Shihua Shen

    2012-01-01

    Differential proteomic analysis and cellular structure observation were employed to analyze the developing seeds at 5,10,15,20,25 and 30 days after flowering.Results revealed that 214 of 355 protein spots with significant changes in abundance were identified through MALDI/TOF-TOF MS.Energy and metabolism related proteins were notable abundant in developing Jatropha curcas seed,accounting for 47% and 36% of the identified differentially expressed proteins respectively.The expressional profiles of energy and metabolic proteins identified in this study ensured a comprehensive overview on the carbon flux to lipid accumulation from the early to late stages of seed development.It seems that glycolysis and OPP are the major pathways for producing carbon flux in developing J.curcas seeds.These data suggested that sugar mobilization from glucose to coenzyme A and its acyl derivative is collaboration between the cytosol and plastids and that temporal control of enzymes and pathways extends beyond transcription.

  20. Proteomics analysis of egg white proteins from different egg varieties.

    Science.gov (United States)

    Wang, Jiapei; Liang, Yue; Omana, Dileep A; Kav, Nat N V; Wu, Jianping

    2012-01-11

    The market of specialty eggs, such as omega-3-enriched eggs, organic eggs, and free-range eggs, is continuously growing. The nutritional composition of egg yolk can be manipulated by feed diet; however, it is not known if there is any difference in the composition of egg white proteins among different egg varieties. The purpose of the study was to compare the egg white proteins among six different egg varieties using proteomics analysis. Egg white proteins were analyzed using two-dimensional gel electrophoresis (2-DE), and 89 protein spots were subjected to LC-MS/MS. A total of 23 proteins, belonging to Gallus gallus , were identified from 72 detected protein spots. A quiescence-specific protein precursor in egg white was identified for the first time in this study. Significant differences in the abundant levels of 19 proteins (from 65 protein spots) were observed among six egg varieties. Four proteins, ovalbumin-related protein Y, cystatin, avidin, and albumin precursor, were not different among these six egg varieties. These findings suggest that the abundance, but not the composition, of egg white proteins varied among the egg varieties.

  1. Proteomic analysis of glycosylphosphatidylinositol-anchored membrane proteins.

    Science.gov (United States)

    Elortza, Felix; Nühse, Thomas S; Foster, Leonard J; Stensballe, Allan; Peck, Scott C; Jensen, Ole N

    2003-12-01

    Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are a functionally and structurally diverse family of post-translationally modified membrane proteins found mostly in the outer leaflet of the plasma membrane in a variety of eukaryotic cells. Although the general role of GPI-APs remains unclear, they have attracted attention because they act as enzymes and receptors in cell adhesion, differentiation, and host-pathogen interactions. GPI-APs may represent potential diagnostic and therapeutic targets in humans and are interesting in plant biotechnology because of their key role in root development. We here present a general mass spectrometry-based proteomic "shave-and-conquer" strategy that specifically targets GPI-APs. Using a combination of biochemical methods, mass spectrometry, and computational sequence analysis we identified six GPI-APs in a Homo sapiens lipid raft-enriched fraction and 44 GPI-APs in an Arabidopsis thaliana membrane preparation, representing the largest experimental dataset of GPI-anchored proteins to date.

  2. Comparative Proteomic Analysis of Rice Shoots Exposed to High Arsenate

    Institute of Scientific and Technical Information of China (English)

    Yanli Liu; Ming Li; Chao Han; Fengxia Wu; Bingkun Tu; Pingfang Yang

    2013-01-01

    Consumption of arsenic contaminated water and cereals is a serious threat to humans all over the world. Rice (Oryza sativa“Nipponbare”), as a main cereal crop, can accumulate arsenic more than 10-fold that of in other cereals. To gain a comprehensive understanding of the response of rice subjected to 100 mM arsenate stress, a comparative proteomic analysis of rice shoots in combination with morphological and biochemical investigations have been performed in this study. The results demonstrated that arsenate suppressed the growth of rice seedlings, destroyed the cellular ultra-structure and changed the homeostasis of reactive oxygen species. Moreover, a total of 38 differentially displayed proteins, which were mainly involved in metabolism, redox and protein-metabolism, were identified. The data suggest the arsenic can inhibit rice growth through negatively affecting chloroplast structure and photosynthesis. In addition, upregulation of the proteins involved in redox and protein metabolism might help the rice to be resistant or tolerant to arsenic toxicity. In general, this study improves our understanding about the rice arsenic responsive mechanism.

  3. Proteomics Analysis for Finding Serum Markers of Ovarian Cancer

    Directory of Open Access Journals (Sweden)

    Yushan Cheng

    2014-01-01

    Full Text Available A combination of peptide ligand library beads (PLLB and 1D gel liquid chromatography-mass spectrometry/mass spectrometry (1DGel-LC-MS/MS was employed to analyze serum samples from patients with ovarian cancer and from healthy controls. Proteomic analysis identified 1200 serum proteins, among which 57 proteins were upregulated and 10 were downregulated in the sera from cancer patients. Retinol binding protein 4 (RBP4 is highly upregulated in the ovarian cancer serum samples. ELISA was employed to measure plasma concentrations of RBP4 in 80 samples from ovarian cancer patients, healthy individuals, myoma patients, and patients with benign ovarian tumor, respectively. The plasma concentrations of RBP4 ranging from 76.91 to 120.08 ng/mL with the mean value 89.13±1.67 ng/mL in ovarian cancer patients are significantly higher than those in healthy individuals (10.85±2.38 ng/mL. Results were further confirmed with immunohistochemistry, demonstrating that RBP4 expression levels in normal ovarian tissue were lower than those in ovarian cancer tissues. Our results suggested that RBP4 is a potential biomarker for diagnostic of screening ovarian cancer.

  4. Dissection of human vitreous body elements for proteomic analysis.

    Science.gov (United States)

    Skeie, Jessica M; Mahajan, Vinit B

    2011-01-23

    The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. (1,2) Abnormal interactions between vitreous substructures and the retina underlie several vitreoretinal diseases, including retinal tear and detachment, macular pucker, macular hole, age-related macular degeneration, vitreomacular traction, proliferative vitreoretinopathy, proliferative diabetic retinopathy, and inherited vitreoretinopathies. (1,2) The molecular composition of the vitreous substructures is not known. Since the vitreous body is transparent with limited surgical access, it has been difficult to study its substructures at the molecular level. We developed a method to separate and preserve these tissues for proteomic and biochemical analysis. The dissection technique in this experimental video shows how to isolate vitreous base, anterior hyaloid, vitreous core, and vitreous cortex from postmortem human eyes. One-dimensional SDS-PAGE analyses of each vitreous component showed that our dissection technique resulted in four unique protein profiles corresponding to each substructure of the human vitreous body. Identification of differentially compartmentalized proteins will reveal candidate molecules underlying various vitreoretinal diseases.

  5. Sodium loading changes urinary protein excretion: a proteomic analysis.

    Science.gov (United States)

    Thongboonkerd, Visith; Klein, Jon B; Pierce, William M; Jevans, Anthony W; Arthur, John M

    2003-06-01

    Plasma sodium concentration is maintained even when sodium intake is altered. Sodium homeostasis may involve changes in renal tubular protein expression that are reflected in the urine. We used proteomic analysis to investigate changes in urinary protein excretion in response to acute sodium loading. Rats were given deionized water followed by hypertonic (2.7%) saline for 28 h each. Urinary protein expression was determined during the final 4 h of each treatment. Acute sodium loading increased urinary sodium excretion (4.53 +/- 1.74 vs. 1.70 +/- 0.27 mmol/day, P = 0.029). Urinary proteins were separated by two-dimensional PAGE and visualized by Sypro ruby staining. Differentially expressed proteins were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry followed by peptide mass fingerprinting. The abundance of a total of 45 protein components was changed after acute sodium loading. Neutral endopeptidase, solute carrier family 3, meprin 1alpha, diphor-1, chaperone heat shock protein 72, vacuolar H(+)-ATPase, ezrin, ezrin/radixin/moesin-binding protein, glutamine synthetase, guanine nucleotide-binding protein, Rho GDI-1, and chloride intracellular channel protein 1 were decreased, whereas albumin and alpha-2u globulin were increased. Some of these proteins have previously been shown to be associated with tubular transport. These data indicate that alterations in the excretion of several urinary proteins occur during acute sodium loading.

  6. Changes in cod muscle proteins during frozen storage revealed by proteome analysis and multivariate data analysis

    DEFF Research Database (Denmark)

    Kjærsgård, Inger Vibeke Holst; Nørrelykke, M.R.; Jessen, Flemming

    2006-01-01

    Multivariate data analysis has been combined with proteomics to enhance the recovery of information from 2-DE of cod muscle proteins during different storage conditions. Proteins were extracted according to 11 different storage conditions and samples were resolved by 2-DE. Data generated by 2-DE...... was subjected to principal component analysis (PCA) and discriminant partial least squares regression (DPLSR). Applying PCA to 2-DE data revealed the samples to form groups according to frozen storage time, whereas differences due to different storage temperatures or chilled storage in modified atmosphere...... light chain 1, 2 and 3, triose-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, aldolase A and two ?-actin fragments, and a nuclease diphosphate kinase B fragment to change in concentration, during frozen storage. Application of proteomics, multivariate data analysis and MS/MS to analyse...

  7. Proteomic analysis of tyrosine phosphorylation during human liver transplantation

    Directory of Open Access Journals (Sweden)

    Boutros Tarek

    2007-01-01

    Full Text Available Abstract Background Ischemia-reperfusion (I/R causes a dramatic reprogramming of cell metabolism during liver transplantation and can be linked to an alteration of the phosphorylation level of several cellular proteins. Over the past two decades, it became clear that tyrosine phosphorylation plays a pivotal role in a variety of important signalling pathways and was linked to a wide spectrum of diseases. Functional profiling of the tyrosine phosphoproteome during liver transplantation is therefore of great biological significance and is likely to lead to the identification of novel targets for drug discovery and provide a basis for novel therapeutic strategies. Results Using liver biopsies collected during the early phases of organ procurement and transplantation, we aimed at characterizing the global patterns of tyrosine phosphorylation during hepatic I/R. A proteomic approach, based on the purification of tyrosine phosphorylated proteins followed by their identification using mass spectrometry, allowed us to identify Nck-1, a SH2/SH3 adaptor, as a potential regulator of I/R injury. Using immunoblot, cell fractionation and immunohistochemistry, we demonstrate that Nck-1 phosphorylation, expression and localization were affected in liver tissue upon I/R. In addition, mass spectrometry identification of Nck-1 binding partners during the course of the transplantation also suggested a dynamic interaction between Nck-1 and actin during I/R. Conclusion Taken together, our data suggest that Nck-1 may play a role in I/R-induced actin reorganization, which was previously reported to be detrimental for the hepatocytes of the transplanted graft. Nck-1 could therefore represent a target of choice for the design of new organ preservation strategies, which could consequently help to reduce post-reperfusion liver damages and improve transplantation outcomes.

  8. Novel small protein identification and quantitative proteomic analysis in Pseudomonas putida KT-­2440

    DEFF Research Database (Denmark)

    Yang, Xiaochen

    .This thesis investigated an industrial bacterium, Pseudomonas putida KT-2440, in two aspects. First, the research focused on discovering novel small proteins (s-proteins) in the bacterium. With large-scale approaches for gene identification, groups of novel s-proteins were identified and validated from...... the genome, transcriptome and proteome of the bacterium. The application of new research approach, ribosome profiling, enabled us to analysis novel open reading frames (ORFs) from different standpoint. Second, by quantitative proteomic approach, the differential expressions of genes were analyzed at proteome...... level under different environmental conditions. The results yield insights intothe adaptation of P. putida KT-2440 in different environments.Based on bioinformatic, proteomic and transcriptomic approaches, global gene expression was analyzed on both transcriptional and translational levels. Our research...

  9. Proteomic analysis of mitochondria: biological and clinical progresses in cancer.

    Science.gov (United States)

    Wang, Yang; Zhang, Jing; Li, Bin; He, Qing-Yu

    2017-10-01

    Mitochondria play important roles in regulating multiple biological processes and signalling pathways in eukaryotic cells, and mitochondrial dysfunction may result in a wide range of serious diseases, including cancer. With improvements in the identification of mitochondrial proteins, mitochondrial proteomics has made great achievements. In particular, this approach has been widely used to compare tumour cells at different stages of malignancy. Therefore, there is an urgent need to identify and characterize the function of mitochondrial proteins in cancer progression and to determine the involved mechanisms. Areas covered: We provide an overview of recent progress related to mitochondrial proteomics in cancer and the application of comparative mitochondrial proteomics in various biological processes, including apoptosis, necroptosis, autophagy and metastasis, as well as clinical progress in cancer. Proteomics-related reports were found using PubMed and Google Scholar databases. Expert commentary: Understanding both post-translational modification and post-translational processing is important in the comprehensive characterization of protein function. The application of comparative mitochondrial proteomics to investigate clinical samples and cancer cells will contribute to our understanding of the molecular interplay of mitochondrial proteins in the development of cancer. This approach will mine more biomarkers for diagnosis and prognosis and improve therapeutic outcomes among cancer patients.

  10. Redox proteomic analysis of the gastrocnemius muscle from adult and old mice

    Directory of Open Access Journals (Sweden)

    Brian McDonagh

    2015-09-01

    Full Text Available The data provides information in support of the research article, “Differential Cysteine Labeling and Global Label-Free Proteomics Reveals an Altered Metabolic State in Skeletal Muscle Aging”, Journal of Proteome Research, 2014, 13 (11, 2008–21 [1]. Raw data is available from ProteomeXchange [2] with identifier PDX001054. The proteome of gastrocnemius muscle from adult and old mice was analyzed by global label-free proteomics and the relative quantification of specific reduced and reversibly oxidized Cysteine (Cys residues was performed using Skyline [3]. Briefly, reduced Cysteine (Cys containing peptides was alkylated using N-ethylmalemide (d0-NEM. Samples were desalted and reversibly oxidized Cys residues were reduced using tris(2-carboxyethylphosphine (TCEP and the newly formed reduced Cys residues were labeled with heavy NEM( d5-NEM. Label-free analysis of the global proteome of adult (n=5 and old (n=4 gastrocnemius muscles was performed using Peaks7™ mass spectrometry data analysis software [4]. Relative quantification of Cys containing peptides that were identified as reduced (d(0 NEM labeled and reversibly oxidized d(5–NEM labeled was performed using the intensity of their precursor ions in Skyline. Results indicate that muscles from old mice show reduced redox flexibility particularly in proteins involved in the generation of precursor metabolites and energy metabolism, indicating a loss in the flexibility of the redox energy response.

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

    Energy Technology Data Exchange (ETDEWEB)

    Clair, Geremy; Piehowski, Paul D.; Nicola, Teodora; Kitzmiller, Joseph A.; Huang, Eric L.; Zink, Erika M.; Sontag, Ryan L.; Orton, Daniel J.; Moore, Ronald J.; Carson, James P.; Smith, Richard D.; Whitsett, Jeffrey A.; Corley, Richard A.; Ambalavanan, Namasivayam; Ansong, Charles

    2016-12-22

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

  12. Transcriptome and proteome analysis of Pinctada margaritifera calcifying mantle and shell: focus on biomineralization

    Directory of Open Access Journals (Sweden)

    Gueguen Yannick

    2010-11-01

    Full Text Available Abstract Background The shell of the pearl-producing bivalve Pinctada margaritifera is composed of an organic cell-free matrix that plays a key role in the dynamic process of biologically-controlled biomineralization. In order to increase genomic resources and identify shell matrix proteins implicated in biomineralization in P. margaritifera, high-throughput Expressed Sequence Tag (EST pyrosequencing was undertaken on the calcifying mantle, combined with a proteomic analysis of the shell. Results We report the functional analysis of 276 738 sequences, leading to the constitution of an unprecedented catalog of 82 P. margaritifera biomineralization-related mantle protein sequences. Components of the current "chitin-silk fibroin gel-acidic macromolecule" model of biomineralization processes were found, in particular a homolog of a biomineralization protein (Pif-177 recently discovered in P. fucata. Among these sequences, we could show the localization of two other biomineralization protein transcripts, pmarg-aspein and pmarg-pearlin, in two distinct areas of the outer mantle epithelium, suggesting their implication in calcite and aragonite formation. Finally, by combining the EST approach with a proteomic mass spectrometry analysis of proteins isolated from the P. margaritifera shell organic matrix, we demonstrated the presence of 30 sequences containing almost all of the shell proteins that have been previously described from shell matrix protein analyses of the Pinctada genus. The integration of these two methods allowed the global composition of biomineralizing tissue and calcified structures to be examined in tandem for the first time. Conclusions This EST study made on the calcifying tissue of P. margaritifera is the first description of pyrosequencing on a pearl-producing bivalve species. Our results provide direct evidence that our EST data set covers most of the diversity of the matrix protein of P. margaritifera shell, but also that the

  13. Transcriptome and proteome analysis of Pinctada margaritifera calcifying mantle and shell: focus on biomineralization.

    Science.gov (United States)

    Joubert, Caroline; Piquemal, David; Marie, Benjamin; Manchon, Laurent; Pierrat, Fabien; Zanella-Cléon, Isabelle; Cochennec-Laureau, Nathalie; Gueguen, Yannick; Montagnani, Caroline

    2010-11-01

    The shell of the pearl-producing bivalve Pinctada margaritifera is composed of an organic cell-free matrix that plays a key role in the dynamic process of biologically-controlled biomineralization. In order to increase genomic resources and identify shell matrix proteins implicated in biomineralization in P. margaritifera, high-throughput Expressed Sequence Tag (EST) pyrosequencing was undertaken on the calcifying mantle, combined with a proteomic analysis of the shell. We report the functional analysis of 276 738 sequences, leading to the constitution of an unprecedented catalog of 82 P. margaritifera biomineralization-related mantle protein sequences. Components of the current "chitin-silk fibroin gel-acidic macromolecule" model of biomineralization processes were found, in particular a homolog of a biomineralization protein (Pif-177) recently discovered in P. fucata. Among these sequences, we could show the localization of two other biomineralization protein transcripts, pmarg-aspein and pmarg-pearlin, in two distinct areas of the outer mantle epithelium, suggesting their implication in calcite and aragonite formation. Finally, by combining the EST approach with a proteomic mass spectrometry analysis of proteins isolated from the P. margaritifera shell organic matrix, we demonstrated the presence of 30 sequences containing almost all of the shell proteins that have been previously described from shell matrix protein analyses of the Pinctada genus. The integration of these two methods allowed the global composition of biomineralizing tissue and calcified structures to be examined in tandem for the first time. This EST study made on the calcifying tissue of P. margaritifera is the first description of pyrosequencing on a pearl-producing bivalve species. Our results provide direct evidence that our EST data set covers most of the diversity of the matrix protein of P. margaritifera shell, but also that the mantle transcripts encode proteins present in P

  14. Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics

    DEFF Research Database (Denmark)

    Blagoev, Blagoy; Ong, S.E.; Kratchmarova, Irina

    2004-01-01

    To study the global dynamics of phosphotyrosine-based signaling events in early growth factor stimulation, we developed a mass spectrometric method that converts temporal changes to differences in peptide isotopic abundance. The proteomes of three cell populations were metabolically encoded with ...

  15. Proteomics analysis of alfalfa response to heat stress.

    Directory of Open Access Journals (Sweden)

    Weimin Li

    Full Text Available The proteome responses to heat stress have not been well understood. In this study, alfalfa (Medicago sativa L. cv. Huaiyin seedlings were exposed to 25 °C (control and 40 °C (heat stress in growth chambers, and leaves were collected at 24, 48 and 72 h after treatment, respectively. The morphological, physiological and proteomic processes were negatively affected under heat stress. Proteins were extracted and separated by two-dimensional polyacrylamide gel electrophoresis (2-DE, and differentially expressed protein spots were identified by mass spectrometry (MS. Totally, 81 differentially expressed proteins were identified successfully by MALDI-TOF/TOF. These proteins were categorized into nine classes: including metabolism, energy, protein synthesis, protein destination/storage, transporters, intracellular traffic, cell structure, signal transduction and disease/defence. Five proteins were further analyzed for mRNA levels. The results of the proteomics analyses provide a better understanding of the molecular basis of heat-stress responses in alfalfa.

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

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    Panga Jaipal Reddy

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

  17. Computational Proteomics: High-throughput Analysis for Systems Biology

    Energy Technology Data Exchange (ETDEWEB)

    Cannon, William R.; Webb-Robertson, Bobbie-Jo M.

    2007-01-03

    High-throughput (HTP) proteomics is a rapidly developing field that offers the global profiling of proteins from a biological system. The HTP technological advances are fueling a revolution in biology, enabling analyses at the scales of entire systems (e.g., whole cells, tumors, or environmental communities). However, simply identifying the proteins in a cell is insufficient for understanding the underlying complexity and operating mechanisms of the overall system. Systems level investigations are relying more and more on computational analyses, especially in the field of proteomics generating large-scale global data.

  18. Dynamic Acclimation to High Light in Arabidopsis thaliana Involves Widespread Reengineering of the Leaf Proteome

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    Matthew A. E. Miller

    2017-07-01

    Full Text Available Leaves of Arabidopsis thaliana transferred from low to high light increase their capacity for photosynthesis, a process of dynamic acclimation. A mutant, gpt2, lacking a chloroplast glucose-6-phosphate/phosphate translocator, is deficient in its ability to acclimate to increased light. Here, we have used a label-free proteomics approach, to perform relative quantitation of 1993 proteins from Arabidopsis wild type and gpt2 leaves exposed to increased light. Data are available via ProteomeXchange with identifier PXD006598. Acclimation to light is shown to involve increases in electron transport and carbon metabolism but no change in the abundance of photosynthetic reaction centers. The gpt2 mutant shows a similar increase in total protein content to wild type but differences in the extent of change of certain proteins, including in the relative abundance of the cytochrome b6f complex and plastocyanin, the thylakoid ATPase and selected Benson-Calvin cycle enzymes. Changes in leaf metabolite content as plants acclimate can be explained by changes in the abundance of enzymes involved in metabolism, which were reduced in gpt2 in some cases. Plants of gpt2 invest more in stress-related proteins, suggesting that their reduced ability to acclimate photosynthetic capacity results in increased stress.

  19. Dynamics in Epistasis Analysis.

    Science.gov (United States)

    Awdeh, Aseel; Phenix, Hilary; Kaern, Mads; Perkins, Theodore

    2017-01-16

    Finding regulatory relationships between genes, including the direction and nature of influence between them, is a fundamental challenge in the field of molecular genetics. One classical approach to this problem is epistasis analysis. Broadly speaking, epistasis analysis infers the regulatory relationships between a pair of genes in a genetic pathway by considering the patterns of change in an observable trait resulting from single and double deletion of genes. While classical epistasis analysis has yielded deep insights on numerous genetic pathways, it is not without limitations. Here, we explore the possibility of dynamic epistasis analysis, in which, in addition to performing genetic perturbations of a pathway, we drive the pathway by a time-varying upstream signal. We explore the theoretical power of dynamical epistasis analysis by conducting an identifiability analysis of Boolean models of genetic pathways, comparing static and dynamic approaches. We find that even relatively simple input dynamics greatly increases the power of epistasis analysis to discriminate alternative network structures. Further, we explore the question of experiment design, and show that a subset of short time-varying signals, which we call dynamic primitives, allow maximum discriminative power with a reduced number of experiments.

  20. MCAM: multiple clustering analysis methodology for deriving hypotheses and insights from high-throughput proteomic datasets.

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    Kristen M Naegle

    2011-07-01

    Full Text Available Advances in proteomic technologies continue to substantially accelerate capability for generating experimental data on protein levels, states, and activities in biological samples. For example, studies on receptor tyrosine kinase signaling networks can now capture the phosphorylation state of hundreds to thousands of proteins across multiple conditions. However, little is known about the function of many of these protein modifications, or the enzymes responsible for modifying them. To address this challenge, we have developed an approach that enhances the power of clustering techniques to infer functional and regulatory meaning of protein states in cell signaling networks. We have created a new computational framework for applying clustering to biological data in order to overcome the typical dependence on specific a priori assumptions and expert knowledge concerning the technical aspects of clustering. Multiple clustering analysis methodology ('MCAM' employs an array of diverse data transformations, distance metrics, set sizes, and clustering algorithms, in a combinatorial fashion, to create a suite of clustering sets. These sets are then evaluated based on their ability to produce biological insights through statistical enrichment of metadata relating to knowledge concerning protein functions, kinase substrates, and sequence motifs. We applied MCAM to a set of dynamic phosphorylation measurements of the ERRB network to explore the relationships between algorithmic parameters and the biological meaning that could be inferred and report on interesting biological predictions. Further, we applied MCAM to multiple phosphoproteomic datasets for the ERBB network, which allowed us to compare independent and incomplete overlapping measurements of phosphorylation sites in the network. We report specific and global differences of the ERBB network stimulated with different ligands and with changes in HER2 expression. Overall, we offer MCAM as a broadly

  1. MCAM: Multiple Clustering Analysis Methodology for Deriving Hypotheses and Insights from High-Throughput Proteomic Datasets

    Science.gov (United States)

    Naegle, Kristen M.; Welsch, Roy E.; Yaffe, Michael B.; White, Forest M.; Lauffenburger, Douglas A.

    2011-01-01

    Advances in proteomic technologies continue to substantially accelerate capability for generating experimental data on protein levels, states, and activities in biological samples. For example, studies on receptor tyrosine kinase signaling networks can now capture the phosphorylation state of hundreds to thousands of proteins across multiple conditions. However, little is known about the function of many of these protein modifications, or the enzymes responsible for modifying them. To address this challenge, we have developed an approach that enhances the power of clustering techniques to infer functional and regulatory meaning of protein states in cell signaling networks. We have created a new computational framework for applying clustering to biological data in order to overcome the typical dependence on specific a priori assumptions and expert knowledge concerning the technical aspects of clustering. Multiple clustering analysis methodology (‘MCAM’) employs an array of diverse data transformations, distance metrics, set sizes, and clustering algorithms, in a combinatorial fashion, to create a suite of clustering sets. These sets are then evaluated based on their ability to produce biological insights through statistical enrichment of metadata relating to knowledge concerning protein functions, kinase substrates, and sequence motifs. We applied MCAM to a set of dynamic phosphorylation measurements of the ERRB network to explore the relationships between algorithmic parameters and the biological meaning that could be inferred and report on interesting biological predictions. Further, we applied MCAM to multiple phosphoproteomic datasets for the ERBB network, which allowed us to compare independent and incomplete overlapping measurements of phosphorylation sites in the network. We report specific and global differences of the ERBB network stimulated with different ligands and with changes in HER2 expression. Overall, we offer MCAM as a broadly

  2. Molecular classification and survival prediction in human gliomas based on proteome analysis.

    Science.gov (United States)

    Iwadate, Yasuo; Sakaida, Tsukasa; Hiwasa, Takaki; Nagai, Yuichiro; Ishikura, Hiroshi; Takiguchi, Masaki; Yamaura, Akira

    2004-04-01

    The biological features of gliomas, which are characterized by highly heterogeneous biological aggressiveness even in the same histological category, would be precisely described by global gene expression data at the protein level. We investigated whether proteome analysis based on two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry can identify differences in protein expression between high- and low-grade glioma tissues. Proteome profiling patterns were compared in 85 tissue samples: 52 glioblastoma multiforme, 13 anaplastic astrocytomas, 10 atrocytomas, and 10 normal brain tissues. We could completely distinguish the normal brain tissues from glioma tissues by cluster analysis based on the proteome profiling patterns. Proteome-based clustering significantly correlated with the patient survival, and we could identify a biologically distinct subset of astrocytomas with aggressive nature. Discriminant analysis extracted a set of 37 proteins differentially expressed based on histological grading. Among them, many of the proteins that were increased in high-grade gliomas were categorized as signal transduction proteins, including small G-proteins. Immunohistochemical analysis confirmed the expression of identified proteins in glioma tissues. The present study shows that proteome analysis is useful to develop a novel system for the prediction of biological aggressiveness of gliomas. The proteins identified here could be novel biomarkers for survival prediction and rational targets for antiglioma therapy.

  3. Proteomic Analysis of Strawberry Leaves Infected with Colletotrichum fragariae

    Institute of Scientific and Technical Information of China (English)

    Xianping Fang; Huasheng Ma; Songlin Raun

    2012-01-01

    Understanding the defense mechanisms used by anthracnose-resistant strawberries against Colletotrichum infection is important for breeding purposes.To characterize cell responses to Colletotrichum infection,proteomes from strawberry seedling leaves that had or had not been infected with Colletotrichum fragariae were characterized at different time points post infection by 2-DE and by MALDI-TOF/TOF MS/MS and database-searching protein identification.Mass spectrometry identified 49 differentially expressed proteins with significant intensity differences (> 1.5-fold,p <0.05) in mock-and C.fragariae-infected leaves at least at one time point.Notably,2-DE analysis revealed that C.fragariae infection increased the expression of well-known and novel pathogen-responsive proteins whose expression patterns tended to correlate with physiological changes in the leaves.Quantitative real-time PCR was used to examine the transcriptional profiles of infected and uninfected strawberry leaves,and western blotting confirmed the induction of β-1,3-glucanase and a lowmolecular-weight heat shock protein in response to C.fragariae infection.During the late phase of infection,proteins involved in the Calvin cycle and glycolysis pathway had suppressed expression.The abundance changes,putative functions,and participation in physiological reactions for the identified proteins produce a pathogenresponsive protein network in C.fragariae-infected strawberry leaves.Together,these findings increase our knowledge of pathogen resistance mechanisms,especially those found in non-model plant species.

  4. Proteomic analysis of regenerating mouse liver following 50% partial hepatectomy

    Directory of Open Access Journals (Sweden)

    Pan Xiaoping

    2009-12-01

    Full Text Available Abstract Background Although 70% (or 2/3 partial hepatectomy (PH is the most studied model for liver regeneration, the hepatic protein expression profile associated with lower volume liver resection (such as 50% PH has not yet been reported. Therefore, the aim of this study was to determine the global protein expression profile of the regenerating mouse liver following 50% PH by differential proteomics, and thereby gaining some insights into the hepatic regeneration mechanism(s under this milder but clinically more relevant condition. Results Proteins from sham-operated mouse livers and livers regenerating for 24 h after 50% PH were separated by SDS-PAGE and analyzed by nanoUPLC-Q-Tof mass spectrometry. Compared to sham-operated group, there were totally 87 differentially expressed proteins (with 50 up-regulated and 37 down-regulated ones identified in the regenerating mouse livers, most of which have not been previously related to liver regeneration. Remarkably, over 25 differentially expressed proteins were located at mitochondria. Several of the mitochondria-resident proteins which play important roles in citric acid cycle, oxidative phosphorylation and ATP production were found to be down-regulated, consistent with the recently-proposed model in which the reduction of ATP content in the remnant liver gives rise to early stress signals that contribute to the onset of liver regeneration. Pathway analysis revealed a central role of c-Myc in the regulation of liver regeneration. Conclusions Our study provides novel evidence for mitochondria as a pivotal organelle that is connected to liver regeneration, and lays the foundation for further studies on key factors and pathways involved in liver regeneration following 50% PH, a condition frequently used for partial liver transplantation and conservative liver resection.

  5. Proteomic analysis of protein profiles in some pathological stages of the human organism

    Directory of Open Access Journals (Sweden)

    Barbara Kossowska

    2009-11-01

    Full Text Available Two-dimensional gel electrophoresis (2-DE is a widely used method for seperation of the proteins of a proteome and it enables their detection in a large concentration range. Sample preparation for isoelectric focusing and SDS-PAGE electrophoresis as well as spot visualization determines the quality of the obtained protein maps. Computer analysis of the proteome maps allows comparison and detection differences in protein profiles. In combination with mass spectrometry (MS it enables the identification of a single protein. Low-abundance proteins of physiological body fluids are considered as the potential source of diagnostic biomarkers. These are obtained by such techniques as affinity chromatography, immunoaffinity, and ultrafiltration. A combination of proteomic and metabonomic analysis provides a collection of new markers which are helpful in modern medical diagnostics.The combination of the 2-DE technique and 1H MRS enables monitoring mild cognitive impairment (MCI and the evolution of Alzheimer disease (AD. Proteome analysis of the liver and red blood cells of patients with diagnosed schizophrenia indicates the importance of analyzing external tissue, not only cerebrospinal fluid, in the diagnosis of this disease. Proteomic techniques enable the identification of new biomarkers in rheumatic disease by analyzing plasma, articular fluid and tissues. New protein biomarkers (in plasma, serum, pancreatic juice, urine enable earlier cancer diagnosis and disease monitoring. Proteome analysis of maternal serum and amniotic fluid creates the possibility detection of protein markers in prenatal tests diagnosing Down’s syndrome. Proteomic studies enable assessment of the influence of environmental contamination on the immunological system.

  6. Transcriptomic and Proteomic Analysis of Arion vulgaris--Proteins for Probably Successful Survival Strategies?

    Science.gov (United States)

    Bulat, Tanja; Smidak, Roman; Sialana, Fernando J; Jung, Gangsoo; Rattei, Thomas; Bilban, Martin; Sattmann, Helmut; Lubec, Gert; Aradska, Jana

    2016-01-01

    The Spanish slug, Arion vulgaris, is considered one of the hundred most invasive species in Central Europe. The immense and very successful adaptation and spreading of A. vulgaris suggest that it developed highly effective mechanisms to deal with infections and natural predators. Current transcriptomic and proteomic studies on gastropods have been restricted mainly to marine and freshwater gastropods. No transcriptomic or proteomic study on A. vulgaris has been carried out so far, and in the current study, the first transcriptomic database from adult specimen of A. vulgaris is reported. To facilitate and enable proteomics in this non-model organism, a mRNA-derived protein database was constructed for protein identification. A gel-based proteomic approach was used to obtain the first generation of a comprehensive slug mantle proteome. A total of 2128 proteins were unambiguously identified; 48 proteins represent novel proteins with no significant homology in NCBI non-redundant database. Combined transcriptomic and proteomic analysis revealed an extensive repertoire of novel proteins with a role in innate immunity including many associated pattern recognition, effector proteins and cytokine-like proteins. The number and diversity in gene families encoding lectins point to a complex defense system, probably as a result of adaptation to a pathogen-rich environment. These results are providing a fundamental and important resource for subsequent studies on molluscs as well as for putative antimicrobial compounds for drug discovery and biomedical applications.

  7. Transcriptomic and Proteomic Analysis of Arion vulgaris—Proteins for Probably Successful Survival Strategies?

    Science.gov (United States)

    Bulat, Tanja; Smidak, Roman; Sialana, Fernando J.; Jung, Gangsoo; Rattei, Thomas; Bilban, Martin; Sattmann, Helmut; Lubec, Gert; Aradska, Jana

    2016-01-01

    The Spanish slug, Arion vulgaris, is considered one of the hundred most invasive species in Central Europe. The immense and very successful adaptation and spreading of A. vulgaris suggest that it developed highly effective mechanisms to deal with infections and natural predators. Current transcriptomic and proteomic studies on gastropods have been restricted mainly to marine and freshwater gastropods. No transcriptomic or proteomic study on A. vulgaris has been carried out so far, and in the current study, the first transcriptomic database from adult specimen of A. vulgaris is reported. To facilitate and enable proteomics in this non-model organism, a mRNA-derived protein database was constructed for protein identification. A gel-based proteomic approach was used to obtain the first generation of a comprehensive slug mantle proteome. A total of 2128 proteins were unambiguously identified; 48 proteins represent novel proteins with no significant homology in NCBI non-redundant database. Combined transcriptomic and proteomic analysis revealed an extensive repertoire of novel proteins with a role in innate immunity including many associated pattern recognition, effector proteins and cytokine-like proteins. The number and diversity in gene families encoding lectins point to a complex defense system, probably as a result of adaptation to a pathogen-rich environment. These results are providing a fundamental and important resource for subsequent studies on molluscs as well as for putative antimicrobial compounds for drug discovery and biomedical applications. PMID:26986963

  8. Transcriptomic and Proteomic Analysis of Arion vulgaris--Proteins for Probably Successful Survival Strategies?

    Directory of Open Access Journals (Sweden)

    Tanja Bulat

    Full Text Available The Spanish slug, Arion vulgaris, is considered one of the hundred most invasive species in Central Europe. The immense and very successful adaptation and spreading of A. vulgaris suggest that it developed highly effective mechanisms to deal with infections and natural predators. Current transcriptomic and proteomic studies on gastropods have been restricted mainly to marine and freshwater gastropods. No transcriptomic or proteomic study on A. vulgaris has been carried out so far, and in the current study, the first transcriptomic database from adult specimen of A. vulgaris is reported. To facilitate and enable proteomics in this non-model organism, a mRNA-derived protein database was constructed for protein identification. A gel-based proteomic approach was used to obtain the first generation of a comprehensive slug mantle proteome. A total of 2128 proteins were unambiguously identified; 48 proteins represent novel proteins with no significant homology in NCBI non-redundant database. Combined transcriptomic and proteomic analysis revealed an extensive repertoire of novel proteins with a role in innate immunity including many associated pattern recognition, effector proteins and cytokine-like proteins. The number and diversity in gene families encoding lectins point to a complex defense system, probably as a result of adaptation to a pathogen-rich environment. These results are providing a fundamental and important resource for subsequent studies on molluscs as well as for putative antimicrobial compounds for drug discovery and biomedical applications.

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

    Directory of Open Access Journals (Sweden)

    Rahman Hazir

    2011-09-01

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

  10. Stressor-induced proteome alterations in zebrafish: a meta-analysis of response patterns.

    Science.gov (United States)

    Groh, Ksenia J; Suter, Marc J-F

    2015-02-01

    Proteomics approaches are being increasingly applied in ecotoxicology on the premise that the identification of specific protein expression changes in response to a particular chemical would allow elucidation of the underlying molecular pathways leading to an adverse effect. This in turn is expected to promote the development of focused testing strategies for specific groups of toxicants. Although both gel-based and gel-free global characterization techniques provide limited proteome coverage, the conclusions regarding the cellular processes affected are still being drawn based on the few changes detected. To investigate how specific the detected responses are, we analyzed a set of studies that characterized proteome alterations induced by various physiological, chemical and biological stressors in zebrafish, a popular model organism. Our analysis highlights several proteins and protein groups, including heat shock and oxidative stress defense proteins, energy metabolism enzymes and cytoskeletal proteins, to be most frequently identified as responding to diverse stressors. In contrast, other potentially more specifically responding protein groups are detected much less frequently. Thus, zebrafish proteome responses to stress reported by different studies appear to depend mostly on the level of stress rather than on the specific stressor itself. This suggests that the most broadly used current proteomics technologies do not provide sufficient proteome coverage to allow in-depth investigation of specific mechanisms of toxicant action. We suggest that the results of any differential proteomics experiment performed with zebrafish should be interpreted keeping in mind the list of the most frequent responders that we have identified. Similar reservations should apply to any other species where proteome responses are analyzed by global proteomics methods. Careful consideration of the reliability and significance of observed changes is necessary in order not to over

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

    Science.gov (United States)

    Yu, Kebing; Salomon, Arthur R

    2009-12-01

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

  12. Low-molecular weight plasma proteome analysis using centrifugal ultrafiltration.

    Science.gov (United States)

    Greening, David W; Simpson, Richard J

    2011-01-01

    The low-molecular weight fraction (LMF) of the human plasma proteome is an invaluable source of biological information, especially in the context of identifying plasma-based biomarkers of disease. This protocol outlines a standardized procedure for the rapid/reproducible LMF profiling of human plasma samples using centrifugal ultrafiltration fractionation, followed by 1D-SDS-PAGE separation and nano-LC-MS/MS. Ultrafiltration is a convective process that uses anisotropic semipermeable membranes to separate macromolecular species on the basis of size. We have optimized centrifugal ultrafiltration for plasma fractionation with respect to buffer and solvent composition, centrifugal force, duration and temperature to facilitate >95% recovery, and enrichment of low-M (r) components from human plasma. Using this protocol, >260 unique peptides can be identified from a single plasma profiling experiment using 100 μL of plasma (Greening and Simpson, J Proteomics 73:637-648, 2010). The efficacy of this method is demonstrated by the identification, for the first time, of several plasma proteins (e.g., protein KIAA0649 (Q9Y4D3), rheumatoid factor D5, serine protease inhibitor A3, and transmembrane adapter protein PAG) previously not reported in extant high-confidence Human Proteome Organization Plasma Proteome Project datasets.

  13. Proteomic analysis of ripening tomato fruit infected by Botrytis cinerea.

    Science.gov (United States)

    Shah, Punit; Powell, Ann L T; Orlando, Ron; Bergmann, Carl; Gutierrez-Sanchez, Gerardo

    2012-04-06

    Botrytis cinerea, a model necrotrophic fungal pathogen that causes gray mold as it infects different organs on more than 200 plant species, is a significant contributor to postharvest rot in fresh fruit and vegetables, including tomatoes. By describing host and pathogen proteomes simultaneously in infected tissues, the plant proteins that provide resistance and allow susceptibility and the pathogen proteins that promote colonization and facilitate quiescence can be identified. This study characterizes fruit and fungal proteins solubilized in the B. cinerea-tomato interaction using shotgun proteomics. Mature green, red ripe wild type and ripening inhibited (rin) mutant tomato fruit were infected with B. cinerea B05.10, and the fruit and fungal proteomes were identified concurrently 3 days postinfection. One hundred eighty-six tomato proteins were identified in common among red ripe and red ripe-equivalent ripening inhibited (rin) mutant tomato fruit infected by B. cinerea. However, the limited infections by B. cinerea of mature green wild type fruit resulted in 25 and 33% fewer defense-related tomato proteins than in red and rin fruit, respectively. In contrast, the ripening stage of genotype of the fruit infected did not affect the secreted proteomes of B. cinerea. The composition of the collected proteins populations and the putative functions of the identified proteins argue for their role in plant-pathogen interactions.

  14. Proteomic Analysis of Male-Fertility Restoration in CMS Onion

    Science.gov (United States)

    The production of hybrid-onion seed is dependent on cytoplasmic-genic male sterility (CMS) systems. For the most commonly used CMS, male-sterile (S) cytoplasm interacts with a dominant allele at one nuclear male-fertility restoration locus (Ms) to condition male fertility. We are using proteomics ...

  15. Global Proteomics Analysis of the Response to Starvation in C. elegans.

    Science.gov (United States)

    Larance, Mark; Pourkarimi, Ehsan; Wang, Bin; Brenes Murillo, Alejandro; Kent, Robert; Lamond, Angus I; Gartner, Anton

    2015-07-01

    Periodic starvation of animals induces large shifts in metabolism but may also influence many other cellular systems and can lead to adaption to prolonged starvation conditions. To date, there is limited understanding of how starvation affects gene expression, particularly at the protein level. Here, we have used mass-spectrometry-based quantitative proteomics to identify global changes in the Caenorhabditis elegans proteome due to acute starvation of young adult animals. Measuring changes in the abundance of over 5,000 proteins, we show that acute starvation rapidly alters the levels of hundreds of proteins, many involved in central metabolic pathways, highlighting key regulatory responses. Surprisingly, we also detect changes in the abundance of chromatin-associated proteins, including specific linker histones, histone variants, and histone posttranslational modifications associated with the epigenetic control of gene expression. To maximize community access to these data, they are presented in an online searchable database, the Encyclopedia of Proteome Dynamics (http://www.peptracker.com/epd/).

  16. Proteomics of the Peroxisome

    OpenAIRE

    2006-01-01

    Genomes provide us with a blue print for the potential of a cell. However, the activity of a cell is expressed in its proteome. Full understanding of the complexity of cells demands a comprehensive view of the proteome; its interactions, activity states and organization. Comprehensive proteomic approaches applied to peroxisomes have yielded new insights into the organelle and its dynamic interplay with other cellular structures. As technologies and methodologies improve proteomics hold the pr...

  17. Survival during long-term starvation: global proteomics analysis of Geobacter sulfurreducens under prolonged electron-acceptor limitation.

    Science.gov (United States)

    Bansal, Reema; Helmus, Ruth A; Stanley, Bruce A; Zhu, Junjia; Liermann, Laura J; Brantley, Susan L; Tien, Ming

    2013-10-04

    The bioavailability of terminal electron acceptors (TEAs) and other substrates affects the efficiency of subsurface bioremediation. While it is often argued that microorganisms exist under "feast or famine", in the laboratory most organisms are studied under "feast" conditions, whereas they typically encounter "famine" in nature. The work described here aims to understand the survival strategies of the anaerobe Geobacter sulfurreduces under TEA-starvation conditions. Cultures were starved for TEA and at various times sampled to perform global comparative proteomic analysis using iTRAQ to obtain insight into the dynamics of change in proteins/enzymes expression associated with change in nutrient availability/environmental stress. Proteins varying in abundance with a high level of statistical significance (p Geobacter under starvation conditions. The cell shuts down anabolic processes and becomes poised, through changes in its membrane proteins, to sense nutrients in the environment, to transport nutrients into the cell, and to detect or utilize TEAs that are encountered. Under TEA-limiting conditions, the cells turned from translucent white to red in color, indicating higher heme content. The increase in heme content supported proteomics results showing an increase in the number of cytochromes involved in membrane electron transport during the survival phase. The cell is also highly reduced with minimal change in energy charge (ATP to total adenine nucleotide ratio). Nonetheless, these proteomic and biochemical results indicate that even under TEA starvation cells remain poised for bioremediation.

  18. Proteomic analysis of pig (Sus scrofa olfactory soluble proteome reveals O-GlcNAcylation of secreted odorant-binding proteins

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    Patricia eNAGNAN-LE MEILLOUR

    2014-12-01

    Full Text Available The diversity of olfactory binding proteins (OBPs is a key point to understand their role in molecular olfaction. Since only few different sequences were characterized in each mammalian species, they have been considered as passive carriers of odors and pheromones. We have explored the soluble proteome of pig nasal mucus, taking benefit of the powerful tools of proteomics. Combining two-dimensional electrophoresis, mass spectrometry and western-blot with specific antibodies, our analyses revealed for the first time that the pig nasal mucus is mainly composed of secreted OBP isoforms, some of them being potentially modified by O-GlcNAcylation. An ortholog gene of the glycosyltransferase responsible of the O-GlcNAc linking on extracellular proteins in Drosophila and Mouse (EOGT was amplified from tissues of pigs of different ages and sex. The sequence was used in a phylogenetic analysis, which evidenced conservation of EOGT in insect and mammalian models studied in molecular olfaction. Extracellular O-GlcNAcylation of secreted OBPs could finely modulate their binding specificities to odors and pheromones. This constitutes a new mechanism for extracellular signaling by OBPs, suggesting that they act as the first step of odor discrimination.

  19. Streamlined Membrane Proteome Preparation for Shotgun Proteomics Analysis with Triton X-100 Cloud Point Extraction and Nanodiamond Solid Phase Extraction

    Directory of Open Access Journals (Sweden)

    Minh D. Pham

    2016-05-01

    Full Text Available While mass spectrometry (MS plays a key role in proteomics research, characterization of membrane proteins (MP by MS has been a challenging task because of the presence of a host of interfering chemicals in the hydrophobic protein extraction process, and the low protease digestion efficiency. We report a sample preparation protocol, two-phase separation with Triton X-100, induced by NaCl, with coomassie blue added for visualizing the detergent-rich phase, which streamlines MP preparation for SDS-PAGE analysis of intact MP and shot-gun proteomic analyses. MP solubilized in the detergent-rich milieu were then sequentially extracted and fractionated by surface-oxidized nanodiamond (ND at three pHs. The high MP affinity of ND enabled extensive washes for removal of salts, detergents, lipids, and other impurities to ensure uncompromised ensuing purposes, notably enhanced proteolytic digestion and down-stream mass spectrometric (MS analyses. Starting with a typical membranous cellular lysate fraction harvested with centrifugation/ultracentrifugation, MP purities of 70%, based on number (not weight of proteins identified by MS, was achieved; the weight-based purity can be expected to be much higher.

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

    KAUST Repository

    Marondedze, Claudius

    2014-08-06

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

  1. Proteome Dynamics in Biobanked Horse Peripheral Blood Derived Lymphocytes (PBL) with Induced Autoimmune Uveitis.

    Science.gov (United States)

    Hauck, Stefanie M; Lepper, Marlen F; Hertl, Michael; Sekundo, Walter; Deeg, Cornelia A

    2017-10-01

    Equine recurrent uveitis is the only spontaneous model for recurrent autoimmune uveitis in humans, where T cells target retinal proteins. Differences between normal and autoaggressive lymphocytes were identified in this study by analyzing peripheral blood derived lymphocytes (PBL) proteomes from the same case with interphotoreceptor retinoid binding protein induced uveitis sampled before (Day 0), during (Day 15), and after uveitic attack (Day 23). Relative protein abundances of PBL were investigated in a quantitative, label-free differential proteome analysis in cells that were kept frozen for 14 years since the initial experiment. Quantitative data could be acquired for 2632 proteins at all three time points. Profound changes (≥2-fold change) in PBL protein abundance were observed when comparing Day 0 with 15, representing acute inflammation (1070 regulated proteins) and Day 0 with 23 (cessation; 1571 regulated). Significant differences applied to proteins with functions in integrin signaling during active uveitis, involving "Erk and pi-3 kinase are necessary for collagen binding in corneal epithelia," "integrins in angiogenesis," and "integrin-linked kinase signaling" pathways. In contrast, at cessation of uveitic attack, significantly changed proteins belonged to pathways of "nongenotropic androgen signaling," "classical complement pathway," and "Amb2 integrin signaling." Several members of respective pathways were earlier shown to be changed in naturally occurring uveitis, underscoring the significance of these findings here and proofing the value of the induced model in mimicking spontaneous autoimmune uveitis. All MS data have been deposited to the ProteomeXchange consortium via the PRIDE partner repository (dataset identifier PXD005580). © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    Science.gov (United States)

    Marondedze, Claudius; Gehring, Christoph; Thomas, Ludivine

    2014-01-01

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

  3. iTRAQ-Based Quantitative Proteomics Analysis of Black Rice Grain Development Reveals Metabolic Pathways Associated with Anthocyanin Biosynthesis

    National Research Council Canada - National Science Library

    Chen, Linghua; Huang, Yining; Xu, Ming; Cheng, Zuxin; Zhang, Dasheng; Zheng, Jingui

    2016-01-01

    .... The aim of the present study was to determine changes in the metabolic pathways that are involved in the dynamic grain proteome during the development of black rice indica cultivar, (Oryza sativa L. indica var. SSP...

  4. Xylem sap proteomics.

    Science.gov (United States)

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

    2014-01-01

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

  5. Analysis of the plasmodium falciparum proteome by high-accuracy mass spectrometry

    DEFF Research Database (Denmark)

    Lasonder, Edwin; Ishihama, Yasushi; Andersen, Jens S;

    2002-01-01

    -accuracy (average deviation less than 0.02 Da at 1,000 Da) mass spectrometric proteome analysis of selected stages of the human malaria parasite Plasmodium falciparum. The analysis revealed 1,289 proteins of which 714 proteins were identified in asexual blood stages, 931 in gametocytes and 645 in gametes. The last...

  6. Data for a proteomic analysis of p53-independent induction of apoptosis by bortezomib

    OpenAIRE

    2014-01-01

    This data article contains data related to the research article entitled, “A proteomic analysis of p53-independent induction of apoptosis by bortezomib in 4T1 breast cancer cell line” by Yerlikaya et al. [1]. The research article presented 2-DE and nLC-MS/MS based proteomic analysis of proteasome inhibitor bortezomib-induced changes in the expression of cellular proteins. The report showed that GRP78 and TCEB2 were over-expressed in response to treatment with bortezomib for 24 h. In addition,...

  7. Proteomic analysis of sea urchin (Strongylocentrotus purpuratus spicule matrix

    Directory of Open Access Journals (Sweden)

    Poustka Albert J

    2010-06-01

    Full Text Available Abstract Background The sea urchin embryo has been an important model organism in developmental biology for more than a century. This is due to its relatively simple construction, translucent appearance, and the possibility to follow the fate of individual cells as development to the pluteus larva proceeds. Because the larvae contain tiny calcitic skeletal elements, the spicules, they are also important model organisms for biomineralization research. Similar to other biominerals the spicule contains an organic matrix, which is thought to play an important role in its formation. However, only few spicule matrix proteins were identified previously. Results Using mass spectrometry-based methods we have identified 231 proteins in the matrix of the S. purpuratus spicule matrix. Approximately two thirds of the identified proteins are either known or predicted to be extracellular proteins or transmembrane proteins with large ectodomains. The ectodomains may have been solubilized by partial proteolysis and subsequently integrated into the growing spicule. The most abundant protein of the spicule matrix is SM50. SM50-related proteins, SM30-related proteins, MSP130 and related proteins, matrix metalloproteases and carbonic anhydrase are among the most abundant components. Conclusions The spicule matrix is a relatively complex mixture of proteins not only containing matrix-specific proteins with a function in matrix assembly or mineralization, but also: 1 proteins possibly important for the formation of the continuous membrane delineating the mineralization space; 2 proteins for secretory processes delivering proteinaceous or non-proteinaceous precursors; 3 or proteins reflecting signaling events at the cell/matrix interface. Comparison of the proteomes of different skeletal matrices allows prediction of proteins of general importance for mineralization in sea urchins, such as SM50, SM30-E, SM29 or MSP130. The comparisons also help point out putative

  8. Proteomic analysis of seed germination under salt stress in soybeans

    Institute of Scientific and Technical Information of China (English)

    Xiao-yan XU; Rui FAN; Rui ZHENG; Chun-mei LI; De-yue YU

    2011-01-01

    Soybean (Glycine max (L.) Merrill) is a salt-sensitive crop,and its production is severely affected by saline soils.Therefore,the response of soybean seeds to salt stress during germination was investigated at both physiological and proteomic levels.The salt-tolerant cultivar Lee68 and salt-sensitive cultivar N2899 were exposed to 100 mmol/L NaCl until radicle protrusion from the seed coat.In both cultivars,the final germination percentage was not affected by salt,but the mean germination times of Lee68 and N2899 were delayed by 0.3 and 1.0 d,respectively,compared with controls.In response to salt stress,the abscisic acid content increased,and gibberellic acid (GA1+3) and isopentenyladenosine decreased.Indole-3-acetic acid increased in Lee68,but remained unchanged in N2899.The proteins extracted from germinated seeds were separated using two-dimensional gel electrophoresis (2-DE),followed by Coomassie brilliant blue G-250 staining.About 350 protein spots from 2-DE gels of pH range 3 to 10 and 650 spots from gels of pH range 4 to 7 were reproducibly resolved,of which 18 protein spots showed changes in abundance as a result of salt stress in both cultivars.After matrix-assisted laser desorption ionization-time of flight-mass spectroscopy (MALDI-TOF-MS) analysis of the differentially expressed proteins,the peptide mass fingerprint was searched against the soybean UniGene database and nine proteins were successfully identified.Ferritin and 20S proteasome subunit β-6 were up-regulated in both cultivars.Glyceraldehyde 3-phosphate dehydrogenase,glutathione S-transferase (GST) 9,GST 10,and seed maturation protein PM36 were down-regulated in Lee68 by salt,but still remained at a certain level.However,these proteins were present in lower levels in control N2899 and were up-regulated under salt stress.The results indicate that these proteins might have important roles in defense mechanisms against salt stress during soybean seed germination.

  9. Toward defining the anatomo-proteomic puzzle of the human brain: An integrative analysis.

    Science.gov (United States)

    Fernandez-Irigoyen, Joaquín; Labarga, Alberto; Zabaleta, Aintzane; de Morentin, Xabier Martínez; Perez-Valderrama, Estela; Zelaya, María Victoria; Santamaria, Enrique

    2015-10-01

    The human brain is exceedingly complex, constituted by billions of neurons and trillions of synaptic connections that, in turn, define ∼900 neuroanatomical subdivisions in the adult brain (Hawrylycz et al. An anatomically comprehensive atlas of the human brain transcriptome. Nature 2012, 489, 391-399). The human brain transcriptome has revealed specific regional transcriptional signatures that are regulated in a spatiotemporal manner, increasing the complexity of the structural and molecular organization of this organ (Kang et al. Spatio-temporal transcriptome of the human brain. Nature 2011, 478, 483-489). During the last decade, neuroproteomics has emerged as a powerful approach to profile neural proteomes using shotgun-based MS, providing complementary information about protein content and function at a global level. Here, we revise recent proteome profiling studies performed in human brain, with special emphasis on proteome mapping of anatomical macrostructures, specific subcellular compartments, and cerebrospinal fluid. Moreover, we have performed an integrative functional analysis of the protein compilation derived from these large-scale human brain proteomic studies in order to obtain a comprehensive view of human brain biology. Finally, we also discuss the potential contribution of our meta-analysis to the Chromosome-centric Human Proteome Project initiative. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Sources of technical variability in quantitative LC-MS proteomics: human brain tissue sample analysis.

    Science.gov (United States)

    Piehowski, Paul D; Petyuk, Vladislav A; Orton, Daniel J; Xie, Fang; Moore, Ronald J; Ramirez-Restrepo, Manuel; Engel, Anzhelika; Lieberman, Andrew P; Albin, Roger L; Camp, David G; Smith, Richard D; Myers, Amanda J

    2013-05-03

    To design a robust quantitative proteomics study, an understanding of both the inherent heterogeneity of the biological samples being studied as well as the technical variability of the proteomics methods and platform is needed. Additionally, accurately identifying the technical steps associated with the largest variability would provide valuable information for the improvement and design of future processing pipelines. We present an experimental strategy that allows for a detailed examination of the variability of the quantitative LC-MS proteomics measurements. By replicating analyses at different stages of processing, various technical components can be estimated and their individual contribution to technical variability can be dissected. This design can be easily adapted to other quantitative proteomics pipelines. Herein, we applied this methodology to our label-free workflow for the processing of human brain tissue. For this application, the pipeline was divided into four critical components: Tissue dissection and homogenization (extraction), protein denaturation followed by trypsin digestion and SPE cleanup (digestion), short-term run-to-run instrumental response fluctuation (instrumental variance), and long-term drift of the quantitative response of the LC-MS/MS platform over the 2 week period of continuous analysis (instrumental stability). From this analysis, we found the following contributions to variability: extraction (72%) > instrumental variance (16%) > instrumental stability (8.4%) > digestion (3.1%). Furthermore, the stability of the platform and its suitability for discovery proteomics studies is demonstrated.

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

    Science.gov (United States)

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

    2013-04-05

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

  12. Sources of Technical Variability in Quantitative LC-MS Proteomics: Human Brain Tissue Sample Analysis.

    Energy Technology Data Exchange (ETDEWEB)

    Piehowski, Paul D.; Petyuk, Vladislav A.; Orton, Daniel J.; Xie, Fang; Moore, Ronald J.; Ramirez Restrepo, Manuel; Engel, Anzhelika; Lieberman, Andrew P.; Albin, Roger L.; Camp, David G.; Smith, Richard D.; Myers, Amanda J.

    2013-05-03

    To design a robust quantitative proteomics study, an understanding of both the inherent heterogeneity of the biological samples being studied as well as the technical variability of the proteomics methods and platform is needed. Additionally, accurately identifying the technical steps associated with the largest variability would provide valuable information for the improvement and design of future processing pipelines. We present an experimental strategy that allows for a detailed examination of the variability of the quantitative LC-MS proteomics measurements. By replicating analyses at different stages of processing, various technical components can be estimated and their individual contribution to technical variability can be dissected. This design can be easily adapted to other quantitative proteomics pipelines. Herein, we applied this methodology to our label-free workflow for the processing of human brain tissue. For this application, the pipeline was divided into four critical components: Tissue dissection and homogenization (extraction), protein denaturation followed by trypsin digestion and SPE clean-up (digestion), short-term run-to-run instrumental response fluctuation (instrumental variance), and long-term drift of the quantitative response of the LC-MS/MS platform over the 2 week period of continuous analysis (instrumental stability). From this analysis, we found the following contributions to variability: extraction (72%) >> instrumental variance (16%) > instrumental stability (8.4%) > digestion (3.1%). Furthermore, the stability of the platform and its’ suitability for discovery proteomics studies is demonstrated.

  13. Comparative bacterial proteomics: analysis of the core genome concept.

    Directory of Open Access Journals (Sweden)

    Stephen J Callister

    Full Text Available While comparative bacterial genomic studies commonly predict a set of genes indicative of common ancestry, experimental validation of the existence of this core genome requires extensive measurement and is typically not undertaken. Enabled by an extensive proteome database developed over six years, we have experimentally verified the expression of proteins predicted from genomic ortholog comparisons among 17 environmental and pathogenic bacteria. More exclusive relationships were observed among the expressed protein content of phenotypically related bacteria, which is indicative of the specific lifestyles associated with these organisms. Although genomic studies can establish relative orthologous relationships among a set of bacteria and propose a set of ancestral genes, our proteomics study establishes expressed lifestyle differences among conserved genes and proposes a set of expressed ancestral traits.

  14. Comparative Bacterial Proteomics: Analysis of the Core Genome Concept

    Energy Technology Data Exchange (ETDEWEB)

    Callister, Stephen J.; McCue, Lee Ann; Turse, Josh E.; Monroe, Matthew E.; Auberry, Kenneth J.; Smith, Richard D.; Adkins, Joshua N.; Lipton, Mary S.

    2008-02-06

    Comparative bacterial genomic studies commonly predict a set of genes indicative of common ancestry. Experimental validation of the existence of this core genome requires extensive measurement and is not typically undertaken. Enabled by an extensive proteome database development over a six year period, we experimentally verified the expression of proteins predicted from genomic ortholog comparisons among 17 environmental and pathogenic bacteria. More exclusive relationships were observed among the expressed protein content of phenotypically related bacteria, which is indicative of the specific lifestyles associated with these organisms. While genomic studies establish relative orthologous relationships among a set of bacteria and propose a set of ancestral genes, our proteomics study establishes expressed lifestyle differences among conserved genes and proposes a set of expressed ancestral traits.

  15. Proteomics analysis of cerebral cortex in Wistar rats

    Institute of Scientific and Technical Information of China (English)

    Xiaofeng ZHAO; Jingrong WEN; Shu WANG; Xuemin SHI

    2008-01-01

    To analyze the protein expression pattern of the cerebral cortex in Wistar rats using the proteomics approach, proteins were separated by two-dimensional gel electrophoresis, stained with Coomassie brilliant blue and digested with trypsin. Then, we analyzed the peptide section using a matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and identified the protein by indexing special database (SwissProt) according to the finger printing of the peptide quality. Eighty-four protein spots were identified, includ-ing metabolic enzymes, skeleton proteins, heat shock pro-teins, antioxidant proteins, signaling proteins, proteasome related proteins, neuron and glial specific proteins and serum associated proteins. The result of this study enriches the database of the proteome in the cerebral cortex of rats and lays a foundation for further research of neurological disorders in rat models.

  16. Proteomic analysis of lamellar bodies isolated from rat lungs

    Directory of Open Access Journals (Sweden)

    Ayalew Sahlu

    2008-06-01

    Full Text Available Abstract Background Lamellar bodies are lysosome-related secretory granules and store lung surfactant in alveolar type II cells. To better understand the mechanisms of surfactant secretion, we carried out proteomic analyses of lamellar bodies isolated from rat lungs. Results With peptide mass fingerprinting by Matrix Assisted Laser Desorption/Ionization – Time of Flight mass spectrometry, 44 proteins were identified with high confidence. These proteins fell into diverse functional categories: surfactant-related, membrane trafficking, calcium binding, signal transduction, cell structure, ion channels, protein processing and miscellaneous. Selected proteins were verified by Western blot and immunohistochemistry. Conclusion This proteomic profiling of lamellar bodies provides a basis for further investigations of functional roles of the identified proteins in lamellar body biogenesis and surfactant secretion.

  17. Comparative Bacterial Proteomics: Analysis of the Core Genome Concept

    Science.gov (United States)

    Callister, Stephen J.; McCue, Lee Ann; Turse, Joshua E.; Monroe, Matthew E.; Auberry, Kenneth J.; Smith, Richard D.; Adkins, Joshua N.; Lipton, Mary S.

    2008-01-01

    While comparative bacterial genomic studies commonly predict a set of genes indicative of common ancestry, experimental validation of the existence of this core genome requires extensive measurement and is typically not undertaken. Enabled by an extensive proteome database developed over six years, we have experimentally verified the expression of proteins predicted from genomic ortholog comparisons among 17 environmental and pathogenic bacteria. More exclusive relationships were observed among the expressed protein content of phenotypically related bacteria, which is indicative of the specific lifestyles associated with these organisms. Although genomic studies can establish relative orthologous relationships among a set of bacteria and propose a set of ancestral genes, our proteomics study establishes expressed lifestyle differences among conserved genes and proposes a set of expressed ancestral traits. PMID:18253490

  18. Proteomic analysis of latex from the rubber-producing plant Taraxacum brevicorniculatum.

    Science.gov (United States)

    Wahler, Daniela; Colby, Thomas; Kowalski, Natalie A; Harzen, Anne; Wotzka, Sandra Y; Hillebrand, Andrea; Fischer, Rainer; Helsper, Johannes; Schmidt, Jürgen; Schulze Gronover, Christian; Prüfer, Dirk

    2012-03-01

    Many plants produce latex, a specialized, metabolically active cytoplasm. This is generally regarded as a defensive trait but latex may also possess additional functions. We investigated the role of latex in the dandelion species Taraxacum brevicorniculatum that contains considerable amounts of high-quality natural rubber by carrying out a comprehensive analysis of the latex proteome. We developed reliable protocols for the preparation of protein samples for one-dimensional gel electrophoresis, two-dimensional gel electrophoresis, and subsequent mass spectrometry analysis, which led to 278 unique identifications. A gene ontology classification system based on comparisons with known Arabidopsis thaliana root proteins showed that dandelion proteins involved in lipid metabolism and transport were enriched in the latex proteome, whereas those involved in stress responses were not. We also found that proteins involved in rubber biosynthesis were distributed among different fractions of the latex proteome.

  19. Is Five Percent Too Small? Analysis of the Overlaps between Disorder, Coiled Coil and Collagen Predictions in Complete Proteomes

    Directory of Open Access Journals (Sweden)

    Zoltán Gáspári

    2014-02-01

    Full Text Available Identification of intrinsic disorder in proteins and proteomes has revealed important novel aspects of protein function and interactions. However, it has been pointed out that several oligomeric fibrillar protein motifs such as coiled coils and collagen triple helical segments can also identified as intrinsically disordered. This feature has not yet been investigated in more detail at the proteome level. The present work aims at the identification and quantification of such overlaps in full proteomes to assess their significance in large-scale studies of protein disorder. It was found that the percentage of cross-predicted residues is around 5% in the human proteome and is generally near that value in other metazoan ones but shows remarkable variation in different organisms. In particular, smaller proteomes are increasingly prone to such cross-predictions, thus, especially the analysis of viral proteomes requires the use of specific prediction tools.

  20. Recent advances in maize nuclear proteomic studies reveal histone modifications.

    Science.gov (United States)

    Casati, Paula

    2012-01-01

    The nucleus of eukaryotic organisms is highly dynamic and complex, containing different types of macromolecules including DNA, RNA, and a wide range of proteins. Novel proteomic applications have led to a better overall determination of nucleus protein content. Although nuclear plant proteomics is only at the initial phase, several studies have been reported and are summarized in this review using different plants species, such as Arabidopsis thaliana, rice, cowpea, onion, garden cress, and barrel clover. These include the description of the total nuclear or phospho-proteome (i.e., Arabidopsis, cowpea, onion), or the analysis of the differential nuclear proteome under different growth environments (i.e., Arabidopsis, rice, cowpea, onion, garden cress, and barrel clover). However, only few reports exist on the analysis of the maize nuclear proteome or its changes under various conditions. This review will present recent data on the study of the nuclear maize proteome, including the analysis of changes in posttranslational modifications in histone proteins.

  1. Proteomic analysis of regenerating mouse liver following 50% partial hepatectomy

    OpenAIRE

    Cao, Hongcui; Yu, Jiong; Xu, Wei; Jia, Xiaofei; Yang, Jinfeng; Pan, Qiaoling; Zhang, Qiyi; Sheng, Guoping; Li, Jun; Pan, Xiaoping; Wang, Yingjie; Li, Lanjuan

    2009-01-01

    Background Although 70% (or 2/3) partial hepatectomy (PH) is the most studied model for liver regeneration, the hepatic protein expression profile associated with lower volume liver resection (such as 50% PH) has not yet been reported. Therefore, the aim of this study was to determine the global protein expression profile of the regenerating mouse liver following 50% PH by differential proteomics, and thereby gaining some insights into the hepatic regeneration mechanism(s) under this milder b...

  2. Quantitative proteomic analysis of the fall armyworm saliva.

    Science.gov (United States)

    Acevedo, Flor E; Stanley, Bruce A; Stanley, Anne; Peiffer, Michelle; Luthe, Dawn S; Felton, Gary W

    2017-07-01

    Lepidopteran larvae secrete saliva on plant tissues during feeding. Components in the saliva may aid in food digestion, whereas other components are recognized by plants as cues to elicit defense responses. Despite the ecological and economical importance of these plant-feeding insects, knowledge of their saliva composition is limited to a few species. In this study, we identified the salivary proteins of larvae of the fall armyworm (FAW), Spodoptera frugiperda; determined qualitative and quantitative differences in the salivary proteome of the two host races-corn and rice strains-of this insect; and identified changes in total protein concentration and relative protein abundance in the saliva of FAW larvae associated with different host plants. Quantitative proteomic analyses were performed using labeling with isobaric tags for relative and absolute quantification followed by liquid chromatography-tandem mass spectrometry. In total, 98 proteins were identified (>99% confidence) in the FAW saliva. These proteins were further categorized into five functional groups: proteins potentially involved in (1) plant defense regulation, (2) herbivore offense, (3) insect immunity, (4) detoxification, (5) digestion, and (6) other functions. Moreover, there were differences in the salivary proteome between the FAW strains that were identified by label-free proteomic analyses. Thirteen differentially identified proteins were present in each strain. There were also differences in the relative abundance of eleven salivary proteins between the two FAW host strains as well as differences within each strain associated with different diets. The total salivary protein concentration was also different for the two strains reared on different host plants. Based on these results, we conclude that the FAW saliva contains a complex mixture of proteins involved in different functions that are specific for each strain and its composition can change plastically in response to diet type

  3. Proteome Analysis of Borrelia burgdorferi Response to Environmental Change

    Energy Technology Data Exchange (ETDEWEB)

    Angel, Thomas E.; Luft, Benjamin J.; Yang, Xiaohua; Nicora, Carrie D.; Camp, David G.; Jacobs, Jon M.; Smith, Richard D.

    2010-11-02

    We examined global changes in protein expression in the B31 strain of Borrelia burgdorferi, in response to two environmental cues (pH and temperature) chosen for their reported similarity to those encountered at different stages of the organism’s life cycle. Multidimensional nano-liquid chromatographic separations coupled with tandem mass spectrometry were used to examine the array of proteins (i.e., the proteome) of B. burgdorferi for different pH and temperature culture conditions. Changes in pH and temperature elicited in vitro adaptations of this spirochete known to cause Lyme disease and led to alterations in protein expression that are associated with increased microbial pathogenesis. We identified 1031 proteins that represent 59% of the annotated genome of B. burgdorferi and elucidated a core proteome of 414 proteins that were present in all environmental conditions investigated. Observed changes in protein abundances indicated varied replicon usage, as well as proteome functional distributions between the in vitro cell culture conditions. Surprisingly, the pH and temperature conditions that mimicked B. burgdorferi residing in the gut of a fed tick showed a marked reduction in protein diversity. Additionally, the results provide us with leading candidates for exploring how B. burgdorferi adapts to and is able to survive in a wide variety of environmental conditions and lay a foundation for planned in situ studies of B. burgdorferi isolated from the tick midgut and infected animals.

  4. Statistical Analysis of Variation in the Human Plasma Proteome

    Directory of Open Access Journals (Sweden)

    Todd H. Corzett

    2010-01-01

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

  5. A Quantitative Proteomic Analysis of In Vitro Assembled Chromatin.

    Science.gov (United States)

    Völker-Albert, Moritz Carl; Pusch, Miriam Caroline; Fedisch, Andreas; Schilcher, Pierre; Schmidt, Andreas; Imhof, Axel

    2016-03-01

    The structure of chromatin is critical for many aspects of cellular physiology and is considered to be the primary medium to store epigenetic information. It is defined by the histone molecules that constitute the nucleosome, the positioning of the nucleosomes along the DNA and the non-histone proteins that associate with it. These factors help to establish and maintain a largely DNA sequence-independent but surprisingly stable structure. Chromatin is extensively disassembled and reassembled during DNA replication, repair, recombination or transcription in order to allow the necessary factors to gain access to their substrate. Despite such constant interference with chromatin structure, the epigenetic information is generally well maintained. Surprisingly, the mechanisms that coordinate chromatin assembly and ensure proper assembly are not particularly well understood. Here, we use label free quantitative mass spectrometry to describe the kinetics of in vitro assembled chromatin supported by an embryo extract prepared from preblastoderm Drosophila melanogaster embryos. The use of a data independent acquisition method for proteome wide quantitation allows a time resolved comparison of in vitro chromatin assembly. A comparison of our in vitro data with proteomic studies of replicative chromatin assembly in vivo reveals an extensive overlap showing that the in vitro system can be used for investigating the kinetics of chromatin assembly in a proteome-wide manner. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Quantitative proteomic analysis of amphotericin B resistance in Leishmania infantum.

    Science.gov (United States)

    Brotherton, Marie-Christine; Bourassa, Sylvie; Légaré, Danielle; Poirier, Guy G; Droit, Arnaud; Ouellette, Marc

    2014-08-01

    Amphotericin B (AmB) in its liposomal form is now considered as either first- or second-line treatment against Leishmania infections in different part of the world. Few cases of AmB resistance have been reported and resistance mechanisms toward AmB are still poorly understood. This paper reports a large-scale comparative proteomic study in the context of AmB resistance. Quantitative proteomics using stable isotope labeling of amino acids in cell culture (SILAC) was used to better characterize cytoplasmic and membrane-enriched (ME) proteomes of the in vitro generated Leishmania infantum AmB resistant mutant AmB1000.1. In total, 97 individual proteins were found as differentially expressed between the mutant and its parental sensitive strain (WT). More than half of these proteins were either metabolic enzymes or involved in transcription or translation processes. Key energetic pathways such as glycolysis and TCA cycle were up-regulated in the mutant. Interestingly, many proteins involved in reactive oxygen species (ROS) scavenging and heat-shock proteins were also up-regulated in the resistant mutant. This work provides a basis for further investigations to understand the roles of proteins differentially expressed in relation with AmB resistance.

  7. Comparative Analysis of Genomics and Proteomics in Bacillus thuringiensis 4.0718

    Science.gov (United States)

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

    2015-01-01

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

  8. Comparative analysis of genomics and proteomics in Bacillus thuringiensis 4.0718.

    Directory of Open Access Journals (Sweden)

    Jie Rang

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

  9. Mass spectrometry-based serum proteome pattern analysis in molecular diagnostics of early stage breast cancer

    Directory of Open Access Journals (Sweden)

    Stobiecki Maciej

    2009-07-01

    Full Text Available Abstract Background Mass spectrometric analysis of the blood proteome is an emerging method of clinical proteomics. The approach exploiting multi-protein/peptide sets (fingerprints detected by mass spectrometry that reflect overall features of a specimen's proteome, termed proteome pattern analysis, have been already shown in several studies to have applicability in cancer diagnostics. We aimed to identify serum proteome patterns specific for early stage breast cancer patients using MALDI-ToF mass spectrometry. Methods Blood samples were collected before the start of therapy in a group of 92 patients diagnosed at stages I and II of the disease, and in a group of age-matched healthy controls (104 women. Serum specimens were purified and the low-molecular-weight proteome fraction was examined using MALDI-ToF mass spectrometry after removal of albumin and other high-molecular-weight serum proteins. Protein ions registered in a mass range between 2,000 and 10,000 Da were analyzed using a new bioinformatic tool created in our group, which included modeling spectra as a sum of Gaussian bell-shaped curves. Results We have identified features of serum proteome patterns that were significantly different between blood samples of healthy individuals and early stage breast cancer patients. The classifier built of three spectral components that differentiated controls and cancer patients had 83% sensitivity and 85% specificity. Spectral components (i.e., protein ions that were the most frequent in such classifiers had approximate m/z values of 2303, 2866 and 3579 Da (a biomarker built from these three components showed 88% sensitivity and 78% specificity. Of note, we did not find a significant correlation between features of serum proteome patterns and established prognostic or predictive factors like tumor size, nodal involvement, histopathological grade, estrogen and progesterone receptor expression. In addition, we observed a significantly (p = 0

  10. Transcriptome and proteome dynamics of a light-dark synchronized bacterial cell cycle.

    Directory of Open Access Journals (Sweden)

    Jacob R Waldbauer

    Full Text Available BACKGROUND: Growth of the ocean's most abundant primary producer, the cyanobacterium Prochlorococcus, is tightly synchronized to the natural 24-hour light-dark cycle. We sought to quantify the relationship between transcriptome and proteome dynamics that underlie this obligate photoautotroph's highly choreographed response to the daily oscillation in energy supply. METHODOLOGY/PRINCIPAL FINDINGS: Using RNA-sequencing transcriptomics and mass spectrometry-based quantitative proteomics, we measured timecourses of paired mRNA-protein abundances for 312 genes every 2 hours over a light-dark cycle. These temporal expression patterns reveal strong oscillations in transcript abundance that are broadly damped at the protein level, with mRNA levels varying on average 2.3 times more than the corresponding protein. The single strongest observed protein-level oscillation is in a ribonucleotide reductase, which may reflect a defense strategy against phage infection. The peak in abundance of most proteins also lags that of their transcript by 2-8 hours, and the two are completely antiphase for some genes. While abundant antisense RNA was detected, it apparently does not account for the observed divergences between expression levels. The redirection of flux through central carbon metabolism from daytime carbon fixation to nighttime respiration is associated with quite small changes in relative enzyme abundances. CONCLUSIONS/SIGNIFICANCE: Our results indicate that expression responses to periodic stimuli that are common in natural ecosystems (such as the diel cycle can diverge significantly between the mRNA and protein levels. Protein expression patterns that are distinct from those of cognate mRNA have implications for the interpretation of transcriptome and metatranscriptome data in terms of cellular metabolism and its biogeochemical impact.

  11. Dynamic changes in the proteome of human peripheral blood mononuclear cells with low dose ionizing radiation.

    Science.gov (United States)

    Nishad, S; Ghosh, Anu

    2016-02-01

    Humans are continually exposed to ionizing radiation from natural as well as anthropogenic sources. Though biological effects of high dose radiation exposures have been well accepted, studies on low-to-moderate dose exposures (in the range of 50-500 mGy) have been strongly debated even as researchers continue to search for elusive 'radiation signatures' in humans. Proteins are considered as dynamic functional players that drive cellular responses. However, there is little proteomic information available in context of human exposure to ionizing radiation. In this study, we determined differential expressed proteins in G0 peripheral blood mononuclear cells (PBMCs) from healthy individuals 1h and 4h after 'ex vivo' exposure with two radiation doses (300 mGy and 1 Gy). Twenty-three proteins were found to be significantly altered in irradiated cells when compared to sham irradiated cells with fold change ± 1.5-fold (p ≤ 0.05), with only three proteins showing ≥ 2.5-fold change, either with dose or with time. Mass spectrometry analyses identified redox sensor protein, chloride intracellular channel protein 1 (CLIC-1), the antioxidant protein, peroxiredoxin-6 and the pro-survival molecular chaperone 78 KDa glucose regulated protein (GRP78) among the 23 modulated proteins. The mean coefficient of variation (CV) for the twenty-three radiation responsive protein spots was found to be 33.7% for 300 mGy and 48.3% for 1 Gy. We thus, conclude that the radiation proteomic response of G0 human PBMCs, which are in the resting stage of the cell cycle, involves moderate upregulation of protective mechanisms, with low inter-individual variability. This study will help further our understanding of cellular effects of low dose acute radiation in humans and contribute toward differential biomarker discovery. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. An individual urinary proteome analysis in normal human beings to define the minimal sample number to represent the normal urinary proteome

    National Research Council Canada - National Science Library

    Liu, Xuejiao; Shao, Chen; Wei, Lilong; Duan, Jindan; Wu, Shuzhen; Li, Xuewang; Li, Mingxi; Sun, Wei

    2012-01-01

    The urinary proteome has been widely used for biomarker discovery. A urinary proteome database from normal humans can provide a background for discovery proteomics and candidate proteins/peptides for targeted proteomics...

  13. An Automated High Throughput Proteolysis and Desalting Platform for Quantitative Proteomic Analysis

    Directory of Open Access Journals (Sweden)

    Albert-Baskar Arul

    2013-06-01

    Full Text Available Proteomics for biomarker validation needs high throughput instrumentation to analyze huge set of clinical samples for quantitative and reproducible analysis at a minimum time without manual experimental errors. Sample preparation, a vital step in proteomics plays a major role in identification and quantification of proteins from biological samples. Tryptic digestion a major check point in sample preparation for mass spectrometry based proteomics needs to be more accurate with rapid processing time. The present study focuses on establishing a high throughput automated online system for proteolytic digestion and desalting of proteins from biological samples quantitatively and qualitatively in a reproducible manner. The present study compares online protein digestion and desalting of BSA with conventional off-line (in-solution method and validated for real time sample for reproducibility. Proteins were identified using SEQUEST data base search engine and the data were quantified using IDEALQ software. The present study shows that the online system capable of handling high throughput samples in 96 well formats carries out protein digestion and peptide desalting efficiently in a reproducible and quantitative manner. Label free quantification showed clear increase of peptide quantities with increase in concentration with much linearity compared to off line method. Hence we would like to suggest that inclusion of this online system in proteomic pipeline will be effective in quantification of proteins in comparative proteomics were the quantification is really very crucial.

  14. Proteomic analysis during larval development and metamorphosis of the spionid polychaete Pseudopolydora vexillosa

    KAUST Repository

    Mok, Flora SY

    2009-12-14

    Background: While the larval-juvenile transition (metamorphosis) in the spionid polychaete Pseudopolydora vexillosa involves gradual morphological changes and does not require substantial development of juvenile organs, the opposite occurs in the barnacle Balanus amphitrite. We hypothesized that the proteome changes during metamorphosis in the spionids are less drastic than that in the barnacles. To test this, proteomes of pre-competent larvae, competent larvae (ready to metamorphose), and juveniles of P. vexillosa were compared using 2-dimensional gel electrophoresis (2-DE), and they were then compared to those of the barnacle.Results: Unlike the significant changes found during barnacle metamorphosis, proteomes of competent P. vexillosa larvae were more similar to those of their juveniles. Pre-competent larvae had significantly fewer protein spots (384 spots), while both competent larvae and juveniles expressed about 660 protein spots each. Proteins up-regulated during competence identified by MALDI-TOF/TOF analysis included a molecular chaperon (calreticulin), a signal transduction regulator (tyrosin activation protein), and a tissue-remodeling enzyme (metallopeptidase).Conclusions: This was the first time to study the protein expression patterns during the metamorphosis of a marine polychaete and to compare the proteomes of marine invertebrates that have different levels of morphological changes during metamorphosis. The findings provide promising initial steps towards the development of a proteome database for marine invertebrate metamorphosis, thus deciphering the possible mechanisms underlying larval metamorphosis in non-model marine organisms. © 2009 Mok et al; licensee BioMed Central Ltd.

  15. Proteomic analysis during larval development and metamorphosis of the spionid polychaete Pseudopolydora vexillosa

    Directory of Open Access Journals (Sweden)

    Qian Pei-Yuan

    2009-12-01

    Full Text Available Abstract Background While the larval-juvenile transition (metamorphosis in the spionid polychaete Pseudopolydora vexillosa involves gradual morphological changes and does not require substantial development of juvenile organs, the opposite occurs in the barnacle Balanus amphitrite. We hypothesized that the proteome changes during metamorphosis in the spionids are less drastic than that in the barnacles. To test this, proteomes of pre-competent larvae, competent larvae (ready to metamorphose, and juveniles of P. vexillosa were compared using 2-dimensional gel electrophoresis (2-DE, and they were then compared to those of the barnacle. Results Unlike the significant changes found during barnacle metamorphosis, proteomes of competent P. vexillosa larvae were more similar to those of their juveniles. Pre-competent larvae had significantly fewer protein spots (384 spots, while both competent larvae and juveniles expressed about 660 protein spots each. Proteins up-regulated during competence identified by MALDI-TOF/TOF analysis included a molecular chaperon (calreticulin, a signal transduction regulator (tyrosin activation protein, and a tissue-remodeling enzyme (metallopeptidase. Conclusions This was the first time to study the protein expression patterns during the metamorphosis of a marine polychaete and to compare the proteomes of marine invertebrates that have different levels of morphological changes during metamorphosis. The findings provide promising initial steps towards the development of a proteome database for marine invertebrate metamorphosis, thus deciphering the possible mechanisms underlying larval metamorphosis in non-model marine organisms.

  16. Data from proteomic analysis of the skin of Chinese giant salamander (Andrias davidianus

    Directory of Open Access Journals (Sweden)

    Xiaofang Geng

    2015-06-01

    Full Text Available The Chinese giant salamander (Andrias davidianus, renowned as a living fossil, is the largest and longest-lived amphibian species in the world. Its skin is rich in collagens, and has developed mucous gland which could secrete a large amount of mucus under the scraping and electric stimulation. The molting is the degraded skin stratum corneum. To establish the functional skin proteome of Chinese giant salamander, two-dimensional gel electrophoresis (2DE and mass spectrometry (MS were applied to detect the composition and relative abundance of the proteins in the skin, mucus and molting. The determination of the general proteome in the skin can potentially serve as a foundation for future studies characterizing the skin proteomes from diseased salamander to provide molecular and mechanistic insights into various disease states and potential therapeutic interventions. Data presented here are also related to the research article “Proteomic analysis of the skin of Chinese giant salamander (Andrias davidianus” in the Journal of Proteomics [1].

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

    Science.gov (United States)

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

    2016-03-01

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

  18. The analysis of proteome changes in sunflower seeds induced by N+ implantation

    Indian Academy of Sciences (India)

    Dong Guijun; Pan Weidong; Liu Gongshe

    2006-06-01

    In this work, the proteomic changes induced by N+ ion implantation were investigated using a sunflower seed model by a two-dimensional electrophoretic analysis. To further understand the changes of total protein irradiated with N+ ion, a proteomic analysis of N+ ion implantation seeds was developed. Among approximately 369 total protein spots displayed in 2-D gels, eight specific proteins were found in non-implanted seeds while four proteins were found in implanted seeds. Six proteins were used for MALDI-TOF MS analysis, of which only two had been reported before. The proteins designated as No. 29 showed 23.4% homology to MADS-box transcriptional factor HAM59, while No. 279 protein had 23.20% identity to homeobox-leucine zipper protein HAHB-4. The analysis of proteome changes induced by N+ implantation could provid a new clue to studing mutation mechanism of ion implantation. To our knowledge, this is the first report about the analysis of proteome changes induced by N+ implantation in sunflower seeds.

  19. Structural dynamics analysis

    Science.gov (United States)

    Housner, J. M.; Anderson, M.; Belvin, W.; Horner, G.

    1985-01-01

    Dynamic analysis of large space antenna systems must treat the deployment as well as vibration and control of the deployed antenna. Candidate computer programs for deployment dynamics, and issues and needs for future program developments are reviewed. Some results for mast and hoop deployment are also presented. Modeling of complex antenna geometry with conventional finite element methods and with repetitive exact elements is considered. Analytical comparisons with experimental results for a 15 meter hoop/column antenna revealed the importance of accurate structural properties including nonlinear joints. Slackening of cables in this antenna is also a consideration. The technology of designing actively damped structures through analytical optimization is discussed and results are presented.

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

    Science.gov (United States)

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

    2008-07-01

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

  1. Data set for the proteomic inventory and quantitative analysis of chicken uterine fluid during eggshell biomineralization

    Directory of Open Access Journals (Sweden)

    Pauline Marie

    2014-12-01

    Full Text Available Chicken eggshell is the protective barrier of the egg. It is a biomineral composed of 95% calcium carbonate on calcitic form and 3.5% organic matrix proteins. Mineralization process occurs in uterus into the uterine fluid. This acellular fluid contains ions and organic matrix proteins precursors which are interacting with the mineral phase and control crystal growth, eggshell structure and mechanical properties. We performed a proteomic approach and identified 308 uterine fluid proteins. Gene Ontology terms enrichments were determined to investigate their potential functions. Mass spectrometry analyses were also combined to label free quantitative analysis to determine the relative abundance of 96 proteins at initiation, rapid growth phase and termination of shell calcification. Sixty four showed differential abundance according to the mineralization stage. Their potential functions have been annotated. The complete proteomic, bioinformatic and functional analyses are reported in Marie et al., J. Proteomics (2015 [1].

  2. Proteomic profile of acute myeloid leukaemia: A review update

    African Journals Online (AJOL)

    Proteome analysis is a complex and dynamic process that encompasses several analytical platforms ... cellular processes, which in turn affect ..... chemical synthesis of new targeted ..... biomarkers: relevant issues on study design & technical.

  3. Integration of gel-based and gel-free proteomic data for functional analysis of proteins through Soybean Proteome Database

    KAUST Repository

    Komatsu, Setsuko

    2017-05-10

    The Soybean Proteome Database (SPD) stores data on soybean proteins obtained with gel-based and gel-free proteomic techniques. The database was constructed to provide information on proteins for functional analyses. The majority of the data is focused on soybean (Glycine max ‘Enrei’). The growth and yield of soybean are strongly affected by environmental stresses such as flooding. The database was originally constructed using data on soybean proteins separated by two-dimensional polyacrylamide gel electrophoresis, which is a gel-based proteomic technique. Since 2015, the database has been expanded to incorporate data obtained by label-free mass spectrometry-based quantitative proteomics, which is a gel-free proteomic technique. Here, the portions of the database consisting of gel-free proteomic data are described. The gel-free proteomic database contains 39,212 proteins identified in 63 sample sets, such as temporal and organ-specific samples of soybean plants grown under flooding stress or non-stressed conditions. In addition, data on organellar proteins identified in mitochondria, nuclei, and endoplasmic reticulum are stored. Furthermore, the database integrates multiple omics data such as genomics, transcriptomics, metabolomics, and proteomics. The SPD database is accessible at http://proteome.dc.affrc.go.jp/Soybean/. Biological significanceThe Soybean Proteome Database stores data obtained from both gel-based and gel-free proteomic techniques. The gel-free proteomic database comprises 39,212 proteins identified in 63 sample sets, such as different organs of soybean plants grown under flooding stress or non-stressed conditions in a time-dependent manner. In addition, organellar proteins identified in mitochondria, nuclei, and endoplasmic reticulum are stored in the gel-free proteomics database. A total of 44,704 proteins, including 5490 proteins identified using a gel-based proteomic technique, are stored in the SPD. It accounts for approximately 80% of all

  4. Integration of gel-based and gel-free proteomic data for functional analysis of proteins through Soybean Proteome Database.

    Science.gov (United States)

    Komatsu, Setsuko; Wang, Xin; Yin, Xiaojian; Nanjo, Yohei; Ohyanagi, Hajime; Sakata, Katsumi

    2017-06-23

    The Soybean Proteome Database (SPD) stores data on soybean proteins obtained with gel-based and gel-free proteomic techniques. The database was constructed to provide information on proteins for functional analyses. The majority of the data is focused on soybean (Glycine max 'Enrei'). The growth and yield of soybean are strongly affected by environmental stresses such as flooding. The database was originally constructed using data on soybean proteins separated by two-dimensional polyacrylamide gel electrophoresis, which is a gel-based proteomic technique. Since 2015, the database has been expanded to incorporate data obtained by label-free mass spectrometry-based quantitative proteomics, which is a gel-free proteomic technique. Here, the portions of the database consisting of gel-free proteomic data are described. The gel-free proteomic database contains 39,212 proteins identified in 63 sample sets, such as temporal and organ-specific samples of soybean plants grown under flooding stress or non-stressed conditions. In addition, data on organellar proteins identified in mitochondria, nuclei, and endoplasmic reticulum are stored. Furthermore, the database integrates multiple omics data such as genomics, transcriptomics, metabolomics, and proteomics. The SPD database is accessible at http://proteome.dc.affrc.go.jp/Soybean/. The Soybean Proteome Database stores data obtained from both gel-based and gel-free proteomic techniques. The gel-free proteomic database comprises 39,212 proteins identified in 63 sample sets, such as different organs of soybean plants grown under flooding stress or non-stressed conditions in a time-dependent manner. In addition, organellar proteins identified in mitochondria, nuclei, and endoplasmic reticulum are stored in the gel-free proteomics database. A total of 44,704 proteins, including 5490 proteins identified using a gel-based proteomic technique, are stored in the SPD. It accounts for approximately 80% of all predicted proteins from

  5. Genomic, proteomic and biochemical analysis of the organohalide respiratory pathway in Desulfitobacterium dehalogenans

    NARCIS (Netherlands)

    Kruse, T.; Pas, van de B.A.; Atteia, A.; Krab, K.; Hagen, W.R.; Goodwin, L.; Chain, P.; Boeren, S.; Maphosa, F.; Schraa, G.; Vos, de W.M.; Oost, van der J.; Smidt, H.; Stams, A.J.M.

    2015-01-01

    Desulfitobacterium dehalogenans is able to grow by organohalide respiration using 3-chloro-4-hydroxyphenyl acetate (Cl-OHPA) as an electron acceptor. We used a combination of genome sequencing, biochemical analysis of redox active components and shotgun proteomics to study elements of the organohali

  6. Chemostat-based proteomic analysis of toluene-affected Pseudomonas putida S12

    NARCIS (Netherlands)

    Volkers, R.J.M.; Jong, A.L. de; Hulst, A.G.; Baar, B.L.M. van; Bont, J.A.M. de; Wery, J.

    2006-01-01

    The aim of this study was to assess the cellular response of the solvent-tolerant Pseudomonas putida S12 to toluene as the single effector. Proteomic analysis (two-dimensional difference-in-gel-electrophoresis) was used to assess the response of P. putida S12 cultured in chemostats. This approach en

  7. Chemostat-based proteomic analysis of toluene-affected Pseudomonas putida S12

    NARCIS (Netherlands)

    Volkers, R.J.M.; Jong, A.L. de; Hulst, A.G.; Baar, B.L.M. van; Bont, J.A.M. de; Wery, J.

    2006-01-01

    The aim of this study was to assess the cellular response of the solvent-tolerant Pseudomonas putida S12 to toluene as the single effector. Proteomic analysis (two-dimensional difference-in-gel-electrophoresis) was used to assess the response of P. putida S12 cultured in chemostats. This approach en

  8. Proteomics reveals the importance of the dynamic redistribution of the subcellular location of proteins in breast cancer cells.

    Science.gov (United States)

    Pinto, Gabriella; Alhaiek, Abdulrab Ahmed M; Godovac-Zimmermann, Jasminka

    2015-02-01

    At the molecular level, living cells are enormously complicated complex adaptive systems in which intertwined genomic, transcriptomic, proteomic and metabolic networks all play a crucial role. At the same time, cells are spatially heterogeneous systems in which subcellular compartmentalization of different functions is ubiquitous and requires efficient cross-compartmental communication. Dynamic redistribution of multitudinous proteins to different subcellular locations in response to cellular functional state is increasingly recognized as a crucial characteristic of cellular function that seems to be at least as important as overall changes in protein abundance. Characterization of the subcellular spatial dynamics of protein distribution is a major challenge for proteomics and recent results with MCF7 breast cancer cells suggest that this may be of particular importance for cancer cells.

  9. Functional Proteomic Analysis of Lipid Raft Kinase Complexes

    Science.gov (United States)

    2009-08-01

    distinct modes. J Cell Sci 2006;119:3833–44. Supplemental Data Proteome-scale Characterization of Human S-acylated Proteins in Lipid Raft...enriched and Non-raft Membrane Domains Wei Yang, Dolores Di Vizio, Marc Kirchner, Hanno Steen, and Michael R. Freeman Supplemental Tables include: 1...Carboxypeptidase M precursor Raft 2 18/443 – 0.5 0.5 1.0 1 1 1.0 0 0 0.0 181 + + IPI00032038 CPT1A Isoform 1 of Carnitine O-palmitoyltransferase I, liver isoform

  10. Dynamic Contingency Analysis Tool

    Energy Technology Data Exchange (ETDEWEB)

    2016-01-14

    The Dynamic Contingency Analysis Tool (DCAT) is an open-platform and publicly available methodology to help develop applications that aim to improve the capabilities of power system planning engineers to assess the impact and likelihood of extreme contingencies and potential cascading events across their systems and interconnections. Outputs from the DCAT will help find mitigation solutions to reduce the risk of cascading outages in technically sound and effective ways. The current prototype DCAT implementation has been developed as a Python code that accesses the simulation functions of the Siemens PSS�E planning tool (PSS/E). It has the following features: It uses a hybrid dynamic and steady-state approach to simulating the cascading outage sequences that includes fast dynamic and slower steady-state events. It integrates dynamic models with protection scheme models for generation, transmission, and load. It models special protection systems (SPSs)/remedial action schemes (RASs) and automatic and manual corrective actions. Overall, the DCAT attempts to bridge multiple gaps in cascading-outage analysis in a single, unique prototype tool capable of automatically simulating and analyzing cascading sequences in real systems using multiprocessor computers.While the DCAT has been implemented using PSS/E in Phase I of the study, other commercial software packages with similar capabilities can be used within the DCAT framework.

  11. Dynamic Contingency Analysis Tool

    Energy Technology Data Exchange (ETDEWEB)

    2016-01-14

    The Dynamic Contingency Analysis Tool (DCAT) is an open-platform and publicly available methodology to help develop applications that aim to improve the capabilities of power system planning engineers to assess the impact and likelihood of extreme contingencies and potential cascading events across their systems and interconnections. Outputs from the DCAT will help find mitigation solutions to reduce the risk of cascading outages in technically sound and effective ways. The current prototype DCAT implementation has been developed as a Python code that accesses the simulation functions of the Siemens PSS/E planning tool (PSS/E). It has the following features: It uses a hybrid dynamic and steady-state approach to simulating the cascading outage sequences that includes fast dynamic and slower steady-state events. It integrates dynamic models with protection scheme models for generation, transmission, and load. It models special protection systems (SPSs)/remedial action schemes (RASs) and automatic and manual corrective actions. Overall, the DCAT attempts to bridge multiple gaps in cascading-outage analysis in a single, unique prototype tool capable of automatically simulating and analyzing cascading sequences in real systems using multiprocessor computers.While the DCAT has been implemented using PSS/E in Phase I of the study, other commercial software packages with similar capabilities can be used within the DCAT framework.

  12. Proteomic analysis of middle and late stages of bread wheat (Triticum aestivum L. grain development

    Directory of Open Access Journals (Sweden)

    Ning eZhang

    2015-09-01

    Full Text Available Proteomic approaches were applied in four grain developmental stages of the Chinese bread wheat Yunong 201 and its ethyl methanesulfonate (EMS mutant line Yunong 3114. 2-DE and tandem MALDI-TOF/TOF-MS analyzed proteome characteristics during middle and late grain development of the Chinese bread wheat Yunong 201 and its EMS mutant line Yunong 3114 with larger grain sizes. We identified 130 differentially accumulated protein spots representing 88 unique proteins, and four main expression patterns displayed a dynamic description of middle and late grain formation. Those identified protein species participated in eight biochemical processes: stress/defense, carbohydrate metabolism, protein synthesis/assembly/degradation, storage proteins, energy production and transportation, photosynthesis, transcription/translation, signal transduction. Comparative proteomic characterization demonstrated 12 protein spots that co-accumulated in the two wheat cultivars with different expression patterns, and 6 cultivar-specific protein spots including serpin, small heat shock protein, beta-amylase, alpha-amylase inhibitor, dimeric alpha-amylase inhibitor precursor, and cold regulated protein. These cultivar-specific protein spots possibly resulted in differential yield-related traits of the two wheat cultivars. Our results provide valuable information for dissection of molecular and genetics basis of yield-related traits in bread wheat and the proteomic characterization in this study could also provide insights in the biology of late grain development.

  13. Peptide-Centric Proteome Analysis: An Alternative Strategy for the Analysis of Tandem Mass Spectrometry Data

    Energy Technology Data Exchange (ETDEWEB)

    Ting, Ying S.; Egertson, Jarrett D.; Payne, Samuel H.; Kim, Sangtae; MacLean, Brendan; Kall, Lukas; Aebersold, Ruedi; Smith, Richard D.; Noble, William; MacCoss, Michael

    2015-09-01

    In mass spectrometry-based bottom-up proteomics, data-independent acquisition (DIA) is an emerging technique due to its comprehensive and unbiased sampling of precursor ions. However, current DIA methods use wide precursor isolation windows, resulting in co- fragmentation and complex mixture spectra. Thus, conventional database searching tools that identify peptides by interpreting individual MS/MS spectra are inherently limited in analyzing DIA data. Here we discuss an alternative approach, peptide-centric analysis, which tests directly for the presence and absence of query peptides. We discuss how peptide-centric analysis resolves some limitations of traditional spectrum-centric analysis, and we outline the benefits of peptide-centric analysis in general.

  14. Proteomics Core

    Data.gov (United States)

    Federal Laboratory Consortium — Proteomics Core is the central resource for mass spectrometry based proteomics within the NHLBI. The Core staff help collaborators design proteomics experiments in a...

  15. Proteomics Core

    Data.gov (United States)

    Federal Laboratory Consortium — Proteomics Core is the central resource for mass spectrometry based proteomics within the NHLBI. The Core staff help collaborators design proteomics experiments in...

  16. Proteome analysis of the rice etioplast: metabolic and regulatory networks and novel protein functions.

    Science.gov (United States)

    von Zychlinski, Anne; Kleffmann, Torsten; Krishnamurthy, Nandini; Sjölander, Kimmen; Baginsky, Sacha; Gruissem, Wilhelm

    2005-08-01

    We report an extensive proteome analysis of rice etioplasts, which were highly purified from dark-grown leaves by a novel protocol using Nycodenz density gradient centrifugation. Comparative protein profiling of different cell compartments from leaf tissue demonstrated the purity of the etioplast preparation by the absence of diagnostic marker proteins of other cell compartments. Systematic analysis of the etioplast proteome identified 240 unique proteins that provide new insights into heterotrophic plant metabolism and control of gene expression. They include several new proteins that were not previously known to localize to plastids. The etioplast proteins were compared with proteomes from Arabidopsis chloroplasts and plastid from tobacco Bright Yellow 2 cells. Together with computational structure analyses of proteins without functional annotations, this comparative proteome analysis revealed novel etioplast-specific proteins. These include components of the plastid gene expression machinery such as two RNA helicases, an RNase II-like hydrolytic exonuclease, and a site 2 protease-like metalloprotease all of which were not known previously to localize to the plastid and are indicative for so far unknown regulatory mechanisms of plastid gene expression. All etioplast protein identifications and related data were integrated into a data base that is freely available upon request.

  17. Protein extraction from Ca-alginate encapsulated plant material for comparative proteomic analysis.

    Science.gov (United States)

    Domżalska, Lucyna; Mikuła, Anna; Rybczyński, Jan J

    2016-10-01

    The extensive use of encapsulation material in biotechnology drove the need to develop analytical techniques for this type of material. This study focuses on the specific problems of protein extraction from Ca-alginate encapsulated plant material. Proteomics is one of the fast-developing analysis categories, specifically for stress resistance and developmental changes in plant material. Sample preparation is a critical step in a two-dimensional gel electrophoresis proteome approach and is essential for good results. The aim was to avoid preliminary manipulations and get good quality material for comparative proteome analysis technique 2DE. The phenol extraction method and the complex method with preliminary TCA precipitation, SDS buffer and phenol phase were compared with respect to the efficiency and quality of the resulting 2DE gel. The most appropriate method turned out to be the TCA/phenol method with the phenol fractioning technique adapted to the gentian cell suspension. It resulted in a high protein concentration and good quality sample that could be analyzed using the standard separation procedures of 2DE and spectrometric identification with high efficiency. The work presented here confirms the possibility of obtaining a sufficient protein sample for effective proteomic analysis from a small number of capsules.

  18. Whole-Proteome Analysis of Twelve Species of Alphaproteobacteria Links Four Pathogens

    Directory of Open Access Journals (Sweden)

    Yunyun Zhou

    2013-11-01

    Full Text Available Thousands of whole-genome and whole-proteome sequences have been made available through advances in sequencing technology, and sequences of millions more organisms will become available in the coming years. This wealth of genetic information will provide numerous opportunities to enhance our understanding of these organisms including a greater understanding of relationships among species. Researchers have used 16S rRNA and other gene sequences to study the evolutionary origins of bacteria, but these strategies do not provide insight into the sharing of genes among bacteria via horizontal transfer. In this work we use an open source software program called pClust to cluster proteins from the complete proteomes of twelve species of Alphaproteobacteria and generate a dendrogram from the resulting orthologous protein clusters. We compare the results with dendrograms constructed using the 16S rRNA gene and multiple sequence alignment of seven housekeeping genes. Analysis of the whole proteomes of these pathogens grouped Rickettsia typhi with three other animal pathogens whereas conventional sequence analysis failed to group these pathogens together. We conclude that whole-proteome analysis can give insight into relationships among species beyond their phylogeny, perhaps reflecting the effects of horizontal gene transfer and potentially providing insight into the functions of shared genes by means of shared phenotypes.

  19. Proteomics based compositional analysis of complex cellulase-hemicellulase mixtures

    Energy Technology Data Exchange (ETDEWEB)

    Chundawat, Shishir P.; Lipton, Mary S.; Purvine, Samuel O.; Uppugundla, Nirmal; Gao, Dahai; Balan, Venkatesh; Dale, Bruce E.

    2011-10-07

    Efficient deconstruction of cellulosic biomass to fermentable sugars for fuel and chemical production is accomplished by a complex mixture of cellulases, hemicellulases and accessory enzymes (e.g., >50 extracellular proteins). Cellulolytic enzyme mixtures, produced industrially mostly using fungi like Trichoderma reesei, are poorly characterized in terms of their protein composition and its correlation to hydrolytic activity on cellulosic biomass. The secretomes of commercial glycosyl hydrolase producing microbes was explored using a proteomics approach with high-throughput quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Here, we show that proteomics based spectral counting approach is a reasonably accurate and rapid analytical technique that can be used to determine protein composition of complex glycosyl hydrolase mixtures that also correlates with the specific activity of individual enzymes present within the mixture. For example, a strong linear correlation was seen between Avicelase activity and total cellobiohydrolase content. Reliable, quantitative and cheaper analytical methods that provide insight into the cellulosic biomass degrading fungal and bacterial secretomes would lead to further improvements towards commercialization of plant biomass derived fuels and chemicals.

  20. Proteomic Analysis of Rat Hippocampus under Simulated Microgravity

    Science.gov (United States)

    Wang, Yun; Li, Yujuan; Zhang, Yongqian; Liu, Yahui; Deng, Yulin

    It has been found that microgravity may lead to impairments in cognitive functions performed by CNS. However, the exact mechanism of effects of microgravity on the learning and memory function in animal nervous system is not elucidated yet. Brain function is mainly mediated by membrane proteins and their dysfunction causes degeneration of the learning and memory. To induce simulated microgravity, the rat tail suspension model was established. Comparative O (18) labeling quantitative proteomic strategy was applied to detect the differentially expressed proteins in rat brain hippocampus. The proteins in membrane fraction from rat hippocampus were digested by trypsin and then the peptides were separated by off-gel for the first dimension with 24 wells device encompassing the pH range of 3 - 10. An off-gel fraction was subjected into LC-ESI-QTOF in triplicate. Preliminary results showed that nearly 77% of the peptides identified were specific to one fraction. 676 proteins were identified among which 108 proteins were found differentially expressed under simulated microgravity. Using the KOBAS server, many enriched pathways, such as metabolic pathway, synaptic vesicle cycle, endocytosis, calcium signaling pathway, and SNAREs pathway were identified. Furthermore, it has been found that neurotransmitter released by Ca (2+) -triggered synaptic vesicles fusion may play key role in neural function. Rab 3A might inhibit the membrane fusion and neurotransmitter release. The protein alteration of the synaptic vesicle cycle may further explain the effects of microgravity on learning and memory function in rats. Key words: Microgravity; proteomics; synaptic vesicle; O (18) ({}) -labeling

  1. Proteome analysis of Acetobacter pasteurianus during acetic acid fermentation.

    Science.gov (United States)

    Andrés-Barrao, Cristina; Saad, Maged M; Chappuis, Marie-Louise; Boffa, Mauro; Perret, Xavier; Ortega Pérez, Ruben; Barja, François

    2012-03-16

    Acetic acid bacteria (AAB) are Gram-negative, strictly aerobic microorganisms that show a unique resistance to ethanol (EtOH) and acetic acid (AcH). Members of the Acetobacter and Gluconacetobacter genera are capable of transforming EtOH into AcH via the alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) enzymes and are used for the industrial production of vinegar. Several mechanisms have been proposed to explain how AAB resist high concentrations of AcH, such as the assimilation of acetate through the tricarboxylic acid (TCA) cycle, the export of acetate by various transporters and modifications of the outer membrane. However, except for a few acetate-specific proteins, little is known about the global proteome responses to AcH. In this study, we used 2D-DIGE to compare the proteome of Acetobacter pasteurianus LMG 1262(T) when growing in glucose or ethanol and in the presence of acetic acid. Interesting protein spots were selected using the ANOVA p-value of 0.05 as threshold and 1.5-fold as the minimal level of differential expression, and a total of 53 proteins were successfully identified. Additionally, the size of AAB was reduced by approximately 30% in length as a consequence of the acidity. A modification in the membrane polysaccharides was also revealed by PATAg specific staining.

  2. Cell wall proteome analysis of Arabidopsis thaliana mature stems.

    Science.gov (United States)

    Duruflé, Harold; Clemente, Hélène San; Balliau, Thierry; Zivy, Michel; Dunand, Christophe; Jamet, Elisabeth

    2017-04-01

    Plant stems carry flowers necessary for species propagation and need to be adapted to mechanical disturbance and environmental factors. The stem cell walls are different from other organs and can modify their rigidity or viscoelastic properties for the integrity and the robustness required to withstand mechanical impacts and environmental stresses. Plant cell wall is composed of complex polysaccharide networks also containing cell wall proteins (CWPs) crucial to perceive and limit the environmental effects. The CWPs are fundamental players in cell wall remodeling processes, and today, only 86 have been identified from the mature stems of the model plant Arabidopsis thaliana. With a destructive method, this study has enlarged its coverage to 302 CWPs. This new proteome is mainly composed of 27.5% proteins acting on polysaccharides, 16% proteases, 11.6% oxido-reductases, 11% possibly related to lipid metabolism and 11% of proteins with interacting domains with proteins or polysaccharides. Compared to stem cell wall proteomes already available (Brachypodium distachyon, Sacharum officinarum, Linum usitatissimum, Medicago sativa), that of A. thaliana stems has a higher proportion of proteins acting on polysaccharides and of proteases, but a lower proportion of oxido-reductases. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Proteomic Analysis of Cucumber Defense Rresponses Induced by Propamocarb

    Institute of Scientific and Technical Information of China (English)

    WU Peng; QIN Zhi-wei; WU Tao; ZHOU Xiu-yan; XIN Ming; GUO Qian-qian

    2013-01-01

    Propamocarb is an agricultural chemical that has been widely used to protect cucumber plants from downy mildew. To understand the mechanisms of cucumber defense responses to propamocarb, we investigated the physiological and proteomic responses of the cucumber line D0351 with propamocarb application. We found that after treatment with propamocarb, the activities of detoxifying enzymes (glutathione reductase, GR; glutathione S-tramsferase, GST) and soluble sugar content of cucumber fruit were signiifcantly increased, but malonaldehyde (MDA) content was signiifcantly reduced. To identify components of propamocarb responsive signaling, we compared the high resolution two-dimensional gel electrophoresis (2-DE) protein proifles of control and propamocarb-treated fruits, and identiifed 18 differentially expressed (13 up-regulated and 5 down-regulated) proteins induced by propamocarb which were determined by matrix-assisted laser desorption/ionization time-of-lfight mass spectrometry (MALDI-TOF-MS). The majority of the proteins had functions related to detoxication, energy and transport, protein biosynthesis, regulating reactions and defending against stresses. A real-time quantitative reverse transcriptional-polymerase chain reaction (qRT-PCR) was used to compare transcript and protein accumulation patterns for 18 candidate proteins, and the expression of 14 was consistent at both transcript and protein levels. The responses of cucumber proteome to propamocarb seemed complex; the identified proteins may play an important role in regulating adaptation activities following exposure to propamocarb. Data presented herein may shed light on understanding cucumber fruit defense responses under propamocarb treatment.

  4. Proteomics analysis of the endogenous, constitutive, leaf SUMOylome.

    Science.gov (United States)

    Colignon, Bertrand; Delaive, Edouard; Dieu, Marc; Demazy, Catherine; Muhovski, Yordan; Wallon, Cindy; Raes, Martine; Mauro, Sergio

    2017-01-06

    SUMOylation is a post-translational modification which regulates a number of critical biological processes in, for example mammals, yeast and plants. In order to fully understand the functional effects of SUMOylation an essential first step is the identification of endogenous targets for SUMOylation. Here we report the results of using a recently developed proteomic approach based on the use of 3D gels to identify the endogenous SUMO targets in leaves of Solanum tuberosum. By using 3D gels we avoid the problem of co-migration of proteins, which is a major limitation of 2D gels, and we enable the use of the highly sensitive CyDye DIGE fluor saturation dyes. Using this new method we have identified 39 individual proteins as probable SUMO targets in leaves of Solanum tuberosum. The advantages of this method compared with other approaches are discussed, and possible future developments are outlined. The authors have no conflicts of interest to declare. All authors have approved the manuscript and agree with submission to Journal of Proteomics. Copyright © 2016. Published by Elsevier B.V.

  5. Gel-free shotgun proteomic analysis of human milk.

    Science.gov (United States)

    Picariello, Gianluca; Ferranti, Pasquale; Mamone, Gianfranco; Klouckova, Iveta; Mechref, Yehia; Novotny, Milos V; Addeo, Francesco

    2012-03-02

    The composition of milk has adapted during the evolution of the species to fulfill the specific nutritional needs of the offspring. Currently, it is widely recognized that milk benefits go beyond mere nutrition and serve as a source of a number of functional components to the newborn, particularly host defense effectors. However, the human milk proteome description is still incomplete, primarily because the detection of low-abundance proteins remains challenging. To overcome the limitations of the classical electrophoresis-based approach, previously separated milk fat globule membrane (MFGM) and whey protein fractions were analyzed by nanoflow-high performance liquid chromatography (HPLC)/Fourier Transform-Ion Cyclotron Resonance (FT-ICR) mass spectrometry (MS). This shotgun strategy showed an as yet unmatched potential to profile low-abundance proteins in human milk. Proteins associated with 301 different gene products were identified, some of which could be clustered into subsets of protein isoforms, thus providing one of the largest protein inventories of human milk. The identified proteins, which were derived from multiple metabolic pathways, are involved in different physiological functions, such as membrane trafficking, cell signaling, fat metabolism and transport, metabolite delivery, protein synthesis/proteolysis or folding, and immunity-related actions. Nevertheless, it appears clear from this study that the overall picture of the human milk proteome is still incomplete, although several protein signatures of milk evolution are emerging.

  6. A label-free proteome analysis strategy for identifying quantitative changes in erythrocyte membranes induced by red cell disorders.

    Science.gov (United States)

    Pesciotta, Esther N; Sriswasdi, Sira; Tang, Hsin-Yao; Mason, Philip J; Bessler, Monica; Speicher, David W

    2012-12-05

    Red blood cells have been extensively studied but many questions regarding membrane properties and pathophysiology remain unanswered. Proteome analysis of red cell membranes is complicated by a very wide dynamic range of protein concentrations as well as the presence of proteins that are very large, very hydrophobic, or heterogeneously glycosylated. This study investigated the removal of other blood cell types, red cell membrane extraction, differing degrees of fractionation using 1-D SDS gels, and label-free quantitative methods to determine optimized conditions for proteomic comparisons of clinical blood samples. The results showed that fractionation of red cell membranes on 1-D SDS gels was more efficient than low-ionic-strength extractions followed by 1-D gel fractionation. When gel lanes were sliced into 30 uniform slices, a good depth of analysis that included the identification of most well-characterized, low-abundance red cell membrane proteins including those present at 500 to 10,000 copies per cell was obtained. Furthermore, the size separation enabled detection of changes due to proteolysis or in vivo protein crosslinking. A combination of Rosetta Elucidator quantitation and subsequent statistical analysis enabled the robust detection of protein differences that could be used to address unresolved questions in red cell disorders. This article is part of a Special Issue entitled: Integrated omics.

  7. Characterization of the mouse pancreatic islet proteome and comparative analysis with other mouse tissues

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-08-01

    The pancreatic islets of Langerhans and insulin-producing beta cells in particular play a central role in the maintenance of glucose homeostasis and the islet dysfunction is associated with the pathogenesis of both type 1 and type 2 diabetes mellitus. To contribute to the understanding of the biology of the pancreatic islets we applied proteomic techniques based on liquid chromatography coupled with mass spectrometry. Here as an initial step we present the first comprehensive proteomic characterization of pancreas islets of the mouse, the commonly used animal model for diabetes research. Two-dimensional SCX LC/RP LC-MS/MS has been applied to characterize of the mouse islet proteome, resulting in the confident identification of 17,350 different tryptic peptides covering 2,612 proteins with at least two unique peptide identifications per protein. The dataset also allowed identification of a number of post-translational modifications including several modifications relevant to oxidative stress and phosphorylation. While many of the identified phosphorylation sites corroborates with previous known sites, the oxidative modifications observed on cysteinyl residues potentially reveal novel information related to the role of oxidation stress in islet functions. Comparative analysis of the islet proteome database with 15 available proteomic datasets from other mouse tissues and cells revealed a set of 68 proteins uniquely detected only in the pancreatic islets. Besides proteins with known functions, like islet secreted peptide hormones, this unique set contains a number of proteins with yet unknown functions. The resulting peptide and protein database will be available at ncrr.pnl.gov web site of the NCRR proteomic center (ncrr.pnl.gov).

  8. Personalized medicine beyond genomics: alternative futures in big data-proteomics, environtome and the social proteome.

    Science.gov (United States)

    Özdemir, Vural; Dove, Edward S; Gürsoy, Ulvi K; Şardaş, Semra; Yıldırım, Arif; Yılmaz, Şenay Görücü; Ömer Barlas, I; Güngör, Kıvanç; Mete, Alper; Srivastava, Sanjeeva

    2017-01-01

    No field in science and medicine today remains untouched by Big Data, and psychiatry is no exception. Proteomics is a Big Data technology and a next generation biomarker, supporting novel system diagnostics and therapeutics in psychiatry. Proteomics technology is, in fact, much older than genomics and dates to the 1970s, well before the launch of the international Human Genome Project. While the genome has long been framed as the master or "elite" executive molecule in cell biology, the proteome by contrast is humble. Yet the proteome is critical for life-it ensures the daily functioning of cells and whole organisms. In short, proteins are the blue-collar workers of biology, the down-to-earth molecules that we cannot live without. Since 2010, proteomics has found renewed meaning and international attention with the launch of the Human Proteome Project and the growing interest in Big Data technologies such as proteomics. This article presents an interdisciplinary technology foresight analysis and conceptualizes the terms "environtome" and "social proteome". We define "environtome" as the entire complement of elements external to the human host, from microbiome, ambient temperature and weather conditions to government innovation policies, stock market dynamics, human values, political power and social norms that collectively shape the human host spatially and temporally. The "social proteome" is the subset of the environtome that influences the transition of proteomics technology to innovative applications in society. The social proteome encompasses, for example, new reimbursement schemes and business innovation models for proteomics diagnostics that depart from the "once-a-life-time" genotypic tests and the anticipated hype attendant to context and time sensitive proteomics tests. Building on the "nesting principle" for governance of complex systems as discussed by Elinor Ostrom, we propose here a 3-tiered organizational architecture for Big Data science such as

  9. Proteomic analysis of Streptomyces coelicolor in response to Ciprofloxacin challenge.

    Science.gov (United States)

    Rao, Aishwarya Anand; Patkari, Minal; Reddy, Panga Jaipal; Srivastava, Rajneesh; Pendharkar, Namita; Rapole, Srikanth; Mehra, Sarika; Srivastava, Sanjeeva

    2014-01-31

    Multi-drug tolerance is an important phenotypic property that complicates treatment of infectious diseases and reshapes drug discovery. Hence a systematic study of the origins and mechanisms of resistance shown by microorganisms is imperative. Since soil-dwelling bacteria are constantly challenged with a myriad of antibiotics, they are potential reservoirs of resistance determinants that can be mobilized into pathogens over a period of time. Elucidating the resistance mechanisms in such bacteria could help future antibiotic discoveries. This research is a preliminary study conducted to determine the effects of ciprofloxacin (CIP) on the intrinsically resistant Gram-positive soil bacterium Streptomyces coelicolor. The effect was investigated by performing 2-DE on total protein extracts of cells exposed to sub-lethal concentrations of ciprofloxacin as compared to the controls. Protein identification by MALDI-TOF/TOF revealed 24 unique differentially expressed proteins, which were statistically significant. The down-regulation of proteins involved in carbohydrate metabolism indicated a shift in the cell physiology towards a state of metabolic shutdown. Furthermore, the observed decline in protein levels involved in transcription and translation machinery, along with depletion of enzymes involved in amino acid biosynthesis and protein folding could be a cellular response to DNA damage caused by CIP, thereby minimizing the effect of defective and energetically wasteful metabolic processes. This could be crucial for the initial survival of the cells before gene level changes could come into play to ensure survival under prolonged adverse conditions. These results are a first attempt towards profiling the proteome of S. coelicolor in response to antibiotic stress. This article is part of a Special Issue entitled: Trends in Microbial Proteomics. Soil-dwelling bacteria could serve as a reservoir of resistance determinants for clinically important bacteria. In this work, we

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

    Science.gov (United States)

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

    2011-08-01

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

  11. Comparative proteomics analysis of placenta from pregnant women with intrahepatic cholestasis of pregnancy.

    Science.gov (United States)

    Zhang, Ting; Guo, Yueshuai; Guo, Xuejiang; Zhou, Tao; Chen, Daozhen; Xiang, Jingying; Zhou, Zuomin

    2013-01-01

    Intrahepatic cholestasis of pregnancy (ICP) usually occurs in the third trimester and associated with increased risks in fetal complications. Currently, the exact cause of this disease is unknown. In this study we aim to investigate the potential proteins in placenta, which may participate in the molecular mechanisms of ICP-related fetal complications using iTRAQ-based proteomics approach. The iTRAQ analysis combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed to separate differentially expressed placental proteins from 4 pregnant women with ICP and 4 healthy pregnant women. Bioinformatics analysis was used to find the relative processes that these differentially expressed proteins were involved in. Three apoptosis related proteins ERp29, PRDX6 and MPO that resulted from iTRAQ-based proteomics were further verified in placenta by Western blotting and immunohistochemistry. Placental apoptosis was also detected by TUNEL assay. Proteomics results showed there were 38 differentially expressed proteins from pregnant women with ICP and healthy pregnant women, 29 were upregulated and 9 were downregulated in placenta from pregnant women with ICP. Bioinformatics analysis showed most of the identified proteins was functionally related to specific cell processes, including apoptosis, oxidative stress, lipid metabolism. The expression levels of ERp29, PRDX6 and MPO were consistent with the proteomics data. The apoptosis index in placenta from ICP patients was significantly increased. This preliminary work provides a better understanding of the proteomic alterations of placenta from pregnant women with ICP and may provide us some new insights into the pathophysiology and potential novel treatment targets for ICP.

  12. Comparative proteomics analysis of placenta from pregnant women with intrahepatic cholestasis of pregnancy.

    Directory of Open Access Journals (Sweden)

    Ting Zhang

    Full Text Available INTRODUCTION: Intrahepatic cholestasis of pregnancy (ICP usually occurs in the third trimester and associated with increased risks in fetal complications. Currently, the exact cause of this disease is unknown. In this study we aim to investigate the potential proteins in placenta, which may participate in the molecular mechanisms of ICP-related fetal complications using iTRAQ-based proteomics approach. METHODS: The iTRAQ analysis combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS was performed to separate differentially expressed placental proteins from 4 pregnant women with ICP and 4 healthy pregnant women. Bioinformatics analysis was used to find the relative processes that these differentially expressed proteins were involved in. Three apoptosis related proteins ERp29, PRDX6 and MPO that resulted from iTRAQ-based proteomics were further verified in placenta by Western blotting and immunohistochemistry. Placental apoptosis was also detected by TUNEL assay. RESULTS: Proteomics results showed there were 38 differentially expressed proteins from pregnant women with ICP and healthy pregnant women, 29 were upregulated and 9 were downregulated in placenta from pregnant women with ICP. Bioinformatics analysis showed most of the identified proteins was functionally related to specific cell processes, including apoptosis, oxidative stress, lipid metabolism. The expression levels of ERp29, PRDX6 and MPO were consistent with the proteomics data. The apoptosis index in placenta from ICP patients was significantly increased. CONCLUSION: This preliminary work provides a better understanding of the proteomic alterations of placenta from pregnant women with ICP and may provide us some new insights into the pathophysiology and potential novel treatment targets for ICP.

  13. Extraction of intracellular protein from Glaciozyma antarctica for proteomics analysis

    Science.gov (United States)

    Faizura, S. Nor; Farahayu, K.; Faizal, A. B. Mohd; Asmahani, A. A. S.; Amir, R.; Nazalan, N.; Diba, A. B. Farah; Muhammad, M. Nor; Munir, A. M. Abdul

    2013-11-01

    Two preparation methods of crude extracts of psychrophilic yeast Glaciozyma antarctica were compared in order to obtain a good recovery of intracellular proteins. Extraction with mechanical procedures using sonication was found to be more effective for obtaining good yield compare to alkaline treatment method. The procedure is simple, rapid, and produce better yield. A total of 52 proteins were identified by combining both extraction methods. Most of the proteins identified in this study involves in the metabolic process including glycolysis pathway, pentose phosphate pathway, pyruyate decarboxylation and also urea cyle. Several chaperons were identified including probable cpr1-cyclophilin (peptidylprolyl isomerase), macrolide-binding protein fkbp12 and heat shock proteins which were postulate to accelerate proper protein folding. Characteristic of the fundamental cellular processes inferred from the expressed-proteome highlight the evolutionary and functional complexity existing in this domain of life.

  14. Centrosome isolation and analysis by mass spectrometry-based proteomics

    DEFF Research Database (Denmark)

    Jakobsen, Lis; Schrøder, Jacob Morville; Larsen, Katja M

    2013-01-01

    Centrioles are microtubule-based scaffolds that are essential for the formation of centrosomes, cilia, and flagella with important functions throughout the cell cycle, in physiology and during development. The ability to purify centriole-containing organelles on a large scale, combined with advan......Centrioles are microtubule-based scaffolds that are essential for the formation of centrosomes, cilia, and flagella with important functions throughout the cell cycle, in physiology and during development. The ability to purify centriole-containing organelles on a large scale, combined...... with advances in protein identification using mass spectrometry-based proteomics, have revealed multiple centriole-associated proteins that are conserved during evolution in eukaryotes. Despite these advances, the molecular basis for the plethora of processes coordinated by cilia and centrosomes is not fully...

  15. Proteomic Analysis of Trypanosoma cruzi Epimastigotes Subjected to Heat Shock

    Directory of Open Access Journals (Sweden)

    Deyanira Pérez-Morales

    2012-01-01

    Full Text Available Trypanosoma cruzi is exposed to sudden temperature changes during its life cycle. Adaptation to these variations is crucial for parasite survival, reproduction, and transmission. Some of these conditions may change the pattern of genetic expression of proteins involved in homeostasis in the course of stress treatment. In the present study, the proteome of T. cruzi epimastigotes subjected to heat shock and epimastigotes grow normally was compared by two-dimensional gel electrophoresis followed by mass spectrometry for protein identification. Twenty-four spots differing in abundance were identified. Of the twenty-four changed spots, nineteen showed a greater intensity and five a lower intensity relative to the control. Several functional categories of the identified proteins were determined: metabolism, cell defense, hypothetical proteins, protein fate, protein synthesis, cellular transport, and cell cycle. Proteins involved in the interaction with the cellular environment were also identified, and the implications of these changes are discussed.

  16. Detection of novel biomarkers of liver cirrhosis by proteomic analysis

    DEFF Research Database (Denmark)

    Mölleken, Christian; Sitek, Barbara; Henkel, Corinna;

    2009-01-01

    liver biopsy allows a reliable evaluation of the course of hepatitis C by grading inflammation and staging fibrosis, and thus serum biomarkers for hepatic fibrosis with high sensitivity and specificity are needed. To identify new candidate biomarkers for hepatic fibrosis, we performed a proteomic......Hepatic cirrhosis is a life-threatening disease arising from different chronic liver disorders. One major cause for hepatic cirrhosis is chronic hepatitis C. Chronic hepatitis C is characterized by a highly variable clinical course, with at least 20% developing liver cirrhosis within 40 years. Only...... approach of microdissected cirrhotic septa and liver parenchyma cells. In cirrhotic septa, we detected an increasing expression of cell structure associated proteins, including actin, prolyl 4-hydroxylase, tropomyosin, calponin, transgelin, and human microfibril-associated protein 4 (MFAP-4). Tropomyosin...

  17. Proteomic tools for the analysis of transient interactions between metalloproteins.

    Science.gov (United States)

    Martínez-Fábregas, Jonathan; Rubio, Silvia; Díaz-Quintana, Antonio; Díaz-Moreno, Irene; De la Rosa, Miguel Á

    2011-05-01

    Metalloproteins play major roles in cell metabolism and signalling pathways. In many cases, they show moonlighting behaviour, acting in different processes, depending on the physiological state of the cell. To understand these multitasking proteins, we need to discover the partners with which they carry out such novel functions. Although many technological and methodological tools have recently been reported for the detection of protein interactions, specific approaches to studying the interactions involving metalloproteins are not yet well developed. The task is even more challenging for metalloproteins, because they often form short-lived complexes that are difficult to detect. In this review, we gather the different proteomic techniques and biointeractomic tools reported in the literature. All of them have shown their applicability to the study of transient and weak protein-protein interactions, and are therefore suitable for metalloprotein interactions.

  18. Proteomic analysis of Taenia solium metacestode excretion-secretion proteins.

    Science.gov (United States)

    Victor, Bjorn; Kanobana, Kirezi; Gabriël, Sarah; Polman, Katja; Deckers, Nynke; Dorny, Pierre; Deelder, André M; Palmblad, Magnus

    2012-06-01

    The metacestode larval stage of Taenia solium is the causal agent of a zoonotic disease called cysticercosis. The disease has an important impact on pork trade (due to porcine cysticercosis) and public health (due to human neurocysticercosis). In order to improve the current diagnostic tools and to get a better understanding of the interaction between T. solium metacestodes and their host, there is a need for more information about the proteins that are released by the parasite. In this study, we used protein sequences from different helminths, 1DE, reversed-phase LC, and MS/MS to analyze the excretion-secretion proteins produced by T. solium metacestodes from infected pigs. This is the first report of the T. solium metacestode excretion-secretion proteome. We report 76 proteins including 27 already described T. solium proteins, 17 host proteins and 32 proteins likely to be of T. solium origin, but identified using sequences from other helminths.

  19. Biomechanical and proteomic analysis of INF- {beta}-treated astrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Vergara, Daniele; Leporatti, Stefano; Maruccio, Giuseppe; Cingolani, Roberto; Rinaldi, Ross [National Nanotechnology Laboratory of CNR-INFM, ISUFI, University of Lecce, Italian Institute of Technology (IIT) Research Unit, via Arnesano, I-73100 Lecce (Italy); Martignago, Roberta; Nuccio, Franco De; Nicolardi, Giuseppe; Maffia, Michele [Department of Biological and Environmental Sciences and Technologies, University of Salento, via Monteroni, I-73100 Lecce (Italy); Bonsegna, Stefania; Santino, Angelo, E-mail: michele.maffia@unile.i, E-mail: ross.rinaldi@unile.i [Institute of Sciences of Food Production CNR, Unit of Lecce I-73100 (Italy)

    2009-11-11

    Astrocytes have a key role in the pathogenesis of several diseases including multiple sclerosis and were proposed as the designed target for immunotherapy. In this study we used atomic force microscopy (AFM) and proteomics methods to analyse and correlate the modifications induced in the viscoleastic properties of astrocytes to the changes induced in protein expression after interferon- {beta} (IFN-{beta}) treatment. Our results indicated that IFN-{beta} treatment resulted in a significant decrease in the Young's modulus, a measure of cell elasticity, in comparison with control cells. The molecular mechanisms that trigger these changes were investigated by 2DE (two-dimensional electrophoresis) and confocal analyses and confirmed by western blotting. Altered proteins were found to be involved in cytoskeleton organization and other important physiological processes.

  20. Proteomic analysis of the cyst stage of Entamoeba histolytica.

    Directory of Open Access Journals (Sweden)

    Ibne Karim M Ali

    Full Text Available The category B agent of bioterrorism, Entamoeba histolytica has a two-stage life cycle: an infective cyst stage, and an invasive trophozoite stage. Due to our inability to effectively induce encystation in vitro, our knowledge about the cyst form remains limited. This also hampers our ability to develop cyst-specific diagnostic tools.Three main aims were (i to identify E. histolytica proteins in cyst samples, (ii to enrich our knowledge about the cyst stage, and (iii to identify candidate proteins to develop cyst-specific diagnostic tools.Cysts were purified from the stool of infected individuals using Percoll (gradient purification. A highly sensitive LC-MS/MS mass spectrometer (Orbitrap was used to identify cyst proteins.A total of 417 non-redundant E. histolytica proteins were identified including 195 proteins that were never detected in trophozoite-derived proteomes or expressed sequence tag (EST datasets, consistent with cyst specificity. Cyst-wall specific glycoproteins Jacob, Jessie and chitinase were positively identified. Antibodies produced against Jacob identified cysts in fecal specimens and have potential utility as a diagnostic reagent. Several protein kinases, small GTPase signaling molecules, DNA repair proteins, epigenetic regulators, and surface associated proteins were also identified. Proteins we identified are likely to be among the most abundant in excreted cysts, and therefore show promise as diagnostic targets.The proteome data generated here are a first for naturally-occurring E. histolytica cysts, and they provide important insights into the infectious cyst form. Additionally, numerous unique candidate proteins were identified which will aid the development of new diagnostic tools for identification of E. histolytica cysts.

  1. Proteomic analysis in allergy and intolerance to wheat products.

    Science.gov (United States)

    Mamone, Gianfranco; Picariello, Gianluca; Addeo, Francesco; Ferranti, Pasquale

    2011-02-01

    Owing to its extensive use in the human diet, wheat is among the most common causes of food-related allergies and intolerances. Allergies to wheat are provoked by ingestion, inhalation or contact with either the soluble or the insoluble gluten proteins in wheat. Gluten proteins, and particularly the gliadin fraction, are also the main factor triggering celiac disease, a common enteropathy induced by ingestion of wheat gluten proteins and related prolamins from oat, rye and barley in genetically susceptible individuals. The role of gliadin and of its derived peptides in eliciting the adverse reactions in celiac disease are still far from being completely explained. Owing to its unique pathogenesis, celiac disease is widely investigated as a model immunogenetic disorder. The structural characterization of the injuring agents, the gluten proteins, assumes a particular significance in order to deepen the understanding of the events that trigger this and similar diseases at the molecular level. Recent developments in proteomics have provided an important contribution to the understanding of several basic aspects of wheat protein-related diseases. These include: the identification of gluten fractions and derived peptides involved in wheat allergy and intolerance, including celiac disease, and the elucidation of their mechanism of toxicity; the development and validation of sensitive and specific methods for detecting trace amounts of gluten proteins in gluten-free foods for intolerant patients; and the formulation of completely new substitute foods and ingredients to replace the gluten-based ones. In this article, the main aspects of current and prospective applications of mass spectrometry and proteomic technologies to the structural characterization of gluten proteins and derived peptides are critically presented, with a focus on issues related to their detection, identification and quantification, which are relevant to the biochemical, immunological and toxicological

  2. Proteomic Analysis of the Cyst Stage of Entamoeba histolytica

    Science.gov (United States)

    Ali, Ibne Karim M.; Haque, Rashidul; Siddique, Abdullah; Kabir, Mamun; Sherman, Nicholas E.; Gray, Sean A.; Cangelosi, Gerard A.; Petri, William A.

    2012-01-01

    Background The category B agent of bioterrorism, Entamoeba histolytica has a two-stage life cycle: an infective cyst stage, and an invasive trophozoite stage. Due to our inability to effectively induce encystation in vitro, our knowledge about the cyst form remains limited. This also hampers our ability to develop cyst-specific diagnostic tools. Aims Three main aims were (i) to identify E. histolytica proteins in cyst samples, (ii) to enrich our knowledge about the cyst stage, and (iii) to identify candidate proteins to develop cyst-specific diagnostic tools. Methods Cysts were purified from the stool of infected individuals using Percoll (gradient) purification. A highly sensitive LC-MS/MS mass spectrometer (Orbitrap) was used to identify cyst proteins. Results A total of 417 non-redundant E. histolytica proteins were identified including 195 proteins that were never detected in trophozoite-derived proteomes or expressed sequence tag (EST) datasets, consistent with cyst specificity. Cyst-wall specific glycoproteins Jacob, Jessie and chitinase were positively identified. Antibodies produced against Jacob identified cysts in fecal specimens and have potential utility as a diagnostic reagent. Several protein kinases, small GTPase signaling molecules, DNA repair proteins, epigenetic regulators, and surface associated proteins were also identified. Proteins we identified are likely to be among the most abundant in excreted cysts, and therefore show promise as diagnostic targets. Major Conclusions The proteome data generated here are a first for naturally-occurring E. histolytica cysts, and they provide important insights into the infectious cyst form. Additionally, numerous unique candidate proteins were identified which will aid the development of new diagnostic tools for identification of E. histolytica cysts. PMID:22590659

  3. Effects of Bacterial Inactivation Methods on Downstream Proteomic Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Andy; Merkley, Eric D.; Clowers, Brian H.; Hutchison, Janine R.; Kreuzer, Helen W.

    2015-05-01

    Inactivation of pathogenic microbial samples is often necessary for the protection of researchers and to comply with local and federal regulations. By its nature, biological inactivation causes changes to microbial samples, potentially affecting observed experimental results. While inactivation induced damage to materials such as DNA has been evaluated, the effect of various inactivation strategies on proteomic data, to our knowledge, has not been discussed. To this end, we inactivated samples of Yersinia pestis and Escherichia coli by autoclave, ethanol, or irradiation treatment to determine how inactivation changes liquid chromatography tandem mass spectrometry data quality as well as apparent protein content of cells. Proteomic datasets obtained from aliquots of samples inactivated by different methods were highly similar, with Pearson correlation coefficients ranging from 0.822 to 0.985 and 0.816 to 0.985 for E. coli and Y. pestis, respectively, suggesting that inactivation had only slight impacts on the set of proteins identified. In addition, spectral quality metrics such as distributions of various database search algorithm scores remained constant across inactivation methods, indicating that inactivation does not appreciably degrade spectral quality. Though overall changes resulting from inactivation were small, there were detectable trends. For example, one-sided Fischer exact tests determined that periplasmic proteins decrease in observed abundance after sample inactivation by autoclaving (α = 1.71x10-2 for E. coli, α = 4.97x10-4 for Y. pestis) and irradiation (α = 9.43x10-7 for E. coli, α = 1.21x10-5 for Y. pestis) when compared to controls that were not inactivated. Based on our data, if sample inactivation is necessary, we recommend inactivation with ethanol treatment with secondary preference given to irradiation.

  4. Application of survival analysis methodology to the quantitative analysis of LC-MS proteomics data

    KAUST Repository

    Tekwe, C. D.

    2012-05-24

    MOTIVATION: Protein abundance in quantitative proteomics is often based on observed spectral features derived from liquid chromatography mass spectrometry (LC-MS) or LC-MS/MS experiments. Peak intensities are largely non-normal in distribution. Furthermore, LC-MS-based proteomics data frequently have large proportions of missing peak intensities due to censoring mechanisms on low-abundance spectral features. Recognizing that the observed peak intensities detected with the LC-MS method are all positive, skewed and often left-censored, we propose using survival methodology to carry out differential expression analysis of proteins. Various standard statistical techniques including non-parametric tests such as the Kolmogorov-Smirnov and Wilcoxon-Mann-Whitney rank sum tests, and the parametric survival model and accelerated failure time-model with log-normal, log-logistic and Weibull distributions were used to detect any differentially expressed proteins. The statistical operating characteristics of each method are explored using both real and simulated datasets. RESULTS: Survival methods generally have greater statistical power than standard differential expression methods when the proportion of missing protein level data is 5% or more. In particular, the AFT models we consider consistently achieve greater statistical power than standard testing procedures, with the discrepancy widening with increasing missingness in the proportions. AVAILABILITY: The testing procedures discussed in this article can all be performed using readily available software such as R. The R codes are provided as supplemental materials. CONTACT: ctekwe@stat.tamu.edu.

  5. Confinement Vessel Dynamic Analysis

    Energy Technology Data Exchange (ETDEWEB)

    R. Robert Stevens; Stephen P. Rojas

    1999-08-01

    A series of hydrodynamic and structural analyses of a spherical confinement vessel has been performed. The analyses used a hydrodynamic code to estimate the dynamic blast pressures at the vessel's internal surfaces caused by the detonation of a mass of high explosive, then used those blast pressures as applied loads in an explicit finite element model to simulate the vessel's structural response. Numerous load cases were considered. Particular attention was paid to the bolted port connections and the O-ring pressure seals. The analysis methods and results are discussed, and comparisons to experimental results are made.

  6. Lipidomic and proteomic analysis of Caenorhabditis elegans lipid droplets and identification of ACS-4 as a lipid droplet-associated protein.

    Science.gov (United States)

    Vrablik, Tracy L; Petyuk, Vladislav A; Larson, Emily M; Smith, Richard D; Watts, Jennifer L

    2015-10-01

    Lipid droplets are cytoplasmic organelles that store neutral lipids for membrane synthesis and energy reserves. In this study, we characterized the lipid and protein composition of purified Caenorhabditis elegans lipid droplets. These lipid droplets are composed mainly of triacylglycerols, surrounded by a phospholipid monolayer composed primarily of phosphatidylcholine and phosphatidylethanolamine. The fatty acid composition of the triacylglycerols is rich in fatty acid species obtained from the dietary Escherichia coli, including cyclopropane fatty acids and cis-vaccenic acid. Unlike other organisms, C. elegans lipid droplets contain very little cholesterol or cholesterol esters. Comparison of the lipid droplet proteomes of wild type and high-fat daf-2 mutant strains shows a very similar proteome in both strains, except that the most abundant protein in the C. elegans lipid droplet proteome, MDT-28, is relatively less abundant in lipid droplets isolated from daf-2 mutants. Functional analysis of lipid droplet proteins identified in our proteomic studies indicated an enrichment of proteins required for growth and fat homeostasis in C. elegans. Finally, we confirmed the localization of one of the newly identified lipid droplet proteins, ACS-4. We found that ACS-4 localizes to the surface of lipid droplets in the C. elegans intestine and skin. This study bolsters C. elegans as a model to study the dynamics and functions of lipid droplets in a multicellular organism.

  7. Desiccation tolerance mechanism in resurrection fern-ally Selaginella tamariscina revealed by physiological and proteomic analysis.

    Science.gov (United States)

    Wang, Xiaonan; Chen, Sixue; Zhang, Heng; Shi, Lei; Cao, Fenglin; Guo, Lihai; Xie, Yongming; Wang, Tai; Yan, Xiufeng; Dai, Shaojun

    2010-12-03

    Drought is one of the most severe limitations to plant growth and productivity. Resurrection plants have evolved a unique capability to tolerate desiccation in vegetative tissues. Fern-ally Selaginella tamariscina (Beauv.) is one of the most primitive vascular resurrection plants, which can survive a desiccated state and recover when water becomes available. To better understand the mechanism of desiccation tolerance, we have applied physiological and proteomic analysis. Samples of S. tamariscina were water-deprived for up to seven days followed by 12 h of rewatering. Our results showed that endogenous abscisic acid (ABA) increased to regulate dehydration-responsive genes/proteins and physiological processes. In the course of dehydration, the contents of osmolytes represented by soluble sugars and proline were increased to maintain cell structure integrity. The activities of four antioxidant enzymes (superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and glutathione reductase (GR)) also increased. In contrast, both the rate of photosynthesis and the chlorophyll content decreased, and plasma membrane integrity was lost. We identified 138 desiccation-responsive two-dimensional electrophoresis (2-DE) spots, representing 103 unique proteins. Hierarchical clustering analysis revealed that 83% of the proteins were down-regulated upon dehydration. They were mainly involved in photosynthesis, carbohydrate and energy metabolism, stress and defense, protein metabolism, signaling, membrane/transport, cell structure, and cell division. The dynamic expression changes of the desiccation-responsive proteins provide strong evidence that cell structure modification, photosynthesis reduction, antioxidant system activation, and protein post-transcriptional/translational modifications are essential to the poikilochlorophyllous fern-ally S. tamariscina in response to dehydration. In addition, our comparative analysis of dehydration-responsive proteins in vegetative tissues

  8. Quantitative Proteomic Analysis of the Hfq-Regulon in Sinorhizobium meliloti 2011

    Science.gov (United States)

    Sobrero, Patricio; Schlüter, Jan-Philip; Lanner, Ulrike; Schlosser, Andreas; Becker, Anke; Valverde, Claudio

    2012-01-01

    Riboregulation stands for RNA-based control of gene expression. In bacteria, small non-coding RNAs (sRNAs) are a major class of riboregulatory elements, most of which act at the post-transcriptional level by base-pairing target mRNA genes. The RNA chaperone Hfq facilitates antisense interactions between target mRNAs and regulatory sRNAs, thus influencing mRNA stability and/or translation rate. In the α-proteobacterium Sinorhizobium meliloti strain 2011, the identification and detection of multiple sRNAs genes and the broadly pleitropic phenotype associated to the absence of a functional Hfq protein both support the existence of riboregulatory circuits controlling gene expression to ensure the fitness of this bacterium in both free living and symbiotic conditions. In order to identify target mRNAs subject to Hfq-dependent riboregulation, we have compared the proteome of an hfq mutant and the wild type S. meliloti by quantitative proteomics following protein labelling with 15N. Among 2139 univocally identified proteins, a total of 195 proteins showed a differential abundance between the Hfq mutant and the wild type strain; 65 proteins accumulated ≥2-fold whereas 130 were downregulated (≤0.5-fold) in the absence of Hfq. This profound proteomic impact implies a major role for Hfq on regulation of diverse physiological processes in S. meliloti, from transport of small molecules to homeostasis of iron and nitrogen. Changes in the cellular levels of proteins involved in transport of nucleotides, peptides and amino acids, and in iron homeostasis, were confirmed with phenotypic assays. These results represent the first quantitative proteomic analysis in S. meliloti. The comparative analysis of the hfq mutant proteome allowed identification of novel strongly Hfq-regulated genes in S. meliloti. PMID:23119037

  9. Quantitative proteomic analysis of the Hfq-regulon in Sinorhizobium meliloti 2011.

    Directory of Open Access Journals (Sweden)

    Patricio Sobrero

    Full Text Available Riboregulation stands for RNA-based control of gene expression. In bacteria, small non-coding RNAs (sRNAs are a major class of riboregulatory elements, most of which act at the post-transcriptional level by base-pairing target mRNA genes. The RNA chaperone Hfq facilitates antisense interactions between target mRNAs and regulatory sRNAs, thus influencing mRNA stability and/or translation rate. In the α-proteobacterium Sinorhizobium meliloti strain 2011, the identification and detection of multiple sRNAs genes and the broadly pleitropic phenotype associated to the absence of a functional Hfq protein both support the existence of riboregulatory circuits controlling gene expression to ensure the fitness of this bacterium in both free living and symbiotic conditions. In order to identify target mRNAs subject to Hfq-dependent riboregulation, we have compared the proteome of an hfq mutant and the wild type S. meliloti by quantitative proteomics following protein labelling with (15N. Among 2139 univocally identified proteins, a total of 195 proteins showed a differential abundance between the Hfq mutant and the wild type strain; 65 proteins accumulated ≥2-fold whereas 130 were downregulated (≤0.5-fold in the absence of Hfq. This profound proteomic impact implies a major role for Hfq on regulation of diverse physiological processes in S. meliloti, from transport of small molecules to homeostasis of iron and nitrogen. Changes in the cellular levels of proteins involved in transport of nucleotides, peptides and amino acids, and in iron homeostasis, were confirmed with phenotypic assays. These results represent the first quantitative proteomic analysis in S. meliloti. The comparative analysis of the hfq mutant proteome allowed identification of novel strongly Hfq-regulated genes in S. meliloti.

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

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Mingqun ..; Kikuchi, Takane; Brewer, Heather M.; Norbeck, Angela D.; Rikihisa, Yasuko

    2011-02-17

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

  11. Detailed tail proteomic analysis of axolotl (Ambystoma mexicanum) using an mRNA-seq reference database.

    Science.gov (United States)

    Demircan, Turan; Keskin, Ilknur; Dumlu, Seda Nilgün; Aytürk, Nilüfer; Avşaroğlu, Mahmut Erhan; Akgün, Emel; Öztürk, Gürkan; Baykal, Ahmet Tarık

    2017-01-01

    Salamander axolotl has been emerging as an important model for stem cell research due to its powerful regenerative capacity. Several advantages, such as the high capability of advanced tissue, organ, and appendages regeneration, promote axolotl as an ideal model system to extend our current understanding on the mechanisms of regeneration. Acknowledging the common molecular pathways between amphibians and mammals, there is a great potential to translate the messages from axolotl research to mammalian studies. However, the utilization of axolotl is hindered due to the lack of reference databases of genomic, transcriptomic, and proteomic data. Here, we introduce the proteome analysis of the axolotl tail section searched against an mRNA-seq database. We translated axolotl mRNA sequences to protein sequences and annotated these to process the LC-MS/MS data and identified 1001 nonredundant proteins. Functional classification of identified proteins was performed by gene ontology searches. The presence of some of the identified proteins was validated by in situ antibody labeling. Furthermore, we have analyzed the proteome expressional changes postamputation at three time points to evaluate the underlying mechanisms of the regeneration process. Taken together, this work expands the proteomics data of axolotl to contribute to its establishment as a fully utilized model.

  12. Proteomic analysis of pRb loss highlights a signature of decreased mitochondrial oxidative phosphorylation.

    Science.gov (United States)

    Nicolay, Brandon N; Danielian, Paul S; Kottakis, Filippos; Lapek, John D; Sanidas, Ioannis; Miles, Wayne O; Dehnad, Mantre; Tschöp, Katrin; Gierut, Jessica J; Manning, Amity L; Morris, Robert; Haigis, Kevin; Bardeesy, Nabeel; Lees, Jacqueline A; Haas, Wilhelm; Dyson, Nicholas J

    2015-09-01

    The retinoblastoma tumor suppressor (pRb) protein associates with chromatin and regulates gene expression. Numerous studies have identified Rb-dependent RNA signatures, but the proteomic effects of Rb loss are largely unexplored. We acutely ablated Rb in adult mice and conducted a quantitative analysis of RNA and proteomic changes in the colon and lungs, where Rb(KO) was sufficient or insufficient to induce ectopic proliferation, respectively. As expected, Rb(KO) caused similar increases in classic pRb/E2F-regulated transcripts in both tissues, but, unexpectedly, their protein products increased only in the colon, consistent with its increased proliferative index. Thus, these protein changes induced by Rb loss are coupled with proliferation but uncoupled from transcription. The proteomic changes in common between Rb(KO) tissues showed a striking decrease in proteins with mitochondrial functions. Accordingly, RB1 inactivation in human cells decreased both mitochondrial mass and oxidative phosphorylation (OXPHOS) function. RB(KO) cells showed decreased mitochondrial respiratory capacity and the accumulation of hypopolarized mitochondria. Additionally, RB/Rb loss altered mitochondrial pyruvate oxidation from (13)C-glucose through the TCA cycle in mouse tissues and cultured cells. Consequently, RB(KO) cells have an enhanced sensitivity to mitochondrial stress conditions. In summary, proteomic analyses provide a new perspective on Rb/RB1 mutation, highlighting the importance of pRb for mitochondrial function and suggesting vulnerabilities for treatment.

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

    Energy Technology Data Exchange (ETDEWEB)

    Sivagnanam, Kumaran [McGill University, Montreal, Quebec; Raghavan, Vijaya G. S. [McGill University, Montreal, Quebec; Shah, Manesh B [ORNL; Hettich, Robert {Bob} L [ORNL; Verberkmoes, Nathan C [ORNL; Lefsrud, Mark G [McGill University, Montreal, Quebec

    2011-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Verberkmoes Nathan C

    2011-10-01

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

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

    Science.gov (United States)

    2011-01-01

    Background Butanol is a second generation biofuel produced by Clostridium acetobutylicum through acetone-butanol-ethanol (ABE) fermentation process. Shotgun proteomics provides a direct approach to study the whole proteome of an organism in depth. This paper focuses on shotgun proteomic profiling of C. acetobutylicum from ABE fermentation using glucose and xylose to understand the functional mechanisms of C. acetobutylicum proteins involved in butanol production. Results We identified 894 different proteins in C. acetobutylicum from ABE fermentation process by two dimensional - liquid chromatography - tandem mass spectrometry (2D-LC-MS/MS) method. This includes 717 proteins from glucose and 826 proteins from the xylose substrate. A total of 649 proteins were found to be common and 22 significantly differentially expressed proteins were identified between glucose and xylose substrates. Conclusion Our results demonstrate that flagellar proteins are highly up-regulated with glucose compared to xylose substrate during ABE fermentation. Chemotactic activity was also found to be lost with the xylose substrate due to the absence of CheW and CheV proteins. This is the first report on the shotgun proteomic analysis of C. acetobutylicum ATCC 824 in ABE fermentation between glucose and xylose substrate from a single time data point and the number of proteins identified here is more than any other study performed on this organism up to this report. PMID:22008648

  16. Evaluation of proteomic search engines for the analysis of histone modifications.

    Science.gov (United States)

    Yuan, Zuo-Fei; Lin, Shu; Molden, Rosalynn C; Garcia, Benjamin A

    2014-10-03

    Identification of histone post-translational modifications (PTMs) is challenging for proteomics search engines. Including many histone PTMs in one search increases the number of candidate peptides dramatically, leading to low search speed and fewer identified spectra. To evaluate database search engines on identifying histone PTMs, we present a method in which one kind of modification is searched each time, for example, unmodified, individually modified, and multimodified, each search result is filtered with false discovery rate less than 1%, and the identifications of multiple search engines are combined to obtain confident results. We apply this method for eight search engines on histone data sets. We find that two search engines, pFind and Mascot, identify most of the confident results at a reasonable speed, so we recommend using them to identify histone modifications. During the evaluation, we also find some important aspects for the analysis of histone modifications. Our evaluation of different search engines on identifying histone modifications will hopefully help those who are hoping to enter the histone proteomics field. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD001118.

  17. Application of meta-transcriptomics and –proteomics to analysis of in situ physiological state

    Directory of Open Access Journals (Sweden)

    Allan eKonopka

    2012-05-01

    Full Text Available Analysis of the growth-limiting factor or environmental stressors affecting microbes in situ is of fundamental importance but analytically difficult. Microbes can reduce in situ limiting nutrient concentrations to sub-micromolar levels, and contaminated ecosystems may contain multiple stressors. The patterns of gene or protein expression by microbes in nature can be used to infer growth limitations, because they are regulated in response to environmental conditions. Experimental studies under controlled conditions in the laboratory provide the physiological underpinnings for developing these physiological indicators. Although regulatory networks may differ among specific microbes, there are some broad principles that can be applied, related to limiting nutrient acquisition, resource allocation, and stress responses. As technologies for transcriptomics and proteomics mature, the capacity to apply these approaches to complex microbial communities will accelerate. In particular, global proteomics reflect expressed catalytic activities. Furthermore, the high mass accuracy of some proteomic approaches allows mapping back to specific microbial strains. For example, at the Rifle IFRC field site in Western Colorado, the physiological status of Fe(III-reducing populations has been tracked over time. Members of a subsurface clade within the Geobacter predominated during carbon amendment to the subsurface environment. At the functional level, proteomic identifications produced inferences regarding (i temporal changes in anabolism and catabolism of acetate, (ii the onset of N2 fixation when N became limiting, and (iii expression of phosphate transporters during periods of intense growth. The application of these approaches in situ can lead to discovery of novel physiological adaptations.

  18. Analysis of Pacific oyster larval proteome and its response to high-CO2

    KAUST Repository

    Dineshram, R.

    2012-10-01

    Most calcifying organisms show depressed metabolic, growth and calcification rates as symptoms to high-CO2 due to ocean acidification (OA) process. Analysis of the global expression pattern of proteins (proteome analysis) represents a powerful tool to examine these physiological symptoms at molecular level, but its applications are inadequate. To address this knowledge gap, 2-DE coupled with mass spectrophotometer was used to compare the global protein expression pattern of oyster larvae exposed to ambient and to high-CO2. Exposure to OA resulted in marked reduction of global protein expression with a decrease or loss of 71 proteins (18% of the expressed proteins in control), indicating a wide-spread depression of metabolic genes expression in larvae reared under OA. This is, to our knowledge, the first proteome analysis that provides insights into the link between physiological suppression and protein down-regulation under OA in oyster larvae. © 2012 Elsevier Ltd.

  19. Quantitative proteomics reveals dynamic responses of Synechocystis sp. PCC 6803 to next-generation biofuel butanol.

    Science.gov (United States)

    Tian, Xiaoxu; Chen, Lei; Wang, Jiangxin; Qiao, Jianjun; Zhang, Weiwen

    2013-01-14

    Butanol is a promising biofuel, and recent metabolic engineering efforts have demonstrated the use of photosynthetic cyanobacterial hosts for its production. However, cyanobacteria have very low tolerance to butanol, limiting the economic viability of butanol production from these renewable producing systems. The existing knowledge of molecular mechanism involved in butanol tolerance in cyanobacteria is very limited. To build a foundation necessary to engineer robust butanol-producing cyanobacterial hosts, in this study, the responses of Synechocystis PCC 6803 to butanol were investigated using a quantitative proteomics approach with iTRAQ - LC-MS/MS technologies. The resulting high-quality dataset consisted of 25,347 peptides corresponding to 1452 unique proteins, a coverage of approximately 40% of the predicted proteins in Synechocystis. Comparative quantification of protein abundances led to the identification of 303 differentially regulated proteins by butanol. Annotation and GO term enrichment analysis showed that multiple biological processes were regulated, suggesting that Synechocystis probably employed multiple and synergistic resistance mechanisms in dealing with butanol stress. Notably, the analysis revealed the induction of heat-shock protein and transporters, along with modification of cell membrane and envelope were the major protection mechanisms against butanol. A conceptual cellular model of Synechocystis PCC 6803 responses to butanol stress was constructed to illustrate the putative molecular mechanisms employed to defend against butanol stress. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. Muscle Tissue Damage Induced by the Venom of Bothrops asper: Identification of Early and Late Pathological Events through Proteomic Analysis

    National Research Council Canada - National Science Library

    Herrera, Cristina; Macêdo, Jéssica Kele A; Feoli, Andrés; Escalante, Teresa; Rucavado, Alexandra; Gutiérrez, José María; Fox, Jay W

    2016-01-01

    The time-course of the pathological effects induced by the venom of the snake Bothrops asper in muscle tissue was investigated by a combination of histology, proteomic analysis of exudates collected...

  1. Analysis of membrane proteome and secretome in cells over-expressing ADAM17 using quantitative proteomics

    Energy Technology Data Exchange (ETDEWEB)

    Kawahara, R.; Simabuco, F.M. [Laboratorio Nacional de Biociencias - LNBIO, Campinas, SP (Brazil); Yokoo, S.; Paes Leme, A.F. [Laboratorio Nacional de Luz Sincrotron (LNLS), Campinas, SP (Brazil); Sherman, N. [University of Virginia, Charlottesville, VA (United States)

    2012-07-01

    Full text: A disintegrin and metalloproteinase (ADAM) protease is involved in proteolytic ectodomain shedding of several membrane-associated proteins and modulation of key cell signaling pathways in the tumor microenvironment. In this study, we examined the effect of over-expressing the full length human ADAM17 in membrane and secreted proteins. To this end, we constructed a stable Flp-In T-RExHEK293 cells expressing ADAM17 by tetracycline induction. These cells were grown in Dulbeccos modified Eagles medium containing light lysine, arginine or heavy, L-Arg-13C615N4 and L-Lys -13C615N2 (SILAC: stable isotope labeling with amino acid in cell culture) media and they were treated with an ADAM17 activator, phorbolester (PMA). Controls such as Flp-In T-RExHEK293 cell without PMA treatment and without ADAM17 cloned were cultivated in light medium. The ADAM17 overexpression was induced with tetracycline 500 ng/ml for 24 hours. Cells in a heavy condition were treated with PMA 50 ng/ml for 1 hour and vehicle DMSO was used as control in a light cell condition. The extracellular media were collected, concentrated and used to evaluate the secretome and a cell surface biotinylation-based approach was used to capture cell surface-associated proteins. The biotinylated proteins were eluted with dithiothreitol, alkylated with iodoacetamide and then digested with trypsin. The resulting peptides were subjected to LC-MS/MS analysis on an ETD enabled Orbitrap Velos instrument. The results showed different proteins up or down regulated in membrane and secretome analysis which might represent potential molecules involved in signaling or ADAM17 regulation events. (author)

  2. Network-based proteomic analysis for postmenopausal osteoporosis in Caucasian females.

    Science.gov (United States)

    Zhang, Lan; Liu, Yao-Zhong; Zeng, Yong; Zhu, Wei; Zhao, Ying-Chun; Zhang, Ji-Gang; Zhu, Jia-Qiang; He, Hao; Shen, Hui; Tian, Qing; Deng, Fei-Yan; Papasian, Christopher J; Deng, Hong-Wen

    2016-01-01

    Menopause is one of the crucial physiological events during the life of a woman. Transition of menopause status is accompanied by increased risks of various health problems such as osteoporosis. Peripheral blood monocytes can differentiate into osteoclasts and produce cytokines important for osteoclast activity. With quantitative proteomics LC-nano-ESI-MS(E) (where MS(E) is elevated-energy MS), we performed protein expression profiling of peripheral blood monocytes in 42 postmenopausal women with discordant bone mineral density (BMD) levels. Traditional comparative analysis showed proteins encoded by four genes (LOC654188, PPIA, TAGLN2, YWHAB) and three genes (LMNB1, ANXA2P2, ANXA2) were significantly down- and upregulated, respectively, in extremely low- versus high-BMD subjects. To study functionally orchestrating groups of detected proteins in the form of networks, we performed weighted gene coexpression network analysis and gene set enrichment analysis. Weighted gene coexpression network analysis showed that the module including the annexin gene family was most significantly correlated with low BMD, and the lipid-binding related GO terms were enriched in this identified module. Gene set enrichment analysis revealed that two significantly enriched gene sets may be involved in postmenopausal BMD variation by regulating pro-inflammatory cytokines activities. To gain more insights into the proteomics data generated, we performed integrative analyses of the datasets available to us at the genome (DNA level), transcriptome (RNA level), and proteome levels jointly.

  3. Comparative Proteomic Analysis of Proteins Expressed in the I-1 and I-2 Internodes of Strawberry Stolons

    Institute of Scientific and Technical Information of China (English)

    Xianping Fang; Huasheng Ma; Songlin Raun

    2012-01-01

    The strawberry (Fragaria ananassa) can reproduce asexually through stolons,including the proximal internode (I-1) and the distal internode (I-2),which has a strong tendency to form adventitious roots at the nodes.Understanding the different development mechanisms of I-1 and I-2 will facilitate nursery plant production.Here we report comparative internode proteomics of strawberry stolons.Proteins from I-1 and I-2 samples were extracted and separated by two-dimensional gel electrophoresis.A total of 164 protein spots from I-1 gels and 200 protein spots from I-2 gels were selected carefully.Out of these spots,a total of 38 and 52 proteins were identified by mass spectrometry in I-1 and I-2,respectively.All the identified proteins were categorized into 8 and 10 groups according to their cellular compartmentalization and biological functionality,respectively.Enzymes necessary for carbohydrate metabolism and photosynthesis dominated in the set of identified proteins.Analysis of protein expression patterns revealed that the ubiquitin-proteasome pathway and sugar-hormone connections may play important roles in the growth and development of I-2.Furthermore,protein-protein interactions studies revealed many proteins were involved in I-2 development mechanism as a dynamic network.Finally,based on our proteomic results,we brought forward a possible schematic representation of mechanism associated with the adventitious root formation of new clonal plants in I-2.

  4. Proteomic analysis of albumin and globulin fractions of pea (Pisum sativum L. seeds

    Directory of Open Access Journals (Sweden)

    Jerzy Dziuba

    2014-06-01

    Full Text Available Background. Proteomic analysis is emerging as a highly useful tool in food research, including studies of food allergies. Two-dimensional gel electrophoresis involving isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis is the most effective method of separating hundreds or even thousands of proteins. In this study, albumin and globulin tractions of pea seeds cv. Ramrod were subjected to proteomic analysis. Selected potentially alergenic proteins were identified based on their molecular weights and isoelectric points. Material and methods. Pea seeds (Pisum sativum L. cv. Ramrod harvested over a period of two years (Plant Breeding Station in Piaski-Szelejewo were used in the experiment. The isolated albumins, globulins and legumin and vicilin fractions of globulins were separated by two-dimensional gel electrophoresis. Proteomic images were analysed in the ImageMaster 2D Platinum program with the use of algorithms from the Melanie application. The relative content, isoelectric points and molecular weights were computed for all identified proteins. Electrophoregrams were analysed by matching spot positions from three independent replications. Results. The proteomes of albumins, globulins and legumin and vicilin fractions of globulins produced up to several hundred spots (proteins. Spots most characteristic of a given fraction were identified by computer analysis and spot matching. The albumin proteome accumulated spots of relatively high intensity over a broad range of pi values of -4.2-8.1 in 3 molecular weight (MW ranges: I - high molecular-weight albumins with MW of -50-110 kDa, II - average molecular-weight albumins with MW of -20-35 kDa, and III - low molecular-weight albumins with MW of -13-17 kDa. 2D gel electrophoregrams revealed the presence of 81 characteristic spots, including 24 characteristic of legumin and 14 - of vicilin. Conclusions. Two-dimensional gel electrophoresis proved to be a useful tool for

  5. Proteomic Analysis of Hylocereus polyrhizus Reveals Metabolic Pathway Changes

    Science.gov (United States)

    Hua, Qingzhu; Zhou, Qianjun; Gan, Susheng; Wu, Jingyu; Chen, Canbin; Li, Jiaqiang; Ye, Yaoxiong; Zhao, Jietang; Hu, Guibing; Qin, Yonghua

    2016-01-01

    Red dragon fruit or red pitaya (Hylocereus polyrhizus) is the only edible fruit that contains betalains. The color of betalains ranges from red and violet to yellow in plants. Betalains may also serve as an important component of health-promoting and disease-preventing functional food. Currently, the biosynthetic and regulatory pathways for betalain production remain to be fully deciphered. In this study, isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analyses were used to reveal the molecular mechanism of betalain biosynthesis in H. polyrhizus fruits at white and red pulp stages, respectively. A total of 1946 proteins were identified as the differentially expressed between the two samples, and 936 of them were significantly highly expressed at the red pulp stage of H. polyrhizus. RNA-seq and iTRAQ analyses showed that some transcripts and proteins were positively correlated; they belonged to “phenylpropanoid biosynthesis”, “tyrosine metabolism”, “flavonoid biosynthesis”, “ascorbate and aldarate metabolism”, “betalains biosynthesis” and “anthocyanin biosynthesis”. In betalains biosynthesis pathway, several proteins/enzymes such as polyphenol oxidase, CYP76AD3 and 4,5-dihydroxy-phenylalanine (DOPA) dioxygenase extradiol-like protein were identified. The present study provides a new insight into the molecular mechanism of the betalain biosynthesis at the posttranscriptional level. PMID:27690004

  6. Proteomic Analysis of Hylocereus polyrhizus Reveals Metabolic Pathway Changes

    Directory of Open Access Journals (Sweden)

    Qingzhu Hua

    2016-09-01

    Full Text Available Red dragon fruit or red pitaya (Hylocereus polyrhizus is the only edible fruit that contains betalains. The color of betalains ranges from red and violet to yellow in plants. Betalains may also serve as an important component of health-promoting and disease-preventing functional food. Currently, the biosynthetic and regulatory pathways for betalain production remain to be fully deciphered. In this study, isobaric tags for relative and absolute quantitation (iTRAQ-based proteomic analyses were used to reveal the molecular mechanism of betalain biosynthesis in H. polyrhizus fruits at white and red pulp stages, respectively. A total of 1946 proteins were identified as the differentially expressed between the two samples, and 936 of them were significantly highly expressed at the red pulp stage of H. polyrhizus. RNA-seq and iTRAQ analyses showed that some transcripts and proteins were positively correlated; they belonged to “phenylpropanoid biosynthesis”, “tyrosine metabolism”, “flavonoid biosynthesis”, “ascorbate and aldarate metabolism”, “betalains biosynthesis” and “anthocyanin biosynthesis”. In betalains biosynthesis pathway, several proteins/enzymes such as polyphenol oxidase, CYP76AD3 and 4,5-dihydroxy-phenylalanine (DOPA dioxygenase extradiol-like protein were identified. The present study provides a new insight into the molecular mechanism of the betalain biosynthesis at the posttranscriptional level.

  7. Comparative proteomic analysis of whole saliva from chronic periodontitis patients.

    Science.gov (United States)

    Gonçalves, Lorena Da Rós; Soares, Márcia Regina; Nogueira, Fábio C S; Garcia, Carlos; Camisasca, Danielle Resende; Domont, Gilberto; Feitosa, Alfredo C R; Pereira, Denise de Abreu; Zingali, Russolina B; Alves, Gilda

    2010-05-07

    Chronic periodontal disease is a chronic inflammatory process affecting tooth supporting tissues in the presence of pathogenic bacterial biofilm. There is some evidence for changes in the protein composition of whole saliva from chronic periodontitis patients, but there have been no studies using a proteomic approach. Hence, the aim of this study was to compare the protein profiles of unstimulated whole saliva from patients with periodontitis and healthy subjects by two complementary approaches (2D-gel electrophoresis and liquid chromatography). Protein spots of interest were analyzed by MALDI-TOF-TOF, and the data was complemented by an ESI-Q-TOF experiment. The analyses revealed that subjects with periodontal disease have increased amounts of blood proteins (serum albumin and hemoglobin) and immunoglobulin, and they have a lower abundance of cystatin compared to the control group. A higher number of protein spots were observed in the periodontitis group, of which most were identified as alpha-amylase. This higher number of alpha-amylase variants seems to be caused by hydrolysis by cysteine proteases under such inflammatory conditions. This approach gives novel insights into alterations of salivary protein in presence of periodontal inflammation and may contribute to the improvement of periodontal diagnosis.

  8. Proteomic analysis of rat cerebral cortex following subchronic acrolein toxicity.

    Science.gov (United States)

    Rashedinia, Marzieh; Lari, Parisa; Abnous, Khalil; Hosseinzadeh, Hossein

    2013-10-01

    Acrolein, a member of reactive α,β-unsaturated aldehydes, is a major environmental pollutant. Acrolein is also produced endogenously as a toxic by-product of lipid peroxidation. Because of high reactivity, acrolein may mediate oxidative damages to cells and tissues. It has been shown to be involved in a wide variety of pathological states including pulmonary, atherosclerosis and neurodegenerative diseases. In this study we employed proteomics approach to investigate the effects of subchronic oral exposures to 3mg/kg of acrolein on protein expression profile in the brain of rats. Moreover effects of acrolein on malondialdehyde (MDA) levels and reduced glutathione (GSH) content were investigated. Our results revealed that treatment with acrolein changed levels of several proteins in diverse physiological process including energy metabolism, cell communication and transport, response to stimulus and metabolic process. Interestingly, several differentially over-expressed proteins, including β-synuclein, enolase and calcineurin, are known to be associated with human neurodegenerative diseases. Changes in the levels of some proteins were confirmed by Western blot. Moreover, acrolein increases the level of MDA, as a lipid peroxidation biomarker and decreased GSH concentrations, as a non-enzyme antioxidant in the brain of acrolein treated rats. These findings suggested that acrolein induces the oxidative stress and lipid peroxidation in the brain, and so that may contribute to the pathophysiology of neurological disorders.

  9. A quantitative proteomic analysis of long-term memory

    Directory of Open Access Journals (Sweden)

    Rosenegger David

    2010-03-01

    Full Text Available Abstract Background Memory is the ability to store, retain, and later retrieve learned information. Long-term memory (LTM formation requires: DNA transcription, RNA translation, and the trafficking of newly synthesized proteins. Several components of these processes have already been identified. However, due to the complexity of the memory formation process, there likely remain many yet to be identified proteins involved in memory formation and persistence. Results Here we use a quantitative proteomic method to identify novel memory-associated proteins in neural tissue taken from animals that were trained in vivo to form a long-term memory. We identified 8 proteins that were significantly up-regulated, and 13 that were significantly down-regulated in the LTM trained animals as compared to two different control groups. In addition we found 19 proteins unique to the trained animals, and 12 unique proteins found only in the control animals. Conclusions These results both confirm the involvement of previously identified memory proteins such as: protein kinase C (PKC, adenylate cyclase (AC, and proteins in the mitogen-activated protein kinase (MAPK pathway. In addition these results provide novel protein candidates (e.g. UHRF1 binding protein on which to base future studies.

  10. Proteomic Analysis of Hylocereus polyrhizus Reveals Metabolic Pathway Changes.

    Science.gov (United States)

    Hua, Qingzhu; Zhou, Qianjun; Gan, Susheng; Wu, Jingyu; Chen, Canbin; Li, Jiaqiang; Ye, Yaoxiong; Zhao, Jietang; Hu, Guibing; Qin, Yonghua

    2016-09-28

    Red dragon fruit or red pitaya (Hylocereus polyrhizus) is the only edible fruit that contains betalains. The color of betalains ranges from red and violet to yellow in plants. Betalains may also serve as an important component of health-promoting and disease-preventing functional food. Currently, the biosynthetic and regulatory pathways for betalain production remain to be fully deciphered. In this study, isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analyses were used to reveal the molecular mechanism of betalain biosynthesis in H. polyrhizus fruits at white and red pulp stages, respectively. A total of 1946 proteins were identified as the differentially expressed between the two samples, and 936 of them were significantly highly expressed at the red pulp stage of H. polyrhizus. RNA-seq and iTRAQ analyses showed that some transcripts and proteins were positively correlated; they belonged to "phenylpropanoid biosynthesis", "tyrosine metabolism", "flavonoid biosynthesis", "ascorbate and aldarate metabolism", "betalains biosynthesis" and "anthocyanin biosynthesis". In betalains biosynthesis pathway, several proteins/enzymes such as polyphenol oxidase, CYP76AD3 and 4,5-dihydroxy-phenylalanine (DOPA) dioxygenase extradiol-like protein were identified. The present study provides a new insight into the molecular mechanism of the betalain biosynthesis at the posttranscriptional level.

  11. Role of salivary and candidal proteins in denture stomatitis: an exploratory proteomic analysis.

    Science.gov (United States)

    Byrd, Warren C; Schwartz-Baxter, Sarah; Carlson, Jim; Barros, Silvana; Offenbacher, Steven; Bencharit, Sompop

    2014-07-29

    Denture stomatitis, inflammation and redness beneath a denture, affects nearly half of all denture wearers. Candidal organisms, the presence of a denture, saliva, and host immunity are the key etiological factors for the condition. The role of salivary proteins in denture stomatitis is not clear. In this study 30 edentulous subjects wearing a maxillary complete denture were recruited. Unstimulated whole saliva from each subject was collected and pooled into two groups (n = 15 each), healthy and stomatitis (Newton classification II and III). Label-free multidimensional liquid chromatography/tandem mass spectrometry (2D-LC-MS/MS) proteomics on two mass spectrometry platforms were used to determine peptide mass differences between control and stomatitis groups. Cluster analysis and principal component analysis were used to determine the differential expression among the groups. The two proteomic platforms identified 97 and 176 proteins (ANOVA; p stomatitis groups. Three proteins including carbonic anhydrase 6, cystatin C, and cystatin SN were found to be the same as previous study. Salivary proteomic profiles of patients with denture stomatitis were found to be uniquely different from controls. Analysis of protein components suggests that certain salivary proteins may predispose some patients to denture stomatitis while others are believed to be involved in the reaction to fungal infection. Analysis of candidal proteins suggests that multiple species of candidal organisms play a role in denture stomatitis.

  12. Differential proteomic analysis highlights metabolic strategies associated with balhimycin production in Amycolatopsis balhimycina chemostat cultivations

    DEFF Research Database (Denmark)

    Gallo, Giuseppe; Alduina, Rosa; Renzone, Giovanni

    2010-01-01

    , used to generate biomass for proteomic analysis, mycelia grew with the same rate and with similar glucose-biomass conversion efficiencies. Global gene expression analysis revealed a differential metabolic adaptation, highlighting strategies for energetic supply and biosynthesis of metabolic...... to balhimycin biosynthesis, and of phoP, phoR, pstS and phoD, known to be associated to Pi limitation stress response. 2D-Differential Gel Electrophoresis (DIGE) and protein identification, performed by mass spectrometry and computer-assisted 2 D reference-map http......://www.unipa.it/ampuglia/Abal-proteome-maps webcite matching, demonstrated a differential expression for proteins involved in many metabolic pathways or cellular processes, including central carbon and phosphate metabolism. Interestingly, proteins playing a key role in generation of primary metabolism intermediates and cofactors required...

  13. Proteomic analysis of rutin-induced secreted proteins from Aspergillus flavus.

    Science.gov (United States)

    Medina, Martha L; Kiernan, Urban A; Francisco, Wilson A

    2004-03-01

    Few studies have been conducted to identify the extracellular proteins and enzymes secreted by filamentous fungi, particularly with respect to dispensable metabolic pathways. Proteomic analysis has proven to be the most powerful method for identification of proteins in complex mixtures and is suitable for the study of the alteration of protein expression under different environmental conditions. The filamentous fungus Aspergillus flavus can degrade the flavonoid rutin as the only source of carbon via an extracellular enzyme system. In this study, a proteomic analysis was used to differentiate and identify the extracellular rutin-induced and non-induced proteins secreted by A. flavus. The secreted proteins were analyzed by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. While 15 rutin-induced proteins and 7 non-induced proteins were identified, more than 90 protein spots remain unidentified, indicating that these proteins are either novel proteins or proteins that have not yet been sequenced.

  14. Dynamic Proteomic Characteristics and Network Integration Revealing Key Proteins for Two Kernel Tissue Developments in Popcorn.

    Directory of Open Access Journals (Sweden)

    Yongbin Dong

    Full Text Available The formation and development of maize kernel is a complex dynamic physiological and biochemical process that involves the temporal and spatial expression of many proteins and the regulation of metabolic pathways. In this study, the protein profiles of the endosperm and pericarp at three important developmental stages were analyzed by isobaric tags for relative and absolute quantification (iTRAQ labeling coupled with LC-MS/MS in popcorn inbred N04. Comparative quantitative proteomic analyses among developmental stages and between tissues were performed, and the protein networks were integrated. A total of 6,876 proteins were identified, of which 1,396 were nonredundant. Specific proteins and different expression patterns were observed across developmental stages and tissues. The functional annotation of the identified proteins revealed the importance of metabolic and cellular processes, and binding and catalytic activities for the development of the tissues. The whole, endosperm-specific and pericarp-specific protein networks integrated 125, 9 and 77 proteins, respectively, which were involved in 54 KEGG pathways and reflected their complex metabolic interactions. Confirmation for the iTRAQ endosperm proteins by two-dimensional gel electrophoresis showed that 44.44% proteins were commonly found. However, the concordance between mRNA level and the protein abundance varied across different proteins, stages, tissues and inbred lines, according to the gene cloning and expression analyses of four relevant proteins with important functions and different expression levels. But the result by western blot showed their same expression tendency for the four proteins as by iTRAQ. These results could provide new insights into the developmental mechanisms of endosperm and pericarp, and grain formation in maize.

  15. Proteomic Analysis of Tardigrades: Towards a Better Understanding of Molecular Mechanisms by Anhydrobiotic Organisms

    OpenAIRE

    Schokraie, Elham; Hotz-Wagenblatt, Agnes; Warnken, Uwe; Mali, Brahim; Frohme, Marcus; Förster, Frank; Dandekar, Thomas; Hengherr, Steffen; Schill, Ralph O.; Schnölzer, Martina

    2010-01-01

    Background Tardigrades are small, multicellular invertebrates which are able to survive times of unfavourable environmental conditions using their well-known capability to undergo cryptobiosis at any stage of their life cycle. Milnesium tardigradum has become a powerful model system for the analysis of cryptobiosis. While some genetic information is already available for Milnesium tardigradum the proteome is still to be discovered. Principal Findings Here we present to the best of our knowled...

  16. Mass spectrometric identification of proteins and characterization of their post-translational modifications in proteome analysis

    DEFF Research Database (Denmark)

    Roepstorff, P; Larsen, Martin Røssel

    2001-01-01

    dominant strategies for identification of proteins from gels based on peptide mass spectrometric fingerprinting and partial sequencing by mass spectrometry are described. After identification of the proteins the next challenge in proteome analysis is characterization of their post-translational...... modifications. The general problems associated with characterization of these directly from gel separated proteins are described and the current state of art for the determination of phosphorylation, glycosylation and proteolytic processing is illustrated....

  17. Ovarian Cancer Proteomic, Phosphoproteomic, and Glycoproteomic Data Released - Office of Cancer Clinical Proteomics Research

    Science.gov (United States)

    National Cancer Institute (NCI) Clinical Proteomic Tumor Analysis Consortium (CPTAC) scientists have just released a comprehensive dataset of the proteomic analysis of high grade serous ovarian tumor samples,

  18. Protein Charge and Mass Contribute to the Spatio-temporal Dynamics of Protein-Protein Interactions in a Minimal Proteome

    Science.gov (United States)

    Xu, Yu; Wang, Hong; Nussinov, Ruth; Ma, Buyong

    2013-01-01

    We constructed and simulated a ‘minimal proteome’ model using Langevin dynamics. It contains 206 essential protein types which were compiled from the literature. For comparison, we generated six proteomes with randomized concentrations. We found that the net charges and molecular weights of the proteins in the minimal genome are not random. The net charge of a protein decreases linearly with molecular weight, with small proteins being mostly positively charged and large proteins negatively charged. The protein copy numbers in the minimal genome have the tendency to maximize the number of protein-protein interactions in the network. Negatively charged proteins which tend to have larger sizes can provide large collision cross-section allowing them to interact with other proteins; on the other hand, the smaller positively charged proteins could have higher diffusion speed and are more likely to collide with other proteins. Proteomes with random charge/mass populations form less stable clusters than those with experimental protein copy numbers. Our study suggests that ‘proper’ populations of negatively and positively charged proteins are important for maintaining a protein-protein interaction network in a proteome. It is interesting to note that the minimal genome model based on the charge and mass of E. Coli may have a larger protein-protein interaction network than that based on the lower organism M. pneumoniae. PMID:23420643

  19. Comparative proteomic analysis of the short-term responses of rice roots and leaves to cadmium.

    Science.gov (United States)

    Lee, Kyunghee; Bae, Dong Won; Kim, Sun Ho; Han, Hay Ju; Liu, Xiaomin; Park, Hyeong Cheol; Lim, Chae Oh; Lee, Sang Yeol; Chung, Woo Sik

    2010-02-15

    Cadmium (Cd) is a non-essential heavy metal that is recognized as a major environmental pollutant. While Cd responses and toxicities in some plant species have been well established, there are few reports about the effects of short-term exposure to Cd on rice, a model monocotyledonous plant, at the proteome level. To investigate the effect of Cd in rice, we monitored the influence of Cd exposure on root and leaf proteomes. After Cd treatment, root and leaf tissues were separately collected and leaf proteins were fractionated with polyethylene glycol. Differentially regulated proteins were selected after image analysis and identified using MALDI-TOF MS. A total of 36 proteins were up- or down-regulated following Cd treatment. As expected, total glutathione levels were significantly decreased in Cd-treated roots, and approximately half of the up-regulated proteins in roots were involved in responses to oxidative stress. These results suggested that prompt antioxidative responses might be necessary for the reduction of Cd-induced oxidative stress in roots but not in leaves. In addition, RNA gel blot analysis showed that the proteins identified in the proteomic analysis were also differentially regulated at the transcriptional level. Collectively, our study provides insights into the integrated molecular mechanisms of early responses to Cd in rice.

  20. From raw data to biological discoveries: a computational analysis pipeline for mass spectrometry-based proteomics.

    Science.gov (United States)

    Lavallée-Adam, Mathieu; Park, Sung Kyu Robin; Martínez-Bartolomé, Salvador; He, Lin; Yates, John R

    2015-11-01

    In the last two decades, computational tools for mass spectrometry-based proteomics data analysis have evolved from a few stand-alone software solutions serving specific goals, such as the identification of amino acid sequences based on mass spectrometry spectra, to large-scale complex pipelines integrating multiple computer programs to solve a collection of problems. This software evolution has been mostly driven by the appearance of novel technologies that allowed the community to tackle complex biological problems, such as the identification of proteins that are differentially expressed in two samples under different conditions. The achievement of such objectives requires a large suite of programs to analyze the intricate mass spectrometry data. Our laboratory addresses complex proteomics questions by producing and using algorithms and software packages. Our current computational pipeline includes, among other things, tools for mass spectrometry raw data processing, peptide and protein identification and quantification, post-translational modification analysis, and protein functional enrichment analysis. In this paper, we describe a suite of software packages we have developed to process mass spectrometry-based proteomics data and we highlight some of the new features of previously published programs as well as tools currently under development. Graphical Abstract ᅟ.

  1. Proteomic analysis of rat cerebral cortex following subchronic acrolein toxicity

    Energy Technology Data Exchange (ETDEWEB)

    Rashedinia, Marzieh; Lari, Parisa [Department of Pharmacodynamics and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad (Iran, Islamic Republic of); Abnous, Khalil, E-mail: Abnouskh@mums.ac.r [Pharmaceutical Research Center, Department of Medicinal Chemistry, Mashhad University of Medical Sciences, Mashhad (Iran, Islamic Republic of); Hosseinzadeh, Hossein, E-mail: Hosseinzadehh@mums.ac.ir [Pharmaceutical Research Center, Department of Pharmacodynamics and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad (Iran, Islamic Republic of)

    2013-10-01

    Acrolein, a member of reactive α,β-unsaturated aldehydes, is a major environmental pollutant. Acrolein is also produced endogenously as a toxic by-product of lipid peroxidation. Because of high reactivity, acrolein may mediate oxidative damages to cells and tissues. It has been shown to be involved in a wide variety of pathological states including pulmonary, atherosclerosis and neurodegenerative diseases. In this study we employed proteomics approach to investigate the effects of subchronic oral exposures to 3 mg/kg of acrolein on protein expression profile in the brain of rats. Moreover effects of acrolein on malondialdehyde (MDA) levels and reduced glutathione (GSH) content were investigated. Our results revealed that treatment with acrolein changed levels of several proteins in diverse physiological process including energy metabolism, cell communication and transport, response to stimulus and metabolic process. Interestingly, several differentially over-expressed proteins, including β-synuclein, enolase and calcineurin, are known to be associated with human neurodegenerative diseases. Changes in the levels of some proteins were confirmed by Western blot. Moreover, acrolein increases the level of MDA, as a lipid peroxidation biomarker and decreased GSH concentrations, as a non-enzyme antioxidant in the brain of acrolein treated rats. These findings suggested that acrolein induces the oxidative stress and lipid peroxidation in the brain, and so that may contribute to the pathophysiology of neurological disorders. - Highlights: • Acrolein intoxication increased lipid peroxidation and deplete GSH in rat brain. • Effect of acrolein on protein levels of cerebral cortex was analyzed by 2DE-PAGE. • Levels of a number of proteins with different biological functions were increased.

  2. multiplierz: an extensible API based desktop environment for proteomics data analysis

    Directory of Open Access Journals (Sweden)

    Webber James T

    2009-10-01

    Full Text Available Abstract Background Efficient analysis of results from mass spectrometry-based proteomics experiments requires access to disparate data types, including native mass spectrometry files, output from algorithms that assign peptide sequence to MS/MS spectra, and annotation for proteins and pathways from various database sources. Moreover, proteomics technologies and experimental methods are not yet standardized; hence a high degree of flexibility is necessary for efficient support of high- and low-throughput data analytic tasks. Development of a desktop environment that is sufficiently robust for deployment in data analytic pipelines, and simultaneously supports customization for programmers and non-programmers alike, has proven to be a significant challenge. Results We describe multiplierz, a flexible and open-source desktop environment for comprehensive proteomics data analysis. We use this framework to expose a prototype version of our recently proposed common API (mzAPI designed for direct access to proprietary mass spectrometry files. In addition to routine data analytic tasks, multiplierz supports generation of information rich, portable spreadsheet-based reports. Moreover, multiplierz is designed around a "zero infrastructure" philosophy, meaning that it can be deployed by end users with little or no system administration support. Finally, access to multiplierz functionality is provided via high-level Python scripts, resulting in a fully extensible data analytic environment for rapid development of custom algorithms and deployment of high-throughput data pipelines. Conclusion Collectively, mzAPI and multiplierz facilitate a wide range of data analysis tasks, spanning technology development to biological annotation, for mass spectrometry-based proteomics research.

  3. Application of nano-LC-MALDI-TOF/TOF-MS for proteomic analysis of microvesicles.

    Science.gov (United States)

    Kasprzyk, Joanna; Stępień, Ewa; Piekoszewski, Wojciech

    2017-03-01

    Activated platelets and platelet derived microvesicles (PMVs) emerged recently to be promising biomarkers. There is no universal procedure to carry out the proteomic analysis on microvesicles. In this study we proposed a nano-liquid chromatography (nano-LC) technique coupled off-line with a spectrometric measurement MALDI-TOF-MS/MS as a throughput and time-saving procedure. In this study we developed a simplified method to evaluate the protein composition of platelet organelles and PMVs. Platelet-rich plasma (PRP) was collected from healthy donors. PMVs were generated from washed and thrombin activated platelets. Activated platelets from every donor were used to compare the PMV proteome. Enzymatic digestion of protein lysate was carried out using Filter Aided Sample Preparation (FASP) method with trypsin as a proteolytic enzyme. Tryptic peptides derived from PMVs and activated platelets were analysed using nano-LC coupled off-line mode with a MALDI-TOF/TOF-MS. PMV and platelet protein identification was performed using the Mascot engine for searching against the Swiss-Prot human database. The precision tolerance was 100ppm for peptide masses and 0.7Da for fragment ion masses. Individual peptide matches with a score above 28 were considered statistically significant. In total, 446 proteins were identified in PMVs and 513 proteins in activated platelets. Among them 190 were specific for activated platelets and 123 were PMV specific. Cellular component analysis of identified proteins revealed that PMVs contained relatively more extracellular proteins than activated platelets (9.6 vs. 6.0 %) and unique synaptic proteins (0.3%). A new simplified bottom-up method for PMV proteome analysis allowed eliminating the drawbacks of the previously used protocols. This approach can be used in PMV proteome identification. Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-11-01

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

  5. Two-dimensional electrophoresis analysis of proteomics based on image feature and mathematical morphology

    Institute of Scientific and Technical Information of China (English)

    SHEN Peng; FAN Xiaohui; ZENG Zhen; CHENG Yiyu

    2005-01-01

    In this paper, a novel method to automatically detect protein spots on a two-dimensional (2-D) electrophoresis gel image is proposed to implement proteomics analysis of complex analyte.On the basis of the identifying spots results based on color variation and spot size features, morphological feature is introduced as a new criterion to distinguish protein spots from non-protein spots.Image-sharpening, edge-detecting and morphological feature extraction methods were consequently combined to detect protein spots on a 2-D electrophoresis gel image subject to strong disturbance.The proposed method was applied to detect the protein spots of proteomic gel images from E.coli cell, human kidney tissue and human serum.The results demonstrated that this method is more accurate and reliable than previous methods such as PDQuest 7.2 and ImageMaster 5.0 software for detecting protein spots on gel images with strong interferences.

  6. Quantitative proteomic analysis of post-translational modifications of human histones

    DEFF Research Database (Denmark)

    Beck, Hans Christian; Nielsen, Eva C; Matthiesen, Rune

    2006-01-01

    software for qualitative and quantitative proteomic analysis of histones extracted from human small cell lung cancer cells. A total of 32 acetylations, methylations, and ubiquitinations were located in the human histones H2A, H2B, H3, and H4, including seven novel modifications. An LC-MSMS-based method....... Deciphering of the histone code is hampered by the lack of analytical methods for monitoring the combinatorial complexity of reversible multisite modifications of histones, including acetylation and methylation. To address this problem, we used LC-MSMS technology and Virtual Expert Mass Spectrometrist...... was applied in a quantitative proteomic study of the dose-response effect of the histone deacetylase inhibitor (HDACi) PXD101 on histone acetylation in human cell cultures. Triplicate LC-MSMS runs at six different HDACi concentrations demonstrated that PXD101 affects acetylation of histones H2A, H2B, H3...

  7. Proteomic analysis for detecting serum biomarkers related to smoking in humans

    Institute of Scientific and Technical Information of China (English)

    XIAO Dan; ZHAO Li-juan; CHU Shui-lian; JING Hang; WANG Chen

    2012-01-01

    Background Smoking is the leading cause of death in the world.This study focused on the difference of the serum proteomic profiling between healthy smokers and nonsmokers in order to find smoking-specific serum biomarkers.Methods Pattern-based proteomic profiling of 100 serum samples (from 50 Chinese male smokers and 50 matched nonsmokers) was performed through magnetic bead fractionation coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis (MALDI-TOF-MS) and resulting data were statistically analyzed by Ciphergen ProteinChip software 3.0.2.Results We found 72 serum peaks were significantly different between smokers and nonsmokers (P <0.05).Marker peaks of mass-to-charge ratio (m/z) 3159.13,7561.03 and 9407.32 were smoking-specific.Conclusion The preliminary data suggested that smoking-specific serum biomarkers could be detected in humans.

  8. APols-aided protein precipitation: a rapid method for concentrating proteins for proteomic analysis.

    Science.gov (United States)

    Ning, Zhibin; Hawley, Brett; Seebun, Deeptee; Figeys, Daniel

    2014-10-01

    Amphipols (APols) are a newly designed and milder class of detergent. They have been used primarily in protein structure analysis for membrane protein trapping and stabilization. We have recently demonstrated that APols can be used as an alternative detergent for proteome extraction and digestion, to achieve a "One-stop" single-tube workflow for proteomics. In this workflow, APols are removed by precipitation after protein digestion without depleting the digested peptides. Here, we took further advantage of this precipitation characteristic of APols to concentrate proteins from diluted samples. In contrast with tryptic peptides, a decrease in pH leads to the unbiased co-precipitation of APols with proteins, including globular hydrophilic proteins. We demonstrated that this precipitation is a combined effect of acid precipitation and the APols' protein interactions. Also, we have been able to demonstrate that APols-aided protein precipitation works well on diluted samples, such as secretome sample, and provides a rapid method for protein concentration.

  9. Preliminary Proteomic Analysis of Tobacco Leaves Influenced by Floral Scent from Rose

    Institute of Scientific and Technical Information of China (English)

    Ping Yu; Yuan Su; Xiaogan Zhou

    2012-01-01

    The volatile organic compounds (VOCs) play an important role in plant communication.There are many VOCs such as:aroma alcohols,esters and terpenes in rose floral scent.The tobacco would be influenced by floral scent from rose is unclear.To understand tobacco response induced by rose floral scent,we carried out proteomic analysis of tobacco leaves influenced by floral scent from rose using 2-DE.Protein profiles showed that 470 ±37 protein spots could be detected in sole tobacco and 319 ±18 in tobacco/rose respectively.Among them,26 protein spots showed 2-fold change in protein expression level,6 protein spots were up-regulated and 20 were down-regulated.We hope to acquire more information to interpret proteomic change of tobacco leaves influenced by floral scent from rose after MS identification above differential expression proteins.

  10. Proteomic Analysis of Lyme Disease: Global Protein Comparison of Three Strains of Borrelia burgdorferi

    Energy Technology Data Exchange (ETDEWEB)

    Jacobs, Jon M.; Yang, Xiaohua; Luft, Benjamin J.; Dunn, John J.; Camp, David G.; Smith, Richard D.

    2005-04-01

    The Borrelia burgdorferi spirochete is the causative agent of Lyme disease, the most common tick-borne disease in the United States. It has been studied extensively to help understand its pathogenicity of infection and how it can persist in different mammalian hosts. We report the proteomic analysis of the archetype B. burgdorferi B31 strain and two other strains (ND40, and JD-1) having different Borrelia pathotypes using strong cation exchange fractionation of proteolytic peptides followed by high-resolution, reversed phase capillary liquid chromatography coupled with ion trap tandem mass spectrometric (LC-MS/MS) analysis. Protein identification was facilitated by the availability of the complete B31 genome sequence. A total of 665 Borrelia proteins were identified representing ~38 % coverage of the theoretical B31 proteome. A significant overlap was observed between the identified proteins in direct comparisons between any two strains (>72%), but distinct differences were observed among identified hypothetical and outer membrane proteins of the three strains. Such a concurrent proteomic overview of three Borrelia strains based upon only the B31 genome sequence is shown to provide significant insights into the presence or absence of specific proteins and a broad overall comparison among strains.

  11. Quantitative proteomic analysis of human lung tumor xenografts treated with the ectopic ATP synthase inhibitor citreoviridin.

    Directory of Open Access Journals (Sweden)

    Yi-Hsuan Wu

    Full Text Available ATP synthase is present on the plasma membrane of several types of cancer cells. Citreoviridin, an ATP synthase inhibitor, selectively suppresses the proliferation and growth of lung cancer without affecting normal cells. However, the global effects of targeting ectopic ATP synthase in vivo have not been well defined. In this study, we performed quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ and provided a comprehensive insight into the complicated regulation by citreoviridin in a lung cancer xenograft model. With high reproducibility of the quantitation, we obtained quantitative proteomic profiling with 2,659 proteins identified. Bioinformatics analysis of the 141 differentially expressed proteins selected by their relative abundance revealed that citreoviridin induces alterations in the expression of glucose metabolism-related enzymes in lung cancer. The up-regulation of enzymes involved in gluconeogenesis and storage of glucose indicated that citreoviridin may reduce the glycolytic intermediates for macromolecule synthesis and inhibit cell proliferation. Using comprehensive proteomics, the results identify metabolic aspects that help explain the antitumorigenic effect of citreoviridin in lung cancer, which may lead to a better understanding of the links between metabolism and tumorigenesis in cancer therapy.

  12. Metabolomics as an extension of proteomic analysis: study of acute kidney injury.

    Science.gov (United States)

    Portilla, Didier; Schnackenberg, Laura; Beger, Richard D

    2007-11-01

    Although proteomics studies the global expression of proteins, metabolomics characterizes and quantifies their end products: the metabolites, produced by an organism under a certain set of conditions. From this perspective it is apparent that proteomics and metabolomics are complementary and when joined allow a fuller appreciation of an organism's phenotype. Our studies using (1)H-nuclear magnetic resonance spectroscopic analysis showed the presence of glucose, amino acids, and trichloroacetic acid cycle metabolites in the urine after 48 hours of cisplatin administration. These metabolic alterations precede changes in serum creatinine. Biochemical studies confirmed the presence of glucosuria, but also showed the accumulation of nonesterified fatty acids, and triglycerides in serum, urine, and kidney tissue, despite increased levels of plasma insulin. These metabolic alterations were ameliorated by the use of fibrates. We propose that the injury-induced metabolic profile may be used as a biomarker of cisplatin-induced nephrotoxicity. These studies serve to illustrate that metabolomic studies add insight into pathophysiology not provided by proteomic analysis alone.

  13. Data set for the proteomics analysis of the endomembrane system from the unicellular Entamoeba histolytica

    Directory of Open Access Journals (Sweden)

    Doranda Perdomo

    2014-12-01

    Full Text Available Entamoeba histolytica is the protozoan parasite agent of amebiasis, an infectious disease of the human intestine and liver. This parasite contact and kills human cells by an active process involving pathogenic factors. Cellular traffic and secretion activities are poorly characterized in E. histolytica. In this work, we took advantage of a wide proteomic analysis to search for principal components of the endomembrane system in E. histolytica. A total of 5683 peptides matching with 1531 proteins (FDR of 1% were identified which corresponds to roughly 20% of the total amebic proteome. Bioinformatics investigations searching for domain homologies (Smart and InterProScan programs and functional descriptions (KEGG and GO terms allowed this data to be organized into distinct categories. This data represents the first in-depth proteomics analysis of subcellular compartments in E. histolytica and allows a detailed map of vesicle traffic components in an ancient single-cell organism that lacks a stereotypical ER and Golgi apparatus to be established. The data are related to [1].

  14. Comparative Proteomic Analysis of Cotton Fiber Development and Protein Extraction Method Comparison in Late Stage Fibers

    Directory of Open Access Journals (Sweden)

    Hana Mujahid

    2016-02-01

    Full Text Available The distinct stages of cotton fiber development and maturation serve as a single-celled model for studying the molecular mechanisms of plant cell elongation, cell wall development and cellulose biosynthesis. However, this model system of plant cell development is compromised for proteomic studies due to a lack of an efficient protein extraction method during the later stages of fiber development, because of a recalcitrant cell wall and the presence of abundant phenolic compounds. Here, we compared the quality and quantities of proteins extracted from 25 dpa (days post anthesis fiber with multiple protein extraction methods and present a comprehensive quantitative proteomic study of fiber development from 10 dpa to 25 dpa. Comparative analysis using a label-free quantification method revealed 287 differentially-expressed proteins in the 10 dpa to 25 dpa fiber developmental period. Proteins involved in cell wall metabolism and regulation, cytoskeleton development and carbohydrate metabolism among other functional categories in four fiber developmental stages were identified. Our studies provide protocols for protein extraction from maturing fiber tissues for mass spectrometry analysis and expand knowledge of the proteomic profile of cotton fiber development.

  15. Proteomic analysis of male 4C germ cell proteins involved in mouse meiosis.

    Science.gov (United States)

    Guo, Xuejiang; Zhang, Ping; Qi, Yujuan; Chen, Wen; Chen, Xiangxiang; Zhou, Zuomin; Sha, Jiahao

    2011-01-01

    Male meiosis is a specialized type of cell division that gives rise to sperm. Errors in this process can result in the generation of aneuploid gametes, which are associated with birth defects and infertility in humans. Until now, there has been a lack of a large-scale identification of proteins involved in male meiosis in mammals. In this study, we report the high-confidence identification of 3625 proteins in mouse male germ cells with 4C DNA content undergoing meiosis I. Of these, 397 were found to be testis specific. Bioinformatics analysis of the proteome led to the identification of 28 proteins known to be essential for male meiosis in mice. We also found 172 proteins that had yeast orthologs known to be essential for meiosis. Chromosome distribution analysis of the proteome showed underrepresentation of the identified proteins on the X chromosome, which may be due to meiotic sex chromosome inactivation. Characterization of the proteome of 4C germ cells from mouse testis provides an inventory of proteins, which is useful for understanding meiosis and the mechanisms of male infertility.

  16. Quantitative proteomic analysis of mice corneal tissues reveals angiogenesis-related proteins involved in corneal neovascularization.

    Science.gov (United States)

    Shen, Minqian; Tao, Yimin; Feng, Yifan; Liu, Xing; Yuan, Fei; Zhou, Hu

    2016-07-01

    Corneal neovascularization (CNV) was induced in Balb/c mice by alkali burns in the central area of the cornea with a diameter of 2.5mm. After fourteen days, the cornea from one eye was collected for histological staining for CNV examination, while the cornea from the other eye of the same mouse was harvested for proteomic analysis. The label-free quantitative proteomic approach was applied to analyze five normal corneal tissues (normal group mice n=5) and five corresponding neovascularized corneal tissues (model group mice n=5). A total of 2124 proteins were identified, and 1682 proteins were quantified from these corneal tissues. Among these quantified proteins, 290 proteins were significantly changed between normal and alkali burned corneal tissues. Of these significantly changed proteins, 35 were reported or predicted as angiogenesis-related proteins. Then, these 35 proteins were analyzed using Ingenuity Pathway Analysis Software, resulting in 26 proteins enriched and connected to each other in the protein-protein interaction network, such as Lcn-2, αB-crystallin and Serpinf1 (PEDF). These three significantly changed proteins were selected for further Western blotting validation. Consistent with the quantitative proteomic results, Western blotting showed that Lcn-2 and αB-crystallin were significantly up-regulated in CNV model, while PEDF was down-regulated. This study provided increased understanding of angiogenesis-related proteins involved in corneal vascular development, which will be useful in the ophthalmic clinic of specifically target angiogenesis.

  17. Comparative proteomic analysis of Bactrocera dorsalis (Hendel) in response to thermal stress.

    Science.gov (United States)

    Wei, Dong; Jia, Fu-Xian; Tian, Chuan-Bei; Tian, Yi; Smagghe, Guy; Dou, Wei; Wang, Jin-Jun

    2015-03-01

    Temperature is one of the most important environmental variables affecting growth, reproduction and distribution of insects. The rise of comparative proteomics provides a powerful tool to explore the response in proteins to thermal stress. As an important worldwide pest, the oriental fruit fly Bactrocera dorsalis causes severe economic losses to crops. To understand the response of B. dorsalis to thermal stress, we performed a comparative proteome analysis of this insect after exposure to extreme low and high temperatures using two-dimensional electrophoresis. Among the separated proteins, 51 diverse protein spots were present differently in response to extreme temperatures. Using tandem mass spectrometry sequencing analysis 39 proteins were successfully identified, which included 13 oxidoreductases, 10 binding proteins, 5 transferases, and 2 each of lyases, isomerases, ligases, and developmental proteins. Subsequently, the expression of these protein transcripts was studied by RT-qPCR to validate the proteomic results. In conclusion, this study provides a first look into the thermal stress response of B. dorsalis at the protein level, and thus it paves the way for further functional studies in the physiological mechanism related to thermal stress. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Characterization of acute renal allograft rejection by proteomic analysis of renal tissue in rat.

    Science.gov (United States)

    Chen, Gang; Huang, Jing-Bin; Mi, Jie; He, Yun-Feng; Wu, Xiao-Hou; Luo, Chun-Li; Liang, Si-Min; Li, Jia-Bing; Tang, Ya-Xiong; Li, Jie

    2012-02-01

    Rapid and reliable biomarkers of renal allograft rejection have not been available. This study aimed to investigate biomarkers in renal allograft tissue using proteomic analysis. Orthotopic kidney transplantations were performed using Fisher (F344) or Lewis rats as donors and Lewis rats as recipients. Syngenic control group (Group I) constituted F344-to-F344 orthotopic kidney allo-transplantations (n = 8); and allogenic group (Group II) consisted of F344-to-Lewis orthotopic kidney allo-transplantations (n = 8). Renal tissues were harvested 7 days after transplantation. Samples were analyzed using 2-D electrophoresis and matrix assisted laser desorption ionization-time of flight mass spectrometry. 6 differentially expressed proteins were identified between allogenic group and syngenic control group. A rat model of acute renal allograft rejection was successfully set up. Differentially expressed proteins in renal allograft tissue of rat were detected using proteomic analysis and might serve as novel diagnostic and therapeutic targets in human. Quantitative proteomics, using MALDL-TOF-MS methodology has the potential to provide a profiling and a deeper understanding of acute renal rejection.

  19. Global Analysis of Nonlinear Dynamics

    CERN Document Server

    Luo, Albert

    2012-01-01

    Global Analysis of Nonlinear Dynamics collects chapters on recent developments in global analysis of non-linear dynamical systems with a particular emphasis on cell mapping methods developed by Professor C.S. Hsu of the University of California, Berkeley. This collection of contributions prepared by a diverse group of internationally recognized researchers is intended to stimulate interests in global analysis of complex and high-dimensional nonlinear dynamical systems, whose global properties are largely unexplored at this time. This book also: Presents recent developments in global analysis of non-linear dynamical systems Provides in-depth considerations and extensions of cell mapping methods Adopts an inclusive style accessible to non-specialists and graduate students Global Analysis of Nonlinear Dynamics is an ideal reference for the community of nonlinear dynamics in different disciplines including engineering, applied mathematics, meteorology, life science, computational science, and medicine.  

  20. Comprehensive proteome analysis of the response of Pseudomonas putida KT2440 to the flavor compound vanillin.

    Science.gov (United States)

    Simon, Oliver; Klaiber, Iris; Huber, Armin; Pfannstiel, Jens

    2014-09-23

    Understanding of the molecular response of bacteria to precursors, products and environmental conditions applied in bioconversions is essential for optimizing whole-cell biocatalysis. To investigate the molecular response of the potential biocatalyst Pseudomonas putida KT2440 to the flavor compound vanillin we applied complementary gel- and LC-MS-based quantitative proteomics approaches. Our comprehensive proteomics survey included cytoplasmic and membrane proteins and led to the identification and quantification of 1614 proteins, corresponding to 30% of the total KT2440 proteome. 662 proteins were altered in abundance during growth on vanillin as sole carbon source as compared to growth on glucose. The proteome response entailed an increased abundance of enzymes involved in vanillin degradation, significant changes in central energy metabolism and an activation of solvent tolerance mechanisms. With respect to vanillin metabolism, particularly enzymes belonging to the β-ketoadipate pathway including a transcriptional regulator and porins specific for vanillin uptake increased in abundance. However, catabolism of vanillin was not dependent on vanillin dehydrogenase (Vdh), as shown by quantitative proteome analysis of a Vdh-deficient KT2440 mutant (GN235). Other aldehyde dehydrogenases that were significantly increased in abundance in response to vanillin may replace Vdh and thus may represent interesting targets for improving vanillin production in P. putida KT2440. The high demand for the flavor compound vanillin by the food and fragrance industry makes natural vanillin from vanilla pods a scarce and expensive resource rendering its biotechnological production economically attractive. Pseudomonas bacteria are metabolically very versatile and accept a broad range of hydrocarbons as carbon source making them suitable candidates for bioconversion processes. This work describes the impact of vanillin on the metabolism of the reference strain P. putida KT2440 on a

  1. Proteomic Analysis of Fruit Bending in Cucumber (Cucumis sativus L.)

    Institute of Scientific and Technical Information of China (English)

    WANG Li-li; ZHANG Peng; QIN Zhi-wei; ZHOU Xiu-yan

    2014-01-01

    In cucumber, fruit shape is an important quality criterion, and fruit bending is known to limit growth, yield, and taste. To investigate the post-transcriptional changes that regulate fruit bending and to better understand the underlying molecular mechanisms, we generated a proteomic proifle of the abdomen and back of cucumber bending fruit. Two-dimensional gel electrophoresis (2-DE) allowed the detection of approximately 900 distinct protein spots in each gel, 32 of which were differentially expressed in the abdomen and back of bending cucumber fruit. Ten of the differentially expressed proteins were analyzed using matrix-assisted laser ionization time of lfight mass spectrometry (MALDI-TOF/MS). A search of primary databases showed that the identiifed proteins are involved in various metabolic processes and cellular responses, including photosynthesis metabolism, energy metabolism, defense and stress response, and regulation. The identiifed proteins included large subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase, which are involved in photosynthesis and photorespiratory metabolism, and isocitrate dehydrogenase, which is involved in the tricarboxylic acid cycle. It is possible that imbalances in catabolic and anabolic processes directly affect the bending of cucumber fruit. The predicted function of the cobalamin-independent methionine synthase isozyme is closely related to ethylene biosynthesis; fruit bending may be regulated by ethylene, or by ethylene signaling crosstalk during fruit development. The 14-3-3 protein is usually considered to be a regulation-related protein, which plays a role in regulating cell hyperplasia, cell differentiation during growth, and apoptosis during senescence. Involvement of guanosine triphosphate (GTP)-binding proteins in signal transmission is known to regulate the development of cells in cucumber fruits and to play a role in fruit shape variation. Patterns of protein expression showed high repeatability. We hypothesize

  2. Proteome analysis of human substantia nigra in Parkinson's disease

    Directory of Open Access Journals (Sweden)

    Werner Cornelius J

    2008-02-01

    Full Text Available Abstract Background Parkinson's disease (PD is the most common neurodegenerative disorder involving the motor system. Although not being the only region involved in PD, affection of the substantia nigra and its projections is responsible for some of the most debilitating features of the disease. To further advance a comprehensive understanding of nigral pathology, we conducted a tissue based comparative proteome study of healthy and diseased human substantia nigra. Results The gross number of differentially regulated proteins in PD was 221. In total, we identified 37 proteins, of which 16 were differentially expressed. Identified differential proteins comprised elements of iron metabolism (H-ferritin and glutathione-related redox metabolism (GST M3, GST P1, GST O1, including novel redox proteins (SH3BGRL. Additionally, many glial or related proteins were found to be differentially regulated in PD (GFAP, GMFB, galectin-1, sorcin, as well as proteins belonging to metabolic pathways sparsely described in PD, such as adenosyl homocysteinase (methylation, aldehyde dehydrogenase 1 and cellular retinol-binding protein 1 (aldehyde metabolism. Further differentially regulated proteins included annexin V, beta-tubulin cofactor A, coactosin-like protein and V-type ATPase subunit 1. Proteins that were similarly expressed in healthy or diseased substantia nigra comprised housekeeping proteins such as COX5A, Rho GDI alpha, actin gamma 1, creatin-kinase B, lactate dehydrogenase B, disulfide isomerase ER-60, Rab GDI beta, methyl glyoxalase 1 (AGE metabolism and glutamine synthetase. Interestingly, also DJ-1 and UCH-L1 were expressed similarly. Furthermore, proteins believed to serve as internal standards were found to be expressed in a constant manner, such as 14-3-3 epsilon and hCRMP-2, thus lending further validity to our results. Conclusion Using an approach encompassing high sensitivity and high resolution, we show that alterations of SN in PD include many

  3. Ultra-deep and quantitative saliva proteome reveals dynamics of the oral microbiome

    DEFF Research Database (Denmark)

    Grassl, Niklas; Kulak, Nils Alexander; Pichler, Garwin

    2016-01-01

    , disruptions in saliva secretion and changes in the oral microbiome contribute to conditions such as tooth decay and respiratory tract infections. Here we set out to quantitatively map the saliva proteome in great depth with a rapid and in-depth mass spectrometry-based proteomics workflow. METHODS: We used...... with next-generation sequencing data from the Human Microbiome Project as well as a comparison to MALDI-TOF mass spectrometry on microbial cultures revealed strong agreement. The oral microbiome differs between individuals and changes drastically upon eating and tooth brushing. CONCLUSION: Rapid shotgun...... and robust technology can now simultaneously characterize the human and microbiome contributions to the proteome of a body fluid and is therefore a valuable complement to genomic studies. This opens new frontiers for the study of host-pathogen interactions and clinical saliva diagnostics....

  4. Proteomic analysis of tardigrades: towards a better understanding of molecular mechanisms by anhydrobiotic organisms.

    Directory of Open Access Journals (Sweden)

    Elham Schokraie

    Full Text Available BACKGROUND: Tardigrades are small, multicellular invertebrates which are able to survive times of unfavourable environmental conditions using their well-known capability to undergo cryptobiosis at any stage of their life cycle. Milnesium tardigradum has become a powerful model system for the analysis of cryptobiosis. While some genetic information is already available for Milnesium tardigradum the proteome is still to be discovered. PRINCIPAL FINDINGS: Here we present to the best of our knowledge the first comprehensive study of Milnesium tardigradum on the protein level. To establish a proteome reference map we developed optimized protocols for protein extraction from tardigrades in the active state and for separation of proteins by high resolution two-dimensional gel electrophoresis. Since only limited sequence information of M. tardigradum on the genome and gene expression level is available to date in public databases we initiated in parallel a tardigrade EST sequencing project to allow for protein identification by electrospray ionization tandem mass spectrometry. 271 out of 606 analyzed protein spots could be identified by searching against the publicly available NCBInr database as well as our newly established tardigrade protein database corresponding to 144 unique proteins. Another 150 spots could be identified in the tardigrade clustered EST database corresponding to 36 unique contigs and ESTs. Proteins with annotated function were further categorized in more detail by their molecular function, biological process and cellular component. For the proteins of unknown function more information could be obtained by performing a protein domain annotation analysis. Our results include proteins like protein member of different heat shock protein families and LEA group 3, which might play important roles in surviving extreme conditions. CONCLUSIONS: The proteome reference map of Milnesium tardigradum provides the basis for further studies in

  5. Identification of azurocidin as a potential periodontitis biomarker by a proteomic analysis of gingival crevicular fluid

    Directory of Open Access Journals (Sweden)

    Lee Jae-Mok

    2011-07-01

    Full Text Available Abstract Background The inflammatory disease periodontitis results in tooth loss and can even lead to diseases of the whole body if not treated. Gingival crevicular fluid (GCF reflects the condition of the gingiva and contains proteins transuded from serum or cells at inflamed sites. In this study, we aimed to discover potential protein biomarkers for periodontitis in GCF proteome using LC-MS/MS. Results We identified 305 proteins from GCF of healthy individuals and periodontitis patients collected using a sterile gel loading tip by ESI-MS/MS coupled to nano-LC. Among these proteins, about 45 proteins were differentially expressed in the GCF proteome of moderate periodontitis patients when compared to the healthy individuals. We first identified azurocidin in the GCF, but not the saliva, as an upregulated protein in the periodontitis patients and verified its increased expression during periodontitis by ELISA using the GCF of the classified periodontitis patients compared to the healthy individuals. In addition, we found that azurocidin inhibited the differentiation of bone marrow-derived macrophages to osteoclasts. Conclusions Our results show that GCF collection using a gel loading tip and subsequent LC-MS/MS analysis following 1D-PAGE proteomic separation are effective for the analysis of the GCF proteome. Our current results also suggest that azurocidin could be a potential biomarker candidate for the early detection of inflammatory periodontal destruction by gingivitis and some chronic periodontitis. Our data also suggest that azurocidin may have an inhibitory role in osteoclast differentiation and, thus, a protective role in alveolar bone loss during the early stages of periodontitis.

  6. Proteomic analysis of tardigrades: towards a better understanding of molecular mechanisms by anhydrobiotic organisms.

    Science.gov (United States)

    Schokraie, Elham; Hotz-Wagenblatt, Agnes; Warnken, Uwe; Mali, Brahim; Frohme, Marcus; Förster, Frank; Dandekar, Thomas; Hengherr, Steffen; Schill, Ralph O; Schnölzer, Martina

    2010-03-03

    Tardigrades are small, multicellular invertebrates which are able to survive times of unfavourable environmental conditions using their well-known capability to undergo cryptobiosis at any stage of their life cycle. Milnesium tardigradum has become a powerful model system for the analysis of cryptobiosis. While some genetic information is already available for Milnesium tardigradum the proteome is still to be discovered. Here we present to the best of our knowledge the first comprehensive study of Milnesium tardigradum on the protein level. To establish a proteome reference map we developed optimized protocols for protein extraction from tardigrades in the active state and for separation of proteins by high resolution two-dimensional gel electrophoresis. Since only limited sequence information of M. tardigradum on the genome and gene expression level is available to date in public databases we initiated in parallel a tardigrade EST sequencing project to allow for protein identification by electrospray ionization tandem mass spectrometry. 271 out of 606 analyzed protein spots could be identified by searching against the publicly available NCBInr database as well as our newly established tardigrade protein database corresponding to 144 unique proteins. Another 150 spots could be identified in the tardigrade clustered EST database corresponding to 36 unique contigs and ESTs. Proteins with annotated function were further categorized in more detail by their molecular function, biological process and cellular component. For the proteins of unknown function more information could be obtained by performing a protein domain annotation analysis. Our results include proteins like protein member of different heat shock protein families and LEA group 3, which might play important roles in surviving extreme conditions. The proteome reference map of Milnesium tardigradum provides the basis for further studies in order to identify and characterize the biochemical mechanisms of

  7. LC-MS/MS based proteomic analysis and functional inference of hypothetical proteins in Desulfovibrio vulgaris.

    Science.gov (United States)

    Zhang, Weiwen; Culley, David E; Gritsenko, Marina A; Moore, Ronald J; Nie, Lei; Scholten, Johannes C M; Petritis, Konstantinos; Strittmatter, Eric F; Camp, David G; Smith, Richard D; Brockman, Fred J

    2006-11-03

    High efficiency capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to examine the proteins extracted from Desulfovibrio vulgaris cells across six treatment conditions. While our previous study provided a proteomic overview of the cellular metabolism based on proteins with known functions [W. Zhang, M.A. Gritsenko, R.J. Moore, D.E. Culley, L. Nie, K. Petritis, E.F. Strittmatter, D.G. Camp II, R.D. Smith, F.J. Brockman, A proteomic view of the metabolism in Desulfovibrio vulgaris determined by liquid chromatography coupled with tandem mass spectrometry, Proteomics 6 (2006) 4286-4299], this study describes the global detection and functional inference for hypothetical D. vulgaris proteins. Using criteria that a given peptide of a protein is identified from at least two out of three independent LC-MS/MS measurements and that for any protein at least two different peptides are identified among the three measurements, 129 open reading frames (ORFs) originally annotated as hypothetical proteins were found to encode expressed proteins. Functional inference for the conserved hypothetical proteins was performed by a combination of several non-homology based methods: genomic context analysis, phylogenomic profiling, and analysis of a combination of experimental information, including peptide detection in cells grown under specific culture conditions and cellular location of the proteins. Using this approach we were able to assign possible functions to 20 conserved hypothetical proteins. This study demonstrated that a combination of proteomics and bioinformatics methodologies can provide verification of the expression of hypothetical proteins and improve genome annotation.

  8. Application of proteomics to ecology and population biology.

    Science.gov (United States)

    Karr, T L

    2008-02-01

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

  9. Assessment and improvement of statistical tools for comparative proteomics analysis of sparse data sets with few experimental replicates

    DEFF Research Database (Denmark)

    Schwämmle, Veit; León, Ileana R.; Jensen, Ole Nørregaard

    2013-01-01

    Large-scale quantitative analyses of biological systems are often performed with few replicate experiments, leading to multiple nonidentical data sets due to missing values. For example, mass spectrometry driven proteomics experiments are frequently performed with few biological or technical...... changes on the peptide level, for example, in phospho-proteomics experiments. In order to assess the extent of this problem and the implications for large-scale proteome analysis, we investigated and optimized the performance of three statistical approaches by using simulated and experimental data sets...... with varying numbers of missing values. We applied three tools, including standard t test, moderated t test, also known as limma, and rank products for the detection of significantly changing features in simulated and experimental proteomics data sets with missing values. The rank product method was improved...

  10. Dynamic Analysis of Shells

    Directory of Open Access Journals (Sweden)

    Charles R. Steele

    1995-01-01

    Full Text Available Shell structures are indispensable in virtually every industry. However, in the design, analysis, fabrication, and maintenance of such structures, there are many pitfalls leading to various forms of disaster. The experience gained by engineers over some 200 years of disasters and brushes with disaster is expressed in the extensive archival literature, national codes, and procedural documentation found in larger companies. However, the advantage of the richness in the behavior of shells is that the way is always open for innovation. In this survey, we present a broad overview of the dynamic response of shell structures. The intention is to provide an understanding of the basic themes behind the detailed codes and stimulate, not restrict, positive innovation. Such understanding is also crucial for the correct computation of shell structures by any computer code. The physics dictates that the thin shell structure offers a challenge for analysis and computation. Shell response can be generally categorized by states of extension, inextensional bending, edge bending, and edge transverse shear. Simple estimates for the magnitudes of stress, deformation, and resonance in the extensional and inextensional states are provided by ring response. Several shell examples demonstrate the different states and combinations. For excitation frequency above the extensional resonance, such as in impact and acoustic excitation, a fine mesh is needed over the entire shell surface. For this range, modal and implicit methods are of limited value. The example of a sphere impacting a rigid surface shows that plastic unloading occurs continuously. Thus, there are no short cuts; the complete material behavior must be included.

  11. Analysis of Biostimulated Microbial Communities from Two Field Experiments Reveals Temporal and Spatial Differences in Proteome Profiles

    Energy Technology Data Exchange (ETDEWEB)

    Callister, Stephen J.; Wilkins, Michael J.; Nicora, Carrie D.; Williams, Kenneth H.; Banfield, Jillian F.; VerBerkmoes, Nathan; Hettich, Robert L.; N' Guessan, A. Lucie; Mouser, Paula; Elifantz, H.; Smith, Richard D.; Lovley, Derek R.; Lipton, Mary S.; Long, Philip E.

    2010-12-01

    Stimulated by acetate-amendment field experiments conducted in 2007 and 2008, anaerobic microbial populations in the aquifer at the Rifle Integrated Field Research Challenge site in Colorado reduced mobile U(VI) to insoluble U(IV). During this period, planktonic biomass was sampled at various time points and used to quantitatively evaluate proteomes, both spatially and temporally to study the dynamics of the microbial community proteome dynamics in relationship to geochemical measurements. As there were no comprehensive genome sequence data available at the time, we systematically evaluated different organisms to generate a "pseudo-metagenome" for proteomics analyses. Proteomics results supported the dominance of Geobacteraceae during biostimulation and revealed a shift from iron reduction to sulfate reduction, evidenced by changes in community membership. Because U(VI) is reduced at a lower rate during sulfate reduction, detecting this shift is important to maintaining the maximum rate of U(VI) reduction. In addition, the comparison of proteome measurements made at the end of the 2007 field experiment to the 2008 field experiment revealed a modified community structure. Importantly, the failure of a community to rebound following the cessation of biostimulation needs to be included in long-term remediation strategies.

  12. Comparative proteomic analysis of primordial follicles from ovaries of immature and aged rats.

    Science.gov (United States)

    Govindaraj, Vijayakumar; Rao, A Jagannadha

    2015-01-01

    Age related decline in reproductive performance in women is well documented and apoptosis has been considered as one of the reasons for the decline of primordial follicle reserve. Recently we observed a decline in the efficiency of DNA repair ability in aged rat primordial follicles as demonstrated by decreased mRNA levels of DNA repair genes BRCA1 and H2AX. In the present study, a two-dimensional electrophoresis (2DE) proteomic approach was employed to identify differentially expressed proteins in primordial follicles isolated from ovaries of immature (∼20 days) and aged (∼400-450 days) rats. Using MALDI-TOF/TOF MS, we identified 13 differentially expressed proteins (p primordial follicles. These proteins are involved in a wide range of biological functions including apoptosis, DNA repair, and the immune system. Interestingly, the differentially expressed proteins such as FIGNL1 (DNA repair) and BOK (apoptotic protein) have not been previously reported in the rat primordial follicles and these proteins can be related to some common features of ovarian aging such as loss of follicle reserve and genome integrity. The quantitative differences of two important proteins BOK and FIGNL1 observed by the proteomic analysis were correlated with the transcript levels, as determined by semi-quantitative RT-PCR. Our results improve the current knowledge about protein factors associated with molecular changes in rat primordial follicles as a function of aging and our understanding of the proteomic processes involved in degenerative changes observed in aging primordial follicles.

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

    Science.gov (United States)

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

    2016-04-01

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

  14. Quantitative and integrated proteome and microRNA analysis of endothelial replicative senescence.

    Science.gov (United States)

    Yentrapalli, Ramesh; Azimzadeh, Omid; Kraemer, Anne; Malinowsky, Katharina; Sarioglu, Hakan; Becker, Karl-Friedrich; Atkinson, Michael J; Moertl, Simone; Tapio, Soile

    2015-08-01

    Age-related changes in vascular functioning are a harbinger of cardiovascular disease but the biological mechanisms during the progression of endothelial senescence have not been studied. We investigated alterations in the proteome and miRNA profiles in the course of replicative senescence using primary human umbilical vein endothelial cells as an in vitro vascular model. Quantitative proteomic profiling from early growth stage to senescence was performed by isotope-coded protein label coupled to LC-ESI-MS/MS analysis. Some proteins consistently changed their expression during the senescence whereas others appeared as deregulated only during the late senescence. The latter was accompanied by alterations in morphology of senescent endothelial cells. MicroRNA expression profiling revealed transient changes in the level of miR-16-5p, miR-28-3p and miR-886-5p in the early senescence, decrease in the level of miR-106b-3p at the late stage, and continuous changes in the expression of miR-181a-5p and miR-376a-3p during the whole senescence process. Integrating data on proteomic and microRNA changes indicated potential crosstalk between specific proteins and non-coding RNAs in the regulation of metabolism, cell cycle progression and cytoskeletal organization in the endothelial senescence. The knowledge of molecular targets that change during the senescence can ultimately contribute to a better understanding and prevention of age-related vascular diseases.

  15. Proteomics analysis of digestive juice from silkworm during Bombyx mori nucleopolyhedrovirus infection.

    Science.gov (United States)

    Hu, Xiaolong; Zhu, Min; Wang, Simei; Zhu, Liyuan; Xue, Renyu; Cao, Guangli; Gong, Chengliang

    2015-08-01

    Previous studies have analyzed the midgut transcriptome and proteome after challenge with Bombyx mori nucleopolyhedrovirus (BmNPV), however little information is available on the digestive juice proteome after BmNPV challenge. This study investigated BmNPV infection-induced protein changes in the digestive juice of silkworms using shotgun proteomics and MS sequencing. From the digestive juice of normal third-day, fifth-instar silkworm larvae, 75 proteins were identified, 44 of which were unknown; from larvae 6 h after inoculation with BmNPV, 106 proteins were identified, of which 39 were unknown. After BmNPV challenge, more secreted proteins appeared that had antiviral and digestive features. GO annotation analysis clustered most proteins in the lumen into catalytic, binding, and metabolic processes. Numerous proteins were reported to have BmNPV interactions. Hsp70 protein cognate, lipase-1, and chlorophyllide A-binding protein precursor were upregulated significantly after BmNPV challenge. Levels of trypsin-like serine protease, beta-1,3-glucanase, catalase, and serine protease transcripts decreased or were not significantly change after BmNPV challenge. Taken together, these findings provided insights into the interaction between host and BmNPV and revealed potential functions of digestive juice after per os BmNPV infection.

  16. Proteomic analysis of the metabolic adaptation of the biocontrol agent Pseudozyma flocculosa leading to glycolipid production

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    Bélanger Richard R

    2010-02-01

    Full Text Available Abstract The yeast-like epiphytic fungus Pseudozyma flocculosa is known to antagonize powdery mildew fungi through proliferation on colonies presumably preceded by the release of an antifungal glycolipid (flocculosin. In culture conditions, P. flocculosa can be induced to produce or not flocculosin through manipulation of the culture medium nutrients. In order to characterize and understand the metabolic changes in P. flocculosa linked to glycolipid production, we conducted a 2-DE proteomic analysis and compared the proteomic profile of P. flocculosa growing under conditions favoring the development of the fungus (control or conducive to flocculosin synthesis (stress. A large number of protein spots (771 were detected in protein extracts of the control treatment compared to only 435 matched protein spots in extracts of the stress cultures, which clearly suggests an important metabolic reorganization in slow-growing cells producing flocculosin. From the latter treatment, we were able to identify 21 protein spots that were either specific to the treatment or up-regulated significantly (2-fold increase. All of them were identified based on similarity between predicted ORF of the newly sequenced genome of P. flocculosa with Ustilago maydis' available annotated sequences. These proteins were associated with the carbon and fatty acid metabolism, and also with the filamentous change of the fungus leading to flocculosin production. This first look into the proteome of P. flocculosa suggests that flocculosin synthesis is elicited in response to specific stress or limiting conditions.

  17. Proteomic analysis of plasma from rats following total parenteral nutrition-induced liver injury.

    Science.gov (United States)

    Tsai, Jai-Jen; Kuo, Hsing-Chun; Lee, Kam-Fai; Tsai, Tung-Hu

    2015-11-01

    Total parenteral nutrition (TPN) is provided as the primary nitrogen source to manage patients with intestinal failure who were not able to sustain themselves on enteral feeds. The most common complication of long-term TPN use is hepatitis. A proteomic approach was used to identify proteins that are differentially expressed in the plasma of rats following TPN-related acute liver injury. Six male rats were randomly assigned to either the saline infusion control group or the TPN infusion group. Our results demonstrate that TPN infusion in rats resulted in hepatic dysfunction and hepatocyte apoptosis. Five proteins that were differentially expressed between TPN infusion and normal rats were determined and validated in vivo. Fascinatingly, the proteomic differential displays, downregulated proteins included peroxiredoxin 2 (PRDX2), alpha-1-antiproteinase (A1AT), and fibrinogen gamma chain (FIBG), which were involved in oxidative stress, inflammatory respondence and cells apoptosis. After TPN infusion, two protein spots showed increased expression, namely, the glucagon receptor (GLR) protein and apolipoprotein A-1 (APOA1), which may mediate the effects of TPN administration on glycogen and lipid metabolism. In this study, proteomic analysis suggested TPN-related acute liver injury could be involved in limiting cellular protection mechanisms against oxidative stress-induced apoptosis. On the basis of the results, we also give molecular evidences replying TPN-related hepatitis.

  18. Comparative Proteomic Analysis of Two Barley Cultivars (Hordeum vulgare L.) with Contrasting Grain Protein Content

    Science.gov (United States)

    Guo, Baojian; Luan, Haiye; Lin, Shen; Lv, Chao; Zhang, Xinzhong; Xu, Rugen

    2016-01-01

    Grain protein contents (GPCs) of barley seeds are significantly different between feed and malting barley cultivars. However, there is still no insight into the proteomic analysis of seed proteins between feed and malting barley cultivars. Also, the genetic control of barley GPC is still unclear. GPCs were measured between mature grains of Yangsimai 3 and Naso Nijo. A proteome profiling of differentially expressed protein was established by using a combination of 2-DE and tandem mass spectrometry. In total, 502 reproducible protein spots in barley seed proteome were detected with a pH range of 4–7 and 6–11, among these 41 protein spots (8.17%) were detected differentially expressed between Yangsimai 3 and Naso Nijo. Thirty-four protein spots corresponding to 23 different proteins were identified, which were grouped into eight categories, including stress, protein degradation and post-translational modification, development, cell, signaling, glycolysis, starch metabolism, and other functions. Among the identified proteins, enolase (spot 274) and small subunit of ADP-glucose pyrophosphorylase (spot 271) are exclusively expressed in barley Yangsimai 3, which may be involved in regulating seed protein expression. In addition, malting quality is characterized by an accumulation of serpin protein, Alpha-amylase/trypsin inhibitor CMb and Alpha-amylase inhibitor BDAI-1. Most noticeably, globulin, an important storage protein in barley seed, undergoes post-translational processing in both cultivars, and also displays different expression patterns. PMID:27200019

  19. In-depth analysis of the chicken egg white proteome using an LTQ Orbitrap Velos

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    Mann Matthias

    2011-02-01

    Full Text Available Abstract Background Hen's egg white has been the subject of intensive chemical, biochemical and food technological research for many decades, because of its importance in human nutrition, its importance as a source of easily accessible model proteins, and its potential use in biotechnological processes. Recently the arsenal of tools used to study the protein components of egg white has been complemented by mass spectrometry-based proteomic technologies. Application of these fast and sensitive methods has already enabled the identification of a large number of new egg white proteins. Recent technological advances may be expected to further expand the egg white protein inventory. Results Using a dual pressure linear ion trap Orbitrap instrument, the LTQ Orbitrap Velos, in conjunction with data analysis in the MaxQuant software package, we identified 158 proteins in chicken egg white with two or more sequence unique peptides. This group of proteins identified with very high confidence included 79 proteins identified in egg white for the first time. In addition, 44 proteins were identified tentatively. Conclusions Our results, apart from identifying many new egg white components, indicate that current mass spectrometry technology is sufficiently advanced to permit direct identification of minor components of proteomes dominated by a few major proteins without resorting to indirect techniques, such as chromatographic depletion or peptide library binding, which change the composition of the proteome.

  20. Proteomic analysis of renal calculi indicates an important role for inflammatory processes in calcium stone formation.

    Science.gov (United States)

    Merchant, Michael L; Cummins, Timothy D; Wilkey, Daniel W; Salyer, Sarah A; Powell, David W; Klein, Jon B; Lederer, Eleanor D

    2008-10-01

    Even though renal stones/calculi occur in approximately 10% of individuals, they are an enormous economic burden to the entire US health system. While the relative metabolic composition of renal calculi is generally known, there is no clear understanding of the genetics of renal stone formation, nor are there clear prognostic indicators of renal stone formation. The application of proteomics to the analysis of renal calculi axiomatically holds that insight into renal stone pathobiology can be gained by a more comprehensive understanding of renal calculus protein composition. We analyzed isolated renal stone matrix proteins with mass spectrometric and immunohistochemical methods identifying 158 proteins with high confidence, including 28 common proteins. The abundant proteins included those identified previously in stones and proteins identified here for the first time, such as myeloid lineage-specific, integral membrane and lipid regulatory proteins. Pathway analyses of all proteins identified suggested that a significant fraction of the most abundant matrix proteins participate in inflammatory processes. These proteomic results support the hypothesis that stone formation induces a cellular inflammatory response and the protein components of this response contribute to the abundant stone matrix proteome.

  1. Proteomic analysis of the alteration of protein expression in the placenta of Down syndrome

    Institute of Scientific and Technical Information of China (English)

    SUN Cheng-juan; YAN Li-yu; WANG Wei; YU Song; WANG Xin; ZHANG Wei-yuan

    2011-01-01

    Background Down syndrome (DS) is the most common form of human aneuploidy,and there is no effective therapy for the chromosomal abnormalities.We aimed to unravel the molecular mechanisms underlying DS and to provide clues to prenatal screening.Methods A series of proteomics-based experiments was conducted using 19 patients with DS fetuses and 17 normal pregnancies.The proteome of placenta was investigated as displayed by two-dimensional difference gel electrophoresis (2D-DIGE),and comparisons were made between placentas that developed under DS and normal pregnancy conditions.Multivariate analysis of the resulting protein patterns revealed DS-specific protein expression.Matrix-assisted laser desorption/ionization (MALDI) time-of-flight/time-of-flight (TOF/TOF) high-resolution tandem mass spectrometer (MS)-based identification was successful for 12 out of 17 selected protein spots.Results Among those,three proteins involved in the resist of reactive oxygen species (ROS) and neurogenesis were more abundant in the DS placenta (superoxide dismutase 1,endoplasmic reticulum protein 29 and heat shock protein beta-1),while peroxiredoxin-6 involved in cell defense mechanism against ROS was expressed at a higher level in the normal pregnancies.Conclusion Knowledge of the DS placenta proteome emphasizes the role of proteins involved in anti-oxidation during DS,and may form the basis of a potential approach to minimize the incidence of DS in the clinical setting.

  2. Characterization of the mouse pancreatic islet proteome and comparative analysis with other mouse tissues.

    Science.gov (United States)

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

    2008-08-01

    The pancreatic islets of Langerhans, and especially the insulin-producing beta cells, play a central role in the maintenance of glucose homeostasis. Alterations in the expression of multiple proteins in the islets that contribute to the maintenance of islet function are likely to underlie the pathogenesis of types 1 and 2 diabetes. To identify proteins that constitute the islet proteome, we provide the first comprehensive proteomic characterization of pancreatic islets for mouse, the most commonly used animal model in diabetes research. Using strong cation exchange fractionation coupled with reversed phase LC-MS/MS we report the confident identification of 17,350 different tryptic peptides covering 2612 proteins having at least two unique peptides per protein. The data set also identified approximately 60 post-translationally modified peptides including oxidative modifications and phosphorylation. While many of the identified phosphorylation sites corroborate those previously known, the oxidative modifications observed on cysteinyl residues reveal potentially novel information suggesting a role for oxidative stress in islet function. Comparative analysis with 15 available proteomic data sets from other mouse tissues and cells revealed a set of 133 proteins predominantly expressed in pancreatic islets. This unique set of proteins, in addition to those with known functions such as peptide hormones secreted from the islets, contains several proteins with as yet unknown functions. The mouse islet protein and peptide database accessible at (http://ncrr.pnl.gov), provides an important reference resource for the research community to facilitate research in the diabetes and metabolism fields.

  3. Recent advances in the analysis of metal hyperaccumulation and hypertolerance in plants using proteomics.

    Science.gov (United States)

    Dalcorso, Giovanni; Fasani, Elisa; Furini, Antonella

    2013-01-01

    Hyperaccumulator/hypertolerant plant species have evolved strategies allowing them to grow in metal-contaminated soils, where they accumulate high concentrations of heavy metals in their shoots without signs of toxicity. The mechanisms that allow enhanced metal uptake, root-to-shoot translocation and detoxification in these species are not fully understood. Complementary approaches such as transcriptomic-based DNA microarrays and proteomics have recently been used to gain insight into the molecular pathways evolved by metal hyperaccumulator/hypertolerant species. Proteomics has the advantage of focusing on the translated portion of the genome and it allows to analyze complex networks of proteins. This review discusses the recent analysis of metal hyperaccumulator/hypertolerant plant species using proteomics. Changes in photosynthetic proteins, sulfur, and glutathione metabolism, transport, biotic and xenobiotic defenses as well as the differential regulation of proteins involved in signaling and secondary metabolism are discussed in relation to metal hyperaccumulation. We also consider the potential contribution of several proteins to the hyperaccumulation phenotype.

  4. Recent advances in the analysis of metal hyperaccumulation and hypertolerance in plants using proteomics

    Directory of Open Access Journals (Sweden)

    Giovanni eDalCorso

    2013-07-01

    Full Text Available Hyperaccumulator/hypertolerant plant species have evolved strategies allowing them to grow in metal-contaminated soils, where they accumulate high concentrations of heavy metals in their shoots without signs of toxicity. The mechanisms that allow enhanced metal uptake, root-to-shoot translocation and detoxification in these species are not fully understood. Complementary approaches such as transcriptomic-based DNA microarrays and proteomics have recently been used to gain insight into the molecular pathways evolved by metal hyperaccumulator/hypertolerant species. Proteomics has the advantage of focusing on the translated portion of the genome and it allows to analyze complex networks of proteins. This review discusses the recent analysis of metal hyperaccumulator/hypertolerant plant species using proteomics. Changes in photosynthetic proteins, sulfur and glutathione metabolism, transport, biotic and xenobiotic defenses as well as the differential regulation of proteins involved in signaling and secondary metabolism are discussed in relation to metal hyperaccumulation. We also consider the potential contribution of several proteins to the hyperaccumulation phenotype.

  5. Improved metabolites of pharmaceutical ingredient grade Ginkgo biloba and the correlated proteomics analysis.

    Science.gov (United States)

    Zheng, Wen; Li, Ximin; Zhang, Lin; Zhang, Yanzhen; Lu, Xiaoping; Tian, Jingkui

    2015-06-01

    Ginkgo biloba is an attractive and traditional medicinal plant, and has been widely used as a phytomedicine in the prevention and treatment of cardiovascular and cerebrovascular diseases. Flavonoids and terpene lactones are the major bioactive components of Ginkgo, whereas the ginkgolic acids (GAs) with strong allergenic properties are strictly controlled. In this study, we tested the content of flavonoids and GAs under ultraviolet-B (UV-B) treatment and performed comparative proteomic analyses to determine the differential proteins that occur upon UV-B radiation. That might play a crucial role in producing flavonoids and GAs. Our phytochemical analyses demonstrated that UV-B irradiation significantly increased the content of active flavonoids, and decreased the content of toxic GAs. We conducted comparative proteomic analysis of both whole leaf and chloroplasts proteins. In total, 27 differential proteins in the whole leaf and 43 differential proteins in the chloroplast were positively identified and functionally annotated. The proteomic data suggested that enhanced UV-B radiation exposure activated antioxidants and stress-responsive proteins as well as reduced the rate of photosynthesis. We demonstrate that UV-B irradiation pharmaceutically improved the metabolic ingredients of Ginkgo, particularly in terms of reducing GAs. With high UV absorption properties, and antioxidant activities, the flavonoids were likely highly induced as protective molecules following UV-B irradiation.

  6. An extensive targeted proteomic analysis of disease-related protein biomarkers in urine from healthy donors.

    Directory of Open Access Journals (Sweden)

    Brian M Nolen

    Full Text Available The analysis of protein biomarkers in urine is expected to lead to advances in a variety of clinical settings. Several characteristics of urine including a low-protein matrix, ease of testing and a demonstrated proteomic stability offer distinct advantages over current widely used biofluids, serum and plasma. Improvements in our understanding of the urine proteome and in methods used in its evaluation will facilitate the clinical development of urinary protein biomarkers. Multiplexed bead-based immunoassays were utilized to evaluate 211 proteins in urines from 103 healthy donors. An additional 25 healthy donors provided serial urine samples over the course of two days in order to assess temporal variation in selected biomarkers. Nearly one-third of the evaluated biomarkers were detected in urine at levels greater than 1 ng/ml, representing a diverse panel of proteins with respect to structure, function and biological role. The presence of several biomarkers in urine was confirmed by western blot. Several methods of data normalization were employed to assess impact on biomarker variability. A complex pattern of correlations with urine creatinine, albumin and beta-2-microglobulin was observed indicating the presence of highly specific mechanisms of renal filtration. Further investigation of the urinary protein biomarkers identified in this preliminary study along with a consideration of the underlying proteomic trends suggested by these findings should lead to an improved capability to identify candidate biomarkers for clinical development.

  7. Total Soluble Protein Extraction for Improved Proteomic Analysis of Transgenic Rice Plant Roots.

    Science.gov (United States)

    Raorane, Manish L; Narciso, Joan O; Kohli, Ajay

    2016-01-01

    With the advent of high-throughput platforms, proteomics has become a powerful tool to search for plant gene products of agronomic relevance. Protein extractions using multistep protocols have been shown to be effective to achieve better proteome profiles than simple, single-step extractions. These protocols are generally efficient for above ground tissues such as leaves. However, each step leads to loss of some amount of proteins. Additionally, compounds such as proteases in the plant tissues lead to protein degradation. While protease inhibitor cocktails are available, these alone do not seem to suffice when roots are included in the plant sample. This is obvious given the lack of high molecular weight (HMW) proteins obtained from samples that include root tissue. For protein/proteome analysis of transgenic plant roots or of seedlings, which include root tissue, such pronounced protein degradation is especially undesirable. A facile protein extraction protocol is presented, which ensures that despite the inclusion of root tissues there is minimal loss in total protein components.

  8. Fast MS/MS acquisition without dynamic exclusion enables precise and accurate quantification of proteome by MS/MS fragment intensity.

    Science.gov (United States)

    Zhang, Shen; Wu, Qi; Shan, Yichu; Zhao, Qun; Zhao, Baofeng; Weng, Yejing; Sui, Zhigang; Zhang, Lihua; Zhang, Yukui

    2016-05-20

    Most currently proteomic studies use data-dependent acquisition with dynamic exclusion to identify and quantify the peptides generated by the digestion of biological sample. Although dynamic exclusion permits more identifications and higher possibility to find low abundant proteins, stochastic and irreproducible precursor ion selection caused by dynamic exclusion limit the quantification capabilities, especially for MS/MS based quantification. This is because a peptide is usually triggered for fragmentation only once due to dynamic exclusion. Therefore the fragment ions used for quantification only reflect the peptide abundances at that given time point. Here, we propose a strategy of fast MS/MS acquisition without dynamic exclusion to enable precise and accurate quantification of proteome by MS/MS fragment intensity. The results showed comparable proteome identification efficiency compared to the traditional data-dependent acquisition with dynamic exclusion, better quantitative accuracy and reproducibility regardless of label-free based quantification or isobaric labeling based quantification. It provides us with new insights to fully explore the potential of modern mass spectrometers. This strategy was applied to the relative quantification of two human disease cell lines, showing great promises for quantitative proteomic applications.

  9. Application of meta-transcriptomics and -proteomics to analysis of in situ physiological state

    Energy Technology Data Exchange (ETDEWEB)

    Konopka, Allan; Wilkins, Michael J.

    2012-05-18

    Analysis of the growth-limiting factor or environmental stressors affecting microbes in situ is of fundamental importance but analytically difficult. Microbes can reduce in situ limiting nutrient concentrations to sub-micromolar levels, and contaminated ecosystems may contain multiple stressors. The patterns of gene or protein expression by microbes in nature can be used to infer growth limitations, because they are regulated in response to environmental conditions. Experimental studies under controlled conditions in the laboratory provide the physiological underpinnings for developing these physiological indicators. Although regulatory networks may differ among specific microbes, there are some broad principles that can be applied, related to limiting nutrient acquisition, resource allocation, and stress responses. As technologies for transcriptomics and proteomics mature, the capacity to apply these approaches to complex microbial communities will accelerate. Global proteomics has the particular advantage that it reflects expressed catalytic activities. Furthermore, the high mass accuracy of some proteomic approaches allows mapping back to specific microbial strains. For example, at the Rifle IFRC field site in Western Colorado, the physiological status of Fe(III)-reducing populations has been tracked over time. Members of a 'subsurface clade' within the Geobacter predominated during carbon amendment to the subsurface environment. At the functional level, proteomic identifications produced inferences regarding (i) temporal changes in anabolism and catabolism of acetate, (ii) the onset of N2 fixation when N became limiting, and (iii) expression of phosphate transporters during periods of intense growth. The application of these approaches in situ can lead to discovery of novel physiological adaptations.

  10. Transcriptomic and Proteomic Analysis of Oenococcus oeni Adaptation to Wine Stress Conditions

    Science.gov (United States)

    Margalef-Català, Mar; Araque, Isabel; Bordons, Albert; Reguant, Cristina; Bautista-Gallego, Joaquín

    2016-01-01

    Oenococcus oeni, the main lactic acid bacteria responsible for malolactic fermentation in wine, has to adapt to stressful conditions, such as low pH and high ethanol content. In this study, the changes in the transcriptome and the proteome of O. oeni PSU-1 during the adaptation period before MLF start have been studied. DNA microarrays were used for the transcriptomic analysis and two complementary proteomic techniques, 2-D DIGE and iTRAQ labeling were used to analyze the proteomic response. One of the most influenced functions in PSU-1 due to inoculation into wine-like medium (WLM) was translation, showing the over-expression of certain ribosomal genes and the corresponding proteins. Amino acid metabolism and transport was also altered and several peptidases were up regulated both at gene and protein level. Certain proteins involved in glutamine and glutamate metabolism showed an increased abundance revealing the key role of nitrogen uptake under stressful conditions. A strong transcriptional inhibition of carbohydrate metabolism related genes was observed. On the other hand, the transcriptional up-regulation of malate transport and citrate consumption was indicative of the use of L-malate and citrate associated to stress response and as an alternative energy source to sugar metabolism. Regarding the stress mechanisms, our results support the relevance of the thioredoxin and glutathione systems in the adaptation of O. oeni to wine related stress. Genes and proteins related to cell wall showed also significant changes indicating the relevance of the cell envelop as protective barrier to environmental stress. The differences found between transcriptomic and proteomic data suggested the relevance of post-transcriptional mechanisms and the complexity of the stress response in O. oeni adaptation. Further research should deepen into the metabolisms mostly altered due to wine conditions to elucidate the role of each mechanism in the O. oeni ability to develop MLF.

  11. Platelet proteomics.

    Science.gov (United States)

    Zufferey, Anne; Fontana, Pierre; Reny, Jean-Luc; Nolli, Severine; Sanchez, Jean-Charles

    2012-01-01

    Platelets are small cell fragments, produced by megakaryocytes, in the bone marrow. They play an important role in hemostasis and diverse thrombotic disorders. They are therefore primary targets of antithrombotic therapies. They are implicated in several pathophysiological pathways, such as inflammation or wound repair. In blood circulation, platelets are activated by several pathways including subendothelial matrix and thrombin, triggering the formation of the platelet plug. Studying their proteome is a powerful approach to understand their biology and function. However, particular attention must be paid to different experimental parameters, such as platelet quality and purity. Several technologies are involved during the platelet proteome processing, yielding information on protein identification, characterization, localization, and quantification. Recent technical improvements in proteomics combined with inter-disciplinary strategies, such as metabolomic, transcriptomics, and bioinformatics, will help to understand platelets biological mechanisms. Therefore, a comprehensive analysis of the platelet proteome under different environmental conditions may contribute to elucidate complex processes relevant to platelet function regarding bleeding disorders or platelet hyperreactivity and identify new targets for antiplatelet therapy.

  12. High-throughput proteome dynamics for discovery of key proteins in sentinel species: Unsuspected vitellogenins diversity in the crustacean Gammarus fossarum.

    Science.gov (United States)

    Trapp, Judith; Armengaud, Jean; Gaillard, Jean-Charles; Pible, Olivier; Chaumot, Arnaud; Geffard, Olivier

    2016-09-02

    In environmental science, omics-based approaches are widely used for the identification of gene products related to stress response. However, when dealing with non-model species, functional prediction of genes is challenging. Indeed, functional predictions are often obtained by sequence similarity searches and functional data from phylogenetically distant organisms, which can lead to inaccurate predictions due to quite different evolutionary scenarios. In oviparous females, vitellogenin production is vital for embryonic development, ensuring population viability. Its abnormal presence in fish male organisms is commonly employed as a biomarker of exposure to xenoestrogens, named endocrine disruptors. Here, in the freshwater amphipod Gammarus fossarum, we identified vitellogenin proteins by means of a proteome temporal dynamics analysis during oogenesis and embryogenesis. This exhaustive approach allows several functional molecular hypotheses in the oogenesis process to be drawn. Moreover, we revealed an unsuspected diversity of molecular players involved in yolk formation as eight proteins originating from different families of the large lipid transfer protein superfamily were identified as "true vitellogenins". In non-model species, next generation sequencing technologies development enables quickly deciphering gene and protein sequences but accuracy of associated functional prediction remains to be established. Here, in the crustacean Gammarus fossarum, a key sentinel species in freshwater biomonitoring, we identified key molecular players involved in the female reproduction by studying the proteome dynamics of ovaries and embryos. An unsuspected diversity of vitellogenin proteins was evidenced. These proteins being vital for offspring development, their high diversity may be advantageous for the organism's reproduction. Phylogenetic analysis showed that some forms are true vitellogenin orthologs while others are included in the apolipoprotein family, a paralogous

  13. Analysis of Biostimulated Microbial Communities from Two Field Experiments Reveals Temporal and Spatial Differences in Proteome Profiles

    Energy Technology Data Exchange (ETDEWEB)

    Callister, Stephen J [Pacific Northwest National Laboratory (PNNL); Wilkins, Mike [University of California, Berkeley; Nicora, Carrie D. [Pacific Northwest National Laboratory (PNNL); Williams, Ken [Lawrence Berkeley National Laboratory (LBNL); Banfield, Jillian F. [University of California, Berkeley; Verberkmoes, Nathan C [ORNL; Hettich, Robert {Bob} L [ORNL; N' Guessan, A. Lucie [University of Massachusetts, Amherst; Mouser, Paula J [University of Massachusetts, Amherst; Elifantz, Hila [University of Massachusetts, Amherst; Smith, Richard D. [Pacific Northwest National Laboratory (PNNL); Lovley, Derek [University of Massachusetts, Amherst; Lipton, Mary S [Pacific Northwest National Laboratory (PNNL); Long, Phil [Pacific Northwest National Laboratory (PNNL)

    2010-01-01

    Stimulated by an acetate-amendment field experiment conducted in 2007, anaerobic microbial populations in the aquifer at the Rifle Integrated Field Research Challenge site in Colorado reduced mobile U(VI) to insoluble U(IV). During this experiment, planktonic biomass was sampled at various time points to quantitatively evaluate proteomes. In 2008, an acetateamended field experiment was again conducted in a similar manner to the 2007 experiment. As there was no comprehensive metagenome sequence available for use in proteomics analysis, we systematically evaluated 12 different organism genome sequences to generate sets of aggregate genomes, or pseudo-metagenomes , for supplying relative quantitative peptide and protein identifications. Proteomics results support previous observations of the dominance of Geobacteraceae during biostimulation using acetate as sole electron donor, and revealed a shift from an early stage of iron reduction to a late stage of iron reduction. Additionally,ashift from iron reduction to sulfate reduction was indicated by changes in the contribution of proteome information contributed by different organism genome sequences within the aggregate set. In addition, the comparison of proteome measurements made between the 2007 field experiment and 2008 field experiment revealed differences in proteome profiles. These differences may be the result of alterations in abundance and population structure within the planktonic biomass samples collected for analysis.

  14. Analysis of biostimulated microbial communities from two field experiments reveals temporal and spatial differences in proteome profiles

    Energy Technology Data Exchange (ETDEWEB)

    Callister, S.J.; Wilkins, M.J.; Nicora, C.D.; Williams, K.H.; Banfield, J.F.; VerBerkmoes, N.C.; Hettich, R.L.; NGuessan, A.L.; Mouser, P.J.; Elifantz, H.; Smith, R.D.; Lovley, D.R.; Lipton, M.S.; Long, P.E.

    2010-07-15

    Stimulated by an acetate-amendment field experiment conducted in 2007, anaerobic microbial populations in the aquifer at the Rifle Integrated Field Research Challenge site in Colorado reduced mobile U(VI) to insoluble U(IV). During this experiment, planktonic biomass was sampled at various time points to quantitatively evaluate proteomes. In 2008, an acetate-amended field experiment was again conducted in a similar manner to the 2007 experiment. As there was no comprehensive metagenome sequence available for use in proteomics analysis, we systematically evaluated 12 different organism genome sequences to generate sets of aggregate genomes, or “pseudo-metagenomes”, for supplying relative quantitative peptide and protein identifications. Proteomics results support previous observations of the dominance of Geobacteraceae during biostimulation using acetate as sole electron donor, and revealed a shift from an early stage of iron reduction to a late stage of iron reduction. Additionally, a shift from iron reduction to sulfate reduction was indicated by changes in the contribution of proteome information contributed by different organism genome sequences within the aggregate set. In addition, the comparison of proteome measurements made between the 2007 field experiment and 2008 field experiment revealed differences in proteome profiles. These differences may be the result of alterations in abundance and population structure within the planktonic biomass samples collected for analysis.

  15. Supplementary Material for: Transcriptome and proteome dynamics in larvae of the barnacle Balanus Amphitrite from the Red Sea

    KAUST Repository

    Chandramouli, Kondethimmanahalli

    2015-01-01

    Abstract Background The barnacle Balanus amphitrite is widely distributed in marine shallow and tidal waters, and has significant economic and ecological importance. Nauplii, the first larval stage of most crustaceans, are extremely abundant in the marine zooplankton. However, a lack of genome information has hindered elucidation of the molecular mechanisms of development, settlement and survival strategies in extreme marine environments. We sequenced and constructed the genome dataset for nauplii to obtain comprehensive larval genetic information. We also investigated iTRAQ-based protein expression patterns to reveal the molecular basis of nauplii development, and to gain information on larval survival strategies in the Red Sea marine environment. Results A nauplii larval transcript dataset, containing 92,117 predicted open reading frames (ORFs), was constructed and used as a reference for the proteome analysis. Genes related to translation, oxidative phosphorylation and cytoskeletal development were highly abundant. We observed remarkable plasticity in the proteome of Red Sea larvae. The proteins associated with development, stress responses and osmoregulation showed the most significant differences between the two larval populations studied. The synergistic overexpression of heat shock and osmoregulatory proteins may facilitate larval survival in intertidal habitats or in extreme environments. Conclusions We presented, for the first time, comprehensive transcriptome and proteome datasets for Red Sea nauplii. The datasets provide a foundation for future investigations focused on the survival mechanisms of other crustaceans in extreme marine environments.

  16. Quantitative proteomics analysis by iTRAQ revealed underlying changes in thermotolerance of Arthrospira platensis.

    Science.gov (United States)

    Chang, Rong; Lv, Bingxin; Li, Bosheng

    2017-08-08

    Growth temperature is a critical factor that affects cultivation of Arthrospira platensis which is a type of cyanobacterium widely known as Spirulina that has significant commercial value. To investigate the molecular mechanism underlying the thermotolerance of Spirulina, differential protein expression profiling was carried out using iTRAQ-based quantitative proteomic analysis. This study only analyzed changes in thylakoids. Among the 2085 proteins quantified, 43 differentially expressed proteins were selected based on the fold change cutoff scores of ≥2 or ≤0.5 for up-regulation or down-regulation, respectively. An analysis of these 43 proteins found that 23% of them are photosynthetic system proteins which include photosynthetic enzymes and pigment proteins. The dynamic change of these proteins indicates that photosynthetic system functions were profoundly affected under heat stress and the light-dependent reactions were probably the most sensitive to temperature changes. Meanwhile, to cope with the low energy production due to impaired photosynthesis there was a considerable down-shift in protein synthesis which is a very energy demanding process. The impaired photosynthesis led to low energy generation that was compensated by a down-shift in translation (the most energy-demanding process) and an up-shift of glycolysis. The reduction of many ribosome proteins may lead to a loss in translation efficiency; therefore, Spirulina may adopted a different mechanism to increase translational elongation under heat stress to compensate for this loss, such as elevate L7/L12 proteins. Changes were also found in the classical heat shock proteins, the ROS scavenging system, DNA-binding proteins, and some membrane proteins. In conclusion, this research demonstrate that heat stress induces profound changes in cellular physiology and shed light on the mechanism of the heat stress response and thermotolerance of Arthrospira platensis. Arthrospira platensis, widely known as

  17. The latest advancements in proteomic two-dimensional gel electrophoresis analysis applied to biological samples.

    Science.gov (United States)

    Santucci, Laura; Bruschi, Maurizio; Ghiggeri, Gian Marco; Candiano, Giovanni

    2015-01-01

    Two-dimensional gel electrophoresis (2DE) is one of the fundamental approaches in proteomics for the separation and visualization of complex protein mixtures. Proteins can be analyzed by 2DE using isoelectric focusing (IEF) in the first dimension, combined to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension, gel staining (silver and Coomassie), image analysis, and 2DE gel database. High-resolution 2DE can resolve up to 5,000 different proteins simultaneously (∼2,000 proteins routinely), and detect and quantify <1 ng of protein per spot. Here, we describe the latest developments for a more complete analysis of biological fluids.

  18. Dynamic proteome profiling of individual proteins in human skeletal muscle after a high-fat diet and resistance exercise.

    Science.gov (United States)

    Camera, Donny M; Burniston, Jatin G; Pogson, Mark A; Smiles, William J; Hawley, John A

    2017-08-30

    It is generally accepted that muscle adaptation to resistance exercise (REX) training is underpinned by contraction-induced, increased rates of protein synthesis and dietary protein availability. By using dynamic proteome profiling (DPP), we investigated the contribution of both synthesis and breakdown to changes in abundance on a protein-by-protein basis in human skeletal muscle. Age-matched, overweight males consumed 9 d of a high-fat, low-carbohydrate diet during which time they either undertook 3 sessions of REX or performed no exercise. Precursor enrichment and the rate of incorporation of deuterium oxide into newly synthesized muscle proteins were determined by mass spectrometry. Ninety proteins were included in the DPP, with 28 proteins exhibiting significant responses to REX. The most common pattern of response was an increase in turnover, followed by an increase in abundance with no detectable increase in protein synthesis. Here, we provide novel evidence that demonstrates that the contribution of synthesis and breakdown to changes in protein abundance induced by REX differ on a protein-by-protein basis. We also highlight the importance of the degradation of individual muscle proteins after exercise in human skeletal muscle.-Camera, D. M., Burniston, J. G., Pogson, M. A., Smiles, W. J., Hawley, J. A. Dynamic proteome profiling of individual proteins in human skeletal muscle after a high-fat diet and resistance exercise. © FASEB.

  19. Proteomics of Foodborne Bacterial Pathogens

    Science.gov (United States)

    Fagerquist, Clifton K.

    This chapter is intended to be a relatively brief overview of proteomic techniques currently in use for the identification and analysis of microorganisms with a special emphasis on foodborne pathogens. The chapter is organized as follows. First, proteomic techniques are introduced and discussed. Second, proteomic applications are presented specifically as they relate to the identification and qualitative/quantitative analysis of foodborne pathogens.

  20. Streamlined Membrane Proteome Preparation for Shotgun Proteomics Analysis with Triton X-100 Cloud Point Extraction and Nanodiamond Solid Phase Extraction

    OpenAIRE

    Pham, Minh D.; Ting-Chun Wen; Hung-Cheng Li; Pei-Hsuan Hsieh; Yet-Ran Chen; Huan-Cheng Chang; Chau-Chung Han

    2016-01-01

    While mass spectrometry (MS) plays a key role in proteomics research, characterization of membrane proteins (MP) by MS has been a challenging task because of the presence of a host of interfering chemicals in the hydrophobic protein extraction process, and the low protease digestion efficiency. We report a sample preparation protocol, two-phase separation with Triton X-100, induced by NaCl, with coomassie blue added for visualizing the detergent-rich phase, which streamlines MP preparation fo...

  1. Muscle Tissue Damage Induced by the Venom of Bothrops asper: Identification of Early and Late Pathological Events through Proteomic Analysis.

    Science.gov (United States)

    Herrera, Cristina; Macêdo, Jéssica Kele A; Feoli, Andrés; Escalante, Teresa; Rucavado, Alexandra; Gutiérrez, José María; Fox, Jay W

    2016-04-01

    The time-course of the pathological effects induced by the venom of the snake Bothrops asper in muscle tissue was investigated by a combination of histology, proteomic analysis of exudates collected in the vicinity of damaged muscle, and immunodetection of extracellular matrix proteins in exudates. Proteomic assay of exudates has become an excellent new methodological tool to detect key biomarkers of tissue alterations for a more integrative perspective of snake venom-induced pathology. The time-course analysis of the intracellular proteins showed an early presence of cytosolic and mitochondrial proteins in exudates, while cytoskeletal proteins increased later on. This underscores the rapid cytotoxic effect of venom, especially in muscle fibers, due to the action of myotoxic phospholipases A2, followed by the action of proteinases in the cytoskeleton of damaged muscle fibers. Similarly, the early presence of basement membrane (BM) and other extracellular matrix (ECM) proteins in exudates reflects the rapid microvascular damage and hemorrhage induced by snake venom metalloproteinases. The presence of fragments of type IV collagen and perlecan one hour after envenoming suggests that hydrolysis of these mechanically/structurally-relevant BM components plays a key role in the genesis of hemorrhage. On the other hand, the increment of some ECM proteins in the exudate at later time intervals is likely a consequence of the action of endogenous matrix metalloproteinases (MMPs) or of de novo synthesis of ECM proteins during tissue remodeling as part of the inflammatory reaction. Our results offer relevant insights for a more integrative and systematic understanding of the time-course dynamics of muscle tissue damage induced by B. asper venom and possibly other viperid venoms.

  2. Muscle Tissue Damage Induced by the Venom of Bothrops asper: Identification of Early and Late Pathological Events through Proteomic Analysis.

    Directory of Open Access Journals (Sweden)

    Cristina Herrera

    2016-04-01

    Full Text Available The time-course of the pathological effects induced by the venom of the snake Bothrops asper in muscle tissue was investigated by a combination of histology, proteomic analysis of exudates collected in the vicinity of damaged muscle, and immunodetection of extracellular matrix proteins in exudates. Proteomic assay of exudates has become an excellent new methodological tool to detect key biomarkers of tissue alterations for a more integrative perspective of snake venom-induced pathology. The time-course analysis of the intracellular proteins showed an early presence of cytosolic and mitochondrial proteins in exudates, while cytoskeletal proteins increased later on. This underscores the rapid cytotoxic effect of venom, especially in muscle fibers, due to the action of myotoxic phospholipases A2, followed by the action of proteinases in the cytoskeleton of damaged muscle fibers. Similarly, the early presence of basement membrane (BM and other extracellular matrix (ECM proteins in exudates reflects the rapid microvascular damage and hemorrhage induced by snake venom metalloproteinases. The presence of fragments of type IV collagen and perlecan one hour after envenoming suggests that hydrolysis of these mechanically/structurally-relevant BM components plays a key role in the genesis of hemorrhage. On the other hand, the increment of some ECM proteins in the exudate at later time intervals is likely a consequence of the action of endogenous matrix metalloproteinases (MMPs or of de novo synthesis of ECM proteins during tissue remodeling as part of the inflammatory reaction. Our results offer relevant insights for a more integrative and systematic understanding of the time-course dynamics of muscle tissue damage induced by B. asper venom and possibly other viperid venoms.

  3. Proteomics Technologies and Challenges

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Proteomics is the study of proteins and their interactions in a cell. With the completion of the Human Genome Project, the emphasis is shifting to the protein compliment of the human organism. Because proteome reflects more accurately on the dynamic state of a cell, tissue, or organism, much is expected from proteomics to yield better disease markers for diagnosis and therapy monitoring. The advent of proteomics technologies for global detection and quantitation of proteins creates new opportunities and challenges for those seeking to gain greater understanding of diseases. High-throughput proteomics technologies combining with advanced bioinformatics are extensively used to identify molecular signatures of diseases based on protein pathways and signaling cascades. Mass spectrometry plays a vital role in proteomics and has become an indispensable tool for molecular and cellular biology. While the potential is great, many challenges and issues remain to be solved, such as mining low abundant proteins and integration of proteomics with genomics and metabolomics data. Nevertheless, proteomics is the foundation for constructing and extracting useful knowledge to biomedical research. In this review, a snapshot of contemporary issues in proteomics technologies is discussed.

  4. Growth Phase-Dependent Proteomes of the Malaysian Isolated Lactococcus lactis Dairy Strain M4 Using Label-Free Qualitative Shotgun Proteomics Analysis

    Directory of Open Access Journals (Sweden)

    Theresa Wan Chen Yap

    2014-01-01

    Full Text Available Lactococcus lactis is the most studied mesophilic fermentative lactic acid bacterium. It is used extensively in the food industry and plays a pivotal role as a cell factory and also as vaccine delivery platforms. The proteome of the Malaysian isolated L. lactis M4 dairy strain, obtained from the milk of locally bred cows, was studied to elucidate the physiological changes occurring between the growth phases of this bacterium. In this study, ultraperformance liquid chromatography nanoflow electrospray ionization tandem mass spectrometry (UPLC- nano-ESI-MSE approach was used for qualitative proteomic analysis. A total of 100 and 121 proteins were identified from the midexponential and early stationary growth phases, respectively, of the L. lactis strain M4. During the exponential phase, the most important reaction was the generation of sufficient energy, whereas, in the early stationary phase, the metabolic energy pathways decreased and the biosynthesis of proteins became more important. Thus, the metabolism of the cells shifted from energy production in the exponential phase to the synthesis of macromolecules in the stationary phase. The resultant proteomes are essential in providing an improved view of the cellular machinery of L. lactis during the transition of growth phases and hence provide insight into various biotechnological applications.

  5. Integrated multifunctional microfluidics for automated proteome analyses.

    Science.gov (United States)

    Osiri, John K; Shadpour, Hamed; Witek, Małgorzata A; Soper, Steven A

    2011-01-01

    Proteomics is a challenging field for realizing totally integrated microfluidic systems for complete proteome processing due to several considerations, including the sheer number of different protein types that exist within most proteomes, the large dynamic range associated with these various protein types, and the diverse chemical nature of the proteins comprising a typical proteome. For example, the human proteome is estimated to have >10(6) different components with a dynamic range of >10(10). The typical processing pipeline for proteomics involves the following steps: (1) selection and/or extraction of the particular proteins to be analyzed; (2) multidimensional separation; (3) proteolytic digestion of the protein sample; and (4) mass spectral identification of either intact proteins (top-down proteomics) or peptide fragments generated from proteolytic digestions (bottom-up proteomics). Although a number of intriguing microfluidic devices have been designed, fabricated and evaluated for carrying out the individual processing steps listed above, work toward building fully integrated microfluidic systems for protein analysis has yet to be realized. In this chapter, information will be provided on the nature of proteomic analysis in terms of the challenges associated with the sample type and the microfluidic devices that have been tested to carry out individual processing steps. These include devices such as those for multidimensional electrophoretic separations, solid-phase enzymatic digestions, and solid-phase extractions, all of which have used microfluidics as the functional platform for their implementation. This will be followed by an in-depth review of microfluidic systems, which are defined as units possessing two or more devices assembled into autonomous systems for proteome processing. In addition, information will be provided on the challenges involved in integrating processing steps into a functional system and the approaches adopted for device

  6. Proteome adaptation in cell reprogramming proceeds via distinct transcriptional networks.

    Science.gov (United States)

    Benevento, Marco; Tonge, Peter D; Puri, Mira C; Hussein, Samer M I; Cloonan, Nicole; Wood, David L; Grimmond, Sean M; Nagy, Andras; Munoz, Javier; Heck, Albert J R

    2014-12-10

    The ectopic expression of Oct4, Klf4, c-Myc and Sox2 (OKMS) transcription factors allows reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). The reprogramming process, which involves a complex network of molecular events, is not yet fully characterized. Here we perform a quantitative mass spectrometry-based analysis to probe in-depth dynamic proteome changes during somatic cell reprogramming. Our data reveal defined waves of proteome resetting, with the first wave occurring 48 h after the activation of the reprogramming transgenes and involving specific biological processes linked to the c-Myc transcriptional network. A second wave of proteome reorganization occurs in a later stage of reprogramming, where we characterize the proteome of two distinct pluripotent cellular populations. In addition, the overlay of our proteome resource with parallel generated -omics data is explored to identify post-transcriptionally regulated proteins involved in key steps during reprogramming.

  7. WaveletQuant, an improved quantification software based on wavelet signal threshold de-noising for labeled quantitative proteomic analysis

    Directory of Open Access Journals (Sweden)

    Li Song

    2010-04-01

    Full Text Available Abstract Background Quantitative proteomics technologies have been developed to comprehensively identify and quantify proteins in two or more complex samples. Quantitative proteomics based on differential stable isotope labeling is one of the proteomics quantification technologies. Mass spectrometric data generated for peptide quantification are often noisy, and peak detection and definition require various smoothing filters to remove noise in order to achieve accurate peptide quantification. Many traditional smoothing filters, such as the moving average filter, Savitzky-Golay filter and Gaussian filter, have been used to reduce noise in MS peaks. However, limitations of these filtering approaches often result in inaccurate peptide quantification. Here we present the WaveletQuant program, based on wavelet theory, for better or alternative MS-based proteomic quantification. Results We developed a novel discrete wavelet transform (DWT and a 'Spatial Adaptive Algorithm' to remove noise and to identify true peaks. We programmed and compiled WaveletQuant using Visual C++ 2005 Express Edition. We then incorporated the WaveletQuant program in the Trans-Proteomic Pipeline (TPP, a commonly used open source proteomics analysis pipeline. Conclusions We showed that WaveletQuant was able to quantify more proteins and to quantify them more accurately than the ASAPRatio, a program that performs quantification in the TPP pipeline, first using known mixed ratios of yeast extracts and then using a data set from ovarian cancer cell lysates. The program and its documentation can be downloaded from our website at http://systemsbiozju.org/data/WaveletQuant.

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

    DEFF Research Database (Denmark)

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

    2011-01-01

    -friendly platform for comprehensive analysis, inspection and visualization of quantitative proteomics data we developed the Graphical Proteomics Data Explorer (GProX)(1). The program requires no special bioinformatics training, as all functions of GProX are accessible within its graphical user-friendly interface...... which will be intuitive to most users. Basic features facilitate the uncomplicated management and organization of large data sets and complex experimental setups as well as the inspection and graphical plotting of quantitative data. These are complemented by readily available high-level analysis options...... such as database querying, clustering based on abundance ratios, feature enrichment tests for e.g. GO terms and pathway analysis tools. A number of plotting options for visualization of quantitative proteomics data is available and most analysis functions in GProX create customizable high quality graphical...

  9. Combined stable isotope, proteomic, metabolomics, and spatial specific analysis to track carbon flow through a hypersaline phototrophic microbial mat

    Science.gov (United States)

    Moran, J.; Cory, A.; Riha, K. M.; Huang, E. L.; Gritsenko, M. A.; Kim, Y. M.; Metz, T. O.; Lipton, M. S.

    2014-12-01

    Tracking labeled substrates through microbial mat systems can help elucidate carbon dynamics, species interactions, and niche partitioning, but the inherent microbial complexity of these systems makes them difficult to probe with single analytical techniques. Here we use a combination of different tools to track three labeled substrates through a benthic phototrophic mat from Hot Lake. Hot Lake is a hypersaline, meromictic lake located in an endorheic basin in north-central Washington which, despite extreme salinity and seasonal water temperatures (> 55 ˚C), hosts dense, phototrophic benthic microbial mats. Cyanobacteria are the dominant CO2-fixing organisms in the system and we seek to understand the spatial and metabolic controls on how the carbon initially fixed by mat cyanobacteria is transferred to associated heterotrophic populations spread throughout the mat strata. We performed ex situ incubations over a complete diel cycle with 13C labeled bicarbonate, acetate, and glucose. Traditional elemental analysis IRMS provided an estimate of bulk label uptake to total biomass and showed that both bicarbonate and acetate were incorporated only during daylight while glucose uptake was nearly constant through the cycle. Spatially resolved isotope analysis using laser ablation IRMS showed distinctive patterns between the different substrates with bicarbonate having highest uptake in the top third of the mat, acetate uptake focused near the mat's center, and glucose showing similar uptake at all mat depths. Proteomic analysis showed a longer lag in substrate conversion to protein than to biomass and a distinct spike in the number of labeled peptides in the bicarbonate incubation near the end of the diel cycle. Proteomic analysis confirmed that photosynthetic organisms showed the highest rates of label conversion to protein but heterotrophic organisms also incorporated label into their peptides. Metabolomic analysis demonstrated the high conversion of organic substrates

  10. Proteomic Analysis of Colorectal Cancer: Prefractionation Strategies Using two-Dimensional Free-Flow Electrophoresis

    Directory of Open Access Journals (Sweden)

    Richard J. Simpson

    2006-04-01

    Full Text Available This review deals with the application of a new prefractionation tool, free-flow electrophoresis (FFE, for proteomic analysis of colorectal cancer (CRC. CRC is a leading cause of cancer death in the Western world. Early detection is the single most important factor influencing outcome of CRC patients. If identified while the disease is still localized, CRC is treatable. To improve outcomes for CRC patients there is a pressing need to identify biomarkers for early detection (diagnostic markers, prognosis (prognostic indicators, tumour responses (predictive markers and disease recurrence (monitoring markers. Despite recent advances in the use of genomic analysis for risk assessment, in the area of biomarker identification genomic methods alone have yet to produce reliable candidate markers for CRC. For this reason, attention is being directed towards proteomics as a complementary analytical tool for biomarker identification. Here we describe a proteomics separation tool, which uses a combination of continuous FFE, a liquid-based isoelectric focusing technique, in the first dimension, followed by rapid reversed-phase HPLC (1–6 min/analysis in the second dimension. We have optimized imaging software to present the FFE/RP-HPLC data in a virtual 2D gel-like format. The advantage of this liquid based fractionation system over traditional gel-based fractionation systems is the ability to fractionate large quantity protein samples. Unlike 2D gels, the method is applicable to both high-Mr proteins and small peptides, which are difficult to separate, and in the case of peptides, are not retained in standard 2D gels.

  11. Proteomic Analysis of Early Mid-Trimester Amniotic Fluid Does Not Predict Spontaneous Preterm Delivery.

    Directory of Open Access Journals (Sweden)

    Maria Hallingström

    Full Text Available The aim of this study was to identify early proteomic biomarkers of spontaneous preterm delivery (PTD in mid-trimester amniotic fluid from asymptomatic women.This is a case-cohort study. Amniotic fluid from mid-trimester genetic amniocentesis (14-19 weeks of gestation was collected from 2008 to 2011. The analysis was conducted in 24 healthy women with subsequent spontaneous PTD (cases and 40 randomly selected healthy women delivering at term (controls. An exploratory phase with proteomics analysis of pooled samples was followed by a verification phase with ELISA of individual case and control samples.The median (interquartile range (IQR: 25th; 75th percentiles gestational age at delivery was 35+5 (33+6-36+6 weeks in women with spontaneous PTD and 40+0 (39+1-40+5 weeks in women who delivered at term. In the exploratory phase, the most pronounced differences were found in C-reactive protein (CRP levels, that were approximately two-fold higher in the pooled case samples than in the pooled control samples. However, we could not verify these differences with ELISA. The median (25th; 75th IQR CRP level was 95.2 ng/mL (64.3; 163.5 in women with spontaneous PTD and 86.0 ng/mL (51.2; 145.8 in women delivering at term (p = 0.37; t-test.Proteomic analysis with mass spectrometry of mid-trimester amniotic fluid suggests CRP as a potential marker of spontaneous preterm delivery, but this prognostic potential was not verified with ELISA.

  12. Comparative proteomic analysis provides insight into cadmium stress responses in brown algae Sargassum fusiforme

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Aiqin; Xu, Tao [Alkali Soil Natural Environmental Science Center, Northeast Forestry University, Key Laboratory of Saline–alkali Vegetation Ecology Restoration in Oil Field, Ministry of Education, Harbin 150040 (China); Zou, Huixi [Zhejiang Provincial Key Laboratory for Subtropical Water Environment and Marine Biological Resources Protection, College of Life and Environmental Science, Wenzhou University, Wenzhou 325035 (China); Pang, Qiuying, E-mail: qiuying@nefu.edu.cn [Alkali Soil Natural Environmental Science Center, Northeast Forestry University, Key Laboratory of Saline–alkali Vegetation Ecology Restoration in Oil Field, Ministry of Education, Harbin 150040 (China)

    2015-06-15

    Highlights: • Proteomic analysis of brown algae response different level Cd stress was performed. • Proteins involved in carbohydrate metabolism were reduced under 1 day Cd stress. • 5 days Cd stress induced glycolysis and citrate cycle related proteins. • Graphic depiction of different metabolic pathways response to Cd stress was framed. - Abstract: Sargassum fusiforme is one of the most widely consumed seaweeds in China, Korea and Japan. In this work, we performed growth analysis and comparative proteomics to investigate the molecular mechanisms of the response to 1 day and 5 days Cd stress in S. fusiforme. Our results showed a significant decrease in growth rate and an increase in Cd ion content in S. fusiforme in response to Cd treatment. Comparative proteomic analysis revealed 25 and 51 differentially expressed protein spots in S. fusiforme under 1 day and 5 days Cd stress, respectively. A great number of these proteins was metabolic enzymes involved in carbohydrate metabolism and energy metabolism. Many proteins involved in the processing of genetic information showed a decrease in abundance under 1 day Cd stress. In contrast, 9 of the identified protein spots primarily involved in genetic information processing and carbohydrate metabolism were greatly enriched under 5 days Cd stress. Overall, our investigation indicated that Cd stress negatively affects the metabolic activity of S. fusiforme through the down-regulation of key metabolic enzymes. In addition, S. fusiforme may adapt to 5 days Cd stress by promoting consumption of photoassimilates through the up-regulation of glycolysis and the citrate cycle to supply energy for survival.

  13. Common Data Analysis Pipeline | Office of Cancer Clinical Proteomics Research

    Science.gov (United States)

    CPTAC supports analyses of the mass spectrometry raw data (mapping of spectra to peptide sequences and protein identification) for the public using a Common Data Analysis Pipeline (CDAP). The data types available on the public portal are described below. A general overview of this pipeline can be downloaded here. Mass Spectrometry Data Formats RAW (Vendor) Format

  14. Proteomic analysis of mitochondria in respiratory epithelial cells infected with human respiratory syncytial virus and functional implications for virus and cell biology.

    Science.gov (United States)

    Munday, Diane C; Howell, Gareth; Barr, John N; Hiscox, Julian A

    2015-03-01

    The aim of this study was to quantitatively characterise the mitochondrial proteome of airway epithelial cells infected with human respiratory syncytial virus (HRSV), a major cause of paediatric illness. Quantitative proteomics, underpinned by stable isotope labelling with amino acids in cell culture, coupled to LC-MS/MS, was applied to mitochondrial fractions prepared from HRSV-infected and mock-infected cells 12 and 24 h post-infection. Datasets were analysed using ingenuity pathway analysis, and the results were validated and characterised using bioimaging, targeted inhibition and gene depletion. The data quantitatively indicated that antiviral signalling proteins converged on mitochondria during HRSV infection. The mitochondrial receptor protein Tom70 was found to act in an antiviral manner, while its chaperone, Hsp90, was confirmed to be a positive viral factor. Proteins associated with different organelles were also co-enriched in the mitochondrial fractions from HRSV-infected cells, suggesting that alterations in organelle dynamics and membrane associations occur during virus infection. Protein and pathway-specific alterations occur to the mitochondrial proteome in a spatial and temporal manner during HRSV infection, suggesting that this organelle may have altered functions. These could be targeted as part of potential therapeutic strategies to disrupt virus biology. © 2014 Royal Pharmaceutical Society.

  15. A quantitative proteomics analysis of MCF7 breast cancer stem and progenitor cell populations.

    Science.gov (United States)

    Nie, Song; McDermott, Sean P; Deol, Yadwinder; Tan, Zhijing; Wicha, Max S; Lubman, David M

    2015-11-01

    Accumulating evidence has demonstrated that breast cancers are initiated and develop from a small population of stem-like cells termed cancer stem cells (CSCs). These cells are hypothesized to mediate tumor metastasis and contribute to therapeutic resistance. However, the molecular regulatory networks responsible for maintaining CSCs in an undifferentiated state have yet to be elucidated. In this study, we used CSC markers to isolate pure breast CSCs fractions (ALDH+ and CD44+CD24- cell populations) and the mature luminal cells (CD49f-EpCAM+) from the MCF7 cell line. Proteomic analysis was performed on these samples and a total of 3304 proteins were identified. A label-free quantitative method was applied to analyze differentially expressed proteins. Using the criteria of greater than twofold changes and p value analysis of differentially expressed proteins by Ingenuity Pathway Analysis (IPA) revealed potential molecular regulatory networks that may regulate CSCs. Selected differential proteins were validated by Western blot assay and immunohistochemical staining. The use of proteomics analysis may increase our understanding of the underlying molecular mechanisms of breast CSCs. This may be of importance in the future development of anti-CSC therapeutics.

  16. Characterization of citrate utilization in Corynebacterium glutamicum by transcriptome and proteome analysis.

    Science.gov (United States)

    Polen, Tino; Schluesener, Daniela; Poetsch, Ansgar; Bott, Michael; Wendisch, Volker F

    2007-08-01

    Corynebacterium glutamicum grows aerobically on a variety of carbohydrates and organic acids as single or combined sources of carbon and energy. To characterize the citrate utilization in C. glutamicum on a genomewide scale, a comparative analysis was carried out by combining transcriptome and proteome analysis. In cells grown on citrate, transcriptome analysis revealed highest expression changes for two different citrate-uptake systems encoded by citM and tctCBA, whereas genes encoding uptake systems for the glucose- (ptsG), sucrose- (ptsS) and fructose- (ptsF) specific PTS components and permeases for gluconate (gntP) and glutamate (gluC) displayed decreased mRNA levels in citrate-grown cells. This pattern was also observed when cells grown in Luria-Bertani (LB) medium plus citrate were compared with cells grown in LB medium, indicating some kind of catabolite repression. Genes encoding enzymes of the tricarboxylic acid cycle (aconitase, succinyl-CoA synthetase, succinate dehydrogenase and fumarase), malic enzyme, PEP carboxykinase, gluconeogenic glyceraldehyde-3-phosphate dehydrogenase and ATP synthase displayed increased expression in cells grown on citrate. Accordingly, proteome analysis revealed elevated protein levels of these enzymes and showed a good correlation with the mRNA levels. In conclusion, this study revealed the citrate stimulon in C. glutamicum and the regulated central metabolic genes when grown on citrate.

  17. Differential Proteomic Analysis of Solanum tuberosum L. Leaves under Drought Stress

    Institute of Scientific and Technical Information of China (English)

    Yuan Su; Ping Yu; Xiaogan Zhou

    2012-01-01

    The research of molecular mechanism with anti-drought in Solanum tuberosum L.is important for breed improvement in potato to avoid yield loss caused by water deficit.Differential proteomics analysis of potato (anti-drought cultivar) leaves under drought stress were carried out using two-dimensional gel electrophoresis.42 differential expression protein spots were analyzed through gel map and identified by MALDI-TOF-TOF/MS.The main function of these proteins were stimulation response,cell development,metabolic and transport adjustment.The experiment can supply theory evidence to explain the anti-drought mechanism of anti-drought potato cultivar with multi-pathways.

  18. Proteomic analysis of Alternaria alternata (Fr.) Keissler responds to COS fumigation.

    Science.gov (United States)

    Liu, Tao; Li, Li; Qu, Haixia; Zhan, Guoping; Liu, Bo; Wang, Yuejin

    2010-01-01

    Carbonyl sulfide (COS) is a new fumigant which has been a potential alternative to methyl bromide and phosphine in many applications. In this study, we investigated the fungitoxicity of COS towards the pathogen of pear black spot disease Alternaria alternata (Fr.) Keissler (A. alternata). Moreover, proteomic analysis and RT-PCR was performed and our results showed that during the fumigation, the regulation of 21 proteins in protein expression and mRNA accumulation levels is involved, which respond to growth inhibition caused by COS. These results provide new clues for the mechanism of the fungitoxicity of COS.

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

    OpenAIRE

    2014-01-01

    Date palm (Phoenix dactylifera) is an economically important fruit tree in the Middle East and North Africa and is characterized by large cultivar diversity, making it a good model for studies on fruit development and other important traits. Here in gel comparative proteomics combined with tandem mass spectrometry were used to study date fruit development and ripening. Total proteins were extracted using a phenol-based protocol. A total of 189 protein spots were differentially regulated (p≤0....

  20. Redox proteomics and the dynamic molecular landscape of the aging brain.

    Science.gov (United States)

    Perluigi, Marzia; Swomley, Aaron M; Butterfield, D Allan

    2014-01-01

    It is well established that the risk to develop neurodegenerative disorders increases with chronological aging. Accumulating studies contributed to characterize the age-dependent changes either at gene and protein expression level which, taken together, show that aging of the human brain results from the combination of the normal decline of multiple biological functions with environmental factors that contribute to defining disease risk of late-life brain disorders. Finding the "way out" of the labyrinth of such complex molecular interactions may help to fill the gap between "normal" brain aging and development of age-dependent diseases. To this purpose, proteomics studies are a powerful tool to better understand where to set the boundary line of healthy aging and age-related disease by analyzing the variation of protein expression levels and the major post translational modifications that determine "protein" physio/pathological fate. Increasing attention has been focused on oxidative modifications due to the crucial role of oxidative stress in aging, in addition to the fact that this type of modification is irreversible and may alter protein function. Redox proteomics studies contributed to decipher the complexity of brain aging by identifying the proteins that were increasingly oxidized and eventually dysfunctional as a function of age. The purpose of this review is to summarize the most important findings obtained by applying proteomics approaches to murine models of aging with also a brief overview of some human studies, in particular those related to dementia.

  1. Proteomic analysis of human substantia nigra identifies novel candidates involved in Parkinson's disease pathogenesis.

    Science.gov (United States)

    Licker, Virginie; Turck, Natacha; Kövari, Enikö; Burkhardt, Karim; Côte, Mélanie; Surini-Demiri, Maria; Lobrinus, Johannes A; Sanchez, Jean-Charles; Burkhard, Pierre R

    2014-03-01

    Parkinson's disease (PD) pathology spreads throughout the brain following a region-specific process predominantly affecting the substantia nigra (SN) pars compacta. SN exhibits a progressive loss of dopaminergic neurons responsible for the major cardinal motor symptoms, along with the occurrence of Lewy bodies in the surviving neurons. To gain new insights into the underlying pathogenic mechanisms in PD, we studied postmortem nigral tissues dissected from pathologically confirmed PD cases (n = 5) and neurologically intact controls (n = 8). Using a high-throughput shotgun proteomic strategy, we simultaneously identified 1795 proteins with concomitant quantitative data. To date, this represents the most extensive catalog of nigral proteins. Of them, 204 proteins displayed significant expression level changes in PD patients versus controls. These were involved in novel or known pathogenic processes including mitochondrial dysfunction, oxidative stress, or cytoskeleton impairment. We further characterized four candidates that might be relevant to PD pathogenesis. We confirmed the differential expression of ferritin-L and seipin by Western blot and demonstrated the neuronal localization of gamma glutamyl hydrolase and nebulette by immunohistochemistry. Our preliminary findings suggest a role for nebulette overexpression in PD neurodegeneration, through mechanisms that may involve cytoskeleton dynamics disruption. All MS data have been deposited in the ProteomeXchange with identifier PXD000427 (http://proteomecentral.proteomexchange.org/dataset/PXD000427).

  2. Parallel analysis of Arabidopsis circadian clock mutants reveals different scales of transcriptome and proteome regulation

    Science.gov (United States)

    Graf, Alexander; Coman, Diana; Walsh, Sean; Flis, Anna; Stitt, Mark; Gruissem, Wilhelm

    2017-01-01

    The circadian clock regulates physiological processes central to growth and survival. To date, most plant circadian clock studies have relied on diurnal transcriptome changes to elucidate molecular connections between the circadian clock and observable phenotypes in wild-type plants. Here, we have integrated RNA-sequencing and protein mass spectrometry data to comparatively analyse the lhycca1, prr7prr9, gi and toc1 circadian clock mutant rosette at the end of day and end of night. Each mutant affects specific sets of genes and proteins, suggesting that the circadian clock regulation is modular. Furthermore, each circadian clock mutant maintains its own dynamically fluctuating transcriptome and proteome profile specific to subcellular compartments. Most of the measured protein levels do not correlate with changes in their corresponding transcripts. Transcripts and proteins that have coordinated changes in abundance are enriched for carbohydrate- and cold-responsive genes. Transcriptome changes in all four circadian clock mutants also affect genes encoding starch degradation enzymes, transcription factors and protein kinases. The comprehensive transcriptome and proteome datasets demonstrate that future system-driven research of the circadian clock requires multi-level experimental approaches. Our work also shows that further work is needed to elucidate the roles of post-translational modifications and protein degradation in the regulation of clock-related processes. PMID:28250106

  3. Comparative proteomic analysis of experimental evolution of the Bacillus cereus-Ketogulonicigenium vulgare co-culture.

    Directory of Open Access Journals (Sweden)

    Qian Ma

    Full Text Available The microbial co-culture system composing of Ketogulonicigenium vulgare and Bacillus cereus was widely adopted in industry for the production of 2-keto-gulonic acid (2-KGA, the precursor of vitamin C. We found serial subcultivation of the co-culture could enhance the yield of 2-KGA by 16% in comparison to that of the ancestral co-culture. To elucidate the evolutionary dynamics and interaction mechanisms of the two microbes, we performed iTRAQ-based quantitative proteomic analyses of the pure cultures of K. vulgare, B. cereus and their co-culture during serial subcultivation. Hierarchy cluster analyses of the proteomic data showed that the expression level of a number of crucial proteins associated with sorbose conversion and oligopeptide transport was significantly enhanced by the experimental evolution. In particular, the expression level of sorbose/sorbosone dehydrogenase was enhanced in the evolved K. vulgare, while the expression level of InhA and the transport efficiency of oligopeptides were increased in the evolved B. cereus. The decreased sporulating protein expression and increased peptide transporter expression observed in evolved B. cereus, together with the increased amino acids synthesis in evolved K. vulgare suggested that serial subcultivation result in enhanced synergistic cooperation between K. vulgare and B. cereus, enabling an increased production of 2-KGA.

  4. Parallel analysis of Arabidopsis circadian clock mutants reveals different scales of transcriptome and proteome regulation.

    Science.gov (United States)

    Graf, Alexander; Coman, Diana; Uhrig, R Glen; Walsh, Sean; Flis, Anna; Stitt, Mark; Gruissem, Wilhelm

    2017-03-01

    The circadian clock regulates physiological processes central to growth and survival. To date, most plant circadian clock studies have relied on diurnal transcriptome changes to elucidate molecular connections between the circadian clock and observable phenotypes in wild-type plants. Here, we have integrated RNA-sequencing and protein mass spectrometry data to comparatively analyse the lhycca1, prr7prr9, gi and toc1 circadian clock mutant rosette at the end of day and end of night. Each mutant affects specific sets of genes and proteins, suggesting that the circadian clock regulation is modular. Furthermore, each circadian clock mutant maintains its own dynamically fluctuating transcriptome and proteome profile specific to subcellular compartments. Most of the measured protein levels do not correlate with changes in their corresponding transcripts. Transcripts and proteins that have coordinated changes in abundance are enriched for carbohydrate- and cold-responsive genes. Transcriptome changes in all four circadian clock mutants also affect genes encoding starch degradation enzymes, transcription factors and protein kinases. The comprehensive transcriptome and proteome datasets demonstrate that future system-driven research of the circadian clock requires multi-level experimental approaches. Our work also shows that further work is needed to elucidate the roles of post-translational modifications and protein degradation in the regulation of clock-related processes. © 2017 The Authors.

  5. The MetaProteomeAnalyzer: a powerful open-source software suite for metaproteomics data analysis and interpretation.

    Science.gov (United States)

    Muth, Thilo; Behne, Alexander; Heyer, Robert; Kohrs, Fabian; Benndorf, Dirk; Hoffmann, Marcus; Lehtevä, Miro; Reichl, Udo; Martens, Lennart; Rapp, Erdmann

    2015-03-06

    The enormous challenges of mass spectrometry-based metaproteomics are primarily related to the analysis and interpretation of the acquired data. This includes reliable identification of mass spectra and the meaningful integration of taxonomic and functional meta-information from samples containing hundreds of unknown species. To ease these difficulties, we developed a dedicated software suite, the MetaProteomeAnalyzer, an intuitive open-source tool for metaproteomics data analysis and interpretation, which includes multiple search engines and the feature to decrease data redundancy by grouping protein hits to so-called meta-proteins. We also designed a graph database back-end for the MetaProteomeAnalyzer to allow seamless analysis of results. The functionality of the MetaProteomeAnalyzer is demonstrated using a sample of a microbial community taken from a biogas plant.

  6. Selecting Sample Preparation Workflows for Mass Spectrometry-Based Proteomic and Phosphoproteomic Analysis of Patient Samples with Acute Myeloid Leukemia

    Directory of Open Access Journals (Sweden)

    Maria Hernandez-Valladares

    2016-08-01

    Full Text Available Global mass spectrometry (MS-based proteomic and phosphoproteomic studies of acute myeloid leukemia (AML biomarkers represent a powerful strategy to identify and confirm proteins and their phosphorylated modifications that could be applied in diagnosis and prognosis, as a support for individual treatment regimens and selection of patients f