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Sample records for yeast quantitative proteome

  1. Optimized protein extraction for quantitative proteomics of yeasts.

    Directory of Open Access Journals (Sweden)

    Tobias von der Haar

    2007-10-01

    Full Text Available The absolute quantification of intracellular protein levels is technically demanding, but has recently become more prominent because novel approaches like systems biology and metabolic control analysis require knowledge of these parameters. Current protocols for the extraction of proteins from yeast cells are likely to introduce artifacts into quantification procedures because of incomplete or selective extraction.We have developed a novel procedure for protein extraction from S. cerevisiae based on chemical lysis and simultaneous solubilization in SDS and urea, which can extract the great majority of proteins to apparent completeness. The procedure can be used for different Saccharomyces yeast species and varying growth conditions, is suitable for high-throughput extraction in a 96-well format, and the resulting extracts can easily be post-processed for use in non-SDS compatible procedures like 2D gel electrophoresis.An improved method for quantitative protein extraction has been developed that removes some of the sources of artefacts in quantitative proteomics experiments, while at the same time allowing novel types of applications.

  2. Global analysis of the yeast osmotic stress response by quantitative proteomics

    DEFF Research Database (Denmark)

    Soufi, Boumediene; Kelstrup, C.D.; Stoehr, G.

    2009-01-01

    a comprehensive, quantitative, and time-resolved analysis using high-resolution mass spectrometry of phospho-proteome and proteome changes in response to osmotic stress in yeast. We identified 5534 unique phosphopeptide variants and 3383 yeast proteins. More than 15% of the detected phosphorylation site status...... changed more than two-fold within 5 minutes of treatment. Many of the corresponding phosphoproteins are involved in the early response to environmental stress. Surprisingly, we find that 158 regulated phosphorylation sites are potential substrates of basophilic kinases as opposed to the classical proline......-directed MAP kinase network implicated in stress response mechanisms such as p38 and HOG pathways. Proteome changes reveal an increase in abundance of more than one hundred proteins after 20 min of salt stress. Many of these are involved in the cellular response to increased osmolarity, which include proteins...

  3. Characterization of global yeast quantitative proteome data generated from the wild-type and glucose repression Saccharomyces cerevisiae strains: The comparison of two quantitative methods

    DEFF Research Database (Denmark)

    Usaite, Renata; Wohlschlegel, James; Venable, John D.

    2008-01-01

    The quantitative proteomic analysis of complex protein mixtures is emerging as a technically challenging but viable systems-level approach for studying cellular function. This study presents a large-scale comparative analysis of protein abundances from yeast protein lysates derived from both wild......-type yeast and yeast strains lacking key components of the Snf1 kinase complex. Four different strains were grown under well-controlled chemostat conditions. Multidimensional protein identification technology followed by quantitation using either spectral counting or stable isotope labeling approaches...... labeling strategy. The stable isotope labeling based quantitative approach was found to be highly reproducible among biological replicates when complex protein mixtures containing small expression changes were analyzed. Where poor correlation between stable isotope labeling and spectral counting was found...

  4. Qualitative and quantitative multiplexed proteomic analysis of complex yeast protein fractions that modulate the assembly of the yeast prion Sup35p.

    Directory of Open Access Journals (Sweden)

    Virginie Redeker

    Full Text Available BACKGROUND: The aggregation of the baker's yeast prion Sup35p is at the origin of the transmissible [PSI(+] trait. We and others have shown that molecular chaperones modulate Sup35p aggregation. However, other protein classes might be involved in [PSI(+] formation. RESULTS: We designed a functional proteomic study that combines two techniques to identify modulators of Sup35p aggregation and describe the changes associated to [PSI(+] formation. The first allows measuring the effect of fractionated Saccharomyces cerevisiae cytosolic extracts from [PSI(+] and [psi(-] yeast cells on Sup35p assembly. The second is a multiplex qualitative and quantitative comparison of protein composition of active and inactive fractions using a gel-free and label-free LC-MS approach. We identify changes in proteins involved in translation, folding, degradation, oxido-reduction and metabolic processes. CONCLUSION: Our functional proteomic study provides the first inventory list of over 300 proteins that directly or indirectly affect Sup35p aggregation and [PSI(+] formation. Our results highlight the complexity of the cellular changes accompanying [PSI(+] formation and pave the way for in vitro studies aimed to document the effect of individual and/or combinations of proteins identified here, susceptible of affecting Sup35p assembly.

  5. [Methods of quantitative proteomics].

    Science.gov (United States)

    Kopylov, A T; Zgoda, V G

    2007-01-01

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

  6. Yeast expression proteomics by high-resolution mass spectrometry

    DEFF Research Database (Denmark)

    Walther, Tobias C; Olsen, Jesper Velgaard; Mann, Matthias

    2010-01-01

    -translational controls contribute majorly to regulation of protein abundance, for example in heat shock stress response. The development of new sample preparation methods, high-resolution mass spectrometry and novel bioinfomatic tools close this gap and allow the global quantitation of the yeast proteome under different...

  7. Magnetoresistive biosensors for quantitative proteomics

    Science.gov (United States)

    Zhou, Xiahan; Huang, Chih-Cheng; Hall, Drew A.

    2017-08-01

    Quantitative proteomics, as a developing method for study of proteins and identification of diseases, reveals more comprehensive and accurate information of an organism than traditional genomics. A variety of platforms, such as mass spectrometry, optical sensors, electrochemical sensors, magnetic sensors, etc., have been developed for detecting proteins quantitatively. The sandwich immunoassay is widely used as a labeled detection method due to its high specificity and flexibility allowing multiple different types of labels. While optical sensors use enzyme and fluorophore labels to detect proteins with high sensitivity, they often suffer from high background signal and challenges in miniaturization. Magnetic biosensors, including nuclear magnetic resonance sensors, oscillator-based sensors, Hall-effect sensors, and magnetoresistive sensors, use the specific binding events between magnetic nanoparticles (MNPs) and target proteins to measure the analyte concentration. Compared with other biosensing techniques, magnetic sensors take advantage of the intrinsic lack of magnetic signatures in biological samples to achieve high sensitivity and high specificity, and are compatible with semiconductor-based fabrication process to have low-cost and small-size for point-of-care (POC) applications. Although still in the development stage, magnetic biosensing is a promising technique for in-home testing and portable disease monitoring.

  8. Mapping out starvation responses in yeast by proteomics

    DEFF Research Database (Denmark)

    Rødkær, Steven Vestergaard; Færgeman, Nils J.; Andersen, Jens S.

    2011-01-01

    that are involved in this positive outcome. Based on that, processes like autophagy, lipid turnover and the generation/clearance of reactive oxygen species (ROS) have all been describe to affect life span, either alone, or in a not fully characterized interplay. The baker’s yeast Saccharomyces cerevisae is by now...... the organism with the best characterized proteome and is therefore the organism of choice in many proteomic studies. Additionally, this single-celled organism exhibits many conserved proteins and pathways of higher animals, thus observations in the yeast might reveal important information applying to other...

  9. Quantitative proteomics of Chlorobaculum tepidum

    DEFF Research Database (Denmark)

    Falkenby, Lasse Gaarde; Szymanska, Monika; Holkenbrink, Carina

    2011-01-01

    Chlorobaculum (Cba.) tepidum is a green sulfur bacterium that oxidizes sulfide, elemental sulfur, and thiosulfate for photosynthetic growth. To gain insight into the sulfur metabolism, the proteome of Cba. tepidum cells sampled under different growth conditions has been quantified using a rapid g...

  10. [Progress in stable isotope labeled quantitative proteomics methods].

    Science.gov (United States)

    Zhou, Yuan; Shan, Yichu; Zhang, Lihua; Zhang, Yukui

    2013-06-01

    Quantitative proteomics is an important research field in post-genomics era. There are two strategies for proteome quantification: label-free methods and stable isotope labeling methods which have become the most important strategy for quantitative proteomics at present. In the past few years, a number of quantitative methods have been developed, which support the fast development in biology research. In this work, we discuss the progress in the stable isotope labeling methods for quantitative proteomics including relative and absolute quantitative proteomics, and then give our opinions on the outlook of proteome quantification methods.

  11. 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)

  12. 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.

  13. Identification of SUMO conjugation sites in the budding yeast proteome

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    Miguel Esteras

    2017-10-01

    Full Text Available Post-translational modification by the small ubiquitin-like modifier (SUMO is an important mechanism regulating protein function. Identification of SUMO conjugation sites on substrates is a challenging task. Here we employed a proteomic method to map SUMO acceptor lysines in budding yeast proteins. We report the identification of 257 lysine residues where SUMO is potentially attached. Amongst the hits, we identified already known SUMO substrates and sites, confirming the success of the approach. In addition, we tested several of the novel substrates using SUMO immunoprecipitation analysis and confirmed that the SUMO acceptor lysines identified in these proteins are indeed bona fide SUMOylation sites. We believe that the collection of SUMO sites presented here is an important resource for future functional studies of SUMOylation in yeast.

  14. Quantitative proteomic assessment of very early cellular signaling events

    DEFF Research Database (Denmark)

    Dengjel, Joern; Akimov, Vyacheslav; Olsen, Jesper V

    2007-01-01

    Technical limitations have prevented proteomic analyses of events occurring less than 30 s after signal initiation. We developed an automated, continuous quench-flow system allowing quantitative proteomic assessment of very early cellular signaling events (qPACE) with a time resolution of 1 s...

  15. The impact of different ale brewer’s yeast strains on the proteome of immature beer

    DEFF Research Database (Denmark)

    Berner, Torben Sune; Jacobsen, Susanne; Arneborg, Nils

    2013-01-01

    BACKGROUND: It is well known that brewer’s yeast affects the taste and aroma of beer. However, the influence of brewer’s yeast on the protein composition of beer is currently unknown. In this study, changes of the proteome of immature beer, i.e. beer that has not been matured after fermentation......, by ale brewer’s yeast strains with different abilities to degrade fermentable sugars were investigated. RESULTS: Beers were fermented from standard hopped wort (13° Plato) using two ale brewer’s yeast (Saccharomyces cerevisiae) strains with different attenuation degrees. Both immature beers had the same....... These three proteins, all derived from yeast, were identified as cell wall associated proteins, that is Exg1 (an exo-β-1,3-glucanase), Bgl2 (an endo-β-1,2-glucanase), and Uth1 (a cell wall biogenesis protein). CONCLUSION: Yeast strain dependent changes in the immature beer proteome were identified, i.e. Bgl2...

  16. The APEX Quantitative Proteomics Tool: Generating protein quantitation estimates from LC-MS/MS proteomics results

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    Saeed Alexander I

    2008-12-01

    Full Text Available Abstract Background Mass spectrometry (MS based label-free protein quantitation has mainly focused on analysis of ion peak heights and peptide spectral counts. Most analyses of tandem mass spectrometry (MS/MS data begin with an enzymatic digestion of a complex protein mixture to generate smaller peptides that can be separated and identified by an MS/MS instrument. Peptide spectral counting techniques attempt to quantify protein abundance by counting the number of detected tryptic peptides and their corresponding MS spectra. However, spectral counting is confounded by the fact that peptide physicochemical properties severely affect MS detection resulting in each peptide having a different detection probability. Lu et al. (2007 described a modified spectral counting technique, Absolute Protein Expression (APEX, which improves on basic spectral counting methods by including a correction factor for each protein (called Oi value that accounts for variable peptide detection by MS techniques. The technique uses machine learning classification to derive peptide detection probabilities that are used to predict the number of tryptic peptides expected to be detected for one molecule of a particular protein (Oi. This predicted spectral count is compared to the protein's observed MS total spectral count during APEX computation of protein abundances. Results The APEX Quantitative Proteomics Tool, introduced here, is a free open source Java application that supports the APEX protein quantitation technique. The APEX tool uses data from standard tandem mass spectrometry proteomics experiments and provides computational support for APEX protein abundance quantitation through a set of graphical user interfaces that partition thparameter controls for the various processing tasks. The tool also provides a Z-score analysis for identification of significant differential protein expression, a utility to assess APEX classifier performance via cross validation, and a

  17. 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

  18. Quantitative proteomics by amino acid labeling in C. elegans

    DEFF Research Database (Denmark)

    Fredens, Julius; Engholm-Keller, Kasper; Giessing, Anders

    2011-01-01

    We demonstrate labeling of Caenorhabditis elegans with heavy isotope-labeled lysine by feeding them with heavy isotope-labeled Escherichia coli. Using heavy isotope-labeled worms and quantitative proteomics methods, we identified several proteins that are regulated in response to loss or RNAi-med......-mediated knockdown of the nuclear hormone receptor 49 in C. elegans. The combined use of quantitative proteomics and selective gene knockdown is a powerful tool for C. elegans biology.......We demonstrate labeling of Caenorhabditis elegans with heavy isotope-labeled lysine by feeding them with heavy isotope-labeled Escherichia coli. Using heavy isotope-labeled worms and quantitative proteomics methods, we identified several proteins that are regulated in response to loss or RNAi...

  19. Guidelines for reporting quantitative mass spectrometry based experiments in proteomics.

    Science.gov (United States)

    Martínez-Bartolomé, Salvador; Deutsch, Eric W; Binz, Pierre-Alain; Jones, Andrew R; Eisenacher, Martin; Mayer, Gerhard; Campos, Alex; Canals, Francesc; Bech-Serra, Joan-Josep; Carrascal, Montserrat; Gay, Marina; Paradela, Alberto; Navajas, Rosana; Marcilla, Miguel; Hernáez, María Luisa; Gutiérrez-Blázquez, María Dolores; Velarde, Luis Felipe Clemente; Aloria, Kerman; Beaskoetxea, Jabier; Medina-Aunon, J Alberto; Albar, Juan P

    2013-12-16

    Mass spectrometry is already a well-established protein identification tool and recent methodological and technological developments have also made possible the extraction of quantitative data of protein abundance in large-scale studies. Several strategies for absolute and relative quantitative proteomics and the statistical assessment of quantifications are possible, each having specific measurements and therefore, different data analysis workflows. The guidelines for Mass Spectrometry Quantification allow the description of a wide range of quantitative approaches, including labeled and label-free techniques and also targeted approaches such as Selected Reaction Monitoring (SRM). The HUPO Proteomics Standards Initiative (HUPO-PSI) has invested considerable efforts to improve the standardization of proteomics data handling, representation and sharing through the development of data standards, reporting guidelines, controlled vocabularies and tooling. In this manuscript, we describe a key output from the HUPO-PSI-namely the MIAPE Quant guidelines, which have developed in parallel with the corresponding data exchange format mzQuantML [1]. The MIAPE Quant guidelines describe the HUPO-PSI proposal concerning the minimum information to be reported when a quantitative data set, derived from mass spectrometry (MS), is submitted to a database or as supplementary information to a journal. The guidelines have been developed with input from a broad spectrum of stakeholders in the proteomics field to represent a true consensus view of the most important data types and metadata, required for a quantitative experiment to be analyzed critically or a data analysis pipeline to be reproduced. It is anticipated that they will influence or be directly adopted as part of journal guidelines for publication and by public proteomics databases and thus may have an impact on proteomics laboratories across the world. This article is part of a Special Issue entitled: Standardization and

  20. 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

  1. Quantitative Proteomic Analysis of Sulfolobus solfataricus Membrane Proteins

    NARCIS (Netherlands)

    Pham, T.K.; Sierocinski, P.; Oost, van der J.; Wright, P.C.

    2010-01-01

    A quantitative proteomic analysis of the membrane of the archaeon Sulfolobus solfataricus P2 using iTRAQ was successfully demonstrated in this technical note. The estimated number of membrane proteins of this organism is 883 (predicted based on Gravy score), corresponding to 30 % of the total

  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. Keywords: Spleen, Rat, Protein extraction, Label-free quantitative proteomics

  3. The Spectra Count Label-free Quantitation in Cancer Proteomics

    OpenAIRE

    Zhou, Weidong; Liotta, Lance A.; Petricoin, Emanuel F.

    2012-01-01

    Mass spectrometry is used routinely for large-scale protein identification from complex biological mixtures. Recently, relative quantitation approach on the basis of spectra count has been applied in several cancer proteomic studies. In this review, we examine the mechanism of this technique and highlight several important parameters associated with its application.

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

    NARCIS (Netherlands)

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

    2013-01-01

    Quantitative and qualitative protein characteristics are regulated at genomic, transcriptomic, and post-transcriptional 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

  5. Quantitative proteome profiling of normal human circulating microparticles

    DEFF Research Database (Denmark)

    Østergaard, Ole; Nielsen, Christoffer T; Iversen, Line V

    2012-01-01

    Circulating microparticles (MPs) are produced as part of normal physiology. Their numbers, origin, and composition change in pathology. Despite this, the normal MP proteome has not yet been characterized with standardized high-resolution methods. We here quantitatively profile the normal MP...... proteome using nano-LC-MS/MS on an LTQ-Orbitrap with optimized sample collection, preparation, and analysis of 12 different normal samples. Analytical and procedural variation were estimated in triply processed samples analyzed in triplicate from two different donors. Label-free quantitation was validated...... by the correlation of cytoskeletal protein intensities with MP numbers obtained by flow cytometry. Finally, the validity of using pooled samples was evaluated using overlap protein identification numbers and multivariate data analysis. Using conservative parameters, 536 different unique proteins were quantitated...

  6. CPTC and NIST-sponsored Yeast Reference Material Now Publicly Available | Office of Cancer Clinical Proteomics Research

    Science.gov (United States)

    The yeast protein extract (RM8323) developed by National Institute of Standards and Technology (NIST) under the auspices of NCI's CPTC initiative is currently available to the public at https://www-s.nist.gov/srmors/view_detail.cfm?srm=8323. The yeast proteome offers researchers a unique biological reference material. RM8323 is the most extensively characterized complex biological proteome and the only one associated with several large-scale studies to estimate protein abundance across a wide concentration range.

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

    Science.gov (United States)

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

    2017-09-01

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

  8. Proteomic approaches for quantitative cancer cell signaling

    DEFF Research Database (Denmark)

    Voellmy, Franziska

    studies in an effort to contribute to the study of signaling dynamics in cancer systems. This thesis is divided into two parts. Part I begins with a brief introduction in the use of omics in systems cancer research with a focus on mass spectrometry as a means to quantitatively measure protein...

  9. Quantitative proteomics of delirium cerebrospinal fluid

    Science.gov (United States)

    Poljak, A; Hill, M; Hall, R J; MacLullich, A M; Raftery, M J; Tai, J; Yan, S; Caplan, G A

    2014-01-01

    Delirium is a common cause and complication of hospitalization in older people, being associated with higher risk of future dementia and progression of existing dementia. However relatively little data are available on which biochemical pathways are dysregulated in the brain during delirium episodes, whether there are protein expression changes common among delirium subjects and whether there are any changes which correlate with the severity of delirium. We now present the first proteomic analysis of delirium cerebrospinal fluid (CSF), and one of few studies exploring protein expression changes in delirium. More than 270 proteins were identified in two delirium cohorts, 16 of which were dysregulated in at least 8 of 17 delirium subjects compared with a mild Alzheimer's disease neurological control group, and 31 proteins were significantly correlated with cognitive scores (mini-mental state exam and acute physiology and chronic health evaluation III). Bioinformatics analyses revealed expression changes in several protein family groups, including apolipoproteins, secretogranins/chromogranins, clotting/fibrinolysis factors, serine protease inhibitors and acute-phase response elements. These data not only provide confirmatory evidence that the inflammatory response is a component of delirium, but also reveal dysregulation of protein expression in a number of novel and unexpected clusters of proteins, in particular the granins. Another surprising outcome of this work is the level of similarity of CSF protein profiles in delirium patients, given the diversity of causes of this syndrome. These data provide additional elements for consideration in the pathophysiology of delirium as well as potential biomarker candidates for delirium diagnosis. PMID:25369144

  10. Dissecting the C. elegans response during infection using quantitative proteomics

    DEFF Research Database (Denmark)

    Simonsen, Karina Trankjær; Møller-Jensen, Jakob; Kristensen, Anders Riis

    2008-01-01

    The adherent invasive E. coli isolated from patients with Crohn’s disease in humans is pathogenic for C. elegans. We show here that when C. elegans feeds on the pathogenic E. coli, the life span is shortened significantly compared to the normal laboratory food, the OP50 E. coli. In this study...... the infection process is followed using GFP-expressing bacteria and persistence assays. A quantitative proteomic approach was used to follow the C. elegans host response during the infection process. C. elegans were metabolic labeled with the stable isotope 15N and samples from three different time points......, many of which also have been found in studies using other pathogens. So far, large-scale investigations of the C. elegans immune response have been performed using micro-arrays. This study is the first to make use of quantitative proteomics to directly follow the protein dynamics during the infection...

  11. 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.

  12. Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway

    DEFF Research Database (Denmark)

    Gruhler, Albrecht; Olsen, Jesper Velgaard; Mohammed, Shabaz

    2005-01-01

    of a detailed molecular view of complex biological processes. We present a quantitative modification-specific proteomic approach that combines stable isotope labeling by amino acids in cell culture (SILAC) for quantitation with IMAC for phosphopeptide enrichment and three stages of mass spectrometry (MS....... Phosphopeptide fractions were analyzed by LC-MS using a linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer. MS/MS and neutral loss-directed MS/MS/MS analysis allowed detection and sequencing of phosphopeptides with exceptional accuracy and specificity. Of more than 700 identified...

  13. Quantitative proteomic analysis of intact plastids.

    Science.gov (United States)

    Shiraya, Takeshi; Kaneko, Kentaro; Mitsui, Toshiaki

    2014-01-01

    Plastids are specialized cell organelles in plant cells that are differentiated into various forms including chloroplasts, chromoplasts, and amyloplasts, and fulfill important functions in maintaining the overall cell metabolism and sensing environmental factors such as sunlight. It is therefore important to grasp the mechanisms of differentiation and functional changes of plastids in order to enhance the understanding of vegetality. In this chapter, details of a method for the extraction of intact plastids that makes analysis possible while maintaining the plastid functions are provided; in addition, a quantitative shotgun method for analyzing the composition and changes in the content of proteins in plastids as a result of environmental impacts is described.

  14. Stable isotope dimethyl labelling for quantitative proteomics and beyond

    Science.gov (United States)

    Hsu, Jue-Liang; Chen, Shu-Hui

    2016-01-01

    Stable-isotope reductive dimethylation, a cost-effective, simple, robust, reliable and easy-to- multiplex labelling method, is widely applied to quantitative proteomics using liquid chromatography-mass spectrometry. This review focuses on biological applications of stable-isotope dimethyl labelling for a large-scale comparative analysis of protein expression and post-translational modifications based on its unique properties of the labelling chemistry. Some other applications of the labelling method for sample preparation and mass spectrometry-based protein identification and characterization are also summarized. This article is part of the themed issue ‘Quantitative mass spectrometry’. PMID:27644970

  15. A quantitative characterization of the yeast heterotrimeric G protein cycle

    Science.gov (United States)

    Yi, Tau-Mu; Kitano, Hiroaki; Simon, Melvin I.

    2003-01-01

    The yeast mating response is one of the best understood heterotrimeric G protein signaling pathways. Yet, most descriptions of this system have been qualitative. We have quantitatively characterized the heterotrimeric G protein cycle in yeast based on direct in vivo measurements. We used fluorescence resonance energy transfer to monitor the association state of cyan fluorescent protein (CFP)-Gα and Gβγ-yellow fluorescent protein (YFP), and we found that receptor-mediated G protein activation produced a loss of fluorescence resonance energy transfer. Quantitative time course and dose–response data were obtained for both wild-type and mutant cells possessing an altered pheromone response. These results paint a quantitative portrait of how regulators such as Sst2p and the C-terminal tail of α-factor receptor modulate the kinetics and sensitivity of G protein signaling. We have explored critical features of the dynamics including the rapid rise and subsequent decline of active G proteins during the early response, and the relationship between the G protein activation dose–response curve and the downstream dose–response curves for cell-cycle arrest and transcriptional induction. Fitting the data to a mathematical model produced estimates of the in vivo rates of heterotrimeric G protein activation and deactivation in yeast. PMID:12960402

  16. Science, marketing and wishful thinking in quantitative proteomics.

    Science.gov (United States)

    Hackett, Murray

    2008-11-01

    In a recent editorial (J. Proteome Res. 2007, 6, 1633) and elsewhere questions have been raised regarding the lack of attention paid to good analytical practice with respect to the reporting of quantitative results in proteomics. Using those comments as a starting point, several issues are discussed that relate to the challenges involved in achieving adequate sampling with MS-based methods in order to generate valid data for large-scale studies. The discussion touches on the relationships that connect sampling depth and the power to detect protein abundance change, conflict of interest, and strategies to overcome bureaucratic obstacles that impede the use of peer-to-peer technologies for transfer and storage of large data files generated in such experiments.

  17. 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.

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

    Science.gov (United States)

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

    2018-05-15

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

  19. Mapping Protein-Protein Interactions by Quantitative Proteomics

    DEFF Research Database (Denmark)

    Dengjel, Joern; Kratchmarova, Irina; Blagoev, Blagoy

    2010-01-01

    spectrometry (MS)-based proteomics in combination with affinity purification protocols has become the method of choice to map and track the dynamic changes in protein-protein interactions, including the ones occurring during cellular signaling events. Different quantitative MS strategies have been used...... to characterize protein interaction networks. In this chapter we describe in detail the use of stable isotope labeling by amino acids in cell culture (SILAC) for the quantitative analysis of stimulus-dependent dynamic protein interactions.......Proteins exert their function inside a cell generally in multiprotein complexes. These complexes are highly dynamic structures changing their composition over time and cell state. The same protein may thereby fulfill different functions depending on its binding partners. Quantitative mass...

  20. Proteomic and genomic characterization of a yeast model for Ogden syndrome

    Science.gov (United States)

    Dörfel, Max J.; Fang, Han; Crain, Jonathan; Klingener, Michael; Weiser, Jake

    2016-01-01

    Abstract Naa10 is an Nα‐terminal acetyltransferase that, in a complex with its auxiliary subunit Naa15, co‐translationally acetylates the α‐amino group of newly synthetized proteins as they emerge from the ribosome. Roughly 40–50% of the human proteome is acetylated by Naa10, rendering this an enzyme one of the most broad substrate ranges known. Recently, we reported an X‐linked disorder of infancy, Ogden syndrome, in two families harbouring a c.109 T > C (p.Ser37Pro) variant in NAA10. In the present study we performed in‐depth characterization of a yeast model of Ogden syndrome. Stress tests and proteomic analyses suggest that the S37P mutation disrupts Naa10 function and reduces cellular fitness during heat shock, possibly owing to dysregulation of chaperone expression and accumulation. Microarray and RNA‐seq revealed a pseudo‐diploid gene expression profile in ΔNaa10 cells, probably responsible for a mating defect. In conclusion, the data presented here further support the disruptive nature of the S37P/Ogden mutation and identify affected cellular processes potentially contributing to the severe phenotype seen in Ogden syndrome. Data are available via GEO under identifier GSE86482 or with ProteomeXchange under identifier PXD004923. © 2016 The Authors. Yeast published by John Wiley & Sons, Ltd. PMID:27668839

  1. Identification of redox-sensitive cysteines in the arabidopsis proteome using OxiTRAQ, a quantitative redox proteomics method

    KAUST Repository

    Liu, Pei; Zhang, Huoming; Wang, Hai; Xia, Yiji

    2014-01-01

    -throughput quantitative proteomic approach termed OxiTRAQ for identifying proteins whose thiols undergo reversible oxidative modifications in Arabidopsis cells subjected to oxidative stress. In this approach, a biotinylated thiol-reactive reagent is used for differential

  2. Comparative proteome and transcriptome analysis of lager brewer's yeast in the autolysis process.

    Science.gov (United States)

    Xu, Weina; Wang, Jinjing; Li, Qi

    2014-12-01

    The autolysis of brewer's yeast during beer production has a significant effect on the quality of the final product. In this work, we performed proteome and transcriptome studies on brewer's yeast to examine changes in protein and mRNA levels in the process of autolysis. Protein and RNA samples of the strain Qing2 at two different autolysis stages were obtained for further study. In all, 49 kinds of proteins were considered to be involved in the autolysis response, eight of which were up-regulated and 41 down-regulated. Seven new kinds of proteins emerged during autolysis. Results of comparative analyses showed that important changes had taken place as an adaptive response to autolysis. Functional analysis showed that carbohydrate and energy metabolism, cellular amino acid metabolic processes, cell response to various stresses (such as oxidative stress, salt stress, and osmotic stress), translation and transcription were repressed by the down-regulation of the corresponding proteins, and starvation and DNA damage responses could be induced. The comparison of data on transcriptomes with proteomes demonstrated that most autolysis-response proteins as well as new proteins showed a general correlation between mRNA and protein levels. Thus these proteins were thought to be transcriptionally regulated. These findings provide important information about how brewer's yeast acts to cope with autolysis at molecular levels, which might enhance global understanding of the autolysis process. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  3. Benchmarking sample preparation/digestion protocols reveals tube-gel being a fast and repeatable method for quantitative proteomics.

    Science.gov (United States)

    Muller, Leslie; Fornecker, Luc; Van Dorsselaer, Alain; Cianférani, Sarah; Carapito, Christine

    2016-12-01

    Sample preparation, typically by in-solution or in-gel approaches, has a strong influence on the accuracy and robustness of quantitative proteomics workflows. The major benefit of in-gel procedures is their compatibility with detergents (such as SDS) for protein solubilization. However, SDS-PAGE is a time-consuming approach. Tube-gel (TG) preparation circumvents this drawback as it involves directly trapping the sample in a polyacrylamide gel matrix without electrophoresis. We report here the first global label-free quantitative comparison between TG, stacking gel (SG), and basic liquid digestion (LD). A series of UPS1 standard mixtures (at 0.5, 1, 2.5, 5, 10, and 25 fmol) were spiked in a complex yeast lysate background. TG preparation allowed more yeast proteins to be identified than did the SG and LD approaches, with mean numbers of 1979, 1788, and 1323 proteins identified, respectively. Furthermore, the TG method proved equivalent to SG and superior to LD in terms of the repeatability of the subsequent experiments, with mean CV for yeast protein label-free quantifications of 7, 9, and 10%. Finally, known variant UPS1 proteins were successfully detected in the TG-prepared sample within a complex background with high sensitivity. All the data from this study are accessible on ProteomeXchange (PXD003841). © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Spiked proteomic standard dataset for testing label-free quantitative software and statistical methods

    Directory of Open Access Journals (Sweden)

    Claire Ramus

    2016-03-01

    Full Text Available This data article describes a controlled, spiked proteomic dataset for which the “ground truth” of variant proteins is known. It is based on the LC-MS analysis of samples composed of a fixed background of yeast lysate and different spiked amounts of the UPS1 mixture of 48 recombinant proteins. It can be used to objectively evaluate bioinformatic pipelines for label-free quantitative analysis, and their ability to detect variant proteins with good sensitivity and low false discovery rate in large-scale proteomic studies. More specifically, it can be useful for tuning software tools parameters, but also testing new algorithms for label-free quantitative analysis, or for evaluation of downstream statistical methods. The raw MS files can be downloaded from ProteomeXchange with identifier http://www.ebi.ac.uk/pride/archive/projects/PXD001819. Starting from some raw files of this dataset, we also provide here some processed data obtained through various bioinformatics tools (including MaxQuant, Skyline, MFPaQ, IRMa-hEIDI and Scaffold in different workflows, to exemplify the use of such data in the context of software benchmarking, as discussed in details in the accompanying manuscript [1]. The experimental design used here for data processing takes advantage of the different spike levels introduced in the samples composing the dataset, and processed data are merged in a single file to facilitate the evaluation and illustration of software tools results for the detection of variant proteins with different absolute expression levels and fold change values.

  5. The temporal analysis of yeast exponential phase using shotgun proteomics as a fermentation monitoring technique.

    Science.gov (United States)

    Huang, Eric L; Orsat, Valérie; Shah, Manesh B; Hettich, Robert L; VerBerkmoes, Nathan C; Lefsrud, Mark G

    2012-09-18

    System biology and bioprocess technology can be better understood using shotgun proteomics as a monitoring system during the fermentation. We demonstrated a shotgun proteomic method to monitor the temporal yeast proteome in early, middle and late exponential phases. Our study identified a total of 1389 proteins combining all 2D-LC-MS/MS runs. The temporal Saccharomyces cerevisiae proteome was enriched with proteolysis, radical detoxification, translation, one-carbon metabolism, glycolysis and TCA cycle. Heat shock proteins and proteins associated with oxidative stress response were found throughout the exponential phase. The most abundant proteins observed were translation elongation factors, ribosomal proteins, chaperones and glycolytic enzymes. The high abundance of the H-protein of the glycine decarboxylase complex (Gcv3p) indicated the availability of glycine in the environment. We observed differentially expressed proteins and the induced proteins at mid-exponential phase were involved in ribosome biogenesis, mitochondria DNA binding/replication and transcriptional activator. Induction of tryptophan synthase (Trp5p) indicated the abundance of tryptophan during the fermentation. As fermentation progressed toward late exponential phase, a decrease in cell proliferation was implied from the repression of ribosomal proteins, transcription coactivators, methionine aminopeptidase and translation-associated proteins. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. Protein functional analysis data in support of comparative proteomics of the pathogenic black yeast Exophiala dermatitidis under different temperature conditions

    Directory of Open Access Journals (Sweden)

    Donatella Tesei

    2015-12-01

    Full Text Available In the current study a comparative proteomic approach was used to investigate the response of the human pathogen black yeast Exophiala dermatitidis toward temperature treatment. Protein functional analysis – based on cellular process GO terms – was performed on the 32 temperature-responsive identified proteins. The bioinformatics analyses and data presented here provided novel insights into the cellular pathways at the base of the fungus temperature tolerance. A detailed analysis and interpretation of the data can be found in “Proteome of tolerance fine-tuning in the human pathogen black yeast Exophiala dermatitidis” by Tesei et al. (2015 [1].

  7. EBprot: Statistical analysis of labeling-based quantitative proteomics data.

    Science.gov (United States)

    Koh, Hiromi W L; Swa, Hannah L F; Fermin, Damian; Ler, Siok Ghee; Gunaratne, Jayantha; Choi, Hyungwon

    2015-08-01

    Labeling-based proteomics is a powerful method for detection of differentially expressed proteins (DEPs). The current data analysis platform typically relies on protein-level ratios, which is obtained by summarizing peptide-level ratios for each protein. In shotgun proteomics, however, some proteins are quantified with more peptides than others, and this reproducibility information is not incorporated into the differential expression (DE) analysis. Here, we propose a novel probabilistic framework EBprot that directly models the peptide-protein hierarchy and rewards the proteins with reproducible evidence of DE over multiple peptides. To evaluate its performance with known DE states, we conducted a simulation study to show that the peptide-level analysis of EBprot provides better receiver-operating characteristic and more accurate estimation of the false discovery rates than the methods based on protein-level ratios. We also demonstrate superior classification performance of peptide-level EBprot analysis in a spike-in dataset. To illustrate the wide applicability of EBprot in different experimental designs, we applied EBprot to a dataset for lung cancer subtype analysis with biological replicates and another dataset for time course phosphoproteome analysis of EGF-stimulated HeLa cells with multiplexed labeling. Through these examples, we show that the peptide-level analysis of EBprot is a robust alternative to the existing statistical methods for the DE analysis of labeling-based quantitative datasets. The software suite is freely available on the Sourceforge website http://ebprot.sourceforge.net/. All MS data have been deposited in the ProteomeXchange with identifier PXD001426 (http://proteomecentral.proteomexchange.org/dataset/PXD001426/). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Proteomic analysis of Rhodotorula mucilaginosa: dealing with the issues of a non-conventional yeast.

    Science.gov (United States)

    Addis, Maria Filippa; Tanca, Alessandro; Landolfo, Sara; Abbondio, Marcello; Cutzu, Raffaela; Biosa, Grazia; Pagnozzi, Daniela; Uzzau, Sergio; Mannazzu, Ilaria

    2016-08-01

    Red yeasts ascribed to the species Rhodotorula mucilaginosa are gaining increasing attention, due to their numerous biotechnological applications, spanning carotenoid production, liquid bioremediation, heavy metal biotransformation and antifungal and plant growth-promoting actions, but also for their role as opportunistic pathogens. Nevertheless, their characterization at the 'omic' level is still scarce. Here, we applied different proteomic workflows to R. mucilaginosa with the aim of assessing their potential in generating information on proteins and functions of biotechnological interest, with a particular focus on the carotenogenic pathway. After optimization of protein extraction, we tested several gel-based (including 2D-DIGE) and gel-free sample preparation techniques, followed by tandem mass spectrometry analysis. Contextually, we evaluated different bioinformatic strategies for protein identification and interpretation of the biological significance of the dataset. When 2D-DIGE analysis was applied, not all spots returned a unambiguous identification and no carotenogenic enzymes were identified, even upon the application of different database search strategies. Then, the application of shotgun proteomic workflows with varying levels of sensitivity provided a picture of the information depth that can be reached with different analytical resources, and resulted in a plethora of information on R. mucilaginosa metabolism. However, also in these cases no proteins related to the carotenogenic pathway were identified, thus indicating that further improvements in sequence databases and functional annotations are strictly needed for increasing the outcome of proteomic analysis of this and other non-conventional yeasts. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  9. Potential spoilage yeasts in winery environments: Characterization and proteomic analysis of Trigonopsis cantarellii.

    Science.gov (United States)

    Portugal, Cauré; Pinto, Luís; Ribeiro, Miguel; Tenorio, Carmen; Igrejas, Gilberto; Ruiz-Larrea, Fernanda

    2015-10-01

    Wine microbiota is complex and includes a wide diversity of yeast species. Few of them are able to survive under the restrictive conditions of dry red wines. In our study we detected and identified seven yeast species of the order Saccharomycetales that can be considered potential spoilers of wines due to physiological traits such as acidogenic metabolism and off-odor generation: Arthroascus schoenii, Candida ishiwadae, Meyerozyma guilliermondii, Pichia holstii, Pichia manshurica, Trigonopsis cantarellii, and Trigonopsis variabilis. Based on the prevalence of T. cantarellii isolates in the wine samples of our study, we further characterized this species, determined molecular and phenotypic features, and performed a proteomic analysis to identify differentially expressed proteins at mid-exponential growth phase in the presence of ethanol in the culture broth. This yeast species is shown to be able to grow in the presence of ethanol by expressing heat shock proteins (Hsp70, Hsp71) and a DNA damage-related protein (Rad24), and to be able to confer spoilage characteristics on wine. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Development and standardization of multiplexed antibody microarrays for use in quantitative proteomics

    Directory of Open Access Journals (Sweden)

    Sorette M

    2004-12-01

    Full Text Available Abstract Background Quantitative proteomics is an emerging field that encompasses multiplexed measurement of many known proteins in groups of experimental samples in order to identify differences between groups. Antibody arrays are a novel technology that is increasingly being used for quantitative proteomics studies due to highly multiplexed content, scalability, matrix flexibility and economy of sample consumption. Key applications of antibody arrays in quantitative proteomics studies are identification of novel diagnostic assays, biomarker discovery in trials of new drugs, and validation of qualitative proteomics discoveries. These applications require performance benchmarking, standardization and specification. Results Six dual-antibody, sandwich immunoassay arrays that measure 170 serum or plasma proteins were developed and experimental procedures refined in more than thirty quantitative proteomics studies. This report provides detailed information and specification for manufacture, qualification, assay automation, performance, assay validation and data processing for antibody arrays in large scale quantitative proteomics studies. Conclusion The present report describes development of first generation standards for antibody arrays in quantitative proteomics. Specifically, it describes the requirements of a comprehensive validation program to identify and minimize antibody cross reaction under highly multiplexed conditions; provides the rationale for the application of standardized statistical approaches to manage the data output of highly replicated assays; defines design requirements for controls to normalize sample replicate measurements; emphasizes the importance of stringent quality control testing of reagents and antibody microarrays; recommends the use of real-time monitors to evaluate sensitivity, dynamic range and platform precision; and presents survey procedures to reveal the significance of biomarker findings.

  11. Transcriptional, proteomic, and metabolic responses to lithium in galactose-grown yeast cells

    DEFF Research Database (Denmark)

    Bro, Christoffer; Regenberg, Birgitte; Lagniel, G.

    2003-01-01

    Lithium is highly toxic to yeast when grown in galactose medium mainly because phosphoglucomutase, a key enzyme of galactose metabolism, is inhibited. We studied the global protein and gene expression profiles of Saccharomyces cerevisiae grown in galactose in different time intervals after addition...... of lithium. These results were related to physiological studies where both secreted and intracellular metabolites were determined. Microarray analysis showed that 664 open reading frames were down-regulated and 725 up-regulated in response to addition of lithium. Genes involved in transcription, translation......-regulated proteins were also identified as being changed on the mRNA level. Functional clusters obtained from proteome data were coincident with transcriptional clusters. Physiological studies showed that acetate, glycerol, and glycogen accumulate in response to lithium, as reflected in expression data, whereas...

  12. Employing proteomic analysis to compare Paracoccidioides lutzii yeast and mycelium cell wall proteins.

    Science.gov (United States)

    Araújo, Danielle Silva; de Sousa Lima, Patrícia; Baeza, Lilian Cristiane; Parente, Ana Flávia Alves; Melo Bailão, Alexandre; Borges, Clayton Luiz; de Almeida Soares, Célia Maria

    2017-11-01

    Paracoccidioidomycosis is an important systemic mycosis caused by thermodimorphic fungi of the Paracoccidioides genus. During the infective process, the cell wall acts at the interface between the fungus and the host. In this way, the cell wall has a key role in growth, environment sensing and interaction, as well as morphogenesis of the fungus. Since the cell wall is absent in mammals, it may present molecules that are described as target sites for new antifungal drugs. Despite its importance, up to now few studies have been conducted employing proteomics in for the identification of cell wall proteins in Paracoccidioides spp. Here, a detailed proteomic approach, including cell wall-fractionation coupled to NanoUPLC-MS E , was used to study and compare the cell wall fractions from Paracoccidioides lutzii mycelia and yeast cells. The analyzed samples consisted of cell wall proteins extracted by hot SDS followed by extraction by mild alkali. In summary, 512 proteins constituting different cell wall fractions were identified, including 7 predicted GPI-dependent cell wall proteins that are potentially involved in cell wall metabolism. Adhesins previously described in Paracoccidioides spp. such as enolase, glyceraldehyde-3-phosphate dehydrogenase were identified. Comparing the proteins in mycelium and yeast cells, we detected some that are common to both fungal phases, such as Ecm33, and some specific proteins, as glucanase Crf1. All of those proteins were described in the metabolism of cell wall. Our study provides an important elucidation of cell wall composition of fractions in Paracoccidioides, opening a way to understand the fungus cell wall architecture. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. ABRF-PRG07: advanced quantitative proteomics study.

    Science.gov (United States)

    Falick, Arnold M; Lane, William S; Lilley, Kathryn S; MacCoss, Michael J; Phinney, Brett S; Sherman, Nicholas E; Weintraub, Susan T; Witkowska, H Ewa; Yates, Nathan A

    2011-04-01

    A major challenge for core facilities is determining quantitative protein differences across complex biological samples. Although there are numerous techniques in the literature for relative and absolute protein quantification, the majority is nonroutine and can be challenging to carry out effectively. There are few studies comparing these technologies in terms of their reproducibility, accuracy, and precision, and no studies to date deal with performance across multiple laboratories with varied levels of expertise. Here, we describe an Association of Biomolecular Resource Facilities (ABRF) Proteomics Research Group (PRG) study based on samples composed of a complex protein mixture into which 12 known proteins were added at varying but defined ratios. All of the proteins were present at the same concentration in each of three tubes that were provided. The primary goal of this study was to allow each laboratory to evaluate its capabilities and approaches with regard to: detection and identification of proteins spiked into samples that also contain complex mixtures of background proteins and determination of relative quantities of the spiked proteins. The results returned by 43 participants were compiled by the PRG, which also collected information about the strategies used to assess overall performance and as an aid to development of optimized protocols for the methodologies used. The most accurate results were generally reported by the most experienced laboratories. Among laboratories that used the same technique, values that were closer to the expected ratio were obtained by more experienced groups.

  14. Data in support of quantitative proteomics to identify potential virulence regulators in Paracoccidioides brasiliensis isolates

    Directory of Open Access Journals (Sweden)

    Alexandre Keiji Tashima

    2015-12-01

    Full Text Available Paracoccidioides genus are the etiologic agents of paracoccidioidomycosis (PCM, a systemic mycosis endemic in Latin America. Few virulence factors have been identified in these fungi. This paper describes support data from the quantitative proteomics of Paracoccidioides brasiliensis attenuated and virulent isolates [1]. The protein compositions of two isolates of the Pb18 strain showing distinct infection profiles were quantitatively assessed by stable isotopic dimethyl labeling and proteomic analysis. The mass spectrometry and the analysis dataset have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with identifier PXD000804.

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

    Science.gov (United States)

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

    2016-01-01

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

  16. Surveying alignment-free features for Ortholog detection in related yeast proteomes by using supervised big data classifiers.

    Science.gov (United States)

    Galpert, Deborah; Fernández, Alberto; Herrera, Francisco; Antunes, Agostinho; Molina-Ruiz, Reinaldo; Agüero-Chapin, Guillermin

    2018-05-03

    The development of new ortholog detection algorithms and the improvement of existing ones are of major importance in functional genomics. We have previously introduced a successful supervised pairwise ortholog classification approach implemented in a big data platform that considered several pairwise protein features and the low ortholog pair ratios found between two annotated proteomes (Galpert, D et al., BioMed Research International, 2015). The supervised models were built and tested using a Saccharomycete yeast benchmark dataset proposed by Salichos and Rokas (2011). Despite several pairwise protein features being combined in a supervised big data approach; they all, to some extent were alignment-based features and the proposed algorithms were evaluated on a unique test set. Here, we aim to evaluate the impact of alignment-free features on the performance of supervised models implemented in the Spark big data platform for pairwise ortholog detection in several related yeast proteomes. The Spark Random Forest and Decision Trees with oversampling and undersampling techniques, and built with only alignment-based similarity measures or combined with several alignment-free pairwise protein features showed the highest classification performance for ortholog detection in three yeast proteome pairs. Although such supervised approaches outperformed traditional methods, there were no significant differences between the exclusive use of alignment-based similarity measures and their combination with alignment-free features, even within the twilight zone of the studied proteomes. Just when alignment-based and alignment-free features were combined in Spark Decision Trees with imbalance management, a higher success rate (98.71%) within the twilight zone could be achieved for a yeast proteome pair that underwent a whole genome duplication. The feature selection study showed that alignment-based features were top-ranked for the best classifiers while the runners-up were

  17. Modification-specific proteomics in plant biology

    DEFF Research Database (Denmark)

    Ytterberg, A Jimmy; Jensen, Ole N

    2010-01-01

    and proteomics. In general, methods for PTM characterization are developed to study yeast and mammalian biology and later adopted to investigate plants. Our point of view is that it is advantageous to enrich for PTMs on the peptide level as part of a quantitative proteomics strategy to not only identify the PTM...

  18. UNiquant, a Program for Quantitative Proteomics Analysis Using Stable Isotope Labeling

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Xin; Tolmachev, Aleksey V.; Shen, Yulei; Liu, Miao; Huang, Lin; Zhang, Zhixin; Anderson, Gordon A.; Smith, Richard D.; Chan, Wing C.; Hinrichs, Steven; Fu, Kai; Ding, Shi-Jian

    2011-03-04

    We present UNiquant, a new software program for analyzing stable isotope labeling (SIL) based quantitative proteomics data. UNiquant surpassed the performance of two other platforms, MaxQuant and Mascot Distiller, using complex proteome mixtures having either known or unknown heavy/light ratios. UNiquant is compatible with a broad spectrum of search engines and SIL methods, providing outstanding peptide pair identification and accurate measurement of the relative peptide/protein abundance.

  19. Proteomics analysis for asymmetric inheritance of preexisting proteins between mother and daughter cells in budding yeast.

    Science.gov (United States)

    Okada, Mitsuhiro; Kusunoki, Shunta; Ishibashi, Yuko; Kito, Keiji

    2017-06-01

    In budding yeast, a mother cell can produce a finite number of daughter cells over its life. The accumulation of a variety of types of damaged components has an impact on the aging process. Asymmetrical inheritance during cell division causes these aberrant intracellular constituents to be retained in mother cells and prevents them from segregating to daughter cells. However, the understanding of asymmetrical inheritance of individual proteins that are damaged or old age, and their relevance to the aging process, has been limited. The aim of this study is to propose a proteomics strategy for asymmetrical inheritance of preexisting proteins between mother and daughter cells. During synchronous culture for one generation, newly synthesized proteins were labeled with stable isotope amino acids to discriminate preexisting proteins originally expressed in mother cells, followed by separation of mother and daughter cells using a conventional method based on biotin labeling. Isotope incorporation ratios for individual proteins were quantified using mass spectrometry. We successfully identified 21 proteins whose preexisting versions were asymmetrically inherited in mother cells, including plasma membrane transporter involved in the aging process and organelle-anchoring proteins related to the stress response to misfolded proteins. Thus, our approach would be useful for making catalog of asymmetrically inherited proteins. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

  20. Dietary Yeast Cell Wall Extract Alters the Proteome of the Skin Mucous Barrier in Atlantic Salmon (Salmo salar: Increased Abundance and Expression of a Calreticulin-Like Protein.

    Directory of Open Access Journals (Sweden)

    Giulia Micallef

    Full Text Available In order to improve fish health and reduce use of chemotherapeutants in aquaculture production, the immunomodulatory effect of various nutritional ingredients has been explored. In salmon, there is evidence that functional feeds can reduce the abundance of sea lice. This study aimed to determine if there were consistent changes in the skin mucus proteome that could serve as a biomarker for dietary yeast cell wall extract. The effect of dietary yeast cell wall extract on the skin mucus proteome of Atlantic salmon was examined using two-dimensional gel electrophoresis. Forty-nine spots showed a statistically significant change in their normalised volumes between the control and yeast cell wall diets. Thirteen spots were successfully identified by peptide fragment fingerprinting and LC-MS/MS and these belonged to a variety of functions and pathways. To assess the validity of the results from the proteome approach, the gene expression of a selection of these proteins was studied in skin mRNA from two different independent feeding trials using yeast cell wall extracts. A calreticulin-like protein increased in abundance at both the protein and transcript level in response to dietary yeast cell wall extract. The calreticulin-like protein was identified as a possible biomarker for yeast-derived functional feeds since it showed the most consistent change in expression in both the mucus proteome and skin transcriptome. The discovery of such a biomarker is expected to quicken the pace of research in the application of yeast cell wall extracts.

  1. Quantitative targeted proteomics for understanding the blood-brain barrier: towards pharmacoproteomics.

    Science.gov (United States)

    Ohtsuki, Sumio; Hirayama, Mio; Ito, Shingo; Uchida, Yasuo; Tachikawa, Masanori; Terasaki, Tetsuya

    2014-06-01

    The blood-brain barrier (BBB) is formed by brain capillary endothelial cells linked together via complex tight junctions, and serves to prevent entry of drugs into the brain. Multiple transporters are expressed at the BBB, where they control exchange of materials between the circulating blood and brain interstitial fluid, thereby supporting and protecting the CNS. An understanding of the BBB is necessary for efficient development of CNS-acting drugs and to identify potential drug targets for treatment of CNS diseases. Quantitative targeted proteomics can provide detailed information on protein expression levels at the BBB. The present review highlights the latest applications of quantitative targeted proteomics in BBB research, specifically to evaluate species and in vivo-in vitro differences, and to reconstruct in vivo transport activity. Such a BBB quantitative proteomics approach can be considered as pharmacoproteomics.

  2. Combinatorial hexapeptide ligand libraries (ProteoMiner): an innovative fractionation tool for differential quantitative clinical proteomics.

    Science.gov (United States)

    Hartwig, Sonja; Czibere, Akos; Kotzka, Jorg; Passlack, Waltraud; Haas, Rainer; Eckel, Jürgen; Lehr, Stefan

    2009-07-01

    Blood serum samples are the major source for clinical proteomics approaches, which aim to identify diagnostically relevant or treatment-response related proteins. But, the presence of very high-abundance proteins and the enormous dynamic range of protein distribution hinders whole serum analysis. An innovative tool to overcome these limitations, utilizes combinatorial hexapeptide ligand libraries (ProteoMiner). Here, we demonstrate that ProteoMiner can be used for comparative and quantitative analysis of complex proteomes. We spiked serum samples with increasing amounts (3 microg to 300 microg) of whole E. coli lysate, processed it with ProteoMiner and performed quantitative analyses of 2D-gels. We found, that the concentration of the spiked bacteria proteome, reflected by the maintained proportional spot intensities, was not altered by ProteoMiner treatment. Therefore, we conclude that the ProteoMiner technology can be used for quantitative analysis of low abundant proteins in complex biological samples.

  3. Quantitative Clinical Chemistry Proteomics (qCCP) using mass spectrometry: general characteristics and application.

    Science.gov (United States)

    Lehmann, Sylvain; Hoofnagle, Andrew; Hochstrasser, Denis; Brede, Cato; Glueckmann, Matthias; Cocho, José A; Ceglarek, Uta; Lenz, Christof; Vialaret, Jérôme; Scherl, Alexander; Hirtz, Christophe

    2013-05-01

    Proteomics studies typically aim to exhaustively detect peptides/proteins in a given biological sample. Over the past decade, the number of publications using proteomics methodologies has exploded. This was made possible due to the availability of high-quality genomic data and many technological advances in the fields of microfluidics and mass spectrometry. Proteomics in biomedical research was initially used in 'functional' studies for the identification of proteins involved in pathophysiological processes, complexes and networks. Improved sensitivity of instrumentation facilitated the analysis of even more complex sample types, including human biological fluids. It is at that point the field of clinical proteomics was born, and its fundamental aim was the discovery and (ideally) validation of biomarkers for the diagnosis, prognosis, or therapeutic monitoring of disease. Eventually, it was recognized that the technologies used in clinical proteomics studies [particularly liquid chromatography-tandem mass spectrometry (LC-MS/MS)] could represent an alternative to classical immunochemical assays. Prior to deploying MS in the measurement of peptides/proteins in the clinical laboratory, it seems likely that traditional proteomics workflows and data management systems will need to adapt to the clinical environment and meet in vitro diagnostic (IVD) regulatory constraints. This defines a new field, as reviewed in this article, that we have termed quantitative Clinical Chemistry Proteomics (qCCP).

  4. 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

    , and H4 in a site-specific and dose-dependent manner. This unbiased analysis revealed that a relative increase in acetylated peptide from the histone variants H2A, H2B, and H4 was accompanied by a relative decrease of dimethylated Lys(57) from histone H2B. The dose-response results obtained...... by quantitative proteomics of histones from HDACi-treated cells were consistent with Western blot analysis of histone acetylation, cytotoxicity, and dose-dependent expression profiles of p21 and cyclin A2. This demonstrates that mass spectrometry-based quantitative proteomic analysis of post-translational...

  5. Quantitative proteome changes in Arabidopsis thaliana suspension-cultured cells in response to plant natriuretic peptides

    KAUST Repository

    Turek, Ilona; Wheeler, Janet I.; Gehring, Christoph A; Irving, Helen R.; Marondedze, Claudius

    2015-01-01

    Proteome changes in the Arabidopsis thaliana suspension cells in response to the A. thaliana plant natriuretic peptide (PNP), AtPNP-A (At2g18660) were assessed using quantitative proteomics employing tandem mass tag (TMT) labeling and tandem mass spectrometry (LC–MS/MS). In this study, we characterized temporal responses of suspension-cultured cells to 1 nM and 10 pM AtPNP-A at 0, 10 and 30 min post-treatment. Both concentrations we found to yield a distinct differential proteome signature. The data shown in this article are associated with the article “Plant natriuretic peptides induce a specific set of proteins diagnostic for an adaptive response to abiotic stress” by Turek et al. (Front. Plant Sci. 5 (2014) 661) and have been deposited to the ProteomeXchange with identifier PXD001386.

  6. Quantitative proteome changes in Arabidopsis thaliana suspension-cultured cells in response to plant natriuretic peptides

    KAUST Repository

    Turek, Ilona

    2015-06-30

    Proteome changes in the Arabidopsis thaliana suspension cells in response to the A. thaliana plant natriuretic peptide (PNP), AtPNP-A (At2g18660) were assessed using quantitative proteomics employing tandem mass tag (TMT) labeling and tandem mass spectrometry (LC–MS/MS). In this study, we characterized temporal responses of suspension-cultured cells to 1 nM and 10 pM AtPNP-A at 0, 10 and 30 min post-treatment. Both concentrations we found to yield a distinct differential proteome signature. The data shown in this article are associated with the article “Plant natriuretic peptides induce a specific set of proteins diagnostic for an adaptive response to abiotic stress” by Turek et al. (Front. Plant Sci. 5 (2014) 661) and have been deposited to the ProteomeXchange with identifier PXD001386.

  7. A proteomic and metabolomic approach for understanding the role of the flor yeast mitochondria in the velum formation.

    Science.gov (United States)

    Moreno-García, Jaime; García-Martínez, Teresa; Moreno, Juan; Millán, M Carmen; Mauricio, Juan Carlos

    2014-02-17

    Saccharomyces cerevisiae "flor" yeast shows a strong tolerance to high ethanol concentrations and develops a velum (biofilm) on the wine surface after the alcoholic fermentation of grape must. This velum remains along several years during the so called "biological aging" process in the elaboration of some special wines carried out in specific regions around the world and it contributes to the typical organoleptic characteristics of these wines. In order to grow in this condition, flor yeast has to elaborate a response where the mitochondrial function is essential. The objective of this study is to elucidate the role of the mitochondria in the response of a flor yeast, S. cerevisiae G1, growing in a controlled velum formation condition. For this purpose, proteome and metabolome were characterized by comparing data with those from an initial fermentative condition used as reference. The obtained proteomic profiles show more mitochondrial proteins related with the ethanol resistance (13), cell respiration (18), mitochondrial genome maintenance (13), and apoptosis (2) detected under the velum formation condition. Also, the finger-printing obtained by means of the exo-metabolites directly related with the quality of fermented beverages and quantified in the velum condition shows important differences from those obtained in the reference condition. Copyright © 2013 Elsevier B.V. All rights reserved.

  8. 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.

  9. Hydroponic isotope labeling of entire plants and high-performance mass spectrometry for quantitative plant proteomics.

    Science.gov (United States)

    Bindschedler, Laurence V; Mills, Davinia J S; Cramer, Rainer

    2012-01-01

    Hydroponic isotope labeling of entire plants (HILEP) combines hydroponic plant cultivation and metabolic labeling with stable isotopes using (15)N-containing inorganic salts to label whole and mature plants. Employing (15)N salts as the sole nitrogen source for HILEP leads to the production of healthy-looking plants which contain (15)N proteins labeled to nearly 100%. Therefore, HILEP is suitable for quantitative plant proteomic analysis, where plants are grown in either (14)N- or (15)N-hydroponic media and pooled when the biological samples are collected for relative proteome quantitation. The pooled (14)N-/(15)N-protein extracts can be fractionated in any suitable way and digested with a protease for shotgun proteomics, using typically reverse phase liquid chromatography nanoelectrospray ionization tandem mass spectrometry (RPLC-nESI-MS/MS). Best results were obtained with a hybrid ion trap/FT-MS mass spectrometer, combining high mass accuracy and sensitivity for the MS data acquisition with speed and high-throughput MS/MS data acquisition, increasing the number of proteins identified and quantified and improving protein quantitation. Peak processing and picking from raw MS data files, protein identification, and quantitation were performed in a highly automated way using integrated MS data analysis software with minimum manual intervention, thus easing the analytical workflow. In this methodology paper, we describe how to grow Arabidopsis plants hydroponically for isotope labeling using (15)N salts and how to quantitate the resulting proteomes using a convenient workflow that does not require extensive bioinformatics skills.

  10. Quantitative monitoring of yeast fermentation using Raman spectroscopy

    DEFF Research Database (Denmark)

    Iversen, Jens A.; Berg, Rolf W.; Ahring, Birgitte K.

    2014-01-01

    of a Saccharomyces cerevisiae fermentation process using a Raman spectroscopy instrument equipped with a robust sapphire ball probe.A method was developed to correct the Raman signal for the attenuation caused by light scattering cell particulate, hence enabling quantification of reaction components and possibly...... measurement of yeast cell concentrations. Extinction of Raman intensities to more than 50 % during fermentation was normalized with approximated extinction expressions using Raman signal of water around 1,627 cm−1 as internal standard to correct for the effect of scattering. Complicated standard multi...... was followed by linear regression. In situ quantification measurements of the fermentation resulted in root mean square errors of prediction (RMSEP) of 2.357, 1.611, and 0.633 g/L for glucose, ethanol, and yeast concentrations, respectively....

  11. Network analysis of quantitative proteomics on asthmatic bronchi: effects of inhaled glucocorticoid treatment

    Directory of Open Access Journals (Sweden)

    Sihlbom Carina

    2011-09-01

    Full Text Available Abstract Background Proteomic studies of respiratory disorders have the potential to identify protein biomarkers for diagnosis and disease monitoring. Utilisation of sensitive quantitative proteomic methods creates opportunities to determine individual patient proteomes. The aim of the current study was to determine if quantitative proteomics of bronchial biopsies from asthmatics can distinguish relevant biological functions and whether inhaled glucocorticoid treatment affects these functions. Methods Endobronchial biopsies were taken from untreated asthmatic patients (n = 12 and healthy controls (n = 3. Asthmatic patients were randomised to double blind treatment with either placebo or budesonide (800 μg daily for 3 months and new biopsies were obtained. Proteins extracted from the biopsies were digested and analysed using isobaric tags for relative and absolute quantitation combined with a nanoLC-LTQ Orbitrap mass spectrometer. Spectra obtained were used to identify and quantify proteins. Pathways analysis was performed using Ingenuity Pathway Analysis to identify significant biological pathways in asthma and determine how the expression of these pathways was changed by treatment. Results More than 1800 proteins were identified and quantified in the bronchial biopsies of subjects. The pathway analysis revealed acute phase response signalling, cell-to-cell signalling and tissue development associations with proteins expressed in asthmatics compared to controls. The functions and pathways associated with placebo and budesonide treatment showed distinct differences, including the decreased association with acute phase proteins as a result of budesonide treatment compared to placebo. Conclusions Proteomic analysis of bronchial biopsy material can be used to identify and quantify proteins using highly sensitive technologies, without the need for pooling of samples from several patients. Distinct pathophysiological features of asthma can be

  12. Identification of redox-sensitive cysteines in the arabidopsis proteome using OxiTRAQ, a quantitative redox proteomics method

    KAUST Repository

    Liu, Pei

    2014-01-28

    Cellular redox status plays a key role in mediating various physiological and developmental processes often through modulating activities of redox-sensitive proteins. Various stresses trigger over-production of reactive oxygen/nitrogen species which lead to oxidative modifications of redox-sensitive proteins. Identification and characterization of redox-sensitive proteins are important steps toward understanding molecular mechanisms of stress responses. Here, we report a high-throughput quantitative proteomic approach termed OxiTRAQ for identifying proteins whose thiols undergo reversible oxidative modifications in Arabidopsis cells subjected to oxidative stress. In this approach, a biotinylated thiol-reactive reagent is used for differential labeling of reduced and oxidized thiols. The biotin-tagged peptides are affinity purified, labeled with iTRAQ reagents, and analyzed using a paralleled HCD-CID fragmentation mode in an LTQ-Orbitrap. With this approach, we identified 195 cysteine-containing peptides from 179 proteins whose thiols underwent oxidative modifications in Arabidopsis cells following the treatment with hydrogen peroxide. A majority of those redox-sensitive proteins, including several transcription factors, were not identified by previous redox proteomics studies. This approach allows identification of the specific redox-regulated cysteine residues, and offers an effective tool for elucidation of redox proteomes. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Annotation of loci from genome-wide association studies using tissue-specific quantitative interaction proteomics

    NARCIS (Netherlands)

    Lundby, Alicia; Rossin, Elizabeth J.; Steffensen, Annette B.; Acha, Moshe Ray; Newton-Cheh, Christopher; Pfeufer, Arne; Lyneh, Stacey N.; Olesen, Soren-Peter; Brunak, Soren; Ellinor, Patrick T.; Jukema, J. Wouter; Trompet, Stella; Ford, Ian; Macfarlane, Peter W.; Krijthe, Bouwe P.; Hofman, Albert; Uitterlinden, Andre G.; Stricker, Bruno H.; Nathoe, Hendrik M.; Spiering, Wilko; Daly, Mark J.; Asselbergs, Ikea W.; van der Harst, Pim; Milan, David J.; de Bakker, Paul I. W.; Lage, Kasper; Olsen, Jesper V.

    Genome-wide association studies (GWAS) have identified thousands of loci associated with complex traits, but it is challenging to pinpoint causal genes in these loci and to exploit subtle association signals. We used tissue-specific quantitative interaction proteomics to map a network of five genes

  14. Analytical performance of reciprocal isotope labeling of proteome digests for quantitative proteomics and its application for comparative studies of aerobic and anaerobic Escherichia coli proteomes

    International Nuclear Information System (INIS)

    Lo, Andy; Weiner, Joel H.; Li, Liang

    2013-01-01

    Graphical abstract: -- Highlights: •Investigating a strategy of reciprocal isotope labeling of comparative samples. •Filtering out incorrect peptide identification or quantification values. •Analyzing the proteome changes of E. coli cells grown aerobically or anaerobically. •Presenting guidelines for reciprocal labeling experimental design. -- Abstract: Due to limited sample amounts, instrument time considerations, and reagent costs, only a small number of replicate experiments are typically performed for quantitative proteome analyses. Generation of reproducible data that can be readily assessed for consistency within a small number of datasets is critical for accurate quantification. We report our investigation of a strategy using reciprocal isotope labeling of two comparative samples as a tool for determining proteome changes. Reciprocal labeling was evaluated to determine the internal consistency of quantified proteome changes from Escherichia coli grown under aerobic and anaerobic conditions. Qualitatively, the peptide overlap between replicate analyses of the same sample and reverse labeled samples were found to be within 8%. Quantitatively, reciprocal analyses showed only a slight increase in average overall inconsistency when compared with replicate analyses (1.29 vs. 1.24-fold difference). Most importantly, reverse labeling was successfully used to identify spurious values resulting from incorrect peptide identifications and poor peak fitting. After removal of 5% of the peptide data with low reproducibility, a total of 275 differentially expressed proteins (>1.50-fold difference) were consistently identified and were then subjected to bioinformatics analysis. General considerations and guidelines for reciprocal labeling experimental design and biological significance of obtained results are discussed

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

    Science.gov (United States)

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

    2012-03-01

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

  16. Will Quantitative Proteomics Redefine Some of the Key Concepts in Skeletal Muscle Physiology?

    Science.gov (United States)

    Gizak, Agnieszka; Rakus, Dariusz

    2016-01-11

    Molecular and cellular biology methodology is traditionally based on the reasoning called "the mechanistic explanation". In practice, this means identifying and selecting correlations between biological processes which result from our manipulation of a biological system. In theory, a successful application of this approach requires precise knowledge about all parameters of a studied system. However, in practice, due to the systems' complexity, this requirement is rarely, if ever, accomplished. Typically, it is limited to a quantitative or semi-quantitative measurements of selected parameters (e.g., concentrations of some metabolites), and a qualitative or semi-quantitative description of expression/post-translational modifications changes within selected proteins. A quantitative proteomics approach gives a possibility of quantitative characterization of the entire proteome of a biological system, in the context of the titer of proteins as well as their post-translational modifications. This enables not only more accurate testing of novel hypotheses but also provides tools that can be used to verify some of the most fundamental dogmas of modern biology. In this short review, we discuss some of the consequences of using quantitative proteomics to verify several key concepts in skeletal muscle physiology.

  17. Will Quantitative Proteomics Redefine Some of the Key Concepts in Skeletal Muscle Physiology?

    Directory of Open Access Journals (Sweden)

    Agnieszka Gizak

    2016-01-01

    Full Text Available Molecular and cellular biology methodology is traditionally based on the reasoning called “the mechanistic explanation”. In practice, this means identifying and selecting correlations between biological processes which result from our manipulation of a biological system. In theory, a successful application of this approach requires precise knowledge about all parameters of a studied system. However, in practice, due to the systems’ complexity, this requirement is rarely, if ever, accomplished. Typically, it is limited to a quantitative or semi-quantitative measurements of selected parameters (e.g., concentrations of some metabolites, and a qualitative or semi-quantitative description of expression/post-translational modifications changes within selected proteins. A quantitative proteomics approach gives a possibility of quantitative characterization of the entire proteome of a biological system, in the context of the titer of proteins as well as their post-translational modifications. This enables not only more accurate testing of novel hypotheses but also provides tools that can be used to verify some of the most fundamental dogmas of modern biology. In this short review, we discuss some of the consequences of using quantitative proteomics to verify several key concepts in skeletal muscle physiology.

  18. Tissue-based quantitative proteome analysis of human hepatocellular carcinoma using tandem mass tags.

    Science.gov (United States)

    Megger, Dominik Andre; Rosowski, Kristin; Ahrens, Maike; Bracht, Thilo; Eisenacher, Martin; Schlaak, Jörg F; Weber, Frank; Hoffmann, Andreas-Claudius; Meyer, Helmut E; Baba, Hideo A; Sitek, Barbara

    2017-03-01

    Human hepatocellular carcinoma (HCC) is a severe malignant disease, and accurate and reliable diagnostic markers are still needed. This study was aimed for the discovery of novel marker candidates by quantitative proteomics. Proteomic differences between HCC and nontumorous liver tissue were studied by mass spectrometry. Among several significantly upregulated proteins, translocator protein 18 (TSPO) and Ras-related protein Rab-1A (RAB1A) were selected for verification by immunohistochemistry in an independent cohort. For RAB1A, a high accuracy for the discrimination of HCC and nontumorous liver tissue was observed. RAB1A was verified to be a potent biomarker candidate for HCC.

  19. A Quantitative Proteomics Approach to Clinical Research with Non-Traditional Samples

    Directory of Open Access Journals (Sweden)

    Rígel Licier

    2016-10-01

    Full Text Available The proper handling of samples to be analyzed by mass spectrometry (MS can guarantee excellent results and a greater depth of analysis when working in quantitative proteomics. This is critical when trying to assess non-traditional sources such as ear wax, saliva, vitreous humor, aqueous humor, tears, nipple aspirate fluid, breast milk/colostrum, cervical-vaginal fluid, nasal secretions, bronco-alveolar lavage fluid, and stools. We intend to provide the investigator with relevant aspects of quantitative proteomics and to recognize the most recent clinical research work conducted with atypical samples and analyzed by quantitative proteomics. Having as reference the most recent and different approaches used with non-traditional sources allows us to compare new strategies in the development of novel experimental models. On the other hand, these references help us to contribute significantly to the understanding of the proportions of proteins in different proteomes of clinical interest and may lead to potential advances in the emerging field of precision medicine.

  20. A Quantitative Proteomics Approach to Clinical Research with Non-Traditional Samples.

    Science.gov (United States)

    Licier, Rígel; Miranda, Eric; Serrano, Horacio

    2016-10-17

    The proper handling of samples to be analyzed by mass spectrometry (MS) can guarantee excellent results and a greater depth of analysis when working in quantitative proteomics. This is critical when trying to assess non-traditional sources such as ear wax, saliva, vitreous humor, aqueous humor, tears, nipple aspirate fluid, breast milk/colostrum, cervical-vaginal fluid, nasal secretions, bronco-alveolar lavage fluid, and stools. We intend to provide the investigator with relevant aspects of quantitative proteomics and to recognize the most recent clinical research work conducted with atypical samples and analyzed by quantitative proteomics. Having as reference the most recent and different approaches used with non-traditional sources allows us to compare new strategies in the development of novel experimental models. On the other hand, these references help us to contribute significantly to the understanding of the proportions of proteins in different proteomes of clinical interest and may lead to potential advances in the emerging field of precision medicine.

  1. An Overview of Advanced SILAC-Labeling Strategies for Quantitative Proteomics.

    Science.gov (United States)

    Terzi, F; Cambridge, S

    2017-01-01

    Comparative, quantitative mass spectrometry of proteins provides great insight to protein abundance and function, but some molecular characteristics related to protein dynamics are not so easily obtained. Because the metabolic incorporation of stable amino acid isotopes allows the extraction of distinct temporal and spatial aspects of protein dynamics, the SILAC methodology is uniquely suited to be adapted for advanced labeling strategies. New SILAC strategies have emerged that allow deeper foraging into the complexity of cellular proteomes. Here, we review a few advanced SILAC-labeling strategies that have been published during last the years. Among them, different subsaturating-labeling as well as dual-labeling schemes are most prominent for a range of analyses including those of neuronal proteomes, secretion, or cell-cell-induced stimulations. These recent developments suggest that much more information can be gained from proteomic analyses if the labeling strategies are specifically tailored toward the experimental design. © 2017 Elsevier Inc. All rights reserved.

  2. Strigolactone-Regulated Proteins Revealed by iTRAQ-Based Quantitative Proteomics in Arabidopsis

    Energy Technology Data Exchange (ETDEWEB)

    Li, Zhou [ORNL; Czarnecki, Olaf [ORNL; Chourey, Karuna [ORNL; Yang, Jun [ORNL; Tuskan, Gerald A [ORNL; Hurst, Gregory {Greg} B [ORNL; Pan, Chongle [ORNL; Chen, Jay [ORNL

    2014-01-01

    Strigolactones (SLs) are a new class of plant hormones. In addition to acting as a key inhibitor of shoot branching, SLs stimulate seed germination of root parasitic plants and promote hyphal branching and root colonization of symbiotic arbuscular mycorrhizal fungi. They also regulate many other aspects of plant growth and development. At the transcription level, SL-regulated genes have been reported. However, nothing is known about the proteome regulated by this new class of plant hormones. Here, a quantitative proteomics approach using an isobaric chemical labeling reagent, iTRAQ, to identify the proteome regulated by SLs in Arabidopsis seedlings is presented. It was found SLs regulate the expression of about three dozens of proteins that have not been previously assigned to SL pathways. These findings provide a new tool to investigate the molecular mechanism of action of SLs.

  3. 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].

  4. 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...... 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...

  5. Studies of the expression of human poly(ADP-ribose) polymerase-1 in Saccharomyces cerevisiae and identification of PARP-1 substrates by yeast proteome microarray screening.

    Science.gov (United States)

    Tao, Zhihua; Gao, Peng; Liu, Hung-Wen

    2009-12-15

    Poly(ADP-ribosyl)ation of various nuclear proteins catalyzed by a family of NAD(+)-dependent enzymes, poly(ADP-ribose) polymerases (PARPs), is an important posttranslational modification reaction. PARP activity has been demonstrated in all types of eukaryotic cells with the exception of yeast, in which the expression of human PARP-1 was shown to lead to retarded cell growth. We investigated the yeast growth inhibition caused by human PARP-1 expression in Saccharomyces cerevisiae. Flow cytometry analysis reveals that PARP-1-expressing yeast cells accumulate in the G(2)/M stage of the cell cycle. Confocal microscopy analysis shows that human PARP-1 is distributed throughout the nucleus of yeast cells but is enriched in the nucleolus. Utilizing yeast proteome microarray screening, we identified 33 putative PARP-1 substrates, six of which are known to be involved in ribosome biogenesis. The poly(ADP-ribosyl)ation of three of these yeast proteins, together with two human homologues, was confirmed by an in vitro PARP-1 assay. Finally, a polysome profile analysis using sucrose gradient ultracentrifugation demonstrated that the ribosome levels in yeast cells expressing PARP-1 are lower than those in control yeast cells. Overall, our data suggest that human PARP-1 may affect ribosome biogenesis by modifying certain nucleolar proteins in yeast. The artificial PARP-1 pathway in yeast may be used as a simple platform to identify substrates and verify function of this important enzyme.

  6. Target identification of natural and traditional medicines with quantitative chemical proteomics approaches.

    Science.gov (United States)

    Wang, Jigang; Gao, Liqian; Lee, Yew Mun; Kalesh, Karunakaran A; Ong, Yong Siang; Lim, Jaehong; Jee, Joo-Eun; Sun, Hongyan; Lee, Su Seong; Hua, Zi-Chun; Lin, Qingsong

    2016-06-01

    Natural and traditional medicines, being a great source of drugs and drug leads, have regained wide interests due to the limited success of high-throughput screening of compound libraries in the past few decades and the recent technology advancement. Many drugs/bioactive compounds exert their functions through interaction with their protein targets, with more and more drugs showing their ability to target multiple proteins, thus target identification has an important role in drug discovery and biomedical research fields. Identifying drug targets not only furthers the understanding of the mechanism of action (MOA) of a drug but also reveals its potential therapeutic applications and adverse side effects. Chemical proteomics makes use of affinity chromatography approaches coupled with mass spectrometry to systematically identify small molecule-protein interactions. Although traditional affinity-based chemical proteomics approaches have made great progress in the identification of cellular targets and elucidation of MOAs of many bioactive molecules, nonspecific binding remains a major issue which may reduce the accuracy of target identification and may hamper the drug development process. Recently, quantitative proteomics approaches, namely, metabolic labeling, chemical labeling, or label-free approaches, have been implemented in target identification to overcome such limitations. In this review, we will summarize and discuss the recent advances in the application of various quantitative chemical proteomics approaches for the identification of targets of natural and traditional medicines. Copyright © 2016. Published by Elsevier Inc.

  7. 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.

  8. Performance of isobaric and isotopic labeling in quantitative plant proteomics

    DEFF Research Database (Denmark)

    Nogueira, Fábio C S; Palmisano, Giuseppe; Schwämmle, Veit

    2012-01-01

    , and quantitation. In the present work, we have used LC-MS to compare an isotopic (ICPL) and isobaric (iTRAQ) chemical labeling technique to quantify proteins in the endosperm of Ricinus communis seeds at three developmental stages (IV, VI, and X). Endosperm proteins of each stage were trypsin-digested in...

  9. Study of monocyte membrane proteome perturbation during lipopolysaccharide-induced tolerance using iTRAQ-based quantitative proteomic approach

    KAUST Repository

    Zhang, Huoming; Zhao, Changqing; Li, Xin; Zhu, Yi; Gan, Chee Sian; Wang, Yong; Ravasi, Timothy; Qian, Pei-Yuan; Wong, Siew Cheng; Sze, Siu Kwan

    2010-01-01

    Human monocytes' exposure to low-level lipopolysaccharide (LPS) induces temporary monocytic insensitivity to subsequent LPS challenge. The underlying mechanism of this phenomenon could have important clinical utilities in preventing and/or treating severe infections. In this study, we used an iTRAQ-based quantitative proteomic approach to comprehensively characterize the membrane proteomes of monocytes before and after LPS exposure. We identified a total of 1651 proteins, of which 53.6% were membrane proteins. Ninety-four percent of the proteins were quantified and 255 proteins were shown to be tightly regulated by LPS. Subcellular location analysis revealed organelle-specific response to LPS exposure: more than 90% of identified mitochondrial membrane proteins were significant downregulated, whereas the majority of proteins from other organelles such as ER, Golgi and ribosome were upregulated. Moreover, we found that the expression of most receptors potentially involved in LPS signal pathway (CD14, toll-like receptor 4, CD11/CD18 complex) were substantially decreased, while the expression of molecules involved in LPS neutralization were enhanced after LPS challenge. Together, these findings could be of significance in understanding the mechanism of LPS tolerance and provide values for designing new approaches for regulating monocytic responses in sepsis patients.

  10. Study of monocyte membrane proteome perturbation during lipopolysaccharide-induced tolerance using iTRAQ-based quantitative proteomic approach

    KAUST Repository

    Zhang, Huoming

    2010-07-02

    Human monocytes\\' exposure to low-level lipopolysaccharide (LPS) induces temporary monocytic insensitivity to subsequent LPS challenge. The underlying mechanism of this phenomenon could have important clinical utilities in preventing and/or treating severe infections. In this study, we used an iTRAQ-based quantitative proteomic approach to comprehensively characterize the membrane proteomes of monocytes before and after LPS exposure. We identified a total of 1651 proteins, of which 53.6% were membrane proteins. Ninety-four percent of the proteins were quantified and 255 proteins were shown to be tightly regulated by LPS. Subcellular location analysis revealed organelle-specific response to LPS exposure: more than 90% of identified mitochondrial membrane proteins were significant downregulated, whereas the majority of proteins from other organelles such as ER, Golgi and ribosome were upregulated. Moreover, we found that the expression of most receptors potentially involved in LPS signal pathway (CD14, toll-like receptor 4, CD11/CD18 complex) were substantially decreased, while the expression of molecules involved in LPS neutralization were enhanced after LPS challenge. Together, these findings could be of significance in understanding the mechanism of LPS tolerance and provide values for designing new approaches for regulating monocytic responses in sepsis patients.

  11. PIQMIe: a web server for semi-quantitative proteomics data management and analysis.

    Science.gov (United States)

    Kuzniar, Arnold; Kanaar, Roland

    2014-07-01

    We 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 peptide and (non-redundant) protein identifications and quantitations from multiple experiments with additional biological information on the protein entries, and makes the linked data available in the form of a light-weight relational database, which enables dedicated data analyses (e.g. in R) and user-driven queries. Using the web interface, users are presented with a concise summary of their proteomics experiments in numerical and graphical forms, as well as with a searchable protein grid and interactive visualization tools to aid in the rapid assessment of the experiments and in the identification of proteins of interest. The web server not only provides data access through a web interface but also supports programmatic access through RESTful web service. The web server is available at http://piqmie.semiqprot-emc.cloudlet.sara.nl or http://www.bioinformatics.nl/piqmie. This website is free and open to all users and there is no login requirement. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  12. Liquid chromatography-mass spectrometry-based quantitative proteomics.

    Science.gov (United States)

    Linscheid, Michael W; Ahrends, Robert; Pieper, Stefan; Kühn, Andreas

    2009-01-01

    During the last decades, molecular sciences revolutionized biomedical research and gave rise to the biotechnology industry. During the next decades, the application of the quantitative sciences--informatics, physics, chemistry, and engineering--to biomedical research brings about the next revolution that will improve human healthcare and certainly create new technologies, since there is no doubt that small changes can have great effects. It is not a question of "yes" or "no," but of "how much," to make best use of the medical options we will have. In this context, the development of accurate analytical methods must be considered a cornerstone, since the understanding of biological processes will be impossible without information about the minute changes induced in cells by interactions of cell constituents with all sorts of endogenous and exogenous influences and disturbances. The first quantitative techniques, which were developed, allowed monitoring relative changes only, but they clearly showed the significance of the information obtained. The recent advent of techniques claiming to quantify proteins and peptides not only relative to each other, but also in an absolute fashion, promised another quantum leap, since knowing the absolute amount will allow comparing even unrelated species and the definition of parameters will permit to model biological systems much more accurate than before. To bring these promises to life, several approaches are under development at this point in time and this review is focused on those developments.

  13. Quantitative proteomics reveals the kinetics of trypsin-catalyzed protein digestion.

    Science.gov (United States)

    Pan, Yanbo; Cheng, Kai; Mao, Jiawei; Liu, Fangjie; Liu, Jing; Ye, Mingliang; Zou, Hanfa

    2014-10-01

    Trypsin is the popular protease to digest proteins into peptides in shotgun proteomics, but few studies have attempted to systematically investigate the kinetics of trypsin-catalyzed protein digestion in proteome samples. In this study, we applied quantitative proteomics via triplex stable isotope dimethyl labeling to investigate the kinetics of trypsin-catalyzed cleavage. It was found that trypsin cleaves the C-terminal to lysine (K) and arginine (R) residues with higher rates for R. And the cleavage sites surrounded by neutral residues could be quickly cut, while those with neighboring charged residues (D/E/K/R) or proline residue (P) could be slowly cut. In a proteome sample, a huge number of proteins with different physical chemical properties coexists. If any type of protein could be preferably digested, then limited digestion could be applied to reduce the sample complexity. However, we found that protein abundance and other physicochemical properties, such as molecular weight (Mw), grand average of hydropathicity (GRAVY), aliphatic index, and isoelectric point (pI) have no notable correlation with digestion priority of proteins.

  14. Identification of cypermethrin induced protein changes in green algae by iTRAQ quantitative proteomics.

    Science.gov (United States)

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

    2016-04-29

    Cypermethrin (CYP) is one of the most widely used pesticides in large scale for agricultural and domestic purpose and the residue often seriously affects aquatic system. Environmental pollutant-induced protein changes in organisms could be detected by proteomics, leading to discovery of potential biomarkers and understanding of mode of action. While proteomics investigations of CYP stress in some animal models have been well studied, few reports about the effects of exposure to CYP on algae proteome were published. To determine CYP effect in algae, the impact of various dosages (0.001μg/L, 0.01μg/L and 1μg/L) of CYP on green algae Chlorella vulgaris for 24h and 96h was investigated by using iTRAQ quantitative proteomics technique. A total of 162 and 198 proteins were significantly altered after CYP exposure for 24h and 96h, respectively. Overview of iTRAQ results indicated that the influence of CYP on algae protein might be dosage-dependent. Functional analysis of differentially expressed proteins showed that CYP could induce protein alterations related to photosynthesis, stress responses and carbohydrate metabolism. This study provides a comprehensive view of complex mode of action of algae under CYP stress and highlights several potential biomarkers for further investigation of pesticide-exposed plant and algae. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Analysis of ribosomal RNA stability in dead cells of wine yeast by quantitative PCR.

    Science.gov (United States)

    Sunyer-Figueres, Merce; Wang, Chunxiao; Mas, Albert

    2018-04-02

    During wine production, some yeasts enter a Viable But Not Culturable (VBNC) state, which may influence the quality and stability of the final wine through remnant metabolic activity or by resuscitation. Culture-independent techniques are used for obtaining an accurate estimation of the number of live cells, and quantitative PCR could be the most accurate technique. As a marker of cell viability, rRNA was evaluated by analyzing its stability in dead cells. The species-specific stability of rRNA was tested in Saccharomyces cerevisiae, as well as in three species of non-Saccharomyces yeast (Hanseniaspora uvarum, Torulaspora delbrueckii and Starmerella bacillaris). High temperature and antimicrobial dimethyl dicarbonate (DMDC) treatments were efficient in lysing the yeast cells. rRNA gene and rRNA (as cDNA) were analyzed over 48 h after cell lysis by quantitative PCR. The results confirmed the stability of rRNA for 48 h after the cell lysis treatments. To sum up, rRNA may not be a good marker of cell viability in the wine yeasts that were tested. Copyright © 2018 Elsevier B.V. All rights reserved.

  16. 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.

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

    Science.gov (United States)

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

    2013-12-16

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

  18. Quantitative proteomic analysis of microdissected oral epithelium for cancer biomarker discovery.

    Science.gov (United States)

    Xiao, Hua; Langerman, Alexander; Zhang, Yan; Khalid, Omar; Hu, Shen; Cao, Cheng-Xi; Lingen, Mark W; Wong, David T W

    2015-11-01

    Specific biomarkers are urgently needed for the detection and progression of oral cancer. The objective of this study was to discover cancer biomarkers from oral epithelium through utilizing high throughput quantitative proteomics approaches. Morphologically malignant, epithelial dysplasia, and adjacent normal epithelial tissues were laser capture microdissected (LCM) from 19 patients and used for proteomics analysis. Total proteins from each group were extracted, digested and then labelled with corresponding isobaric tags for relative and absolute quantitation (iTRAQ). Labelled peptides from each sample were combined and analyzed by liquid chromatography-mass spectrometry (LC-MS/MS) for protein identification and quantification. In total, 500 proteins were identified and 425 of them were quantified. When compared with adjacent normal oral epithelium, 17 and 15 proteins were consistently up-regulated or down-regulated in malignant and epithelial dysplasia, respectively. Half of these candidate biomarkers were discovered for oral cancer for the first time. Cornulin was initially confirmed in tissue protein extracts and was further validated in tissue microarray. Its presence in the saliva of oral cancer patients was also explored. Myoglobin and S100A8 were pre-validated by tissue microarray. These data demonstrated that the proteomic biomarkers discovered through this strategy are potential targets for oral cancer detection and salivary diagnostics. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Quantitative Proteomics for the Comprehensive Analysis of Stress Responses of Lactobacillus paracasei subsp. paracasei F19.

    Science.gov (United States)

    Schott, Ann-Sophie; Behr, Jürgen; Geißler, Andreas J; Kuster, Bernhard; Hahne, Hannes; Vogel, Rudi F

    2017-10-06

    Lactic acid bacteria are broadly employed as starter cultures in the manufacture of foods. Upon technological preparation, they are confronted with drying stress that amalgamates numerous stress conditions resulting in losses of fitness and survival. To better understand and differentiate physiological stress responses, discover general and specific markers for the investigated stress conditions, and predict optimal preconditioning for starter cultures, we performed a comprehensive genomic and quantitative proteomic analysis of a commonly used model system, Lactobacillus paracasei subsp. paracasei TMW 1.1434 (isogenic with F19) under 11 typical stress conditions, including among others oxidative, osmotic, pH, and pressure stress. We identified and quantified >1900 proteins in triplicate analyses, representing 65% of all genes encoded in the genome. The identified genes were thoroughly annotated in terms of subcellular localization prediction and biological functions, suggesting unbiased and comprehensive proteome coverage. In total, 427 proteins were significantly differentially expressed in at least one condition. Most notably, our analysis suggests that optimal preconditioning toward drying was predicted to be alkaline and high-pressure stress preconditioning. Taken together, we believe the presented strategy may serve as a prototypic example for the analysis and utility of employing quantitative-mass-spectrometry-based proteomics to study bacterial physiology.

  20. Quantitative proteomic characterization of redox-dependent post-translational modifications on protein cysteines

    Energy Technology Data Exchange (ETDEWEB)

    Duan, Jicheng; Gaffrey, Matthew J.; Qian, Wei-Jun

    2017-01-01

    Protein cysteine thiols play a crucial role in redox signaling, regulation of enzymatic activity and protein function, and maintaining redox homeostasis in living systems. The unique chemical reactivity of thiol groups makes cysteine susceptible to oxidative modifications by reactive oxygen and nitrogen species to form a broad array of reversible and irreversible protein post-translational modifications (PTMs). The reversible modifications in particular are one of the major components of redox signaling and are involved in regulation of various cellular processes under physiological and pathological conditions. The biological significance of these redox PTMs in health and diseases has been increasingly recognized. Herein, we review the recent advances of quantitative proteomic approaches for investigating redox PTMs in complex biological systems, including the general considerations of sample processing, various chemical or affinity enrichment strategies, and quantitative approaches. We also highlight a number of redox proteomic approaches that enable effective profiling of redox PTMs for addressing specific biological questions. Although some technological limitations remain, redox proteomics is paving the way towards a better understanding of redox signaling and regulation in human health and diseases.

  1. Quantitative proteomics and dynamic imaging of the nucleolus reveal distinct responses to UV and ionizing radiation.

    Science.gov (United States)

    Moore, Henna M; Bai, Baoyan; Boisvert, François-Michel; Latonen, Leena; Rantanen, Ville; Simpson, Jeremy C; Pepperkok, Rainer; Lamond, Angus I; Laiho, Marikki

    2011-10-01

    The nucleolus is a nuclear organelle that coordinates rRNA transcription and ribosome subunit biogenesis. Recent proteomic analyses have shown that the nucleolus contains proteins involved in cell cycle control, DNA processing and DNA damage response and repair, in addition to the many proteins connected with ribosome subunit production. Here we study the dynamics of nucleolar protein responses in cells exposed to stress and DNA damage caused by ionizing and ultraviolet (UV) radiation in diploid human fibroblasts. We show using a combination of imaging and quantitative proteomics methods that nucleolar substructure and the nucleolar proteome undergo selective reorganization in response to UV damage. The proteomic responses to UV include alterations of functional protein complexes such as the SSU processome and exosome, and paraspeckle proteins, involving both decreases and increases in steady state protein ratios, respectively. Several nonhomologous end-joining proteins (NHEJ), such as Ku70/80, display similar fast responses to UV. In contrast, nucleolar proteomic responses to IR are both temporally and spatially distinct from those caused by UV, and more limited in terms of magnitude. With the exception of the NHEJ and paraspeckle proteins, where IR induces rapid and transient changes within 15 min of the damage, IR does not alter the ratios of most other functional nucleolar protein complexes. The rapid transient decrease of NHEJ proteins in the nucleolus indicates that it may reflect a response to DNA damage. Our results underline that the nucleolus is a specific stress response organelle that responds to different damage and stress agents in a unique, damage-specific manner.

  2. The proteome of human liver peroxisomes: identification of five new peroxisomal constituents by a label-free quantitative proteomics survey.

    Directory of Open Access Journals (Sweden)

    Thomas Gronemeyer

    Full Text Available The peroxisome is a key organelle of low abundance that fulfils various functions essential for human cell metabolism. Severe genetic diseases in humans are caused by defects in peroxisome biogenesis or deficiencies in the function of single peroxisomal proteins. To improve our knowledge of this important cellular structure, we studied for the first time human liver peroxisomes by quantitative proteomics. Peroxisomes were isolated by differential and Nycodenz density gradient centrifugation. A label-free quantitative study of 314 proteins across the density gradient was accomplished using high resolution mass spectrometry. By pairing statistical data evaluation, cDNA cloning and in vivo colocalization studies, we report the association of five new proteins with human liver peroxisomes. Among these, isochorismatase domain containing 1 protein points to the existence of a new metabolic pathway and hydroxysteroid dehydrogenase like 2 protein is likely involved in the transport or β-oxidation of fatty acids in human peroxisomes. The detection of alcohol dehydrogenase 1A suggests the presence of an alternative alcohol-oxidizing system in hepatic peroxisomes. In addition, lactate dehydrogenase A and malate dehydrogenase 1 partially associate with human liver peroxisomes and enzyme activity profiles support the idea that NAD(+ becomes regenerated during fatty acid β-oxidation by alternative shuttling processes in human peroxisomes involving lactate dehydrogenase and/or malate dehydrogenase. Taken together, our data represent a valuable resource for future studies of peroxisome biochemistry that will advance research of human peroxisomes in health and disease.

  3. Optimized Clinical Use of RNALater and FFPE Samples for Quantitative Proteomics

    DEFF Research Database (Denmark)

    Bennike, Tue Bjerg; Kastaniegaard, Kenneth; Padurariu, Simona

    2015-01-01

    Introduction and Objectives The availability of patient samples is essential for clinical proteomic research. Biobanks worldwide store mainly samples stabilized in RNAlater as well as formalin-fixed and paraffin embedded (FFPE) biopsies. Biobank material is a potential source for clinical...... we compare to FFPE and frozen samples being the control. Methods From the sigmoideum of two healthy participants’ twenty-four biopsies were extracted using endoscopy. The biopsies was stabilized either by being directly frozen, RNAlater, FFPE or incubated for 30 min at room temperature prior to FFPE...... information. Conclusion We have demonstrated that quantitative proteome analysis and pathway mapping of samples stabilized in RNAlater as well as by FFPE is feasible with minimal impact on the quality of protein quantification and post-translational modifications....

  4. Quantitative Proteomics Identifies Activation of Hallmark Pathways of Cancer in Patient Melanoma.

    Science.gov (United States)

    Byrum, Stephanie D; Larson, Signe K; Avaritt, Nathan L; Moreland, Linley E; Mackintosh, Samuel G; Cheung, Wang L; Tackett, Alan J

    2013-03-01

    Molecular pathways regulating melanoma initiation and progression are potential targets of therapeutic development for this aggressive cancer. Identification and molecular analysis of these pathways in patients has been primarily restricted to targeted studies on individual proteins. Here, we report the most comprehensive analysis of formalin-fixed paraffin-embedded human melanoma tissues using quantitative proteomics. From 61 patient samples, we identified 171 proteins varying in abundance among benign nevi, primary melanoma, and metastatic melanoma. Seventy-three percent of these proteins were validated by immunohistochemistry staining of malignant melanoma tissues from the Human Protein Atlas database. Our results reveal that molecular pathways involved with tumor cell proliferation, motility, and apoptosis are mis-regulated in melanoma. These data provide the most comprehensive proteome resource on patient melanoma and reveal insight into the molecular mechanisms driving melanoma progression.

  5. Annotation of loci from genome-wide association studies using tissue-specific quantitative interaction proteomics

    DEFF Research Database (Denmark)

    Lundby, Alicia; Rossin, Elizabeth J.; Steffensen, Annette B.

    2014-01-01

    Genome-wide association studies (GWAS) have identified thousands of loci associated with complex traits, but it is challenging to pinpoint causal genes in these loci and to exploit subtle association signals. We used tissue-specific quantitative interaction proteomics to map a network of five genes...... involved in the Mendelian disorder long QT syndrome (LOTS). We integrated the LOTS network with GWAS loci from the corresponding common complex trait, QT-interval variation, to identify candidate genes that were subsequently confirmed in Xenopus laevis oocytes and zebrafish. We used the LOTS protein...... network to filter weak GWAS signals by identifying single-nucleotide polymorphisms (SNPs) in proximity to genes in the network supported by strong proteomic evidence. Three SNPs passing this filter reached genome-wide significance after replication genotyping. Overall, we present a general strategy...

  6. Characterization of ubiquitination dependent dynamics in growth factor receptor signaling by quantitative proteomics

    DEFF Research Database (Denmark)

    Akimov, Vyacheslav; Rigbolt, Kristoffer T G; Nielsen, Mogens M

    2011-01-01

    Protein ubiquitination is a dynamic reversible post-translational modification that plays a key role in the regulation of numerous cellular processes including signal transduction, endocytosis, cell cycle control, DNA repair and gene transcription. The conjugation of the small protein ubiquitin...... investigating ubiquitination on a proteomic scale, mainly due to the inherited complexity and heterogeneity of ubiquitination. We describe here a quantitative proteomics strategy based on the specificity of ubiquitin binding domains (UBDs) and Stable Isotope Labeling by Amino acids in Cell culture (SILAC...... as ubiquitination-dependent events in signaling pathways. In addition to a detailed seven time-point profile of EGFR ubiquitination over 30 minutes of ligand stimulation, our data determined prominent involvement of Lysine-63 ubiquitin branching in EGF signaling. Furthermore, we found two centrosomal proteins, PCM1...

  7. Dominant Epistasis Between Two Quantitative Trait Loci Governing Sporulation Efficiency in Yeast Saccharomyces cerevisiae

    Science.gov (United States)

    Bergman, Juraj; Mitrikeski, Petar T.

    2015-01-01

    Summary Sporulation efficiency in the yeast Saccharomyces cerevisiae is a well-established model for studying quantitative traits. A variety of genes and nucleotides causing different sporulation efficiencies in laboratory, as well as in wild strains, has already been extensively characterised (mainly by reciprocal hemizygosity analysis and nucleotide exchange methods). We applied a different strategy in order to analyze the variation in sporulation efficiency of laboratory yeast strains. Coupling classical quantitative genetic analysis with simulations of phenotypic distributions (a method we call phenotype modelling) enabled us to obtain a detailed picture of the quantitative trait loci (QTLs) relationships underlying the phenotypic variation of this trait. Using this approach, we were able to uncover a dominant epistatic inheritance of loci governing the phenotype. Moreover, a molecular analysis of known causative quantitative trait genes and nucleotides allowed for the detection of novel alleles, potentially responsible for the observed phenotypic variation. Based on the molecular data, we hypothesise that the observed dominant epistatic relationship could be caused by the interaction of multiple quantitative trait nucleotides distributed across a 60--kb QTL region located on chromosome XIV and the RME1 locus on chromosome VII. Furthermore, we propose a model of molecular pathways which possibly underlie the phenotypic variation of this trait. PMID:27904371

  8. Differential Proteome Analysis of a Flor Yeast Strain under Biofilm Formation.

    Science.gov (United States)

    Moreno-García, Jaime; Mauricio, Juan Carlos; Moreno, Juan; García-Martínez, Teresa

    2017-03-28

    Several Saccharomyces cerevisiae strains (flor yeasts) form a biofilm (flor velum) on the surface of Sherry wines after fermentation, when glucose is depleted. This flor velum is fundamental to biological aging of these particular wines. In this study, we identify abundant proteins in the formation of the biofilm of an industrial flor yeast strain. A database search to enrich flor yeast "biological process" and "cellular component" according to Gene Ontology Terminology (GO Terms) and, "pathways" was carried out. The most abundant proteins detected were largely involved in respiration, translation, stress damage prevention and repair, amino acid metabolism (glycine, isoleucine, leucine and arginine), glycolysis/gluconeogenesis and biosynthesis of vitamin B9 (folate). These proteins were located in cellular components as in the peroxisome, mitochondria, vacuole, cell wall and extracellular region; being these two last directly related with the flor formation. Proteins like Bgl2p, Gcv3p, Hyp2p, Mdh1p, Suc2p and Ygp1p were quantified in very high levels. This study reveals some expected processes and provides new and important information for the design of conditions and genetic constructions of flor yeasts for improving the cellular survival and, thus, to optimize biological aging of Sherry wine production.

  9. Genetic and proteomic evidences support the localization of yeast enolase in the cell surface

    DEFF Research Database (Denmark)

    López-Villar, Elena; Monteoliva, Lucía; Larsen, Martin Røssel

    2006-01-01

    Although enolase, other glycolytic enzymes, and a variety of cytoplasmic proteins lacking an N-terminal secretion signal have been widely described as located at the cell surface in yeast and in mammalian cells, their presence in this external location is still controversial. Here, we report that...

  10. iTRAQ-Based Quantitative Proteomics of Developing and Ripening Muscadine Grape Berry

    Science.gov (United States)

    Kambiranda, Devaiah; Katam, Ramesh; Basha, Sheikh M.; Siebert, Shalom

    2014-01-01

    Grapes are among the widely cultivated fruit crops in the world. Grape berries like other nonclimacteric fruits undergo a complex set of dynamic, physical, physiological, and biochemical changes during ripening. Muscadine grapes are widely cultivated in the southern United States for fresh fruit and wine. To date, changes in the metabolites composition of muscadine grapes have been well documented; however, the molecular changes during berry development and ripening are not fully known. The aim of this study was to investigate changes in the berry proteome during ripening in muscadine grape cv. Noble. Isobaric tags for relative and absolute quantification (iTRAQ) MS/MS was used to detect statistically significant changes in the berry proteome. A total of 674 proteins were detected, and 76 were differentially expressed across four time points in muscadine berry. Proteins obtained were further analyzed to provide information about its potential functions during ripening. Several proteins involved in abiotic and biotic stimuli and sucrose and hexose metabolism were upregulated during berry ripening. Quantitative real-time PCR analysis validated the protein expression results for nine proteins. Identification of vicilin-like antimicrobial peptides indicates additional disease tolerance proteins are present in muscadines for berry protection during ripening. The results provide new information for characterization and understanding muscadine berry proteome and grape ripening. PMID:24251720

  11. Toward improved peptide feature detection in quantitative proteomics using stable isotope labeling.

    Science.gov (United States)

    Nilse, Lars; Sigloch, Florian Christoph; Biniossek, Martin L; Schilling, Oliver

    2015-08-01

    Reliable detection of peptides in LC-MS data is a key algorithmic step in the analysis of quantitative proteomics experiments. While highly abundant peptides can be detected reliably by most modern software tools, there is much less agreement on medium and low-intensity peptides in a sample. The choice of software tools can have a big impact on the quantification of proteins, especially for proteins that appear in lower concentrations. However, in many experiments, it is precisely this region of less abundant but substantially regulated proteins that holds the biggest potential for discoveries. This is particularly true for discovery proteomics in the pharmacological sector with a specific interest in key regulatory proteins. In this viewpoint article, we discuss how the development of novel software algorithms allows us to study this region of the proteome with increased confidence. Reliable results are one of many aspects to be considered when deciding on a bioinformatics software platform. Deployment into existing IT infrastructures, compatibility with other software packages, scalability, automation, flexibility, and support need to be considered and are briefly addressed in this viewpoint article. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Quantitative proteomic analyses of the microbial degradation of estrone under various background nitrogen and carbon conditions.

    Science.gov (United States)

    Du, Zhe; Chen, Yinguang; Li, Xu

    2017-10-15

    Microbial degradation of estrogenic compounds can be affected by the nitrogen source and background carbon in the environment. However, the underlying mechanisms are not well understood. The objective of this study was to elucidate the molecular mechanisms of estrone (E1) biodegradation at the protein level under various background nitrogen (nitrate or ammonium) and carbon conditions (no background carbon, acetic acid, or humic acid as background carbon) by a newly isolated bacterial strain. The E1 degrading bacterial strain, Hydrogenophaga atypica ZD1, was isolated from river sediments and its proteome was characterized under various experimental conditions using quantitative proteomics. Results show that the E1 degradation rate was faster when ammonium was used as the nitrogen source than with nitrate. The degradation rate was also faster when either acetic acid or humic acid was present in the background. Proteomics analyses suggested that the E1 biodegradation products enter the tyrosine metabolism pathway. Compared to nitrate, ammonium likely promoted E1 degradation by increasing the activities of the branched-chain-amino-acid aminotransferase (IlvE) and enzymes involved in the glutamine synthetase-glutamine oxoglutarate aminotransferase (GS-GOGAT) pathway. The increased E1 degradation rate with acetic acid or humic acid in the background can also be attributed to the up-regulation of IlvE. Results from this study can help predict and explain E1 biodegradation kinetics under various environmental conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Relative Quantitative Proteomic Analysis of Brucella abortus Reveals Metabolic Adaptation to Multiple Environmental Stresses.

    Science.gov (United States)

    Zai, Xiaodong; Yang, Qiaoling; Yin, Ying; Li, Ruihua; Qian, Mengying; Zhao, Taoran; Li, Yaohui; Zhang, Jun; Fu, Ling; Xu, Junjie; Chen, Wei

    2017-01-01

    Brucella spp. are facultative intracellular pathogens that cause chronic brucellosis in humans and animals. The virulence of Brucella primarily depends on its successful survival and replication in host cells. During invasion of the host tissue, Brucella is simultaneously subjected to a variety of harsh conditions, including nutrient limitation, low pH, antimicrobial defenses, and extreme levels of reactive oxygen species (ROS) via the host immune response. This suggests that Brucella may be able to regulate its metabolic adaptation in response to the distinct stresses encountered during its intracellular infection of the host. An investigation into the differential proteome expression patterns of Brucella grown under the relevant stress conditions may contribute toward a better understanding of its pathogenesis and adaptive response. Here, we utilized a mass spectrometry-based label-free relative quantitative proteomics approach to investigate and compare global proteomic changes in B. abortus in response to eight different stress treatments. The 3 h short-term in vitro single-stress and multi-stress conditions mimicked the in vivo conditions of B. abortus under intracellular infection, with survival rates ranging from 3.17 to 73.17%. The proteomic analysis identified and quantified a total of 2,272 proteins and 74% of the theoretical proteome, thereby providing wide coverage of the B. abortus proteome. By including eight distinct growth conditions and comparing these with a control condition, we identified a total of 1,221 differentially expressed proteins (DEPs) that were significantly changed under the stress treatments. Pathway analysis revealed that most of the proteins were involved in oxidative phosphorylation, ABC transporters, two-component systems, biosynthesis of secondary metabolites, the citrate cycle, thiamine metabolism, and nitrogen metabolism; constituting major response mechanisms toward the reconstruction of cellular homeostasis and metabolic

  14. Relative Quantitative Proteomic Analysis of Brucella abortus Reveals Metabolic Adaptation to Multiple Environmental Stresses

    Directory of Open Access Journals (Sweden)

    Xiaodong Zai

    2017-11-01

    Full Text Available Brucella spp. are facultative intracellular pathogens that cause chronic brucellosis in humans and animals. The virulence of Brucella primarily depends on its successful survival and replication in host cells. During invasion of the host tissue, Brucella is simultaneously subjected to a variety of harsh conditions, including nutrient limitation, low pH, antimicrobial defenses, and extreme levels of reactive oxygen species (ROS via the host immune response. This suggests that Brucella may be able to regulate its metabolic adaptation in response to the distinct stresses encountered during its intracellular infection of the host. An investigation into the differential proteome expression patterns of Brucella grown under the relevant stress conditions may contribute toward a better understanding of its pathogenesis and adaptive response. Here, we utilized a mass spectrometry-based label-free relative quantitative proteomics approach to investigate and compare global proteomic changes in B. abortus in response to eight different stress treatments. The 3 h short-term in vitro single-stress and multi-stress conditions mimicked the in vivo conditions of B. abortus under intracellular infection, with survival rates ranging from 3.17 to 73.17%. The proteomic analysis identified and quantified a total of 2,272 proteins and 74% of the theoretical proteome, thereby providing wide coverage of the B. abortus proteome. By including eight distinct growth conditions and comparing these with a control condition, we identified a total of 1,221 differentially expressed proteins (DEPs that were significantly changed under the stress treatments. Pathway analysis revealed that most of the proteins were involved in oxidative phosphorylation, ABC transporters, two-component systems, biosynthesis of secondary metabolites, the citrate cycle, thiamine metabolism, and nitrogen metabolism; constituting major response mechanisms toward the reconstruction of cellular

  15. Quantitative proteomics reveals the central changes of wheat in response to powdery mildew.

    Science.gov (United States)

    Fu, Ying; Zhang, Hong; Mandal, Siddikun Nabi; Wang, Changyou; Chen, Chunhuan; Ji, Wanquan

    2016-01-01

    Powdery mildew (Pm), caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most important crop diseases, causing severe economic losses to wheat production worldwide. However, there are few reports about the proteomic response to Bgt infection in resistant wheat. Hence, quantitative proteomic analysis of N9134, a resistant wheat line, was performed to explore the molecular mechanism of wheat in defense against Bgt. Comparing the leaf proteins of Bgt-inoculated N9134 with that of mock-inoculated controls, a total of 2182 protein-species were quantified by iTRAQ at 24, 48 and 72h postinoculation (hpi) with Bgt, of which 394 showed differential accumulation. These differentially accumulated protein-species (DAPs) mainly included pathogenesis-related (PR) polypeptides, oxidative stress responsive proteins and components involved in primary metabolic pathways. KEGG enrichment analysis showed that phenylpropanoid biosynthesis, phenylalanine metabolism and photosynthesis-antenna proteins were the key pathways in response to Bgt infection. InterProScan 5 and the Gibbs Motif Sampler cluster 394 DAPs into eight conserved motifs, which shared leucine repeats and histidine sites in the sequence motifs. Moreover, eight separate protein-protein interaction (PPI) networks were predicted from STRING database. This study provides a powerful platform for further exploration of the molecular mechanism underlying resistant wheat responding to Bgt. Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a destructive pathogenic disease in wheat-producing regions worldwide, resulting in severe yield reductions. Although many resistant wheat varieties have been cultivated, there are few reports about the proteomic response to Bgt infection in resistant wheat. Therefore, an iTRAQ-based quantitative proteomic analysis of a resistant wheat line (N9134) in response to Bgt infection has been performed. This paper provides new insights into the underlying molecular

  16. Proteomic evolution of a wine yeast during the first hours of fermentation.

    Science.gov (United States)

    Salvadó, Zoel; Chiva, Rosana; Rodríguez-Vargas, Sonia; Rández-Gil, Francisca; Mas, Albert; Guillamón, José Manuel

    2008-11-01

    The inoculation of active dry wine yeast (ADWY) is one of the most common practices in winemaking. This inoculation exposes the yeast cells to strong osmotic, acidic and thermal stresses, and adaptation to the new medium is crucial for successful fermentation. We have analysed the changes that occur in the ADWY protein profile in the first hours after inoculation under enological-like conditions at a low temperature. Protein changes mainly included enzymes of the nitrogen and carbon metabolism and proteins related to the cellular stress response. Most of the enzymes of the lower part of the glycolysis showed an increase in their concentration 4 and 24 h after inoculation, indicating an increase in glycolytic flux and in ATP production. However, the shift from respiration to fermentation was not immediate in the inoculation because some mitochondrial proteins involved in oxidative metabolism were induced in the first hours after inoculation. Inoculation in this fresh medium also reduced the cellular concentration of stress proteins produced during industrial production of the ADWY. The only exception was Cys3p, which might be involved in glutathione synthesis as a response to oxidative stress. A better understanding of the yeast stress response to rehydration and inoculation will lead to improvements in the handling efficiency of ADWY in winemaking and presumably to better control of fermentation startup.

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

    Science.gov (United States)

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

    2017-04-01

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

  18. A knowledge-based T2-statistic to perform pathway analysis for quantitative proteomic data.

    Science.gov (United States)

    Lai, En-Yu; Chen, Yi-Hau; Wu, Kun-Pin

    2017-06-01

    Approaches to identify significant pathways from high-throughput quantitative data have been developed in recent years. Still, the analysis of proteomic data stays difficult because of limited sample size. This limitation also leads to the practice of using a competitive null as common approach; which fundamentally implies genes or proteins as independent units. The independent assumption ignores the associations among biomolecules with similar functions or cellular localization, as well as the interactions among them manifested as changes in expression ratios. Consequently, these methods often underestimate the associations among biomolecules and cause false positives in practice. Some studies incorporate the sample covariance matrix into the calculation to address this issue. However, sample covariance may not be a precise estimation if the sample size is very limited, which is usually the case for the data produced by mass spectrometry. In this study, we introduce a multivariate test under a self-contained null to perform pathway analysis for quantitative proteomic data. The covariance matrix used in the test statistic is constructed by the confidence scores retrieved from the STRING database or the HitPredict database. We also design an integrating procedure to retain pathways of sufficient evidence as a pathway group. The performance of the proposed T2-statistic is demonstrated using five published experimental datasets: the T-cell activation, the cAMP/PKA signaling, the myoblast differentiation, and the effect of dasatinib on the BCR-ABL pathway are proteomic datasets produced by mass spectrometry; and the protective effect of myocilin via the MAPK signaling pathway is a gene expression dataset of limited sample size. Compared with other popular statistics, the proposed T2-statistic yields more accurate descriptions in agreement with the discussion of the original publication. We implemented the T2-statistic into an R package T2GA, which is available at https

  19. iTRAQ-Based Quantitative Proteomic Analysis of the Initiation of Head Regeneration in Planarians.

    Directory of Open Access Journals (Sweden)

    Xiaofang Geng

    Full Text Available The planarian Dugesia japonica has amazing ability to regenerate a head from the anterior ends of the amputated stump with maintenance of the original anterior-posterior polarity. Although planarians present an attractive system for molecular investigation of regeneration and research has focused on clarifying the molecular mechanism of regeneration initiation in planarians at transcriptional level, but the initiation mechanism of planarian head regeneration (PHR remains unclear at the protein level. Here, a global analysis of proteome dynamics during the early stage of PHR was performed using isobaric tags for relative and absolute quantitation (iTRAQ-based quantitative proteomics strategy, and our data are available via ProteomeXchange with identifier PXD002100. The results showed that 162 proteins were differentially expressed at 2 h and 6 h following amputation. Furthermore, the analysis of expression patterns and functional enrichment of the differentially expressed proteins showed that proteins involved in muscle contraction, oxidation reduction and protein synthesis were up-regulated in the initiation of PHR. Moreover, ingenuity pathway analysis showed that predominant signaling pathways such as ILK, calcium, EIF2 and mTOR signaling which were associated with cell migration, cell proliferation and protein synthesis were likely to be involved in the initiation of PHR. The results for the first time demonstrated that muscle contraction and ILK signaling might played important roles in the initiation of PHR at the global protein level. The findings of this research provide a molecular basis for further unraveling the mechanism of head regeneration initiation in planarians.

  20. Prediction of quantitative phenotypes based on genetic networks: a case study in yeast sporulation

    Directory of Open Access Journals (Sweden)

    Shen Li

    2010-09-01

    Full Text Available Abstract Background An exciting application of genetic network is to predict phenotypic consequences for environmental cues or genetic perturbations. However, de novo prediction for quantitative phenotypes based on network topology is always a challenging task. Results Using yeast sporulation as a model system, we have assembled a genetic network from literature and exploited Boolean network to predict sporulation efficiency change upon deleting individual genes. We observe that predictions based on the curated network correlate well with the experimentally measured values. In addition, computational analysis reveals the robustness and hysteresis of the yeast sporulation network and uncovers several patterns of sporulation efficiency change caused by double gene deletion. These discoveries may guide future investigation of underlying mechanisms. We have also shown that a hybridized genetic network reconstructed from both temporal microarray data and literature is able to achieve a satisfactory prediction accuracy of the same quantitative phenotypes. Conclusions This case study illustrates the value of predicting quantitative phenotypes based on genetic network and provides a generic approach.

  1. Two-dimensional gel electrophoresis data for proteomic profiling of Sporothrix yeast cells

    Directory of Open Access Journals (Sweden)

    Anderson Messias Rodrigues

    2015-03-01

    Full Text Available Sporotrichosis is a chronic infection of the skin and subcutaneous tissues of human and other mammals caused by a complex of cryptic dimorphic fungi in the plant-associated order Ophiostomatales. With major differences between routes of transmission, Sporothrix infections are emerging as new threat in tropical and subtropical areas, particularly in form of outbreaks. The mechanisms underlying the pathogenesis and invasion of Sporothrix spp. are still poorly understood and many virulence factors remain unidentified. In this scenario, a global analysis of proteins expressed by clinical Sporothrix species combined with the identification of seroreactive proteins is overdue. Optimization of sample preparation and electrophoresis conditions are key steps toward reproducibility of gel-based proteomics assays. We provide the data generated using an efficient protocol of protein extraction for rapid and large-scale proteome analysis using two-dimensional gel electrophoresis. The protocol was established and optimized for pathogenic and non-pathogenic Sporothrix spp. including Sporothrix brasiliensis (CBS 132990, Sporothrix schenckii sensu stricto (CBS 132974, Sporothrix globosa (CBS 132922, and Sporothrix mexicana (CBS 120341. The data, supplied in this article, are related to the research article entitled “Immunoproteomic analysis reveals a convergent humoral response signature in the Sporothrix schenckii complex” (Rodrigues et al., 2014 [1].

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

    Directory of Open Access Journals (Sweden)

    Ganglong Yang

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

  3. Quantitative and temporal proteome analysis of butyrate-treated colorectal cancer cells.

    Science.gov (United States)

    Tan, Hwee Tong; Tan, Sandra; Lin, Qingsong; Lim, Teck Kwang; Hew, Choy Leong; Chung, Maxey C M

    2008-06-01

    Colorectal cancer is one of the most common cancers in developed countries, and its incidence is negatively associated with high dietary fiber intake. Butyrate, a short-chain fatty acid fermentation by-product of fiber induces cell maturation with the promotion of growth arrest, differentiation, and/or apoptosis of cancer cells. The stimulation of cell maturation by butyrate in colonic cancer cells follows a temporal progression from the early phase of growth arrest to the activation of apoptotic cascades. Previously we performed two-dimensional DIGE to identify differentially expressed proteins induced by 24-h butyrate treatment of HCT-116 colorectal cancer cells. Herein we used quantitative proteomics approaches using iTRAQ (isobaric tags for relative and absolute quantitation), a stable isotope labeling methodology that enables multiplexing of four samples, for a temporal study of HCT-116 cells treated with butyrate. In addition, cleavable ICAT, which selectively tags cysteine-containing proteins, was also used, and the results complemented those obtained from the iTRAQ strategy. Selected protein targets were validated by real time PCR and Western blotting. A model is proposed to illustrate our findings from this temporal analysis of the butyrate-responsive proteome that uncovered several integrated cellular processes and pathways involved in growth arrest, apoptosis, and metastasis. These signature clusters of butyrate-regulated pathways are potential targets for novel chemopreventive and therapeutic drugs for treatment of colorectal cancer.

  4. Quantitative Proteomic Analysis of Hepatic Tissue of T2DM Rhesus Macaque

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    Tingfu Du

    2017-01-01

    Full Text Available Type 2 diabetes mellitus (T2DM is a metabolic disorder that severely affects human health, but the pathogenesis of the disease remains unknown. The high-fat/high-sucrose diets combined with streptozotocin- (STZ- induced nonhuman primate animal model of diabetes are a valuable research source of T2DM. Here, we present a study of a STZ rhesus macaque model of T2DM that utilizes quantitative iTRAQ-based proteomic method. We compared the protein profiles in the liver of STZ-treated macaques as well as age-matched healthy controls. We identified 171 proteins differentially expressed in the STZ-treated groups, about 70 of which were documented as diabetes-related gene in previous studies. Pathway analyses indicated that the biological functions of differentially expressed proteins were related to glycolysis/gluconeogenesis, fatty acid metabolism, complements, and coagulation cascades. Expression change in tryptophan metabolism pathway was also found in this study which may be associations with diabetes. This study is the first to explore genome-wide protein expression in hepatic tissue of diabetes macaque model using HPLC-Q-TOF/MS technology. In addition to providing potential T2DM biomarkers, this quantitative proteomic study may also shed insights regarding the molecular pathogenesis of T2DM.

  5. Label free quantitative proteomics analysis on the cisplatin resistance in ovarian cancer cells.

    Science.gov (United States)

    Wang, F; Zhu, Y; Fang, S; Li, S; Liu, S

    2017-05-20

    Quantitative proteomics has been made great progress in recent years. Label free quantitative proteomics analysis based on the mass spectrometry is widely used. Using this technique, we determined the differentially expressed proteins in the cisplatin-sensitive ovarian cancer cells COC1 and cisplatin-resistant cells COC1/DDP before and after the application of cisplatin. Using the GO analysis, we classified those proteins into different subgroups bases on their cellular component, biological process, and molecular function. We also used KEGG pathway analysis to determine the key signal pathways that those proteins were involved in. There are 710 differential proteins between COC1 and COC1/DDP cells, 783 between COC1 and COC1/DDP cells treated with cisplatin, 917 between the COC1/DDP cells and COC1/DDP cells treated with LaCl3, 775 between COC1/DDP cells treated with cisplatin and COC1/DDP cells treated with cisplatin and LaCl3. Among the same 411 differentially expressed proteins in cisplatin-sensitive COC1 cells and cisplain-resistant COC1/DDP cells before and after cisplatin treatment, 14% of them were localized on the cell membrane. According to the KEGG results, differentially expressed proteins were classified into 21 groups. The most abundant proteins were involved in spliceosome. This study lays a foundation for deciphering the mechanism for drug resistance in ovarian tumor.

  6. Quantitative proteomics and terminomics to elucidate the role of ubiquitination and proteolysis in adaptive immunity.

    Science.gov (United States)

    Klein, Theo; Viner, Rosa I; Overall, Christopher M

    2016-10-28

    Adaptive immunity is the specialized defence mechanism in vertebrates that evolved to eliminate pathogens. Specialized lymphocytes recognize specific protein epitopes through antigen receptors to mount potent immune responses, many of which are initiated by nuclear factor-kappa B activation and gene transcription. Most, if not all, pathways in adaptive immunity are further regulated by post-translational modification (PTM) of signalling proteins, e.g. phosphorylation, citrullination, ubiquitination and proteolytic processing. The importance of PTMs is reflected by genetic or acquired defects in these pathways that lead to a dysfunctional immune response. Here we discuss the state of the art in targeted proteomics and systems biology approaches to dissect the PTM landscape specifically regarding ubiquitination and proteolysis in B- and T-cell activation. Recent advances have occurred in methods for specific enrichment and targeted quantitation. Together with improved instrument sensitivity, these advances enable the accurate analysis of often rare PTM events that are opaque to conventional proteomics approaches, now rendering in-depth analysis and pathway dissection possible. We discuss published approaches, including as a case study the profiling of the N-terminome of lymphocytes of a rare patient with a genetic defect in the paracaspase protease MALT1, a key regulator protease in antigen-driven signalling, which was manifested by elevated linear ubiquitination.This article is part of the themed issue 'Quantitative mass spectrometry'. © 2016 The Authors.

  7. Quantitative proteomic analysis of ibuprofen-degrading Patulibacter sp. strain I11

    DEFF Research Database (Denmark)

    Almeida, Barbara; Kjeldal, Henrik; Lolas, Ihab Bishara Yousef

    2013-01-01

    was identified and quantified by gel based shotgun-proteomics. In total 251 unique proteins were quantitated using this approach. Biological process and pathway analysis indicated a number of proteins that were up-regulated in response to active degradation of ibuprofen, some of them are known to be involved...... in the degradation of aromatic compounds. Data analysis revealed that several of these proteins are likely involved in ibuprofen degradation by Patulibacter sp. strain I11.......Ibuprofen is the third most consumed pharmaceutical drug in the world. Several isolates have been shown to degrade ibuprofen, but very little is known about the biochemistry of this process. This study investigates the degradation of ibuprofen by Patulibacter sp. strain I11 by quantitative...

  8. Detection of Nuclear Protein Profile Changes by Human Metapneumovirus M2-2 Protein Using Quantitative Differential Proteomics

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    Yuping Ren

    2017-12-01

    Full Text Available Human metapneumovirus (hMPV is a leading cause of lower respiratory infection in pediatric populations globally. This study examined proteomic profile changes in A549 cells infected with hMPV and two attenuated mutants with deleted PDZ domain-binding motif(s in the M2-2 protein. These motifs are involved in the interruption of antiviral signaling, namely the interaction between the TNF receptor associated factor (TRAF and mitochondrial antiviral-signaling (MAVS proteins. The aim of this study was to provide insight into the overall and novel impact of M2-2 motifs on cellular responses via an unbiased comparison. Tandem mass tagging, stable isotope labeling, and high-resolution mass spectrometry were used for quantitative proteomic analysis. Using quantitative proteomics and Venn analysis, 1248 common proteins were detected in all infected samples of both technical sets. Hierarchical clustering of the differentiated proteome displayed distinct proteomic signatures that were controlled by the motif(s. Bioinformatics and experimental analysis confirmed the differentiated proteomes, revealed novel cellular biological events, and implicated key pathways controlled by hMPV M2-2 PDZ domain-binding motif(s. This provides further insight for evaluating M2-2 mutants as potent vaccine candidates.

  9. Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) Applied to Quantitative Proteomics of Bacillus subtilis

    DEFF Research Database (Denmark)

    Soufi, Boumediene; Kumar, C.; Gnad, F.

    2010-01-01

    We applied stable isotope labeling by amino acids in cell culture (SILAC) to large-scale quantitative proteomics analyses of the model bacterium Bacillus subtilis in two physiological conditions: growth on succinate and growth under phosphate starvation. Using a B. subtilis strain auxotrophic...... of the most comprehensive quantitative proteomics studies in bacteria, covering more than 75% of the B. subtilis genes expressed in the log phase of growth. Furthermore, we detect and quantify dynamics of 35 Ser/Thr/Tyr phosphorylation sites under growth on succinate, and 10 phosphorylation sites under...

  10. High confidence proteomic analysis of yeast LDs identifies additional droplet proteins and reveals connections to dolichol synthesis and sterol acetylation.

    Science.gov (United States)

    Currie, Erin; Guo, Xiuling; Christiano, Romain; Chitraju, Chandramohan; Kory, Nora; Harrison, Kenneth; Haas, Joel; Walther, Tobias C; Farese, Robert V

    2014-07-01

    Accurate protein inventories are essential for understanding an organelle's functions. The lipid droplet (LD) is a ubiquitous intracellular organelle with major functions in lipid storage and metabolism. LDs differ from other organelles because they are bounded by a surface monolayer, presenting unique features for protein targeting to LDs. Many proteins of varied functions have been found in purified LD fractions by proteomics. While these studies have become increasingly sensitive, it is often unclear which of the identified proteins are specific to LDs. Here we used protein correlation profiling to identify 35 proteins that specifically enrich with LD fractions of Saccharomyces cerevisiae Of these candidates, 30 fluorophore-tagged proteins localize to LDs by microscopy, including six proteins, several with human orthologs linked to diseases, which we newly identify as LD proteins (Cab5, Rer2, Say1, Tsc10, YKL047W, and YPR147C). Two of these proteins, Say1, a sterol deacetylase, and Rer2, a cis-isoprenyl transferase, are enzymes involved in sterol and polyprenol metabolism, respectively, and we show their activities are present in LD fractions. Our results provide a highly specific list of yeast LD proteins and reveal that the vast majority of these proteins are involved in lipid metabolism. Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.

  11. Quantitative proteomics and network analysis of SSA1 and SSB1 deletion mutants reveals robustness of chaperone HSP70 network in Saccharomyces cerevisiae.

    Science.gov (United States)

    Jarnuczak, Andrew F; Eyers, Claire E; Schwartz, Jean-Marc; Grant, Christopher M; Hubbard, Simon J

    2015-09-01

    Molecular chaperones play an important role in protein homeostasis and the cellular response to stress. In particular, the HSP70 chaperones in yeast mediate a large volume of protein folding through transient associations with their substrates. This chaperone interaction network can be disturbed by various perturbations, such as environmental stress or a gene deletion. Here, we consider deletions of two major chaperone proteins, SSA1 and SSB1, from the chaperone network in Sacchromyces cerevisiae. We employ a SILAC-based approach to examine changes in global and local protein abundance and rationalise our results via network analysis and graph theoretical approaches. Although the deletions result in an overall increase in intracellular protein content, correlated with an increase in cell size, this is not matched by substantial changes in individual protein concentrations. Despite the phenotypic robustness to deletion of these major hub proteins, it cannot be simply explained by the presence of paralogues. Instead, network analysis and a theoretical consideration of folding workload suggest that the robustness to perturbation is a product of the overall network structure. This highlights how quantitative proteomics and systems modelling can be used to rationalise emergent network properties, and how the HSP70 system can accommodate the loss of major hubs. © 2015 The Authors. PROTEOMICS published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Quantitative Proteomic Analysis of the Response to Zinc, Magnesium, and Calcium Deficiency in Specific Cell Types of Arabidopsis Roots

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    Yoichiro Fukao

    2016-01-01

    Full Text Available The proteome profiles of specific cell types have recently been investigated using techniques such as fluorescence activated cell sorting and laser capture microdissection. However, quantitative proteomic analysis of specific cell types has not yet been performed. In this study, to investigate the response of the proteome to zinc, magnesium, and calcium deficiency in specific cell types of Arabidopsis thaliana roots, we performed isobaric tags for relative and absolute quantification (iTRAQ-based quantitative proteomics using GFP-expressing protoplasts collected by fluorescence-activated cell sorting. Protoplasts were collected from the pGL2-GFPer and pMGP-GFPer marker lines for epidermis or inner cell lines (pericycle, endodermis, and cortex, respectively. To increase the number of proteins identified, iTRAQ-labeled peptides were separated into 24 fractions by OFFGFEL electrophoresis prior to high-performance liquid chromatography coupled with mass spectrometry analysis. Overall, 1039 and 737 proteins were identified and quantified in the epidermal and inner cell lines, respectively. Interestingly, the expression of many proteins was decreased in the epidermis by mineral deficiency, although a weaker effect was observed in inner cell lines such as the pericycle, endodermis, and cortex. Here, we report for the first time the quantitative proteomics of specific cell types in Arabidopsis roots.

  13. Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes

    Science.gov (United States)

    Trinkle-Mulcahy, Laura; Boulon, Séverine; Lam, Yun Wah; Urcia, Roby; Boisvert, François-Michel; Vandermoere, Franck; Morrice, Nick A.; Swift, Sam; Rothbauer, Ulrich; Leonhardt, Heinrich; Lamond, Angus

    2008-01-01

    The identification of interaction partners in protein complexes is a major goal in cell biology. Here we present a reliable affinity purification strategy to identify specific interactors that combines quantitative SILAC-based mass spectrometry with characterization of common contaminants binding to affinity matrices (bead proteomes). This strategy can be applied to affinity purification of either tagged fusion protein complexes or endogenous protein complexes, illustrated here using the well-characterized SMN complex as a model. GFP is used as the tag of choice because it shows minimal nonspecific binding to mammalian cell proteins, can be quantitatively depleted from cell extracts, and allows the integration of biochemical protein interaction data with in vivo measurements using fluorescence microscopy. Proteins binding nonspecifically to the most commonly used affinity matrices were determined using quantitative mass spectrometry, revealing important differences that affect experimental design. These data provide a specificity filter to distinguish specific protein binding partners in both quantitative and nonquantitative pull-down and immunoprecipitation experiments. PMID:18936248

  14. Quantitative proteomics study of larval settlement in the barnacle Balanus amphitrite

    KAUST Repository

    Chen, Zhang-Fan; Zhang, Huoming; Wang, Hao; Matsumura, Kiyotaka; Wong, Yue Him; Ravasi, Timothy; Qian, Pei-Yuan

    2014-01-01

    Barnacles are major sessile components of the intertidal areas worldwide, and also one of the most dominant fouling organisms in fouling communities. Larval settlement has a crucial ecological effect not only on the distribution of the barnacle population but also intertidal community structures. However, the molecular mechanisms involved in the transition process from the larval to the juvenile stage remain largely unclear. In this study, we carried out comparative proteomic profiles of stage II nauplii, stage VI nauplii, cyprids, and juveniles of the barnacle Balanus amphitrite using label-free quantitative proteomics, followed by the measurement of the gene expression levels of candidate proteins. More than 700 proteins were identified at each stage; 80 were significantly up-regulated in cyprids and 95 in juveniles vs other stages. Specifically, proteins involved in energy and metabolism, the nervous system and signal transduction were significantly up-regulated in cyprids, whereas proteins involved in cytoskeletal remodeling, transcription and translation, cell proliferation and differentiation, and biomineralization were up-regulated in juveniles, consistent with changes associated with larval metamorphosis and tissue remodeling in juveniles. These findings provided molecular evidence for the morphological, physiological and biological changes that occur during the transition process from the larval to the juvenile stages in B. amphitrite. © 2014 Chen et al.

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

    Science.gov (United States)

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

    2015-02-01

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

  16. iTRAQ Quantitative Proteomic Analysis of Vitreous from Patients with Retinal Detachment

    Directory of Open Access Journals (Sweden)

    Fátima Milhano Santos

    2018-04-01

    Full Text Available Rhegmatogenous retinal detachment (RRD is a potentially blinding condition characterized by a physical separation between neurosensory retina and retinal pigment epithelium. Quantitative proteomics can help to understand the changes that occur at the cellular level during RRD, providing additional information about the molecular mechanisms underlying its pathogenesis. In the present study, iTRAQ labeling was combined with two-dimensional LC-ESI-MS/MS to find expression changes in the proteome of vitreous from patients with RRD when compared to control samples. A total of 150 proteins were found differentially expressed in the vitreous of patients with RRD, including 96 overexpressed and 54 underexpressed. Several overexpressed proteins, several such as glycolytic enzymes (fructose-bisphosphate aldolase A, gamma-enolase, and phosphoglycerate kinase 1, glucose transporters (GLUT-1, growth factors (metalloproteinase inhibitor 1, and serine protease inhibitors (plasminogen activator inhibitor 1 are regulated by HIF-1, which suggests that HIF-1 signaling pathway can be triggered in response to RRD. Also, the accumulation of photoreceptor proteins, including phosducin, rhodopsin, and s-arrestin, and vimentin in vitreous may indicate that photoreceptor degeneration occurs in RRD. Also, the accumulation of photoreceptor proteins, including phosducin, rhodopsin, and s-arrestin, and vimentin in vitreous may indicate that photoreceptor degeneration occurs in RRD. Nevertheless, the differentially expressed proteins found in this study suggest that different mechanisms are activated after RRD to promote the survival of retinal cells through complex cellular responses.

  17. Quantitative Proteomics Analysis of the cAMP/Protein Kinase A Signaling Pathway

    Science.gov (United States)

    2012-01-01

    To define the proteins whose expression is regulated by cAMP and protein kinase A (PKA), we used a quantitative proteomics approach in studies of wild-type (WT) and kin- (PKA-null) S49 murine T lymphoma cells. We also compared the impact of endogenous increases in the level of cAMP [by forskolin (Fsk) and the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX)] or by a cAMP analogue (8-CPT-cAMP). We identified 1056 proteins in WT and kin- S49 cells and found that 8-CPT-cAMP and Fsk with IBMX produced differences in protein expression. WT S49 cells had a correlation coefficient of 0.41 between DNA microarray data and the proteomics analysis in cells incubated with 8-CPT-cAMP for 24 h and a correlation coefficient of 0.42 between the DNA microarray data obtained at 6 h and the changes in protein expression after incubation with 8-CPT-cAMP for 24 h. Glutathione reductase (Gsr) had a higher level of basal expression in kin- S49 cells than in WT cells. Consistent with this finding, kin- cells are less sensitive to cell killing and generation of malondialdehyde than are WT cells incubated with H2O2. Cyclic AMP acting via PKA thus has a broad impact on protein expression in mammalian cells, including in the regulation of Gsr and oxidative stress. PMID:23110364

  18. Quantitative Proteomics Reveals Membrane Protein-Mediated Hypersaline Sensitivity and Adaptation in Halophilic Nocardiopsis xinjiangensis.

    Science.gov (United States)

    Zhang, Yao; Li, Yanchang; Zhang, Yongguang; Wang, Zhiqiang; Zhao, Mingzhi; Su, Na; Zhang, Tao; Chen, Lingsheng; Wei, Wei; Luo, Jing; Zhou, Yanxia; Xu, Yongru; Xu, Ping; Li, Wenjun; Tao, Yong

    2016-01-04

    The genus Nocardiopsis is one of the most dominant Actinobacteria that survives in hypersaline environments. However, the adaptation mechanisms for halophilism are still unclear. Here, we performed isobaric tags for relative and absolute quantification based quantitative proteomics to investigate the functions of the membrane proteome after salt stress. A total of 683 membrane proteins were identified and quantified, of which 126 membrane proteins displayed salt-induced changes in abundance. Intriguingly, bioinformatics analyses indicated that these differential proteins showed two expression patterns, which were further validated by phenotypic changes and functional differences. The majority of ABC transporters, secondary active transporters, cell motility proteins, and signal transduction kinases were up-regulated with increasing salt concentration, whereas cell differentiation, small molecular transporter (ions and amino acids), and secondary metabolism proteins were significantly up-regulated at optimum salinity, but down-regulated or unchanged at higher salinity. The small molecule transporters and cell differentiation-related proteins acted as sensing proteins that played a more important biological role at optimum salinity. However, the ABC transporters for compatible solutes, Na(+)-dependent transporters, and cell motility proteins acted as adaptive proteins that actively counteracted higher salinity stress. Overall, regulation of membrane proteins may provide a major protection strategy against hyperosmotic stress.

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

    Science.gov (United States)

    Tekwe, Carmen D; Carroll, Raymond J; Dabney, Alan R

    2012-08-01

    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. 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. 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. ctekwe@stat.tamu.edu.

  20. Nucleophosmin in the pathogenesis of arsenic-related bladder carcinogenesis revealed by quantitative proteomics

    International Nuclear Information System (INIS)

    Chen Shuhui; Wang Yiwen; Hsu Jueliang; Chang Hongyi; Wang Chiyun; Shen Potsun; Chiang Chiwu; Chuang Jingjing; Tsai Hungwen; Gu Powen; Chang Fangchih; Liu Hsiaosheng; Chow Nanhaw

    2010-01-01

    To investigate the molecular mechanisms of arsenic (As)-associated carcinogenesis, we performed proteomic analysis on E7 immortalized human uroepithelial cells after treatment with As in vitro. Quantitative proteomics was performed using stable isotope dimethyl labeling coupled with two-dimensional liquid chromatography peptide separation and mass spectrometry (MS)/MS analysis. Among 285 proteins, a total of 26 proteins were upregulated (ratio > 2.0) and 18 proteins were downregulated (ratio < 0.65) by As treatment, which are related to nucleotide binding, lipid metabolism, protein folding, protein biosynthesis, transcription, DNA repair, cell cycle control, and signal transduction. This study reports the potential significance of nucleophosmin (NPM) in the As-related bladder carcinogenesis. NPM was universally expressed in all of uroepithelial cell lines examined, implying that NPM may play a role in human bladder carcinogenesis. Upregulation of NPM tends to be dose- and time-dependent after As treatment. Expression of NPM was associated with cell proliferation, migration and anti-apoptosis. On the contrary, soy isoflavones inhibited the expression of NPM in vitro. The results suggest that NPM may play a role in the As-related bladder carcinogenesis, and soybean-based foods may have potential in the suppression of As/NPM-related tumorigenesis.

  1. Quantitative proteomics of Spodoptera frugiperda cells during growth and baculovirus infection.

    Directory of Open Access Journals (Sweden)

    Nuno Carinhas

    Full Text Available Baculovirus infection of Spodoptera frugiperda cells is a system of choice to produce a range of recombinant proteins, vaccines and, potentially, gene therapy vectors. While baculovirus genomes are well characterized, the genome of S. frugiperda is not sequenced and the virus-host molecular interplay is sparsely known. Herein, we describe the application of stable isotope labeling by amino acids in cell culture (SILAC to obtain the first comparative proteome quantitation of S. frugiperda cells during growth and early baculovirus infection. The proteome coverage was maximized by compiling a search database with protein annotations from insect species. Of interest were differentially proteins related to energy metabolism, endoplasmic reticulum and oxidative stress, yet not investigated in the scope of baculovirus infection. Further, the reduced expression of key viral-encoded proteins early in the infection cycle is suggested to be related with decreased viral replication at high cell density culture. These findings have implications for virological research and improvement of baculovirus-based bioprocesses.

  2. Quantitative proteomics study of larval settlement in the barnacle Balanus amphitrite

    KAUST Repository

    Chen, Zhang-Fan

    2014-02-13

    Barnacles are major sessile components of the intertidal areas worldwide, and also one of the most dominant fouling organisms in fouling communities. Larval settlement has a crucial ecological effect not only on the distribution of the barnacle population but also intertidal community structures. However, the molecular mechanisms involved in the transition process from the larval to the juvenile stage remain largely unclear. In this study, we carried out comparative proteomic profiles of stage II nauplii, stage VI nauplii, cyprids, and juveniles of the barnacle Balanus amphitrite using label-free quantitative proteomics, followed by the measurement of the gene expression levels of candidate proteins. More than 700 proteins were identified at each stage; 80 were significantly up-regulated in cyprids and 95 in juveniles vs other stages. Specifically, proteins involved in energy and metabolism, the nervous system and signal transduction were significantly up-regulated in cyprids, whereas proteins involved in cytoskeletal remodeling, transcription and translation, cell proliferation and differentiation, and biomineralization were up-regulated in juveniles, consistent with changes associated with larval metamorphosis and tissue remodeling in juveniles. These findings provided molecular evidence for the morphological, physiological and biological changes that occur during the transition process from the larval to the juvenile stages in B. amphitrite. © 2014 Chen et al.

  3. Label-free quantitative proteome analysis of the surface-bound salivary pellicle.

    Science.gov (United States)

    Delius, Judith; Trautmann, Simone; Médard, Guillaume; Kuster, Bernhard; Hannig, Matthias; Hofmann, Thomas

    2017-04-01

    The salivary pellicle, covering natural as well as restored tooth surfaces in the oral cavity as an immobilized protein-rich layer, acts as an important physico-chemical and biological mediator at the tooth-saliva-interface. For the first time, the pellicle's proteome of individual volunteers were analyzed separately on three consecutive days and the relative protein abundance determined by a label-free quantitative nano-LC-MS/MS approach. A total of 72 major proteins were identified in the initial pellicles formed intraorally on dental ceramic specimens already after 3min with high inter-individual and inter-day consistency. In comparison, significant differences in protein abundance were evident between subjects, thus indicating unique individual pellicle profiles. Furthermore, the relative protein abundance in pellicles was compared to the proteome pattern in the corresponding saliva samples of the same individuals to provide first data on significantly enriched and depleted salivary proteins (p <0.05) within the surface-bound salivary pellicle. Our findings reveal the initial adsorption of salivary proteins at the solid-liquid interface to be a rapid, highly selective, and reproducible process leading to the immobilization of a broad range of protective proteins and enzymes on the substratum surface within a few minutes. This provides evidence that the pellicle layer might be physiologically functional even without further maturation. Copyright © 2017 Elsevier B.V. All rights reserved.

  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. iTRAQ-based Quantitative Proteomics Study in Patients with Refractory Mycoplasma pneumoniae Pneumonia.

    Science.gov (United States)

    Yu, Jia-Lu; Song, Qi-Fang; Xie, Zhi-Wei; Jiang, Wen-Hui; Chen, Jia-Hui; Fan, Hui-Feng; Xie, Ya-Ping; Lu, Gen

    2017-09-25

    Mycoplasma pneumoniae (MP) is a leading cause of community-acquired pneumonia in children and young adults. Although MP pneumonia is usually benign and self-limited, in some cases it can develop into life-threating refractory MP pneumonia (RMPP). However, the pathogenesis of RMPP is poorly understood. The identification and characterization of proteins related to RMPP could provide a proof of principle to facilitate appropriate diagnostic and therapeutic strategies for treating paients with MP. In this study, we used a quantitative proteomic technique (iTRAQ) to analyze MP-related proteins in serum samples from 5 patients with RMPP, 5 patients with non-refractory MP pneumonia (NRMPP), and 5 healthy children. Functional classification, sub-cellular localization, and protein interaction network analysis were carried out based on protein annotation through evolutionary relationship (PANTHER) and Cytoscape analysis. A total of 260 differentially expressed proteins were identified in the RMPP and NRMPP groups. Compared to the control group, the NRMPP and RMPP groups showed 134 (70 up-regulated and 64 down-regulated) and 126 (63 up-regulated and 63 down-regulated) differentially expressed proteins, respectively. The complex functional classification and protein interaction network of the identified proteins reflected the complex pathogenesis of RMPP. Our study provides the first comprehensive proteome map of RMPP-related proteins from MP pneumonia. These profiles may be useful as part of a diagnostic panel, and the identified proteins provide new insights into the pathological mechanisms underlying RMPP.

  6. Deciphering the molecular mechanisms underlying sea urchin reversible adhesion: A quantitative proteomics approach.

    Science.gov (United States)

    Lebesgue, Nicolas; da Costa, Gonçalo; Ribeiro, Raquel Mesquita; Ribeiro-Silva, Cristina; Martins, Gabriel G; Matranga, Valeria; Scholten, Arjen; Cordeiro, Carlos; Heck, Albert J R; Santos, Romana

    2016-04-14

    Marine bioadhesives have unmatched performances in wet environments, being an inspiration for biomedical applications. In sea urchins specialized adhesive organs, tube feet, mediate reversible adhesion, being composed by a disc, producing adhesive and de-adhesive secretions, and a motile stem. After tube foot detachment, the secreted adhesive remains bound to the substratum as a footprint. Sea urchin adhesive is composed by proteins and sugars, but so far only one protein, Nectin, was shown to be over-expressed as a transcript in tube feet discs, suggesting its involvement in sea urchin adhesion. Here we use high-resolution quantitative mass-spectrometry to perform the first study combining the analysis of the differential proteome of an adhesive organ, with the proteome of its secreted adhesive. This strategy allowed us to identify 163 highly over-expressed disc proteins, specifically involved in sea urchin reversible adhesion; to find that 70% of the secreted adhesive components fall within five protein groups, involved in exocytosis and microbial protection; and to provide evidences that Nectin is not only highly expressed in tube feet discs but is an actual component of the adhesive. These results give an unprecedented insight into the molecular mechanisms underlying sea urchin adhesion, and opening new doors to develop wet-reliable, reversible, and ecological biomimetic adhesives. Sea urchins attach strongly but in a reversible manner to substratum, being a valuable source of inspiration for industrial and biomedical applications. Yet, the molecular mechanisms governing reversible adhesion are still poorly studied delaying the engineering of biomimetic adhesives. We used the latest mass spectrometry techniques to analyze the differential proteome of an adhesive organ and the proteome of its secreted adhesive, allowing us to uncover the key players in sea urchin reversible adhesion. We demonstrate, that Nectin, a protein previously pointed out as potentially

  7. Quantitative analysis of oyster larval proteome provides new insights into the effects of multiple climate change stressors

    KAUST Repository

    Dineshram, Ramadoss; Chandramouli, Kondethimmanahalli; Ko, Ginger Wai Kuen; Zhang, Huoming; Qian, Pei-Yuan; Ravasi, Timothy; Thiyagarajan, Vengatesen

    2016-01-01

    might be affected in a future ocean, we examined changes in the proteome of metamorphosing larvae under multiple stressors: decreased pH (pH 7.4), increased temperature (30 °C), and reduced salinity (15 psu). Quantitative protein expression profiling

  8. Deep Proteomics of Mouse Skeletal Muscle Enables Quantitation of Protein Isoforms, Metabolic Pathways, and Transcription Factors*

    Science.gov (United States)

    Deshmukh, Atul S.; Murgia, Marta; Nagaraj, Nagarjuna; Treebak, Jonas T.; Cox, Jürgen; Mann, Matthias

    2015-01-01

    Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging because of highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art MS workflow and a strategy to map identifications from the C2C12 cell line model to tissues, we identified a total of 10,218 proteins, including skeletal muscle specific transcription factors like myod1 and myogenin and circadian clock proteins. We obtain absolute abundances for proteins expressed in a muscle cell line and skeletal muscle, which should serve as a valuable resource. Quantitation of protein isoforms of glucose uptake signaling pathways and in glucose and lipid metabolic pathways provides a detailed metabolic map of the cell line compared with tissue. This revealed unexpectedly complex regulation of AMP-activated protein kinase and insulin signaling in muscle tissue at the level of enzyme isoforms. PMID:25616865

  9. Quantitative Analysis of Human Pluripotency and Neural Specification by In-Depth (PhosphoProteomic Profiling

    Directory of Open Access Journals (Sweden)

    Ilyas Singec

    2016-09-01

    Full Text Available Controlled differentiation of human embryonic stem cells (hESCs can be utilized for precise analysis of cell type identities during early development. We established a highly efficient neural induction strategy and an improved analytical platform, and determined proteomic and phosphoproteomic profiles of hESCs and their specified multipotent neural stem cell derivatives (hNSCs. This quantitative dataset (nearly 13,000 proteins and 60,000 phosphorylation sites provides unique molecular insights into pluripotency and neural lineage entry. Systems-level comparative analysis of proteins (e.g., transcription factors, epigenetic regulators, kinase families, phosphorylation sites, and numerous biological pathways allowed the identification of distinct signatures in pluripotent and multipotent cells. Furthermore, as predicted by the dataset, we functionally validated an autocrine/paracrine mechanism by demonstrating that the secreted protein midkine is a regulator of neural specification. This resource is freely available to the scientific community, including a searchable website, PluriProt.

  10. iTRAQ-Based Quantitative Proteomics Identifies Potential Regulatory Proteins Involved in Chicken Eggshell Brownness.

    Directory of Open Access Journals (Sweden)

    Guangqi Li

    Full Text Available Brown eggs are popular in many countries and consumers regard eggshell brownness as an important indicator of egg quality. However, the potential regulatory proteins and detailed molecular mechanisms regulating eggshell brownness have yet to be clearly defined. In the present study, we performed quantitative proteomics analysis with iTRAQ technology in the shell gland epithelium of hens laying dark and light brown eggs to investigate the candidate proteins and molecular mechanisms underlying variation in chicken eggshell brownness. The results indicated 147 differentially expressed proteins between these two groups, among which 65 and 82 proteins were significantly up-regulated in the light and dark groups, respectively. Functional analysis indicated that in the light group, the down-regulated iron-sulfur cluster assembly protein (Iba57 would decrease the synthesis of protoporphyrin IX; furthermore, the up-regulated protein solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator, member 5 (SLC25A5 and down-regulated translocator protein (TSPO would lead to increased amounts of protoporphyrin IX transported into the mitochondria matrix to form heme with iron, which is supplied by ovotransferrin protein (TF. In other words, chickens from the light group produce less protoporphyrin IX, which is mainly used for heme synthesis. Therefore, the exported protoporphyrin IX available for eggshell deposition and brownness is reduced in the light group. The current study provides valuable information to elucidate variation of chicken eggshell brownness, and demonstrates the feasibility and sensitivity of iTRAQ-based quantitative proteomics analysis in providing useful insights into the molecular mechanisms underlying brown eggshell pigmentation.

  11. Simple preparation of plant epidermal tissue for laser microdissection and downstream quantitative proteome and carbohydrate analysis

    Directory of Open Access Journals (Sweden)

    Christian eFalter

    2015-03-01

    Full Text Available The outwardly directed cell wall and associated plasma membrane of epidermal cells represent the first layers of plant defense against intruding pathogens. Cell wall modifications and the formation of defense structures at sites of attempted pathogen penetration are decisive for plant defense. A precise isolation of these stress-induced structures would allow a specific analysis of regulatory mechanism and cell wall adaption. However, methods for large-scale epidermal tissue preparation from the model plant Arabidopsis thaliana, which would allow proteome and cell wall analysis of complete, laser-microdissected epidermal defense structures, have not been provided. We developed the adhesive tape – liquid cover glass technique for simple leaf epidermis preparation from A. thaliana, which is also applicable on grass leaves. This method is compatible with subsequent staining techniques to visualize stress-related cell wall structures, which were precisely isolated from the epidermal tissue layer by laser microdissection coupled to laser pressure catapulting. We successfully demonstrated that these specific epidermal tissue samples could be used for quantitative downstream proteome and cell wall analysis. The development of the adhesive tape – liquid cover glass technique for simple leaf epidermis preparation and the compatibility to laser microdissection and downstream quantitative analysis opens new possibilities in the precise examination of stress- and pathogen-related cell wall structures in epidermal cells. Because the developed tissue processing is also applicable on A. thaliana, well-established, model pathosystems that include the interaction with powdery mildews can be studied to determine principal regulatory mechanisms in plant-microbe interaction with their potential outreach into crop breeding.

  12. Improving data quality and preserving HCD-generated reporter ions with EThcD for isobaric tag-based quantitative proteomics and proteome-wide PTM studies

    International Nuclear Information System (INIS)

    Yu, Qing; Shi, Xudong; Feng, Yu; Kent, K. Craig; Li, Lingjun

    2017-01-01

    Mass spectrometry (MS)-based isobaric labeling has undergone rapid development in recent years due to its capability for high throughput quantitation. Apart from its originally designed use with collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD), isobaric tagging technique could also work with electron-transfer dissociation (ETD), which provides complementarity to CID and is preferred in sequencing peptides with post-translational modifications (PTMs). However, ETD suffers from long reaction time, reduced duty cycle and bias against peptides with lower charge states. In addition, common fragmentation mechanism in ETD results in altered reporter ion production, decreased multiplexing capability, and even loss of quantitation capability for some of the isobaric tags, including custom-designed dimethyl leucine (DiLeu) tags. Here, we demonstrate a novel electron-transfer/higher-energy collision dissociation (EThcD) approach that preserves original reporter ion channels, mitigates bias against lower charge states, improves sensitivity, and significantly improves data quality for quantitative proteomics and proteome-wide PTM studies. Systematic optimization was performed to achieve a balance between data quality and sensitivity. We provide direct comparison of EThcD with ETD and HCD for DiLeu- and TMT-labeled HEK cell lysate and IMAC enriched phosphopeptides. Results demonstrate improved data quality and phosphorylation localization accuracy while preserving sufficient reporter ion production. Biological studies were performed to investigate phosphorylation changes in a mouse vascular smooth muscle cell line treated with four different conditions. Overall, EThcD exhibits superior performance compared to conventional ETD and offers distinct advantages compared to HCD in isobaric labeling based quantitative proteomics and quantitative PTM studies. - Highlights: • EThcD was optimized for isobaric tag-labeled peptides for quantitative

  13. Improving data quality and preserving HCD-generated reporter ions with EThcD for isobaric tag-based quantitative proteomics and proteome-wide PTM studies

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Qing [School of Pharmacy, University of Wisconsin, Madison, WI 53705 (United States); Shi, Xudong [Department of Surgery, School of Medicine and Public Health, University of Wisconsin, Madison, WI 53705 (United States); Feng, Yu [School of Pharmacy, University of Wisconsin, Madison, WI 53705 (United States); Kent, K. Craig [Department of Surgery, School of Medicine and Public Health, University of Wisconsin, Madison, WI 53705 (United States); Li, Lingjun, E-mail: lingjun.li@wisc.edu [School of Pharmacy, University of Wisconsin, Madison, WI 53705 (United States); Department of Chemistry, University of Wisconsin, Madison, WI 53706 (United States)

    2017-05-22

    Mass spectrometry (MS)-based isobaric labeling has undergone rapid development in recent years due to its capability for high throughput quantitation. Apart from its originally designed use with collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD), isobaric tagging technique could also work with electron-transfer dissociation (ETD), which provides complementarity to CID and is preferred in sequencing peptides with post-translational modifications (PTMs). However, ETD suffers from long reaction time, reduced duty cycle and bias against peptides with lower charge states. In addition, common fragmentation mechanism in ETD results in altered reporter ion production, decreased multiplexing capability, and even loss of quantitation capability for some of the isobaric tags, including custom-designed dimethyl leucine (DiLeu) tags. Here, we demonstrate a novel electron-transfer/higher-energy collision dissociation (EThcD) approach that preserves original reporter ion channels, mitigates bias against lower charge states, improves sensitivity, and significantly improves data quality for quantitative proteomics and proteome-wide PTM studies. Systematic optimization was performed to achieve a balance between data quality and sensitivity. We provide direct comparison of EThcD with ETD and HCD for DiLeu- and TMT-labeled HEK cell lysate and IMAC enriched phosphopeptides. Results demonstrate improved data quality and phosphorylation localization accuracy while preserving sufficient reporter ion production. Biological studies were performed to investigate phosphorylation changes in a mouse vascular smooth muscle cell line treated with four different conditions. Overall, EThcD exhibits superior performance compared to conventional ETD and offers distinct advantages compared to HCD in isobaric labeling based quantitative proteomics and quantitative PTM studies. - Highlights: • EThcD was optimized for isobaric tag-labeled peptides for quantitative

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

    International Nuclear Information System (INIS)

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

    2015-01-01

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

  15. Quality Assessments of Long-Term Quantitative Proteomic Analysis of Breast Cancer Xenograft Tissues

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Jian-Ying; Chen, Lijun; Zhang, Bai; Tian, Yuan; Liu, Tao; Thomas, Stefani N.; Chen, Li; Schnaubelt, Michael; Boja, Emily; Hiltket, Tara; Kinsinger, Christopher; Rodriguez, Henry; Davies, Sherri; Li, Shunqiang; Snider, Jacqueline E.; Erdmann-Gilmore, Petra; Tabb, David L.; Townsend, Reid; Ellis, Matthew; Rodland, Karin D.; Smith, Richard D.; Carr, Steven A.; Zhang, Zhen; Chan, Daniel W.; Zhang, Hui

    2017-09-21

    The identification of protein biomarkers requires large-scale analysis of human specimens to achieve statistical significance. In this study, we evaluated the long-term reproducibility of an iTRAQ (isobaric tags for relative and absolute quantification) based quantitative proteomics strategy using one channel for universal normalization across all samples. A total of 307 liquid chromatography tandem mass spectrometric (LC-MS/MS) analyses were completed, generating 107 one-dimensional (1D) LC-MS/MS datasets and 8 offline two-dimensional (2D) LC-MS/MS datasets (25 fractions for each set) for human-in-mouse breast cancer xenograft tissues representative of basal and luminal subtypes. Such large-scale studies require the implementation of robust metrics to assess the contributions of technical and biological variability in the qualitative and quantitative data. Accordingly, we developed a quantification confidence score based on the quality of each peptide-spectrum match (PSM) to remove quantification outliers from each analysis. After combining confidence score filtering and statistical analysis, reproducible protein identification and quantitative results were achieved from LC-MS/MS datasets collected over a 16 month period.

  16. Quantitative proteomic characterization of the lung extracellular matrix in chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis.

    Science.gov (United States)

    Åhrman, Emma; Hallgren, Oskar; Malmström, Lars; Hedström, Ulf; Malmström, Anders; Bjermer, Leif; Zhou, Xiao-Hong; Westergren-Thorsson, Gunilla; Malmström, Johan

    2018-03-01

    Remodeling of the extracellular matrix (ECM) is a common feature in lung diseases such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Here, we applied a sequential tissue extraction strategy to describe disease-specific remodeling of human lung tissue in disease, using end-stages of COPD and IPF. Our strategy was based on quantitative comparison of the disease proteomes, with specific focus on the matrisome, using data-independent acquisition and targeted data analysis (SWATH-MS). Our work provides an in-depth proteomic characterization of human lung tissue during impaired tissue remodeling. In addition, we show important quantitative and qualitative effects of the solubility of matrisome proteins. COPD was characterized by a disease-specific increase in ECM regulators, metalloproteinase inhibitor 3 (TIMP3) and matrix metalloproteinase 28 (MMP-28), whereas for IPF, impairment in cell adhesion proteins, such as collagen VI and laminins, was most prominent. For both diseases, we identified increased levels of proteins involved in the regulation of endopeptidase activity, with several proteins belonging to the serpin family. The established human lung quantitative proteome inventory and the construction of a tissue-specific protein assay library provides a resource for future quantitative proteomic analyses of human lung tissues. We present a sequential tissue extraction strategy to determine changes in extractability of matrisome proteins in end-stage COPD and IPF compared to healthy control tissue. Extensive quantitative analysis of the proteome changes of the disease states revealed altered solubility of matrisome proteins involved in ECM regulators and cell-ECM communication. The results highlight disease-specific remodeling mechanisms associated with COPD and IPF. Copyright © 2018 Elsevier B.V. All rights reserved.

  17. AUTOMATED ANALYSIS OF QUANTITATIVE IMAGE DATA USING ISOMORPHIC FUNCTIONAL MIXED MODELS, WITH APPLICATION TO PROTEOMICS DATA.

    Science.gov (United States)

    Morris, Jeffrey S; Baladandayuthapani, Veerabhadran; Herrick, Richard C; Sanna, Pietro; Gutstein, Howard

    2011-01-01

    Image data are increasingly encountered and are of growing importance in many areas of science. Much of these data are quantitative image data, which are characterized by intensities that represent some measurement of interest in the scanned images. The data typically consist of multiple images on the same domain and the goal of the research is to combine the quantitative information across images to make inference about populations or interventions. In this paper, we present a unified analysis framework for the analysis of quantitative image data using a Bayesian functional mixed model approach. This framework is flexible enough to handle complex, irregular images with many local features, and can model the simultaneous effects of multiple factors on the image intensities and account for the correlation between images induced by the design. We introduce a general isomorphic modeling approach to fitting the functional mixed model, of which the wavelet-based functional mixed model is one special case. With suitable modeling choices, this approach leads to efficient calculations and can result in flexible modeling and adaptive smoothing of the salient features in the data. The proposed method has the following advantages: it can be run automatically, it produces inferential plots indicating which regions of the image are associated with each factor, it simultaneously considers the practical and statistical significance of findings, and it controls the false discovery rate. Although the method we present is general and can be applied to quantitative image data from any application, in this paper we focus on image-based proteomic data. We apply our method to an animal study investigating the effects of opiate addiction on the brain proteome. Our image-based functional mixed model approach finds results that are missed with conventional spot-based analysis approaches. In particular, we find that the significant regions of the image identified by the proposed method

  18. Proteome reference map of Lactobacillus acidophilus NCFM and quantitative proteomics towards understanding the prebiotic action of lactitol

    DEFF Research Database (Denmark)

    Majumder, Avishek; Sultan, Abida; Jersie-Christensen, Rosa Rakownikow

    2011-01-01

    subunit, galactokinase, galactose‐1‐phosphate uridylyltransferase and UDP‐glucose‐4‐epimerase, which all are potentially involved in lactitol metabolism. This first comprehensive proteome analysis of L. acidophilus NCFM provides insights into protein abundance changes elicited by the prebiotic lactitol....

  19. Dataset for the proteomic inventory and quantitative analysis of the breast cancer hypoxic secretome associated with osteotropism

    DEFF Research Database (Denmark)

    Cox, T.R.; Schoof, Erwin; Gartland, A.

    2015-01-01

    secretomes are known to be active mediators of both local and distant host cells and play an important role in the progression and dissemination of cancer. Here we have quantitatively profiled both the composition of breast cancer secretomes associated with osteotropism, and their modulation under normoxic...... and hypoxic conditions. We detect and quantify 162 secretome proteins across all conditions which show differential hypoxic induction and association with osteotropism. Mass Spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD000397...

  20. Quantitative 3D imaging of yeast by hard X-ray tomography.

    Science.gov (United States)

    Zheng, Ting; Li, Wenjie; Guan, Yong; Song, Xiangxia; Xiong, Ying; Liu, Gang; Tian, Yangchao

    2012-05-01

    Full-field hard X-ray tomography could be used to obtain three-dimensional (3D) nanoscale structures of biological samples. The image of the fission yeast, Schizosaccharomyces pombe, was clearly visualized based on Zernike phase contrast imaging technique and heavy metal staining method at a spatial resolution better than 50 nm at the energy of 8 keV. The distributions and shapes of the organelles during the cell cycle were clearly visualized and two types of organelle were distinguished. The results for cells during various phases were compared and the ratios of organelle volume to cell volume can be analyzed quantitatively. It showed that the ratios remained constant between growth and division phase and increased strongly in stationary phase, following the shape and size of two types of organelles changes. Our results demonstrated that hard X-ray microscopy was a complementary method for imaging and revealing structural information for biological samples. Copyright © 2011 Wiley Periodicals, Inc.

  1. Recent advances on multidimensional liquid chromatography–mass spectrometry for proteomics: From qualitative to quantitative analysis—A review

    International Nuclear Information System (INIS)

    Wu Qi; Yuan Huiming; Zhang Lihua; Zhang Yukui

    2012-01-01

    Highlights: ► We discuss progress of MDLC–MS systems in qualitative and quantitative proteomics. ► Both “Top-down” and “bottom-up” strategies are discussed in detail. ► On-line integrations of stable isotope labeling process are highlighted. ► This review gives insights into further directions for higher level integration. - Abstract: With the acceleration of proteome research, increasing attention has been paid to multidimensional liquid chromatography–mass spectrometry (MDLC–MS) due to its high peak capacity and separation efficiency. Recently, many efforts have been put to improve MDLC-based strategies including “top-down” and “bottom-up” to enable highly sensitive qualitative and quantitative analysis of proteins, as well as accelerate the whole analytical procedure. Integrated platforms with combination of sample pretreatment, multidimensional separations and identification were also developed to achieve high throughput and sensitive detection of proteomes, facilitating highly accurate and reproducible quantification. This review summarized the recent advances of such techniques and their applications in qualitative and quantitative analysis of proteomes.

  2. Dataset for the proteomic inventory and quantitative analysis of the breast cancer hypoxic secretome associated with osteotropism

    Directory of Open Access Journals (Sweden)

    Thomas R. Cox

    2015-12-01

    Full Text Available The cancer secretome includes all of the macromolecules secreted by cells into their microenvironment. Cancer cell secretomes are significantly different to that of normal cells reflecting the changes that normal cells have undergone during their transition to malignancy. More importantly, cancer secretomes are known to be active mediators of both local and distant host cells and play an important role in the progression and dissemination of cancer. Here we have quantitatively profiled both the composition of breast cancer secretomes associated with osteotropism, and their modulation under normoxic and hypoxic conditions. We detect and quantify 162 secretome proteins across all conditions which show differential hypoxic induction and association with osteotropism. Mass Spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD000397 and the complete proteomic, bioinformatic and biological analyses are reported in Cox et al. (2015 [1].

  3. Quantitative proteomic analysis of the rice (Oryza sativa L. salt response.

    Directory of Open Access Journals (Sweden)

    Jianwen Xu

    Full Text Available Salt stress is one of most serious limiting factors for crop growth and production. An isobaric Tags for Relative and Absolute Quantitation (iTRAQ approach was used to analyze proteomic changes in rice shoots under salt stress in this study. A total of 56 proteins were significantly altered and 16 of them were enriched in the pathways of photosynthesis, antioxidant and oxidative phosphorylation. Among these 16 proteins, peroxiredoxin Q and photosystem I subunit D were up-regulated, while thioredoxin M-like, thioredoxin x, thioredoxin peroxidase, glutathione S-transferase F3, PSI subunit H, light-harvesting antenna complex I subunits, chloroplast chaperonin, vacuolar ATP synthase subunit H, and ATP synthase delta chain were down-regulated. Moreover, physiological data including total antioxidant capacity, peroxiredoxin activity, chlorophyll a/b content, glutathione S-transferase activity, reduced glutathione content and ATPase activity were consistent with changes in the levels of these proteins. The levels of the mRNAs encoding these proteins were also analyzed by real-time quantitative reverse transcription PCR, and approximately 86% of the results were consistent with the iTRAQ data. Importantly, our data suggest the important role of PSI in balancing energy supply and ROS generation under salt stress. This study provides information for an improved understanding of the function of photosynthesis and PSI in the salt-stress response of rice.

  4. Quantitative evaluation of the mitochondrial proteomes of Drosophila melanogaster adapted to extreme oxygen conditions.

    Directory of Open Access Journals (Sweden)

    Songyue Yin

    Full Text Available Mitochondria are the primary organelles that consume oxygen and provide energy for cellular activities. To investigate the mitochondrial mechanisms underlying adaptation to extreme oxygen conditions, we generated Drosophila strains that could survive in low- or high-oxygen environments (LOF or HOF, respectively, examined their mitochondria at the ultrastructural level via transmission electron microscopy, studied the activity of their respiratory chain complexes, and quantitatively analyzed the protein abundance responses of the mitochondrial proteomes using Isobaric tag for relative and absolute quantitation (iTRAQ. A total of 718 proteins were identified with high confidence, and 55 and 75 mitochondrial proteins displayed significant differences in abundance in LOF and HOF, respectively, compared with the control flies. Importantly, these differentially expressed mitochondrial proteins are primarily involved in respiration, calcium regulation, the oxidative response, and mitochondrial protein translation. A correlation analysis of the changes in the levels of the mRNAs corresponding to differentially regulated mitochondrial proteins revealed two sets of proteins with different modes of regulation (transcriptional vs. post-transcriptional in both LOF and HOF. We believe that these findings will not only enhance our understanding of the mechanisms underlying adaptation to extreme oxygen conditions in Drosophila but also provide a clue in studying human disease induced by altered oxygen tension in tissues and cells.

  5. Determining protein complex connectivity using a probabilistic deletion network derived from quantitative proteomics.

    Science.gov (United States)

    Sardiu, Mihaela E; Gilmore, Joshua M; Carrozza, Michael J; Li, Bing; Workman, Jerry L; Florens, Laurence; Washburn, Michael P

    2009-10-06

    Protein complexes are key molecular machines executing a variety of essential cellular processes. Despite the availability of genome-wide protein-protein interaction studies, determining the connectivity between proteins within a complex remains a major challenge. Here we demonstrate a method that is able to predict the relationship of proteins within a stable protein complex. We employed a combination of computational approaches and a systematic collection of quantitative proteomics data from wild-type and deletion strain purifications to build a quantitative deletion-interaction network map and subsequently convert the resulting data into an interdependency-interaction model of a complex. We applied this approach to a data set generated from components of the Saccharomyces cerevisiae Rpd3 histone deacetylase complexes, which consists of two distinct small and large complexes that are held together by a module consisting of Rpd3, Sin3 and Ume1. The resulting representation reveals new protein-protein interactions and new submodule relationships, providing novel information for mapping the functional organization of a complex.

  6. Determining protein complex connectivity using a probabilistic deletion network derived from quantitative proteomics.

    Directory of Open Access Journals (Sweden)

    Mihaela E Sardiu

    2009-10-01

    Full Text Available Protein complexes are key molecular machines executing a variety of essential cellular processes. Despite the availability of genome-wide protein-protein interaction studies, determining the connectivity between proteins within a complex remains a major challenge. Here we demonstrate a method that is able to predict the relationship of proteins within a stable protein complex. We employed a combination of computational approaches and a systematic collection of quantitative proteomics data from wild-type and deletion strain purifications to build a quantitative deletion-interaction network map and subsequently convert the resulting data into an interdependency-interaction model of a complex. We applied this approach to a data set generated from components of the Saccharomyces cerevisiae Rpd3 histone deacetylase complexes, which consists of two distinct small and large complexes that are held together by a module consisting of Rpd3, Sin3 and Ume1. The resulting representation reveals new protein-protein interactions and new submodule relationships, providing novel information for mapping the functional organization of a complex.

  7. The Arabidopsis thaliana Cyclic-Nucleotide-Dependent Response – a Quantitative Proteomic and Phosphoproteomic Analysis

    KAUST Repository

    Alqurashi, May M.

    2013-11-01

    Protein phosphorylation governs many regulatory pathways and an increasing number of kinases, proteins that transfer phosphate groups, are in turn activated by cyclic nucleotides. One of the cyclic nucleotides, cyclic adenosine monophosphate (cAMP), has been shown to be a second messenger in abiotic and biotic stress responses. However, little is known about the precise role of cAMP in plants and in the down-stream activation of kinases, and hence cAMP-dependent phosphorylation. To increase our understanding of the role of cAMP, proteomic and phosphoproteomic profiles of Arabidopsis thaliana suspension culture cells were analyzed before and after treatment of cells with two different concentrations of 8-Bromo-cAMP (1 µM and 100 nM) and over a time-course of one hour. A comparative quantitative analysis was undertaken using two- dimensional gel electrophoresis and the Delta 2D software (DECODON) followed by protein spot identification by tandem mass spectrometry combined with Mascot and Scaffold. Differentially expressed proteins and regulated phosphoproteins were categorized according to their biological function using bioinformatics tools. The results revealed that the treatment with 1 µM and 100 nM 8-Bromo-cAMP was sufficient to induce specific concentration- and time-dependent changes at the proteome and phosphoproteome levels. In particular, different phosphorylation patterns were observed overtime preferentially affecting proteins in a number of functional categories, notably phosphatases, proteins that remove phosphate groups. This suggests that cAMP both transiently activates and deactivates proteins through specific phosphorylation events and provides new insight into biological mechanisms and functions at the systems level.

  8. Quantitative Proteomics Reveals the Regulatory Networks of Circular RNA CDR1as in Hepatocellular Carcinoma Cells.

    Science.gov (United States)

    Yang, Xue; Xiong, Qian; Wu, Ying; Li, Siting; Ge, Feng

    2017-10-06

    Circular RNAs (circRNAs), a class of widespread endogenous RNAs, play crucial roles in diverse biological processes and are potential biomarkers in diverse human diseases and cancers. Cerebellar-degeneration-related protein 1 antisense RNA (CDR1as), an oncogenic circRNA, is involved in human tumorigenesis and is dysregulated in hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying CDR1as functions in HCC remain unclear. Here we explored the functions of CDR1as and searched for CDR1as-regulated proteins in HCC cells. A quantitative proteomics strategy was employed to globally identify CDR1as-regulated proteins in HCC cells. In total, we identified 330 differentially expressed proteins (DEPs) upon enhanced CDR1as expression in HepG2 cells, indicating that they could be proteins regulated by CDR1as. Bioinformatic analysis revealed that many DEPs were involved in cell proliferation and the cell cycle. Further functional studies of epidermal growth factor receptor (EGFR) found that CDR1as exerts its effects on cell proliferation at least in part through the regulation of EGFR expression. We further confirmed that CDR1as could inhibit the expression of microRNA-7 (miR-7). EGFR is a validated target of miR-7; therefore, CDR1as may exert its function by regulating EGFR expression via targeting miR-7 in HCC cells. Taken together, we revealed novel functions and underlying mechanisms of CDR1as in HCC cells. This study serves as the first proteome-wide analysis of a circRNA-regulated protein in cells and provides a reliable and highly efficient method for globally identifying circRNA-regulated proteins.

  9. Quantitative Proteomics Analysis Identifies Mitochondria as Therapeutic Targets of Multidrug-Resistance in Ovarian Cancer

    Science.gov (United States)

    Chen, Xiulan; Wei, Shasha; Ma, Ying; Lu, Jie; Niu, Gang; Xue, Yanhong; Chen, Xiaoyuan; Yang, Fuquan

    2014-01-01

    Doxorubicin is a widely used chemotherapeutic agent for the treatment of a variety of solid tumors. However, resistance to this anticancer drug is a major obstacle to the effective treatment of tumors. As mitochondria play important roles in cell life and death, we anticipate that mitochondria may be related to drug resistance. Here, stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomic strategy was applied to compare mitochondrial protein expression in doxorubicin sensitive OVCAR8 cells and its doxorubicin-resistant variant NCI_ADR/RES cells. A total of 2085 proteins were quantified, of which 122 proteins displayed significant changes in the NCI_ADR/RES cells. These proteins participated in a variety of cell processes including cell apoptosis, substance metabolism, transport, detoxification and drug metabolism. Then qRT-PCR and western blot were applied to validate the differentially expressed proteins quantified by SILAC. Further functional studies with RNAi demonstrated TOP1MT, a mitochondrial protein participated in DNA repair, was involved in doxorubicin resistance in NCI_ADR/RES cells. Besides the proteomic study, electron microscopy and fluorescence analysis also observed that mitochondrial morphology and localization were greatly altered in NCI_ADR/RES cells. Mitochondrial membrane potential was also decreased in NCI_ADR/RES cells. All these results indicate that mitochondrial function is impaired in doxorubicin-resistant cells and mitochondria play an important role in doxorubicin resistance. This research provides some new information about doxorubicin resistance, indicating that mitochondria could be therapeutic targets of doxorubicin resistance in ovarian cancer cells. PMID:25285166

  10. Quantitative Estimation of Yeast on Maxillary Denture in Patients with Denture Stomatitis and the Effect of Chlorhexidine Gluconate in Reduction of Yeast

    Directory of Open Access Journals (Sweden)

    Jaykumar R Gade

    2011-01-01

    Full Text Available Denture stomatitis is a condition associated with wearing of a denture. The predisposing factor leading to denture stomatitis could be poor oral hygiene, ill-fitting denture and relief areas. Around 30 patients with denture stomatitis were advised to rinse with chlorhexidine gluconate mouthwash for 14 days and were directed to immerse the upper denture in the chlorhexidine solution for 8 hours. The samples were collected by scraping maxillary denture in saline at three intervals, prior to, at the end of 24 hours and after 14 days of treatment, then were inoculated and quantitative estimation of the yeast growth on Sabouraud′s dextrose agar plate was done. It was observed that after a period of 14 days, there was a reduction in the growth of yeast and also improvement in the clinical picture of the oral mucosa

  11. A qualitative and quantitative evaluation of the peptide characteristics of microwave- and ultrasound-assisted digestion in discovery and targeted proteomic analyses.

    Science.gov (United States)

    Guo, Zhengguang; Cheng, Jie; Sun, Haidan; Sun, Wei

    2017-08-30

    Fast digestion methods can dramatically accelerate enzyme digestion and increase the throughput of proteomic analysis. However, the peptide characteristics of fast digestion methods and their performance in discovery and targeted proteomic analysis must be systematically evaluated. Three digestion methods, including overnight digestion, microwave-assisted protein enzymatic digestion (MAPED), and high-intensity focused ultrasonic-assisted enzymatic digestion (HIFUSAED), in trypsin or in trypsin/Lys-C were comprehensively compared in both discovery and targeted proteomics analysis using the HeLa cell proteome. In discovery proteomic analysis, the highest numbers of peptides and proteins were identified when the sample was digested via the MAPED method with trypsin/Lys-C. The fast digestion methods showed a higher mis-cleavage rate and a lower semi-tryptic rate than the overnight digestion method. In both label-free quantitative analysis and targeted proteomic analysis, both fully cleaved peptides (FCPs) and mis-cleaved peptides (MCPs) from the fast digestion methods and the overnight digestion method showed good reproducibility if they showed good abundance. When both the FCPs and MCPs were included in the analysis, the MAPED with trypsin/Lys-C method showed the best results for both discovery proteomic analysis and relative quantitative targeted proteomic analysis. These results will be beneficial for the application of fast digestion methods to proteomics. Copyright © 2017 John Wiley & Sons, Ltd.

  12. PANDA-view: An easy-to-use tool for statistical analysis and visualization of quantitative proteomics data.

    Science.gov (United States)

    Chang, Cheng; Xu, Kaikun; Guo, Chaoping; Wang, Jinxia; Yan, Qi; Zhang, Jian; He, Fuchu; Zhu, Yunping

    2018-05-22

    Compared with the numerous software tools developed for identification and quantification of -omics data, there remains a lack of suitable tools for both downstream analysis and data visualization. To help researchers better understand the biological meanings in their -omics data, we present an easy-to-use tool, named PANDA-view, for both statistical analysis and visualization of quantitative proteomics data and other -omics data. PANDA-view contains various kinds of analysis methods such as normalization, missing value imputation, statistical tests, clustering and principal component analysis, as well as the most commonly-used data visualization methods including an interactive volcano plot. Additionally, it provides user-friendly interfaces for protein-peptide-spectrum representation of the quantitative proteomics data. PANDA-view is freely available at https://sourceforge.net/projects/panda-view/. 1987ccpacer@163.com and zhuyunping@gmail.com. Supplementary data are available at Bioinformatics online.

  13. Quantitative proteomics reveals the mechanism and consequence of gliotoxin-mediated dysregulation of the methionine cycle in Aspergillus niger

    OpenAIRE

    Manzanares-Miralles, Lara; Bayram, Ozgur; Sarikaya-Bayram, Ozlem; Smith, Elizabeth B.; Dolan, Stephen K.; Jones, Gary W.; Doyle, Sean

    2016-01-01

    Gliotoxin (GT) is a redox-active metabolite, produced by Aspergillus fumigatus,which inhibits the growth of other fungi. Here we demonstrate how Aspergillus niger responds to GT exposure. Quantitative proteomics revealed that GT dysregulated the abundance of 378 proteins including those involved in methionine metabolism and induced de novo abundance of two S-adenosylmethionine (SAM)-dependent methyltransferases. Increased abundance of enzymes S-adenosylhomocysteinase (p = 0.0018) ...

  14. Deep proteomics of mouse skeletal muscle enables quantitation of protein isoforms, metabolic pathways, and transcription factors.

    Science.gov (United States)

    Deshmukh, Atul S; Murgia, Marta; Nagaraj, Nagarjuna; Treebak, Jonas T; Cox, Jürgen; Mann, Matthias

    2015-04-01

    Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging because of highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art MS workflow and a strategy to map identifications from the C2C12 cell line model to tissues, we identified a total of 10,218 proteins, including skeletal muscle specific transcription factors like myod1 and myogenin and circadian clock proteins. We obtain absolute abundances for proteins expressed in a muscle cell line and skeletal muscle, which should serve as a valuable resource. Quantitation of protein isoforms of glucose uptake signaling pathways and in glucose and lipid metabolic pathways provides a detailed metabolic map of the cell line compared with tissue. This revealed unexpectedly complex regulation of AMP-activated protein kinase and insulin signaling in muscle tissue at the level of enzyme isoforms. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Quantitative Proteomic Profiling the Molecular Signatures of Annexin A5 in Lung Squamous Carcinoma Cells.

    Science.gov (United States)

    Sun, Bing; Bai, Yuxin; Zhang, Liyuan; Gong, Linlin; Qi, Xiaoyu; Li, Huizhen; Wang, Faming; Chi, Xinming; Jiang, Yulin; Shao, Shujuan

    Lung cancer remains the leading cancer killer around the world. It's crucial to identify newer mechanism-based targets to effectively manage lung cancer. Annexin A5 (ANXA5) is a protein kinase C inhibitory protein and calcium dependent phospholipid-binding protein, which may act as an endogenous regulator of various pathophysiological processes. However, its molecular mechanism in lung cancer remains poorly understood. This study was designed to determine the mechanism of ANXA5 in lung cancer with a hope to obtain useful information to provide a new therapeutic target. We used a stable isotope dimethyl labeling based quantitative proteomic method to identify differentially expressed proteins in NSCLC cell lines after ANXA5 transfection. Out of 314 proteins, we identified 26 and 44 proteins that were down- and up-regulated upon ANXA5 modulation, respectively. The IPA analysis revealed that glycolysis and gluconeogenesis were the predominant pathways modulated by ANXA5. Multiple central nodes, namely HSPA5, FN1, PDIA6, ENO1, ALDOA, JUP and KRT6A appeared to occupy regulatory nodes in the protein-protein networks upon ANXA5 modulation. Taken together, ANXA5 appears to have pleotropic effects, as it modulates multiple key signaling pathways, supporting the potential usefulness of ANXA5 as a potential target in lung cancer. This study might provide a new insight into the mechanism of ANXA5 in lung cancer.

  16. Quantitative Proteomic Profiling the Molecular Signatures of Annexin A5 in Lung Squamous Carcinoma Cells.

    Directory of Open Access Journals (Sweden)

    Bing Sun

    Full Text Available Lung cancer remains the leading cancer killer around the world. It's crucial to identify newer mechanism-based targets to effectively manage lung cancer. Annexin A5 (ANXA5 is a protein kinase C inhibitory protein and calcium dependent phospholipid-binding protein, which may act as an endogenous regulator of various pathophysiological processes. However, its molecular mechanism in lung cancer remains poorly understood. This study was designed to determine the mechanism of ANXA5 in lung cancer with a hope to obtain useful information to provide a new therapeutic target. We used a stable isotope dimethyl labeling based quantitative proteomic method to identify differentially expressed proteins in NSCLC cell lines after ANXA5 transfection. Out of 314 proteins, we identified 26 and 44 proteins that were down- and up-regulated upon ANXA5 modulation, respectively. The IPA analysis revealed that glycolysis and gluconeogenesis were the predominant pathways modulated by ANXA5. Multiple central nodes, namely HSPA5, FN1, PDIA6, ENO1, ALDOA, JUP and KRT6A appeared to occupy regulatory nodes in the protein-protein networks upon ANXA5 modulation. Taken together, ANXA5 appears to have pleotropic effects, as it modulates multiple key signaling pathways, supporting the potential usefulness of ANXA5 as a potential target in lung cancer. This study might provide a new insight into the mechanism of ANXA5 in lung cancer.

  17. 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.

  18. Quantitative proteomics identify molecular targets that are crucial in larval settlement and metamorphosis of bugula neritina

    KAUST Repository

    Zhang, Huoming

    2011-01-07

    The marine invertebrate Bugula neritina has a biphasic life cycle that consists of a swimming larval stage and a sessile juvenile and adult stage. The attachment of larvae to the substratum and their subsequent metamorphosis have crucial ecological consequences. Despite many studies on this species, little is known about the molecular mechanism of these processes. Here, we report a comparative study of swimming larvae and metamorphosing individuals at 4 and 24 h postattachment using label-free quantitative proteomics. We identified more than 1100 proteins at each stage, 61 of which were differentially expressed. Specifically, proteins involved in energy metabolism and structural molecules were generally down-regulated, whereas proteins involved in transcription and translation, the extracellular matrix, and calcification were strongly up-regulated during metamorphosis. Many tightly regulated novel proteins were also identified. Subsequent analysis of the temporal and spatial expressions of some of the proteins and an assay of their functions indicated that they may have key roles in metamorphosis of B. neritina. These findings not only provide molecular evidence with which to elucidate the substantial changes in morphology and physiology that occur during larval attachment and metamorphosis but also identify potential targets for antifouling treatment. © 2011 American Chemical Society.

  19. Quantitative Proteomic Analysis of Serum Exosomes from Patients with Locally Advanced Pancreatic Cancer Undergoing Chemoradiotherapy.

    Science.gov (United States)

    An, Mingrui; Lohse, Ines; Tan, Zhijing; Zhu, Jianhui; Wu, Jing; Kurapati, Himabindu; Morgan, Meredith A; Lawrence, Theodore S; Cuneo, Kyle C; Lubman, David M

    2017-04-07

    Pancreatic cancer is the third leading cause of cancer-related death in the USA. Despite extensive research, minimal improvements in patient outcomes have been achieved. Early identification of treatment response and metastasis would be valuable to determine the appropriate therapeutic course for patients. In this work, we isolated exosomes from the serum of 10 patients with locally advanced pancreatic cancer at serial time points over a course of therapy, and quantitative analysis was performed using the iTRAQ method. We detected approximately 700-800 exosomal proteins per sample, several of which have been implicated in metastasis and treatment resistance. We compared the exosomal proteome of patients at different time points during treatment to healthy controls and identified eight proteins that show global treatment-specific changes. We then tested the effect of patient-derived exosomes on the migration of tumor cells and found that patient-derived exosomes, but not healthy controls, induce cell migration, supporting their role in metastasis. Our data show that exosomes can be reliably extracted from patient serum and analyzed for protein content. The differential loading of exosomes during a course of therapy suggests that exosomes may provide novel insights into the development of treatment resistance and metastasis.

  20. Quantitative comparison of a human cancer cell surface proteome between interphase and mitosis.

    Science.gov (United States)

    Özlü, Nurhan; Qureshi, Mohammad H; Toyoda, Yusuke; Renard, Bernhard Y; Mollaoglu, Gürkan; Özkan, Nazlı E; Bulbul, Selda; Poser, Ina; Timm, Wiebke; Hyman, Anthony A; Mitchison, Timothy J; Steen, Judith A

    2015-01-13

    The cell surface is the cellular compartment responsible for communication with the environment. The interior of mammalian cells undergoes dramatic reorganization when cells enter mitosis. These changes are triggered by activation of the CDK1 kinase and have been studied extensively. In contrast, very little is known of the cell surface changes during cell division. We undertook a quantitative proteomic comparison of cell surface-exposed proteins in human cancer cells that were tightly synchronized in mitosis or interphase. Six hundred and twenty-eight surface and surface-associated proteins in HeLa cells were identified; of these, 27 were significantly enriched at the cell surface in mitosis and 37 in interphase. Using imaging techniques, we confirmed the mitosis-selective cell surface localization of protocadherin PCDH7, a member of a family with anti-adhesive roles in embryos. We show that PCDH7 is required for development of full mitotic rounding pressure at the onset of mitosis. Our analysis provided basic information on how cell cycle progression affects the cell surface. It also provides potential pharmacodynamic biomarkers for anti-mitotic cancer chemotherapy. © 2014 The Authors.

  1. Label-Free Quantitative Proteomic Analysis of Harmless and Pathogenic Strains of Infectious Microalgae, Prototheca spp.

    Directory of Open Access Journals (Sweden)

    Jayaseelan Murugaiyan

    2016-12-01

    Full Text Available Microalgae of the genus Prototheca (P. spp are associated with rare algal infections of invertebrates termed protothecosis. Among the seven generally accepted species, P. zopfii genotype 2 (GT2 is associated with a severe form of bovine mastitis while P. blaschkeae causes the mild and sub-clinical form of mastitis. The reason behind the infectious nature of P. zopfii GT2, while genotype 1 (GT1 remains non-infectious, is not known. Therefore, in the present study we investigated the protein expression level difference between the genotypes of P. zopfii and P. blaschkeae. Cells were cultured to the mid-exponential phase, harvested, and processed for LC-MS analysis. Peptide data was acquired on an LTQ Orbitrap Velos, raw spectra were quantitatively analyzed with MaxQuant software and matching with the reference database of Chlorella variabilis and Auxenochlorella protothecoides resulted in the identification of 226 proteins. Comparison of an environmental strain with infectious strains resulted in the identification of 51 differentially expressed proteins related to carbohydrate metabolism, energy production and protein translation. The expression level of Hsp70 proteins and their role in the infectious process is worth further investigation. All mass spectrometry data are available via ProteomeXchange with identifier PXD005305.

  2. Quantitative proteome analysis reveals the correlation between endocytosis-associated proteins and hepatocellular carcinoma dedifferentiation.

    Science.gov (United States)

    Naboulsi, Wael; Bracht, Thilo; Megger, Dominik A; Reis, Henning; Ahrens, Maike; Turewicz, Michael; Eisenacher, Martin; Tautges, Stephanie; Canbay, Ali E; Meyer, Helmut E; Weber, Frank; Baba, Hideo A; Sitek, Barbara

    2016-11-01

    The majority of poorly differentiated hepatocellular carcinomas (HCCs) develop from well-differentiated tumors. Endocytosis is a cellular function which is likely to take part in this development due to its important role in regulating the abundances of vital signaling receptors. Here, we aimed to investigate the abundance of endocytosis-associated proteins in HCCs with various differentiation grades. Therefore, we analyzed 36 tissue specimens from HCC patients via LC-MS/MS-based label-free quantitative proteomics including 19 HCC tissue samples with different degrees of histological grades and corresponding non-tumorous tissue controls. As a result, 277 proteins were differentially regulated between well-differentiated tumors and controls. In moderately and poorly differentiated tumors, 278 and 1181 proteins, respectively, were significantly differentially regulated compared to non-tumorous tissue. We explored the regulated proteins based on their functions and identified thirty endocytosis-associated proteins, mostly overexpressed in poorly differentiated tumors. These included proteins that have been shown to be up-regulated in HCC like clathrin heavy chain-1 (CLTC) as well as unknown proteins, such as secretory carrier-associated membrane protein 3 (SCAMP3). The abundances of SCAMP3 and CLTC were immunohistochemically examined in tissue sections of 84 HCC patients. We demonstrate the novel association of several endocytosis-associated proteins, in particular, SCAMP3 with HCC progression. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Quantitative proteomics of the tonoplast reveals a role for glycolytic enzymes in salt tolerance.

    Science.gov (United States)

    Barkla, Bronwyn J; Vera-Estrella, Rosario; Hernández-Coronado, Marcela; Pantoja, Omar

    2009-12-01

    To examine the role of the tonoplast in plant salt tolerance and identify proteins involved in the regulation of transporters for vacuolar Na(+) sequestration, we exploited a targeted quantitative proteomics approach. Two-dimensional differential in-gel electrophoresis analysis of free flow zonal electrophoresis separated tonoplast fractions from control, and salt-treated Mesembryanthemum crystallinum plants revealed the membrane association of glycolytic enzymes aldolase and enolase, along with subunits of the vacuolar H(+)-ATPase V-ATPase. Protein blot analysis confirmed coordinated salt regulation of these proteins, and chaotrope treatment indicated a strong tonoplast association. Reciprocal coimmunoprecipitation studies revealed that the glycolytic enzymes interacted with the V-ATPase subunit B VHA-B, and aldolase was shown to stimulate V-ATPase activity in vitro by increasing the affinity for ATP. To investigate a physiological role for this association, the Arabidopsis thaliana cytoplasmic enolase mutant, los2, was characterized. These plants were salt sensitive, and there was a specific reduction in enolase abundance in the tonoplast from salt-treated plants. Moreover, tonoplast isolated from mutant plants showed an impaired ability for aldolase stimulation of V-ATPase hydrolytic activity. The association of glycolytic proteins with the tonoplast may not only channel ATP to the V-ATPase, but also directly upregulate H(+)-pump activity.

  4. Proteomic analysis of astrocytic secretion that regulates neurogenesis using quantitative amine-specific isobaric tagging

    Energy Technology Data Exchange (ETDEWEB)

    Yan, Hu; Zhou, Wenhao [Children' s Hospital of Fudan University, 399 Wanyuan Road, Shanghai 201102 (China); Wei, Liming; Zhong, Fan [Institutes of Biomedical Sciences, Fudan University, 138 Yixueyuan Roda, Shanghai 200032 (China); Yang, Yi, E-mail: yyang@shmu.edu.cn [Children' s Hospital of Fudan University, 399 Wanyuan Road, Shanghai 201102 (China)

    2010-01-08

    Astrocytes are essential components of neurogenic niches that affect neurogenesis through membrane association and/or the release of soluble factors. To identify factors released from astrocytes that could regulate neural stem cell differentiation and proliferation, we used mild oxygen-glucose deprivation (OGD) to inhibit the secretory capacity of astrocytes. Using the Transwell co-culture system, we found that OGD-treated astrocytes could not promote neural stem cell differentiation and proliferation. Next, isobaric tagging for the relative and absolute quantitation (iTRAQ) proteomics techniques was performed to identify the proteins in the supernatants of astrocytes (with or without OGD). Through a multi-step analysis and gene ontology classification, 130 extracellular proteins were identified, most of which were involved in neuronal development, the inflammatory response, extracellular matrix composition and supportive functions. Of these proteins, 44 had never been reported to be produced by astrocytes. Using ProteinPilot software analysis, we found that 60 extracellular proteins were significantly altered (27 upregulated and 33 downregulated) in the supernatant of OGD-treated astrocytes. Among these proteins, 7 have been reported to be able to regulate neurogenesis, while others may have the potential to regulate neurogenesis. This study profiles the major proteins released by astrocytes, which play important roles in the modulation of neurogenesis.

  5. Quantitative proteomic analysis of wheat cultivars with differing drought stress tolerance

    Directory of Open Access Journals (Sweden)

    Kristina L Ford

    2011-09-01

    Full Text Available Using a series of multiplexed experiments we studied the quantitative changes in protein abundance of three Australian bread wheat cultivars (Triticum aestivum L. in response to a drought stress. Three cultivars differing in their ability to maintain grain yield during drought, Kukri (intolerant, Excalibur (tolerant and RAC875 (tolerant, were grown in the glasshouse with cyclic drought treatment that mimicked conditions in the field. Proteins were isolated from leaves of mature plants and isobaric tags were used to follow changes in the relative protein abundance of 159 proteins. This is the first shotgun proteomics study in wheat, providing important insights into protein responses to drought as well as identifying the largest number of wheat proteins (1,299 in a single study. The changes in the three cultivars at the different time points reflected their differing physiological responses to drought, with the two drought tolerant varieties (Excalibur and RAC875 differing in their protein responses. Excalibur lacked significant changes in proteins during the initial onset of the water deficit in contrast to RAC875 that had a large number of significant changes. All three cultivars had changes consistent with an increase in oxidative stress metabolism and ROS scavenging capacity seen through increases in superoxide dismutases and catalases as well as ROS avoidance through the decreases in proteins involved in photosynthesis and the Calvin cycle.

  6. Development and application of a quantitative multiplexed small GTPase activity assay using targeted proteomics.

    Science.gov (United States)

    Zhang, Cheng-Cheng; Li, Ru; Jiang, Honghui; Lin, Shujun; Rogalski, Jason C; Liu, Kate; Kast, Juergen

    2015-02-06

    Small GTPases are a family of key signaling molecules that are ubiquitously expressed in various types of cells. Their activity is often analyzed by western blot, which is limited by its multiplexing capability, the quality of isoform-specific antibodies, and the accuracy of quantification. To overcome these issues, a quantitative multiplexed small GTPase activity assay has been developed. Using four different binding domains, this assay allows the binding of up to 12 active small GTPase isoforms simultaneously in a single experiment. To accurately quantify the closely related small GTPase isoforms, a targeted proteomic approach, i.e., selected/multiple reaction monitoring, was developed, and its functionality and reproducibility were validated. This assay was successfully applied to human platelets and revealed time-resolved coactivation of multiple small GTPase isoforms in response to agonists and differential activation of these isoforms in response to inhibitor treatment. This widely applicable approach can be used for signaling pathway studies and inhibitor screening in many cellular systems.

  7. Comparison of Pancreas Juice Proteins from Cancer Versus Pancreatitis Using Quantitative Proteomic Analysis

    Science.gov (United States)

    Chen, Ru; Pan, Sheng; Cooke, Kelly; Moyes, Kara White; Bronner, Mary P.; Goodlett, David R.; Aebersold, Ruedi; Brentnall, Teresa A.

    2008-01-01

    Objectives Pancreatitis is an inflammatory condition of the pancreas. However, it often shares many molecular features with pancreatic cancer. Biomarkers present in pancreatic cancer frequently occur in the setting of pancreatitis. The efforts to develop diagnostic biomarkers for pancreatic cancer have thus been complicated by the false-positive involvement of pancreatitis. Methods In an attempt to develop protein biomarkers for pancreatic cancer, we previously use quantitative proteomics to identify and quantify the proteins from pancreatic cancer juice. Pancreatic juice is a rich source of proteins that are shed by the pancreatic ductal cells. In this study, we used a similar approach to identify and quantify proteins from pancreatitis juice. Results In total, 72 proteins were identified and quantified in the comparison of pancreatic juice from pancreatitis patients versus pooled normal control juice. Nineteen of the juice proteins were overexpressed, and 8 were underexpressed in pancreatitis juice by at least 2-fold compared with normal pancreatic juice. Of these 27 differentially expressed proteins in pancreatitis, 9 proteins were also differentially expressed in the pancreatic juice from pancreatic cancer patient. Conclusions Identification of these differentially expressed proteins from pancreatitis juice provides useful information for future study of specific pancreatitis-associated proteins and to eliminate potential false-positive biomarkers for pancreatic cancer. PMID:17198186

  8. Proteomic analysis of astrocytic secretion that regulates neurogenesis using quantitative amine-specific isobaric tagging

    International Nuclear Information System (INIS)

    Yan, Hu; Zhou, Wenhao; Wei, Liming; Zhong, Fan; Yang, Yi

    2010-01-01

    Astrocytes are essential components of neurogenic niches that affect neurogenesis through membrane association and/or the release of soluble factors. To identify factors released from astrocytes that could regulate neural stem cell differentiation and proliferation, we used mild oxygen-glucose deprivation (OGD) to inhibit the secretory capacity of astrocytes. Using the Transwell co-culture system, we found that OGD-treated astrocytes could not promote neural stem cell differentiation and proliferation. Next, isobaric tagging for the relative and absolute quantitation (iTRAQ) proteomics techniques was performed to identify the proteins in the supernatants of astrocytes (with or without OGD). Through a multi-step analysis and gene ontology classification, 130 extracellular proteins were identified, most of which were involved in neuronal development, the inflammatory response, extracellular matrix composition and supportive functions. Of these proteins, 44 had never been reported to be produced by astrocytes. Using ProteinPilot software analysis, we found that 60 extracellular proteins were significantly altered (27 upregulated and 33 downregulated) in the supernatant of OGD-treated astrocytes. Among these proteins, 7 have been reported to be able to regulate neurogenesis, while others may have the potential to regulate neurogenesis. This study profiles the major proteins released by astrocytes, which play important roles in the modulation of neurogenesis.

  9. Systematic and quantitative comparison of digest efficiency and specificity reveals the impact of trypsin quality on MS-based proteomics.

    Science.gov (United States)

    Burkhart, Julia Maria; Schumbrutzki, Cornelia; Wortelkamp, Stefanie; Sickmann, Albert; Zahedi, René Peiman

    2012-02-02

    Trypsin is the most frequently used proteolytic enzyme in mass spectrometry-based proteomics. Beside its good availability, it also offers some major advantages such as an optimal average peptide length of ~14 amino acids, and typically the presence of at least two defined positive charges at the N-terminus as well as the C-terminal Arg/Lys, rendering tryptic peptides well suited for CID-based LC-MS/MS. Here, we conducted a systematic study of different types of commercially available trypsin in order to qualitatively and quantitatively compare cleavage specificity, efficiency as well as reproducibility and the potential impact on quantitation and proteome coverage. We present a straightforward strategy applied to complex digests of human platelets, comprising (1) digest controls using a monolithic column HPLC-setup, (2) SCX enrichment of semitryptic/nonspecific peptides, (3) targeted MRM analysis of corresponding full cleavage/missed cleavage peptide pairs as well as (4) LC-MS analyses of complete digests with a three-step data interpretation. Thus, differences in digest performance can be readily assessed, rendering these procedures extremely beneficial to quality control not only the trypsin of choice, but also to effectively compare as well as optimize different digestion conditions and to evaluate the reproducibility of a dedicated digest protocol for all kinds of quantitative proteome studies. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. Investigating the Effect of Growth Phase on the Surface-Layer Associated Proteome of Lactobacillus acidophilus Using Quantitative Proteomics

    Directory of Open Access Journals (Sweden)

    Courtney Klotz

    2017-11-01

    Full Text Available Bacterial surface-layers (S-layers are semi-porous crystalline arrays that self-assemble to form the outermost layer of some cell envelopes. S-layers have been shown to act as scaffolding structures for the display of auxiliary proteins externally. These S-layer associated proteins have recently gained attention in probiotics due to their direct physical contact with the intestinal mucosa and potential role in cell proliferation, adhesion, and immunomodulation. A number of studies have attempted to catalog the S-layer associated proteome of Lactobacillus acidophilus NCFM under a single condition. However, due to the versatility of the cell surface, we chose to employ a multiplexing-based approach with the intention of accurately contrasting multiple conditions. In this study, a previously described lithium chloride isolation protocol was used to release proteins bound to the L. acidophilus S-layer during logarithmic and early stationary growth phases. Protein quantification values were obtained via TMT (tandem mass tag labeling combined with a triple-stage mass spectrometry (MS3 method. Results showed significant growth stage-dependent alterations to the surface-associated proteome while simultaneously highlighting the sensitivity and reproducibility of the technology. Thus, this study establishes a framework for quantifying condition-dependent changes to cell surface proteins that can easily be applied to other S-layer forming bacteria.

  11. Potential protein biomarkers for burning mouth syndrome discovered by quantitative proteomics.

    Science.gov (United States)

    Ji, Eoon Hye; Diep, Cynthia; Liu, Tong; Li, Hong; Merrill, Robert; Messadi, Diana; Hu, Shen

    2017-01-01

    Burning mouth syndrome (BMS) is a chronic pain disorder characterized by severe burning sensation in normal looking oral mucosa. Diagnosis of BMS remains to be a challenge to oral healthcare professionals because the method for definite diagnosis is still uncertain. In this study, a quantitative saliva proteomic analysis was performed in order to identify target proteins in BMS patients' saliva that may be used as biomarkers for simple, non-invasive detection of the disease. By using isobaric tags for relative and absolute quantitation labeling and liquid chromatography-tandem mass spectrometry to quantify 1130 saliva proteins between BMS patients and healthy control subjects, we found that 50 proteins were significantly changed in the BMS patients when compared to the healthy control subjects ( p ≤ 0.05, 39 up-regulated and 11 down-regulated). Four candidates, alpha-enolase, interleukin-18 (IL-18), kallikrein-13 (KLK13), and cathepsin G, were selected for further validation. Based on enzyme-linked immunosorbent assay measurements, three potential biomarkers, alpha-enolase, IL-18, and KLK13, were successfully validated. The fold changes for alpha-enolase, IL-18, and KLK13 were determined as 3.6, 2.9, and 2.2 (burning mouth syndrome vs. control), and corresponding receiver operating characteristic values were determined as 0.78, 0.83, and 0.68, respectively. Our findings indicate that testing of the identified protein biomarkers in saliva might be a valuable clinical tool for BMS detection. Further validation studies of the identified biomarkers or additional candidate biomarkers are needed to achieve a multi-marker prediction model for improved detection of BMS with high sensitivity and specificity.

  12. MSQuant, an Open Source Platform for Mass Spectrometry-Based Quantitative Proteomics

    DEFF Research Database (Denmark)

    Mortensen, Peter; Gouw, Joost W; Olsen, Jesper V

    2010-01-01

    Mass spectrometry-based proteomics critically depends on algorithms for data interpretation. A current bottleneck in the rapid advance of proteomics technology is the closed nature and slow development cycle of vendor-supplied software solutions. We have created an open source software environment...

  13. Evaluation of six sample preparation procedures for qualitative and quantitative proteomics analysis of milk fat globule membrane.

    Science.gov (United States)

    Yang, Yongxin; Anderson, Elizabeth; Zhang, Sheng

    2018-04-12

    Proteomic analysis of membrane proteins is challenged by the proteins solubility and detergent incompatibility with MS analysis. No single perfect protocol can be used to comprehensively characterize the proteome of membrane fraction. Here, we used cow milk fat globule membrane (MFGM) proteome analysis to assess six sample preparation procedures including one in-gel and five in-solution digestion approaches prior to LC-MS/MS analysis. The largest number of MFGM proteins were identified by suspension trapping (S-Trap) and filter-aided sample preparation (FASP) methods, followed by acetone precipitation without clean-up of tryptic peptides method. Protein identifications with highest average coverage was achieved by Chloroform/MeOH, in-gel and S-Trap methods. Most distinct proteins were identified by FASP method, followed by S-Trap. Analyses by Venn diagram, principal-component analysis, hierarchical clustering and the abundance ranking of quantitative proteins highlight differences in the MFGM fraction by the all sample preparation procedures. These results reveal the biased proteins/peptides loss occurred in each protocol. In this study, we found several novel proteins that were not observed previously by in-depth proteomics characterization of MFGM fraction in milk. Thus, a combination of multiple procedures with orthologous properties of sample preparation was demonstrated to improve the protein sequence coverage and expression level accuracy of membrane samples. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Accounting for the Multiple Natures of Missing Values in Label-Free Quantitative Proteomics Data Sets to Compare Imputation Strategies.

    Science.gov (United States)

    Lazar, Cosmin; Gatto, Laurent; Ferro, Myriam; Bruley, Christophe; Burger, Thomas

    2016-04-01

    Missing values are a genuine issue in label-free quantitative proteomics. Recent works have surveyed the different statistical methods to conduct imputation and have compared them on real or simulated data sets and recommended a list of missing value imputation methods for proteomics application. Although insightful, these comparisons do not account for two important facts: (i) depending on the proteomics data set, the missingness mechanism may be of different natures and (ii) each imputation method is devoted to a specific type of missingness mechanism. As a result, we believe that the question at stake is not to find the most accurate imputation method in general but instead the most appropriate one. We describe a series of comparisons that support our views: For instance, we show that a supposedly "under-performing" method (i.e., giving baseline average results), if applied at the "appropriate" time in the data-processing pipeline (before or after peptide aggregation) on a data set with the "appropriate" nature of missing values, can outperform a blindly applied, supposedly "better-performing" method (i.e., the reference method from the state-of-the-art). This leads us to formulate few practical guidelines regarding the choice and the application of an imputation method in a proteomics context.

  15. Quantitative proteome analysis of plasma microparticles for the characterization of HCV-induced hepatic cirrhosis and hepatocellular carcinoma.

    Science.gov (United States)

    Taleb, Raghda Saad Zaghloul; Moez, Pacint; Younan, Doreen; Eisenacher, Martin; Tenbusch, Matthias; Sitek, Barbara; Bracht, Thilo

    2017-12-01

    Hepatocellular carcinoma (HCC) is the most common primary malignant liver tumor and a leading cause of cancer-related deaths worldwide. Cirrhosis induced by hepatitis-C virus (HCV) infection is the most critical risk factor for HCC. However, the mechanism of HCV-induced carcinogenesis is not fully understood. Plasma microparticles (PMP) contribute to numerous physiological and pathological processes and contain proteins whose composition correlates to the respective pathophysiological conditions. We analyzed PMP from 22 HCV-induced cirrhosis patients, 16 HCV-positive HCC patients with underlying cirrhosis and 18 healthy controls. PMP were isolated using ultracentrifugation and analyzed via label-free LC-MS/MS. We identified 840 protein groups and quantified 507 proteins. 159 proteins were found differentially abundant between the three experimental groups. PMP in both disease entities displayed remarkable differences in the proteome composition compared to healthy controls. Conversely, the proteome difference between both diseases was minimal. GO analysis revealed that PMP isolated from both diseases were significantly enriched in proteins involved in complement activation, while endopeptidase activity was downregulated exclusively in HCC patients. This study reports for the first time a quantitative proteome analysis for PMP from patients with HCV-induced cirrhosis and HCC. Data are available via ProteomeXchange with identifier PXD005777. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Delineating Rearrangements in Single Yeast Artificial Chromosomes by Quantitative DNA Fiber Mapping

    Energy Technology Data Exchange (ETDEWEB)

    Weier, Heinz-Ulrich G.; Greulich-Bode, Karin M.; Wu, Jenny; Duell, Thomas

    2009-09-18

    Cloning of large chunks of human genomic DNA in recombinant systems such as yeast or bacterial artificial chromosomes has greatly facilitated the construction of physical maps, the positional cloning of disease genes or the preparation of patient-specific DNA probes for diagnostic purposes. For this process to work efficiently, the DNA cloning process and subsequent clone propagation need to maintain stable inserts that are neither deleted nor otherwise rearranged. Some regions of the human genome; however, appear to have a higher propensity than others to rearrange in any host system. Thus, techniques to detect and accurately characterize such rearrangements need to be developed. We developed a technique termed 'Quantitative DNA Fiber Mapping (QDFM)' that allows accurate tagging of sequence elements of interest with near kilobase accuracy and optimized it for delineation of rearrangements in recombinant DNA clones. This paper demonstrates the power of this microscopic approach by investigating YAC rearrangements. In our examples, high-resolution physical maps for regions within the immunoglobulin lambda variant gene cluster were constructed for three different YAC clones carrying deletions of 95 kb and more. Rearrangements within YACs could be demonstrated unambiguously by pairwise mapping of cosmids along YAC DNA molecules. When coverage by YAC clones was not available, distances between cosmid clones were estimated by hybridization of cosmids onto DNA fibers prepared from human genomic DNA. In addition, the QDFM technology provides essential information about clone stability facilitating closure of the maps of the human genome as well as those of model organisms.

  17. Quantitative Genome-Wide Analysis of Yeast Deletion Strain Sensitivities to Oxidative and Chemical Stress

    Directory of Open Access Journals (Sweden)

    Stanley Fields

    2006-03-01

    Full Text Available Understanding the actions of drugs and toxins in a cell is of critical importance to medicine, yet many of the molecular events involved in chemical resistance are relatively uncharacterized. In order to identify the cellular processes and pathways targeted by chemicals, we took advantage of the haploid Saccharomyces cerevisiae deletion strains (Winzeler et al., 1999. Although ~4800 of the strains are viable, the loss of a gene in a pathway affected by a drug can lead to a synthetic lethal effect in which the combination of a deletion and a normally sublethal dose of a chemical results in loss of viability. WE carried out genome-wide screens to determine quantitative sensitivities of the deletion set to four chemicals: hydrogen peroxide, menadione, ibuprofen and mefloquine. Hydrogen peroxide and menadione induce oxidative stress in the cell, whereas ibuprofen and mefloquine are toxic to yeast by unknown mechanisms. Here we report the sensitivities of 659 deletion strains that are sensitive to one or more of these four compounds, including 163 multichemicalsensitive strains, 394 strains specific to hydrogen peroxide and/or menadione, 47 specific to ibuprofen and 55 specific to mefloquine.We correlate these results with data from other large-scale studies to yield novel insights into cellular function.

  18. Transcriptome and quantitative proteome analysis reveals molecular processes associated with larval metamorphosis in the polychaete pseudopolydora vexillosa

    KAUST Repository

    Chandramouli, Kondethimmanahalli; Sun, Jin; Mok, FloraSy; Liu, Lingli; Qiu, Jianwen; Ravasi, Timothy; Qian, Peiyuan

    2013-01-01

    Larval growth of the polychaete worm Pseudopolydora vexillosa involves the formation of segment-specific structures. When larvae attain competency to settle, they discard swimming chaetae and secrete mucus. The larvae build tubes around themselves and metamorphose into benthic juveniles. Understanding the molecular processes, which regulate this complex and unique transition, remains a major challenge because of the limited molecular information available. To improve this situation, we conducted high-throughput RNA sequencing and quantitative proteome analysis of the larval stages of P. vexillosa. Based on gene ontology (GO) analysis, transcripts related to cellular and metabolic processes, binding, and catalytic activities were highly represented during larval-adult transition. Mitogen-activated protein kinase (MAPK), calcium-signaling, Wnt/β-catenin, and notch signaling metabolic pathways were enriched in transcriptome data. Quantitative proteomics identified 107 differentially expressed proteins in three distinct larval stages. Fourteen and 53 proteins exhibited specific differential expression during competency and metamorphosis, respectively. Dramatic up-regulation of proteins involved in signaling, metabolism, and cytoskeleton functions were found during the larval-juvenile transition. Several proteins involved in cell signaling, cytoskeleton and metabolism were up-regulated, whereas proteins related to transcription and oxidative phosphorylation were down-regulated during competency. The integration of high-throughput RNA sequencing and quantitative proteomics allowed a global scale analysis of larval transcripts/proteins associated molecular processes in the metamorphosis of polychaete worms. Further, transcriptomic and proteomic insights provide a new direction to understand the fundamental mechanisms that regulate larval metamorphosis in polychaetes. © 2013 American Chemical Society.

  19. Transcriptome and quantitative proteome analysis reveals molecular processes associated with larval metamorphosis in the polychaete pseudopolydora vexillosa

    KAUST Repository

    Chandramouli, Kondethimmanahalli

    2013-03-01

    Larval growth of the polychaete worm Pseudopolydora vexillosa involves the formation of segment-specific structures. When larvae attain competency to settle, they discard swimming chaetae and secrete mucus. The larvae build tubes around themselves and metamorphose into benthic juveniles. Understanding the molecular processes, which regulate this complex and unique transition, remains a major challenge because of the limited molecular information available. To improve this situation, we conducted high-throughput RNA sequencing and quantitative proteome analysis of the larval stages of P. vexillosa. Based on gene ontology (GO) analysis, transcripts related to cellular and metabolic processes, binding, and catalytic activities were highly represented during larval-adult transition. Mitogen-activated protein kinase (MAPK), calcium-signaling, Wnt/β-catenin, and notch signaling metabolic pathways were enriched in transcriptome data. Quantitative proteomics identified 107 differentially expressed proteins in three distinct larval stages. Fourteen and 53 proteins exhibited specific differential expression during competency and metamorphosis, respectively. Dramatic up-regulation of proteins involved in signaling, metabolism, and cytoskeleton functions were found during the larval-juvenile transition. Several proteins involved in cell signaling, cytoskeleton and metabolism were up-regulated, whereas proteins related to transcription and oxidative phosphorylation were down-regulated during competency. The integration of high-throughput RNA sequencing and quantitative proteomics allowed a global scale analysis of larval transcripts/proteins associated molecular processes in the metamorphosis of polychaete worms. Further, transcriptomic and proteomic insights provide a new direction to understand the fundamental mechanisms that regulate larval metamorphosis in polychaetes. © 2013 American Chemical Society.

  20. Exploring potential virulence regulators in Paracoccidioides brasiliensis isolates of varying virulence through quantitative proteomics.

    Science.gov (United States)

    Castilho, Daniele G; Chaves, Alison F A; Xander, Patricia; Zelanis, André; Kitano, Eduardo S; Serrano, Solange M T; Tashima, Alexandre K; Batista, Wagner L

    2014-10-03

    Few virulence factors have been identified for Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis. In this study, we quantitatively evaluated the protein composition of P. brasiliensis in the yeast phase using minimal and rich media to obtain a better understanding of its virulence and to gain new insights into pathogen adaptation strategies. This analysis was performed on two isolates of the Pb18 strain showing distinct infection profiles in B10.A mice. Using liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis, we identified and quantified 316 proteins in minimal medium, 29 of which were overexpressed in virulent Pb18. In rich medium, 29 out of 295 proteins were overexpressed in the virulent fungus. Three proteins were found to be up-regulated in both media, suggesting the potential roles of these proteins in virulence regulation in P. brasiliensis. Moreover, genes up-regulated in virulent Pb18 showed an increase in its expression after the recovery of virulence of attenuated Pb18. Proteins up-regulated in both isolates were grouped according to their functional categories. Virulent Pb18 undergoes metabolic reorganization and increased expression of proteins involved in fermentative respiration. This approach allowed us to identify potential virulence regulators and provided a foundation for achieving a molecular understanding of how Paracoccidioides modulates the host-pathogen interaction to its advantage.

  1. Quantitative proteomic study of Aspergillus Fumigatus secretome revealed deamidation of secretory enzymes.

    Science.gov (United States)

    Adav, Sunil S; Ravindran, Anita; Sze, Siu Kwan

    2015-04-24

    Aspergillus sp. plays an essential role in lignocellulosic biomass recycling and is also exploited as cell factories for the production of industrial enzymes. This study profiled the secretome of Aspergillus fumigatus when grown with cellulose, xylan and starch by high throughput quantitative proteomics using isobaric tags for relative and absolute quantification (iTRAQ). Post translational modifications (PTMs) of proteins play a critical role in protein functions. However, our understanding of the PTMs in secretory proteins is limited. Here, we present the identification of PTMs such as deamidation of secreted proteins of A. fumigatus. This study quantified diverse groups of extracellular secreted enzymes and their functional classification revealed cellulases and glycoside hydrolases (32.9%), amylases (0.9%), hemicellulases (16.2%), lignin degrading enzymes (8.1%), peptidases and proteases (11.7%), chitinases, lipases and phosphatases (7.6%), and proteins with unknown function (22.5%). The comparison of quantitative iTRAQ results revealed that cellulose and xylan stimulates expression of specific cellulases and hemicellulases, and their abundance level as a function of substrate. In-depth data analysis revealed deamidation as a major PTM of key cellulose hydrolyzing enzymes like endoglucanases, cellobiohydrolases and glucosidases. Hemicellulose degrading endo-1,4-beta-xylanase, monosidases, xylosidases, lignin degrading laccase, isoamyl alcohol oxidase and oxidoreductases were also found to be deamidated. The filamentous fungi play an essential role in lignocellulosic biomass recycling and fungal strains belonging to Aspergillus were also exploited as cell factories for the production of organic acids, pharmaceuticals, and industrially important enzymes. In this study, extracellular proteins secreted by thermophilic A. fumigatus when grown with cellulose, xylan and starch were profiled using isobaric tags for relative and absolute quantification (iTRAQ) by

  2. Quantitative, high-resolution proteomics for data-driven systems biology

    DEFF Research Database (Denmark)

    Cox, J.; Mann, M.

    2011-01-01

    Systems biology requires comprehensive data at all molecular levels. Mass spectrometry (MS)-based proteomics has emerged as a powerful and universal method for the global measurement of proteins. In the most widespread format, it uses liquid chromatography (LC) coupled to high-resolution tandem...... primary structure of proteins including posttranslational modifications, to localize proteins to organelles, and to determine protein interactions. Here, we describe the principles of analysis and the areas of biology where proteomics can make unique contributions. The large-scale nature of proteomics...... data and its high accuracy pose special opportunities as well as challenges in systems biology that have been largely untapped so far....

  3. Region and cell-type resolved quantitative proteomic map of the human heart

    DEFF Research Database (Denmark)

    Doll, Sophia; Dreßen, Martina; Geyer, Philipp E

    2017-01-01

    The heart is a central human organ and its diseases are the leading cause of death worldwide, but an in-depth knowledge of the identity and quantity of its constituent proteins is still lacking. Here, we determine the healthy human heart proteome by measuring 16 anatomical regions and three major...... cardiac cell types by high-resolution mass spectrometry-based proteomics. From low microgram sample amounts, we quantify over 10,700 proteins in this high dynamic range tissue. We combine copy numbers per cell with protein organellar assignments to build a model of the heart proteome at the subcellular...

  4. Integrative Analysis of Subcellular Quantitative Proteomics Studies Reveals Functional Cytoskeleton Membrane-Lipid Raft Interactions in Cancer.

    Science.gov (United States)

    Shah, Anup D; Inder, Kerry L; Shah, Alok K; Cristino, Alexandre S; McKie, Arthur B; Gabra, Hani; Davis, Melissa J; Hill, Michelle M

    2016-10-07

    Lipid rafts are dynamic membrane microdomains that orchestrate molecular interactions and are implicated in cancer development. To understand the functions of lipid rafts in cancer, we performed an integrated analysis of quantitative lipid raft proteomics data sets modeling progression in breast cancer, melanoma, and renal cell carcinoma. This analysis revealed that cancer development is associated with increased membrane raft-cytoskeleton interactions, with ∼40% of elevated lipid raft proteins being cytoskeletal components. Previous studies suggest a potential functional role for the raft-cytoskeleton in the action of the putative tumor suppressors PTRF/Cavin-1 and Merlin. To extend the observation, we examined lipid raft proteome modulation by an unrelated tumor suppressor opioid binding protein cell-adhesion molecule (OPCML) in ovarian cancer SKOV3 cells. In agreement with the other model systems, quantitative proteomics revealed that 39% of OPCML-depleted lipid raft proteins are cytoskeletal components, with microfilaments and intermediate filaments specifically down-regulated. Furthermore, protein-protein interaction network and simulation analysis showed significantly higher interactions among cancer raft proteins compared with general human raft proteins. Collectively, these results suggest increased cytoskeleton-mediated stabilization of lipid raft domains with greater molecular interactions as a common, functional, and reversible feature of cancer cells.

  5. Quantitative Analysis of Human Salivary Gland-Derived Intact Proteome Using Top-Down Mass Spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Si; Brown, Joseph N.; Tolic, Nikola; Meng, Da; Liu, Xiaowen; Zhang, Haizhen; Zhao, Rui; Moore, Ronald J.; Pevzner, Pavel A.; Smith, Richard D.; Pasa-Tolic, Ljiljana

    2014-05-31

    There are several notable challenges inherent to fully characterizing the entirety of the human saliva proteome using bottom-up approaches, including polymorphic isoforms, post-translational modifications, unique splice variants, deletions, and truncations. To address these challenges, we have developed a top-down based liquid chromatography-mass spectrometry (LC-MS) approach, which cataloged 20 major human salivary proteins with a total of 83 proteoforms, containing a broad range of post-translational modifications. Among these proteins, several previously reported disease biomarker proteins were identified at the intact protein level, such as beta-2 microglobulin (B2M). In addition, intact glycosylated proteoforms of several saliva proteins were also characterized, including intact N-glycosylated protein prolactin inducible protein (PIP) and O-glycosylated acidic protein rich protein (aPRP). These characterized proteoforms constitute an intact saliva proteoform database, which was used for quantitative comparison of intact salivary proteoforms among six healthy individuals. Human parotid (PS) and submandibular/sublingual gland (SMSL) secretion samples (2 μg of protein each) from six healthy individuals were compared using RPLC coupled with the 12T FTICR mass spectrometer. Significantly different protein and PTM patterns were resolved with high reproducibility between PS and SMSL glands. The results from this study provide further insight into the potential mechanisms of PTM pathways in oral glandular secretion, expanding our knowledge of this complex yet easily accessible fluid. Intact protein LC-MS approach presented herein can potentially be applied for rapid and accurate identification of biomarkers from only a few microliters of human glandular saliva.

  6. Quantitative proteome and phosphoproteome analyses of Streptomyces coelicolor reveal proteins and phosphoproteins modulating differentiation and secondary metabolism

    DEFF Research Database (Denmark)

    Rioseras, Beatriz; Sliaha, Pavel V; Gorshkov, Vladimir

    2018-01-01

    identified and quantified 3461 proteins corresponding to 44.3% of the S. coelicolor proteome across three developmental stages: vegetative hypha (MI); secondary metabolite producing hyphae (MII); and sporulating hyphae. A total of 1350 proteins exhibited more than 2-fold expression changes during....../Thr/Tyr kinases, making this genus an outstanding model for the study of bacterial protein phosphorylation events. We used mass spectrometry based quantitative proteomics and phosphoproteomics to characterize bacterial differentiation and activation of secondary metabolism of Streptomyces coelicolor. We...... the bacterial differentiation process. These proteins include 136 regulators (transcriptional regulators, transducers, Ser/Thr/Tyr kinases, signalling proteins), as well as 542 putative proteins with no clear homology to known proteins which are likely to play a role in differentiation and secondary metabolism...

  7. iTRAQ-based quantitative proteomic analysis reveals proteomic changes in leaves of cultivated tobacco (Nicotiana tabacum) in response to drought stress.

    Science.gov (United States)

    Xie, He; Yang, Da-Hai; Yao, Heng; Bai, Ge; Zhang, Yi-Han; Xiao, Bing-Guang

    2016-01-15

    Drought is one of the most severe forms of abiotic stresses that threaten the survival of plants, including crops. In turn, plants dramatically change their physiology to increase drought tolerance, including reconfiguration of proteomes. Here, we studied drought-induced proteomic changes in leaves of cultivated tobacco (Nicotiana tabacum), a solanaceous plant, using the isobaric tags for relative and absolute quantitation (iTRAQ)-based protein labeling technology. Of identified 5570 proteins totally, drought treatment increased and decreased abundance of 260 and 206 proteins, respectively, compared with control condition. Most of these differentially regulated proteins are involved in photosynthesis, metabolism, and stress and defense. Although abscisic acid (ABA) levels greatly increased in drought-treated tobacco leaves, abundance of detected ABA biosynthetic enzymes showed no obvious changes. In contrast, heat shock proteins (HSPs), thioredoxins, ascorbate-, glutathione-, and hydrogen peroxide (H2O2)-related proteins were up- or down-regulated in drought-treated tobacco leaves, suggesting that chaperones and redox signaling are important for tobacco tolerance to drought, and it is likely that redox-induced posttranslational modifications play an important role in modulating protein activity. This study not only provides a comprehensive dataset on overall protein changes in drought-treated tobacco leaves, but also shed light on the mechanism by which solanaceous plants adapt to drought stress. Copyright © 2015 Yunnan Academy of Tobacco Agricultural Sciences. Published by Elsevier Inc. All rights reserved.

  8. Elevated host lipid metabolism revealed by iTRAQ-based quantitative proteomic analysis of cerebrospinal fluid of tuberculous meningitis patients

    Energy Technology Data Exchange (ETDEWEB)

    Mu, Jun [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Chongqing Key Laboratory of Neurobiology, Chongqing (China); Yang, Yongtao [Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Chongqing Key Laboratory of Neurobiology, Chongqing (China); Department of Neurology, Yongchuan Hospital of Chongqing Medical University, Chongqing (China); Chen, Jin; Cheng, Ke; Li, Qi; Wei, Yongdong; Zhu, Dan; Shao, Weihua; Zheng, Peng [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Chongqing Key Laboratory of Neurobiology, Chongqing (China); Xie, Peng, E-mail: xiepeng@cqmu.edu.cn [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Chongqing Key Laboratory of Neurobiology, Chongqing (China); Department of Neurology, Yongchuan Hospital of Chongqing Medical University, Chongqing (China)

    2015-10-30

    Purpose: Tuberculous meningitis (TBM) remains to be one of the most deadly infectious diseases. The pathogen interacts with the host immune system, the process of which is largely unknown. Various cellular processes of Mycobacterium tuberculosis (MTB) centers around lipid metabolism. To determine the lipid metabolism related proteins, a quantitative proteomic study was performed here to identify differential proteins in the cerebrospinal fluid (CSF) obtained from TBM patients (n = 12) and healthy controls (n = 12). Methods: CSF samples were desalted, concentrated, labelled with isobaric tags for relative and absolute quantitation (iTRAQ™), and analyzed by multi-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gene ontology and proteomic phenotyping analysis of the differential proteins were conducted using Database for Annotation, Visualization, and Integrated Discovery (DAVID) Bioinformatics Resources. ApoE and ApoB were selected for validation by ELISA. Results: Proteomic phenotyping of the 4 differential proteins was invloved in the lipid metabolism. ELISA showed significantly increased ApoB levels in TBM subjects compared to healthy controls. Area under the receiver operating characteristic curve analysis demonstrated ApoB levels could distinguish TBM subjects from healthy controls and viral meningitis subjects with 89.3% sensitivity and 92% specificity. Conclusions: CSF lipid metabolism disregulation, especially elevated expression of ApoB, gives insights into the pathogenesis of TBM. Further evaluation of these findings in larger studies including anti-tuberculosis medicated and unmedicated patient cohorts with other center nervous system infectious diseases is required for successful clinical translation. - Highlights: • The first proteomic study on the cerebrospinal fluid of tuberculous meningitis patients using iTRAQ. • Identify 4 differential proteins invloved in the lipid metabolism. • Elevated expression of ApoB gives

  9. Elevated host lipid metabolism revealed by iTRAQ-based quantitative proteomic analysis of cerebrospinal fluid of tuberculous meningitis patients

    International Nuclear Information System (INIS)

    Mu, Jun; Yang, Yongtao; Chen, Jin; Cheng, Ke; Li, Qi; Wei, Yongdong; Zhu, Dan; Shao, Weihua; Zheng, Peng; Xie, Peng

    2015-01-01

    Purpose: Tuberculous meningitis (TBM) remains to be one of the most deadly infectious diseases. The pathogen interacts with the host immune system, the process of which is largely unknown. Various cellular processes of Mycobacterium tuberculosis (MTB) centers around lipid metabolism. To determine the lipid metabolism related proteins, a quantitative proteomic study was performed here to identify differential proteins in the cerebrospinal fluid (CSF) obtained from TBM patients (n = 12) and healthy controls (n = 12). Methods: CSF samples were desalted, concentrated, labelled with isobaric tags for relative and absolute quantitation (iTRAQ™), and analyzed by multi-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gene ontology and proteomic phenotyping analysis of the differential proteins were conducted using Database for Annotation, Visualization, and Integrated Discovery (DAVID) Bioinformatics Resources. ApoE and ApoB were selected for validation by ELISA. Results: Proteomic phenotyping of the 4 differential proteins was invloved in the lipid metabolism. ELISA showed significantly increased ApoB levels in TBM subjects compared to healthy controls. Area under the receiver operating characteristic curve analysis demonstrated ApoB levels could distinguish TBM subjects from healthy controls and viral meningitis subjects with 89.3% sensitivity and 92% specificity. Conclusions: CSF lipid metabolism disregulation, especially elevated expression of ApoB, gives insights into the pathogenesis of TBM. Further evaluation of these findings in larger studies including anti-tuberculosis medicated and unmedicated patient cohorts with other center nervous system infectious diseases is required for successful clinical translation. - Highlights: • The first proteomic study on the cerebrospinal fluid of tuberculous meningitis patients using iTRAQ. • Identify 4 differential proteins invloved in the lipid metabolism. • Elevated expression of ApoB gives

  10. Clinical proteomics

    DEFF Research Database (Denmark)

    Albrethsen, Jakob; Frederiksen, Hanne; Johannsen, Trine Holm

    2018-01-01

    Clinical proteomics aims to deliver cost-effective multiplexing of potentially hundreds of diagnostic proteins, including distinct protein isoforms. The analytical strategy known as targeted proteomics is particularly promising because it is compatible with robust mass spectrometry (MS)-platforms...... standards and calibrants. The present challenge is to examine if targeted proteomics of IGF-I can truly measure up to the routine performance that must be expected from a clinical testing platform.......Clinical proteomics aims to deliver cost-effective multiplexing of potentially hundreds of diagnostic proteins, including distinct protein isoforms. The analytical strategy known as targeted proteomics is particularly promising because it is compatible with robust mass spectrometry (MS......)-platforms already implemented in many clinical laboratories for routine quantitation of small molecules (i.e. uHPLC coupled to triple-quadrupole MS). Progress in targeted proteomics of circulating insulin-like growth factor 1 (IGF-I) have provided valuable insights about tryptic peptides, transitions, internal...

  11. Quantitative elementary mode analysis of metabolic pathways: the example of yeast glycolysis

    Directory of Open Access Journals (Sweden)

    Kanehisa Minoru

    2006-04-01

    Full Text Available Abstract Background Elementary mode analysis of metabolic pathways has proven to be a valuable tool for assessing the properties and functions of biochemical systems. However, little comprehension of how individual elementary modes are used in real cellular states has been achieved so far. A quantitative measure of fluxes carried by individual elementary modes is of great help to identify dominant metabolic processes, and to understand how these processes are redistributed in biological cells in response to changes in environmental conditions, enzyme kinetics, or chemical concentrations. Results Selecting a valid decomposition of a flux distribution onto a set of elementary modes is not straightforward, since there is usually an infinite number of possible such decompositions. We first show that two recently introduced decompositions are very closely related and assign the same fluxes to reversible elementary modes. Then, we show how such decompositions can be used in combination with kinetic modelling to assess the effects of changes in enzyme kinetics on the usage of individual metabolic routes, and to analyse the range of attainable states in a metabolic system. This approach is illustrated by the example of yeast glycolysis. Our results indicate that only a small subset of the space of stoichiometrically feasible steady states is actually reached by the glycolysis system, even when large variation intervals are allowed for all kinetic parameters of the model. Among eight possible elementary modes, the standard glycolytic route remains dominant in all cases, and only one other elementary mode is able to gain significant flux values in steady state. Conclusion These results indicate that a combination of structural and kinetic modelling significantly constrains the range of possible behaviours of a metabolic system. All elementary modes are not equal contributors to physiological cellular states, and this approach may open a direction toward a

  12. Detection and quantitative determination by PIXE of the mutagen Sn2+ in yeast cells

    International Nuclear Information System (INIS)

    Viau, C.M.; Yoneama, M.-L.; Dias, J.F.; Pungartnik, C.; Brendel, M.; Henriques, J.A.P.

    2006-01-01

    The main goal of this work was to determine the concentration of Sn 2+ ions in cells of the yeast Saccharomyces cerevisiae and to correlate their quantity with the genotoxicity of intracellularly accumulated metal ions. The intracellular metal content of yeast cells was determined by PIXE (particle-induced X-ray emission) after cell exposure to SnCl 2 . To that end, a thick target protocol was developed for PIXE analysis. The samples were irradiated with a 2 MeV proton beam, while the induced X-rays were detected with a high-purity germanium detector. The results of the toxicity of SnCl 2 and the PIXE analysis performed with two different yeast strains (haploid and diploid) suggest that the exposure of haploid and diploid yeast to Sn 2+ induces DNA lesions and that the absorption depends on the genetic background of each strain

  13. Global analysis of the yeast lipidome by quantitative shotgun mass spectrometry

    DEFF Research Database (Denmark)

    Ejsing, Christer S.; Sampaio, Julio L; Surendranath, Vineeth

    2009-01-01

    95% coverage of the yeast lipidome achieved with 125-fold improvement in sensitivity compared with previous approaches. Comparative lipidomics demonstrated that growth temperature and defects in lipid biosynthesis induce ripple effects throughout the molecular composition of the yeast lipidome....... This work serves as a resource for molecular characterization of eukaryotic lipidomes, and establishes shotgun lipidomics as a powerful platform for complementing biochemical studies and other systems-level approaches....

  14. Strategy for the extraction of yeast DNA from artisan agave must for quantitative PCR analysis.

    Science.gov (United States)

    Kirchmayr, Manuel Reinhart; Segura-Garcia, Luis Eduardo; Flores-Berrios, Ericka Patricia; Gschaedler, Anne

    2011-11-01

    An efficient method for the direct extraction of yeast genomic DNA from agave must was developed. The optimized protocol, which was based on silica-adsorption of DNA on microcolumns, included an enzymatic cell wall degradation step followed by prolonged lysis with hot detergent. The resulting extracts were suitable templates for subsequent qPCR assays that quantified mixed yeast populations in artisan Mexican mezcal fermentations. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  15. Quantitative proteomic analysis for novel biomarkers of buccal squamous cell carcinoma arising in background of oral submucous fibrosis

    International Nuclear Information System (INIS)

    Liu, Wen; Zeng, Lijuan; Li, Ning; Wang, Fei; Jiang, Canhua; Guo, Feng; Chen, Xinqun; Su, Tong; Xu, Chunjiao; Zhang, Shanshan; Fang, Changyun

    2016-01-01

    In South and Southeast Asian, the majority of buccal squamous cell carcinoma (BSCC) can arise from oral submucous fibrosis (OSF). BSCCs develop in OSF that are often not completely resected, causing local relapse. The aim of our study was to find candidate protein biomarkers to detect OSF and predict prognosis in BSCCs by quantitative proteomics approaches. We compared normal oral mucosa (NBM) and paired biopsies of BSCC and OSF by quantitative proteomics using isobaric tags for relative and absolute quantification (iTRAQ) to discover proteins with differential expression. Gene Ontology and KEGG networks were analyzed. The prognostic value of biomarkers was evaluated in 94 BSCCs accompanied with OSF. Significant associations were assessed by Kaplan-Meier survival and Cox-proportional hazards analysis. In total 30 proteins were identified with significantly different expression (false discovery rate < 0.05) among three tissues. Two consistently upregulated proteins, ANXA4 and FLNA, were validated. The disease-free survival was negatively associated with the expression of ANXA4 (hazard ratio, 3.4; P = 0.000), FLNA (hazard ratio, 2.1; P = 0.000) and their combination (hazard ratio, 8.8; P = 0.002) in BSCCs. The present study indicates that iTRAQ quantitative proteomics analysis for tissues of BSCC and OSF is a reliable strategy. A significantly up-regulated ANXA4 and FLNA could be not only candidate biomarkers for BSCC prognosis but also potential targets for its therapy. The online version of this article (doi:10.1186/s12885-016-2650-1) contains supplementary material, which is available to authorized users

  16. New insights into the mechanisms of acetic acid resistance in Acetobacter pasteurianus using iTRAQ-dependent quantitative proteomic analysis.

    Science.gov (United States)

    Xia, Kai; Zang, Ning; Zhang, Junmei; Zhang, Hong; Li, Yudong; Liu, Ye; Feng, Wei; Liang, Xinle

    2016-12-05

    Acetobacter pasteurianus is the main starter in rice vinegar manufacturing due to its remarkable abilities to resist and produce acetic acid. Although several mechanisms of acetic acid resistance have been proposed and only a few effector proteins have been identified, a comprehensive depiction of the biological processes involved in acetic acid resistance is needed. In this study, iTRAQ-based quantitative proteomic analysis was adopted to investigate the whole proteome of different acidic titers (3.6, 7.1 and 9.3%, w/v) of Acetobacter pasteurianus Ab3 during the vinegar fermentation process. Consequently, 1386 proteins, including 318 differentially expressed proteins (p150 proteins were differentially expressed. Specifically, proteins involved in amino acid metabolic processes and fatty acid biosynthesis were differentially expressed, which may contribute to the acetic acid resistance of Acetobacter. Transcription factors, two component systems and toxin-antitoxin systems were implicated in the modulatory network at multiple levels. In addition, the identification of proteins involved in redox homeostasis, protein metabolism, and the cell envelope suggested that the whole cellular system is mobilized in response to acid stress. These findings provide a differential proteomic profile of acetic acid resistance in Acetobacter pasteurianus and have potential application to highly acidic rice vinegar manufacturing. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Quantitative analysis of oyster larval proteome provides new insights into the effects of multiple climate change stressors

    KAUST Repository

    Dineshram, Ramadoss

    2016-03-19

    The metamorphosis of planktonic larvae of the Pacific oyster (Crassostrea gigas) underpins their complex life-history strategy by switching on the molecular machinery required for sessile life and building calcite shells. Metamorphosis becomes a survival bottleneck, which will be pressured by different anthropogenically induced climate change-related variables. Therefore, it is important to understand how metamorphosing larvae interact with emerging climate change stressors. To predict how larvae might be affected in a future ocean, we examined changes in the proteome of metamorphosing larvae under multiple stressors: decreased pH (pH 7.4), increased temperature (30 °C), and reduced salinity (15 psu). Quantitative protein expression profiling using iTRAQ-LC-MS/MS identified more than 1300 proteins. Decreased pH had a negative effect on metamorphosis by down-regulating several proteins involved in energy production, metabolism, and protein synthesis. However, warming switched on these down-regulated pathways at pH 7.4. Under multiple stressors, cell signaling, energy production, growth, and developmental pathways were up-regulated, although metamorphosis was still reduced. Despite the lack of lethal effects, significant physiological responses to both individual and interacting climate change related stressors were observed at proteome level. The metamorphosing larvae of the C. gigas population in the Yellow Sea appear to have adequate phenotypic plasticity at the proteome level to survive in future coastal oceans, but with developmental and physiological costs. © 2016 John Wiley & Sons Ltd.

  18. Quantitative proteomics reveals the mechanism and consequence of gliotoxin-mediated dysregulation of the methionine cycle in Aspergillus niger.

    Science.gov (United States)

    Manzanares-Miralles, Lara; Sarikaya-Bayram, Özlem; Smith, Elizabeth B; Dolan, Stephen K; Bayram, Özgür; Jones, Gary W; Doyle, Sean

    2016-01-10

    Gliotoxin (GT) is a redox-active metabolite, produced by Aspergillus fumigatus, which inhibits the growth of other fungi. Here we demonstrate how Aspergillus niger responds to GT exposure. Quantitative proteomics revealed that GT dysregulated the abundance of 378 proteins including those involved in methionine metabolism and induced de novo abundance of two S-adenosylmethionine (SAM)-dependent methyltransferases. Increased abundance of enzymes S-adenosylhomocysteinase (p=0.0018) required for homocysteine generation from S-adenosylhomocysteine (SAH), and spermidine synthase (p=0.0068), involved in the recycling of Met, was observed. Analysis of Met-related metabolites revealed significant increases in the levels of Met and adenosine, in correlation with proteomic data. Methyltransferase MT-II is responsible for bisthiobis(methylthio)gliotoxin (BmGT) formation, deletion of MT-II abolished BmGT formation and led to increased GT sensitivity in A. niger. Proteomic analysis also revealed that GT exposure also significantly (pniger. Thus, it provides new opportunities to exploit the response of GT-naïve fungi to GT. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. iTRAQ-based quantitative proteomic analysis of midgut in silkworm infected with Bombyx mori cytoplasmic polyhedrosis virus.

    Science.gov (United States)

    Gao, Kun; Deng, Xiang-Yuan; Shang, Meng-Ke; Qin, Guang-Xing; Hou, Cheng-Xiang; Guo, Xi-Jie

    2017-01-30

    Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) specifically infects the epithelial cells in the midgut of silkworm and causes them to death, which negatively affects the sericulture industry. In order to determine the midgut response at the protein levels to the virus infection, differential proteomes of the silkworm midgut responsive to BmCPV infection were identified with isobaric tags for relative and absolute quantitation (iTRAQ) labeling followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). 193, 408, 189 differentially expressed proteins (DEPs) were reliably quantified by iTRAQ analysis in the midgut of BmCPV-infected and control larvae at 24, 48, 72h post infection (hpi) respectively. KEGG enrichment analysis showed that Oxidative phosphorylation, amyotrophic lateral sclerosis, Toll-like receptor signaling pathway, steroid hormone biosynthesis were the significant pathways (Q value≤0.05) both at 24 and 48hpi. qRT-PCR was used to further verify gene transcription of 30 DEPs from iTRAQ, showing that the regulations of 24 genes at the transcript level were consistent with those at the proteomic level. Moreover, the cluster analysis of the three time groups showed that there were seven co-regulated DEPs including BGIBMGA002620-PA, which was a putative p62/sequestosome-1 protein in silkworm. It was upregulated at both the mRNA level and the proteomic level and may play an important role in regulating the autophagy and apoptosis (especially apoptosis) induced by BmCPV infection. This was the first report using an iTRAQ approach to analyze proteomes of the silkworm midgut against BmCPV infection, which contributes to understanding the defense mechanisms of silkworm midgut to virus infection. The domesticated silkworm, Bombyx mori, is renowned for silk production as well as being a traditional lepidopteron model insect served as a subject for morphological, genetic, physiological, and developmental studies. Bombyx mori cytoplasmic polyhedrosis

  20. The membrane proteome of Medicago truncatula roots displays qualitative and quantitative changes in response to arbuscular mycorrhizal symbiosis.

    Science.gov (United States)

    Abdallah, Cosette; Valot, Benoit; Guillier, Christelle; Mounier, Arnaud; Balliau, Thierry; Zivy, Michel; van Tuinen, Diederik; Renaut, Jenny; Wipf, Daniel; Dumas-Gaudot, Eliane; Recorbet, Ghislaine

    2014-08-28

    Arbuscular mycorrhizal (AM) symbiosis that associates roots of most land plants with soil-borne fungi (Glomeromycota), is characterized by reciprocal nutritional benefits. Fungal colonization of plant roots induces massive changes in cortical cells where the fungus differentiates an arbuscule, which drives proliferation of the plasma membrane. Despite the recognized importance of membrane proteins in sustaining AM symbiosis, the root microsomal proteome elicited upon mycorrhiza still remains to be explored. In this study, we first examined the qualitative composition of the root membrane proteome of Medicago truncatula after microsome enrichment and subsequent in depth analysis by GeLC-MS/MS. The results obtained highlighted the identification of 1226 root membrane protein candidates whose cellular and functional classifications predispose plastids and protein synthesis as prevalent organelle and function, respectively. Changes at the protein abundance level between the membrane proteomes of mycorrhizal and nonmycorrhizal roots were further monitored by spectral counting, which retrieved a total of 96 proteins that displayed a differential accumulation upon AM symbiosis. Besides the canonical markers of the periarbuscular membrane, new candidates supporting the importance of membrane trafficking events during mycorrhiza establishment/functioning were identified, including flotillin-like proteins. The data have been deposited to the ProteomeXchange with identifier PXD000875. During arbuscular mycorrhizal symbiosis, one of the most widespread mutualistic associations in nature, the endomembrane system of plant roots is believed to undergo qualitative and quantitative changes in order to sustain both the accommodation process of the AM fungus within cortical cells and the exchange of nutrients between symbionts. Large-scale GeLC-MS/MS proteomic analysis of the membrane fractions from mycorrhizal and nonmycorrhizal roots of M. truncatula coupled to spectral counting

  1. Social network architecture of human immune cells unveiled by quantitative proteomics.

    Science.gov (United States)

    Rieckmann, Jan C; Geiger, Roger; Hornburg, Daniel; Wolf, Tobias; Kveler, Ksenya; Jarrossay, David; Sallusto, Federica; Shen-Orr, Shai S; Lanzavecchia, Antonio; Mann, Matthias; Meissner, Felix

    2017-05-01

    The immune system is unique in its dynamic interplay between numerous cell types. However, a system-wide view of how immune cells communicate to protect against disease has not yet been established. We applied high-resolution mass-spectrometry-based proteomics to characterize 28 primary human hematopoietic cell populations in steady and activated states at a depth of >10,000 proteins in total. Protein copy numbers revealed a specialization of immune cells for ligand and receptor expression, thereby connecting distinct immune functions. By integrating total and secreted proteomes, we discovered fundamental intercellular communication structures and previously unknown connections between cell types. Our publicly accessible (http://www.immprot.org/) proteomic resource provides a framework for the orchestration of cellular interplay and a reference for altered communication associated with pathology.

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

    Science.gov (United States)

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

    2018-02-28

    Nanoscale or single-cell technologies are critical for biomedical applications. However, current mass spectrometry (MS)-based proteomic approaches require samples comprising a minimum of thousands of cells to provide in-depth profiling. Here, we report the development of a nanoPOTS (nanodroplet processing in one pot for trace samples) platform for small cell population proteomics analysis. NanoPOTS enhances the efficiency and recovery of sample processing by downscaling processing volumes to 3000 proteins are consistently identified from as few as 10 cells. Furthermore, we demonstrate quantification of ~2400 proteins from single human pancreatic islet thin sections from type 1 diabetic and control donors, illustrating the application of nanoPOTS for spatially resolved proteome measurements from clinical tissues.

  3. Streptococcus mutans Protein Synthesis during Mixed-Species Biofilm Development by High-Throughput Quantitative Proteomics

    Science.gov (United States)

    Klein, Marlise I.; Xiao, Jin; Lu, Bingwen; Delahunty, Claire M.; Yates, John R.; Koo, Hyun

    2012-01-01

    Biofilms formed on tooth surfaces are comprised of mixed microbiota enmeshed in an extracellular matrix. Oral biofilms are constantly exposed to environmental changes, which influence the microbial composition, matrix formation and expression of virulence. Streptococcus mutans and sucrose are key modulators associated with the evolution of virulent-cariogenic biofilms. In this study, we used a high-throughput quantitative proteomics approach to examine how S. mutans produces relevant proteins that facilitate its establishment and optimal survival during mixed-species biofilms development induced by sucrose. Biofilms of S. mutans, alone or mixed with Actinomyces naeslundii and Streptococcus oralis, were initially formed onto saliva-coated hydroxyapatite surface under carbohydrate-limiting condition. Sucrose (1%, w/v) was then introduced to cause environmental changes, and to induce biofilm accumulation. Multidimensional protein identification technology (MudPIT) approach detected up to 60% of proteins encoded by S. mutans within biofilms. Specific proteins associated with exopolysaccharide matrix assembly, metabolic and stress adaptation processes were highly abundant as the biofilm transit from earlier to later developmental stages following sucrose introduction. Our results indicate that S. mutans within a mixed-species biofilm community increases the expression of specific genes associated with glucan synthesis and remodeling (gtfBC, dexA) and glucan-binding (gbpB) during this transition (Pmutans up-regulates specific adaptation mechanisms to cope with acidic environments (F1F0-ATPase system, fatty acid biosynthesis, branched chain amino acids metabolism), and molecular chaperones (GroEL). Interestingly, the protein levels and gene expression are in general augmented when S. mutans form mixed-species biofilms (vs. single-species biofilms) demonstrating fundamental differences in the matrix assembly, survival and biofilm maintenance in the presence of other

  4. Systematic analysis of asymmetric partitioning of yeast proteome between mother and daughter cells reveals “aging factors” and mechanism of lifespan asymmetry

    Science.gov (United States)

    Yang, Jing; McCormick, Mark A.; Zheng, Jiashun; Xie, Zhengwei; Tsuchiya, Mitsuhiro; Tsuchiyama, Scott; El-Samad, Hana; Ouyang, Qi; Kaeberlein, Matt; Kennedy, Brian K.; Li, Hao

    2015-01-01

    Budding yeast divides asymmetrically, giving rise to a mother cell that progressively ages and a daughter cell with full lifespan. It is generally assumed that mother cells retain damaged, lifespan limiting materials (“aging factors”) through asymmetric division. However, the identity of these aging factors and the mechanisms through which they limit lifespan remain poorly understood. Using a flow cytometry-based, high-throughput approach, we quantified the asymmetric partitioning of the yeast proteome between mother and daughter cells during cell division, discovering 74 mother-enriched and 60 daughter-enriched proteins. While daughter-enriched proteins are biased toward those needed for bud construction and genome maintenance, mother-enriched proteins are biased towards those localized in the plasma membrane and vacuole. Deletion of 23 of the 74 mother-enriched proteins leads to lifespan extension, a fraction that is about six times that of the genes picked randomly from the genome. Among these lifespan-extending genes, three are involved in endosomal sorting/endosome to vacuole transport, and three are nitrogen source transporters. Tracking the dynamic expression of specific mother-enriched proteins revealed that their concentration steadily increases in the mother cells as they age, but is kept relatively low in the daughter cells via asymmetric distribution. Our results suggest that some mother-enriched proteins may increase to a concentration that becomes deleterious and lifespan-limiting in aged cells, possibly by upsetting homeostasis or leading to aberrant signaling. Our study provides a comprehensive resource for analyzing asymmetric cell division and aging in yeast, which should also be valuable for understanding similar phenomena in other organisms. PMID:26351681

  5. Systematic analysis of asymmetric partitioning of yeast proteome between mother and daughter cells reveals "aging factors" and mechanism of lifespan asymmetry.

    Science.gov (United States)

    Yang, Jing; McCormick, Mark A; Zheng, Jiashun; Xie, Zhengwei; Tsuchiya, Mitsuhiro; Tsuchiyama, Scott; El-Samad, Hana; Ouyang, Qi; Kaeberlein, Matt; Kennedy, Brian K; Li, Hao

    2015-09-22

    Budding yeast divides asymmetrically, giving rise to a mother cell that progressively ages and a daughter cell with full lifespan. It is generally assumed that mother cells retain damaged, lifespan limiting materials ("aging factors") through asymmetric division. However, the identity of these aging factors and the mechanisms through which they limit lifespan remain poorly understood. Using a flow cytometry-based, high-throughput approach, we quantified the asymmetric partitioning of the yeast proteome between mother and daughter cells during cell division, discovering 74 mother-enriched and 60 daughter-enriched proteins. While daughter-enriched proteins are biased toward those needed for bud construction and genome maintenance, mother-enriched proteins are biased towards those localized in the plasma membrane and vacuole. Deletion of 23 of the 74 mother-enriched proteins leads to lifespan extension, a fraction that is about six times that of the genes picked randomly from the genome. Among these lifespan-extending genes, three are involved in endosomal sorting/endosome to vacuole transport, and three are nitrogen source transporters. Tracking the dynamic expression of specific mother-enriched proteins revealed that their concentration steadily increases in the mother cells as they age, but is kept relatively low in the daughter cells via asymmetric distribution. Our results suggest that some mother-enriched proteins may increase to a concentration that becomes deleterious and lifespan-limiting in aged cells, possibly by upsetting homeostasis or leading to aberrant signaling. Our study provides a comprehensive resource for analyzing asymmetric cell division and aging in yeast, which should also be valuable for understanding similar phenomena in other organisms.

  6. Assigning Quantitative Function to Post-Translational Modifications Reveals Multiple Sites of Phosphorylation That Tune Yeast Pheromone Signaling Output

    Energy Technology Data Exchange (ETDEWEB)

    Pincus, David; Ryan, Christopher J.; Smith, Richard D.; Brent, Roger; Resnekov, Orna; Hakimi, Mohamed Ali

    2013-03-12

    Cell signaling systems transmit information by post-­translationally modifying signaling proteins, often via phosphorylation. While thousands of sites of phosphorylation have been identified in proteomic studies, the vast majority of sites have no known function. Assigning functional roles to the catalog of uncharacterized phosphorylation sites is a key research challenge. Here we present a general approach to address this challenge and apply it to a prototypical signaling pathway, the pheromone response pathway in Saccharomyces cerevisiae. The pheromone pathway includes a mitogen activated protein kinase (MAPK) cascade activated by a G-­protein coupled receptor (GPCR). We used mass spectrometry-based proteomics to identify sites whose phosphorylation changed when the system was active, and evolutionary conservation to assign priority to a list of candidate MAPK regulatory sites. We made targeted alterations in those sites, and measured the effects of the mutations on pheromone pathway output in single cells. Our work identified six new sites that quantitatively tuned system output. We developed simple computational models to find system architectures that recapitulated the quantitative phenotypes of the mutants. Our results identify a number of regulated phosphorylation events that contribute to adjust the input-­output relationship of this model eukaryotic signaling system. We believe this combined approach constitutes a general means not only to reveal modification sites required to turn a pathway on and off, but also those required for more subtle quantitative effects that tune pathway output. Our results further suggest that relatively small quantitative influences from individual regulatory phosphorylation events endow signaling systems with plasticity that evolution may exploit to quantitatively tailor signaling outcomes.

  7. Quantitative Proteomic Analysis Reveals that Antioxidation Mechanisms Contribute to Cold Tolerance in Plantain (Musa paradisiaca L.; ABB Group) Seedlings*

    Science.gov (United States)

    Yang, Qiao-Song; Wu, Jun-Hua; Li, Chun-Yu; Wei, Yue-Rong; Sheng, Ou; Hu, Chun-Hua; Kuang, Rui-Bin; Huang, Yong-Hong; Peng, Xin-Xiang; McCardle, James A.; Chen, Wei; Yang, Yong; Rose, Jocelyn K. C.; Zhang, Sheng; Yi, Gan-Jun

    2012-01-01

    Banana and its close relative, plantain are globally important crops and there is considerable interest in optimizing their cultivation. Plantain has superior cold tolerance compared with banana and a thorough understanding of the molecular mechanisms and responses of plantain to cold stress has great potential value for developing cold tolerant banana cultivars. In this study, we used iTRAQ-based comparative proteomic analysis to investigate the temporal responses of plantain to cold stress. Plantain seedlings were exposed for 0, 6, and 24 h of cold stress at 8 °C and subsequently allowed to recover for 24 h at 28 °C. A total of 3477 plantain proteins were identified, of which 809 showed differential expression from the three treatments. The majority of differentially expressed proteins were predicted to be involved in oxidation-reduction, including oxylipin biosynthesis, whereas others were associated with photosynthesis, photorespiration, and several primary metabolic processes, such as carbohydrate metabolic process and fatty acid beta-oxidation. Western blot analysis and enzyme activity assays were performed on seven differentially expressed, cold-response candidate plantain proteins to validate the proteomics data. Similar analyses of the seven candidate proteins were performed in cold-sensitive banana to examine possible functional conservation, and to compare the results to equivalent responses between the two species. Consistent results were achieved by Western blot and enzyme activity assays, demonstrating that the quantitative proteomics data collected in this study are reliable. Our results suggest that an increase of antioxidant capacity through adapted ROS scavenging capability, reduced production of ROS, and decreased lipid peroxidation contribute to molecular mechanisms for the increased cold tolerance in plantain. To the best of our knowledge, this is the first report of a global investigation on molecular responses of plantain to cold stress by

  8. Quantitative proteomic analysis reveals that antioxidation mechanisms contribute to cold tolerance in plantain (Musa paradisiaca L.; ABB Group) seedlings.

    Science.gov (United States)

    Yang, Qiao-Song; Wu, Jun-Hua; Li, Chun-Yu; Wei, Yue-Rong; Sheng, Ou; Hu, Chun-Hua; Kuang, Rui-Bin; Huang, Yong-Hong; Peng, Xin-Xiang; McCardle, James A; Chen, Wei; Yang, Yong; Rose, Jocelyn K C; Zhang, Sheng; Yi, Gan-Jun

    2012-12-01

    Banana and its close relative, plantain are globally important crops and there is considerable interest in optimizing their cultivation. Plantain has superior cold tolerance compared with banana and a thorough understanding of the molecular mechanisms and responses of plantain to cold stress has great potential value for developing cold tolerant banana cultivars. In this study, we used iTRAQ-based comparative proteomic analysis to investigate the temporal responses of plantain to cold stress. Plantain seedlings were exposed for 0, 6, and 24 h of cold stress at 8 °C and subsequently allowed to recover for 24 h at 28 °C. A total of 3477 plantain proteins were identified, of which 809 showed differential expression from the three treatments. The majority of differentially expressed proteins were predicted to be involved in oxidation-reduction, including oxylipin biosynthesis, whereas others were associated with photosynthesis, photorespiration, and several primary metabolic processes, such as carbohydrate metabolic process and fatty acid beta-oxidation. Western blot analysis and enzyme activity assays were performed on seven differentially expressed, cold-response candidate plantain proteins to validate the proteomics data. Similar analyses of the seven candidate proteins were performed in cold-sensitive banana to examine possible functional conservation, and to compare the results to equivalent responses between the two species. Consistent results were achieved by Western blot and enzyme activity assays, demonstrating that the quantitative proteomics data collected in this study are reliable. Our results suggest that an increase of antioxidant capacity through adapted ROS scavenging capability, reduced production of ROS, and decreased lipid peroxidation contribute to molecular mechanisms for the increased cold tolerance in plantain. To the best of our knowledge, this is the first report of a global investigation on molecular responses of plantain to cold stress by

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

    Science.gov (United States)

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

    2018-01-16

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

  10. Comparative and quantitative proteomics reveal the adaptive strategies of oyster larvae to ocean acidification

    KAUST Repository

    Dineshram, R.; Q., Quan; Sharma, Rakesh; Chandramouli, Kondethimmanahalli; Yalamanchili, Hari Krishna; Chu, Ivan; Thiyagarajan, Vengatesen

    2015-01-01

    © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Decreasing pH due to anthropogenic CO2 inputs, called ocean acidification (OA), can make coastal environments unfavorable for oysters. This is a serious socioeconomical issue for China which supplies >70% of the world's edible oysters. Here, we present an iTRAQ-based protein profiling approach for the detection and quantification of proteome changes under OA in the early life stage of a commercially important oyster, Crassostrea hongkongensis. Availability of complete genome sequence for the pacific oyster (Crassostrea gigas) enabled us to confidently quantify over 1500 proteins in larval oysters. Over 7% of the proteome was altered in response to OA at pHNBS 7.6. Analysis of differentially expressed proteins and their associated functional pathways showed an upregulation of proteins involved in calcification, metabolic processes, and oxidative stress, each of which may be important in physiological adaptation of this species to OA. The downregulation of cytoskeletal and signal transduction proteins, on the other hand, might have impaired cellular dynamics and organelle development under OA. However, there were no significant detrimental effects in developmental processes such as metamorphic success. Implications of the differentially expressed proteins and metabolic pathways in the development of OA resistance in oyster larvae are discussed. The MS proteomics data have been deposited to the ProteomeXchange with identifiers PXD002138 (http://proteomecentral.proteomexchange.org/dataset/PXD002138).

  11. Comparative and quantitative proteomics reveal the adaptive strategies of oyster larvae to ocean acidification

    KAUST Repository

    Dineshram, R.

    2015-10-28

    © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Decreasing pH due to anthropogenic CO2 inputs, called ocean acidification (OA), can make coastal environments unfavorable for oysters. This is a serious socioeconomical issue for China which supplies >70% of the world\\'s edible oysters. Here, we present an iTRAQ-based protein profiling approach for the detection and quantification of proteome changes under OA in the early life stage of a commercially important oyster, Crassostrea hongkongensis. Availability of complete genome sequence for the pacific oyster (Crassostrea gigas) enabled us to confidently quantify over 1500 proteins in larval oysters. Over 7% of the proteome was altered in response to OA at pHNBS 7.6. Analysis of differentially expressed proteins and their associated functional pathways showed an upregulation of proteins involved in calcification, metabolic processes, and oxidative stress, each of which may be important in physiological adaptation of this species to OA. The downregulation of cytoskeletal and signal transduction proteins, on the other hand, might have impaired cellular dynamics and organelle development under OA. However, there were no significant detrimental effects in developmental processes such as metamorphic success. Implications of the differentially expressed proteins and metabolic pathways in the development of OA resistance in oyster larvae are discussed. The MS proteomics data have been deposited to the ProteomeXchange with identifiers PXD002138 (http://proteomecentral.proteomexchange.org/dataset/PXD002138).

  12. 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

    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...

  13. Deep proteomics of mouse skeletal muscle enables quantitation of protein isoforms, metabolic pathways and transcription factors

    DEFF Research Database (Denmark)

    Deshmukh, Atul S; Murgia, Marta; Nagaraja, Nagarjuna

    2015-01-01

    Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging due to highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art mass...

  14. Towards high throughput and spatiotemporal proteomics : analytical workflows and quantitative label-free mass spectrometry

    NARCIS (Netherlands)

    Mostovenko, Ekaterina

    2013-01-01

    A large part of modern biology is dedicated to the functional annotation and interpretation of genetic information and its influence on the subject’s phenotype. Proteomics describes the state of the system from the perspective of expression, structure, localization, interaction and function of the

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

    Science.gov (United States)

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

    2017-07-01

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

  16. A Proteome-wide, Quantitative Survey of In Vivo Ubiquitylation Sites Reveals Widespread Regulatory Roles

    DEFF Research Database (Denmark)

    Wagner, Sebastian Alexander; Beli, Petra; Weinert, Brian Tate

    2011-01-01

    Post-translational modification of proteins by ubiquitin is a fundamentally important regulatory mechanism. However, proteome-wide analysis of endogenous ubiquitylation remains a challenging task, and almost always has relied on cells expressing affinity tagged ubiquitin. Here we combine single...

  17. Cell wall staining with Trypan blue enables quantitative analysis of morphological changes in yeast cells

    DEFF Research Database (Denmark)

    Liesche, Johannes; Marek, Magdalena; Günther-Pomorski, Thomas

    2015-01-01

    staining with fluorescent dyes is a valuable tool. Furthermore, cell wall staining is used to facilitate sub-cellular localization experiments with fluorescently-labeled proteins and the detection of yeast cells in non-fungal host tissues. Here, we report staining of Saccharomyces cerevisiae cell wall......Yeast cells are protected by a cell wall that plays an important role in the exchange of substances with the environment. The cell wall structure is dynamic and can adapt to different physiological states or environmental conditions. For the investigation of morphological changes, selective...... with Trypan Blue, which emits strong red fluorescence upon binding to chitin and yeast glucan; thereby, it facilitates cell wall analysis by confocal and super-resolution microscopy. The staining pattern of Trypan Blue was similar to that of the widely used UV-excitable, blue fluorescent cell wall stain...

  18. Elevated host lipid metabolism revealed by iTRAQ-based quantitative proteomic analysis of cerebrospinal fluid of tuberculous meningitis patients.

    Science.gov (United States)

    Mu, Jun; Yang, Yongtao; Chen, Jin; Cheng, Ke; Li, Qi; Wei, Yongdong; Zhu, Dan; Shao, Weihua; Zheng, Peng; Xie, Peng

    2015-10-30

    Tuberculous meningitis (TBM) remains to be one of the most deadly infectious diseases. The pathogen interacts with the host immune system, the process of which is largely unknown. Various cellular processes of Mycobacterium tuberculosis (MTB) centers around lipid metabolism. To determine the lipid metabolism related proteins, a quantitative proteomic study was performed here to identify differential proteins in the cerebrospinal fluid (CSF) obtained from TBM patients (n = 12) and healthy controls (n = 12). CSF samples were desalted, concentrated, labelled with isobaric tags for relative and absolute quantitation (iTRAQ™), and analyzed by multi-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gene ontology and proteomic phenotyping analysis of the differential proteins were conducted using Database for Annotation, Visualization, and Integrated Discovery (DAVID) Bioinformatics Resources. ApoE and ApoB were selected for validation by ELISA. Proteomic phenotyping of the 4 differential proteins was invloved in the lipid metabolism. ELISA showed significantly increased ApoB levels in TBM subjects compared to healthy controls. Area under the receiver operating characteristic curve analysis demonstrated ApoB levels could distinguish TBM subjects from healthy controls and viral meningitis subjects with 89.3% sensitivity and 92% specificity. CSF lipid metabolism disregulation, especially elevated expression of ApoB, gives insights into the pathogenesis of TBM. Further evaluation of these findings in larger studies including anti-tuberculosis medicated and unmedicated patient cohorts with other center nervous system infectious diseases is required for successful clinical translation. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Comparison of the proteomes of three yeast wild type strains: CEN.PK2, FY1679 and W303

    DEFF Research Database (Denmark)

    Rogowska-Wrzesinska, A.; Mose Larsen, P.; Blomberg, A.

    2001-01-01

    Yeast deletion strains created during gene function analysis projects very often show drastic phenotypic differences depending on the genetic background used. These results indicate the existence of important molecular differences between the CEN.PK2, FY1679 and W303 wild type strains...

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

    Science.gov (United States)

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

    2013-01-01

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

  1. Quantitative proteomics links metabolic pathways to specific developmental stages of the plant-pathogenic oomycete Phytophthora capsici.

    Science.gov (United States)

    Pang, Zhili; Srivastava, Vaibhav; Liu, Xili; Bulone, Vincent

    2017-04-01

    The oomycete Phytophthora capsici is a plant pathogen responsible for important losses to vegetable production worldwide. Its asexual reproduction plays an important role in the rapid propagation and spread of the disease in the field. A global proteomics study was conducted to compare two key asexual life stages of P. capsici, i.e. the mycelium and cysts, to identify stage-specific biochemical processes. A total of 1200 proteins was identified using qualitative and quantitative proteomics. The transcript abundance of some of the enriched proteins was also analysed by quantitative real-time polymerase chain reaction. Seventy-three proteins exhibited different levels of abundance between the mycelium and cysts. The proteins enriched in the mycelium are mainly associated with glycolysis, the tricarboxylic acid (or citric acid) cycle and the pentose phosphate pathway, providing the energy required for the biosynthesis of cellular building blocks and hyphal growth. In contrast, the proteins that are predominant in cysts are essentially involved in fatty acid degradation, suggesting that the early infection stage of the pathogen relies primarily on fatty acid degradation for energy production. The data provide a better understanding of P. capsici biology and suggest potential metabolic targets at the two different developmental stages for disease control. © 2016 BSPP AND JOHN WILEY & SONS LTD.

  2. Quantitative changes in proteins responsible for flavonoid and anthocyanin biosynthesis in strawberry fruit at different ripening stages: A targeted quantitative proteomic investigation employing multiple reaction monitoring.

    Science.gov (United States)

    Song, Jun; Du, Lina; Li, Li; Kalt, Wilhelmina; Palmer, Leslie Campbell; Fillmore, Sherry; Zhang, Ying; Zhang, ZhaoQi; Li, XiHong

    2015-06-03

    To better understand the regulation of flavonoid and anthocyanin biosynthesis, a targeted quantitative proteomic investigation employing LC-MS with multiple reaction monitoring was conducted on two strawberry cultivars at three ripening stages. This quantitative proteomic workflow was improved through an OFFGEL electrophoresis to fractionate peptides from total protein digests. A total of 154 peptide transitions from 47 peptides covering 21 proteins and isoforms related to anthocyanin biosynthesis were investigated. The normalized protein abundance, which was measured using isotopically-labeled standards, was significantly changed concurrently with increased anthocyanin content and advanced fruit maturity. The protein abundance of phenylalanine ammonia-lyase; anthocyanidin synthase, chalcone isomerase; flavanone 3-hydroxylase; dihydroflavonol 4-reductase, UDP-glucose:flavonoid-3-O-glucosyltransferase, cytochrome c and cytochrome C oxidase subunit 2, was all significantly increased in fruit of more advanced ripeness. An interaction between cultivar and maturity was also shown with respect to chalcone isomerase. The good correlation between protein abundance and anthocyanin content suggested that a metabolic control point may exist for anthocyanin biosynthesis. This research provides insights into the process of anthocyanin formation in strawberry fruit at the level of protein concentration and reveals possible candidates in the regulation of anthocyanin formation during fruit ripening. To gain insight into the molecular mechanisms contributing to flavonoids and anthocyanin biosynthesis and regulation of strawberry fruit during ripening is challenging due to limited molecular biology tools and established hypothesis. Our targeted proteomic approach employing LC-MS/MS analysis and MRM technique to quantify proteins in relation to flavonoids and anthocyanin biosynthesis and regulation in strawberry fruit during fruit ripening is novel. The identification of peptides

  3. Quantitation & Case-Study-Driven Inquiry to Enhance Yeast Fermentation Studies

    Science.gov (United States)

    Grammer, Robert T.

    2012-01-01

    We propose a procedure for the assay of fermentation in yeast in microcentrifuge tubes that is simple and rapid, permitting assay replicates, descriptive statistics, and the preparation of line graphs that indicate reproducibility. Using regression and simple derivatives to determine initial velocities, we suggest methods to compare the effects of…

  4. Condenser: a statistical aggregation tool for multi-sample quantitative proteomic data from Matrix Science Mascot Distiller™.

    Science.gov (United States)

    Knudsen, Anders Dahl; Bennike, Tue; Kjeldal, Henrik; Birkelund, Svend; Otzen, Daniel Erik; Stensballe, Allan

    2014-05-30

    We describe Condenser, a freely available, comprehensive open-source tool for merging multidimensional quantitative proteomics data from the Matrix Science Mascot Distiller Quantitation Toolbox into a common format ready for subsequent bioinformatic analysis. A number of different relative quantitation technologies, such as metabolic (15)N and amino acid stable isotope incorporation, label-free and chemical-label quantitation are supported. The program features multiple options for curative filtering of the quantified peptides, allowing the user to choose data quality thresholds appropriate for the current dataset, and ensure the quality of the calculated relative protein abundances. Condenser also features optional global normalization, peptide outlier removal, multiple testing and calculation of t-test statistics for highlighting and evaluating proteins with significantly altered relative protein abundances. Condenser provides an attractive addition to the gold-standard quantitative workflow of Mascot Distiller, allowing easy handling of larger multi-dimensional experiments. Source code, binaries, test data set and documentation are available at http://condenser.googlecode.com/. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Identification of autophagosome-associated proteins and regulators by quantitative proteomic analysis and genetic screens

    DEFF Research Database (Denmark)

    Dengjel, Jörn; Høyer-Hansen, Maria; Nielsen, Maria

    2012-01-01

    Autophagy is one of the major intracellular catabolic pathways, but little is known about the composition of autophagosomes. To study the associated proteins, we isolated autophagosomes from human breast cancer cells using two different biochemical methods and three stimulus types: amino acid dep...... regulators of autophagy, including subunits of the retromer complex. The combined spatiotemporal proteomic and genetic data sets presented here provide a basis for further characterization of autophagosome biogenesis and cargo selection....

  6. Multiple testing corrections in quantitative proteomics: A useful but blunt tool.

    Science.gov (United States)

    Pascovici, Dana; Handler, David C L; Wu, Jemma X; Haynes, Paul A

    2016-09-01

    Multiple testing corrections are a useful tool for restricting the FDR, but can be blunt in the context of low power, as we demonstrate by a series of simple simulations. Unfortunately, in proteomics experiments low power can be common, driven by proteomics-specific issues like small effects due to ratio compression, and few replicates due to reagent high cost, instrument time availability and other issues; in such situations, most multiple testing corrections methods, if used with conventional thresholds, will fail to detect any true positives even when many exist. In this low power, medium scale situation, other methods such as effect size considerations or peptide-level calculations may be a more effective option, even if they do not offer the same theoretical guarantee of a low FDR. Thus, we aim to highlight in this article that proteomics presents some specific challenges to the standard multiple testing corrections methods, which should be employed as a useful tool but not be regarded as a required rubber stamp. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Quantitative proteomics reveals differential biological processes in healthy neonatal cord neutrophils and adult neutrophils

    KAUST Repository

    Zhu, Jiang; Zhang, Huoming; Guo, Tiannan; Li, Wenying; Li, Huiyu; Zhu, Yi; Huang, Shiang

    2014-01-01

    Neonatal neutrophils are characterized by the immaturity of bactericidal mechanisms that contributes largely to neonatal mortality. However, underlying molecular mechanism associated with the immaturity remains incompletely understood. In this study, we performed comparative proteomic analysis on neonatal neutrophils derived from human cord blood and adult peripheral neutrophils. A total of 1332 proteins were identified and quantified, and 127 proteins were characterized as differentially expressed between adult and cord neutrophils. The differentially expressed proteins are mapped in KEGG pathways into five clusters and indicated impaired functions of neonatal neutrophils in proteasome, lysosome, phagosome, and leukocyte transendothelial migration. In particular, many proteins associated with NETosis, a critical mechanism for antimicrobial process and auto-clearance, were also found to be downregulated in cord neutrophils. This study represents a first comparative proteome profiling of neonatal and adult neutrophils, and provides a global view of differentially expressed proteome for enhancing our understanding of their various functional difference. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Data-Independent Acquisition-Based Quantitative Proteomic Analysis Reveals Potential Biomarkers of Kidney Cancer.

    Science.gov (United States)

    Song, Yimeng; Zhong, Lijun; Zhou, Juntuo; Lu, Min; Xing, Tianying; Ma, Lulin; Shen, Jing

    2017-12-01

    Renal cell carcinoma (RCC) is a malignant and metastatic cancer with 95% mortality, and clear cell RCC (ccRCC) is the most observed among the five major subtypes of RCC. Specific biomarkers that can distinguish cancer tissues from adjacent normal tissues should be developed to diagnose this disease in early stages and conduct a reliable prognostic evaluation. Data-independent acquisition (DIA) strategy has been widely employed in proteomic analysis because of various advantages, including enhanced protein coverage and reliable data acquisition. In this study, a DIA workflow is constructed on a quadrupole-Orbitrap LC-MS platform to reveal dysregulated proteins between ccRCC and adjacent normal tissues. More than 4000 proteins are identified, 436 of these proteins are dysregulated in ccRCC tissues. Bioinformatic analysis reveals that multiple pathways and Gene Ontology items are strongly associated with ccRCC. The expression levels of L-lactate dehydrogenase A chain, annexin A4, nicotinamide N-methyltransferase, and perilipin-2 examined through RT-qPCR, Western blot, and immunohistochemistry confirm the validity of the proteomic analysis results. The proposed DIA workflow yields optimum time efficiency and data reliability and provides a good choice for proteomic analysis in biological and clinical studies, and these dysregulated proteins might be potential biomarkers for ccRCC diagnosis. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Quantitative proteomics reveals differential biological processes in healthy neonatal cord neutrophils and adult neutrophils

    KAUST Repository

    Zhu, Jiang

    2014-06-11

    Neonatal neutrophils are characterized by the immaturity of bactericidal mechanisms that contributes largely to neonatal mortality. However, underlying molecular mechanism associated with the immaturity remains incompletely understood. In this study, we performed comparative proteomic analysis on neonatal neutrophils derived from human cord blood and adult peripheral neutrophils. A total of 1332 proteins were identified and quantified, and 127 proteins were characterized as differentially expressed between adult and cord neutrophils. The differentially expressed proteins are mapped in KEGG pathways into five clusters and indicated impaired functions of neonatal neutrophils in proteasome, lysosome, phagosome, and leukocyte transendothelial migration. In particular, many proteins associated with NETosis, a critical mechanism for antimicrobial process and auto-clearance, were also found to be downregulated in cord neutrophils. This study represents a first comparative proteome profiling of neonatal and adult neutrophils, and provides a global view of differentially expressed proteome for enhancing our understanding of their various functional difference. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. A systematic evaluation of normalization methods in quantitative label-free proteomics.

    Science.gov (United States)

    Välikangas, Tommi; Suomi, Tomi; Elo, Laura L

    2018-01-01

    To date, mass spectrometry (MS) data remain inherently biased as a result of reasons ranging from sample handling to differences caused by the instrumentation. Normalization is the process that aims to account for the bias and make samples more comparable. The selection of a proper normalization method is a pivotal task for the reliability of the downstream analysis and results. Many normalization methods commonly used in proteomics have been adapted from the DNA microarray techniques. Previous studies comparing normalization methods in proteomics have focused mainly on intragroup variation. In this study, several popular and widely used normalization methods representing different strategies in normalization are evaluated using three spike-in and one experimental mouse label-free proteomic data sets. The normalization methods are evaluated in terms of their ability to reduce variation between technical replicates, their effect on differential expression analysis and their effect on the estimation of logarithmic fold changes. Additionally, we examined whether normalizing the whole data globally or in segments for the differential expression analysis has an effect on the performance of the normalization methods. We found that variance stabilization normalization (Vsn) reduced variation the most between technical replicates in all examined data sets. Vsn also performed consistently well in the differential expression analysis. Linear regression normalization and local regression normalization performed also systematically well. Finally, we discuss the choice of a normalization method and some qualities of a suitable normalization method in the light of the results of our evaluation. © The Author 2016. Published by Oxford University Press.

  11. Quantitative proteomic analysis of serum from pregnant women carrying a fetus with conotruncal heart defect using isobaric tags for relative and absolute quantitation (iTRAQ labeling.

    Directory of Open Access Journals (Sweden)

    Ying Zhang

    Full Text Available To identify differentially expressed proteins from serum of pregnant women carrying a conotruncal heart defects (CTD fetus, using proteomic analysis.The study was conducted using a nested case-control design. The 5473 maternal serum samples were collected at 14-18 weeks of gestation. The serum from 9 pregnant women carrying a CTD fetus, 10 with another CHD (ACHD fetus, and 11 with a normal fetus were selected from the above samples, and analyzed by using isobaric tags for relative and absolute quantitation (iTRAQ coupled with two-dimensional liquid chromatography-tandem mass spectrometry(2D LC-MS/MS. The differentially expressed proteins identified by iTRAQ were further validated with Western blot.A total of 105 unique proteins present in the three groups were identified, and relative expression data were obtained for 92 of them with high confidence by employing the iTRAQ-based experiments. The downregulation of gelsolin in maternal serum of fetus with CTD was further verified by Western blot.The identification of differentially expressed protein gelsolin in the serum of the pregnant women carrying a CTD fetus by using proteomic technology may be able to serve as a foundation to further explore the biomarker for detection of CTD fetus from the maternal serum.

  12. A label-free quantitative shotgun proteomics analysis of rice grain development

    Directory of Open Access Journals (Sweden)

    Koh Hee-Jong

    2011-09-01

    Full Text Available Abstract Background Although a great deal of rice proteomic research has been conducted, there are relatively few studies specifically addressing the rice grain proteome. The existing rice grain proteomic researches have focused on the identification of differentially expressed proteins or monitoring protein expression patterns during grain filling stages. Results Proteins were extracted from rice grains 10, 20, and 30 days after flowering, as well as from fully mature grains. By merging all of the identified proteins in this study, we identified 4,172 non-redundant proteins with a wide range of molecular weights (from 5.2 kDa to 611 kDa and pI values (from pH 2.9 to pH 12.6. A Genome Ontology category enrichment analysis for the 4,172 proteins revealed that 52 categories were enriched, including the carbohydrate metabolic process, transport, localization, lipid metabolic process, and secondary metabolic process. The relative abundances of the 1,784 reproducibly identified proteins were compared to detect 484 differentially expressed proteins during rice grain development. Clustering analysis and Genome Ontology category enrichment analysis revealed that proteins involved in the metabolic process were enriched through all stages of development, suggesting that proteome changes occurred even in the desiccation phase. Interestingly, enrichments of proteins involved in protein folding were detected in the desiccation phase and in fully mature grain. Conclusion This is the first report conducting comprehensive identification of rice grain proteins. With a label free shotgun proteomic approach, we identified large number of rice grain proteins and compared the expression patterns of reproducibly identified proteins during rice grain development. Clustering analysis, Genome Ontology category enrichment analysis, and the analysis of composite expression profiles revealed dynamic changes of metabolisms during rice grain development. Interestingly, we

  13. Quantitative proteomic analysis of cabernet sauvignon grape cells exposed to thermal stresses reveals alterations in sugar and phenylpropanoid metabolism.

    Science.gov (United States)

    George, Iniga S; Pascovici, Dana; Mirzaei, Mehdi; Haynes, Paul A

    2015-09-01

    Grapes (Vitis vinifera) are a valuable fruit crop and wine production is a major industry. Global warming and expanded range of cultivation will expose grapes to more temperature stresses in future. Our study investigated protein level responses to abiotic stresses, with particular reference to proteomic changes induced by the impact of four different temperature stress regimes, including both hot and cold temperatures, on cultured grape cells. Cabernet Sauvignon cell suspension cultures grown at 26°C were subjected to 14 h of exposure to 34 and 42°C for heat stress, and 18 and 10°C for cold stress. Cells from the five temperatures were harvested in biological triplicates and label-free quantitative shotgun proteomic analysis was performed. A total of 2042 non-redundant proteins were identified from the five temperature points. Fifty-five proteins were only detected in extreme heat stress conditions (42°C) and 53 proteins were only detected at extreme cold stress conditions (10°C). Gene Ontology (GO) annotations of differentially expressed proteins provided insights into the metabolic pathways that are involved in temperature stress in grape cells. Sugar metabolism displayed switching between alternative and classical pathways during temperature stresses. Additionally, nine proteins involved in the phenylpropanoid pathway were greatly increased in abundance at extreme cold stress, and were thus found to be cold-responsive proteins. All MS data have been deposited in the ProteomeXchange with identifier PXD000977 (http://proteomecentral.proteomexchange.org/dataset/PXD000977). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Quantitative multiplexed proteomics of Taenia solium cysts obtained from the skeletal muscle and central nervous system of pigs.

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    José Navarrete-Perea

    2017-09-01

    Full Text Available In human and porcine cysticercosis caused by the tapeworm Taenia solium, the larval stage (cysts can infest several tissues including the central nervous system (CNS and the skeletal muscles (SM. The cyst's proteomics changes associated with the tissue localization in the host tissues have been poorly studied. Quantitative multiplexed proteomics has the power to evaluate global proteome changes in response to different conditions. Here, using a TMT-multiplexed strategy we identified and quantified over 4,200 proteins in cysts obtained from the SM and CNS of pigs, of which 891 were host proteins. To our knowledge, this is the most extensive intermixing of host and parasite proteins reported for tapeworm infections.Several antigens in cysticercosis, i.e., GP50, paramyosin and a calcium-binding protein were enriched in skeletal muscle cysts. Our results suggested the occurrence of tissue-enriched antigen that could be useful in the improvement of the immunodiagnosis for cysticercosis. Using several algorithms for epitope detection, we selected 42 highly antigenic proteins enriched for each tissue localization of the cysts. Taking into account the fold changes and the antigen/epitope contents, we selected 10 proteins and produced synthetic peptides from the best epitopes. Nine peptides were recognized by serum antibodies of cysticercotic pigs, suggesting that those peptides are antigens. Mixtures of peptides derived from SM and CNS cysts yielded better results than mixtures of peptides derived from a single tissue location, however the identification of the 'optimal' tissue-enriched antigens remains to be discovered. Through machine learning technologies, we determined that a reliable immunodiagnostic test for porcine cysticercosis required at least five different antigenic determinants.

  15. Quantitative multiplexed proteomics of Taenia solium cysts obtained from the skeletal muscle and central nervous system of pigs.

    Science.gov (United States)

    Navarrete-Perea, José; Isasa, Marta; Paulo, Joao A; Corral-Corral, Ricardo; Flores-Bautista, Jeanette; Hernández-Téllez, Beatriz; Bobes, Raúl J; Fragoso, Gladis; Sciutto, Edda; Soberón, Xavier; Gygi, Steven P; Laclette, Juan P

    2017-09-01

    In human and porcine cysticercosis caused by the tapeworm Taenia solium, the larval stage (cysts) can infest several tissues including the central nervous system (CNS) and the skeletal muscles (SM). The cyst's proteomics changes associated with the tissue localization in the host tissues have been poorly studied. Quantitative multiplexed proteomics has the power to evaluate global proteome changes in response to different conditions. Here, using a TMT-multiplexed strategy we identified and quantified over 4,200 proteins in cysts obtained from the SM and CNS of pigs, of which 891 were host proteins. To our knowledge, this is the most extensive intermixing of host and parasite proteins reported for tapeworm infections.Several antigens in cysticercosis, i.e., GP50, paramyosin and a calcium-binding protein were enriched in skeletal muscle cysts. Our results suggested the occurrence of tissue-enriched antigen that could be useful in the improvement of the immunodiagnosis for cysticercosis. Using several algorithms for epitope detection, we selected 42 highly antigenic proteins enriched for each tissue localization of the cysts. Taking into account the fold changes and the antigen/epitope contents, we selected 10 proteins and produced synthetic peptides from the best epitopes. Nine peptides were recognized by serum antibodies of cysticercotic pigs, suggesting that those peptides are antigens. Mixtures of peptides derived from SM and CNS cysts yielded better results than mixtures of peptides derived from a single tissue location, however the identification of the 'optimal' tissue-enriched antigens remains to be discovered. Through machine learning technologies, we determined that a reliable immunodiagnostic test for porcine cysticercosis required at least five different antigenic determinants.

  16. The mzqLibrary--An open source Java library supporting the HUPO-PSI quantitative proteomics standard.

    Science.gov (United States)

    Qi, Da; Zhang, Huaizhong; Fan, Jun; Perkins, Simon; Pisconti, Addolorata; Simpson, Deborah M; Bessant, Conrad; Hubbard, Simon; Jones, Andrew R

    2015-09-01

    The mzQuantML standard has been developed by the Proteomics Standards Initiative for capturing, archiving and exchanging quantitative proteomic data, derived from mass spectrometry. It is a rich XML-based format, capable of representing data about two-dimensional features from LC-MS data, and peptides, proteins or groups of proteins that have been quantified from multiple samples. In this article we report the development of an open source Java-based library of routines for mzQuantML, called the mzqLibrary, and associated software for visualising data called the mzqViewer. The mzqLibrary contains routines for mapping (peptide) identifications on quantified features, inference of protein (group)-level quantification values from peptide-level values, normalisation and basic statistics for differential expression. These routines can be accessed via the command line, via a Java programming interface access or a basic graphical user interface. The mzqLibrary also contains several file format converters, including import converters (to mzQuantML) from OpenMS, Progenesis LC-MS and MaxQuant, and exporters (from mzQuantML) to other standards or useful formats (mzTab, HTML, csv). The mzqViewer contains in-built routines for viewing the tables of data (about features, peptides or proteins), and connects to the R statistical library for more advanced plotting options. The mzqLibrary and mzqViewer packages are available from https://code.google.com/p/mzq-lib/. © 2015 The Authors. PROTEOMICS Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Cell wall staining with Trypan Blue enables quantitative analysis of morphological changes in yeast cells

    Directory of Open Access Journals (Sweden)

    Johannes eLiesche

    2015-02-01

    Full Text Available Yeast cells are protected by a cell wall that plays an important role in the exchange of substances with the environment. The cell wall structure is dynamic and can adapt to different physiological states or environmental conditions. For the investigation of morphological changes, selective staining with fluorescent dyes is a valuable tool. Furthermore, cell wall staining is used to facilitate sub-cellular localization experiments with fluorescently-labeled proteins and the detection of yeast cells in non-fungal host tissues. Here, we report staining of Saccharomyces cerevisiae cell wall with Trypan Blue, which emits strong red fluorescence upon binding to chitin and yeast glucan; thereby, it facilitates cell wall analysis by confocal and super-resolution microscopy. The staining pattern of Trypan Blue was similar to that of the widely used UV-excitable, blue fluorescent cell wall stain Calcofluor White. Trypan Blue staining facilitated quantification of cell size and cell wall volume when utilizing the optical sectioning capacity of a confocal microscope. This enabled the quantification of morphological changes during growth under anaerobic conditions and in the presence of chemicals, demonstrating the potential of this approach for morphological investigations or screening assays.

  18. Cell wall staining with Trypan blue enables quantitative analysis of morphological changes in yeast cells.

    Science.gov (United States)

    Liesche, Johannes; Marek, Magdalena; Günther-Pomorski, Thomas

    2015-01-01

    Yeast cells are protected by a cell wall that plays an important role in the exchange of substances with the environment. The cell wall structure is dynamic and can adapt to different physiological states or environmental conditions. For the investigation of morphological changes, selective staining with fluorescent dyes is a valuable tool. Furthermore, cell wall staining is used to facilitate sub-cellular localization experiments with fluorescently-labeled proteins and the detection of yeast cells in non-fungal host tissues. Here, we report staining of Saccharomyces cerevisiae cell wall with Trypan Blue, which emits strong red fluorescence upon binding to chitin and yeast glucan; thereby, it facilitates cell wall analysis by confocal and super-resolution microscopy. The staining pattern of Trypan Blue was similar to that of the widely used UV-excitable, blue fluorescent cell wall stain Calcofluor White. Trypan Blue staining facilitated quantification of cell size and cell wall volume when utilizing the optical sectioning capacity of a confocal microscope. This enabled the quantification of morphological changes during growth under anaerobic conditions and in the presence of chemicals, demonstrating the potential of this approach for morphological investigations or screening assays.

  19. Quantitative Proteomic Analysis of Mouse Embryonic Fibroblasts and Induced Pluripotent Stem Cells Using 16O /18O labeling

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Xin; Tian, Changhai; Liu, Miao; Wang, Yongxiang; Tolmachev, Aleksey V.; Sharma, Seema; Yu, Fang; Fu, Kai; Zheng, Jialin; Ding, Shi-Jian

    2012-04-06

    Induced pluripotent stem cells (iPSC) hold great promise for regenerative medicine as well as for investigations into the pathogenesis and treatment of various diseases. Understanding of key intracellular signaling pathways and protein targets that control development of iPSC from somatic cells is essential for designing new approaches to improve reprogramming efficiency. Here we report the development and application of an integrated quantitative proteomics platform for investigating differences in protein expressions between mouse embryonic fibroblasts (MEF) and MEF-derived iPSC. This platform consists of 16O/18O labeling, multidimensional peptide separation coupled with tandem mass spectrometry, and data analysis with UNiquant software. Using this platform a total of 2,481 proteins were identified and quantified from the 16O/18O-labeled MEF-iPSC proteome mixtures with a false discovery rate of 0.01. Among them, 218 proteins were significantly upregulated, while 247 proteins were significantly downregulated in iPSC compared to MEF. Many nuclear proteins, including Hdac1, Dnmt1, Pcna, Ccnd1, Smarcc1, and subunits in DNA replication and RNA polymerase II complex were found to be enhanced in iPSC. Protein network analysis revealed that Pcna functions as a hub orchestrating complicated mechanisms including DNA replication, epigenetic inheritance (Dnmt1) and chromatin remodeling (Smarcc1) to reprogram MEF and maintain stemness of iPSC.

  20. Generation of accurate peptide retention data for targeted and data independent quantitative LC-MS analysis: Chromatographic lessons in proteomics.

    Science.gov (United States)

    Krokhin, Oleg V; Spicer, Vic

    2016-12-01

    The emergence of data-independent quantitative LC-MS/MS analysis protocols further highlights the importance of high-quality reproducible chromatographic procedures. Knowing, controlling and being able to predict the effect of multiple factors that alter peptide RP-HPLC separation selectivity is critical for successful data collection for the construction of ion libraries. Proteomic researchers have often regarded RP-HPLC as a "black box", while vast amount of research on peptide separation is readily available. In addition to obvious parameters, such as the type of ion-pairing modifier, stationary phase and column temperature, we describe the "mysterious" effects of gradient slope, column size and flow rate on peptide separation selectivity. Retention time variations due to these parameters are governed by the linear solvent strength (LSS) theory on a peptide level by the value of its slope S in the basic LSS equation-a parameter that can be accurately predicted. Thus, the application of shallower gradients, higher flow rates, or smaller columns will each increases the relative retention of peptides with higher S-values (long species with multiple positively charged groups). Simultaneous changes to these parameters that each drive shifts in separation selectivity in the same direction should be avoided. The unification of terminology represents another pressing issue in this field of applied proteomics that should be addressed to facilitate further progress. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. SAFER, an Analysis Method of Quantitative Proteomic Data, Reveals New Interactors of the C. elegans Autophagic Protein LGG-1.

    Science.gov (United States)

    Yi, Zhou; Manil-Ségalen, Marion; Sago, Laila; Glatigny, Annie; Redeker, Virginie; Legouis, Renaud; Mucchielli-Giorgi, Marie-Hélène

    2016-05-06

    Affinity purifications followed by mass spectrometric analysis are used to identify protein-protein interactions. Because quantitative proteomic data are noisy, it is necessary to develop statistical methods to eliminate false-positives and identify true partners. We present here a novel approach for filtering false interactors, named "SAFER" for mass Spectrometry data Analysis by Filtering of Experimental Replicates, which is based on the reproducibility of the replicates and the fold-change of the protein intensities between bait and control. To identify regulators or targets of autophagy, we characterized the interactors of LGG1, a ubiquitin-like protein involved in autophagosome formation in C. elegans. LGG-1 partners were purified by affinity, analyzed by nanoLC-MS/MS mass spectrometry, and quantified by a label-free proteomic approach based on the mass spectrometric signal intensity of peptide precursor ions. Because the selection of confident interactions depends on the method used for statistical analysis, we compared SAFER with several statistical tests and different scoring algorithms on this set of data. We show that SAFER recovers high-confidence interactors that have been ignored by the other methods and identified new candidates involved in the autophagy process. We further validated our method on a public data set and conclude that SAFER notably improves the identification of protein interactors.

  2. Protein content and functional characteristics of serum-purified exosomes from patients with colorectal cancer revealed by quantitative proteomics.

    Science.gov (United States)

    Chen, Yanyu; Xie, Yong; Xu, Lai; Zhan, Shaohua; Xiao, Yi; Gao, Yanpan; Wu, Bin; Ge, Wei

    2017-02-15

    Tumor cells of colorectal cancer (CRC) release exosomes into the circulation. These exosomes can mediate communication between cells and affect various tumor-related processes in their target cells. We present a quantitative proteomics analysis of the exosomes purified from serum of patients with CRC and normal volunteers; data are available via ProteomeXchange with identifier PXD003875. We identified 918 proteins with an overlap of 725 Gene IDs in the Exocarta proteins list. Compared with the serum-purified exosomes (SPEs) of normal volunteers, we found 36 proteins upregulated and 22 proteins downregulated in the SPEs of CRC patients. Bioinformatics analysis revealed that upregulated proteins are involved in processes that modulate the pretumorigenic microenvironment for metastasis. In contrast, differentially expressed proteins (DEPs) that play critical roles in tumor growth and cell survival were principally downregulated. Our study demonstrates that SPEs of CRC patients play a pivotal role in promoting the tumor invasiveness, but have minimal influence on putative alterations in tumor survival or proliferation. According to bioinformatics analysis, we speculate that the protein contents of exosomes might be associated with whether they are involved in premetastatic niche establishment or growth and survival of metastatic tumor cells. This information will be helpful in elucidating the pathophysiological functions of tumor-derived exosomes, and aid in the development of CRC diagnostics and therapeutics. © 2016 UICC.

  3. Identification of indicator proteins associated with flooding injury in soybean seedlings using label-free quantitative proteomics.

    Science.gov (United States)

    Nanjo, Yohei; Nakamura, Takuji; Komatsu, Setsuko

    2013-11-01

    Flooding injury is one of the abiotic constraints on soybean growth. An experimental system established for evaluating flooding injury in soybean seedlings indicated that the degree of injury is dependent on seedling density in floodwater. Dissolved oxygen levels in the floodwater were decreased by the seedlings and correlated with the degree of injury. To understand the molecular mechanism responsible for the injury, proteomic alterations in soybean seedlings that correlated with severity of stress were analyzed using label-free quantitative proteomics. The analysis showed that the abundance of proteins involved in cell wall modification, such as polygalacturonase inhibitor-like and expansin-like B1-like proteins, which may be associated with the defense system, increased dependence on stress at both the protein and mRNA levels in all organs during flooding. The manner of alteration in abundance of these proteins was distinct from those of other responsive proteins. Furthermore, proteins also showing specific changes in abundance in the root tip included protein phosphatase 2A subunit-like proteins, which are possibly involved in flooding-induced root tip cell death. Additionally, decreases in abundance of cell wall synthesis-related proteins, such as cinnamyl-alcohol dehydrogenase and cellulose synthase-interactive protein-like proteins, were identified in hypocotyls of seedlings grown for 3 days after flooding, and these proteins may be associated with suppression of growth after flooding. These flooding injury-associated proteins can be defined as indicator proteins for severity of flooding stress in soybean.

  4. Structural and metabolic transitions of C4 leaf development and differentiation defined by microscopy and quantitative proteomics in maize.

    Science.gov (United States)

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

    2010-11-01

    C(4) grasses, such as maize (Zea mays), have high photosynthetic efficiency through combined biochemical and structural adaptations. C(4) photosynthesis is established along the developmental axis of the leaf blade, leading from an undifferentiated leaf base just above the ligule into highly specialized mesophyll cells (MCs) and bundle sheath cells (BSCs) at the tip. To resolve the kinetics of maize leaf development and C(4) differentiation and to obtain a systems-level understanding of maize leaf formation, the accumulation profiles of proteomes of the leaf and the isolated BSCs with their vascular bundle along the developmental gradient were determined using large-scale mass spectrometry. This was complemented by extensive qualitative and quantitative microscopy analysis of structural features (e.g., Kranz anatomy, plasmodesmata, cell wall, and organelles). More than 4300 proteins were identified and functionally annotated. Developmental protein accumulation profiles and hierarchical cluster analysis then determined the kinetics of organelle biogenesis, formation of cellular structures, metabolism, and coexpression patterns. Two main expression clusters were observed, each divided in subclusters, suggesting that a limited number of developmental regulatory networks organize concerted protein accumulation along the leaf gradient. The coexpression with BSC and MC markers provided strong candidates for further analysis of C(4) specialization, in particular transporters and biogenesis factors. Based on the integrated information, we describe five developmental transitions that provide a conceptual and practical template for further analysis. An online protein expression viewer is provided through the Plant Proteome Database.

  5. Quantitative Proteomic Analysis of Optimal Cutting Temperature (OCT) Embedded Core-Needle Biopsy of Lung Cancer

    Science.gov (United States)

    Zhao, Xiaozheng; Huffman, Kenneth E.; Fujimoto, Junya; Canales, Jamie Rodriguez; Girard, Luc; Nie, Guangjun; Heymach, John V.; Wistuba, Igacio I.; Minna, John D.; Yu, Yonghao

    2017-10-01

    With recent advances in understanding the genomic underpinnings and oncogenic drivers of pathogenesis in different subtypes, it is increasingly clear that proper pretreatment diagnostics are essential for the choice of appropriate treatment options for non-small cell lung cancer (NSCLC). Tumor tissue preservation in optimal cutting temperature (OCT) compound is commonly used in the surgical suite. However, proteins recovered from OCT-embedded specimens pose a challenge for LC-MS/MS experiments, due to the large amounts of polymers present in OCT. Here we present a simple workflow for whole proteome analysis of OCT-embedded NSCLC tissue samples, which involves a simple trichloroacetic acid precipitation step. Comparisons of protein recovery between frozen versus OCT-embedded tissue showed excellent consistency with more than 9200 proteins identified. Using an isobaric labeling strategy, we quantified more than 5400 proteins in tumor versus normal OCT-embedded core needle biopsy samples. Gene ontology analysis indicated that a number of proliferative as well as squamous cell carcinoma (SqCC) marker proteins were overexpressed in the tumor, consistent with the patient's pathology based diagnosis of "poorly differentiated SqCC". Among the most downregulated proteins in the tumor sample, we noted a number of proteins with potential immunomodulatory functions. Finally, interrogation of the aberrantly expressed proteins using a candidate approach and cross-referencing with publicly available databases led to the identification of potential druggable targets in DNA replication and DNA damage repair pathways. We conclude that our approach allows LC-MS/MS proteomic analyses on OCT-embedded lung cancer specimens, opening the way to bring powerful proteomics into the clinic. [Figure not available: see fulltext.

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

    Science.gov (United States)

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

    2015-08-03

    Modern proteomics has proven instrumental in our understanding of the molecular deregulations associated with the development and progression of cancer. Herein, we profile membrane-enriched proteome of tumor and adjacent normal tissues from eight CRC patients using label-free nanoLC-MS/MS-based quantitative proteomics and advanced pathway analysis. Of the 948 identified proteins, 184 proteins were differentially expressed (P1.5) between the tumor and non-tumor tissue (69 up-regulated and 115 down-regulated in tumor tissues). The CRC tumor and non-tumor tissues clustered tightly in separate groups using hierarchical cluster analysis of the differentially expressed proteins, indicating a strong CRC-association of this proteome subset. Specifically, cancer associated proteins such as FN1, TNC, DEFA1, ITGB2, MLEC, CDH17, EZR and pathways including actin cytoskeleton and RhoGDI signaling were deregulated. Stage-specific proteome signatures were identified including up-regulated ribosomal proteins and down-regulated annexin proteins in early stage CRC. Finally, EGFR(+) CRC tissues showed an EGFR-dependent down-regulation of cell adhesion molecules, relative to EGFR(-) tissues. Taken together, this study provides a detailed map of the altered proteome and associated protein pathways in CRC, which enhances our mechanistic understanding of CRC biology and opens avenues for a knowledge-driven search for candidate CRC protein markers. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Quantitative proteomics and transcriptomics addressing the estrogen receptor subtype-mediated effects in T47D breast cancer cells exposed to the phytoestrogen genistein

    NARCIS (Netherlands)

    Sotoca Covaleda, A.M.; Sollewijn Gelpke, M.D.; Boeren, S.; Ström, A.; Gustafsson, J.A.; Murk, A.J.; Rietjens, I.M.C.M.; Vervoort, J.J.M.

    2011-01-01

    The present study addresses, by transcriptomics and quantitative SILAC-based proteomics, the estrogen receptor alpha (ER) and beta (ERß)-mediated effects on gene and protein expression in T47D breast cancer cells exposed to the phytoestrogen genistein. Using the T47D human breast cancer cell line

  8. Study of early leaf senescence in Arabidopsis thaliana by quantitative proteomics using reciprocal N-14/N-15 Labeling and difference gel electrophoresis

    NARCIS (Netherlands)

    Hebeler, Romano; Oeljeklaus, Silke; Reidegeld, Kai E.; Eisenacher, Martin; Stephan, Christian; Sitek, Barbara; Stuehler, Kai; Meyer, Helmut E.; Sturre, Marcel J. G.; Dijkwel, Paul P.; Warscheid, Bettina

    Leaf senescence represents the final stage of leaf development and is associated with fundamental changes on the level of the proteome. For the quantitative analysis of changes in protein abundance related to early leaf senescence, we designed an elaborate double and reverse labeling strategy

  9. Identification of targets of miR-200b by a SILAC-based quantitative proteomic approach

    Directory of Open Access Journals (Sweden)

    Arivusudar Marimuthu

    2014-09-01

    Full Text Available miRNAs regulate gene expression by binding to cognate mRNAs causing mRNA degradation or translational repression. Mass spectrometry-based proteomic analysis is being widely used to identify miRNA targets. The miR-200b miRNA cluster is often overexpressed in multiple cancer types, but the identity of the targets remains elusive. Using SILAC-based analysis, we examined the effects of overexpression of a miR-200b mimic or a control miRNA in fibrosarcoma cells. We identified around 300 potential targets of miR-200b based on a change in the expression of protein levels. We validated a subset of potential targets at the transcript level using quantitative PCR.

  10. Comprehensive quantitative comparison of the membrane proteome, phosphoproteome, and sialiome of human embryonic and neural stem cells

    DEFF Research Database (Denmark)

    Melo-Braga, Marcella Nunes; Schulz, Melanie; Liu, Qiuyue

    2014-01-01

    Human embryonic stem cells (hESCs) can differentiate into neural stem cells (NSCs), which can further be differentiated into neurons and glia cells. Therefore, these cells have huge potential as source for treatment of neurological diseases. Membrane-associated proteins are very important......ESCs and NSCs as well as to investigate potential new markers for these two cell stages, we performed large-scale quantitative membrane-proteomic of hESCs and NSCs. This approach employed membrane purification followed by peptide dimethyl labeling and peptide enrichment to study the membrane subproteome as well...... in which 78% of phosphopeptides were identified with ≥99% confidence in site assignment and 1810 unique formerly sialylated N-linked glycopeptides. Several proteins were identified as significantly regulated in hESCs and NSC, including proteins involved in the early embryonic and neural development...

  11. Integrative Analysis Using Proteome and Transcriptome Data From Yeast to Unravel Regulatory Patterns at Post-Transcriptional Level

    DEFF Research Database (Denmark)

    Olivares Hernandez, Roberto; Usaite, Renata; Nielsen, Jens

    2010-01-01

    In this stud) we combined proteome and transcriptome data from six different published dataset to identify patterns that can provide new insight into the reasons for these deviations By using a categorization method and integrating genome-scale information we found that the relation between protein and mRNA...... is related to the gene function We could further identify that for genes belonging to amino acid biosynthetic pathways there is no translational regulation, meaning that there is generally a good correlation between mRNA and protein levels We also found that there is generally translational control for large...... proteins and there also evidence for a role of conserved motifs m the 3' untranslated regions in the mRNA-protein correlation, probably by controlling the level of mRNA Biotechnol Bioeng 2010,107 865-875...

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

    Science.gov (United States)

    2013-01-01

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

  13. Quantitative proteomics reveals that peroxidases play key roles in post-flooding recovery in soybean roots.

    Science.gov (United States)

    Khan, Mudassar Nawaz; Sakata, Katsumi; Hiraga, Susumu; Komatsu, Setsuko

    2014-12-05

    Soybean is an important legume crop that exhibits markedly reduced growth and yields under flooding conditions. To unravel the mechanisms involved in recovery after flooding in soybean root, gel-free proteomic analysis was performed. Morphological analysis revealed that growth suppression was more severe with increased flooding duration. Out of a total of 1645 and 1707 identified proteins, 73 and 21 proteins were changed significantly during the recovery stage following 2 and 4 days flooding, respectively. Based on the proteomic, clustering, and in silico protein-protein interaction analyses, six key enzymes were analyzed at the mRNA level. Lipoxygenase 1, which was increased at the protein level during the recovery period, was steadily down-regulated at the mRNA level. The peroxidase superfamily protein continuously increased in abundance during the course of recovery and was up-regulated at the mRNA level. HAD acid phosphatase was decreased at the protein level and down-regulated at the transcript level, while isoflavone reductase and an unknown protein were increased at both the protein and mRNA levels. Consistent with these findings, the enzymatic activity of peroxidase was decreased under flooding stress but increased significantly during the recovery sage. These results suggest that peroxidases might play key roles in post-flooding recovery in soybean roots through the scavenging of toxic radicals.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2018-02-28

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

  15. Triple SILAC quantitative proteomic analysis reveals differential abundance of cell signaling proteins between normal and lung cancer-derived exosomes.

    Science.gov (United States)

    Clark, David J; Fondrie, William E; Yang, Austin; Mao, Li

    2016-02-05

    Exosomes are 30-100 nm sized membrane vesicles released by cells into the extracellular space that mediate intercellular communication via transfer of proteins and other biological molecules. To better understand the role of these microvesicles in lung carcinogenesis, we employed a Triple SILAC quantitative proteomic strategy to examine the differential protein abundance between exosomes derived from an immortalized normal bronchial epithelial cell line and two non-small cell lung cancer (NSCLC) cell lines harboring distinct activating mutations in the cell signaling molecules: Kirsten rat sarcoma viral oncogene homolog (KRAS) or epidermal growth factor receptor (EGFR). In total, we were able to quantify 721 exosomal proteins derived from the three cell lines. Proteins associated with signal transduction, including EGFR, GRB2 and SRC, were enriched in NSCLC exosomes, and could actively regulate cell proliferation in recipient cells. This study's investigation of the NSCLC exosomal proteome has identified enriched protein cargo that can contribute to lung cancer progression, which may have potential clinical implications in biomarker development for patients with NSCLC. The high mortality associated with lung cancer is a result of late-stage diagnosis of the disease. Current screening techniques used for early detection of lung cancer lack the specificity for accurate diagnosis. Exosomes are nano-sized extracellular vesicles, and the increased abundance of select protein cargo in exosomes derived from cancer cells may be used for diagnostic purposes. In this paper, we applied quantitative proteomic analysis to elucidate abundance differences in exosomal protein cargo between two NSCLC cell lines with distinctive oncogene mutations and an immortalized normal bronchial epithelial cell line. This study revealed proteins associated with cell adhesion, the extracellular matrix, and a variety of signaling molecules were enriched in NSCLC exosomes. The present data reveals

  16. LFQuant: a label-free fast quantitative analysis tool for high-resolution LC-MS/MS proteomics data.

    Science.gov (United States)

    Zhang, Wei; Zhang, Jiyang; Xu, Changming; Li, Ning; Liu, Hui; Ma, Jie; Zhu, Yunping; Xie, Hongwei

    2012-12-01

    Database searching based methods for label-free quantification aim to reconstruct the peptide extracted ion chromatogram based on the identification information, which can limit the search space and thus make the data processing much faster. The random effect of the MS/MS sampling can be remedied by cross-assignment among different runs. Here, we present a new label-free fast quantitative analysis tool, LFQuant, for high-resolution LC-MS/MS proteomics data based on database searching. It is designed to accept raw data in two common formats (mzXML and Thermo RAW), and database search results from mainstream tools (MASCOT, SEQUEST, and X!Tandem), as input data. LFQuant can handle large-scale label-free data with fractionation such as SDS-PAGE and 2D LC. It is easy to use and provides handy user interfaces for data loading, parameter setting, quantitative analysis, and quantitative data visualization. LFQuant was compared with two common quantification software packages, MaxQuant and IDEAL-Q, on the replication data set and the UPS1 standard data set. The results show that LFQuant performs better than them in terms of both precision and accuracy, and consumes significantly less processing time. LFQuant is freely available under the GNU General Public License v3.0 at http://sourceforge.net/projects/lfquant/. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Quantitative proteomics analysis using 2D-PAGE to investigate the effects of cigarette smoke and aerosol of a prototypic modified risk tobacco product on the lung proteome in C57BL/6 mice.

    Science.gov (United States)

    Elamin, Ashraf; Titz, Bjoern; Dijon, Sophie; Merg, Celine; Geertz, Marcel; Schneider, Thomas; Martin, Florian; Schlage, Walter K; Frentzel, Stefan; Talamo, Fabio; Phillips, Blaine; Veljkovic, Emilija; Ivanov, Nikolai V; Vanscheeuwijck, Patrick; Peitsch, Manuel C; Hoeng, Julia

    2016-08-11

    Smoking is associated with several serious diseases, such as lung cancer and chronic obstructive pulmonary disease (COPD). Within our systems toxicology framework, we are assessing whether potential modified risk tobacco products (MRTP) can reduce smoking-related health risks compared to conventional cigarettes. In this article, we evaluated to what extent 2D-PAGE/MALDI MS/MS (2D-PAGE) can complement the iTRAQ LC-MS/MS results from a previously reported mouse inhalation study, in which we assessed a prototypic MRTP (pMRTP). Selected differentially expressed proteins identified by both LC-MS/MS and 2D-PAGE approaches were further verified using reverse-phase protein microarrays. LC-MS/MS captured the effects of cigarette smoke (CS) on the lung proteome more comprehensively than 2D-PAGE. However, an integrated analysis of both proteomics data sets showed that 2D-PAGE data complement the LC-MS/MS results by supporting the overall trend of lower effects of pMRTP aerosol than CS on the lung proteome. Biological effects of CS exposure supported by both methods included increases in immune-related, surfactant metabolism, proteasome, and actin cytoskeleton protein clusters. Overall, while 2D-PAGE has its value, especially as a complementary method for the analysis of effects on intact proteins, LC-MS/MS approaches will likely be the method of choice for proteome analysis in systems toxicology investigations. Quantitative proteomics is anticipated to play a growing role within systems toxicology assessment frameworks in the future. To further understand how different proteomics technologies can contribute to toxicity assessment, we conducted a quantitative proteomics analysis using 2D-PAGE and isobaric tag-based LC-MS/MS approaches and compared the results produced from the 2 approaches. Using a prototypic modified risk tobacco product (pMRTP) as our test item, we show compared with cigarette smoke, how 2D-PAGE results can complement and support LC-MS/MS data, demonstrating

  18. Multidimensional electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) for quantitative analysis of the proteome and phosphoproteome in clinical and biomedical research.

    Science.gov (United States)

    Loroch, Stefan; Schommartz, Tim; Brune, Wolfram; Zahedi, René Peiman; Sickmann, Albert

    2015-05-01

    Quantitative proteomics and phosphoproteomics have become key disciplines in understanding cellular processes. Fundamental research can be done using cell culture providing researchers with virtually infinite sample amounts. In contrast, clinical, pre-clinical and biomedical research is often restricted to minute sample amounts and requires an efficient analysis with only micrograms of protein. To address this issue, we generated a highly sensitive workflow for combined LC-MS-based quantitative proteomics and phosphoproteomics by refining an ERLIC-based 2D phosphoproteomics workflow into an ERLIC-based 3D workflow covering the global proteome as well. The resulting 3D strategy was successfully used for an in-depth quantitative analysis of both, the proteome and the phosphoproteome of murine cytomegalovirus-infected mouse fibroblasts, a model system for host cell manipulation by a virus. In a 2-plex SILAC experiment with 150 μg of a tryptic digest per condition, the 3D strategy enabled the quantification of ~75% more proteins and even ~134% more peptides compared to the 2D strategy. Additionally, we could quantify ~50% more phosphoproteins by non-phosphorylated peptides, concurrently yielding insights into changes on the levels of protein expression and phosphorylation. Beside its sensitivity, our novel three-dimensional ERLIC-strategy has the potential for semi-automated sample processing rendering it a suitable future perspective for clinical, pre-clinical and biomedical research. Copyright © 2015. Published by Elsevier B.V.

  19. Quantitative proteomic studies in resistance mechanisms of Eimeria tenella against polyether ionophores.

    Science.gov (United States)

    Thabet, Ahmed; Honscha, Walther; Daugschies, Arwid; Bangoura, Berit

    2017-05-01

    Polyether ionophores are widely used to treat and control coccidiosis in chickens. Widespread use of anticoccidials resulted in worldwide resistance. Mechanisms of resistance development and expansion are complex and poorly understood. Relative proteomic quantification using LC-MS/MS was used to compare sensitive reference strains (Ref-1, Ref-2) with putatively resistant and moderately sensitive field strains (FS-R, FS-mS) of Eimeria tenella after isotopic labelling with tandem mass tags (TMT). Ninety-seven proteins were identified, and 25 of them were regulated. Actin was significantly upregulated in resistant strains in comparison with their sensitive counterparts. On the other hand, microneme protein (MIC4) was downregulated in resistant strains. Optimization of labelling E. tenella sporozoites by TMT might identify further proteins that play a role in the obvious complex mechanism leading to resistance against Monensin.

  20. Serum quantitative proteomic analysis reveals potential zinc-associated biomarkers for nonbacterial prostatitis.

    Science.gov (United States)

    Yang, Xiaoli; Li, Hongtao; Zhang, Chengdong; Lin, Zhidi; Zhang, Xinhua; Zhang, Youjie; Yu, Yanbao; Liu, Kun; Li, Muyan; Zhang, Yuening; Lv, Wenxin; Xie, Yuanliang; Lu, Zheng; Wu, Chunlei; Teng, Ruobing; Lu, Shaoming; He, Min; Mo, Zengnan

    2015-10-01

    Prostatitis is one of the most common urological problems afflicting adult men. The etiology and pathogenesis of nonbacterial prostatitis, which accounts for 90-95% of cases, is largely unknown. As serum proteins often indicate the overall pathologic status of patients, we hypothesized that protein biomarkers of prostatitis might be identified by comparing the serum proteomes of patients with and without nonbacterial prostatitis. All untreated samples were collected from subjects attending the Fangchenggang Area Male Health and Examination Survey (FAMHES). We profiled pooled serum samples from four carefully selected groups of patients (n = 10/group) representing the various categories of nonbacterial prostatitis (IIIa, IIIb, and IV) and matched healthy controls using a mass spectrometry-based 4-plex iTRAQ proteomic approach. More than 160 samples were validated by ELISA. Overall, 69 proteins were identified. Among them, 42, 52, and 37 proteins were identified with differential expression in Category IIIa, IIIb, and IV prostatitis, respectively. The 19 common proteins were related to immunity and defense, ion binding, transport, and proteolysis. Two zinc-binding proteins, superoxide dismutase 3 (SOD3), and carbonic anhydrase I (CA1), were significantly higher in all types of prostatitis than in the control. A receiver operating characteristic curve estimated sensitivities of 50.4 and 68.1% and specificities of 92.1 and 83.8% for CA1 and SOD3, respectively, in detecting nonbacterial prostatitis. The serum CA1 concentration was inversely correlated to the zinc concentration in expressed-prostatic secretions. Our findings suggest that SOD3 and CA1 are potential diagnostic markers of nonbacterial prostatitis, although further large-scale studies are required. The molecular profiles of nonbacterial prostatitis pathogenesis may lay a foundation for discovery of new therapies. © 2015 Wiley Periodicals, Inc.

  1. Discovery of prognostic biomarker candidates of lacunar infarction by quantitative proteomics of microvesicles enriched plasma.

    Directory of Open Access Journals (Sweden)

    Arnab Datta

    Full Text Available Lacunar infarction (LACI is a subtype of acute ischemic stroke affecting around 25% of all ischemic stroke cases. Despite having an excellent recovery during acute phase, certain LACI patients have poor mid- to long-term prognosis due to the recurrence of vascular events or a decline in cognitive functions. Hence, blood-based biomarkers could be complementary prognostic and research tools.Plasma was collected from forty five patients following a non-disabling LACI along with seventeen matched control subjects. The LACI patients were monitored prospectively for up to five years for the occurrence of adverse outcomes and grouped accordingly (i.e., LACI-no adverse outcome, LACI-recurrent vascular event, and LACI-cognitive decline without any recurrence of vascular events. Microvesicles-enriched fractions isolated from the pooled plasma of four groups were profiled by an iTRAQ-guided discovery approach to quantify the differential proteome. The data have been deposited to the ProteomeXchange with identifier PXD000748. Bioinformatics analysis and data mining revealed up-regulation of brain-specific proteins including myelin basic protein, proteins of coagulation cascade (e.g., fibrinogen alpha chain, fibrinogen beta chain and focal adhesion (e.g., integrin alpha-IIb, talin-1, and filamin-A while albumin was down-regulated in both groups of patients with adverse outcome.This data set may offer important insight into the mechanisms of poor prognosis and provide candidate prognostic biomarkers for validation on larger cohort of individual LACI patients.

  2. Quantitative Proteomics of an Amphibian Pathogen, Batrachochytrium dendrobatidis, following Exposure to Thyroid Hormone.

    Science.gov (United States)

    Thekkiniath, Jose; Zabet-Moghaddam, Masoud; Kottapalli, Kameswara Rao; Pasham, Mithun R; San Francisco, Susan; San Francisco, Michael

    2015-01-01

    Batrachochytrium dendrobatidis (Bd), a chytrid fungus, has increasingly been implicated as a major factor in the worldwide decline of amphibian populations. The fungus causes chytridiomycosis in susceptible species leading to massive die-offs of adult amphibians. Although Bd infects the keratinized mouthparts of tadpoles and negatively affects foraging behavior, these infections are non-lethal. An important morphogen controlling amphibian metamorphosis is thyroid hormone (T3). Tadpoles may be infected with Bd and the fungus may be exposed to T3 during metamorphosis. We hypothesize that exposure of Bd to T3 may induce the expression of factors associated with host colonization and pathogenicity. We utilized a proteomics approach to better understand the dynamics of the Bd-T3 interaction. Using liquid chromatography-mass spectrometry (LC-MS), we generated a data set of a large number of cytoplasmic and membrane proteins following exposure of Bd to T3. From these data, we identified a total of 263 proteins whose expression was significantly changed following T3 exposure. We provide evidence for expression of an array of proteins that may play key roles in both genomic and non-genomic actions of T3 in Bd. Additionally, our proteomics study shows an increase in several proteins including proteases and a class of uncommon crinkler and crinkler-like effector proteins suggesting their importance in Bd pathogenicity as well as those involved in metabolism and energy transfer, protein fate, transport and stress responses. This approach provides insights into the mechanistic basis of the Bd-amphibian interaction following T3 exposure.

  3. Quantitative Proteomics of an Amphibian Pathogen, Batrachochytrium dendrobatidis, following Exposure to Thyroid Hormone.

    Directory of Open Access Journals (Sweden)

    Jose Thekkiniath

    Full Text Available Batrachochytrium dendrobatidis (Bd, a chytrid fungus, has increasingly been implicated as a major factor in the worldwide decline of amphibian populations. The fungus causes chytridiomycosis in susceptible species leading to massive die-offs of adult amphibians. Although Bd infects the keratinized mouthparts of tadpoles and negatively affects foraging behavior, these infections are non-lethal. An important morphogen controlling amphibian metamorphosis is thyroid hormone (T3. Tadpoles may be infected with Bd and the fungus may be exposed to T3 during metamorphosis. We hypothesize that exposure of Bd to T3 may induce the expression of factors associated with host colonization and pathogenicity. We utilized a proteomics approach to better understand the dynamics of the Bd-T3 interaction. Using liquid chromatography-mass spectrometry (LC-MS, we generated a data set of a large number of cytoplasmic and membrane proteins following exposure of Bd to T3. From these data, we identified a total of 263 proteins whose expression was significantly changed following T3 exposure. We provide evidence for expression of an array of proteins that may play key roles in both genomic and non-genomic actions of T3 in Bd. Additionally, our proteomics study shows an increase in several proteins including proteases and a class of uncommon crinkler and crinkler-like effector proteins suggesting their importance in Bd pathogenicity as well as those involved in metabolism and energy transfer, protein fate, transport and stress responses. This approach provides insights into the mechanistic basis of the Bd-amphibian interaction following T3 exposure.

  4. Longitudinal Multiplexed Measurement of Quantitative Proteomic Signatures in Mouse Lymphoma Models Using Magneto-Nanosensors.

    Science.gov (United States)

    Lee, Jung-Rok; Appelmann, Iris; Miething, Cornelius; Shultz, Tyler O; Ruderman, Daniel; Kim, Dokyoon; Mallick, Parag; Lowe, Scott W; Wang, Shan X

    2018-01-01

    Cancer proteomics is the manifestation of relevant biological processes in cancer development. Thus, it reflects the activities of tumor cells, host-tumor interactions, and systemic responses to cancer therapy. To understand the causal effects of tumorigenesis or therapeutic intervention, longitudinal studies are greatly needed. However, most of the conventional mouse experiments are unlikely to accommodate frequent collection of serum samples with a large enough volume for multiple protein assays towards single-object analysis. Here, we present a technique based on magneto-nanosensors to longitudinally monitor the protein profiles in individual mice of lymphoma models using a small volume of a sample for multiplex assays. Methods: Drug-sensitive and -resistant cancer cell lines were used to develop the mouse models that render different outcomes upon the drug treatment. Two groups of mice were inoculated with each cell line, and treated with either cyclophosphamide or vehicle solution. Serum samples taken longitudinally from each mouse in the groups were measured with 6-plex magneto-nanosensor cytokine assays. To find the origin of IL-6, experiments were performed using IL-6 knock-out mice. Results: The differences in serum IL-6 and GCSF levels between the drug-treated and untreated groups were revealed by the magneto-nanosensor measurement on individual mice. Using the multiplex assays and mouse models, we found that IL-6 is secreted by the host in the presence of tumor cells upon the drug treatment. Conclusion: The multiplex magneto-nanosensor assays enable longitudinal proteomic studies on mouse tumor models to understand tumor development and therapy mechanisms more precisely within a single biological object.

  5. High-performance hybrid Orbitrap mass spectrometers for quantitative proteome analysis

    DEFF Research Database (Denmark)

    Williamson, James C; Edwards, Alistair V G; Verano-Braga, Thiago

    2016-01-01

    We present basic workups and quantitative comparisons for two current generation Orbitrap mass spectrometers, the Q Exactive Plus and Orbitrap Fusion Tribrid, which are widely considered two of the highest performing instruments on the market. We assessed the performance of two quantitative methods...... on both instruments, namely label-free quantitation and stable isotope labeling using isobaric tags, for studying the heat shock response in Escherichia coli. We investigated the recently reported MS3 method on the Fusion instrument and the potential of MS3-based reporter ion isolation Synchronous...... Precursor Selection (SPS) and its impact on quantitative accuracy. We confirm that the label-free approach offers a more linear response with a wider dynamic range than MS/MS-based isobaric tag quantitation and that the MS3/SPS approach alleviates but does not eliminate dynamic range compression. We...

  6. Differential effects of a post-anthesis fertilizer regimen on the wheat flour proteome determined by quantitative 2-DE

    Directory of Open Access Journals (Sweden)

    Altenbach Susan B

    2011-08-01

    Full Text Available Abstract Background Mineral nutrition during wheat grain development has large effects on wheat flour protein content and composition, which in turn affect flour quality and immunogenic potential for a commodity of great economic value. However, it has been difficult to define the precise effects of mineral nutrition on protein composition because of the complexity of the wheat flour proteome. Recent improvements in the identification of flour proteins by tandem mass spectrometry (MS/MS and the availability of a comprehensive proteome map of flour from the US wheat Butte 86 now make it possible to document changes in the proportions of individual flour proteins that result from the application of mineral nutrition. Results Plants of Triticum aestivum 'Butte 86' were grown with or without post-anthesis fertilization (PAF and quantitative 2-dimensional gel electrophoresis (2-DE was used to analyze protein composition of the resulting flour. Significant changes in the proportions of 54 unique proteins were observed as a result of the treatment. Most omega-gliadins, high molecular weight glutenin subunits (HMW-GS and serpins as well as some alpha-gliadins increased in proportion with PAF. In contrast, alpha-amylase/protease inhibitors, farinins, purinins and puroindolines decreased in proportion. Decreases were also observed in several low molecular weight glutenin subunits (LMW-GS, globulins, defense proteins and enzymes. The ratio of HMW-GS to LMW-GS in the flour increased from 0.61 to 0.95 and the ratio of gliadins to glutenins increased from 1.02 to 1.30 with PAF. Because flour protein content doubled with PAF from 7 to 14%, most protein types actually increased in absolute amount (μg/mg flour protein. Data further suggest that flour proteins change with PAF according to their content of sulfur-containing amino acids Cys + Met. Conclusions A 2-DE approach revealed changes in the wheat flour proteome due to PAF that are important for flour

  7. Multiplex N-terminome analysis of MMP-2 and MMP-9 substrate degradomes by iTRAQ-TAILS quantitative proteomics.

    Science.gov (United States)

    Prudova, Anna; auf dem Keller, Ulrich; Butler, Georgina S; Overall, Christopher M

    2010-05-01

    Proteolysis is a major protein posttranslational modification that, by altering protein structure, affects protein function and, by truncating the protein sequence, alters peptide signatures of proteins analyzed by proteomics. To identify such modified and shortened protease-generated neo-N-termini on a proteome-wide basis, we developed a whole protein isobaric tag for relative and absolute quantitation (iTRAQ) labeling method that simultaneously labels and blocks all primary amines including protein N- termini and lysine side chains. Blocking lysines limits trypsin cleavage to arginine, which effectively elongates the proteolytically truncated peptides for improved MS/MS analysis and peptide identification. Incorporating iTRAQ whole protein labeling with terminal amine isotopic labeling of substrates (iTRAQ-TAILS) to enrich the N-terminome by negative selection of the blocked mature original N-termini and neo-N-termini has many advantages. It enables simultaneous characterization of the natural N-termini of proteins, their N-terminal modifications, and proteolysis product and cleavage site identification. Furthermore, iTRAQ-TAILS also enables multiplex N-terminomics analysis of up to eight samples and allows for quantification in MS2 mode, thus preventing an increase in spectral complexity and extending proteome coverage by signal amplification of low abundance proteins. We compared the substrate degradomes of two closely related matrix metalloproteinases, MMP-2 (gelatinase A) and MMP-9 (gelatinase B), in fibroblast secreted proteins. Among 3,152 unique N-terminal peptides identified corresponding to 1,054 proteins, we detected 201 cleavage products for MMP-2 and unexpectedly only 19 for the homologous MMP-9 under identical conditions. Novel substrates identified and biochemically validated include insulin-like growth factor binding protein-4, complement C1r component A, galectin-1, dickkopf-related protein-3, and thrombospondin-2. Hence, N-terminomics analyses

  8. On-Beads Digestion in Conjunction with Data-Dependent Mass Spectrometry: A Shortcut to Quantitative and Dynamic Interaction Proteomics

    Directory of Open Access Journals (Sweden)

    Benedetta Turriziani

    2014-04-01

    Full Text Available With the advent of the “-omics” era, biological research has shifted from functionally analyzing single proteins to understanding how entire protein networks connect and adapt to environmental cues. Frequently, pathological processes are initiated by a malfunctioning protein network rather than a single protein. It is therefore crucial to investigate the regulation of proteins in the context of a pathway first and signaling network second. In this study, we demonstrate that a quantitative interaction proteomic approach, combining immunoprecipitation, in-solution digestion and label-free quantification mass spectrometry, provides data of high accuracy and depth. This protocol is applicable, both to tagged, exogenous and untagged, endogenous proteins. Furthermore, it is fast, reliable and, due to a label-free quantitation approach, allows the comparison of multiple conditions. We further show that we are able to generate data in a medium throughput fashion and that we can quantify dynamic interaction changes in signaling pathways in response to mitogenic stimuli, making our approach a suitable method to generate data for system biology approaches.

  9. ExSTA: External Standard Addition Method for Accurate High-Throughput Quantitation in Targeted Proteomics Experiments.

    Science.gov (United States)

    Mohammed, Yassene; Pan, Jingxi; Zhang, Suping; Han, Jun; Borchers, Christoph H

    2018-03-01

    Targeted proteomics using MRM with stable-isotope-labeled internal-standard (SIS) peptides is the current method of choice for protein quantitation in complex biological matrices. Better quantitation can be achieved with the internal standard-addition method, where successive increments of synthesized natural form (NAT) of the endogenous analyte are added to each sample, a response curve is generated, and the endogenous concentration is determined at the x-intercept. Internal NAT-addition, however, requires multiple analyses of each sample, resulting in increased sample consumption and analysis time. To compare the following three methods, an MRM assay for 34 high-to-moderate abundance human plasma proteins is used: classical internal SIS-addition, internal NAT-addition, and external NAT-addition-generated in buffer using NAT and SIS peptides. Using endogenous-free chicken plasma, the accuracy is also evaluated. The internal NAT-addition outperforms the other two in precision and accuracy. However, the curves derived by internal vs. external NAT-addition differ by only ≈3.8% in slope, providing comparable accuracies and precision with good CV values. While the internal NAT-addition method may be "ideal", this new external NAT-addition can be used to determine the concentration of high-to-moderate abundance endogenous plasma proteins, providing a robust and cost-effective alternative for clinical analyses or other high-throughput applications. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Quantitative Proteomics of the Root of Transgenic Wheat Expressing TaBWPR-1.2 Genes in Response to Waterlogging

    Directory of Open Access Journals (Sweden)

    Emdadul Haque

    2014-11-01

    Full Text Available Once candidate genes are available, the application of genetic transformation plays a major part to study their function in plants for adaptation to respective environmental stresses, including waterlogging (WL. The introduction of stress-inducible genes into wheat remains difficult because of low transformation and plant regeneration efficiencies and expression variability and instability. Earlier, we found two cDNAs encoding WL stress-responsive wheat pathogenesis-related proteins 1.2 (TaBWPR-1.2, TaBWPR-1.2#2 and TaBWPR-1.2#13. Using microprojectile bombardment, both cDNAs were introduced into “Bobwhite”. Despite low transformation efficiency, four independent T2 homozygous lines for each gene were isolated, where transgenes were ubiquitously and variously expressed. The highest transgene expression was obtained in Ubi:TaBWPR-1.2#2 L#11a and Ubi:TaBWPR-1.2#13 L#4a. Using quantitative proteomics, the root proteins of L#11a were analyzed to explore possible physiological pathways regulated by TaBWPR-1.2 under normal and waterlogged conditions. In L#11a, the abundance of proteasome subunit alpha type-3 decreased under normal conditions, whereas that of ferredoxin precursor and elongation factor-2 increased under waterlogged conditions in comparison with normal plants. Proteomic results suggest that L#11a is one of the engineered wheat plants where TaBWPR-1.2#2 is most probably involved in proteolysis, protein synthesis and alteration in the energy pathway in root tissues via the above proteins in order to gain metabolic adjustment to WL.

  11. Quantitative proteomic analysis of extracellular matrix extracted from mono- and dual-species biofilms of Fusobacterium nucleatum and Porphyromonas gingivalis.

    Science.gov (United States)

    Mohammed, Marwan Mansoor Ali; Pettersen, Veronika Kuchařová; Nerland, Audun H; Wiker, Harald G; Bakken, Vidar

    2017-04-01

    The Gram-negative bacteria Fusobacterium nucleatum and Porphyromonas gingivalis are members of a complex dental biofilm associated with periodontal disease. In this study, we cultured F. nucleatum and P. gingivalis as mono- and dual-species biofilms, and analyzed the protein composition of the biofilms extracellular polymeric matrix (EPM) by high-resolution liquid chromatography-tandem mass spectrometry. Label-free quantitative proteomic analysis was used for identification of proteins and sequence-based functional characterization for their classification and prediction of possible roles in EPM. We identified 542, 93 and 280 proteins in the matrix of F. nucleatum, P. gingivalis, and the dual-species biofilm, respectively. Nearly 70% of all EPM proteins in the dual-species biofilm originated from F. nucleatum, and a majority of these were cytoplasmic proteins, suggesting an enhanced lysis of F. nucleatum cells. The proteomic analysis also indicated an interaction between the two species: 22 F. nucleatum proteins showed differential levels between the mono and dual-species EPMs, and 11 proteins (8 and 3 from F. nucleatum and P. gingivalis, respectively) were exclusively detected in the dual-species EPM. Oxidoreductases and chaperones were among the most abundant proteins identified in all three EPMs. The biofilm matrices in addition contained several known and hypothetical virulence proteins, which can mediate adhesion to the host cells and disintegration of the periodontal tissues. This study demonstrated that the biofilm matrix of two important periodontal pathogens consists of a multitude of proteins whose amounts and functionalities vary largely. Relatively high levels of several of the detected proteins might facilitate their potential use as targets for the inhibition of biofilm development. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Proteomic analysis of a high aluminum tolerant yeast Rhodotorula taiwanensis RS1 in response to aluminum stress.

    Science.gov (United States)

    Wang, Chao; Wang, Chang Yi; Zhao, Xue Qiang; Chen, Rong Fu; Lan, Ping; Shen, Ren Fang

    2013-10-01

    Rhodotorula taiwanensis RS1 is a high-aluminum (Al)-tolerant yeast that can survive in Al concentrations up to 200mM. The mechanisms for the high Al tolerance of R. taiwanensis RS1 are not well understood. To investigate the molecular mechanisms underlying Al tolerance and toxicity in R. taiwanensis RS1, Al toxicity-induced changes in the total soluble protein profile were analyzed using two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry. A total of 33 differentially expressed proteins responding to Al stress were identified from approximately 850 reproducibly detected proteins. Among them, the abundance of 29 proteins decreased and 4 increased. In the presence of 100mM Al, the abundance of proteins involved in DNA transcription, protein translation, DNA defense, Golgi functions and glucose metabolism was decreased. By contrast, Al treatment led to increased abundance of malate dehydrogenase, which correlated with increased malate dehydrogenase activity and the accumulation of intracellular citrate, suggesting that Al-induced intracellular citrate could play an important role in detoxification of Al in R. taiwanensis RS1. © 2013.

  13. Quantitative Tissue Proteomics Analysis Reveals Versican as Potential Biomarker for Early-Stage Hepatocellular Carcinoma.

    Science.gov (United States)

    Naboulsi, Wael; Megger, Dominik A; Bracht, Thilo; Kohl, Michael; Turewicz, Michael; Eisenacher, Martin; Voss, Don Marvin; Schlaak, Jörg F; Hoffmann, Andreas-Claudius; Weber, Frank; Baba, Hideo A; Meyer, Helmut E; Sitek, Barbara

    2016-01-04

    Hepatocellular carcinoma (HCC) is one of the most aggressive tumors, and the treatment outcome of this disease is improved when the cancer is diagnosed at an early stage. This requires biomarkers allowing an accurate and early tumor diagnosis. To identify potential markers for such applications, we analyzed a patient cohort consisting of 50 patients (50 HCC and 50 adjacent nontumorous tissue samples as controls) using two independent proteomics approaches. We performed label-free discovery analysis on 19 HCC and corresponding tissue samples. The data were analyzed considering events known to take place in early events of HCC development, such as abnormal regulation of Wnt/b-catenin and activation of receptor tyrosine kinases (RTKs). 31 proteins were selected for verification experiments. For this analysis, the second set of the patient cohort (31 HCC and corresponding tissue samples) was analyzed using selected (multiple) reaction monitoring (SRM/MRM). We present the overexpression of ATP-dependent RNA helicase (DDX39), Fibulin-5 (FBLN5), myristoylated alanine-rich C-kinase substrate (MARCKS), and Serpin H1 (SERPINH1) in HCC for the first time. We demonstrate Versican core protein (VCAN) to be significantly associated with well differentiated and low-stage HCC. We revealed for the first time the evidence of VCAN as a potential biomarker for early-HCC diagnosis.

  14. High-Throughput Quantitative Proteomic Analysis of Dengue Virus Type 2 Infected A549 Cells

    Science.gov (United States)

    Chiu, Han-Chen; Hannemann, Holger; Heesom, Kate J.; Matthews, David A.; Davidson, Andrew D.

    2014-01-01

    Disease caused by dengue virus is a global health concern with up to 390 million individuals infected annually worldwide. There are no vaccines or antiviral compounds available to either prevent or treat dengue disease which may be fatal. To increase our understanding of the interaction of dengue virus with the host cell, we analyzed changes in the proteome of human A549 cells in response to dengue virus type 2 infection using stable isotope labelling in cell culture (SILAC) in combination with high-throughput mass spectrometry (MS). Mock and infected A549 cells were fractionated into nuclear and cytoplasmic extracts before analysis to identify proteins that redistribute between cellular compartments during infection and reduce the complexity of the analysis. We identified and quantified 3098 and 2115 proteins in the cytoplasmic and nuclear fractions respectively. Proteins that showed a significant alteration in amount during infection were examined using gene enrichment, pathway and network analysis tools. The analyses revealed that dengue virus infection modulated the amounts of proteins involved in the interferon and unfolded protein responses, lipid metabolism and the cell cycle. The SILAC-MS results were validated for a select number of proteins over a time course of infection by Western blotting and immunofluorescence microscopy. Our study demonstrates for the first time the power of SILAC-MS for identifying and quantifying novel changes in cellular protein amounts in response to dengue virus infection. PMID:24671231

  15. High-throughput quantitative proteomic analysis of dengue virus type 2 infected A549 cells.

    Directory of Open Access Journals (Sweden)

    Han-Chen Chiu

    Full Text Available Disease caused by dengue virus is a global health concern with up to 390 million individuals infected annually worldwide. There are no vaccines or antiviral compounds available to either prevent or treat dengue disease which may be fatal. To increase our understanding of the interaction of dengue virus with the host cell, we analyzed changes in the proteome of human A549 cells in response to dengue virus type 2 infection using stable isotope labelling in cell culture (SILAC in combination with high-throughput mass spectrometry (MS. Mock and infected A549 cells were fractionated into nuclear and cytoplasmic extracts before analysis to identify proteins that redistribute between cellular compartments during infection and reduce the complexity of the analysis. We identified and quantified 3098 and 2115 proteins in the cytoplasmic and nuclear fractions respectively. Proteins that showed a significant alteration in amount during infection were examined using gene enrichment, pathway and network analysis tools. The analyses revealed that dengue virus infection modulated the amounts of proteins involved in the interferon and unfolded protein responses, lipid metabolism and the cell cycle. The SILAC-MS results were validated for a select number of proteins over a time course of infection by Western blotting and immunofluorescence microscopy. Our study demonstrates for the first time the power of SILAC-MS for identifying and quantifying novel changes in cellular protein amounts in response to dengue virus infection.

  16. Quantitative Proteomic Analysis of Mosquito C6/36 Cells Reveals Host Proteins Involved in Zika Virus Infection.

    Science.gov (United States)

    Xin, Qi-Lin; Deng, Cheng-Lin; Chen, Xi; Wang, Jun; Wang, Shao-Bo; Wang, Wei; Deng, Fei; Zhang, Bo; Xiao, Gengfu; Zhang, Lei-Ke

    2017-06-15

    Zika virus (ZIKV) is an emerging arbovirus belonging to the genus Flavivirus of the family Flaviviridae During replication processes, flavivirus manipulates host cell systems to facilitate its replication, while the host cells activate antiviral responses. Identification of host proteins involved in the flavivirus replication process may lead to the discovery of antiviral targets. The mosquitoes Aedes aegypti and Aedes albopictus are epidemiologically important vectors for ZIKV, and effective restrictions of ZIKV replication in mosquitoes will be vital in controlling the spread of virus. In this study, an iTRAQ-based quantitative proteomic analysis of ZIKV-infected Aedes albopictus C6/36 cells was performed to investigate host proteins involved in the ZIKV infection process. A total of 3,544 host proteins were quantified, with 200 being differentially regulated, among which CHCHD2 can be upregulated by ZIKV infection in both mosquito C6/36 and human HeLa cells. Our further study indicated that CHCHD2 can promote ZIKV replication and inhibit beta interferon (IFN-β) production in HeLa cells, suggesting that ZIKV infection may upregulate CHCHD2 to inhibit IFN-I production and thus promote virus replication. Bioinformatics analysis of regulated host proteins highlighted several ZIKV infection-regulated biological processes. Further study indicated that the ubiquitin proteasome system (UPS) plays roles in the ZIKV entry process and that an FDA-approved inhibitor of the 20S proteasome, bortezomib, can inhibit ZIKV infection in vivo Our study illustrated how host cells respond to ZIKV infection and also provided a candidate drug for the control of ZIKV infection in mosquitoes and treatment of ZIKV infection in patients. IMPORTANCE ZIKV infection poses great threats to human health, and there is no FDA-approved drug available for the treatment of ZIKV infection. During replication, ZIKV manipulates host cell systems to facilitate its replication, while host cells activate

  17. Quantitative interaction proteomics and genome-wide profiling of epigenetic histone marks and their readers

    DEFF Research Database (Denmark)

    Vermeulen, Michiel; Eberl, H Christian; Matarese, Filomena

    2010-01-01

    Trimethyl-lysine (me3) modifications on histones are the most stable epigenetic marks and they control chromatin-mediated regulation of gene expression. Here, we determine proteins that bind these marks by high-accuracy, quantitative mass spectrometry. These chromatin "readers" are assigned...

  18. Label-Free Quantitative Proteomics of Embryogenic and Non-Embryogenic Callus during Sugarcane Somatic Embryogenesis.

    Directory of Open Access Journals (Sweden)

    Angelo Schuabb Heringer

    Full Text Available The development of somatic cells in to embryogenic cells occurs in several stages and ends in somatic embryo formation, though most of these biochemical and molecular changes have yet to be elucidated. Somatic embryogenesis coupled with genetic transformation could be a biotechnological tool to improve potential crop yields potential in sugarcane cultivars. The objective of this study was to observe somatic embryo development and to identify differentially expressed proteins in embryogenic (E and non-embryogenic (NE callus during maturation treatment. E and NE callus were cultured on maturation culture medium supplemented with different concentrations (0.0, 0.75, 1.5 and 2.0 g L(-1 of activated charcoal (AC. Somatic embryo formation and differential protein expression were evaluated at days 0 and 21 using shotgun proteomic analyses. Treatment with 1.5 g L(-1 AC resulted in higher somatic embryo maturation rates (158 somatic embryos in 14 days in E callus but has no effect in NE callus. A total of 752 co-expressed proteins were identified through the SUCEST (The Sugarcane EST Project, including many housekeeping proteins. E callus showed 65 exclusive proteins on day 0, including dehydrogenase, desiccation-related protein, callose synthase 1 and nitric oxide synthase. After 21 days on maturation treatment, 14 exclusive proteins were identified in E callus, including catalase and secreted protein. NE callus showed 23 exclusive proteins on day 0 and 10 exclusive proteins after 21 days on maturation treatment, including many proteins related to protein degradation. The induction of maturation leads to somatic embryo development, which likely depends on the expression of specific proteins throughout the process, as seen in E callus under maturation treatment. On the other hand, some exclusive proteins can also specifically prevent of somatic embryos development, as seen in the NE callus.

  19. Protein profiling in hepatocellular carcinoma by label-free quantitative proteomics in two west African populations.

    Directory of Open Access Journals (Sweden)

    Haddy K S Fye

    Full Text Available Hepatocellular Carcinoma is the third most common cause of cancer related death worldwide, often diagnosed by measuring serum AFP; a poor performance stand-alone biomarker. With the aim of improving on this, our study focuses on plasma proteins identified by Mass Spectrometry in order to investigate and validate differences seen in the respective proteomes of controls and subjects with LC and HCC.Mass Spectrometry analysis using liquid chromatography electro spray ionization quadrupole time-of-flight was conducted on 339 subjects using a pooled expression profiling approach. ELISA assays were performed on four significantly differentially expressed proteins to validate their expression profiles in subjects from the Gambia and a pilot group from Nigeria. Results from this were collated for statistical multiplexing using logistic regression analysis.Twenty-six proteins were identified as differentially expressed between the three subject groups. Direct measurements of four; hemopexin, alpha-1-antitrypsin, apolipoprotein A1 and complement component 3 confirmed their change in abundance in LC and HCC versus control patients. These trends were independently replicated in the pilot validation subjects from Nigeria. The statistical multiplexing of these proteins demonstrated performance comparable to or greater than ALT in identifying liver cirrhosis or carcinogenesis. This exercise also proposed preliminary cut offs with achievable sensitivity, specificity and AUC statistics greater than reported AFP averages.The validated changes of expression in these proteins have the potential for development into high-performance tests usable in the diagnosis and or monitoring of HCC and LC patients. The identification of sustained expression trends strengthens the suggestion of these four proteins as worthy candidates for further investigation in the context of liver disease. The statistical combinations also provide a novel inroad of analyses able to propose

  20. Label-Free Quantitative Analysis of Mitochondrial Proteomes Using the Multienzyme Digestion-Filter Aided Sample Preparation (MED-FASP) and "Total Protein Approach".

    Science.gov (United States)

    Wiśniewski, Jacek R

    2017-01-01

    Determination of proteome composition and measuring of changes in protein titers provide important information with a substantial value for studying mitochondria.This chapter describes a workflow for the quantitative analysis of mitochondrial proteome with a focus on sample preparation and quantitative analysis of the data. The workflow involves the multienzyme digestion-filter aided sample preparation (MED-FASP) protocol enabling efficient extraction of proteins and high rate of protein-to-peptide conversion. Consecutive protein digestion with Lys C and trypsin enables generation of peptide fractions with minimal overlap, largely increases the number of identified proteins, and extends their sequence coverage. Abundances of proteins identified by multiple peptides can be assessed by the "Total Protein Approach."

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

    DEFF Research Database (Denmark)

    Jeppesen, Dennis Kjølhede; Nawrocki, Arkadiusz; Jensen, Steffen Grann

    2014-01-01

    Cancer cells secrete soluble factors and various extracellular vesicles, including exosomes, into their tissue microenvironment. The secretion of exosomes is speculated to facilitate local invasion and metastatic spread. Here, we used an in vivo metastasis model of human bladder carcinoma cell line...... T24 without metastatic capacity and its two isogenic derivate cell lines SLT4 and FL3, which form metastases in the lungs and liver of mice, respectively. Cultivation in CLAD1000 bioreactors rather than conventional culture flasks resulted in a 13-16-fold increased exosome yield and facilitated...... quantitative proteomics of fractionated exosomes. Exosomes from T24, SLT4, and FL3 cells were partitioned into membrane and luminal fractions and changes in protein abundance related to the gain of metastatic capacity were identified by quantitative iTRAQ- proteomics. We identified several proteins linked...

  2. Quantitative Proteomics of the Tonoplast Reveals a Role for Glycolytic Enzymes in Salt Tolerance[C][W

    Science.gov (United States)

    Barkla, Bronwyn J.; Vera-Estrella, Rosario; Hernández-Coronado, Marcela; Pantoja, Omar

    2009-01-01

    To examine the role of the tonoplast in plant salt tolerance and identify proteins involved in the regulation of transporters for vacuolar Na+ sequestration, we exploited a targeted quantitative proteomics approach. Two-dimensional differential in-gel electrophoresis analysis of free flow zonal electrophoresis separated tonoplast fractions from control, and salt-treated Mesembryanthemum crystallinum plants revealed the membrane association of glycolytic enzymes aldolase and enolase, along with subunits of the vacuolar H+-ATPase V-ATPase. Protein blot analysis confirmed coordinated salt regulation of these proteins, and chaotrope treatment indicated a strong tonoplast association. Reciprocal coimmunoprecipitation studies revealed that the glycolytic enzymes interacted with the V-ATPase subunit B VHA-B, and aldolase was shown to stimulate V-ATPase activity in vitro by increasing the affinity for ATP. To investigate a physiological role for this association, the Arabidopsis thaliana cytoplasmic enolase mutant, los2, was characterized. These plants were salt sensitive, and there was a specific reduction in enolase abundance in the tonoplast from salt-treated plants. Moreover, tonoplast isolated from mutant plants showed an impaired ability for aldolase stimulation of V-ATPase hydrolytic activity. The association of glycolytic proteins with the tonoplast may not only channel ATP to the V-ATPase, but also directly upregulate H+-pump activity. PMID:20028841

  3. Quantitative proteomics of seed filling in castor: comparison with soybean and rapeseed reveals differences between photosynthetic and nonphotosynthetic seed metabolism.

    Science.gov (United States)

    Houston, Norma L; Hajduch, Martin; Thelen, Jay J

    2009-10-01

    Seed maturation or seed filling is a phase of development that plays a major role in the storage reserve composition of a seed. In many plant seeds photosynthesis plays a major role in this process, although oilseeds, such as castor (Ricinus communis), are capable of accumulating oil without the benefit of photophosphorylation to augment energy demands. To characterize seed filling in castor, a systematic quantitative proteomics study was performed. Two-dimensional gel electrophoresis was used to resolve and quantify Cy-dye-labeled proteins expressed at 2, 3, 4, 5, and 6 weeks after flowering in biological triplicate. Expression profiles for 660 protein spot groups were established, and of these, 522 proteins were confidently identified by liquid chromatography-tandem mass spectrometry by mining against the castor genome. Identified proteins were classified according to function, and the most abundant groups of proteins were involved in protein destination and storage (34%), energy (19%), and metabolism (15%). Carbon assimilatory pathways in castor were compared with previous studies of photosynthetic oilseeds, soybean (Glycine max) and rapeseed (Brassica napus). These comparisons revealed differences in abundance and number of protein isoforms at numerous steps in glycolysis. One such difference was the number of enolase isoforms and their sum abundance; castor had approximately six times as many isoforms as soy and rapeseed. Furthermore, Rubisco was 11-fold less prominent in castor compared to rapeseed. These and other differences suggest some aspects of carbon flow, carbon recapture, as well as ATP and NADPH production in castor differs from photosynthetic oilseeds.

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

    Science.gov (United States)

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

    2011-04-01

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

  5. Isobaric Tags for Relative and Absolute Quantitation-Based Proteomic Analysis of Patent and Constricted Ductus Arteriosus Tissues Confirms the Systemic Regulation of Ductus Arteriosus Closure.

    Science.gov (United States)

    Hong, Haifa; Ye, Lincai; Chen, Huiwen; Xia, Yu; Liu, Yue; Liu, Jinfen; Lu, Yanan; Zhang, Haibo

    2015-08-01

    We aimed to evaluate global changes in protein expression associated with patency by undertaking proteomic analysis of human constricted and patent ductus arteriosus (DA). Ten constricted and 10 patent human DAs were excised from infants with ductal-dependent heart disease during surgery. Using isobaric tags for relative and absolute quantitation-based quantitative proteomics, 132 differentially expressed proteins were identified. Of 132 proteins, voltage-gated sodium channel 1.3 (SCN3A), myosin 1d (Myo1d), Rho GTPase activating protein 26 (ARHGAP26), and retinitis pigmentosa 1 (RP1) were selected for validation by Western blot and quantitative real-time polymerase chain reaction analyses. Significant upregulation of SCN3A, Myo1d, and RP1 messenger RNA, and protein levels was observed in the patent DA group (all P ≤ 0.048). ARHGAP26 messenger RNA and protein levels were decreased in patent DA tissue (both P ≤ 0.018). Immunohistochemistry analysis revealed that Myo1d, ARHGAP26, and RP1 were specifically expressed in the subendothelial region of constricted DAs; however, diffuse expression of these proteins was noted in the patent group. Proteomic analysis revealed global changes in the expression of proteins that regulate oxygen sensing, ion channels, smooth muscle cell migration, nervous system, immune system, and metabolism, suggesting a basis for the systemic regulation of DA patency by diverse signaling pathways, which will be confirmed in further studies.

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

    Science.gov (United States)

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

    2016-01-01

    Black rice (Oryza sativa L.), whose pericarp is rich in anthocyanins (ACNs), is considered as a healthier alternative to white rice. Molecular species of ACNs in black rice have been well documented in previous studies; however, information about the metabolic mechanisms underlying ACN biosynthesis during black rice grain development is unclear. 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). Isobaric tags for relative and absolute quantification (iTRAQ) MS/MS were employed to identify statistically significant alterations in the grain proteome. Approximately 928 proteins were detected, of which 230 were differentially expressed throughout 5 successive developmental stages, starting from 3 to 20 days after flowering (DAF). The greatest number of differentially expressed proteins was observed on 7 and 10 DAF, including 76 proteins that were upregulated and 39 that were downregulated. The biological process analysis of gene ontology revealed that the 230 differentially expressed proteins could be sorted into 14 functional groups. Proteins in the largest group were related to metabolic process, which could be integrated into multiple biochemical pathways. Specifically, proteins with a role in ACN biosynthesis, sugar synthesis, and the regulation of gene expression were upregulated, particularly from the onset of black rice grain development and during development. In contrast, the expression of proteins related to signal transduction, redox homeostasis, photosynthesis and N-metabolism decreased during grain maturation. Finally, 8 representative genes encoding different metabolic proteins were verified via quantitative real-time polymerase chain reaction (qRT-PCR) analysis, these genes had differed in transcriptional and translational expression during grain development. Expression analyses of

  7. DNA barcoding analysis of more than 9 000 yeast isolates contributes to quantitative thresholds for yeast species and genera delimitation

    NARCIS (Netherlands)

    Vu, D; Groenewald, M; Szöke, S; Cardinali, G; Eberhardt, U; Stielow, B; de Vries, M; Verkleij, G J M; Crous, P W; Boekhout, T; Robert, V

    DNA barcoding is a global initiative for species identification through sequencing of short DNA sequence markers. Sequences of two loci, ITS and LSU, were generated as barcode data for all (ca. 9k) yeast strains included in the CBS collection, originally assigned to ca. 2 000 species. Taxonomic

  8. Quantitative Proteomics Reveals Ecophysiological Effects of Light and Silver Stress on the Mixotrophic Protist Poterioochromonas malhamensis.

    Directory of Open Access Journals (Sweden)

    Daniela Beisser

    Full Text Available Aquatic environments are heavily impacted by human activities including climate warming and the introduction of xenobiotics. Due to the application of silver nanoparticles as bactericidal agent the introduction of silver into the environment strongly has increased during the past years. Silver ions affect the primary metabolism of algae, in particular photosynthesis. Mixotrophic algae are an interesting test case as they do not exclusively rely on photosynthesis which may attenuate the harmful effect of silver. In order to study the effect of silver ions on mixotrophs, cultures of the chrysophyte Poterioochromonas malhamensis were treated in a replicate design in light and darkness with silver nitrate at a sub-lethal concentration. At five time points samples were taken for the identification and quantitation of proteins by mass spectrometry. In our analysis, relative quantitative protein mass spectrometry has shown to be a useful tool for functional analyses in conjunction with transcriptome reference sequences. A total of 3,952 proteins in 63 samples were identified and quantified, mapping to 4,829 transcripts of the sequenced and assembled transcriptome. Among them, 720 and 104 proteins performing various cellular functions were differentially expressed after eight days in light versus darkness and after three days of silver treatment, respectively. Specifically pathways of the energy and primary carbon metabolism were differentially affected by light and the utilization of expensive reactions hints to an energy surplus of P. malhamensis under light conditions. The excess energy is not invested in growth, but in the synthesis of storage metabolites. The effects of silver were less explicit, observable especially in the dark treatments where the light effect could not mask coinciding but weaker effects of silver. Photosynthesis, particularly the light harvesting complexes, and several sulphur containing enzymes were affected presumably due to

  9. Comparison of two label-free global quantitation methods, APEX and 2D gel electrophoresis, applied to the Shigella dysenteriae proteome

    Directory of Open Access Journals (Sweden)

    Fleischmann Robert D

    2009-06-01

    Full Text Available Abstract The in vitro stationary phase proteome of the human pathogen Shigella dysenteriae serotype 1 (SD1 was quantitatively analyzed in Coomassie Blue G250 (CBB-stained 2D gels. More than four hundred and fifty proteins, of which 271 were associated with distinct gel spots, were identified. In parallel, we employed 2D-LC-MS/MS followed by the label-free computationally modified spectral counting method APEX for absolute protein expression measurements. Of the 4502 genome-predicted SD1 proteins, 1148 proteins were identified with a false positive discovery rate of 5% and quantitated using 2D-LC-MS/MS and APEX. The dynamic range of the APEX method was approximately one order of magnitude higher than that of CBB-stained spot intensity quantitation. A squared Pearson correlation analysis revealed a reasonably good correlation (R2 = 0.67 for protein quantities surveyed by both methods. The correlation was decreased for protein subsets with specific physicochemical properties, such as low Mr values and high hydropathy scores. Stoichiometric ratios of subunits of protein complexes characterized in E. coli were compared with APEX quantitative ratios of orthologous SD1 protein complexes. A high correlation was observed for subunits of soluble cellular protein complexes in several cases, demonstrating versatile applications of the APEX method in quantitative proteomics.

  10. A multiplex quantitative proteomics strategy for protein biomarker studies in urinary exosomes.

    Science.gov (United States)

    Raj, Delfin A A; Fiume, Immacolata; Capasso, Giovambattista; Pocsfalvi, Gabriella

    2012-06-01

    Urinary exosomes have received considerable attention as a potential biomarker source for the diagnosis of renal diseases. Notwithstanding, their use in protein biomarker research is hampered by the lack of efficient methods for vesicle isolation, lysis, and protein quantification. Here we report an improved ultracentrifugation-based method that facilitates the solubilization and removal of major impurities associated with urinary exosomes. A double-cushion sucrose/D(2)O centrifugation step was used after a two-step differential centrifugation to separate exosomes from the heavier vesicles. After the removal of uromodulin, 378 and 79 unique proteins were identified, respectively, in low- and high-density fractions. Comparison of our data with two previously published data sets helped to define proteins commonly found in urinary exosomes. Lysis, protein extraction, and in-solution digestion of exosomes were then optimized for MudPIT application. More than a hundred exosomal proteins were quantified by four-plex iTRAQ analysis of single and pooled samples from two different age groups. For healthy men, six proteins (TSN1, PODXL, IDHC, PPAP, ACBP, and ANXA5) showed significant expression differences between exosome pools of those aged 25-50 and 50-70 years old. Thus, exosomes isolated by our method provide the basis for the development of robust quantitative methods for protein biomarker research.

  11. Genome-wide quantitative trait loci mapping of the human cerebrospinal fluid proteome.

    Science.gov (United States)

    Sasayama, Daimei; Hattori, Kotaro; Ogawa, Shintaro; Yokota, Yuuki; Matsumura, Ryo; Teraishi, Toshiya; Hori, Hiroaki; Ota, Miho; Yoshida, Sumiko; Kunugi, Hiroshi

    2017-01-01

    Cerebrospinal fluid (CSF) is virtually the only one accessible source of proteins derived from the central nervous system (CNS) of living humans and possibly reflects the pathophysiology of a variety of neuropsychiatric diseases. However, little is known regarding the genetic basis of variation in protein levels of human CSF. We examined CSF levels of 1,126 proteins in 133 subjects and performed a genome-wide association analysis of 514,227 single nucleotide polymorphisms (SNPs) to detect protein quantitative trait loci (pQTLs). To be conservative, Spearman's correlation was used to identify an association between genotypes of SNPs and protein levels. A total of 421 cis and 25 trans SNP-protein pairs were significantly correlated at a false discovery rate (FDR) of less than 0.01 (nominal P genome-wide association studies. The present findings suggest that genetic variations play an important role in the regulation of protein expression in the CNS. The obtained database may serve as a valuable resource to understand the genetic bases for CNS protein expression pattern in humans. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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

    Science.gov (United States)

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

    2018-01-10

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

  13. Response of Human Osteoblast to n-HA/PEEK—Quantitative Proteomic Study of Bio-effects of Nano-Hydroxyapatite Composite

    Science.gov (United States)

    Zhao, Minzhi; Li, Haiyun; Liu, Xiaochen; Wei, Jie; Ji, Jianguo; Yang, Shu; Hu, Zhiyuan; Wei, Shicheng

    2016-03-01

    Nano-sized hydroxyapatite (n-HA) is considered as a bio-active material, which is often mixed into bone implant material, polyetheretherketone (PEEK). To reveal the global protein expression modulations of osteoblast in response to direct contact with the PEEK composite containing high level (40%) nano-sized hydroxyapatite (n-HA/PEEK) and explain its comprehensive bio-effects, quantitative proteomic analysis was conducted on human osteoblast-like cells MG-63 cultured on n-HA/PEEK in comparison with pure PEEK. Results from quantitative proteomic analysis showed that the most enriched categories in the up-regulated proteins were related to calcium ion processes and associated functions while the most enriched categories in the down-regulated proteins were related to RNA process. This enhanced our understanding to the molecular mechanism of the promotion of the cell adhesion and differentiation with the inhibition of the cell proliferation on n-HA/PEEK composite. It also exhibited that although the calcium ion level of incubate environment hadn’t increased, merely the calcium fixed on the surface of material had influence to intracellular calcium related processes, which was also reflect by the higher intracellular Ca2+ concentration of n-HA/PEEK. This study could lead to more comprehensive cognition to the versatile biocompatibility of composite materials. It further proves that proteomics is useful in new bio-effect discovery.

  14. Response of Human Osteoblast to n-HA/PEEK—Quantitative Proteomic Study of Bio-effects of Nano-Hydroxyapatite Composite

    Science.gov (United States)

    Zhao, Minzhi; Li, Haiyun; Liu, Xiaochen; Wei, Jie; Ji, Jianguo; Yang, Shu; Hu, Zhiyuan; Wei, Shicheng

    2016-01-01

    Nano-sized hydroxyapatite (n-HA) is considered as a bio-active material, which is often mixed into bone implant material, polyetheretherketone (PEEK). To reveal the global protein expression modulations of osteoblast in response to direct contact with the PEEK composite containing high level (40%) nano-sized hydroxyapatite (n-HA/PEEK) and explain its comprehensive bio-effects, quantitative proteomic analysis was conducted on human osteoblast-like cells MG-63 cultured on n-HA/PEEK in comparison with pure PEEK. Results from quantitative proteomic analysis showed that the most enriched categories in the up-regulated proteins were related to calcium ion processes and associated functions while the most enriched categories in the down-regulated proteins were related to RNA process. This enhanced our understanding to the molecular mechanism of the promotion of the cell adhesion and differentiation with the inhibition of the cell proliferation on n-HA/PEEK composite. It also exhibited that although the calcium ion level of incubate environment hadn’t increased, merely the calcium fixed on the surface of material had influence to intracellular calcium related processes, which was also reflect by the higher intracellular Ca2+ concentration of n-HA/PEEK. This study could lead to more comprehensive cognition to the versatile biocompatibility of composite materials. It further proves that proteomics is useful in new bio-effect discovery. PMID:26956660

  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 a...

  16. Quantitative proteomic analysis of ofloxacin resistant and sensitive clinical isolates of Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Xiang-yu HUANG

    2014-10-01

    Full Text Available Objective To identify the proteins related to ofloxacin (OFX resistance of Mycobacterium tuberculosis (MTB. Methods Standard MTB H37Rv strain, clinical isolates of OFX resistant strain (OFXR and sensitive strain (OFXS were obtained from the Chinese Center for Disease Control and Prevention, and they were cultured in Sauton's medium, and then inactivated by 60Co. Whole cellular proteins were extracted from OFXR, OFXS and H37Rv strain of MTB, respectively. The peptides were labeled, separated and identified by isobaric tags of relative and absolute quantitation (iTRAQ combined with Nano LCMS/MS technology. Results One hundred and seventy-five and 134 differential expression proteins were identified in MTB OFXR compared with MTB OFXS and H37Rv, respectively. One hundred and four common differential expression proteins were identified in MTB OFXR compared with both MTB OFXS and H37Rv. The isoelectric point and theoretic relative molecular mass of differential expression proteins were widely distributed. The majority of the common differential expression proteins were involved in intermediary metabolism, respiration, and lipid metabolism. Twelve common differential expression proteins showed significant differences (the ratio>1.2 or <0.55 in MTB OFXR, including Rv0106, Rv0895, Rv2185c, Rv3248c and Rv3841 up-regulation and Rv2524c, Rv2986c, Rv3118 and Rv3597c down-regulation. Conclusion iTRAQ has been used to identify the common differential expression proteins in MTB OFXR compared with both MTB OFXS and H37Rv, which provides a basis for further study of the mechanism of OFX-resistance. DOI: 10.11855/j.issn.0577-7402.2014.09.06

  17. mapDIA: Preprocessing and statistical analysis of quantitative proteomics data from data independent acquisition mass spectrometry.

    Science.gov (United States)

    Teo, Guoshou; Kim, Sinae; Tsou, Chih-Chiang; Collins, Ben; Gingras, Anne-Claude; Nesvizhskii, Alexey I; Choi, Hyungwon

    2015-11-03

    Data independent acquisition (DIA) mass spectrometry is an emerging technique that offers more complete detection and quantification of peptides and proteins across multiple samples. DIA allows fragment-level quantification, which can be considered as repeated measurements of the abundance of the corresponding peptides and proteins in the downstream statistical analysis. However, few statistical approaches are available for aggregating these complex fragment-level data into peptide- or protein-level statistical summaries. In this work, we describe a software package, mapDIA, for statistical analysis of differential protein expression using DIA fragment-level intensities. The workflow consists of three major steps: intensity normalization, peptide/fragment selection, and statistical analysis. First, mapDIA offers normalization of fragment-level intensities by total intensity sums as well as a novel alternative normalization by local intensity sums in retention time space. Second, mapDIA removes outlier observations and selects peptides/fragments that preserve the major quantitative patterns across all samples for each protein. Last, using the selected fragments and peptides, mapDIA performs model-based statistical significance analysis of protein-level differential expression between specified groups of samples. Using a comprehensive set of simulation datasets, we show that mapDIA detects differentially expressed proteins with accurate control of the false discovery rates. We also describe the analysis procedure in detail using two recently published DIA datasets generated for 14-3-3β dynamic interaction network and prostate cancer glycoproteome. The software was written in C++ language and the source code is available for free through SourceForge website http://sourceforge.net/projects/mapdia/.This article is part of a Special Issue entitled: Computational Proteomics. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Quantitative proteomic analysis by iTRAQ® for the identification of candidate biomarkers in ovarian cancer serum

    Directory of Open Access Journals (Sweden)

    Higgins LeeAnn

    2010-06-01

    Full Text Available Abstract Background Ovarian cancer is the most lethal gynecologic malignancy, with the majority of cases diagnosed at an advanced stage when treatments are less successful. Novel serum protein markers are needed to detect ovarian cancer in its earliest stage; when detected early, survival rates are over 90%. The identification of new serum biomarkers is hindered by the presence of a small number of highly abundant proteins that comprise approximately 95% of serum total protein. In this study, we used pooled serum depleted of the most highly abundant proteins to reduce the dynamic range of proteins, and thereby enhance the identification of serum biomarkers using the quantitative proteomic method iTRAQ®. Results Medium and low abundance proteins from 6 serum pools of 10 patients each from women with serous ovarian carcinoma, and 6 non-cancer control pools were labeled with isobaric tags using iTRAQ® to determine the relative abundance of serum proteins identified by MS. A total of 220 unique proteins were identified and fourteen proteins were elevated in ovarian cancer compared to control serum pools, including several novel candidate ovarian cancer biomarkers: extracellular matrix protein-1, leucine-rich alpha-2 glycoprotein-1, lipopolysaccharide binding protein-1, and proteoglycan-4. Western immunoblotting validated the relative increases in serum protein levels for several of the proteins identified. Conclusions This study provides the first analysis of immunodepleted serum in combination with iTRAQ® to measure relative protein expression in ovarian cancer patients for the pursuit of serum biomarkers. Several candidate biomarkers were identified which warrant further development.

  19. Quantitative proteomic analysis of human testis reveals system-wide molecular and cellular pathways associated with non-obstructive azoospermia.

    Science.gov (United States)

    Alikhani, Mehdi; Mirzaei, Mehdi; Sabbaghian, Marjan; Parsamatin, Pouria; Karamzadeh, Razieh; Adib, Samane; Sodeifi, Niloofar; Gilani, Mohammad Ali Sadighi; Zabet-Moghaddam, Masoud; Parker, Lindsay; Wu, Yunqi; Gupta, Vivek; Haynes, Paul A; Gourabi, Hamid; Baharvand, Hossein; Salekdeh, Ghasem Hosseini

    2017-06-06

    Male infertility accounts for half of the infertility problems experienced by couples. Azoospermia, having no measurable level of sperm in seminal fluid, is one of the known conditions resulting in male infertility. In order to elucidate the complex molecular mechanisms causing male azoospermia, label-free quantitative shotgun proteomics was carried out on testicular tissue specimens from patients with obstructive azoospermia and non-obstructive azoospermia, including maturation arrest (MA) and Sertoli cell only syndrome (SCOS). The abundance of 520 proteins was significantly changed across three groups of samples. We were able to identify several functional biological pathways enriched in azoospermia samples and confirm selected differentially abundant proteins, using multiple histological methods. The results revealed that cell cycle and proteolysis, and RNA splicing were the most significant biological processes impaired by the substantial suppression of proteins related to the aforementioned categories in SCOS tissues. In the MA patient testes, generation of precursor metabolites and energy as well as oxidation-reduction were the most significantly altered processes. Novel candidate proteins identified in this study include key transcription factors, many of which have not previously been shown to be associated with azoospermia. Our findings can provide substantial insights into the molecular regulation of spermatogenesis and human reproduction. The obtained data showed a drastic suppression of proteins involved in spliceosome, cell cycle and proteasome proteins, as well as energy and metabolic production in Sertoli cell only syndrome testis tissue, and to a lesser extent in maturation arrest samples. Moreover, we identified new transcription factors that are highly down-regulated in SCOS and MA patients, thus helping to understand the molecular complexity of spermatogenesis in male infertility. Our findings provide novel candidate protein targets associated

  20. Quantitative Proteomics Reveals Dynamic Interactions of the Minichromosome Maintenance Complex (MCM) in the Cellular Response to Etoposide Induced DNA Damage.

    Science.gov (United States)

    Drissi, Romain; Dubois, Marie-Line; Douziech, Mélanie; Boisvert, François-Michel

    2015-07-01

    The minichromosome maintenance complex (MCM) proteins are required for processive DNA replication and are a target of S-phase checkpoints. The eukaryotic MCM complex consists of six proteins (MCM2-7) that form a heterohexameric ring with DNA helicase activity, which is loaded on chromatin to form the pre-replication complex. Upon entry in S phase, the helicase is activated and opens the DNA duplex to recruit DNA polymerases at the replication fork. The MCM complex thus plays a crucial role during DNA replication, but recent work suggests that MCM proteins could also be involved in DNA repair. Here, we employed a combination of stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics with immunoprecipitation of green fluorescent protein-tagged fusion proteins to identify proteins interacting with the MCM complex, and quantify changes in interactions in response to DNA damage. Interestingly, the MCM complex showed very dynamic changes in interaction with proteins such as Importin7, the histone chaperone ASF1, and the Chromodomain helicase DNA binding protein 3 (CHD3) following DNA damage. These changes in interactions were accompanied by an increase in phosphorylation and ubiquitination on specific sites on the MCM proteins and an increase in the co-localization of the MCM complex with γ-H2AX, confirming the recruitment of these proteins to sites of DNA damage. In summary, our data indicate that the MCM proteins is involved in chromatin remodeling in response to DNA damage. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Quantitative proteomics reveals divergent responses in Apis mellifera worker and drone pupae to parasitization by Varroa destructor.

    Science.gov (United States)

    Surlis, Carla; Carolan, James C; Coffey, Mary; Kavanagh, Kevin

    Varroa destructor is a haemophagous ectoparasite of honeybees and is considered a major causal agent of colony losses in Europe and North America. Although originating in Eastern Asia where it parasitizes Apis cerana, it has shifted hosts to the western honeybee Apis mellifera on which it has a greater deleterious effect on the individual and colony level. To investigate this important host-parasite interaction and to determine whether Varroa causes different effects on different castes we conducted a label free quantitative proteomic analysis of Varroa-parasitized and non-parasitized drone and worker Apis mellifera pupae. 1195 proteins were identified in total, of which 202 and 250 were differentially abundant in parasitized drone and worker pupae, respectively. Both parasitized drone and worker pupae displayed reduced abundance in proteins associated with the cuticle, lipid transport and innate immunity. Proteins involved in metabolic processes were more abundant in both parasitized castes although the response in workers was more pronounced. A number of caste specific responses were observed including differential abundance of numerous cytoskeletal and muscle proteins, which were of higher abundance in parasitized drones in comparison to parasitized workers. Proteins involved in fatty acid and carbohydrate metabolism were more abundant in parasitized workers as were a large number of ribosomal proteins highlighting either potentially divergent responses to Varroa or a different strategy by the mite when parasitizing the different castes. This data improves our understanding of this interaction and may provide a basis for future studies into improvements to therapy and control of Varroasis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Quantitative Characterization of Major Hepatic UDP-Glucuronosyltransferase Enzymes in Human Liver Microsomes: Comparison of Two Proteomic Methods and Correlation with Catalytic Activity.

    Science.gov (United States)

    Achour, Brahim; Dantonio, Alyssa; Niosi, Mark; Novak, Jonathan J; Fallon, John K; Barber, Jill; Smith, Philip C; Rostami-Hodjegan, Amin; Goosen, Theunis C

    2017-10-01

    Quantitative characterization of UDP-glucuronosyltransferase (UGT) enzymes is valuable in glucuronidation reaction phenotyping, predicting metabolic clearance and drug-drug interactions using extrapolation exercises based on pharmacokinetic modeling. Different quantitative proteomic workflows have been employed to quantify UGT enzymes in various systems, with reports indicating large variability in expression, which cannot be explained by interindividual variability alone. To evaluate the effect of methodological differences on end-point UGT abundance quantification, eight UGT enzymes were quantified in 24 matched liver microsomal samples by two laboratories using stable isotope-labeled (SIL) peptides or quantitative concatemer (QconCAT) standard, and measurements were assessed against catalytic activity in seven enzymes ( n = 59). There was little agreement between individual abundance levels reported by the two methods; only UGT1A1 showed strong correlation [Spearman rank order correlation (Rs) = 0.73, P quantitative proteomic data should be validated against catalytic activity whenever possible. In addition, metabolic reaction phenotyping exercises should consider spurious abundance-activity correlations to avoid misleading conclusions. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  3. Mastitomics, the integrated omics of bovine milk in an experimental model of Streptococcus uberis mastitis: 2. Label-free relative quantitative proteomics.

    Science.gov (United States)

    Mudaliar, Manikhandan; Tassi, Riccardo; Thomas, Funmilola C; McNeilly, Tom N; Weidt, Stefan K; McLaughlin, Mark; Wilson, David; Burchmore, Richard; Herzyk, Pawel; Eckersall, P David; Zadoks, Ruth N

    2016-08-16

    Mastitis, inflammation of the mammary gland, is the most common and costly disease of dairy cattle in the western world. It is primarily caused by bacteria, with Streptococcus uberis as one of the most prevalent causative agents. To characterize the proteome during Streptococcus uberis mastitis, an experimentally induced model of intramammary infection was used. Milk whey samples obtained from 6 cows at 6 time points were processed using label-free relative quantitative proteomics. This proteomic analysis complements clinical, bacteriological and immunological studies as well as peptidomic and metabolomic analysis of the same challenge model. A total of 2552 non-redundant bovine peptides were identified, and from these, 570 bovine proteins were quantified. Hierarchical cluster analysis and principal component analysis showed clear clustering of results by stage of infection, with similarities between pre-infection and resolution stages (0 and 312 h post challenge), early infection stages (36 and 42 h post challenge) and late infection stages (57 and 81 h post challenge). Ingenuity pathway analysis identified upregulation of acute phase protein pathways over the course of infection, with dominance of different acute phase proteins at different time points based on differential expression analysis. Antimicrobial peptides, notably cathelicidins and peptidoglycan recognition protein, were upregulated at all time points post challenge and peaked at 57 h, which coincided with 10 000-fold decrease in average bacterial counts. The integration of clinical, bacteriological, immunological and quantitative proteomics and other-omic data provides a more detailed systems level view of the host response to mastitis than has been achieved previously.

  4. Quantitative label-free proteomics for discovery of biomarkers in cerebrospinal fluid: assessment of technical and inter-individual variation.

    Directory of Open Access Journals (Sweden)

    Richard J Perrin

    Full Text Available Biomarkers are required for pre-symptomatic diagnosis, treatment, and monitoring of neurodegenerative diseases such as Alzheimer's disease. Cerebrospinal fluid (CSF is a favored source because its proteome reflects the composition of the brain. Ideal biomarkers have low technical and inter-individual variability (subject variance among control subjects to minimize overlaps between clinical groups. This study evaluates a process of multi-affinity fractionation (MAF and quantitative label-free liquid chromatography tandem mass spectrometry (LC-MS/MS for CSF biomarker discovery by (1 identifying reparable sources of technical variability, (2 assessing subject variance and residual technical variability for numerous CSF proteins, and (3 testing its ability to segregate samples on the basis of desired biomarker characteristics.Fourteen aliquots of pooled CSF and two aliquots from six cognitively normal individuals were randomized, enriched for low-abundance proteins by MAF, digested endoproteolytically, randomized again, and analyzed by nano-LC-MS. Nano-LC-MS data were time and m/z aligned across samples for relative peptide quantification. Among 11,433 aligned charge groups, 1360 relatively abundant ones were annotated by MS2, yielding 823 unique peptides. Analyses, including Pearson correlations of annotated LC-MS ion chromatograms, performed for all pairwise sample comparisons, identified several sources of technical variability: i incomplete MAF and keratins; ii globally- or segmentally-decreased ion current in isolated LC-MS analyses; and iii oxidized methionine-containing peptides. Exclusion of these sources yielded 609 peptides representing 81 proteins. Most of these proteins showed very low coefficients of variation (CV<5% whether they were quantified from the mean of all or only the 2 most-abundant peptides. Unsupervised clustering, using only 24 proteins selected for high subject variance, yielded perfect segregation of pooled and

  5. Substrate analysis of the Pneumocystis carinii protein kinases PcCbk1 and PcSte20 using yeast proteome microarrays provides a novel method for Pneumocystis signalling biology.

    Science.gov (United States)

    Kottom, Theodore J; Limper, Andrew H

    2011-10-01

    Pneumocystis carinii (Pc) undergoes morphological transitions between cysts and trophic forms. We have previously described two Pc serine/threonine kinases, termed PcCbk1 and PcSte20, with PcSte20 belonging to a family of kinases involved in yeast mating, while PcCbk1 is a member of a group of protein kinases involved in regulation of cell cycle, shape, and proliferation. As Pc remains genetically intractable, knowledge on specific substrates phosphorylated by these kinases remains limited. Utilizing the phylogenetic relatedness of Pc to Saccharomyces cerevisiae, we interrogated a yeast proteome microarray containing >4000 purified protein based peptides, leading to the identification of 18 potential PcCbk1 and 15 PcSte20 substrates (Z-score > 3.0). A number of these potential protein substrates are involved in bud site selection, polarized growth, and response to mating α factor and pseudohyphal and invasive growth. Full-length open reading frames suggested by the PcCbk1 and PcSte20 protoarrays were amplified and expressed. These five proteins were used as substrates for PcCbk1 or PcSte20, with each being highly phosphorylated by the respective kinase. Finally, to demonstrate the utility of this method to identify novel PcCbk1 and PcSte20 substrates, we analysed DNA sequence data from the partially complete Pc genome database and detected partial sequence information of potential PcCbk1 kinase substrates PcPxl1 and PcInt1. We additionally identified the potential PcSte20 kinase substrate PcBdf2. Full-length Pc substrates were cloned and expressed in yeast, and shown to be phosphorylated by the respective Pc kinases. In conclusion, the yeast protein microarray represents a novel crossover technique for identifying unique potential Pc kinase substrates. Copyright © 2011 John Wiley & Sons, Ltd.

  6. A targeted proteomics approach to the quantitative analysis of ERK/Bcl-2-mediated anti-apoptosis and multi-drug resistance in breast cancer.

    Science.gov (United States)

    Yang, Ting; Xu, Feifei; Sheng, Yuan; Zhang, Wen; Chen, Yun

    2016-10-01

    Apoptosis suppression caused by overexpression of anti-apoptotic proteins is a central factor to the acquisition of multi-drug resistance (MDR) in breast cancer. As a highly conserved anti-apoptotic protein, Bcl-2 can initiate an anti-apoptosis response via an ERK1/2-mediated pathway. However, the details therein are still far from completely understood and a quantitative description of the associated proteins in the biological context may provide more insights into this process. Following our previous attempts in the quantitative analysis of MDR mechanisms, liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based targeted proteomics was continually employed here to describe ERK/Bcl-2-mediated anti-apoptosis. A targeted proteomics assay was developed and validated first for the simultaneous quantification of ERK1/2 and Bcl-2. In particular, ERK isoforms (i.e., ERK1 and ERK2) and their differential phosphorylated forms including isobaric ones were distinguished. Using this assay, differential protein levels and site-specific phosphorylation stoichiometry were observed in parental drug-sensitive MCF-7/WT cancer cells and drug-resistant MCF-7/ADR cancer cells and breast tissue samples from two groups of patients who were either suspected or diagnosed to have drug resistance. In addition, quantitative analysis of the time course of both ERK1/2 and Bcl-2 in doxorubicin (DOX)-treated MCF-7/WT cells confirmed these findings. Overall, we propose that targeted proteomics can be used generally to resolve more complex cellular events.

  7. A comparative proteomics method for multiple samples based on a 18O-reference strategy and a quantitation and identification-decoupled strategy.

    Science.gov (United States)

    Wang, Hongbin; Zhang, Yongqian; Gui, Shuqi; Zhang, Yong; Lu, Fuping; Deng, Yulin

    2017-08-15

    Comparisons across large numbers of samples are frequently necessary in quantitative proteomics. Many quantitative methods used in proteomics are based on stable isotope labeling, but most of these are only useful for comparing two samples. For up to eight samples, the iTRAQ labeling technique can be used. For greater numbers of samples, the label-free method has been used, but this method was criticized for low reproducibility and accuracy. An ingenious strategy has been introduced, comparing each sample against a 18 O-labeled reference sample that was created by pooling equal amounts of all samples. However, it is necessary to use proportion-known protein mixtures to investigate and evaluate this new strategy. Another problem for comparative proteomics of multiple samples is the poor coincidence and reproducibility in protein identification results across samples. In present study, a method combining 18 O-reference strategy and a quantitation and identification-decoupled strategy was investigated with proportion-known protein mixtures. The results obviously demonstrated that the 18 O-reference strategy had greater accuracy and reliability than other previously used comparison methods based on transferring comparison or label-free strategies. By the decoupling strategy, the quantification data acquired by LC-MS and the identification data acquired by LC-MS/MS are matched and correlated to identify differential expressed proteins, according to retention time and accurate mass. This strategy made protein identification possible for all samples using a single pooled sample, and therefore gave a good reproducibility in protein identification across multiple samples, and allowed for optimizing peptide identification separately so as to identify more proteins. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Candida albicans induces pro-inflammatory and anti-apoptotic signals in macrophages as revealed by quantitative proteomics and phosphoproteomics

    DEFF Research Database (Denmark)

    Reales-Calderón, Jose Antonio; Sylvester, Marc; Strijbis, Karin

    2013-01-01

    Macrophages play a pivotal role in the prevention of Candida albicans infections. Yeast recognition and phagocytosis by macrophages is mediated by Pattern Recognition Receptors (PRRs) that initiate downstream signal transduction cascades by protein phosphorylation and dephosphorylation. We exposed...

  9. Deciphering of the Human Interferon-Regulated Proteome by Mass Spectrometry-Based Quantitative Analysis Reveals Extent and Dynamics of Protein Induction and Repression.

    Science.gov (United States)

    Megger, Dominik A; Philipp, Jos; Le-Trilling, Vu Thuy Khanh; Sitek, Barbara; Trilling, Mirko

    2017-01-01

    Interferons (IFNs) are pleotropic cytokines secreted upon encounter of pathogens and tumors. Applying their antipathogenic, antiproliferative, and immune stimulatory capacities, recombinant IFNs are frequently prescribed as drugs to treat different diseases. IFNs act by changing the gene expression profile of cells. Due to characteristics such as rapid gene induction and signaling, IFNs also represent prototypical model systems for various aspects of biomedical research (e.g., signal transduction). In regard to the signaling and activated promoters, IFNs can be subdivided into two groups. Here, alterations of the cellular proteome of human cells treated with IFNα and IFNγ were elucidated in a time-resolved manner by quantitative proteome analysis. The majority of protein regulations were strongly IFN type and time dependent. In addition to the expected upregulation of IFN-responsive proteins, an astonishing number of proteins became profoundly repressed especially by IFNγ. Thus, our comprehensive analysis revealed important insights into the human IFN-regulated proteome and its dynamics of protein induction and repression. Interestingly, the new class of IFN-repressed genes comprises known host factors for highly relevant pathogens such as HIV, dengue virus, and hepatitis C virus.

  10. Deciphering of the Human Interferon-Regulated Proteome by Mass Spectrometry-Based Quantitative Analysis Reveals Extent and Dynamics of Protein Induction and Repression

    Directory of Open Access Journals (Sweden)

    Dominik A. Megger

    2017-09-01

    Full Text Available Interferons (IFNs are pleotropic cytokines secreted upon encounter of pathogens and tumors. Applying their antipathogenic, antiproliferative, and immune stimulatory capacities, recombinant IFNs are frequently prescribed as drugs to treat different diseases. IFNs act by changing the gene expression profile of cells. Due to characteristics such as rapid gene induction and signaling, IFNs also represent prototypical model systems for various aspects of biomedical research (e.g., signal transduction. In regard to the signaling and activated promoters, IFNs can be subdivided into two groups. Here, alterations of the cellular proteome of human cells treated with IFNα and IFNγ were elucidated in a time-resolved manner by quantitative proteome analysis. The majority of protein regulations were strongly IFN type and time dependent. In addition to the expected upregulation of IFN-responsive proteins, an astonishing number of proteins became profoundly repressed especially by IFNγ. Thus, our comprehensive analysis revealed important insights into the human IFN-regulated proteome and its dynamics of protein induction and repression. Interestingly, the new class of IFN-repressed genes comprises known host factors for highly relevant pathogens such as HIV, dengue virus, and hepatitis C virus.

  11. Quantitative Proteomics Analysis of Altered Protein Expression in the Placental Villous Tissue of Early Pregnancy Loss Using Isobaric Tandem Mass Tags

    Directory of Open Access Journals (Sweden)

    Xiaobei Ni

    2014-01-01

    Full Text Available Many pregnant women suffer miscarriages during early gestation, but the description of these early pregnancy losses (EPL can be somewhat confusing because of the complexities of early development. Thus, the identification of proteins with different expression profiles related to early pregnancy loss is essential for understanding the comprehensive pathophysiological mechanism. In this study, we report a gel-free tandem mass tags- (TMT- labeling based proteomic analysis of five placental villous tissues from patients with early pregnancy loss and five from normal pregnant women. The application of this method resulted in the identification of 3423 proteins and 19647 peptides among the patient group and the matched normal control group. Qualitative and quantitative proteomic analysis revealed 51 proteins to be differentially abundant between the two groups (≥1.2-fold, Student's t-test, P<0.05. To obtain an overview of the biological functions of the proteins whose expression levels altered significantly in EPL group, gene ontology analysis was performed. We also investigated the twelve proteins with a difference over 1.5-fold using pathways analysis. Our results demonstrate that the gel-free TMT-based proteomic approach allows the quantification of differences in protein expression levels, which is useful for obtaining molecular insights into early pregnancy loss.

  12. Time-resolved quantitative proteome profiling of host-pathogen interactions: the response of Staphylococcus aureus RN1HG to internalisation by human airway epithelial cells.

    Science.gov (United States)

    Schmidt, Frank; Scharf, Sandra S; Hildebrandt, Petra; Burian, Marc; Bernhardt, Jörg; Dhople, Vishnu; Kalinka, Julia; Gutjahr, Melanie; Hammer, Elke; Völker, Uwe

    2010-08-01

    Staphylococcus aureus is a versatile gram-positive pathogen that gains increasing importance due to the rapid spreading of resistances. Functional genomics technologies can provide new insights into the adaptational network of this bacterium and its response to environmental challenges. While functional genomics technologies, including proteomics, have been extensively used to study these phenomena in shake flask cultures, studies of bacteria from in vivo settings lack behind. Particularly for proteomics studies, the major bottleneck is the lack of sufficient proteomic coverage for low numbers of cells. In this study, we introduce a workflow that combines a pulse-chase stable isotope labelling by amino acids in cell culture approach with high capacity cell sorting, on-membrane digestion, and high-sensitivity MS to detect and quantitatively monitor several hundred S. aureus proteins from a few million internalised bacteria. This workflow has been used in a proof-of-principle experiment to reveal changes in levels of proteins with a function in protection against oxidative damage and adaptation of cell wall synthesis in strain RN1HG upon internalisation by S9 human bronchial epithelial cells.

  13. Identification of Host Defense-Related Proteins Using Label-Free Quantitative Proteomic Analysis of Milk Whey from Cows with Staphylococcus aureus Subclinical Mastitis

    Directory of Open Access Journals (Sweden)

    Shaimaa Abdelmegid

    2017-12-01

    Full Text Available Staphylococcus aureus is the most common contagious pathogen associated with bovine subclinical mastitis. Current diagnosis of S. aureus mastitis is based on bacteriological culture of milk samples and somatic cell counts, which lack either sensitivity or specificity. Identification of milk proteins that contribute to host defense and their variable responses to pathogenic stimuli would enable the characterization of putative biomarkers of subclinical mastitis. To accomplish this, milk whey samples from healthy and mastitic dairy cows were analyzed using a label-free quantitative proteomics approach. In total, 90 proteins were identified, of which 25 showed significant differential abundance between healthy and mastitic samples. In silico functional analyses indicated the involvement of the differentially abundant proteins in biological mechanisms and signaling pathways related to host defense including pathogen-recognition, direct antimicrobial function, and the acute-phase response. This proteomics and bioinformatics analysis not only facilitates the identification of putative biomarkers of S. aureus subclinical mastitis but also recapitulates previous findings demonstrating the abundance of host defense proteins in intramammary infection. All mass spectrometry data are available via ProteomeXchange with identifier PXD007516.

  14. Quantitative Phospho-proteomic Analysis of TNFα/NFκB Signaling Reveals a Role for RIPK1 Phosphorylation in Suppressing Necrotic Cell Death.

    Science.gov (United States)

    Mohideen, Firaz; Paulo, Joao A; Ordureau, Alban; Gygi, Steve P; Harper, J Wade

    2017-07-01

    TNFα is a potent inducer of inflammation due to its ability to promote gene expression, in part via the NFκB pathway. Moreover, in some contexts, TNFα promotes Caspase-dependent apoptosis or RIPK1/RIPK3/MLKL-dependent necrosis. Engagement of the TNF Receptor Signaling Complex (TNF-RSC), which contains multiple kinase activities, promotes phosphorylation of several downstream components, including TAK1, IKKα/IKKβ, IκBα, and NFκB. However, immediate downstream phosphorylation events occurring in response to TNFα signaling are poorly understood at a proteome-wide level. Here we use Tandem Mass Tagging-based proteomics to quantitatively characterize acute TNFα-mediated alterations in the proteome and phosphoproteome with or without inhibition of the cIAP-dependent survival arm of the pathway with a SMAC mimetic. We identify and quantify over 8,000 phosphorylated peptides, among which are numerous known sites in the TNF-RSC, NFκB, and MAP kinase signaling systems, as well as numerous previously unrecognized phosphorylation events. Functional analysis of S320 phosphorylation in RIPK1 demonstrates a role for this event in suppressing its kinase activity, association with CASPASE-8 and FADD proteins, and subsequent necrotic cell death during inflammatory TNFα stimulation. This study provides a resource for further elucidation of TNFα-dependent signaling pathways. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Quantitative proteomics and systems analysis of cultured H9C2 cardiomyoblasts during differentiation over time supports a 'function follows form' model of differentiation.

    Science.gov (United States)

    Kankeu, Cynthia; Clarke, Kylie; Van Haver, Delphi; Gevaert, Kris; Impens, Francis; Dittrich, Anna; Roderick, H Llewelyn; Passante, Egle; Huber, Heinrich J

    2018-05-17

    The rat cardiomyoblast cell line H9C2 has emerged as a valuable tool for studying cardiac development, mechanisms of disease and toxicology. We present here a rigorous proteomic analysis that monitored the changes in protein expression during differentiation of H9C2 cells into cardiomyocyte-like cells over time. Quantitative mass spectrometry followed by gene ontology (GO) enrichment analysis revealed that early changes in H9C2 differentiation are related to protein pathways of cardiac muscle morphogenesis and sphingolipid synthesis. These changes in the proteome were followed later in the differentiation time-course by alterations in the expression of proteins involved in cation transport and beta-oxidation. Studying the temporal profile of the H9C2 proteome during differentiation in further detail revealed eight clusters of co-regulated proteins that can be associated with early, late, continuous and transient up- and downregulation. Subsequent reactome pathway analysis based on these eight clusters further corroborated and detailed the results of the GO analysis. Specifically, this analysis confirmed that proteins related to pathways in muscle contraction are upregulated early and transiently, and proteins relevant to extracellular matrix organization are downregulated early. In contrast, upregulation of proteins related to cardiac metabolism occurs at later time points. Finally, independent validation of the proteomics results by immunoblotting confirmed hereto unknown regulators of cardiac structure and ionic metabolism. Our results are consistent with a 'function follows form' model of differentiation, whereby early and transient alterations of structural proteins enable subsequent changes that are relevant to the characteristic physiology of cardiomyocytes.

  16. Quantitative iTRAQ-Based Proteomic Identification of Candidate Biomarkers for Diabetic Nephropathy in Plasma of Type 1 Diabetic Patients

    DEFF Research Database (Denmark)

    Overgaard, Anne Julie; Thingholm, Tine Engberg; Larsen, Martin R

    2010-01-01

    INTRODUCTION: As part of a clinical proteomics programme focused on diabetes and its complications, it was our goal to investigate the proteome of plasma in order to find improved candidate biomarkers to predict diabetic nephropathy. METHODS: Proteins derived from plasma from a cross-sectional co...... nephropathy; however, they need to be confirmed in a longitudinal cohort. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12014-010-9053-0) contains supplementary material, which is available to authorized users....

  17. New Protocol Based on UHPLC-MS/MS for Quantitation of Metabolites in Xylose-Fermenting Yeasts

    Science.gov (United States)

    Campos, Christiane Gonçalves; Veras, Henrique César Teixeira; de Aquino Ribeiro, José Antônio; Costa, Patrícia Pinto Kalil Gonçalves; Araújo, Katiúscia Pereira; Rodrigues, Clenilson Martins; de Almeida, João Ricardo Moreira; Abdelnur, Patrícia Verardi

    2017-12-01

    Xylose fermentation is a bottleneck in second-generation ethanol production. As such, a comprehensive understanding of xylose metabolism in naturally xylose-fermenting yeasts is essential for prospection and construction of recombinant yeast strains. The objective of the current study was to establish a reliable metabolomics protocol for quantification of key metabolites of xylose catabolism pathways in yeast, and to apply this protocol to Spathaspora arborariae. Ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) was used to quantify metabolites, and afterwards, sample preparation was optimized to examine yeast intracellular metabolites. S. arborariae was cultivated using xylose as a carbon source under aerobic and oxygen-limited conditions. Ion pair chromatography (IPC) and hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) were shown to efficiently quantify 14 and 5 metabolites, respectively, in a more rapid chromatographic protocol than previously described. Thirteen and eleven metabolites were quantified in S. arborariae under aerobic and oxygen-limited conditions, respectively. This targeted metabolomics protocol is shown here to quantify a total of 19 metabolites, including sugars, phosphates, coenzymes, monosaccharides, and alcohols, from xylose catabolism pathways (glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle) in yeast. Furthermore, to our knowledge, this is the first time that intracellular metabolites have been quantified in S. arborariae after xylose consumption. The results indicated that fine control of oxygen levels during fermentation is necessary to optimize ethanol production by S. arborariae. The protocol presented here may be applied to other yeast species and could support yeast genetic engineering to improve second generation ethanol production. [Figure not available: see fulltext.

  18. Quantitative proteome profiling of human myoma and myometrium tissue reveals kinase expression signatures with potential for therapeutic intervention

    NARCIS (Netherlands)

    Lemeer, Simone; Gholami, Amin Moghaddas; Wu, Zhixiang; Kuster, Bernhard

    2015-01-01

    Uterine leiomyomas are benign tumors affecting a large proportion of the female population. Despite the very high prevalence, the molecular basis for understanding the onset and development of the disease are still poorly understood. In this study, we profiled the proteomes and kinomes of leiomyoma

  19. Automation of dimethylation after guanidination labeling chemistry and its compatibility with common buffers and surfactants for mass spectrometry-based shotgun quantitative proteome analysis

    Energy Technology Data Exchange (ETDEWEB)

    Lo, Andy; Tang, Yanan; Chen, Lu; Li, Liang, E-mail: Liang.Li@ualberta.ca

    2013-07-25

    Graphical abstract: -- Highlights: •Dimethylation after guanidination (2MEGA) uses inexpensive reagents for isotopic labeling of peptides. •2MEGA can be optimized and automated for labeling peptides with high efficiency. •2MEGA is compatible with several commonly used cell lysis and protein solubilization reagents. •The automated 2MEGA labeling method can be used to handle a variety of protein samples for relative proteome quantification. -- Abstract: Isotope labeling liquid chromatography–mass spectrometry (LC–MS) is a major analytical platform for quantitative proteome analysis. Incorporation of isotopes used to distinguish samples plays a critical role in the success of this strategy. In this work, we optimized and automated a chemical derivatization protocol (dimethylation after guanidination, 2MEGA) to increase the labeling reproducibility and reduce human intervention. We also evaluated the reagent compatibility of this protocol to handle biological samples in different types of buffers and surfactants. A commercially available liquid handler was used for reagent dispensation to minimize analyst intervention and at least twenty protein digest samples could be prepared in a single run. Different front-end sample preparation methods for protein solubilization (SDS, urea, Rapigest™, and ProteaseMAX™) and two commercially available cell lysis buffers were evaluated for compatibility with the automated protocol. It was found that better than 94% desired labeling could be obtained in all conditions studied except urea, where the rate was reduced to about 92% due to carbamylation on the peptide amines. This work illustrates the automated 2MEGA labeling process can be used to handle a wide range of protein samples containing various reagents that are often encountered in protein sample preparation for quantitative proteome analysis.

  20. ProteomicsDB.

    Science.gov (United States)

    Schmidt, Tobias; Samaras, Patroklos; Frejno, Martin; Gessulat, Siegfried; Barnert, Maximilian; Kienegger, Harald; Krcmar, Helmut; Schlegl, Judith; Ehrlich, Hans-Christian; Aiche, Stephan; Kuster, Bernhard; Wilhelm, Mathias

    2018-01-04

    ProteomicsDB (https://www.ProteomicsDB.org) is a protein-centric in-memory database for the exploration of large collections of quantitative mass spectrometry-based proteomics data. ProteomicsDB was first released in 2014 to enable the interactive exploration of the first draft of the human proteome. To date, it contains quantitative data from 78 projects totalling over 19k LC-MS/MS experiments. A standardized analysis pipeline enables comparisons between multiple datasets to facilitate the exploration of protein expression across hundreds of tissues, body fluids and cell lines. We recently extended the data model to enable the storage and integrated visualization of other quantitative omics data. This includes transcriptomics data from e.g. NCBI GEO, protein-protein interaction information from STRING, functional annotations from KEGG, drug-sensitivity/selectivity data from several public sources and reference mass spectra from the ProteomeTools project. The extended functionality transforms ProteomicsDB into a multi-purpose resource connecting quantification and meta-data for each protein. The rich user interface helps researchers to navigate all data sources in either a protein-centric or multi-protein-centric manner. Several options are available to download data manually, while our application programming interface enables accessing quantitative data systematically. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  1. Yeast Monitoring of Wine Mixed or Sequential Fermentations Made by Native Strains from D.O. “Vinos de Madrid” Using Real-Time Quantitative PCR

    Science.gov (United States)

    García, Margarita; Esteve-Zarzoso, Braulio; Crespo, Julia; Cabellos, Juan M.; Arroyo, Teresa

    2017-01-01

    There is an increasing trend toward understanding the impact of non-Saccharomyces yeasts on the winemaking process. Although Saccharomyces cerevisiae is the predominant species at the end of fermentation, it has been recognized that the presence of non-Saccharomyces species during alcoholic fermentation can produce an improvement in the quality and complexity of the final wines. A previous work was developed for selecting the best combinations between S. cerevisiae and five non-Saccharomyces (Torulaspora delbrueckii, Schizosaccharomyces pombe, Candida stellata, Metschnikowia pulcherrima, and Lachancea thermotolorans) native yeast strains from D.O. “Vinos de Madrid” at the laboratory scale. The best inoculation strategies between S. cerevisiae and non-Saccharomyces strains were chosen to analyze, by real-time quantitative PCR (qPCR) combined with the use of specific primers, the dynamics of inoculated populations throughout the fermentation process at the pilot scale using the Malvar white grape variety. The efficiency of the qPCR system was verified independently of the samples matrix, founding the inoculated yeast species throughout alcoholic fermentation. Finally, we can validate the positive effect of selected co-cultures in the Malvar wine quality, highlighting the sequential cultures of T. delbrueckii CLI 918/S. cerevisiae CLI 889 and C. stellata CLI 920/S. cerevisiae CLI 889 and, mixed and sequential cultures of L. thermotolerans 9-6C combined with S. cerevisiae CLI 889. PMID:29326669

  2. Yeast Monitoring of Wine Mixed or Sequential Fermentations Made by Native Strains from D.O. “Vinos de Madrid” Using Real-Time Quantitative PCR

    Directory of Open Access Journals (Sweden)

    Margarita García

    2017-12-01

    Full Text Available There is an increasing trend toward understanding the impact of non-Saccharomyces yeasts on the winemaking process. Although Saccharomyces cerevisiae is the predominant species at the end of fermentation, it has been recognized that the presence of non-Saccharomyces species during alcoholic fermentation can produce an improvement in the quality and complexity of the final wines. A previous work was developed for selecting the best combinations between S. cerevisiae and five non-Saccharomyces (Torulaspora delbrueckii, Schizosaccharomyces pombe, Candida stellata, Metschnikowia pulcherrima, and Lachancea thermotolorans native yeast strains from D.O. “Vinos de Madrid” at the laboratory scale. The best inoculation strategies between S. cerevisiae and non-Saccharomyces strains were chosen to analyze, by real-time quantitative PCR (qPCR combined with the use of specific primers, the dynamics of inoculated populations throughout the fermentation process at the pilot scale using the Malvar white grape variety. The efficiency of the qPCR system was verified independently of the samples matrix, founding the inoculated yeast species throughout alcoholic fermentation. Finally, we can validate the positive effect of selected co-cultures in the Malvar wine quality, highlighting the sequential cultures of T. delbrueckii CLI 918/S. cerevisiae CLI 889 and C. stellata CLI 920/S. cerevisiae CLI 889 and, mixed and sequential cultures of L. thermotolerans 9-6C combined with S. cerevisiae CLI 889.

  3. Identification of Thioredoxin Disulfide Targets Using a Quantitative Proteomics Approach Based on Isotope-Coded Affinity Tags

    DEFF Research Database (Denmark)

    Hägglund, Per; Bunkenborg, Jakob; Maeda, Kenji

    2008-01-01

    Thioredoxin (Trx) is a ubiquitous protein disulfide reductase involved in a wide range of cellular redox processes. A large number of putative target proteins have been identified using proteomics approaches, but insight into target specificity at the molecular level is lacking since the reactivity...... of Trx toward individual disulfides has not been quantified. Here, a novel proteomics procedure is described for quantification of Trx-mediated target disulfide reduction based on thiol-specific differential labeling with the iodoacetamide-based isotope-coded affinity tag (ICAT) reagents. Briefly......, protein extract of embryos from germinated barley seeds was treated +/- Trx, and thiols released from target protein disulfides were irreversibly blocked with iodoacetamide. The remaining cysteine residues in the Trx-treated and the control (-Trx) samples were then chemically reduced and labeled...

  4. Assigning Significance in Label-Free Quantitative Proteomics to Include Single-Peptide-Hit Proteins with Low Replicates

    OpenAIRE

    Li, Qingbo

    2010-01-01

    When sample replicates are limited in a label-free proteomics experiment, selecting differentially regulated proteins with an assignment of statistical significance remains difficult for proteins with a single-peptide hit or a small fold-change. This paper aims to address this issue. An important component of the approach employed here is to utilize the rule of Minimum number of Permuted Significant Pairings (MPSP) to reduce false positives. The MPSP rule generates permuted sample pairings fr...

  5. Label-Free Quantitative Proteomic Analysis of Puccinia psidii Uredospores Reveals Differences of Fungal Populations Infecting Eucalyptus and Guava.

    Science.gov (United States)

    Quecine, Maria Carolina; Leite, Thiago Falda; Bini, Andressa Peres; Regiani, Thais; Franceschini, Lívia Maria; Budzinski, Ilara Gabriela Frasson; Marques, Felipe Garbelini; Labate, Mônica Teresa Veneziano; Guidetti-Gonzalez, Simone; Moon, David Henry; Labate, Carlos Alberto

    2016-01-01

    Puccinia psidii sensu lato (s.l.) is the causal agent of eucalyptus and guava rust, but it also attacks a wide range of plant species from the myrtle family, resulting in a significant genetic and physiological variability among populations accessed from different hosts. The uredospores are crucial to P. psidii dissemination in the field. Although they are important for the fungal pathogenesis, their molecular characterization has been poorly studied. In this work, we report the first in-depth proteomic analysis of P. psidii s.l. uredospores from two contrasting populations: guava fruits (PpGuava) and eucalyptus leaves (PpEucalyptus). NanoUPLC-MSE was used to generate peptide spectra that were matched to the UniProt Puccinia genera sequences (UniProt database) resulting in the first proteomic analysis of the phytopathogenic fungus P. psidii. Three hundred and fourty proteins were detected and quantified using Label free proteomics. A significant number of unique proteins were found for each sample, others were significantly more or less abundant, according to the fungal populations. In PpGuava population, many proteins correlated with fungal virulence, such as malate dehydrogenase, proteossomes subunits, enolases and others were increased. On the other hand, PpEucalyptus proteins involved in biogenesis, protein folding and translocation were increased, supporting the physiological variability of the fungal populations according to their protein reservoirs and specific host interaction strategies.

  6. Label-Free Quantitative Proteomic Analysis of Puccinia psidii Uredospores Reveals Differences of Fungal Populations Infecting Eucalyptus and Guava.

    Directory of Open Access Journals (Sweden)

    Maria Carolina Quecine

    Full Text Available Puccinia psidii sensu lato (s.l. is the causal agent of eucalyptus and guava rust, but it also attacks a wide range of plant species from the myrtle family, resulting in a significant genetic and physiological variability among populations accessed from different hosts. The uredospores are crucial to P. psidii dissemination in the field. Although they are important for the fungal pathogenesis, their molecular characterization has been poorly studied. In this work, we report the first in-depth proteomic analysis of P. psidii s.l. uredospores from two contrasting populations: guava fruits (PpGuava and eucalyptus leaves (PpEucalyptus. NanoUPLC-MSE was used to generate peptide spectra that were matched to the UniProt Puccinia genera sequences (UniProt database resulting in the first proteomic analysis of the phytopathogenic fungus P. psidii. Three hundred and fourty proteins were detected and quantified using Label free proteomics. A significant number of unique proteins were found for each sample, others were significantly more or less abundant, according to the fungal populations. In PpGuava population, many proteins correlated with fungal virulence, such as malate dehydrogenase, proteossomes subunits, enolases and others were increased. On the other hand, PpEucalyptus proteins involved in biogenesis, protein folding and translocation were increased, supporting the physiological variability of the fungal populations according to their protein reservoirs and specific host interaction strategies.

  7. Data Generated by Quantitative Liquid Chromatography-Mass Spectrometry Proteomics Are Only the Start and Not the Endpoint: Optimization of Quantitative Concatemer-Based Measurement of Hepatic Uridine-5'-Diphosphate-Glucuronosyltransferase Enzymes with Reference to Catalytic Activity.

    Science.gov (United States)

    Achour, Brahim; Dantonio, Alyssa; Niosi, Mark; Novak, Jonathan J; Al-Majdoub, Zubida M; Goosen, Theunis C; Rostami-Hodjegan, Amin; Barber, Jill

    2018-06-01

    Quantitative proteomic methods require optimization at several stages, including sample preparation, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and data analysis, with the final analysis stage being less widely appreciated by end-users. Previously reported measurement of eight uridine-5'-diphospho-glucuronosyltransferases (UGT) generated by two laboratories [using stable isotope-labeled (SIL) peptides or quantitative concatemer (QconCAT)] reflected significant disparity between proteomic methods. Initial analysis of QconCAT data showed lack of correlation with catalytic activity for several UGTs (1A4, 1A6, 1A9, 2B15) and moderate correlations for UGTs 1A1, 1A3, and 2B7 ( R s = 0.40-0.79, P data analysis, starting from unprocessed LC-MS/MS data, was undertaken, with the aim of improving accuracy, defined by correlation against activity. Three main criteria were found to be important: choice of monitored peptides and fragments, correction for isotope-label incorporation, and abundance normalization using fractional protein mass. Upon optimization, abundance-activity correlations improved significantly for six UGTs ( R s = 0.53-0.87, P data analysis strategy and indicates, using examples, the significance of systematic data processing following acquisition. The proposed strategy offers significant improvement on existing guidelines applicable to clinically relevant proteins quantified using QconCAT. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

  8. Aggregation of polyQ proteins is increased upon yeast aging and affected by Sir2 and Hsf1: novel quantitative biochemical and microscopic assays.

    Directory of Open Access Journals (Sweden)

    Aviv Cohen

    Full Text Available Aging-related neurodegenerative disorders, such as Parkinson's, Alzheimer's and Huntington's diseases, are characterized by accumulation of protein aggregates in distinct neuronal cells that eventually die. In Huntington's disease, the protein huntingtin forms aggregates, and the age of disease onset is inversely correlated to the length of the protein's poly-glutamine tract. Using quantitative assays to estimate microscopically and capture biochemically protein aggregates, here we study in Saccharomyces cerevisiae aging-related aggregation of GFP-tagged, huntingtin-derived proteins with different polyQ lengths. We find that the short 25Q protein never aggregates whereas the long 103Q version always aggregates. However, the mid-size 47Q protein is soluble in young logarithmically growing yeast but aggregates as the yeast cells enter the stationary phase and age, allowing us to plot an "aggregation timeline". This aging-dependent aggregation was associated with increased cytotoxicity. We also show that two aging-related genes, SIR2 and HSF1, affect aggregation of the polyQ proteins. In Δsir2 strain the aging-dependent aggregation of the 47Q protein is aggravated, while overexpression of the transcription factor Hsf1 attenuates aggregation. Thus, the mid-size 47Q protein and our quantitative aggregation assays provide valuable tools to unravel the roles of genes and environmental conditions that affect aging-related aggregation.

  9. Identification of p90 Ribosomal S6 Kinase 2 as a Novel Host Protein in HBx Augmenting HBV Replication by iTRAQ-Based Quantitative Comparative Proteomics.

    Science.gov (United States)

    Yan, Li-Bo; Yu, You-Jia; Zhang, Qing-Bo; Tang, Xiao-Qiong; Bai, Lang; Huang, FeiJun; Tang, Hong

    2018-05-01

    The aim of this study was to screen for novel host proteins that play a role in HBx augmenting Hepatitis B virus (HBV) replication. Three HepG2 cell lines stably harboring different functional domains of HBx (HBx, HBx-Cm6, and HBx-Cm16) were cultured. ITRAQ technology integrated with LC-MS/MS analysis was applied to identify the proteome differences among these three cell lines. In brief, a total of 70 different proteins were identified among HepG2-HBx, HepG2-HBx-Cm6, and HepG2-HBx-Cm16 by double repetition. Several differentially expressed proteins, including p90 ribosomal S6 kinase 2 (RSK2), were further validated. RSK2 was expressed at higher levels in HepG2-HBx and HepG2-HBx-Cm6 compared with HepG2-HBx-Cm16. Furthermore, levels of HBV replication intermediates were decreased after silencing RSK2 in HepG2.2.15. An HBx-minus HBV mutant genome led to decreased levels of HBV replication intermediates and these decreases were restored to levels similar to wild-type HBV by transient ectopic expression of HBx. After silencing RSK2 expression, the levels of HBV replication intermediates synthesized from the HBx-minus HBV mutant genome were not restored to levels that were observed with wild-type HBV by transient HBx expression. Based on iTRAQ quantitative comparative proteomics, RSK2 was identified as a novel host protein that plays a role in HBx augmenting HBV replication. © 2018 The Authors. Proteomics - Clinical Application Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Non-biased enrichment does not improve quantitative proteomic delineation of reovirus T3D-infected HeLa cell protein alterations

    Directory of Open Access Journals (Sweden)

    Jieyuan eJiang

    2012-09-01

    Full Text Available Mass spectrometry-based methods have allowed elucidation of alterations in complex proteomes, such as eukaryotic cells. Such studies have identified and measured relative abundances of thousands of host proteins after cells are infected with a virus. One of the potential limitations in such studies is that generally only the most abundant proteins are identified, leaving the deep richness of the cellular proteome largely unexplored. We differentially labeled HeLa cells with light and heavy stable isotopic forms of lysine and arginine (SILAC and infected cells with reovirus strain T3D. Cells were harvested at 24 hours post-infection. Heavy-labeled infected and light-labeled mock-infected cells were mixed together 1:1. Cells were then divided into cytosol and nuclear fractions and each fraction analyzed, both by standard 2D-HPLC/MS, and also after each fraction had been reacted with a random hexapeptide library (Proteominer® beads to attempt to enrich for low-abundance cellular proteins. A total of 2736 proteins were identified by 2 or more peptides at >99% confidence, of which 66 were significantly up-regulated and 67 were significantly down-regulated. Up-regulated proteins included those involved in antimicrobial and antiviral responses, GTPase activity, nucleotide binding, interferon signaling, and enzymes associated with energy generation. Down-regulated proteins included those involved in cell and biological adhesion, regulation of cell proliferation, structural molecule activity, and numerous molecular binding activities. Comparisons of the r2 correlations, degree of dataset overlap, and numbers of peptides detected suggest that non-biased enrichment approaches may not provide additional data to allow deeper quantitative and comparative mining of complex proteomes.

  11. Quantitative iTRAQ LC-MS/MS proteomics reveals metabolic responses to biofuel ethanol in cyanobacterial Synechocystis sp. PCC 6803.

    Science.gov (United States)

    Qiao, Jianjun; Wang, Jiangxin; Chen, Lei; Tian, Xiaoxu; Huang, Siqiang; Ren, Xiaoyue; Zhang, Weiwen

    2012-11-02

    Recent progress in metabolic engineering has led to autotrophic production of ethanol in various cyanobacterial hosts. However, cyanobacteria are known to be sensitive to ethanol, which restricts further efforts to increase ethanol production levels in these renewable host systems. To understand the mechanisms of ethanol tolerance so that engineering more robust cyanobacterial hosts can be possible, in this study, the responses of model cyanobacterial Synechocystis sp. PCC 6803 to ethanol were determined using a quantitative proteomics approach with iTRAQ LC-MS/MS technologies. The resulting high-quality proteomic data set consisted of 24,887 unique peptides corresponding to 1509 identified proteins, a coverage of approximately 42% of the predicted proteins in the Synechocystis genome. Using a cutoff of 1.5-fold change and a p-value less than 0.05, 135 and 293 unique proteins with differential abundance levels were identified between control and ethanol-treated samples at 24 and 48 h, respectively. Functional analysis showed that the Synechocystis cells employed a combination of induced common stress response, modifications of cell membrane and envelope, and induction of multiple transporters and cell mobility-related proteins as protection mechanisms against ethanol toxicity. Interestingly, our proteomic analysis revealed that proteins related to multiple aspects of photosynthesis were up-regulated in the ethanol-treated Synechocystis cells, consistent with increased chlorophyll a concentration in the cells upon ethanol exposure. The study provided the first comprehensive view of the complicated molecular mechanisms against ethanol stress and also provided a list of potential gene targets for further engineering ethanol tolerance in Synechocystis PCC 6803.

  12. The Coming Age of Complete, Accurate, and Ubiquitous Proteomes

    DEFF Research Database (Denmark)

    Mann, M.; Kulak, N.A.; Nagaraj, N.

    2013-01-01

    High-resolution mass spectrometry (MS)-based proteomics has progressed tremendously over the years. For model organisms like yeast, we can now quantify complete proteomes in just a few hours. Developments discussed in this Perspective will soon enable complete proteome analysis of mammalian cells...

  13. Proteomics: Protein Identification Using Online Databases

    Science.gov (United States)

    Eurich, Chris; Fields, Peter A.; Rice, Elizabeth

    2012-01-01

    Proteomics is an emerging area of systems biology that allows simultaneous study of thousands of proteins expressed in cells, tissues, or whole organisms. We have developed this activity to enable high school or college students to explore proteomic databases using mass spectrometry data files generated from yeast proteins in a college laboratory…

  14. Differential quantitative proteomics of Porphyromonas gingivalis by linear ion trap mass spectrometry: Non-label methods comparison, q-values and LOWESS curve fitting

    Science.gov (United States)

    Xia, Qiangwei; Wang, Tiansong; Park, Yoonsuk; Lamont, Richard J.; Hackett, Murray

    2007-01-01

    Differential analysis of whole cell proteomes by mass spectrometry has largely been applied using various forms of stable isotope labeling. While metabolic stable isotope labeling has been the method of choice, it is often not possible to apply such an approach. Four different label free ways of calculating expression ratios in a classic "two-state" experiment are compared: signal intensity at the peptide level, signal intensity at the protein level, spectral counting at the peptide level, and spectral counting at the protein level. The quantitative data were mined from a dataset of 1245 qualitatively identified proteins, about 56% of the protein encoding open reading frames from Porphyromonas gingivalis, a Gram-negative intracellular pathogen being studied under extracellular and intracellular conditions. Two different control populations were compared against P. gingivalis internalized within a model human target cell line. The q-value statistic, a measure of false discovery rate previously applied to transcription microarrays, was applied to proteomics data. For spectral counting, the most logically consistent estimate of random error came from applying the locally weighted scatter plot smoothing procedure (LOWESS) to the most extreme ratios generated from a control technical replicate, thus setting upper and lower bounds for the region of experimentally observed random error.

  15. Quantitative proteomics reveals altered expression of extracellular matrix related proteins of human primary dermal fibroblasts in response to sulfated hyaluronan and collagen applied as artificial extracellular matrix.

    Science.gov (United States)

    Müller, Stephan A; van der Smissen, Anja; von Feilitzsch, Margarete; Anderegg, Ulf; Kalkhof, Stefan; von Bergen, Martin

    2012-12-01

    Fibroblasts are the main matrix producing cells of the dermis and are also strongly regulated by their matrix environment which can be used to improve and guide skin wound healing processes. Here, we systematically investigated the molecular effects on primary dermal fibroblasts in response to high-sulfated hyaluronan [HA] (hsHA) by quantitative proteomics. The comparison of non- and high-sulfated HA revealed regulation of 84 of more than 1,200 quantified proteins. Based on gene enrichment we found that sulfation of HA alters extracellular matrix remodeling. The collagen degrading enzymes cathepsin K, matrix metalloproteinases-2 and -14 were found to be down-regulated on hsHA. Additionally protein expression of thrombospondin-1, decorin, collagen types I and XII were reduced, whereas the expression of trophoblast glycoprotein and collagen type VI were slightly increased. This study demonstrates that global proteomics provides a valuable tool for revealing proteins involved in molecular effects of growth substrates for further material optimization.

  16. Quantitative proteomic analysis of Streptomyces coelicolor development demonstrates that onset of secondary metabolism coincides with hyphae differentiation

    DEFF Research Database (Denmark)

    Manteca, Angel; Sanchez, Jesus; Jung, Hye Ryung

    2010-01-01

    mycelial stages: an early compartmentalized vegetative mycelium (first mycelium, MI), and a multinucleated reproductive mycelium (second mycelium, MII), arising after PCD processes. In the present study, we made a detailed proteomic analysis of the distinct developmental stages of solid confluent...... Streptomyces coelicolor cultures using iTRAQ labelling and LC-MS/MS. A new experimental approach was developed to obtain homogeneous samples at each developmental stage (temporal protein analysis) and also to obtain membrane and cytosolic protein fractions (spatial protein analysis). A total of 345 proteins...

  17. Quantitative analysis of oyster larval proteome provides new insights into the effects of multiple climate change stressors, supplement to: Dineshram, R; Chandramouli, K; Ko, W K Ginger; Zhang, Huoming; Qian, Pei Yuan; Ravasi, Timothy; Thiyagarajan, Vengatesen (2016): Quantitative analysis of oyster larval proteome provides new insights into the effects of multiple climate change stressors. Global Change Biology, 22(6), 2054-2068

    KAUST Repository

    Dineshram, R

    2016-01-01

    The metamorphosis of planktonic larvae of the Pacific oyster (Crassostrea gigas) underpins their complex life-history strategy by switching on the molecular machinery required for sessile life and building calcite shells. Metamorphosis becomes a survival bottleneck, which will be pressured by different anthropogenically induced climate change-related variables. Therefore, it is important to understand how metamorphosing larvae interact with emerging climate change stressors. To predict how larvae might be affected in a future ocean, we examined changes in the proteome of metamorphosing larvae under multiple stressors: decreased pH (pH 7.4), increased temperature (30 °C), and reduced salinity (15 psu). Quantitative protein expression profiling using iTRAQ-LC-MS/MS identified more than 1300 proteins. Decreased pH had a negative effect on metamorphosis by down-regulating several proteins involved in energy production, metabolism, and protein synthesis. However, warming switched on these down-regulated pathways at pH 7.4. Under multiple stressors, cell signaling, energy production, growth, and developmental pathways were up-regulated, although metamorphosis was still reduced. Despite the lack of lethal effects, significant physiological responses to both individual and interacting climate change related stressors were observed at proteome level. The metamorphosing larvae of the C. gigas population in the Yellow Sea appear to have adequate phenotypic plasticity at the proteome level to survive in future coastal oceans, but with developmental and physiological costs.

  18. Quantitative Proteome Analysis Reveals Increased Content of Basement Membrane Proteins in Arteries from Patients with Type 2 Diabetes and Lower Levels among Metformin Users

    DEFF Research Database (Denmark)

    Rørdam Preil, Simone; Kristensen, Lars P; Beck, Hans C

    2015-01-01

    hypothesized that metformin intake influences the protein composition. METHODS AND RESULTS: -We analyzed non-atherosclerotic repair arteries gathered at coronary by-pass operations from 30 patients with type 2 diabetes, as well as from 30 age- and gender-matched non-diabetic individuals. Quantitative proteome......BACKGROUND: -The increased risk of cardiovascular diseases (CVD) in type 2 diabetes has been extensively documented, but the origins of the association remain largely unknown. We sought to determine changes in protein expressions in arterial tissue from patients with type 2 diabetes and moreover...... analysis was done by iTRAQ-labelling and LC-MS/MS analysis on individual arterial samples. The amounts of the basement membrane (BM) components, alpha-1- and alpha-2- type IV collagen, gamma-1- and beta-2-laminin were significantly increased in patients with diabetes. Moreover, the expressions of basement...

  19. Stable isotope labeling by amino acids in cell culture (SILAC) and quantitative comparison of the membrane proteomes of self-renewing and differentiating human embryonic stem cells

    DEFF Research Database (Denmark)

    Prokhorova, Tatyana A; Rigbolt, Kristoffer T G; Johansen, Pia T

    2009-01-01

    Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful quantitative proteomics platform for comprehensive characterization of complex biological systems. However, the potential of SILAC-based approaches has not been fully utilized in human embryonic stem cell (hESC) research...... embryonic stem cell lines. Of the 811 identified membrane proteins, six displayed significantly higher expression levels in the undifferentiated state compared with differentiating cells. This group includes the established marker CD133/Prominin-1 as well as novel candidates for hESC surface markers......: Glypican-4, Neuroligin-4, ErbB2, receptor-type tyrosine-protein phosphatase zeta (PTPRZ), and Glycoprotein M6B. Our study also revealed 17 potential markers of hESC differentiation as their corresponding protein expression levels displayed a dramatic increase in differentiated embryonic stem cell...

  20. Integration analysis of quantitative proteomics and transcriptomics data identifies potential targets of frizzled-8 protein-related antiproliferative factor in vivo.

    Science.gov (United States)

    Yang, Wei; Kim, Yongsoo; Kim, Taek-Kyun; Keay, Susan K; Kim, Kwang Pyo; Steen, Hanno; Freeman, Michael R; Hwang, Daehee; Kim, Jayoung

    2012-12-01

    What's known on the subject? and What does the study add? Interstitial cystitis (IC) is a prevalent and debilitating pelvic disorder generally accompanied by chronic pain combined with chronic urinating problems. Over one million Americans are affected, especially middle-aged women. However, its aetiology or mechanism remains unclear. No efficient drug has been provided to patients. Several urinary biomarker candidates have been identified for IC; among the most promising is antiproliferative factor (APF), whose biological activity is detectable in urine specimens from >94% of patients with both ulcerative and non-ulcerative IC. The present study identified several important mediators of the effect of APF on bladder cell physiology, suggesting several candidate drug targets against IC. In an attempt to identify potential proteins and genes regulated by APF in vivo, and to possibly expand the APF-regulated network identified by stable isotope labelling by amino acids in cell culture (SILAC), we performed an integration analysis of our own SILAC data and the microarray data of Gamper et al. (2009) BMC Genomics 10: 199. Notably, two of the proteins (i.e. MAPKSP1 and GSPT1) that are down-regulated by APF are involved in the activation of mTORC1, suggesting that the mammalian target of rapamycin (mTOR) pathway is potentially a critical pathway regulated by APF in vivo. Several components of the mTOR pathway are currently being studied as potential therapeutic targets in other diseases. Our analysis suggests that this pathway might also be relevant in the design of diagnostic tools and medications targeting IC. • To enhance our understanding of the interstitial cystitis urine biomarker antiproliferative factor (APF), as well as interstitial cystitis biology more generally at the systems level, we reanalyzed recently published large-scale quantitative proteomics and in vivo transcriptomics data sets using an integration analysis tool that we have developed. • To

  1. Research resource: new and diverse substrates for the insulin receptor isoform a revealed by quantitative proteomics after stimulation with igf-ii or insulin

    DEFF Research Database (Denmark)

    Morcavallo, Alaide; Gaspari, Marco; Pandini, Giuseppe

    2011-01-01

    progression. We hypothesized that IGF-II binding to the IR-A elicits a unique signaling pathway. In order to obtain an unbiased evaluation of IR-A substrates differentially involved after IGF-II and insulin stimulation, we performed quantitative proteomics of IR-A substrates recruited to tyrosine......-phosphorylated protein complexes using stable isotope labeling with amino acids in cell culture in combination with antiphosphotyrosine antibody pull down and mass spectrometry. Using cells expressing only the human IR-A and lacking the IGF-I receptor, we identified 38 IR-A substrates. Only 10 were known IR mediators......, whereas 28 substrates were not previously related to IR signaling. Eleven substrates were recruited by stimulation with both ligands: two equally recruited by IGF-II and insulin, three more strongly recruited by IGF-II, and six more strongly recruited by insulin. Moreover, 14 substrates were recruited...

  2. Salinity-Induced Palmella Formation Mechanism in Halotolerant Algae Dunaliella salina Revealed by Quantitative Proteomics and Phosphoproteomics

    Directory of Open Access Journals (Sweden)

    Sijia Wei

    2017-05-01

    Full Text Available Palmella stage is critical for some unicellular algae to survive in extreme environments. The halotolerant algae Dunaliella salina is a good single-cell model for studying plant adaptation to high salinity. To investigate the molecular adaptation mechanism in salinity shock-induced palmella formation, we performed a comprehensive physiological, proteomics and phosphoproteomics study upon palmella formation of D. salina using dimethyl labeling and Ti4+-immobilized metal ion affinity chromatography (IMAC proteomic approaches. We found that 151 salinity-responsive proteins and 35 salinity-responsive phosphoproteins were involved in multiple signaling and metabolic pathways upon palmella formation. Taken together with photosynthetic parameters and enzyme activity analyses, the patterns of protein accumulation and phosphorylation level exhibited the mechanisms upon palmella formation, including dynamics of cytoskeleton and cell membrane curvature, accumulation and transport of exopolysaccharides, photosynthesis and energy supplying (i.e., photosystem II stability and activity, cyclic electron transport, and C4 pathway, nuclear/chloroplastic gene expression regulation and protein processing, reactive oxygen species homeostasis, and salt signaling transduction. The salinity-responsive protein–protein interaction (PPI networks implied that signaling and protein synthesis and fate are crucial for modulation of these processes. Importantly, the 3D structure of phosphoprotein clearly indicated that the phosphorylation sites of eight proteins were localized in the region of function domain.

  3. A guide through the computational analysis of isotope-labeled mass spectrometry-based quantitative proteomics data: an application study

    Directory of Open Access Journals (Sweden)

    Haußmann Ute

    2011-06-01

    Full Text Available Abstract Background Mass spectrometry-based proteomics has reached a stage where it is possible to comprehensively analyze the whole proteome of a cell in one experiment. Here, the employment of stable isotopes has become a standard technique to yield relative abundance values of proteins. In recent times, more and more experiments are conducted that depict not only a static image of the up- or down-regulated proteins at a distinct time point but instead compare developmental stages of an organism or varying experimental conditions. Results Although the scientific questions behind these experiments are of course manifold, there are, nevertheless, two questions that commonly arise: 1 which proteins are differentially regulated regarding the selected experimental conditions, and 2 are there groups of proteins that show similar abundance ratios, indicating that they have a similar turnover? We give advice on how these two questions can be answered and comprehensively compare a variety of commonly applied computational methods and their outcomes. Conclusions This work provides guidance through the jungle of computational methods to analyze mass spectrometry-based isotope-labeled datasets and recommends an effective and easy-to-use evaluation strategy. We demonstrate our approach with three recently published datasets on Bacillus subtilis 12 and Corynebacterium glutamicum 3. Special focus is placed on the application and validation of cluster analysis methods. All applied methods were implemented within the rich internet application QuPE 4. Results can be found at http://qupe.cebitec.uni-bielefeld.de.

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

    Directory of Open Access Journals (Sweden)

    Yangyong Lv

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

  5. Quantitative Analysis of Proteome Modulations in Alveolar Epithelial Type II Cells in Response to Pulmonary Aspergillus fumigatus Infection.

    Science.gov (United States)

    Seddigh, Pegah; Bracht, Thilo; Molinier-Frenkel, Válerie; Castellano, Flavia; Kniemeyer, Olaf; Schuster, Marc; Weski, Juliane; Hasenberg, Anja; Kraus, Andreas; Poschet, Gernot; Hager, Thomas; Theegarten, Dirk; Opitz, Christiane A; Brakhage, Axel A; Sitek, Barbara; Hasenberg, Mike; Gunzer, Matthias

    2017-12-01

    The ubiquitous mold Aspergillus fumigatus threatens immunosuppressed patients as inducer of lethal invasive aspergillosis. A. fumigatus conidia are airborne and reach the alveoli, where they encounter alveolar epithelial cells (AEC). Previous studies reported the importance of the surfactant-producing AEC II during A. fumigatus infection via in vitro experiments using cell lines. We established a negative isolation protocol yielding untouched primary murine AEC II with a purity >90%, allowing ex vivo analyses of the cells, which encountered the mold in vivo By label-free proteome analysis of AEC II isolated from mice 24h after A. fumigatus or mock infection we quantified 2256 proteins and found 154 proteins to be significantly differentially abundant between both groups (ANOVA p value ≤ 0.01, ratio of means ≥1.5 or ≤0.67, quantified with ≥2 peptides). Most of these proteins were higher abundant in the infected condition and reflected a comprehensive activation of AEC II on interaction with A. fumigatus This was especially represented by proteins related to oxidative phosphorylation, hence energy production. However, the most strongly induced protein was the l-amino acid oxidase (LAAO) Interleukin 4 induced 1 (IL4I1) with a 42.9 fold higher abundance (ANOVA p value 2.91 -10 ). IL4I1 has previously been found in B cells, macrophages, dendritic cells and rare neurons. Increased IL4I1 abundance in AEC II was confirmed by qPCR, Western blot and immunohistology. Furthermore, A. fumigatus infected lungs showed high levels of IL4I1 metabolic products. Importantly, higher IL4I1 abundance was also confirmed in lung tissue from human aspergilloma. Because LAAO are key enzymes for bactericidal product generation, AEC II might actively participate in pathogen defense. We provide insights into proteome changes of primary AEC II thereby opening new avenues to analyze the molecular changes of this central lung cell on infectious threats. Data are available via Proteome

  6. Quantitative proteomic analysis of host--pathogen interactions: a study of Acinetobacter baumannii responses to host airways.

    Science.gov (United States)

    Méndez, Jose Antonio; Mateos, Jesús; Beceiro, Alejandro; Lopez, María; Tomás, María; Poza, Margarita; Bou, Germán

    2015-05-30

    Acinetobacter baumannii is a major health problem. The most common infection caused by A. baumannii is hospital acquired pneumonia, and the associated mortality rate is approximately 50%. Neither in vivo nor ex vivo expression profiling has been performed at the proteomic or transcriptomic level for pneumonia caused by A. baumannii. In this study, we characterized the proteome of A. baumannii under conditions that simulate those found in the airways, to gain some insight into how A. baumannii adapts to the host and to improve knowledge about the pathogenesis and virulence of this bacterium. A clinical strain of A. baumannii was grown under different conditions: in the presence of bronchoalveolar lavage fluid from infected rats, of RAW 264.7 cells to simulate conditions in the respiratory tract and in control conditions. We used iTRAQ labelling and LC-MALDI-TOF/TOF to investigate how A. baumannii responds on exposure to macrophages/BALF. 179 proteins showed differential expression. In both models, proteins involved in the following processes were over-expressed: (i) pathogenesis and virulence (OmpA, YjjK); (ii) cell wall/membrane/envelope biogenesis (MurC); (iii) energy production and conversion (acetyl-CoA hydrolase); and (iv) translation (50S ribosomal protein L9). Proteins involved in the following were under-expressed: (i) lipid metabolism (short-chain dehydrogenase); (ii) amino acid metabolism and transport (aspartate aminotransferase); (iii) unknown function (DNA-binding protein); and (iv) inorganic ion transport and metabolism (hydroperoxidase). We observed alterations in cell wall synthesis and identified 2 upregulated virulence-associated proteins with >15 peptides/protein in both ex vivo models (OmpA and YjjK), suggesting that these proteins are fundamental for pathogenesis and virulence in the airways. This study is the first comprehensive overview of the ex vivo proteome of A. baumannii and is an important step towards identification of diagnostic

  7. Data for iTRAQ-based quantitative proteomics analysis of Brassica napus leaves in response to chlorophyll deficiency

    Directory of Open Access Journals (Sweden)

    Pu Chu

    2015-03-01

    Full Text Available The essential pigment chlorophyll (Chl plays important roles in light harvesting and energy transfer during photosynthesis. Here we present the data from a comparative proteomic analysis of chlorophyll-deficient Brassica napus mutant cde1 and its corresponding wild-type using the iTRAQ approach (Pu Chu et al., 2014 [1]. The distribution of length and number of peptides, mass and sequence coverage of proteins identified was calculated, and the repeatability of the replicates was analyzed. A total of 443 differentially expressed proteins were identified in B. napus leaves, including 228 down-accumulated proteins mainly involved in photosynthesis, porphyrin and chlorophyll metabolism, biosynthesis of secondary metabolites, carbon fixation and 215 up-accumulated proteins that enriched in the spliceosome, mRNA surveillance and RNA degradation.

  8. Quantitative Proteomics Analysis of VEGF-Responsive Endothelial Protein S-Nitrosylation Using Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and LC-MS/MS.

    Science.gov (United States)

    Zhang, Hong-Hai; Lechuga, Thomas J; Chen, Yuezhou; Yang, Yingying; Huang, Lan; Chen, Dong-Bao

    2016-05-01

    Adduction of a nitric oxide moiety (NO•) to cysteine(s), termed S-nitrosylation (SNO), is a novel mechanism for NO to regulate protein function directly. However, the endothelial SNO-protein network that is affected by endogenous and exogenous NO is obscure. This study was designed to develop a quantitative proteomics approach using stable isotope labeling by amino acids in cell culture for comparing vascular endothelial growth factor (VEGFA)- and NO donor-responsive endothelial nitroso-proteomes. Primary placental endothelial cells were labeled with "light" (L-(12)C6 (14)N4-Arg and L-(12)C6 (14)N2-Lys) or "heavy" (L-(13)C6 (15)N4-Arg and L-(13)C6 (15)N2-Lys) amino acids. The light cells were treated with an NO donor nitrosoglutathione (GSNO, 1 mM) or VEGFA (10 ng/ml) for 30 min, while the heavy cells received vehicle as control. Equal amounts of cellular proteins from the light (GSNO or VEGFA treated) and heavy cells were mixed for labeling SNO-proteins by the biotin switch technique and then trypsin digested. Biotinylated SNO-peptides were purified for identifying SNO-proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Ratios of light to heavy SNO-peptides were calculated for determining the changes of the VEGFA- and GSNO-responsive endothelial nitroso-proteomes. A total of 387 light/heavy pairs of SNO-peptides were identified, corresponding to 213 SNO-proteins that include 125 common and 27 VEGFA- and 61 GSNO-responsive SNO-proteins. The specific SNO-cysteine(s) in each SNO-protein were simultaneously identified. Pathway analysis revealed that SNO-proteins are involved in various endothelial functions, including proliferation, motility, metabolism, and protein synthesis. We collectively conclude that endogenous NO on VEGFA stimulation and exogenous NO from GSNO affect common and different SNO-protein networks, implicating SNO as a critical mechanism for VEGFA stimulation of angiogenesis. © 2016 by the Society for the Study of Reproduction

  9. Data set for the proteomic inventory and quantitative analysis of chicken eggshell matrix proteins during the primary events of eggshell mineralization and the active growth phase of calcification.

    Science.gov (United States)

    Marie, Pauline; Labas, Valérie; Brionne, Aurélien; Harichaux, Grégoire; Hennequet-Antier, Christelle; Rodriguez-Navarro, Alejandro B; Nys, Yves; Gautron, Joël

    2015-09-01

    Chicken eggshell is a biomineral composed of 95% calcite calcium carbonate mineral and of 3.5% organic matrix proteins. The assembly of mineral and its structural organization is controlled by its organic matrix. In a recent study [1], we have used quantitative proteomic, bioinformatic and functional analyses to explore the distribution of 216 eggshell matrix proteins at four key stages of shell mineralization defined as: (1) widespread deposition of amorphous calcium carbonate (ACC), (2) ACC transformation into crystalline calcite aggregates, (3) formation of larger calcite crystal units and (4) rapid growth of calcite as columnar structure with preferential crystal orientation. The current article detailed the quantitative analysis performed at the four stages of shell mineralization to determine the proteins which are the most abundant. Additionally, we reported the enriched GO terms and described the presence of 35 antimicrobial proteins equally distributed at all stages to keep the egg free of bacteria and of 81 proteins, the function of which could not be ascribed.

  10. Quantitative proteomics identifies Gemin5, a scaffolding protein involved in ribonucleoprotein assembly, as a novel partner for eukaryotic initiation factor 4E

    DEFF Research Database (Denmark)

    Fierro-Monti, Ivo; Mohammed, Shabaz; Matthiesen, Rune

    2006-01-01

    Protein complexes are dynamic entities; identification and quantitation of their components is critical in elucidating functional roles under specific cellular conditions. We report the first quantitative proteomic analysis of the human cap-binding protein complex. Components and proteins......-starved tumorigenic human mesenchymal stromal cells, attested to their activated translational states. The WD-repeat, scaffolding-protein Gemin5 was identified as a novel eIF4E binding partner, which interacted directly with eIF4E through a motif (YXXXXLPhi) present in a number of eIF4E-interacting partners. Elevated...... levels of Gemin5:eIF4E complexes were found in phorbol ester treated HEK293 cells. Gemin5 and eIF4E co-localized to cytoplasmic P-bodies in human osteosarcoma U2OS cells. Interaction between eIF4E and Gemin5 and their co-localization to the P-bodies, may serve to recruit capped mRNAs to these RNP...

  11. Data set for the proteomic inventory and quantitative analysis of chicken eggshell matrix proteins during the primary events of eggshell mineralization and the active growth phase of calcification

    Directory of Open Access Journals (Sweden)

    Pauline Marie

    2015-09-01

    Full Text Available Chicken eggshell is a biomineral composed of 95% calcite calcium carbonate mineral and of 3.5% organic matrix proteins. The assembly of mineral and its structural organization is controlled by its organic matrix. In a recent study [1], we have used quantitative proteomic, bioinformatic and functional analyses to explore the distribution of 216 eggshell matrix proteins at four key stages of shell mineralization defined as: (1 widespread deposition of amorphous calcium carbonate (ACC, (2 ACC transformation into crystalline calcite aggregates, (3 formation of larger calcite crystal units and (4 rapid growth of calcite as columnar structure with preferential crystal orientation. The current article detailed the quantitative analysis performed at the four stages of shell mineralization to determine the proteins which are the most abundant. Additionally, we reported the enriched GO terms and described the presence of 35 antimicrobial proteins equally distributed at all stages to keep the egg free of bacteria and of 81 proteins, the function of which could not be ascribed.

  12. Qupe--a Rich Internet Application to take a step forward in the analysis of mass spectrometry-based quantitative proteomics experiments.

    Science.gov (United States)

    Albaum, Stefan P; Neuweger, Heiko; Fränzel, Benjamin; Lange, Sita; Mertens, Dominik; Trötschel, Christian; Wolters, Dirk; Kalinowski, Jörn; Nattkemper, Tim W; Goesmann, Alexander

    2009-12-01

    The goal of present -omics sciences is to understand biological systems as a whole in terms of interactions of the individual cellular components. One of the main building blocks in this field of study is proteomics where tandem mass spectrometry (LC-MS/MS) in combination with isotopic labelling techniques provides a common way to obtain a direct insight into regulation at the protein level. Methods to identify and quantify the peptides contained in a sample are well established, and their output usually results in lists of identified proteins and calculated relative abundance values. The next step is to move ahead from these abstract lists and apply statistical inference methods to compare measurements, to identify genes that are significantly up- or down-regulated, or to detect clusters of proteins with similar expression profiles. We introduce the Rich Internet Application (RIA) Qupe providing comprehensive data management and analysis functions for LC-MS/MS experiments. Starting with the import of mass spectra data the system guides the experimenter through the process of protein identification by database search, the calculation of protein abundance ratios, and in particular, the statistical evaluation of the quantification results including multivariate analysis methods such as analysis of variance or hierarchical cluster analysis. While a data model to store these results has been developed, a well-defined programming interface facilitates the integration of novel approaches. A compute cluster is utilized to distribute computationally intensive calculations, and a web service allows to interchange information with other -omics software applications. To demonstrate that Qupe represents a step forward in quantitative proteomics analysis an application study on Corynebacterium glutamicum has been carried out. Qupe is implemented in Java utilizing Hibernate, Echo2, R and the Spring framework. We encourage the usage of the RIA in the sense of the 'software as a

  13. Single-cell-type quantitative proteomic and ionomic analysis of epidermal bladder cells from the halophyte model plant Mesembryanthemum crystallinum to identify salt-responsive proteins.

    Science.gov (United States)

    Barkla, Bronwyn J; Vera-Estrella, Rosario; Raymond, Carolyn

    2016-05-10

    Epidermal bladder cells (EBC) are large single-celled, specialized, and modified trichomes found on the aerial parts of the halophyte Mesembryanthemum crystallinum. Recent development of a simple but high throughput technique to extract the contents from these cells has provided an opportunity to conduct detailed single-cell-type analyses of their molecular characteristics at high resolution to gain insight into the role of these cells in the salt tolerance of the plant. In this study, we carry out large-scale complementary quantitative proteomic studies using both a label (DIGE) and label-free (GeLC-MS) approach to identify salt-responsive proteins in the EBC extract. Additionally we perform an ionomics analysis (ICP-MS) to follow changes in the amounts of 27 different elements. Using these methods, we were able to identify 54 proteins and nine elements that showed statistically significant changes in the EBC from salt-treated plants. GO enrichment analysis identified a large number of transport proteins but also proteins involved in photosynthesis, primary metabolism and Crassulacean acid metabolism (CAM). Validation of results by western blot, confocal microscopy and enzyme analysis helped to strengthen findings and further our understanding into the role of these specialized cells. As expected EBC accumulated large quantities of sodium, however, the most abundant element was chloride suggesting the sequestration of this ion into the EBC vacuole is just as important for salt tolerance. This single-cell type omics approach shows that epidermal bladder cells of M. crystallinum are metabolically active modified trichomes, with primary metabolism supporting cell growth, ion accumulation, compatible solute synthesis and CAM. Data are available via ProteomeXchange with identifier PXD004045.

  14. Development of quantitative proteomics using iTRAQ based on the immunological response of Galleria mellonella larvae challenged with Fusarium oxysporum microconidia.

    Directory of Open Access Journals (Sweden)

    Amalia Muñoz-Gómez

    Full Text Available Galleria mellonella has emerged as a potential invertebrate model for scrutinizing innate immunity. Larvae are easy to handle in host-pathogen assays. We undertook proteomics research in order to understand immune response in a heterologous host when challenged with microconidia of Fusarium oxysporum. The aim of this study was to investigate hemolymph proteins that were differentially expressed between control and immunized larvae sets, tested with F. oxysporum at two temperatures. The iTRAQ approach allowed us to observe the effects of immune challenges in a lucid and robust manner, identifying more than 50 proteins, 17 of them probably involved in the immune response. Changes in protein expression were statistically significant, especially when temperature was increased because this was notoriously affected by F. oxysporum 104 or 106 microconidia/mL. Some proteins were up-regulated upon immune fungal microconidia challenge when temperature changed from 25 to 37°C. After analysis of identified proteins by bioinformatics and meta-analysis, results revealed that they were involved in transport, immune response, storage, oxide-reduction and catabolism: 20 from G. mellonella, 20 from the Lepidoptera species and 19 spread across bacteria, protista, fungi and animal species. Among these, 13 proteins and 2 peptides were examined for their immune expression, and the hypothetical 3D structures of 2 well-known proteins, unannotated for G. mellonella, i.e., actin and CREBP, were resolved using peptides matched with Bombyx mori and Danaus plexippus, respectively. The main conclusion in this study was that iTRAQ tool constitutes a consistent method to detect proteins associated with the innate immune system of G. mellonella in response to infection caused by F. oxysporum. In addition, iTRAQ was a reliable quantitative proteomic approach to detect and quantify the expression levels of immune system proteins and peptides, in particular, it was found that 104

  15. Quantitative proteomics analysis reveals perturbation of lipid metabolic pathways in the liver of Atlantic cod (Gadus morhua) treated with PCB 153.

    Science.gov (United States)

    Yadetie, Fekadu; Oveland, Eystein; Døskeland, Anne; Berven, Frode; Goksøyr, Anders; Karlsen, Odd André

    2017-04-01

    PCB 153 is one of the most abundant PCB congeners detected in biological samples. It is a persistent compound that is still present in the environment despite the ban on production and use of PCBs in the late 1970s. It has strong tendencies to bioaccumulate and biomagnify in biota, and studies have suggested that it is an endocrine and metabolic disruptor. In order to study mechanisms of toxicity, we exposed Atlantic cod (Gadus morhua) to various doses of PCB 153 (0, 0.5, 2 and 8mg/kg body weight) for two weeks and examined the effects on expression of liver proteins using label-free quantitative proteomics. Label-free liquid chromatography-mass spectrometry analysis of the liver proteome resulted in the quantification of 1272 proteins, of which 78 proteins were differentially regulated in the PCB 153-treated dose groups compared to the control group. Functional enrichment analysis showed that pathways significantly affected are related to lipid metabolism, cytoskeletal remodeling, cell cycle and cell adhesion. Importantly, the main effects appear to be on lipid metabolism, with up-regulation of enzymes in the de novo fatty acid synthesis pathway, consistent with previous transcriptomics results. Increased plasma triglyceride levels were also observed in the PCB 153 treated fish, in agreement with the induction of the lipogenic genes and proteins. The results suggest that PCB 153 perturbs lipid metabolism in the Atlantic cod liver. Elevated levels of lipogenic enzymes and plasma triglycerides further suggest increased synthesis of fatty acids and triglycerides. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Quantitative proteomic analysis of the inhibitory effects of CIL-102 on viability and invasiveness in human glioma cells

    Energy Technology Data Exchange (ETDEWEB)

    Teng, Chih-Chuan [Institute of Nursing and Department of Nursing, Chang Gung University of Science and Technology, Taiwan (China); Chronic Diseases and Health Promotion Research Center, CGUST, Taiwan (China); Institute of Basic Medicine Science, National Cheng Kung University, Tainan, Taiwan (China); Kuo, Hsing-Chun [Institute of Nursing and Department of Nursing, Chang Gung University of Science and Technology, Taiwan (China); Chronic Diseases and Health Promotion Research Center, CGUST, Taiwan (China); Department of Medical Research China Medical University Hospital, Taichung, Taiwan (China); Sze, Chun-I, E-mail: szec@mail.ncku.edu.tw [Institute of Basic Medicine Science, Department of Cell Biology and Anatomy and Pathology, National Cheng Kung University, Tainan, Taiwan (China)

    2013-11-01

    CIL-102 (1-[4-(furo[2,3-b]quinolin-4-ylamino)phenyl]ethanone), the major active agent of the alkaloid derivative, has been demonstrated to exert anticancer effects. Herein, we present an investigation focused on the identification of the target(s) of CIL-102's action and the mechanism of its action in apoptotic and anti-invasive pathways. Proteomic approaches were used to purify and identify the protein substrates using 2D difference gel electrophoresis (2D SDS-PAGE) to assess changes in the expression of relevant protein treatment with CIL-102 that resulted in the inhibition of viability and invasion. Our results demonstrate that CIL-102 treatment of U87 cells decreased cell proliferation and invasiveness. CIL-102 dose-dependent induction of apoptosis and inhibitory invasiveness were accompanied by sustained phosphorylation of JNK1/2 and p70S6K as well as generation of the reactive oxygen species. In addition, differential proteins displayed between CIL-102-treated and untreated U87 were determined and validated. There were 11 differentially expressed proteins between the CIL-102-treated and untreated groups. Furthermore, we demonstrated that CIL-102 inhibited cancer cell proliferation and reduced anti-invasion properties by up-regulating the levels of FUMH (Fumarate hydratase). The investigation demonstrated that there was an increase in the cellular levels of FUMH in the CIL-102 reduction in viability and invasion via the activation of JNK1/2 and mTOR signaling modules. NAC administration and shRNA FUMH conferred resistance to CIL-102-inhibited HIF1α and MMP-2 levels via inhibition of JNK1/2 and mTOR activation. We concluded that CIL-102-induced an apoptosis cascade and decreased aggressiveness in astrocytoma cells by modulation of mitochondria function, providing a new mechanism for CIL-102 treatment. - Highlights: • We found the effect of CIL-102 on neuroblastoma cells. • Fumarate hydratase as a CIL-102's target by proteomic differential

  17. Quantitative proteomic analysis of the inhibitory effects of CIL-102 on viability and invasiveness in human glioma cells

    International Nuclear Information System (INIS)

    Teng, Chih-Chuan; Kuo, Hsing-Chun; Sze, Chun-I

    2013-01-01

    CIL-102 (1-[4-(furo[2,3-b]quinolin-4-ylamino)phenyl]ethanone), the major active agent of the alkaloid derivative, has been demonstrated to exert anticancer effects. Herein, we present an investigation focused on the identification of the target(s) of CIL-102's action and the mechanism of its action in apoptotic and anti-invasive pathways. Proteomic approaches were used to purify and identify the protein substrates using 2D difference gel electrophoresis (2D SDS-PAGE) to assess changes in the expression of relevant protein treatment with CIL-102 that resulted in the inhibition of viability and invasion. Our results demonstrate that CIL-102 treatment of U87 cells decreased cell proliferation and invasiveness. CIL-102 dose-dependent induction of apoptosis and inhibitory invasiveness were accompanied by sustained phosphorylation of JNK1/2 and p70S6K as well as generation of the reactive oxygen species. In addition, differential proteins displayed between CIL-102-treated and untreated U87 were determined and validated. There were 11 differentially expressed proteins between the CIL-102-treated and untreated groups. Furthermore, we demonstrated that CIL-102 inhibited cancer cell proliferation and reduced anti-invasion properties by up-regulating the levels of FUMH (Fumarate hydratase). The investigation demonstrated that there was an increase in the cellular levels of FUMH in the CIL-102 reduction in viability and invasion via the activation of JNK1/2 and mTOR signaling modules. NAC administration and shRNA FUMH conferred resistance to CIL-102-inhibited HIF1α and MMP-2 levels via inhibition of JNK1/2 and mTOR activation. We concluded that CIL-102-induced an apoptosis cascade and decreased aggressiveness in astrocytoma cells by modulation of mitochondria function, providing a new mechanism for CIL-102 treatment. - Highlights: • We found the effect of CIL-102 on neuroblastoma cells. • Fumarate hydratase as a CIL-102's target by proteomic differential displays. • CIL

  18. The effect of 24S-hydroxycholesterol on cholesterol homeostasis in neurons: quantitative changes to the cortical neuron proteome.

    Science.gov (United States)

    Wang, Yuqin; Muneton, Sabina; Sjövall, Jan; Jovanovic, Jasmina N; Griffiths, William J

    2008-04-01

    In humans, the brain represents only about 2% of the body's mass but contains about one-quarter of the body's free cholesterol. Cholesterol is synthesized de novo in brain and removed by metabolism to oxysterols. 24S-Hydoxycholesterol represents the major metabolic product of cholesterol in brain, being formed via the cytochrome P450 (CYP) enzyme CYP46A1. CYP46A1 is expressed exclusively in brain, normally by neurons. In this study, we investigated the effect of 24S-hydroxycholesterol on the proteome of rat cortical neurons. With the use of two-dimensional liquid chromatography linked to nanoelectrospray tandem mass spectrometry, over 1040 proteins were identified including members of the cholesterol, isoprenoid and fatty acid synthesis pathways. With the use of stable isotope labeling technology, the protein expression patterns of enzymes in these pathways were investigated. 24S-Hydroxycholesterol was found to down-regulate the expression of members of the cholesterol/isoprenoid synthesis pathways including 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (EC 2.3.3.10), diphosphomevalonate decarboxylase (EC 4.1.1.33), isopentenyl-diphosphate delta isomerase (EC 5.3.3.2), farnesyl-diphosphate synthase (Geranyl trans transferase, EC 2.5.1.10), and dedicated sterol synthesis enzymes, farnesyl-diphosphate farnesyltransferase 1 (squalene synthase, EC 2.5.1.21) and methylsterol monooxygenase (EC 1.14.13.72). The expression of many enzymes in the cholesterol/isoprenoid and fatty acid synthesis pathways are regulated by the membrane-bound transcription factors named sterol regulatory element-binding proteins (SREBPs), which themselves are both transcriptionally and post-transcriptionally regulated. The current proteomic data indicates that 24S-hydroxycholesterol down-regulates cholesterol synthesis in neurons, possibly, in a post-transcriptional manner through SREBP-2. In contrast to cholesterol metabolism, enzymes responsible for the synthesis of fatty acids were not

  19. Genetics Coupled to Quantitative Intact Proteomics Links Heritable Aphid and Endosymbiont Protein Expression to Circulative Polerovirus Transmission▿ †

    Science.gov (United States)

    Cilia, M.; Tamborindeguy, C.; Fish, T.; Howe, K.; Thannhauser, T. W.; Gray, S.

    2011-01-01

    Yellow dwarf viruses in the family Luteoviridae, which are the causal agents of yellow dwarf disease in cereal crops, are each transmitted most efficiently by different species of aphids in a circulative manner that requires the virus to interact with a multitude of aphid proteins. Aphid proteins differentially expressed in F2 Schizaphis graminum genotypes segregating for the ability to transmit Cereal yellow dwarf virus-RPV (CYDV-RPV) were identified using two-dimensional difference gel electrophoresis (DIGE) coupled to either matrix-assisted laser desorption ionization-tandem mass spectrometry or online nanoscale liquid chromatography coupled to electrospray tandem mass spectrometry. A total of 50 protein spots, containing aphid proteins and proteins from the aphid's obligate and maternally inherited bacterial endosymbiont, Buchnera, were identified as differentially expressed between transmission-competent and refractive aphids. Surprisingly, in virus transmission-competent F2 genotypes, the isoelectric points of the Buchnera proteins did not match those in the maternal Buchnera proteome as expected, but instead they aligned with the Buchnera proteome of the transmission-competent paternal parent. Among the aphid proteins identified, many were involved in energy metabolism, membrane trafficking, lipid signaling, and the cytoskeleton. At least eight aphid proteins were expressed as heritable, isoelectric point isoform pairs, one derived from each parental lineage. In the F2 genotypes, the expression of aphid protein isoforms derived from the competent parental lineage aligned with the virus transmission phenotype with high precision. Thus, these isoforms are candidate biomarkers for CYDV-RPV transmission in S. graminum. Our combined genetic and DIGE approach also made it possible to predict where several of the proteins may be expressed in refractive aphids with different barriers to transmission. Twelve proteins were predicted to act in the hindgut of the aphid

  20. Genetics coupled to quantitative intact proteomics links heritable aphid and endosymbiont protein expression to circulative polerovirus transmission.

    Science.gov (United States)

    Cilia, M; Tamborindeguy, C; Fish, T; Howe, K; Thannhauser, T W; Gray, S

    2011-03-01

    Yellow dwarf viruses in the family Luteoviridae, which are the causal agents of yellow dwarf disease in cereal crops, are each transmitted most efficiently by different species of aphids in a circulative manner that requires the virus to interact with a multitude of aphid proteins. Aphid proteins differentially expressed in F2 Schizaphis graminum genotypes segregating for the ability to transmit Cereal yellow dwarf virus-RPV (CYDV-RPV) were identified using two-dimensional difference gel electrophoresis (DIGE) coupled to either matrix-assisted laser desorption ionization-tandem mass spectrometry or online nanoscale liquid chromatography coupled to electrospray tandem mass spectrometry. A total of 50 protein spots, containing aphid proteins and proteins from the aphid's obligate and maternally inherited bacterial endosymbiont, Buchnera, were identified as differentially expressed between transmission-competent and refractive aphids. Surprisingly, in virus transmission-competent F2 genotypes, the isoelectric points of the Buchnera proteins did not match those in the maternal Buchnera proteome as expected, but instead they aligned with the Buchnera proteome of the transmission-competent paternal parent. Among the aphid proteins identified, many were involved in energy metabolism, membrane trafficking, lipid signaling, and the cytoskeleton. At least eight aphid proteins were expressed as heritable, isoelectric point isoform pairs, one derived from each parental lineage. In the F2 genotypes, the expression of aphid protein isoforms derived from the competent parental lineage aligned with the virus transmission phenotype with high precision. Thus, these isoforms are candidate biomarkers for CYDV-RPV transmission in S. graminum. Our combined genetic and DIGE approach also made it possible to predict where several of the proteins may be expressed in refractive aphids with different barriers to transmission. Twelve proteins were predicted to act in the hindgut of the aphid

  1. Quantitative evaluation of yeast's requirement for glycerol formation in very high ethanol performance fed-batch process

    Directory of Open Access Journals (Sweden)

    Nevoigt Elke

    2010-05-01

    Full Text Available Abstract Background Glycerol is the major by-product accounting for up to 5% of the carbon in Saccharomyces cerevisiae ethanolic fermentation. Decreasing glycerol formation may redirect part of the carbon toward ethanol production. However, abolishment of glycerol formation strongly affects yeast's robustness towards different types of stress occurring in an industrial process. In order to assess whether glycerol production can be reduced to a certain extent without jeopardising growth and stress tolerance, the yeast's capacity to synthesize glycerol was adjusted by fine-tuning the activity of the rate-controlling enzyme glycerol 3-phosphate dehydrogenase (GPDH. Two engineered strains whose specific GPDH activity was significantly reduced by two different degrees were comprehensively characterized in a previously developed Very High Ethanol Performance (VHEP fed-batch process. Results The prototrophic strain CEN.PK113-7D was chosen for decreasing glycerol formation capacity. The fine-tuned reduction of specific GPDH activity was achieved by replacing the native GPD1 promoter in the yeast genome by previously generated well-characterized TEF promoter mutant versions in a gpd2Δ background. Two TEF promoter mutant versions were selected for this study, resulting in a residual GPDH activity of 55 and 6%, respectively. The corresponding strains were referred to here as TEFmut7 and TEFmut2. The genetic modifications were accompanied to a strong reduction in glycerol yield on glucose; the level of reduction compared to the wild-type was 61% in TEFmut7 and 88% in TEFmut2. The overall ethanol production yield on glucose was improved from 0.43 g g-1 in the wild type to 0.44 g g-1 measured in TEFmut7 and 0.45 g g-1 in TEFmut2. Although maximal growth rate in the engineered strains was reduced by 20 and 30%, for TEFmut7 and TEFmut2 respectively, strains' ethanol stress robustness was hardly affected; i.e. values for final ethanol concentration (117 ± 4 g

  2. IBT-based quantitative proteomics identifies potential regulatory proteins involved in pigmentation of purple sea cucumber, Apostichopus japonicus.

    Science.gov (United States)

    Xing, Lili; Sun, Lina; Liu, Shilin; Li, Xiaoni; Zhang, Libin; Yang, Hongsheng

    2017-09-01

    Sea cucumbers are an important economic species and exhibit high yield value among aquaculture animals. Purple sea cucumbers are very rare and beautiful and have stable hereditary patterns. In this study, isobaric tags (IBT) were first used to reveal the molecular mechanism of pigmentation in the body wall of the purple sea cucumber. We analyzed the proteomes of purple sea cucumber in early pigmentation stage (Pa), mid pigmentation stage (Pb) and late pigmentation stage (Pc), resulting in the identification of 5580 proteins, including 1099 differentially expressed proteins in Pb: Pa and 339 differentially expressed proteins in Pc: Pb. GO and KEGG analyses revealed possible differentially expressed proteins, including"melanogenesis", "melanosome", "melanoma", "pigment-biosynthetic process", "Epidermis development", "Ras-signaling pathway", "Wnt-signaling pathway", "response to UV light", and "tyrosine metabolism", involved in pigment synthesis and regulation in purple sea cucumbers. The large number of differentially expressed proteins identified here should be highly useful in further elucidating the mechanisms underlying pigmentation in sea cucumbers. Furthermore, these results may also provide the base for further identification of proteins involved in resistance mechanisms against melanoma, albinism, UV damage, and other diseases in sea cucumbers. Copyright © 2017. Published by Elsevier Inc.

  3. Implementation of a Multiplex and Quantitative Proteomics Platform for Assessing Protein Lysates Using DNA-Barcoded Antibodies.

    Science.gov (United States)

    Lee, Jinho; Geiss, Gary K; Demirkan, Gokhan; Vellano, Christopher P; Filanoski, Brian; Lu, Yiling; Ju, Zhenlin; Yu, Shuangxing; Guo, Huifang; Bogatzki, Lisa Y; Carter, Warren; Meredith, Rhonda K; Krishnamurthy, Savitri; Ding, Zhiyong; Beechem, Joseph M; Mills, Gordon B

    2018-06-01

    Molecular analysis of tumors forms the basis for personalized cancer medicine and increasingly guides patient selection for targeted therapy. Future opportunities for personalized medicine are highlighted by the measurement of protein expression levels via immunohistochemistry, protein arrays, and other approaches; however, sample type, sample quantity, batch effects, and "time to result" are limiting factors for clinical application. Here, we present a development pipeline for a novel multiplexed DNA-labeled antibody platform which digitally quantifies protein expression from lysate samples. We implemented a rigorous validation process for each antibody and show that the platform is amenable to multiple protocols covering nitrocellulose and plate-based methods. Results are highly reproducible across technical and biological replicates, and there are no observed "batch effects" which are common for most multiplex molecular assays. Tests from basal and perturbed cancer cell lines indicate that this platform is comparable to orthogonal proteomic assays such as Reverse-Phase Protein Array, and applicable to measuring the pharmacodynamic effects of clinically-relevant cancer therapeutics. Furthermore, we demonstrate the potential clinical utility of the platform with protein profiling from breast cancer patient samples to identify molecular subtypes. Together, these findings highlight the potential of this platform for enhancing our understanding of cancer biology in a clinical translation setting. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Quantitative proteomics revealed partial fungistatic mechanism of ammonia against conidial germination of nematode-trapping fungus Arthrobotrys oligospora ATCC24927.

    Science.gov (United States)

    Liu, Tong; Tian, Dong-Wei; Zou, Li-Juan; Liu, Fang-Yu; Can, Qi-Yan; Yang, Jin-Kui; Xu, Jian-Ping; Huang, Xiao-Wei; Xi, Jia-Qin; Zhu, Ming-Liang; Mo, Ming-He; Zhang, Ke-Qin

    2018-05-01

    Ammonia is one of the fungistatic factors in soil that can suppress conidial germination, but the molecular mechanism underlying the suppression is unknown. In this study, the proteomes of fungistatic conidia, fresh conidia and germinated conidia of Arthrobotrys oligospora ATCC24927 were determined and quantified. The protein expression profile of fungistatic conidia was significantly different from those in the other two conditions. 281 proteins were down expressed in fungistatic conidia and characterized by GO annotation. Gene transcription analysis and inhibition of puromycin (a protein translation inhibitor) on conidial germination suggested that down expression of 33 protein translation related proteins might well result in repression of protein synthesis and inhibition of conidial germination. In addition, 16 down-expressed proteins were mapped to the Ras/mitogen-activated protein (Ras/MAP) regulatory networks which regulate conidial DNA synthesis. The conidial DNA synthesis was found to be definitely inhibited under by ammonia, and function studies of two Ras/MAP proteins by using knock-out strains provided partial evidence that Ras/MAP pathway regulate the conidial germination. These results suggested that down-expression of Ras/MAP related proteins might result in inhibition of DNA synthesis and finally result in inhibition conidial germination. This study revealed partial fungistatic mechanism of ammonia against conidial germination. Copyright © 2018 Elsevier Ltd. All rights reserved.

  5. Use of the KlADH3 promoter for the quantitative production of the murine PDE5A isoforms in the yeast Kluyveromyces lactis.

    Science.gov (United States)

    Cardarelli, Silvia; Giorgi, Mauro; Naro, Fabio; Malatesta, Francesco; Biagioni, Stefano; Saliola, Michele

    2017-09-22

    Phosphodiesterases (PDE) are a superfamily of enzymes that hydrolyse cyclic nucleotides (cAMP/cGMP), signal molecules in transduction pathways regulating crucial aspects of cell life. PDEs regulate the intensity and duration of the cyclic nucleotides signal modulating the downstream biological effect. Due to this critical role associated with the extensive distribution and multiplicity of isozymes, the 11 mammalian families (PDE1 to PDE11) constitute key therapeutic targets. PDE5, one of these cGMP-specific hydrolysing families, is the molecular target of several well known drugs used to treat erectile dysfunction and pulmonary hypertension. Kluyveromyces lactis, one of the few yeasts capable of utilizing lactose, is an attractive host alternative to Saccharomyces cerevisiae for heterologous protein production. Here we established K. lactis as a powerful host for the quantitative production of the murine PDE5 isoforms. Using the promoter of the highly expressed KlADH3 gene, multicopy plasmids were engineered to produce the native and recombinant Mus musculus PDE5 in K. lactis. Yeast cells produced large amounts of the purified A1, A2 and A3 isoforms displaying K m , V max and Sildenafil inhibition values similar to those of the native murine enzymes. PDE5 whose yield was nearly 1 mg/g wet weight biomass for all three isozymes (30 mg/L culture), is well tolerated by K. lactis cells without major growth deficiencies and interferences with the endogenous cAMP/cGMP signal transduction pathways. To our knowledge, this is the first time that the entire PDE5 isozymes family containing both regulatory and catalytic domains has been produced at high levels in a heterologous eukaryotic organism. K. lactis has been shown to be a very promising host platform for large scale production of mammalian PDEs for biochemical and structural studies and for the development of new specific PDE inhibitors for therapeutic applications in many pathologies.

  6. Quantitation of yeast total proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer for uniform loading.

    Science.gov (United States)

    Sheen, Hyukho

    2016-04-01

    Proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer are difficult to quantitate due to SDS and reducing agents being in the buffer. Although acetone precipitation has long been used to clean up proteins from detergents and salts, previous studies showed that protein recovery from acetone precipitation varies from 50 to 100% depending on the samples tested. Here, this article shows that acetone precipitates proteins highly efficiently from SDS-PAGE sample buffer and that quantitative recovery is achieved in 5 min at room temperature. Moreover, precipitated proteins are resolubilized with urea/guanidine, rather than with SDS. Thus, the resolubilized samples are readily quantifiable with Bradford reagent without using SDS-compatible assays. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Identification of Proteins Related to Epigenetic Regulation in the Malignant Transformation of Aberrant Karyotypic Human Embryonic Stem Cells by Quantitative Proteomics

    Science.gov (United States)

    Sun, Yi; Yang, Yixuan; Zeng, Sicong; Tan, Yueqiu; Lu, Guangxiu; Lin, Ge

    2014-01-01

    Previous reports have demonstrated that human embryonic stem cells (hESCs) tend to develop genomic alterations and progress to a malignant state during long-term in vitro culture. This raises concerns of the clinical safety in using cultured hESCs. However, transformed hESCs might serve as an excellent model to determine the process of embryonic stem cell transition. In this study, ITRAQ-based tandem mass spectrometry was used to quantify normal and aberrant karyotypic hESCs proteins from simple to more complex karyotypic abnormalities. We identified and quantified 2583 proteins, and found that the expression levels of 316 proteins that represented at least 23 functional molecular groups were significantly different in both normal and abnormal hESCs. Dysregulated protein expression in epigenetic regulation was further verified in six pairs of hESC lines in early and late passage. In summary, this study is the first large-scale quantitative proteomic analysis of the malignant transformation of aberrant karyotypic hESCs. The data generated should serve as a useful reference of stem cell-derived tumor progression. Increased expression of both HDAC2 and CTNNB1 are detected as early as the pre-neoplastic stage, and might serve as prognostic markers in the malignant transformation of hESCs. PMID:24465727

  8. Quantitative Proteomic Analysis of Staphylococcus aureus Treated With Punicalagin, a Natural Antibiotic From Pomegranate That Disrupts Iron Homeostasis and Induces SOS.

    Science.gov (United States)

    Cooper, Bret; Islam, Nazrul; Xu, Yunfeng; Beard, Hunter S; Garrett, Wesley M; Gu, Ganyu; Nou, Xiangwu

    2018-05-01

    Staphylococcus aureus, a bacterial, food-borne pathogen of humans, can contaminate raw fruits and vegetables. While physical and chemical methods are available to control S. aureus, scientists are searching for inhibitory phytochemicals from plants. One promising compound from pomegranate is punicalagin, a natural antibiotic. To get a broader understanding of the inhibitory effect of punicalagin on S. aureus growth, high-throughput mass spectrometry and quantitative isobaric labeling was used to investigate the proteome of S. aureus after exposure to a sublethal dose of punicalagin. Nearly half of the proteins encoded by the small genome were interrogated, and nearly half of those exhibited significant changes in accumulation. Punicalagin treatment altered the accumulation of proteins and enzymes needed for iron acquisition, and it altered amounts of enzymes for glycolysis, citric acid cycling, protein biosynthesis, and purine and pyrimidine biosynthesis. Punicalagin treatment also induced an SOS cellular response to damaged DNA. Transcriptional comparison of marker genes shows that the punicalagin-induced iron starvation and SOS responses resembles those produced by EDTA and ciprofloxacin. These results show that punicalagin adversely alters bacterial growth by disrupting iron homeostasis and that it induces SOS, possibly through DNA biosynthesis inhibition. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. SWATH2stats: An R/Bioconductor Package to Process and Convert Quantitative SWATH-MS Proteomics Data for Downstream Analysis Tools.

    Science.gov (United States)

    Blattmann, Peter; Heusel, Moritz; Aebersold, Ruedi

    2016-01-01

    SWATH-MS is an acquisition and analysis technique of targeted proteomics that enables measuring several thousand proteins with high reproducibility and accuracy across many samples. OpenSWATH is popular open-source software for peptide identification and quantification from SWATH-MS data. For downstream statistical and quantitative analysis there exist different tools such as MSstats, mapDIA and aLFQ. However, the transfer of data from OpenSWATH to the downstream statistical tools is currently technically challenging. Here we introduce the R/Bioconductor package SWATH2stats, which allows convenient processing of the data into a format directly readable by the downstream analysis tools. In addition, SWATH2stats allows annotation, analyzing the variation and the reproducibility of the measurements, FDR estimation, and advanced filtering before submitting the processed data to downstream tools. These functionalities are important to quickly analyze the quality of the SWATH-MS data. Hence, SWATH2stats is a new open-source tool that summarizes several practical functionalities for analyzing, processing, and converting SWATH-MS data and thus facilitates the efficient analysis of large-scale SWATH/DIA datasets.

  10. Label-Free Quantitative Proteomic Analysis of Chitosan Oligosaccharide-Treated Rice Infected with Southern Rice Black-Streaked Dwarf Virus.

    Science.gov (United States)

    Yang, Anming; Yu, Lu; Chen, Zhuo; Zhang, Shanxue; Shi, Jing; Zhao, Xiaozhen; Yang, Yuanyou; Hu, Deyu; Song, Baoan

    2017-05-18

    Southern rice black-streaked dwarf virus (SRBSDV) has spread from thesouth of China to the north of Vietnam in the past few years and severelyinfluenced rice production. Its long incubation period and early symptoms are not evident; thus, controlling it is difficult. Chitosan oligosaccharide (COS) is a green plant immunomodulator. Early studies showed that preventing and controlling SRBSDV have a certain effect and reduce disease infection rate, but its underlying controlling and preventing mechanism is unclear. In this study, label-free proteomics was used to analyze differentially expressed proteins in rice after COS treatment. The results showed that COS can up-regulate the plant defense-related proteins and down-regulate the protein expression levels of SRBSDV. Meanwhile, quantitative real-time PCR test results showed that COS can improve defense gene expression in rice. Moreover, COS can enhance the defense enzymatic activities of peroxidase, superoxide dismutase and catalase through mitogen-activated protein kinase signaling cascade pathway, and enhance the rice disease resistance.

  11. Quantitative Proteomics Analysis Reveals Novel Insights into Mechanisms of Action of Long Noncoding RNA Hox Transcript Antisense Intergenic RNA (HOTAIR) in HeLa Cells*

    Science.gov (United States)

    Zheng, Peng; Xiong, Qian; Wu, Ying; Chen, Ying; Chen, Zhuo; Fleming, Joy; Gao, Ding; Bi, Lijun; Ge, Feng

    2015-01-01

    Long noncoding RNAs (lncRNAs), which have emerged in recent years as a new and crucial layer of gene regulators, regulate various biological processes such as carcinogenesis and metastasis. HOTAIR (Hox transcript antisense intergenic RNA), a lncRNA overexpressed in most human cancers, has been shown to be an oncogenic lncRNA. Here, we explored the role of HOTAIR in HeLa cells and searched for proteins regulated by HOTAIR. To understand the mechanism of action of HOTAIR from a systems perspective, we employed a quantitative proteomic strategy to systematically identify potential targets of HOTAIR. The expression of 170 proteins was significantly dys-regulated after inhibition of HOTAIR, implying that they could be potential targets of HOTAIR. Analysis of this data at the systems level revealed major changes in proteins involved in diverse cellular components, including the cytoskeleton and the respiratory chain. Further functional studies on vimentin (VIM), a key protein involved in the cytoskeleton, revealed that HOTAIR exerts its effects on migration and invasion of HeLa cells, at least in part, through the regulation of VIM expression. Inhibition of HOTAIR leads to mitochondrial dysfunction and ultrastructural alterations, suggesting a novel role of HOTAIR in maintaining mitochondrial function in cancer cells. Our results provide novel insights into the mechanisms underlying the function of HOTAIR in cancer cells. We expect that the methods used in this study will become an integral part of functional studies of lncRNAs. PMID:25762744

  12. Proteomics - new analytical approaches

    International Nuclear Information System (INIS)

    Hancock, W.S.

    2001-01-01

    for high resolution peptide mapping (hydrophobicity and ionic charge). This presentation will describe the development of new instrumentation based on the combination of capillary-based separation systems, including reversed phase and ion exchange HPLC integrated with on-line mass spectrometry. This approach can be used for stand-alone shot-gun sequencing or integrated with 2D-gel proteomic studies. Examples will include global studies such as the yeast proteome and focused studies on selected protein extracts such as secreted and nuclear proteins from mammalian cells

  13. Quantitative proteomic profiling of membrane proteins from the mouse brain cortex, hippocampus, and cerebellum using the HysTag reagent: mapping of neurotransmitter receptors and ion channels

    DEFF Research Database (Denmark)

    Olsen, Jesper V; Nielsen, Peter Aa; Andersen, Jens R

    2007-01-01

    of recently developed methods for isolation of membrane proteins from 10-20 mg brain tissue [Nielsen, P.Aa., Olsen, J.V., Podtelejnokov, A.V., Andersen, J.R., Mann, M., Wisniewski, J.R., 2005. Proteomic mapping of brain plasma membrane proteins. Mol. Cell. Proteomics 4, 402--408] and the Hys...

  14. Yeast ecology of Kombucha fermentation.

    Science.gov (United States)

    Teoh, Ai Leng; Heard, Gillian; Cox, Julian

    2004-09-01

    Kombucha is a traditional fermentation of sweetened tea, involving a symbiosis of yeast species and acetic acid bacteria. Despite reports of different yeast species being associated with the fermentation, little is known of the quantitative ecology of yeasts in Kombucha. Using oxytetracycline-supplemented malt extract agar, yeasts were isolated from four commercially available Kombucha products and identified using conventional biochemical and physiological tests. During the fermentation of each of the four products, yeasts were enumerated from both the cellulosic pellicle and liquor of the Kombucha. The number and diversity of species varied between products, but included Brettanomyces bruxellensis, Candida stellata, Schizosaccharomyces pombe, Torulaspora delbrueckii and Zygosaccharomyces bailii. While these yeast species are known to occur in Kombucha, the enumeration of each species present throughout fermentation of each of the four Kombucha cultures demonstrated for the first time the dynamic nature of the yeast ecology. Kombucha fermentation is, in general, initiated by osmotolerant species, succeeded and ultimately dominated by acid-tolerant species.

  15. Application of targeted quantitative proteomics analysis in human cerebrospinal fluid using a liquid chromatography matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometer (LC MALDI TOF/TOF) platform.

    Science.gov (United States)

    Pan, Sheng; Rush, John; Peskind, Elaine R; Galasko, Douglas; Chung, Kathryn; Quinn, Joseph; Jankovic, Joseph; Leverenz, James B; Zabetian, Cyrus; Pan, Catherine; Wang, Yan; Oh, Jung Hun; Gao, Jean; Zhang, Jianpeng; Montine, Thomas; Zhang, Jing

    2008-02-01

    Targeted quantitative proteomics by mass spectrometry aims to selectively detect one or a panel of peptides/proteins in a complex sample and is particularly appealing for novel biomarker verification/validation because it does not require specific antibodies. Here, we demonstrated the application of targeted quantitative proteomics in searching, identifying, and quantifying selected peptides in human cerebrospinal spinal fluid (CSF) using a matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometer (MALDI TOF/TOF)-based platform. The approach involved two major components: the use of isotopic-labeled synthetic peptides as references for targeted identification and quantification and a highly selective mass spectrometric analysis based on the unique characteristics of the MALDI instrument. The platform provides high confidence for targeted peptide detection in a complex system and can potentially be developed into a high-throughput system. Using the liquid chromatography (LC) MALDI TOF/TOF platform and the complementary identification strategy, we were able to selectively identify and quantify a panel of targeted peptides in the whole proteome of CSF without prior depletion of abundant proteins. The effectiveness and robustness of the approach associated with different sample complexity, sample preparation strategies, as well as mass spectrometric quantification were evaluated. Other issues related to chromatography separation and the feasibility for high-throughput analysis were also discussed. Finally, we applied targeted quantitative proteomics to analyze a subset of previously identified candidate markers in CSF samples of patients with Parkinson's disease (PD) at different stages and Alzheimer's disease (AD) along with normal controls.

  16. Wine yeast phenomics: A standardized fermentation method for assessing quantitative traits of Saccharomyces cerevisiae strains in enological conditions

    Science.gov (United States)

    Bernard, Margaux; Trujillo, Marine; Prodhomme, Duyên; Barbe, Jean-Christophe; Gibon, Yves; Marullo, Philippe

    2018-01-01

    This work describes the set up of a small scale fermentation methodology for measuring quantitative traits of hundreds of samples in an enological context. By using standardized screw cap vessels, the alcoholic fermentation kinetics of Saccharomyces cerevisiae strains were measured by following their weight loss over the time. This dispositive was coupled with robotized enzymatic assays for measuring metabolites of enological interest in natural grape juices. Despite the small volume used, kinetic parameters and fermentation end products measured are similar with those observed in larger scale vats. The vessel used also offers the possibility to assay 32 volatiles compounds using a headspace solid-phase micro-extraction coupled to gas chromatography and mass spectrometry. The vessel shaking applied strongly impacted most of the phenotypes investigated due to oxygen transfer occuring in the first hours of the alcoholic fermentation. The impact of grape must and micro-oxygenation was investigated illustrating some relevant genetic x environmental interactions. By phenotyping a wide panel of commercial wine starters in five grape juices, broad phenotypic correlations between kinetics and metabolic end products were evidentiated. Moreover, a multivariate analysis illustrates that some grape musts are more able than others to discriminate commercial strains since some are less robust to environmental changes. PMID:29351285

  17. Wine yeast phenomics: A standardized fermentation method for assessing quantitative traits of Saccharomyces cerevisiae strains in enological conditions.

    Science.gov (United States)

    Peltier, Emilien; Bernard, Margaux; Trujillo, Marine; Prodhomme, Duyên; Barbe, Jean-Christophe; Gibon, Yves; Marullo, Philippe

    2018-01-01

    This work describes the set up of a small scale fermentation methodology for measuring quantitative traits of hundreds of samples in an enological context. By using standardized screw cap vessels, the alcoholic fermentation kinetics of Saccharomyces cerevisiae strains were measured by following their weight loss over the time. This dispositive was coupled with robotized enzymatic assays for measuring metabolites of enological interest in natural grape juices. Despite the small volume used, kinetic parameters and fermentation end products measured are similar with those observed in larger scale vats. The vessel used also offers the possibility to assay 32 volatiles compounds using a headspace solid-phase micro-extraction coupled to gas chromatography and mass spectrometry. The vessel shaking applied strongly impacted most of the phenotypes investigated due to oxygen transfer occuring in the first hours of the alcoholic fermentation. The impact of grape must and micro-oxygenation was investigated illustrating some relevant genetic x environmental interactions. By phenotyping a wide panel of commercial wine starters in five grape juices, broad phenotypic correlations between kinetics and metabolic end products were evidentiated. Moreover, a multivariate analysis illustrates that some grape musts are more able than others to discriminate commercial strains since some are less robust to environmental changes.

  18. Core Data of Yeast Interacting Proteins Database (Original Version) - Yeast Interacting Proteins Database | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available y are in the reverse direction. *1 A comprehensive two-hybrid analysis to explore the yeast protein interact...s. 2000 Jan 1;28(1):73-6. *2 The yeast proteome database (YPD) and Caenorhabditis elegans proteome database (WormPD): comprehensive...000 Jan 1;28(1):73-6. *3 A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisia

  19. Quantitative Proteomic Analysis of Venoms from Russian Vipers of Pelias Group: Phospholipases A₂ are the Main Venom Components.

    Science.gov (United States)

    Kovalchuk, Sergey I; Ziganshin, Rustam H; Starkov, Vladislav G; Tsetlin, Victor I; Utkin, Yuri N

    2016-04-12

    Venoms of most Russian viper species are poorly characterized. Here, by quantitative chromato-mass-spectrometry, we analyzed protein and peptide compositions of venoms from four Vipera species (V. kaznakovi, V. renardi, V. orlovi and V. nikolskii) inhabiting different regions of Russia. In all these species, the main components were phospholipases A₂, their content ranging from 24% in V. orlovi to 65% in V. nikolskii. Altogether, enzyme content in venom of V. nikolskii reached ~85%. Among the non-enzymatic proteins, the most abundant were disintegrins (14%) in the V. renardi venom, C-type lectin like (12.5%) in V. kaznakovi, cysteine-rich venom proteins (12%) in V. orlovi and venom endothelial growth factors (8%) in V. nikolskii. In total, 210 proteins and 512 endogenous peptides were identified in the four viper venoms. They represented 14 snake venom protein families, most of which were found in the venoms of Vipera snakes previously. However, phospholipase B and nucleotide degrading enzymes were reported here for the first time. Compositions of V. kaznakovi and V. orlovi venoms were described for the first time and showed the greatest similarity among the four venoms studied, which probably reflected close relationship between these species within the "kaznakovi" complex.

  20. Characterization of yeast extracellular vesicles: evidence for the participation of different pathways of cellular traffic in vesicle biogenesis.

    Directory of Open Access Journals (Sweden)

    Débora L Oliveira

    2010-06-01

    Full Text Available Extracellular vesicles in yeast cells are involved in the molecular traffic across the cell wall. In yeast pathogens, these vesicles have been implicated in the transport of proteins, lipids, polysaccharide and pigments to the extracellular space. Cellular pathways required for the biogenesis of yeast extracellular vesicles are largely unknown.We characterized extracellular vesicle production in wild type (WT and mutant strains of the model yeast Saccharomyces cerevisiae using transmission electron microscopy in combination with light scattering analysis, lipid extraction and proteomics. WT cells and mutants with defective expression of Sec4p, a secretory vesicle-associated Rab GTPase essential for Golgi-derived exocytosis, or Snf7p, which is involved in multivesicular body (MVB formation, were analyzed in parallel. Bilayered vesicles with diameters at the 100-300 nm range were found in extracellular fractions from yeast cultures. Proteomic analysis of vesicular fractions from the cells aforementioned and additional mutants with defects in conventional secretion pathways (sec1-1, fusion of Golgi-derived exocytic vesicles with the plasma membrane; bos1-1, vesicle targeting to the Golgi complex or MVB functionality (vps23, late endosomal trafficking revealed a complex and interrelated protein collection. Semi-quantitative analysis of protein abundance revealed that mutations in both MVB- and Golgi-derived pathways affected the composition of yeast extracellular vesicles, but none abrogated vesicle production. Lipid analysis revealed that mutants with defects in Golgi-related components of the secretory pathway had slower vesicle release kinetics, as inferred from intracellular accumulation of sterols and reduced detection of these lipids in vesicle fractions in comparison with WT cells.Our results suggest that both conventional and unconventional pathways of secretion are required for biogenesis of extracellular vesicles, which demonstrate the

  1. Role of DNA Repair Factor Xeroderma Pigmentosum Protein Group C in Response to Replication Stress As Revealed by DNA Fragile Site Affinity Chromatography and Quantitative Proteomics.

    Science.gov (United States)

    Beresova, Lucie; Vesela, Eva; Chamrad, Ivo; Voller, Jiri; Yamada, Masayuki; Furst, Tomas; Lenobel, Rene; Chroma, Katarina; Gursky, Jan; Krizova, Katerina; Mistrik, Martin; Bartek, Jiri

    2016-12-02

    Replication stress (RS) fuels genomic instability and cancer development and may contribute to aging, raising the need to identify factors involved in cellular responses to such stress. Here, we present a strategy for identification of factors affecting the maintenance of common fragile sites (CFSs), which are genomic loci that are particularly sensitive to RS and suffer from increased breakage and rearrangements in tumors. A DNA probe designed to match the high flexibility island sequence typical for the commonly expressed CFS (FRA16D) was used as specific DNA affinity bait. Proteins significantly enriched at the FRA16D fragment under normal and replication stress conditions were identified using stable isotope labeling of amino acids in cell culture-based quantitative mass spectrometry. The identified proteins interacting with the FRA16D fragment included some known CFS stabilizers, thereby validating this screening approach. Among the hits from our screen so far not implicated in CFS maintenance, we chose Xeroderma pigmentosum protein group C (XPC) for further characterization. XPC is a key factor in the DNA repair pathway known as global genomic nucleotide excision repair (GG-NER), a mechanism whose several components were enriched at the FRA16D fragment in our screen. Functional experiments revealed defective checkpoint signaling and escape of DNA replication intermediates into mitosis and the next generation of XPC-depleted cells exposed to RS. Overall, our results provide insights into an unexpected biological role of XPC in response to replication stress and document the power of proteomics-based screening strategies to elucidate mechanisms of pathophysiological significance.

  2. Proteomics dataset

    DEFF Research Database (Denmark)

    Bennike, Tue Bjerg; Carlsen, Thomas Gelsing; Ellingsen, Torkell

    2017-01-01

    The datasets presented in this article are related to the research articles entitled “Neutrophil Extracellular Traps in Ulcerative Colitis: A Proteome Analysis of Intestinal Biopsies” (Bennike et al., 2015 [1]), and “Proteome Analysis of Rheumatoid Arthritis Gut Mucosa” (Bennike et al., 2017 [2])...... been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD001608 for ulcerative colitis and control samples, and PXD003082 for rheumatoid arthritis samples....

  3. The protein expression landscape of mitosis and meiosis in diploid budding yeast.

    Science.gov (United States)

    Becker, Emmanuelle; Com, Emmanuelle; Lavigne, Régis; Guilleux, Marie-Hélène; Evrard, Bertrand; Pineau, Charles; Primig, Michael

    2017-03-06

    Saccharomyces cerevisiae is an established model organism for the molecular analysis of fundamental biological processes. The genomes of numerous strains have been sequenced, and the transcriptome and proteome ofmajor phases during the haploid and diploid yeast life cycle have been determined. However, much less is known about dynamic changes of the proteome when cells switch from mitotic growth to meiotic development. We report a quantitative protein profiling analysis of yeast cell division and differentiation based on mass spectrometry. Information about protein levels was integrated with strand-specific tiling array expression data. We identified a total of 2366 proteins in at least one condition, including 175 proteins showing a statistically significant>5-fold change across the sample set, and 136 proteins detectable in sporulating but not respiring cells. We correlate protein expression patterns with biological processes and molecular function by Gene Ontology term enrichment, chemoprofiling, transcription interference and the formation of double stranded RNAs by overlapping sense/antisense transcripts. Our work provides initial quantitative insight into protein expression in diploid respiring and differentiating yeast cells. Critically, it associates developmentally regulated induction of antisense long noncoding RNAs and double stranded RNAs with fluctuating protein concentrations during growth and development. This integrated genomics analysis helps better understand how the transcriptome and the proteome correlate in diploid yeast cells undergoing mitotic growth in the presence of acetate (respiration) versus meiotic differentiation (Meiosis I and II). The study (i) provides quantitative expression data for 2366 proteins and their cognate mRNAs in at least one sample, (ii) shows strongly fluctuating protein levels during growth and differentiation for 175 cases, and (iii) identifies 136 proteins absent in mitotic but present in meiotic yeast cells. We

  4. 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...... 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...... for detailed functional and comparative analysis of the dynamic plasma membrane proteome....

  5. The proteome response to amyloid protein expression in vivo.

    Directory of Open Access Journals (Sweden)

    Ricardo A Gomes

    Full Text Available Protein misfolding disorders such as Alzheimer, Parkinson and transthyretin amyloidosis are characterized by the formation of protein amyloid deposits. Although the nature and location of the aggregated proteins varies between different diseases, they all share similar molecular pathways of protein unfolding, aggregation and amyloid deposition. Most effects of these proteins are likely to occur at the proteome level, a virtually unexplored reality. To investigate the effects of an amyloid protein expression on the cellular proteome, we created a yeast expression system using human transthyretin (TTR as a model amyloidogenic protein. We used Saccharomyces cerevisiae, a living test tube, to express native TTR (non-amyloidogenic and the amyloidogenic TTR variant L55P, the later forming aggregates when expressed in yeast. Differential proteome changes were quantitatively analyzed by 2D-differential in gel electrophoresis (2D-DIGE. We show that the expression of the amyloidogenic TTR-L55P causes a metabolic shift towards energy production, increased superoxide dismutase expression as well as of several molecular chaperones involved in protein refolding. Among these chaperones, members of the HSP70 family and the peptidyl-prolyl-cis-trans isomerase (PPIase were identified. The latter is highly relevant considering that it was previously found to be a TTR interacting partner in the plasma of ATTR patients but not in healthy or asymptomatic subjects. The small ubiquitin-like modifier (SUMO expression is also increased. Our findings suggest that refolding and degradation pathways are activated, causing an increased demand of energetic resources, thus the metabolic shift. Additionally, oxidative stress appears to be a consequence of the amyloidogenic process, posing an enhanced threat to cell survival.

  6. Full Data of Yeast Interacting Proteins Database (Original Version) - Yeast Interacting Proteins Database | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available List Contact us Yeast Interacting Proteins Database Full Data of Yeast Interacting Proteins Database (Origin...al Version) Data detail Data name Full Data of Yeast Interacting Proteins Database (Original Version) DOI 10....18908/lsdba.nbdc00742-004 Description of data contents The entire data in the Yeast Interacting Proteins Database...eir interactions are required. Several sources including YPD (Yeast Proteome Database, Costanzo, M. C., Hoga...ematic name in the SGD (Saccharomyces Genome Database; http://www.yeastgenome.org /). Bait gene name The gen

  7. Quantitative proteomics of heavy metal exposure in Arabidopsis thaliana reveals alterations in one-carbon metabolism enzymes upon exposure to zinc.

    Science.gov (United States)

    Barkla, Bronwyn J; Vera-Estrella, Rosario; Miranda-Vergara, María Cristina; Pantoja, Omar

    2014-12-05

    Plant zinc (Zn) homeostasis must be tightly regulated as the requirement for this micronutrient necessitates its uptake. However, excessive Zn can lead to toxicity and the plant must respond rapidly and effectively within its capacity to minimize damage. To detect mechanisms that may be important for coping with excess Zn we carried out a quantitative proteomics approach using 2D-DIGE to identify Zn-responsive proteins in microsomal fractions from leaves of 4day, 200μM Zn-treated, Arabidopsis thaliana plants. Of the eight proteins which showed significant changes in abundance in the Zn-treated samples and which met all of the selection criteria following statistical analysis, six were successfully identified by LC-MS/MS with 2 or more unique peptides. Three of the proteins were found to be involved in the one-carbon metabolism pathway; including glycine decarboxylase P protein, serine hydroxymethyltransferase (SHMT) and methionine synthase, all of which showed reduced abundance in the Zn-treated samples. Western blot analysis confirmed the decrease in SHMT, while changes in metal tolerance protein indicated plants were most likely actively sequestering Zn. Interestingly, excess Zn led to increased petiole length, a phenotype which may reflect the reduced levels of methionine, a key product of the one-carbon metabolism pathway. Metal contamination is becoming an increasingly common environmental problem. High levels of zinc can be found in certain soils naturally or as a result of long-term anthropogenic activity which leads to its accumulation; i.e. use of fertilizers or industrial waste. The study of metal tolerant plants, particularly those classified as hyperaccumulators has been driven by the potential use of these plants for bioremediation purposes. However, the effects of heavy metal exposure on sensitive plants and the different cellular processes that are affected have received significantly less attention. We are interested in identifying proteins in A

  8. Proteomics dataset

    DEFF Research Database (Denmark)

    Bennike, Tue Bjerg; Carlsen, Thomas Gelsing; Ellingsen, Torkell

    2017-01-01

    patients (Morgan et al., 2012; Abraham and Medzhitov, 2011; Bennike, 2014) [8–10. Therefore, we characterized the proteome of colon mucosa biopsies from 10 inflammatory bowel disease ulcerative colitis (UC) patients, 11 gastrointestinal healthy rheumatoid arthritis (RA) patients, and 10 controls. We...... been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD001608 for ulcerative colitis and control samples, and PXD003082 for rheumatoid arthritis samples....

  9. Evaluation of Rice Resistance to Southern Rice Black-Streaked Dwarf Virus and Rice Ragged Stunt Virus through Combined Field Tests, Quantitative Real-Time PCR, and Proteome Analysis.

    Science.gov (United States)

    Wang, Zhenchao; Yu, Lu; Jin, Linhong; Wang, Wenli; Zhao, Qi; Ran, Longlu; Li, Xiangyang; Chen, Zhuo; Guo, Rong; Wei, Yongtian; Yang, Zhongcheng; Liu, Enlong; Hu, Deyu; Song, Baoan

    2017-02-22

    Diseases caused by southern rice black-streaked dwarf virus (SRBSDV) and rice ragged stunt virus (RRSV) considerably decrease grain yield. Therefore, determining rice cultivars with high resistance to SRBSDV and RRSV is necessary. In this study, rice cultivars with high resistance to SRBSDV and RRSV were evaluated through field trials in Shidian and Mangshi county, Yunnan province, China. SYBR Green I-based quantitative real-time polymerase chain reaction (qRT-PCR) analysis was used to quantitatively detect virus gene expression levels in different rice varieties. The following parameters were applied to evaluate rice resistance: acre yield (A.Y.), incidence of infected plants (I.I.P.), virus load (V.L.), disease index (D.I.), and insect quantity (I.Q.) per 100 clusters. Zhongzheyou1 (Z1) and Liangyou2186 (L2186) were considered the most suitable varieties with integrated higher A.Y., lower I.I.P., V.L., D.I. and I.Q. In order to investigate the mechanism of rice resistance, comparative label-free shotgun liquid chromatography tandem-mass spectrometry (LC-MS/MS) proteomic approaches were applied to comprehensively describe the proteomics of rice varieties' SRBSDV tolerance. Systemic acquired resistance (SAR)-related proteins in Z1 and L2186 may result in the superior resistance of these varieties compared with Fengyouxiangzhan (FYXZ).

  10. Quantitative real-time PCR as a sensitive protein-protein interaction quantification method and a partial solution for non-accessible autoactivator and false-negative molecule analysis in the yeast two-hybrid system.

    Science.gov (United States)

    Maier, Richard H; Maier, Christina J; Hintner, Helmut; Bauer, Johann W; Onder, Kamil

    2012-12-01

    Many functional proteomic experiments make use of high-throughput technologies such as mass spectrometry combined with two-dimensional polyacrylamide gel electrophoresis and the yeast two-hybrid (Y2H) system. Currently there are even automated versions of the Y2H system available that can be used for proteome-wide research. The Y2H system has the capacity to deliver a profusion of Y2H positive colonies from a single library screen. However, subsequent analysis of these numerous primary candidates with complementary methods can be overwhelming. Therefore, a method to select the most promising candidates with strong interaction properties might be useful to reduce the number of candidates requiring further analysis. The method described here offers a new way of quantifying and rating the performance of positive Y2H candidates. The novelty lies in the detection and measurement of mRNA expression instead of proteins or conventional Y2H genetic reporters. This method correlates well with the direct genetic reporter readouts usually used in the Y2H system, and has greater sensitivity for detecting and quantifying protein-protein interactions (PPIs) than the conventional Y2H system, as demonstrated by detection of the Y2H false-negative PPI of RXR/PPARG. Approximately 20% of all proteins are not suitable for the Y2H system, the so-called autoactivators. A further advantage of this method is the possibility to evaluate molecules that usually cannot be analyzed in the Y2H system, exemplified by a VDR-LXXLL motif peptide interaction. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. Analysis of mass spectrometry data in proteomics

    DEFF Research Database (Denmark)

    Matthiesen, Rune; Jensen, Ole N

    2008-01-01

    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...... 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...... some of the basic concepts and current approaches to the analysis of MS and MS/MS data in proteomics....

  12. Flor Yeast: New Perspectives Beyond Wine Aging

    Science.gov (United States)

    Legras, Jean-Luc; Moreno-Garcia, Jaime; Zara, Severino; Zara, Giacomo; Garcia-Martinez, Teresa; Mauricio, Juan C.; Mannazzu, Ilaria; Coi, Anna L.; Bou Zeidan, Marc; Dequin, Sylvie; Moreno, Juan; Budroni, Marilena

    2016-01-01

    The most important dogma in white-wine production is the preservation of the wine aroma and the limitation of the oxidative action of oxygen. In contrast, the aging of Sherry and Sherry-like wines is an aerobic process that depends on the oxidative activity of flor strains of Saccharomyces cerevisiae. Under depletion of nitrogen and fermentable carbon sources, these yeast produce aggregates of floating cells and form an air–liquid biofilm on the wine surface, which is also known as velum or flor. This behavior is due to genetic and metabolic peculiarities that differentiate flor yeast from other wine yeast. This review will focus first on the most updated data obtained through the analysis of flor yeast with -omic tools. Comparative genomics, proteomics, and metabolomics of flor and wine yeast strains are shedding new light on several features of these special yeast, and in particular, they have revealed the extent of proteome remodeling imposed by the biofilm life-style. Finally, new insights in terms of promotion and inhibition of biofilm formation through small molecules, amino acids, and di/tri-peptides, and novel possibilities for the exploitation of biofilm immobilization within a fungal hyphae framework, will be discussed. PMID:27148192

  13. Quantitative proteomics by 2DE and MALDI MS/MS uncover the effects of organic and conventional cropping methods on vegetable products

    DEFF Research Database (Denmark)

    Nawrocki, Arkadiusz; Thorup-Kristensen, Kristian; Jensen, Ole Nørregaard

    2011-01-01

    overexpressed in conventionally grown cabbage. Proteins involved in metabolism of carbohydrates, polypeptides and secondary metabolites were affected by different cropping regimes in carrots. The proteomes of conventionally grown vegetables varied from organically grown vegetables to a larger extent than...... of slurry, in accordance to regulations of organic farming and O2, in which nutrient supply was based mainly on autumn green manures. Proteins were extracted from lyophilized plant tissues into a buffer containing high concentrations of urea/thiourea, two detergents and reducing agent. This approach allowed...... short handling times of fresh plant materials. In the case of cabbage samples, the abundance levels of 58 out of more than 1300 quantified protein spots varied significantly between conventional farming and any of the organic cropping systems. Proteome profiles were also very similar between carrot root...

  14. Genomes to Proteomes

    Energy Technology Data Exchange (ETDEWEB)

    Panisko, Ellen A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Grigoriev, Igor [USDOE Joint Genome Inst., Walnut Creek, CA (United States); Daly, Don S. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Webb-Robertson, Bobbie-Jo [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Baker, Scott E. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2009-03-01

    annotation and the generation of high quality gene models, the setup and execution of global proteomics experiments that are quantitative and statistically rigorous and finally add biological context to proteomics.

  15. The Seed Proteome Web Portal

    Directory of Open Access Journals (Sweden)

    Marc eGalland

    2012-06-01

    Full Text Available The Seed Proteome Web Portal (SPWP; http://www.seedproteome.com/ gives access to information both on quantitative seed proteomic data and on seed-related protocols. Firstly, the SPWP provides access to the 475 different Arabidopsis seed proteins annotated from 2 dimensional electrophoresis (2DE maps. Quantitative data are available for each protein according to their accumulation profile during the germination process. These proteins can be retrieved either in list format or directly on scanned 2DE maps. These proteomic data reveal that 40% of seed proteins maintain a stable abundance over germination, up to radicle protrusion. During sensu stricto germination (24 h upon imbibition about 50% of the proteins display quantitative variations, exhibiting an increased abundance (35% or a decreasing abundance (15%. Moreover, during radicle protrusion (24 h to 48 h upon imbibition, 41% proteins display quantitative variations with an increased (23% or a decreasing abundance (18%. In addition, an analysis of the seed proteome revealed the importance of protein post-translational modifications as demonstrated by the poor correlation (r2 = 0.29 between the theoretical (predicted from Arabidopsis genome and the observed protein isoelectric points. Secondly, the SPWP is a relevant technical resource for protocols specifically dedicated to Arabidopsis seed proteome studies. Concerning 2D electrophoresis, the user can find efficient procedures for sample preparation, electrophoresis coupled with gel analysis and protein identification by mass spectrometry, which we have routinely used during the last 12 years. Particular applications such as the detection of oxidized proteins or de novo synthetized proteins radiolabeled by [35S]-methionine are also given in great details. Future developments of this portal will include proteomic data from studies such as dormancy release and protein turnover through de novo protein synthesis analyses during germination.

  16. Biomarker discovery in low-grade breast cancer using isobaric stable isotope tags and two-dimensional liquid chromatography-tandem mass spectrometry (iTRAQ-2DLC-MS/MS) based quantitative proteomic analysis.

    Science.gov (United States)

    Bouchal, Pavel; Roumeliotis, Theodoros; Hrstka, Roman; Nenutil, Rudolf; Vojtesek, Borivoj; Garbis, Spiros D

    2009-01-01

    The present pilot study constitutes a proof-of-principle in the use of a quantitative LC-MS/MS based proteomic method for the comparative analysis of representative low-grade breast primary tumor tissues with and without metastases and metastasis in lymph node relative to the nonmetastatic tumor type. The study method incorporated iTRAQ stable isotope labeling, two-dimensional liquid chromatography, nanoelectrospray ionization and high resolution tandem mass spectrometry using the hybrid QqTOF platform (iTRAQ-2DLC-MS/MS). The principal aims of this study were (1) to define the protein spectrum obtainable using this approach, and (2) to highlight potential candidates for verification and validation studies focused on biomarkers involved in metastatic processes in breast cancer. The study resulted in the reproducible identification of 605 nonredundant proteins (p biomarker discovery program.

  17. Comparative Proteomic Profiling of Mycobacterium bovis and BCG Vaccine Strains

    KAUST Repository

    Gao, Ge

    2013-01-01

    , Danish, Phipps and Birkhaug by Tandem Mass Tag® (TMT®)-labeling quantitative proteomic approach. In total, 420 proteins were identified and 377 of them were quantitated for their relative abundance. We reported the number and relationship of differential

  18. Quantitative characterization of pyrimidine dimer excision from UV-irradiated DNA (excision capacity) by cell-free extracts of the yeast Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Bekker, M.L.; Kaboev, O.K.; Akhmedov, A.T.; Luchkina, L.A.

    1984-01-01

    Cell-free extracts from wild-type yeast (RAD + ) and from rad mutants belonging to the RAD3 epistatic group (rad1-1, rad2-1, rad3-1, rad4-1) contain activities catalyzing the excision of pyrimidine dimers (PD) from purified ultraviolet-irradiated DNA which was not pre-treated with exogenous UV-endonuclease. The level of these activities in cell-free extracts from rad mutants did not differ from that in wild-type extract and was close to the in vivo excision capacity of the latter calculated from the LD 37 (about 10 4 PD per haploid genome). (Auth.)

  19. A hybrid approach to protein differential expression in mass spectrometry-based proteomics

    KAUST Repository

    Wang, X.; Anderson, G. A.; Smith, R. D.; Dabney, A. R.

    2012-01-01

    MOTIVATION: Quantitative mass spectrometry-based proteomics involves statistical inference on protein abundance, based on the intensities of each protein's associated spectral peaks. However, typical MS-based proteomics datasets have substantial

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

    Science.gov (United States)

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

    2017-12-01

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

  1. Effect of in vitro digested cod liver oil of different quality on oxidative, proteomic and inflammatory responses in the yeast Saccharomyces cerevisiae and human monocyte-derived dendritic cells

    DEFF Research Database (Denmark)

    Larsson, Karin; Istenič, Katja; Wulff, Tune

    2015-01-01

    BACKGROUND: Upon oxidation of the polyunsaturated fatty acids in fish oil, either before ingestion or, as recently shown, during the gastro-intestinal passage, a cascade of potentially cytotoxic peroxidation products, such as malondialdehyde and 4-hydroxy-2-hexenal, can form. In this study, we di...... in yeast and immunomodulation of dendritic cells....

  2. Yeast Genomics for Bread, Beer, Biology, Bucks and Breath

    Science.gov (United States)

    Sakharkar, Kishore R.; Sakharkar, Meena K.

    The rapid advances and scale up of projects in DNA sequencing dur ing the past two decades have produced complete genome sequences of several eukaryotic species. The versatile genetic malleability of the yeast, and the high degree of conservation between its cellular processes and those of human cells have made it a model of choice for pioneering research in molecular and cell biology. The complete sequence of yeast genome has proven to be extremely useful as a reference towards the sequences of human and for providing systems to explore key gene functions. Yeast has been a ‘legendary model’ for new technologies and gaining new biological insights into basic biological sciences and biotechnology. This chapter describes the awesome power of yeast genetics, genomics and proteomics in understanding of biological function. The applications of yeast as a screening tool to the field of drug discovery and development are highlighted and the traditional importance of yeast for bakers and brewers is discussed.

  3. Efficient Isolation and Quantitative Proteomic Analysis of Cancer Cell Plasma Membrane Proteins for Identification of Metastasis-Associated Cell Surface Markers

    DEFF Research Database (Denmark)

    Lund, Rikke; Leth-Larsen, Rikke; Jensen, Ole N

    2009-01-01

    Cell surface membrane proteins are involved in central processes such as cell signaling, cell-cell interactions, ion and solute transport, and they seem to play a pivotal role in several steps of the metastatic process of cancer cells. The low abundance and hydrophobic nature of cell surface...... membrane proteins complicate their purification and identification by MS. We used two isogenic cell lines with opposite metastatic capabilities in nude mice to optimize cell surface membrane protein purification and to identify potential novel markers of metastatic cancer. The cell surface membrane...... proteins were isolated by centrifugation/ultracentrifugation steps, followed by membrane separation using a Percoll/sucrose density gradient. The gradient fractions containing the cell surface membrane proteins were identified by enzymatic assays. Stable isotope labeling of the proteome of the metastatic...

  4. Quantitative proteomics and transcriptomics reveals metabolic differences in attracting and non-attracting human-in-mouse glioma stem cell xenografts and stromal cells

    Directory of Open Access Journals (Sweden)

    Norelle C. Wildburger

    2015-09-01

    Full Text Available Bone marrow-derived human mesenchymal stem cells (BM-hMSCs show promise as cell-based delivery vehicles for anti-glioma therapeutics, due to innate tropism for gliomas. However, in clinically relevant human-in-mouse glioma stem cell xenograft models, BM-hMSCs tropism is variable. We compared the proteomic profile of cancer and stromal cells in GSCXs that attract BM-hMSCs (“attractors” with those to do not (“non-attractors” to identify pathways that may modulate BM-hMSC homing, followed by targeted transcriptomics. The results provide the first link between fatty acid metabolism, glucose metabolism, ROS, and N-glycosylation patterns in attractors. Reciprocal expression of these pathways in the stromal cells suggests microenvironmental cross-talk.

  5. iTRAQ quantitative proteomics-based identification of cell adhesion as a dominant phenotypic modulation in thrombin-stimulated human aortic endothelial cells.

    Science.gov (United States)

    Wang, Huang-Joe; Chen, Sung-Fang; Lo, Wan-Yu

    2015-05-01

    The phenotypic changes in thrombin-stimulated endothelial cells include alterations in permeability, cell shape, vasomotor tone, leukocyte trafficking, migration, proliferation, and angiogenesis. Previous studies regarding the pleotropic effects of thrombin on the endothelium used human umbilical vein endothelial cells (HUVECs)-cells derived from fetal tissue that does not exist in adults. Only a few groups have used screening approaches such as microarrays to profile the global effects of thrombin on endothelial cells. Moreover, the proteomic changes of thrombin-stimulated human aortic endothelial cells (HAECs) have not been elucidated. HAECs were stimulated with 2 units/mL thrombin for 5h and their proteome was investigated using isobaric tags for the relative and absolute quantification (iTRAQ) and the MetaCore(TM) software. A total of 627 (experiment A) and 622 proteins (experiment B) were quantified in the duplicated iTRAQ analyses. MetaCore(TM) pathway analysis identified cell adhesion as a dominant phenotype in thrombin-stimulated HAECs. Replicated iTRAQ data revealed that "Cell adhesion_Chemokines and adhesion," "Cell adhesion_Histamine H1 receptor signaling in the interruption of cell barrier integrity," and "Cell adhesion_Integrin-mediated cell adhesion and migration" were among the top 10 statistically significant pathways. The cell adhesion phenotype was verified by increased THP-1 adhesion to thrombin-stimulated HAECs. In addition, the expression of ICAM-1, VCAM-1, and SELE was significantly upregulated in thrombin-stimulated HAECs. Several regulatory pathways are altered in thrombin-stimulated HAECs, with cell adhesion being the dominant altered phenotype. Our findings show the feasibility of the iTRAQ technique for evaluating cellular responses to acute stimulation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Quantitative proteome-level analysis of paulownia witches’ broom disease with methyl methane sulfonate assistance reveals diverse metabolic changes during the infection and recovery processes

    Directory of Open Access Journals (Sweden)

    Zhe Wang

    2017-07-01

    Full Text Available Paulownia witches’ broom (PaWB disease caused by phytoplasma is a fatal disease that leads to considerable economic losses. Although there are a few reports describing studies of PaWB pathogenesis, the molecular mechanisms underlying phytoplasma pathogenicity in Paulownia trees remain uncharacterized. In this study, after building a transcriptome database containing 67,177 sequences, we used isobaric tags for relative and absolute quantification (iTRAQ to quantify and analyze the proteome-level changes among healthy P. fortunei (PF, PaWB-infected P. fortunei (PFI, and PaWB-infected P. fortunei treated with 20 mg L−1 or 60 mg L−1 methyl methane sulfonate (MMS (PFI-20 and PFI-60, respectively. A total of 2,358 proteins were identified. We investigated the proteins profiles in PF vs. PFI (infected process and PFI-20 vs. PFI-60 (recovered process, and further found that many of the MMS-response proteins mapped to “photosynthesis” and “ribosome” pathways. Based on our comparison scheme, 36 PaWB-related proteins were revealed. Among them, 32 proteins were classified into three functional groups: (1 carbohydrate and energy metabolism, (2 protein synthesis and degradation, and (3 stress resistance. We then investigated the PaWB-related proteins involved in the infected and recovered processes, and discovered that carbohydrate and energy metabolism was inhibited, and protein synthesis and degradation decreased, as the plant responded to PaWB. Our observations may be useful for characterizing the proteome-level changes that occur at different stages of PaWB disease. The data generated in this study may serve as a valuable resource for elucidating the pathogenesis of PaWB disease during phytoplasma infection and recovery stages.

  7. Quantitative proteomics reveals new insights into calcium-mediated resistance mechanisms in Aspergillus flavus against the antifungal protein PgAFP in cheese.

    Science.gov (United States)

    Delgado, Josué; Owens, Rebecca A; Doyle, Sean; Núñez, Félix; Asensio, Miguel A

    2017-09-01

    The ability of Aspergillus flavus to produce aflatoxins in dairy products presents a potential hazard. The antifungal protein PgAFP from Penicillium chrysogenum inhibits various foodborne toxigenic fungi, including Aspergillus flavus. However, PgAFP did not inhibit A. flavus growth in cheese, which was related to the associated cation content. CaCl 2 increased A. flavus permeability and prevented PgAFP-mediated inhibition in potato dextrose broth (PDB). PgAFP did not elicit any additional increase in permeability of CaCl 2 -incubated A. flavus. Furthermore, PgAFP did not alter metabolic capability, chitin deposition, or hyphal viability of A. flavus grown with CaCl 2 . Comparative proteomic analysis after PgAFP treatment of A. flavus in calcium-enriched PDB revealed increased abundance of 125 proteins, including oxidative stress-related proteins, as determined by label-free mass spectrometry (MS)-based proteomics. Seventy proteins were found at lower abundance, with most involved in metabolic pathways and biosynthesis of secondary metabolites. These changes do not support the blockage of potential PgAFP receptors in A. flavus by calcium as the main cause of the protective role. A. flavus resistance appears to be mediated by calcineurin, G-protein, and γ-glutamyltranspeptidase that combat oxidative stress and impede apoptosis. These findings could serve to design strategies to improve PgAFP activity against aflatoxigenic moulds in dairy products. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Comparison of a Label-Free Quantitative Proteomic Method Based on Peptide Ion Current Area to the Isotope Coded Affinity Tag Method

    Directory of Open Access Journals (Sweden)

    Young Ah Goo

    2008-01-01

    Full Text Available Recently, several research groups have published methods for the determination of proteomic expression profiling by mass spectrometry without the use of exogenously added stable isotopes or stable isotope dilution theory. These so-called label-free, methods have the advantage of allowing data on each sample to be acquired independently from all other samples to which they can later be compared in silico for the purpose of measuring changes in protein expression between various biological states. We developed label free software based on direct measurement of peptide ion current area (PICA and compared it to two other methods, a simpler label free method known as spectral counting and the isotope coded affinity tag (ICAT method. Data analysis by these methods of a standard mixture containing proteins of known, but varying, concentrations showed that they performed similarly with a mean squared error of 0.09. Additionally, complex bacterial protein mixtures spiked with known concentrations of standard proteins were analyzed using the PICA label-free method. These results indicated that the PICA method detected all levels of standard spiked proteins at the 90% confidence level in this complex biological sample. This finding confirms that label-free methods, based on direct measurement of the area under a single ion current trace, performed as well as the standard ICAT method. Given the fact that the label-free methods provide ease in experimental design well beyond pair-wise comparison, label-free methods such as our PICA method are well suited for proteomic expression profiling of large numbers of samples as is needed in clinical analysis.

  9. Maillard Proteomics: Opening New Pages

    Directory of Open Access Journals (Sweden)

    Alena Soboleva

    2017-12-01

    Full Text Available Protein glycation is a ubiquitous non-enzymatic post-translational modification, formed by reaction of protein amino and guanidino groups with carbonyl compounds, presumably reducing sugars and α-dicarbonyls. Resulting advanced glycation end products (AGEs represent a highly heterogeneous group of compounds, deleterious in mammals due to their pro-inflammatory effect, and impact in pathogenesis of diabetes mellitus, Alzheimer’s disease and ageing. The body of information on the mechanisms and pathways of AGE formation, acquired during the last decades, clearly indicates a certain site-specificity of glycation. It makes characterization of individual glycation sites a critical pre-requisite for understanding in vivo mechanisms of AGE formation and developing adequate nutritional and therapeutic approaches to reduce it in humans. In this context, proteomics is the methodology of choice to address site-specific molecular changes related to protein glycation. Therefore, here we summarize the methods of Maillard proteomics, specifically focusing on the techniques providing comprehensive structural and quantitative characterization of glycated proteome. Further, we address the novel break-through areas, recently established in the field of Maillard research, i.e., in vitro models based on synthetic peptides, site-based diagnostics of metabolism-related diseases (e.g., diabetes mellitus, proteomics of anti-glycative defense, and dynamics of plant glycated proteome during ageing and response to environmental stress.

  10. Quantitative Proteomics Analysis of VEGF-Responsive Endothelial Protein S-Nitrosylation Using Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and LC-MS/MS1

    Science.gov (United States)

    Zhang, Hong-Hai; Lechuga, Thomas J.; Chen, Yuezhou; Yang, Yingying; Huang, Lan; Chen, Dong-Bao

    2016-01-01

    Adduction of a nitric oxide moiety (NO•) to cysteine(s), termed S-nitrosylation (SNO), is a novel mechanism for NO to regulate protein function directly. However, the endothelial SNO-protein network that is affected by endogenous and exogenous NO is obscure. This study was designed to develop a quantitative proteomics approach using stable isotope labeling by amino acids in cell culture for comparing vascular endothelial growth factor (VEGFA)- and NO donor-responsive endothelial nitroso-proteomes. Primary placental endothelial cells were labeled with “light” (L-12C614N4-Arg and L-12C614N2-Lys) or “heavy” (L-13C615N4-Arg and L-13C615N2-Lys) amino acids. The light cells were treated with an NO donor nitrosoglutathione (GSNO, 1 mM) or VEGFA (10 ng/ml) for 30 min, while the heavy cells received vehicle as control. Equal amounts of cellular proteins from the light (GSNO or VEGFA treated) and heavy cells were mixed for labeling SNO-proteins by the biotin switch technique and then trypsin digested. Biotinylated SNO-peptides were purified for identifying SNO-proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Ratios of light to heavy SNO-peptides were calculated for determining the changes of the VEGFA- and GSNO-responsive endothelial nitroso-proteomes. A total of 387 light/heavy pairs of SNO-peptides were identified, corresponding to 213 SNO-proteins that include 125 common and 27 VEGFA- and 61 GSNO-responsive SNO-proteins. The specific SNO-cysteine(s) in each SNO-protein were simultaneously identified. Pathway analysis revealed that SNO-proteins are involved in various endothelial functions, including proliferation, motility, metabolism, and protein synthesis. We collectively conclude that endogenous NO on VEGFA stimulation and exogenous NO from GSNO affect common and different SNO-protein networks, implicating SNO as a critical mechanism for VEGFA stimulation of angiogenesis. PMID:27075618

  11. Prediction of skin anti-aging clinical benefits of an association of ingredients from marine and maritime origins: Ex vivo evaluation using a label-free quantitative proteomic and customized data processing approach.

    Science.gov (United States)

    Hameury, Sebastien; Borderie, Laurent; Monneuse, Jean-Marc; Skorski, Gilbert; Pradines, Dominique

    2018-05-23

    The application of ingredients from marine and maritime origins is increasingly common in skin care products, driven by consumer expectations for natural ingredients. However, these ingredients are typically studied for a few isolated in vitro activities. The purpose of this study was to carry out a comprehensive evaluation of the activity on the skin of an association of ingredients from marine and maritime origins using label-free quantitative proteomic analysis, in order to predict the clinical benefits if used in a skin care product. An aqueous gel containing 6.1% of ingredients from marine and maritime origins (amino acid-enriched giant kelp extract, trace element-enriched seawater, dedifferentiated sea fennel cells) was topically applied on human skin explants. The skin explants' proteome was analyzed in a label-free manner by high-performance liquid nano-chromatography coupled with tandem mass spectrometry. A specific data processing pipeline (CORAVALID) providing an objective and comprehensive interpretation of the statistically relevant biological activities processed the results. Compared to untreated skin explants, 64 proteins were significantly regulated by the gel treatment (q-value ≤ 0.05). Computer data processing revealed an activity of the ingredients on the epidermis and the dermis. These significantly regulated proteins are involved in gene expression, cell survival and metabolism, inflammatory processes, dermal extracellular matrix synthesis, melanogenesis and keratinocyte proliferation, migration, and differentiation. These results suggest that the tested ingredients could help to preserve a healthy epidermis and dermis, and possibly to prevent the visible signs of skin aging. © 2018 The Authors. Journal of Cosmetic Dermatology Published by Wiley Periodicals, Inc.

  12. Comprehensive proteomic analysis of human pancreatic juice

    DEFF Research Database (Denmark)

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

    2004-01-01

    Proteomic technologies provide an excellent means for analysis of body fluids for cataloging protein constituents and identifying biomarkers for early detection of cancers. The biomarkers currently available for pancreatic cancer, such as CA19-9, lack adequate sensitivity and specificity...... contributing to late diagnosis of this deadly disease. In this study, we carried out a comprehensive characterization of the "pancreatic juice proteome" in patients with pancreatic adenocarcinoma. Pancreatic juice was first fractionated by 1-dimensional gel electrophoresis and subsequently analyzed by liquid...... in this study could be directly assessed for their potential as biomarkers for pancreatic cancer by quantitative proteomics methods or immunoassays....

  13. Reconstructing the regulatory circuit of cell fate determination in yeast mating response.

    Science.gov (United States)

    Shao, Bin; Yuan, Haiyu; Zhang, Rongfei; Wang, Xuan; Zhang, Shuwen; Ouyang, Qi; Hao, Nan; Luo, Chunxiong

    2017-07-01

    Massive technological advances enabled high-throughput measurements of proteomic changes in biological processes. However, retrieving biological insights from large-scale protein dynamics data remains a challenging task. Here we used the mating differentiation in yeast Saccharomyces cerevisiae as a model and developed integrated experimental and computational approaches to analyze the proteomic dynamics during the process of cell fate determination. When exposed to a high dose of mating pheromone, the yeast cell undergoes growth arrest and forms a shmoo-like morphology; however, at intermediate doses, chemotropic elongated growth is initialized. To understand the gene regulatory networks that control this differentiation switch, we employed a high-throughput microfluidic imaging system that allows real-time and simultaneous measurements of cell growth and protein expression. Using kinetic modeling of protein dynamics, we classified the stimulus-dependent changes in protein abundance into two sources: global changes due to physiological alterations and gene-specific changes. A quantitative framework was proposed to decouple gene-specific regulatory modes from the growth-dependent global modulation of protein abundance. Based on the temporal patterns of gene-specific regulation, we established the network architectures underlying distinct cell fates using a reverse engineering method and uncovered the dose-dependent rewiring of gene regulatory network during mating differentiation. Furthermore, our results suggested a potential crosstalk between the pheromone response pathway and the target of rapamycin (TOR)-regulated ribosomal biogenesis pathway, which might underlie a cell differentiation switch in yeast mating response. In summary, our modeling approach addresses the distinct impacts of the global and gene-specific regulation on the control of protein dynamics and provides new insights into the mechanisms of cell fate determination. We anticipate that our

  14. Quantitative proteomics unravels that the post-transcriptional regulator Crc modulates the generation of vesicles and secreted virulence determinants of Pseudomonas aeruginosa.

    Science.gov (United States)

    Reales-Calderón, Jose Antonio; Corona, Fernando; Monteoliva, Lucía; Gil, Concha; Martínez, Jose Luis

    2015-09-08

    Recent research indicates that the post-transcriptional regulator Crc modulates susceptibility to antibiotics and virulence in Pseudomonas aeruginosa. Several P. aeruginosa virulence factors are secreted or engulfed in vesicles. To decipher the Crc modulation of P. aeruginosa virulence, we constructed a crc deficient mutant and measure the proteome associated extracellular vesicles and the vesicle-free secretome using iTRAQ. Fifty vesicle-associated proteins were more abundant and 14 less abundant in the crc-defective strain, whereas 37 were more abundant and 17 less abundant in the vesicle-free secretome. Among them, virulence determinants, such as ToxA, protease IV, azurin, chitin-binding protein, PlcB and Hcp1, were less abundant in the crc-defective mutant. Transcriptomic analysis revealed that some of the observed changes were post-transcriptional and, thus, could be attributed to a direct Crc regulatory role; whereas, for other differentially secreted proteins, the regulatory role was likely indirect. We also observed that the crc mutant presented an impaired vesicle-associated secretion of quorum sensing signal molecules and less cytotoxicity than its wild-type strain. Our results offer new insights into the mechanisms by which Crc regulates P. aeruginosa virulence, through the modulation of vesicle formation and secretion of both virulence determinants and quorum sensing signals. This article is part of a Special Issue entitled: HUPO 2014. Published by Elsevier B.V.

  15. Quantitative Analysis of the Proteome Response to the Histone Deacetylase Inhibitor (HDACi) Vorinostat in Niemann-Pick Type C1 disease.

    Science.gov (United States)

    Subramanian, Kanagaraj; Rauniyar, Navin; Lavalleé-Adam, Mathieu; Yates, John R; Balch, William E

    2017-11-01

    Niemann-Pick type C (NPC) disease is an inherited, progressive neurodegenerative disorder principally caused by mutations in the NPC1 gene. NPC disease is characterized by the accumulation of unesterified cholesterol in the late endosomes (LE) and lysosomes (Ly) (LE/Ly). Vorinostat, a histone deacetylase inhibitor (HDACi), restores cholesterol homeostasis in fibroblasts derived from NPC patients; however, the exact mechanism by which Vorinostat restores cholesterol level is not known yet. In this study, we performed comparative proteomic profiling of the response of NPC1 I1061T fibroblasts to Vorinostat. After stringent statistical criteria to filter identified proteins, we observed 202 proteins that are differentially expressed in Vorinostat-treated fibroblasts. These proteins are members of diverse cellular pathways including the endomembrane dependent protein folding-stability-degradation-trafficking axis, energy metabolism, and lipid metabolism. Our study shows that treatment of NPC1 I1061T fibroblasts with Vorinostat not only enhances pathways promoting the folding, stabilization and trafficking of NPC1 (I1061T) mutant to the LE/Ly, but alters the expression of lysosomal proteins, specifically the lysosomal acid lipase (LIPA) involved in the LIPA->NPC2->NPC1 based flow of cholesterol from the LE/Ly lumen to the LE/Ly membrane. We posit that the Vorinostat may modulate numerous pathways that operate in an integrated fashion through epigenetic and post-translational modifications reflecting acetylation/deacetylation balance to help manage the defective NPC1 fold, the function of the LE/Ly system and/or additional cholesterol metabolism/distribution pathways, that could globally contribute to improved mitigation of NPC1 disease in the clinic based on as yet uncharacterized principles of cellular metabolism dictating cholesterol homeostasis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Improving the phenotype predictions of a yeast genome-scale metabolic model by incorporating enzymatic constraints

    DEFF Research Database (Denmark)

    Sanchez, Benjamin J.; Zhang, Xi-Cheng; Nilsson, Avlant

    2017-01-01

    , which act as limitations on metabolic fluxes, are not taken into account. Here, we present GECKO, a method that enhances a GEM to account for enzymes as part of reactions, thereby ensuring that each metabolic flux does not exceed its maximum capacity, equal to the product of the enzyme's abundance...... and turnover number. We applied GECKO to a Saccharomyces cerevisiae GEM and demonstrated that the new model could correctly describe phenotypes that the previous model could not, particularly under high enzymatic pressure conditions, such as yeast growing on different carbon sources in excess, coping...... with stress, or overexpressing a specific pathway. GECKO also allows to directly integrate quantitative proteomics data; by doing so, we significantly reduced flux variability of the model, in over 60% of metabolic reactions. Additionally, the model gives insight into the distribution of enzyme usage between...

  17. Proteome stability analysis of snap frozen, RNAlater preserved, and formalin-fixed paraffin-embedded human colon mucosal biopsies

    DEFF Research Database (Denmark)

    Bennike, Tue Bjerg; Kastaniegaard, Kenneth; Padurariu, Simona

    2016-01-01

    Large repositories of well characterized RNAlater preserved samples and formalin-fixed, paraffin-embedded samples have been generated worldwide. However, the impact on the proteome of the preservation methods remain poorly described. Therefore, we analyzed the impact on the proteome of preserving...... throughput gel free quantitative proteomics. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE: PXD002029....

  18. Mass Spectrometry Instrumentation in Proteomics

    DEFF Research Database (Denmark)

    Sprenger, Richard Remko; Roepstorff, Peter

    2012-01-01

    Mass spectrometry has evolved into a crucial technology for the field of proteomics, enabling the comprehensive study of proteins in biological systems. Innovative developments have yielded flexible and versatile mass spectrometric tools, including quadrupole time-of-flight, linear ion trap......, Orbitrap and ion mobility instruments. Together they offer various and complementary capabilities in terms of ionization, sensitivity, speed, resolution, mass accuracy, dynamic range and methods of fragmentation. Mass spectrometers can acquire qualitative and quantitative information on a large scale...

  19. Tissue-based map of the human proteome

    DEFF Research Database (Denmark)

    Uhlén, Mathias; Fagerberg, Linn; Hallström, Björn M.

    2015-01-01

    Resolving the molecular details of proteome variation in the different tissues and organs of the human body will greatly increase our knowledge of human biology and disease. Here, we present a map of the human tissue proteome based on an integrated omics approach that involves quantitative transc...

  20. Quantitative proteomics identifies altered O-GlcNAcylation of structural, synaptic and memory-associated proteins in Alzheimer's disease: Brain protein O-GlcNAcylation in Alzheimer's disease

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Sheng [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA USA; Yang, Feng [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA USA; Petyuk, Vladislav A. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA USA; Shukla, Anil K. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA USA; Monroe, Matthew E. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA USA; Gritsenko, Marina A. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA USA; Rodland, Karin D. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA USA; Smith, Richard D. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA USA; Qian, Wei-Jun [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA USA; Gong, Cheng-Xin [New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York USA; Liu, Tao [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA USA

    2017-07-28

    Protein modification by O-linked beta-N-acetylglucosamine (O-GlcNAc) is emerging as an important factor in the pathogenesis of sporadic Alzheimer’s disease. Herein we report the most comprehensive, quantitative proteomics analysis for protein O-GlcNAcylation in post-mortem human brains with and without Alzheimer’s using isobaric tandem mass tags labeling, chemoenzymatic photocleavage enrichment and liquid chromatography coupled to mass spectrometry. A total of 1,850 O-GlcNAc peptides covering 1,094 O-GlcNAcylation sites were identified from 530 proteins in the human brain. 128 O-GlcNAc peptides covering 78 proteins were altered significantly in Alzheimer’s brain as compared to controls (q<0.05). Moreover, alteration of the O-GlcNAc peptide abundance could be attributed more to O-GlcNAcylation level than to protein level changes. The altered O-GlcNAcylated proteins belong to several structural and functional categories, including synaptic proteins, cytoskeleton proteins, and memory-associated proteins. These findings suggest that dysregulation of O-GlcNAcylation of multiple brain proteins may be involved in the development of sporadic Alzheimer’s disease.

  1. Study of the role of the covalently linked cell wall protein (Ccw14p) and yeast glycoprotein (Ygp1p) within biofilm formation in a flor yeast strain.

    Science.gov (United States)

    Moreno-García, J; Coi, A L; Zara, G; García-Martínez, T; Mauricio, J C; Budroni, M

    2018-03-01

    Flor yeasts are Saccharomyces cerevisiae strains noted by their ability to create a type of biofilm in the air-liquid interface of some wines, known as 'flor' or 'velum', for which certain proteins play an essential role. Following a proteomic study of a flor yeast strain, we deleted the CCW14 (covalently linked cell wall protein) and YGP1 (yeast glycoprotein) genes-codifying for two cell surface glycoproteins-in a haploid flor yeast strain and we reported that both influence the weight of the biofilm as well as cell adherence (CCW14).

  2. High-resolution proteomic profiling of spider venom: expanding the toxin diversity of Phoneutria nigriventer venom.

    Science.gov (United States)

    Liberato, Tarcísio; Troncone, Lanfranco Ranieri Paolo; Yamashiro, Edson T; Serrano, Solange M T; Zelanis, André

    2016-03-01

    Here we present a proteomic characterization of Phoneutria nigriventer venom. A shotgun proteomic approach allowed the identification, for the first time, of O-glycosyl hydrolases (chitinases) in P. nigriventer venom. The electrophoretic profiles under nonreducing and reducing conditions, and protein identification by mass spectrometry, indicated the presence of oligomeric toxin structures in the venom. Complementary proteomic approaches allowed for a qualitative and semi-quantitative profiling of P. nigriventer venom complexity, expanding its known venom proteome diversity.

  3. Prions in yeast

    OpenAIRE

    Bezdíčka, Martin

    2013-01-01

    The thesis describes yeast prions and their biological effects on yeast in general. It defines the basic characteristics of yeast prions, that distinguish prions from other proteins. The thesis introduces various possibilities of prion formation, and propagation as well as specific types of yeast prions, including various functions of most studied types of prions. The thesis also focuses on chaperones that affect the state of yeast prions in cells. Lastly, the thesis indicates similarities be...

  4. Mining the granule proteome

    DEFF Research Database (Denmark)

    Albrethsen, Jakob; Goetze, Jens P; Johnsen, Anders H

    2015-01-01

    Proteomics of secretory granules is an emerging strategy for identifying secreted proteins, including potentially novel candidate biomarkers and peptide hormones. In addition, proteomics can provide information about the abundance, localization and structure (post-translational modification) of g...

  5. Quantitative Proteome Analysis of Mouse Liver Lysosomes Provides Evidence for Mannose 6-phosphate-independent Targeting Mechanisms of Acid Hydrolases in Mucolipidosis II.

    Science.gov (United States)

    Markmann, Sandra; Krambeck, Svenja; Hughes, Christopher J; Mirzaian, Mina; Aerts, Johannes M F G; Saftig, Paul; Schweizer, Michaela; Vissers, Johannes P C; Braulke, Thomas; Damme, Markus

    2017-03-01

    The efficient receptor-mediated targeting of soluble lysosomal proteins to lysosomes requires the modification with mannose 6-phosphate (M6P) residues. Although the absence of M6P results in misrouting and hypersecretion of lysosomal enzymes in many cells, normal levels of lysosomal enzymes have been reported in liver of patients lacking the M6P-generating phosphotransferase (PT). The identity of lysosomal proteins depending on M6P has not yet been comprehensively analyzed. In this study we purified lysosomes from liver of PT-defective mice and 67 known soluble lysosomal proteins were identified that illustrated quantitative changes using an ion mobility-assisted data-independent label-free LC-MS approach. After validation of various differentially expressed lysosomal components by Western blotting and enzyme activity assays, the data revealed a small number of lysosomal proteins depending on M6P, including neuraminidase 1, cathepsin F, Npc2, and cathepsin L, whereas the majority reach lysosomes by alternative pathways. These data were compared with findings on cultured hepatocytes and liver sinusoid endothelial cells isolated from the liver of wild-type and PT-defective mice. Our findings show that the relative expression, targeting efficiency and lysosomal localization of lysosomal proteins tested in cultured hepatic cells resemble their proportion in isolated liver lysosomes. Hypersecretion of newly synthesized nonphosphorylated lysosomal proteins suggest that secretion-recapture mechanisms contribute to maintain major lysosomal functions in liver. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Comparative Proteomic Analysis of Tolerance and Adaptation of Ethanologenic Saccharomyces cerevisiae to Furfural, a Lignocellulosic Inhibitory Compound▿ †

    Science.gov (United States)

    Lin, Feng-Ming; Qiao, Bin; Yuan, Ying-Jin

    2009-01-01

    The molecular mechanism involved in tolerance and adaptation of ethanologenic Saccharomyces cerevisiae to inhibitors (such as furfural, acetic acid, and phenol) represented in lignocellulosic hydrolysate is still unclear. Here, 18O-labeling-aided shotgun comparative proteome analysis was applied to study the global protein expression profiles of S. cerevisiae under conditions of treatment of furfural compared with furfural-free fermentation profiles. Proteins involved in glucose fermentation and/or the tricarboxylic acid cycle were upregulated in cells treated with furfural compared with the control cells, while proteins involved in glycerol biosynthesis were downregulated. Differential levels of expression of alcohol dehydrogenases were observed. On the other hand, the levels of NADH, NAD+, and NADH/NAD+ were reduced whereas the levels of ATP and ADP were increased. These observations indicate that central carbon metabolism, levels of alcohol dehydrogenases, and the redox balance may be related to tolerance of ethanologenic yeast for and adaptation to furfural. Furthermore, proteins involved in stress response, including the unfolded protein response, oxidative stress, osmotic and salt stress, DNA damage and nutrient starvation, were differentially expressed, a finding that was validated by quantitative real-time reverse transcription-PCR to further confirm that the general stress responses are essential for cellular defense against furfural. These insights into the response of yeast to the presence of furfural will benefit the design and development of inhibitor-tolerant ethanologenic yeast by metabolic engineering or synthetic biology. PMID:19363068

  7. Application of mass spectrometry-based proteomics for biomarker discovery in neurological disorders

    Directory of Open Access Journals (Sweden)

    Venugopal Abhilash

    2009-01-01

    Full Text Available Mass spectrometry-based quantitative proteomics has emerged as a powerful approach that has the potential to accelerate biomarker discovery, both for diagnostic as well as therapeutic purposes. Proteomics has traditionally been synonymous with 2D gels but is increasingly shifting to the use of gel-free systems and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS. Quantitative proteomic approaches have already been applied to investigate various neurological disorders, especially in the context of identifying biomarkers from cerebrospinal fluid and serum. This review highlights the scope of different applications of quantitative proteomics in understanding neurological disorders with special emphasis on biomarker discovery.

  8. Quantitative Proteomics of Gut-Derived Th1 and Th1/Th17 Clones Reveal the Presence of CD28+ NKG2D- Th1 Cytotoxic CD4+ T cells.

    Science.gov (United States)

    Riaz, Tahira; Sollid, Ludvig Magne; Olsen, Ingrid; de Souza, Gustavo Antonio

    2016-03-01

    T-helper cells are differentiated from CD4+ T cells and are traditionally characterized by inflammatory or immunosuppressive responses in contrast to cytotoxic CD8+ T cells. Mass-spectrometry studies on T-helper cells are rare. In this study, we aimed to identify the proteomes of human Th1 and Th1/Th17 clones derived from intestinal biopsies of Crohn's disease patients and to identify differentially expressed proteins between the two phenotypes. Crohn's disease is an inflammatory bowel disease, with predominantly Th1- and Th17-mediated response where cells of the "mixed" phenotype Th1/Th17 have also been commonly found. High-resolution mass spectrometry was used for protein identification and quantitation. In total, we identified 7401 proteins from Th1 and Th1/Th17 clones, where 334 proteins were differentially expressed. Major differences were observed in cytotoxic proteins that were overrepresented in the Th1 clones. The findings were validated by flow cytometry analyses using staining with anti-granzyme B and anti-perforin and by a degranulation assay, confirming higher cytotoxic features of Th1 compared with Th1/Th17 clones. By testing a larger panel of T-helper cell clones from seven different Crohn's disease patients, we concluded that only a subgroup of the Th1 cell clones had cytotoxic features, and these expressed the surface markers T-cell-specific surface glycoprotein CD28 and were negative for expression of natural killer group 2 member D. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. [Proteomics and transfusion medicine].

    Science.gov (United States)

    Lion, N; Prudent, M; Crettaz, D; Tissot, J-D

    2011-04-01

    The term "proteomics" covers tools and techniques that are used to analyze and characterize complex mixtures of proteins from various biological samples. In this short review, a typical proteomic approach, related to the study of particular and illustrative situation related to transfusion medicine is reported. This "case report" will allow the reader to be familiar with a practical proteomic approach of a real situation, and will permit to describe the tools that are usually used in proteomic labs, and, in a second part, to present various proteomic applications in transfusion medicine. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  10. Proteomics methods applied to malaria: Plasmodium falciparum

    International Nuclear Information System (INIS)

    Cuesta Astroz, Yesid; Segura Latorre, Cesar

    2012-01-01

    Malaria is a parasitic disease that has a high impact on public health in developing countries. The sequencing of the plasmodium falciparum genome and the development of proteomics have enabled a breakthrough in understanding the biology of the parasite. Proteomics have allowed to characterize qualitatively and quantitatively the parasite s expression of proteins and has provided information on protein expression under conditions of stress induced by antimalarial. Given the complexity of their life cycle, this takes place in the vertebrate host and mosquito vector. It has proven difficult to characterize the protein expression during each stage throughout the infection process in order to determine the proteome that mediates several metabolic, physiological and energetic processes. Two dimensional electrophoresis, liquid chromatography and mass spectrometry have been useful to assess the effects of antimalarial on parasite protein expression and to characterize the proteomic profile of different p. falciparum stages and organelles. The purpose of this review is to present state of the art tools and advances in proteomics applied to the study of malaria, and to present different experimental strategies used to study the parasite's proteome in order to show the advantages and disadvantages of each one.

  11. Population FBA predicts metabolic phenotypes in yeast.

    Directory of Open Access Journals (Sweden)

    Piyush Labhsetwar

    2017-09-01

    Full Text Available Using protein counts sampled from single cell proteomics distributions to constrain fluxes through a genome-scale model of metabolism, Population flux balance analysis (Population FBA successfully described metabolic heterogeneity in a population of independent Escherichia coli cells growing in a defined medium. We extend the methodology to account for correlations in protein expression arising from the co-regulation of genes and apply it to study the growth of independent Saccharomyces cerevisiae cells in two different growth media. We find the partitioning of flux between fermentation and respiration predicted by our model agrees with recent 13C fluxomics experiments, and that our model largely recovers the Crabtree effect (the experimentally known bias among certain yeast species toward fermentation with the production of ethanol even in the presence of oxygen, while FBA without proteomics constraints predicts respirative metabolism almost exclusively. The comparisons to the 13C study showed improvement upon inclusion of the correlations and motivated a technique to systematically identify inconsistent kinetic parameters in the literature. The minor secretion fluxes for glycerol and acetate are underestimated by our method, which indicate a need for further refinements to the metabolic model. For yeast cells grown in synthetic defined (SD medium, the calculated broad distribution of growth rates matches experimental observations from single cell studies, and we characterize several metabolic phenotypes within our modeled populations that make use of diverse pathways. Fast growing yeast cells are predicted to perform significant amount of respiration, use serine-glycine cycle and produce ethanol in mitochondria as opposed to slow growing cells. We use a genetic algorithm to determine the proteomics constraints necessary to reproduce the growth rate distributions seen experimentally. We find that a core set of 51 constraints are essential but

  12. Reconciling proteomics with next generation sequencing

    NARCIS (Netherlands)

    Low, Teck Yew; Heck, Albert Jr

    2015-01-01

    Both genomics and proteomics technologies have matured in the last decade to a level where they are able to deliver system-wide data on the qualitative and quantitative abundance of their respective molecular entities, that is DNA/RNA and proteins. A next logical step is the collective use of these

  13. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics

    DEFF Research Database (Denmark)

    Ong, S.E.; Blagoev, B.; Kratchmarova, I.

    2002-01-01

    Quantitative proteomics has traditionally been performed by two-dimensional gel electrophoresis, but recently, mass spectrometric methods based on stable isotope quantitation have shown great promise for the simultaneous and automated identification and quantitation of complex protein mixtures. H...

  14. Data from proteome analysis of Lasiodiplodia theobromae (Botryosphaeriaceae

    Directory of Open Access Journals (Sweden)

    Carla C. Uranga

    2017-08-01

    Full Text Available Trunk disease fungi are a global problem affecting many economically important fruiting trees. The Botryosphaeriaceae are a family of trunk disease fungi that require detailed biochemical characterization in order to gain insight into their pathogenicity. The application of a modified Folch extraction to protein extraction from the Botryosphaeriaceae Lasiodiplodia theobromae generated an unprecedented data set of protein identifications from fragmentation analysis and de novo peptide sequencing of its proteome. This article contains data from protein identifications obtained from a database-dependent fragmentation analysis using three different proteomics algorithms (MSGF, Comet and X! Tandem via the SearchGUI proteomics pipeline program and de novo peptide sequencing. Included are data sets of gene ontology annotations using an all-Uniprot ontology database, as well as a Saccharomyces cerevisiae-only and a Candida albicans-only ontology database, in order to discern between those proteins involved in common functions with S. cerevisiae and those in common with the pathogenic yeast C. albicans. Our results reveal the proteome of L. theobromae contains more ontological categories in common to C. albicans, yet possesses a much wider metabolic repertoire than any of the yeasts studied in this work. Many novel proteins of interest were identified for further biochemical characterization and annotation efforts, as further discussed in the article referencing this article (1. Interactive Cytoscape networks of molecular functions of identified peptides using an all-Uniprot ontological database are included. Data, including raw data, are available via ProteomeXchange with identifier PXD005283.

  15. Vaginal yeast infection

    Science.gov (United States)

    Yeast infection - vagina; Vaginal candidiasis; Monilial vaginitis ... Most women have a vaginal yeast infection at some time. Candida albicans is a common type of fungus. It is often found in small amounts ...

  16. Proteomic screening for amyloid proteins.

    Directory of Open Access Journals (Sweden)

    Anton A Nizhnikov

    Full Text Available Despite extensive study, progress in elucidation of biological functions of amyloids and their role in pathology is largely restrained due to the lack of universal and reliable biochemical methods for their discovery. All biochemical methods developed so far allowed only identification of glutamine/asparagine-rich amyloid-forming proteins or proteins comprising amyloids that form large deposits. In this article we present a proteomic approach which may enable identification of a broad range of amyloid-forming proteins independently of specific features of their sequences or levels of expression. This approach is based on the isolation of protein fractions enriched with amyloid aggregates via sedimentation by ultracentrifugation in the presence of strong ionic detergents, such as sarkosyl or SDS. Sedimented proteins are then separated either by 2D difference gel electrophoresis or by SDS-PAGE, if they are insoluble in the buffer used for 2D difference gel electrophoresis, after which they are identified by mass-spectrometry. We validated this approach by detection of known yeast prions and mammalian proteins with established capacity for amyloid formation and also revealed yeast proteins forming detergent-insoluble aggregates in the presence of human huntingtin with expanded polyglutamine domain. Notably, with one exception, all these proteins contained glutamine/asparagine-rich stretches suggesting that their aggregates arose due to polymerization cross-seeding by human huntingtin. Importantly, though the approach was developed in a yeast model, it can easily be applied to any organism thus representing an efficient and universal tool for screening for amyloid proteins.

  17. Mass spectrometry analysis of proteome-wide proteolytic post-translational degradation of proteins

    OpenAIRE

    Shen, Yufeng; Hixson, Kim K.; Tolić, Nikola; Camp, David G.; Purvine, Samuel O.; Moore, Ronald J.; Smith, Richard D.

    2008-01-01

    Protein proteolytic degradation is an essential component to proper cell function and its life cycle. Here, we study the protein degradation in yeast Saccharomyces cerevisiae cells on a proteome-wide scale by detection of the intermediate peptides produced from the intracellular degradation of proteins using sequencing-based tandem mass spectrometry. By tracing the detected ~1,100 peptides and their ~200 protein substrate origins we obtain evidence for new insights into the proteome-wide prot...

  18. Protein interaction networks by proteome peptide scanning.

    Directory of Open Access Journals (Sweden)

    Christiane Landgraf

    2004-01-01

    Full Text Available A substantial proportion of protein interactions relies on small domains binding to short peptides in the partner proteins. Many of these interactions are relatively low affinity and transient, and they impact on signal transduction. However, neither the number of potential interactions mediated by each domain nor the degree of promiscuity at a whole proteome level has been investigated. We have used a combination of phage display and SPOT synthesis to discover all the peptides in the yeast proteome that have the potential to bind to eight SH3 domains. We first identified the peptides that match a relaxed consensus, as deduced from peptides selected by phage display experiments. Next, we synthesized all the matching peptides at high density on a cellulose membrane, and we probed them directly with the SH3 domains. The domains that we have studied were grouped by this approach into five classes with partially overlapping specificity. Within the classes, however, the domains display a high promiscuity and bind to a large number of common targets with comparable affinity. We estimate that the yeast proteome contains as few as six peptides that bind to the Abp1 SH3 domain with a dissociation constant lower than 100 microM, while it contains as many as 50-80 peptides with corresponding affinity for the SH3 domain of Yfr024c. All the targets of the Abp1 SH3 domain, identified by this approach, bind to the native protein in vivo, as shown by coimmunoprecipitation experiments. Finally, we demonstrate that this strategy can be extended to the analysis of the entire human proteome. We have developed an approach, named WISE (whole interactome scanning experiment, that permits rapid and reliable identification of the partners of any peptide recognition module by peptide scanning of a proteome. Since the SPOT synthesis approach is semiquantitative and provides an approximation of the dissociation constants of the several thousands of interactions that are

  19. False-Positive Rate Determination of Protein Target Discovery using a Covalent Modification- and Mass Spectrometry-Based Proteomics Platform

    Science.gov (United States)

    Strickland, Erin C.; Geer, M. Ariel; Hong, Jiyong; Fitzgerald, Michael C.

    2014-01-01

    Detection and quantitation of protein-ligand binding interactions is important in many areas of biological research. Stability of proteins from rates of oxidation (SPROX) is an energetics-based technique for identifying the proteins targets of ligands in complex biological mixtures. Knowing the false-positive rate of protein target discovery in proteome-wide SPROX experiments is important for the correct interpretation of results. Reported here are the results of a control SPROX experiment in which chemical denaturation data is obtained on the proteins in two samples that originated from the same yeast lysate, as would be done in a typical SPROX experiment except that one sample would be spiked with the test ligand. False-positive rates of 1.2-2.2 % and analysis of the isobaric mass tag (e.g., iTRAQ®) reporter ions used for peptide quantitation. Our results also suggest that technical replicates can be used to effectively eliminate such false positives that result from this random error, as is demonstrated in a SPROX experiment to identify yeast protein targets of the drug, manassantin A. The impact of ion purity in the tandem mass spectral analyses and of background oxidation on the false-positive rate of protein target discovery using SPROX is also discussed.

  20. Comparative proteome analysis of Brettanomyces bruxellensis under hydroxycinnamic acid growth

    Directory of Open Access Journals (Sweden)

    Lourdes Carmona

    2016-09-01

    Conclusions: The proteomic profile of B. bruxellensis cultivated in the presence of p-coumaric acid in synthetic wine, agrees with the hypothesis of metabolic flux regulation, allowing a better conditioning to an adverse environment. This study involved the translational level of B. bruxellensis in the production of ethylphenols and corroborated that this yeast presented an advantage in these stress conditions. Thus, this work will allow an understanding of the regulation and processes involved in the production of ethyl-derivate compounds by B. bruxellensis. Furthermore, it allows the development of newer and better techniques for spoilage yeast control.

  1. Yeast for virus research

    Science.gov (United States)

    Zhao, Richard Yuqi

    2017-01-01

    Budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe) are two popular model organisms for virus research. They are natural hosts for viruses as they carry their own indigenous viruses. Both yeasts have been used for studies of plant, animal and human viruses. Many positive sense (+) RNA viruses and some DNA viruses replicate with various levels in yeasts, thus allowing study of those viral activities during viral life cycle. Yeasts are single cell eukaryotic organisms. Hence, many of the fundamental cellular functions such as cell cycle regulation or programed cell death are highly conserved from yeasts to higher eukaryotes. Therefore, they are particularly suited to study the impact of those viral activities on related cellular activities during virus-host interactions. Yeasts present many unique advantages in virus research over high eukaryotes. Yeast cells are easy to maintain in the laboratory with relative short doubling time. They are non-biohazardous, genetically amendable with small genomes that permit genome-wide analysis of virologic and cellular functions. In this review, similarities and differences of these two yeasts are described. Studies of virologic activities such as viral translation, viral replication and genome-wide study of virus-cell interactions in yeasts are highlighted. Impacts of viral proteins on basic cellular functions such as cell cycle regulation and programed cell death are discussed. Potential applications of using yeasts as hosts to carry out functional analysis of small viral genome and to develop high throughput drug screening platform for the discovery of antiviral drugs are presented. PMID:29082230

  2. Quantitative Non-canonical Amino Acid Tagging (QuaNCAT) Proteomics Identifies Distinct Patterns of Protein Synthesis Rapidly Induced by Hypertrophic Agents in Cardiomyocytes, Revealing New Aspects of Metabolic Remodeling*

    Science.gov (United States)

    Liu, Rui; Kenney, Justin W.; Manousopoulou, Antigoni; Johnston, Harvey E.; Kamei, Makoto; Woelk, Christopher H.; Xie, Jianling; Schwarzer, Michael; Proud, Christopher G.

    2016-01-01

    Cardiomyocytes undergo growth and remodeling in response to specific pathological or physiological conditions. In the former, myocardial growth is a risk factor for cardiac failure and faster protein synthesis is a major factor driving cardiomyocyte growth. Our goal was to quantify the rapid effects of different pro-hypertrophic stimuli on the synthesis of specific proteins in ARVC and to determine whether such effects are caused by alterations on mRNA abundance or the translation of specific mRNAs. Cardiomyocytes have very low rates of protein synthesis, posing a challenging problem in terms of studying changes in the synthesis of specific proteins, which also applies to other nondividing primary cells. To study the rates of accumulation of specific proteins in these cells, we developed an optimized version of the Quantitative Noncanonical Amino acid Tagging LC/MS proteomic method to label and selectively enrich newly synthesized proteins in these primary cells while eliminating the suppressive effects of pre-existing and highly abundant nonisotope-tagged polypeptides. Our data revealed that a classical pathologic (phenylephrine; PE) and the recently identified insulin stimulus that also contributes to the development of pathological cardiac hypertrophy (insulin), both increased the synthesis of proteins involved in, e.g. glycolysis, the Krebs cycle and beta-oxidation, and sarcomeric components. However, insulin increased synthesis of many metabolic enzymes to a greater extent than PE. Using a novel validation method, we confirmed that synthesis of selected candidates is indeed up-regulated by PE and insulin. Synthesis of all proteins studied was up-regulated by signaling through mammalian target of rapamycin complex 1 without changes in their mRNA levels, showing the key importance of translational control in the rapid effects of hypertrophic stimuli. Expression of PKM2 was up-regulated in rat hearts following TAC. This isoform possesses specific regulatory

  3. Mass Spectrometry for Translational Proteomics: Progress and Clinical Implications

    Energy Technology Data Exchange (ETDEWEB)

    Baker, Erin Shammel; Liu, Tao; Petyuk, Vladislav A.; Burnum-Johnson, Kristin E.; Ibrahim, Yehia M.; Anderson, Gordon A.; Smith, Richard D.

    2012-08-31

    Mass spectrometry (MS)-based proteomics measurements have become increasingly utilized in a wide range of biological and biomedical applications, and have significantly enhanced the understanding of the complex and dynamic nature of the proteome and its connections to biology and diseases. While some MS techniques such as those for targeted analysis are increasingly applied with great success, others such as global quantitative analysis (for e.g. biomarker discovery) are more challenging and continue to be developed and refined to provide the desired throughput, sensitivity and/ or specificity. New MS capabilities and proteomics-based pipelines/strategies also keep enhancing for the advancement of clinical proteomics applications such as protein biomarker discovery and validation. Herein, we provide a brief review to summarize the current state of MS-based proteomics with respect to its advantages and present limitations, while highlighting its potential in future clinical applications.

  4. The Proteome of Primary Prostate Cancer

    DEFF Research Database (Denmark)

    Iglesias-Gato, Diego; Wikström, Pernilla; Tyanova, Stefka

    2016-01-01

    for disease aggressiveness. DESIGN, SETTING, AND PARTICIPANTS: Mass spectrometry was used for genome-scale quantitative proteomic profiling of 28 prostate tumors (Gleason score 6-9) and neighboring nonmalignant tissue in eight cases, obtained from formalin-fixed paraffin-embedded prostatectomy samples. Two...... changes occurring during prostate cancer (PCa) initiation and progression can result in clinically relevant discoveries. OBJECTIVES: To study cellular processes altered in PCa using system-wide quantitative analysis of changes in protein expression in clinical samples and to identify prognostic biomarkers......BACKGROUND: Clinical management of the prostate needs improved prognostic tests and treatment strategies. Because proteins are the u