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Sample records for quantitative phosphoproteomics reveals

  1. A quantitative map of the liver mitochondrial phosphoproteome reveals posttranslational control of ketogenesis.

    Science.gov (United States)

    Grimsrud, Paul A; Carson, Joshua J; Hebert, Alex S; Hubler, Shane L; Niemi, Natalie M; Bailey, Derek J; Jochem, Adam; Stapleton, Donald S; Keller, Mark P; Westphall, Michael S; Yandell, Brian S; Attie, Alan D; Coon, Joshua J; Pagliarini, David J

    2012-11-07

    Mitochondria are dynamic organelles that play a central role in a diverse array of metabolic processes. Elucidating mitochondrial adaptations to changing metabolic demands and the pathogenic alterations that underlie metabolic disorders represent principal challenges in cell biology. Here, we performed multiplexed quantitative mass spectrometry-based proteomics to chart the remodeling of the mouse liver mitochondrial proteome and phosphoproteome during both acute and chronic physiological transformations in more than 50 mice. Our analyses reveal that reversible phosphorylation is widespread in mitochondria, and is a key mechanism for regulating ketogenesis during the onset of obesity and type 2 diabetes. Specifically, we have demonstrated that phosphorylation of a conserved serine on Hmgcs2 (S456) significantly enhances its catalytic activity in response to increased ketogenic demand. Collectively, our work describes the plasticity of this organelle at high resolution and provides a framework for investigating the roles of proteome restructuring and reversible phosphorylation in mitochondrial adaptation.

  2. Time-resolved quantitative phosphoproteomics

    DEFF Research Database (Denmark)

    Verano-Braga, Thiago; Schwämmle, Veit; Sylvester, Marc

    2012-01-01

    proteins involved in the Ang-(1-7) signaling, we performed a mass spectrometry-based time-resolved quantitative phosphoproteome study of human aortic endothelial cells (HAEC) treated with Ang-(1-7). We identified 1288 unique phosphosites on 699 different proteins with 99% certainty of correct peptide...

  3. Strategies for quantitation of phosphoproteomic data

    DEFF Research Database (Denmark)

    Palmisano, Giuseppe; Thingholm, Tine Engberg

    2010-01-01

    Recent developments in phosphoproteomic sample-preparation techniques and sensitive mass spectrometry instrumentation have led to large-scale identifications of phosphoproteins and phosphorylation sites from highly complex samples. This has facilitated the implementation of different quantitation...... will be on different quantitation strategies. Methods for metabolic labeling, chemical modification and label-free quantitation and their applicability or inapplicability in phosphoproteomic studies are discussed....

  4. Quantitative phosphoproteomics to characterize signaling networks

    DEFF Research Database (Denmark)

    Rigbolt, Kristoffer T G; Blagoev, Blagoy

    2012-01-01

    Reversible protein phosphorylation is involved in the regulation of most, if not all, major cellular processes via dynamic signal transduction pathways. During the last decade quantitative phosphoproteomics have evolved from a highly specialized area to a powerful and versatile platform for analy......Reversible protein phosphorylation is involved in the regulation of most, if not all, major cellular processes via dynamic signal transduction pathways. During the last decade quantitative phosphoproteomics have evolved from a highly specialized area to a powerful and versatile platform...... and quantify thousands of phosphorylations, thus providing extensive overviews of the cellular signaling networks. As a result of these developments quantitative phosphoproteomics have been applied to study processes as diverse as immunology, stem cell biology and DNA damage. Here we review the developments...... in phosphoproteomics technology that have facilitated the application of phosphoproteomics to signaling networks and introduce examples of recent system-wide applications of quantitative phosphoproteomics. Despite the great advances in phosphoproteomics technology there are still several outstanding issues and we...

  5. Quantitative phosphoproteomic study of pressure-overloaded mouse heart reveals dynamin-related protein 1 as a modulator of cardiac hypertrophy.

    Science.gov (United States)

    Chang, Yu-Wang; Chang, Ya-Ting; Wang, Qinchuan; Lin, Jim Jung-Ching; Chen, Yu-Ju; Chen, Chien-Chang

    2013-11-01

    Pressure-overload stress to the heart causes pathological cardiac hypertrophy, which increases the risk of cardiac morbidity and mortality. However, the detailed signaling pathways induced by pressure overload remain unclear. Here we used phosphoproteomics to delineate signaling pathways in the myocardium responding to acute pressure overload and chronic hypertrophy in mice. Myocardial samples at 4 time points (10, 30, 60 min and 2 weeks) after transverse aortic banding (TAB) in mice underwent quantitative phosphoproteomics assay. Temporal phosphoproteomics profiles showed 360 phosphorylation sites with significant regulation after TAB. Multiple mechanical stress sensors were activated after acute pressure overload. Gene ontology analysis revealed differential phosphorylation between hearts with acute pressure overload and chronic hypertrophy. Most interestingly, analysis of the cardiac hypertrophy pathway revealed phosphorylation of the mitochondrial fission protein dynamin-related protein 1 (DRP1) by prohypertrophic kinases. Phosphorylation of DRP1 S622 was confirmed in TAB-treated mouse hearts and phenylephrine (PE)-treated rat neonatal cardiomyocytes. TAB-treated mouse hearts showed phosphorylation-mediated mitochondrial translocation of DRP1. Inhibition of DRP1 with the small-molecule inhibitor mdivi-1 reduced the TAB-induced hypertrophic responses. Mdivi-1 also prevented PE-induced hypertrophic growth and oxygen consumption in rat neonatal cardiomyocytes. We reveal the signaling responses of the heart to pressure stress in vivo and in vitro. DRP1 may be important in the development of cardiac hypertrophy.

  6. Quantitative Label-Free Phosphoproteomics Reveals Differentially Regulated Protein Phosphorylation Involved in West Nile Virus-Induced Host Inflammatory Response.

    Science.gov (United States)

    Zhang, Hao; Sun, Jun; Ye, Jing; Ashraf, Usama; Chen, Zheng; Zhu, Bibo; He, Wen; Xu, Qiuping; Wei, Yanming; Chen, Huanchun; Fu, Zhen F; Liu, Rong; Cao, Shengbo

    2015-12-01

    West Nile virus (WNV) can cause neuro-invasive and febrile illness that may be fatal to humans. The production of inflammatory cytokines is key to mediating WNV-induced immunopathology in the central nervous system. Elucidating the host factors utilized by WNV for productive infection would provide valuable insights into the evasion strategies used by this virus. Although attempts have been made to determine these host factors, proteomic data depicting WNV-host protein interactions are limited. We applied liquid chromatography-tandem mass spectrometry for label-free, quantitative phosphoproteomics to systematically investigate the global phosphorylation events induced by WNV infection. Quantifiable changes to 1,657 phosphoproteins were found; of these, 626 were significantly upregulated and 227 were downregulated at 12 h postinfection. The phosphoproteomic data were subjected to gene ontology enrichment analysis, which returned the inflammation-related spliceosome, ErbB, mitogen-activated protein kinase, nuclear factor kappa B, and mechanistic target of rapamycin signaling pathways. We used short interfering RNAs to decrease the levels of glycogen synthase kinase-3 beta, bifunctional polynucleotide phosphatase/kinase, and retinoblastoma 1 and found that the activity of nuclear factor kappa B (p65) is significantly decreased in WNV-infected U251 cells, which in turn led to markedly reduced inflammatory cytokine production. Our results provide a better understanding of the host response to WNV infection and highlight multiple targets for the development of antiviral and anti-inflammatory therapies.

  7. Global effects of kinase inhibitors on signaling networks revealed by quantitative phosphoproteomics

    DEFF Research Database (Denmark)

    Pan, Cuiping; Olsen, Jesper V; Daub, Henrik;

    2009-01-01

    to identify the direct targets of kinase inhibitors upon affinity purification from cellular extracts. Here we introduce a complementary approach to evaluate the effects of kinase inhibitors on the entire cell signaling network. We used triple labeling SILAC (stable isotope labeling by amino acids in cell...... culture) to compare cellular phosphorylation levels for control, epidermal growth factor stimulus, and growth factor combined with kinase inhibitors. Of thousands of phosphopeptides, less than 10% had a response pattern indicative of targets of U0126 and SB202190, two widely used MAPK inhibitors....... Interestingly, 83% of the growth factor-induced phosphorylation events were affected by either or both inhibitors, showing quantitatively that early signaling processes are predominantly transmitted through the MAPK cascades. In contrast to MAPK inhibitors, dasatinib, a clinical drug directed against BCR...

  8. Label-free quantitative phosphoproteomics with novel pairwise abundance normalization reveals synergistic RAS and CIP2A signaling.

    Science.gov (United States)

    Kauko, Otto; Laajala, Teemu Daniel; Jumppanen, Mikael; Hintsanen, Petteri; Suni, Veronika; Haapaniemi, Pekka; Corthals, Garry; Aittokallio, Tero; Westermarck, Jukka; Imanishi, Susumu Y

    2015-08-17

    Hyperactivated RAS drives progression of many human malignancies. However, oncogenic activity of RAS is dependent on simultaneous inactivation of protein phosphatase 2A (PP2A) activity. Although PP2A is known to regulate some of the RAS effector pathways, it has not been systematically assessed how these proteins functionally interact. Here we have analyzed phosphoproteomes regulated by either RAS or PP2A, by phosphopeptide enrichment followed by mass-spectrometry-based label-free quantification. To allow data normalization in situations where depletion of RAS or PP2A inhibitor CIP2A causes a large uni-directional change in the phosphopeptide abundance, we developed a novel normalization strategy, named pairwise normalization. This normalization is based on adjusting phosphopeptide abundances measured before and after the enrichment. The superior performance of the pairwise normalization was verified by various independent methods. Additionally, we demonstrate how the selected normalization method influences the downstream analyses and interpretation of pathway activities. Consequently, bioinformatics analysis of RAS and CIP2A regulated phosphoproteomes revealed a significant overlap in their functional pathways. This is most likely biologically meaningful as we observed a synergistic survival effect between CIP2A and RAS expression as well as KRAS activating mutations in TCGA pan-cancer data set, and synergistic relationship between CIP2A and KRAS depletion in colony growth assays.

  9. Quantitative phosphoproteomic analysis reveals system-wide signaling pathways downstream of SDF-1/CXCR4 in breast cancer stem cells.

    Science.gov (United States)

    Yi, Tingfang; Zhai, Bo; Yu, Yonghao; Kiyotsugu, Yoshikawa; Raschle, Thomas; Etzkorn, Manuel; Seo, Hee-Chan; Nagiec, Michal; Luna, Rafael E; Reinherz, Ellis L; Blenis, John; Gygi, Steven P; Wagner, Gerhard

    2014-05-27

    Breast cancer is the leading cause of cancer-related mortality in women worldwide, with an estimated 1.7 million new cases and 522,000 deaths around the world in 2012 alone. Cancer stem cells (CSCs) are essential for tumor reoccurrence and metastasis which is the major source of cancer lethality. G protein-coupled receptor chemokine (C-X-C motif) receptor 4 (CXCR4) is critical for tumor metastasis. However, stromal cell-derived factor 1 (SDF-1)/CXCR4-mediated signaling pathways in breast CSCs are largely unknown. Using isotope reductive dimethylation and large-scale MS-based quantitative phosphoproteome analysis, we examined protein phosphorylation induced by SDF-1/CXCR4 signaling in breast CSCs. We quantified more than 11,000 phosphorylation sites in 2,500 phosphoproteins. Of these phosphosites, 87% were statistically unchanged in abundance in response to SDF-1/CXCR4 stimulation. In contrast, 545 phosphosites in 266 phosphoproteins were significantly increased, whereas 113 phosphosites in 74 phosphoproteins were significantly decreased. SDF-1/CXCR4 increases phosphorylation in 60 cell migration- and invasion-related proteins, of them 43 (>70%) phosphoproteins are unrecognized. In addition, SDF-1/CXCR4 upregulates the phosphorylation of 44 previously uncharacterized kinases, 8 phosphatases, and 1 endogenous phosphatase inhibitor. Using computational approaches, we performed system-based analyses examining SDF-1/CXCR4-mediated phosphoproteome, including construction of kinase-substrate network and feedback regulation loops downstream of SDF-1/CXCR4 signaling in breast CSCs. We identified a previously unidentified SDF-1/CXCR4-PKA-MAP2K2-ERK signaling pathway and demonstrated the feedback regulation on MEK, ERK1/2, δ-catenin, and PPP1Cα in SDF-1/CXCR4 signaling in breast CSCs. This study gives a system-wide view of phosphorylation events downstream of SDF-1/CXCR4 signaling in breast CSCs, providing a resource for the study of CSC-targeted cancer therapy.

  10. The current state of the art of quantitative phosphoproteomics and its applications to diabetes research

    Energy Technology Data Exchange (ETDEWEB)

    Chan, Chi Yuet X’avia; Gritsenko, Marina A.; Smith, Richard D.; Qian, Wei-Jun

    2016-03-17

    Protein phosphorylation is a fundamental regulatory mechanism in many cellular processes and aberrant perturbation of phosphorylation has been revealed in various human diseases. Kinases and their cognate inhibitors have been hotspot for drug development. Therefore, the emerging tools, which enable a system-wide quantitative profiling of phosphoproteome, would offer a powerful impetus in unveiling novel signaling pathways, drug targets and/or biomarkers for the disease of interest. In this review, we will highlight recent advances in phosphoproteomics, the current state-of-the-art of the technologies, and the challenges and future perspectives of this research area. Finally, we will underscore some exemplary applications of phosphoproteomics in diabetes research.

  11. 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.

  12. Quantitative Phosphoproteomics Analysis of ERBB3/ERBB4 Signaling.

    Science.gov (United States)

    Wandinger, Sebastian K; Lahortiga, Idoya; Jacobs, Kris; Klammer, Martin; Jordan, Nicole; Elschenbroich, Sarah; Parade, Marc; Jacoby, Edgar; Linders, Joannes T M; Brehmer, Dirk; Cools, Jan; Daub, Henrik

    2016-01-01

    The four members of the epidermal growth factor receptor (EGFR/ERBB) family form homo- and heterodimers which mediate ligand-specific regulation of many key cellular processes in normal and cancer tissues. While signaling through the EGFR has been extensively studied on the molecular level, signal transduction through ERBB3/ERBB4 heterodimers is less well understood. Here, we generated isogenic mouse Ba/F3 cells that express full-length and functional membrane-integrated ERBB3 and ERBB4 or ERBB4 alone, to serve as a defined cellular model for biological and phosphoproteomics analysis of ERBB3/ERBB4 signaling. ERBB3 co-expression significantly enhanced Ba/F3 cell proliferation upon neuregulin-1 (NRG1) treatment. For comprehensive signaling studies we performed quantitative mass spectrometry (MS) experiments to compare the basal ERBB3/ERBB4 cell phosphoproteome to NRG1 treatment of ERBB3/ERBB4 and ERBB4 cells. We employed a workflow comprising differential isotope labeling with mTRAQ reagents followed by chromatographic peptide separation and final phosphopeptide enrichment prior to MS analysis. Overall, we identified 9686 phosphorylation sites which could be confidently localized to specific residues. Statistical analysis of three replicate experiments revealed 492 phosphorylation sites which were significantly changed in NRG1-treated ERBB3/ERBB4 cells. Bioinformatics data analysis recapitulated regulation of mitogen-activated protein kinase and Akt pathways, but also indicated signaling links to cytoskeletal functions and nuclear biology. Comparative assessment of NRG1-stimulated ERBB4 Ba/F3 cells revealed that ERBB3 did not trigger defined signaling pathways but more broadly enhanced phosphoproteome regulation in cells expressing both receptors. In conclusion, our data provide the first global picture of ERBB3/ERBB4 signaling and provide numerous potential starting points for further mechanistic studies.

  13. Quantitative Phosphoproteomics Analysis of ERBB3/ERBB4 Signaling.

    Directory of Open Access Journals (Sweden)

    Sebastian K Wandinger

    Full Text Available The four members of the epidermal growth factor receptor (EGFR/ERBB family form homo- and heterodimers which mediate ligand-specific regulation of many key cellular processes in normal and cancer tissues. While signaling through the EGFR has been extensively studied on the molecular level, signal transduction through ERBB3/ERBB4 heterodimers is less well understood. Here, we generated isogenic mouse Ba/F3 cells that express full-length and functional membrane-integrated ERBB3 and ERBB4 or ERBB4 alone, to serve as a defined cellular model for biological and phosphoproteomics analysis of ERBB3/ERBB4 signaling. ERBB3 co-expression significantly enhanced Ba/F3 cell proliferation upon neuregulin-1 (NRG1 treatment. For comprehensive signaling studies we performed quantitative mass spectrometry (MS experiments to compare the basal ERBB3/ERBB4 cell phosphoproteome to NRG1 treatment of ERBB3/ERBB4 and ERBB4 cells. We employed a workflow comprising differential isotope labeling with mTRAQ reagents followed by chromatographic peptide separation and final phosphopeptide enrichment prior to MS analysis. Overall, we identified 9686 phosphorylation sites which could be confidently localized to specific residues. Statistical analysis of three replicate experiments revealed 492 phosphorylation sites which were significantly changed in NRG1-treated ERBB3/ERBB4 cells. Bioinformatics data analysis recapitulated regulation of mitogen-activated protein kinase and Akt pathways, but also indicated signaling links to cytoskeletal functions and nuclear biology. Comparative assessment of NRG1-stimulated ERBB4 Ba/F3 cells revealed that ERBB3 did not trigger defined signaling pathways but more broadly enhanced phosphoproteome regulation in cells expressing both receptors. In conclusion, our data provide the first global picture of ERBB3/ERBB4 signaling and provide numerous potential starting points for further mechanistic studies.

  14. Quantitative phosphoproteomics of CXCL12 (SDF-1 signaling.

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    Jason A Wojcechowskyj

    Full Text Available CXCL12 (SDF-1 is a chemokine that binds to and signals through the seven transmembrane receptor CXCR4. The CXCL12/CXCR4 signaling axis has been implicated in both cancer metastases and human immunodeficiency virus type 1 (HIV-1 infection and a more complete understanding of CXCL12/CXCR4 signaling pathways may support efforts to develop therapeutics for these diseases. Mass spectrometry-based phosphoproteomics has emerged as an important tool in studying signaling networks in an unbiased fashion. We employed stable isotope labeling with amino acids in cell culture (SILAC quantitative phosphoproteomics to examine the CXCL12/CXCR4 signaling axis in the human lymphoblastic CEM cell line. We quantified 4,074 unique SILAC pairs from 1,673 proteins and 89 phosphopeptides were deemed CXCL12-responsive in biological replicates. Several well established CXCL12-responsive phosphosites such as AKT (pS473 and ERK2 (pY204 were confirmed in our study. We also validated two novel CXCL12-responsive phosphosites, stathmin (pS16 and AKT1S1 (pT246 by Western blot. Pathway analysis and comparisons with other phosphoproteomic datasets revealed that genes from CXCL12-responsive phosphosites are enriched for cellular pathways such as T cell activation, epidermal growth factor and mammalian target of rapamycin (mTOR signaling, pathways which have previously been linked to CXCL12/CXCR4 signaling. Several of the novel CXCL12-responsive phosphoproteins from our study have also been implicated with cellular migration and HIV-1 infection, thus providing an attractive list of potential targets for the development of cancer metastasis and HIV-1 therapeutics and for furthering our understanding of chemokine signaling regulation by reversible phosphorylation.

  15. Quantitative label-free phosphoproteomics of six different life stages of the late blight pathogen Phytophthora infestans reveals abundant phosphorylation of members of the CRN effector family.

    Science.gov (United States)

    Resjö, Svante; Ali, Ashfaq; Meijer, Harold J G; Seidl, Michael F; Snel, Berend; Sandin, Marianne; Levander, Fredrik; Govers, Francine; Andreasson, Erik

    2014-04-04

    The oomycete Phytophthora infestans is the causal agent of late blight in potato and tomato. Since the underlying processes that govern pathogenicity and development in P. infestans are largely unknown, we have performed a large-scale phosphoproteomics study of six different P. infestans life stages. We have obtained quantitative data for 2922 phosphopeptides and compared their abundance. Life-stage-specific phosphopeptides include ATP-binding cassette transporters and a kinase that only occurs in appressoria. In an extended data set, we identified 2179 phosphorylation sites and deduced 22 phosphomotifs. Several of the phosphomotifs matched consensus sequences of kinases that occur in P. infestans but not Arabidopsis. In addition, we detected tyrosine phosphopeptides that are potential targets of kinases resembling mammalian tyrosine kinases. Among the phosphorylated proteins are members of the RXLR and Crinkler effector families. The latter are phosphorylated in several life stages and at multiple positions, in sites that are conserved between different members of the Crinkler family. This indicates that proteins in the Crinkler family have functions beyond their putative role as (necrosis-inducing) effectors. This phosphoproteomics data will be instrumental for studies on oomycetes and host-oomycete interactions. The data sets have been deposited to ProteomeXchange (identifier PXD000433).

  16. Quantitative phosphoproteomics dissection of seven-transmembrane receptor signaling using full and biased agonists

    DEFF Research Database (Denmark)

    Christensen, Gitte L; Kelstrup, Christian D; Lyngsø, Christina;

    2010-01-01

    , we performed a global quantitative phosphoproteomics analysis of the AT(1)R signaling network. We analyzed ligand-stimulated SILAC (stable isotope labeling by amino acids in cell culture) cells by high resolution (LTQ-Orbitrap) MS and compared the phosphoproteomes of the AT(1)R agonist angiotensin II......(q)-dependent and -independent AT(1)R signaling. This study provides substantial novel insight into angiotensin II signal transduction and is the first study dissecting the differences between a full agonist and a biased agonist from a 7TMR on a systems-wide scale. Importantly, it reveals a previously unappreciated diversity...

  17. Quantitative Phosphoproteomic Analysis of T-Cell Receptor Signaling.

    Science.gov (United States)

    Ahsan, Nagib; Salomon, Arthur R

    2017-01-01

    TCR signaling critically depends on protein phosphorylation across many proteins. Localization of each phosphorylation event relative to the T-cell receptor (TCR) and canonical T-cell signaling proteins will provide clues about the structure of TCR signaling networks. Quantitative phosphoproteomic analysis by mass spectrometry provides a wide-scale view of cellular phosphorylation networks. However, analysis of phosphorylation by mass spectrometry is still challenging due to the relative low abundance of phosphorylated proteins relative to all proteins and the extraordinary diversity of phosphorylation sites across the proteome. Highly selective enrichment of phosphorylated peptides is essential to provide the most comprehensive view of the phosphoproteome. Optimization of phosphopeptide enrichment methods coupled with highly sensitive mass spectrometry workflows significantly improves the sequencing depth of the phosphoproteome to over 10,000 unique phosphorylation sites from complex cell lysates. Here we describe a step-by-step method for phosphoproteomic analysis that has achieved widespread success for identification of serine, threonine, and tyrosine phosphorylation. Reproducible quantification of relative phosphopeptide abundance is provided by intensity-based label-free quantitation. An ideal set of mass spectrometry analysis parameters is also provided that optimize the yield of identified sites. We also provide guidelines for the bioinformatic analysis of this type of data to assess the quality of the data and to comply with proteomic data reporting requirements.

  18. Characterization of early autophagy signaling by quantitative phosphoproteomics

    DEFF Research Database (Denmark)

    Rigbolt, Kristoffer Tg; Zarei, Mostafa; Sprenger, Adrian;

    2014-01-01

    Under conditions of nutrient shortage autophagy is the primary cellular mechanism ensuring availability of substrates for continuous biosynthesis. Subjecting cells to starvation or rapamycin efficiently induces autophagy by inhibiting the MTOR signaling pathway triggering increased autophagic flux....... To elucidate the regulation of early signaling events upon autophagy induction, we applied quantitative phosphoproteomics characterizing the temporal phosphorylation dynamics after starvation and rapamycin treatment. We obtained a comprehensive atlas of phosphorylation kinetics within the first 30 min upon...

  19. Dissection of the insulin signaling pathway via quantitative phosphoproteomics

    DEFF Research Database (Denmark)

    Krüger, Marcus; Kratchmarova, Irina; Blagoev, Blagoy

    2008-01-01

    The insulin signaling pathway is of pivotal importance in metabolic diseases, such as diabetes, and in cellular processes, such as aging. Insulin activates a tyrosine phosphorylation cascade that branches to create a complex network affecting multiple biological processes. To understand the full ...... the calcium transporting ATPase SERCA2, supporting a connection to calcium signaling. The combination of quantitative phosphoproteomics with cell culture models provides a powerful strategy to dissect the insulin signaling pathways in intact cells....

  20. Quantitative phosphoproteomic analysis using iTRAQ method.

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    Asano, Tomoya; Nishiuchi, Takumi

    2014-01-01

    The MAPK (mitogen-activated kinase) cascade plays important roles in plant perception of and reaction to developmental and environmental cues. Phosphoproteomics are useful to identify target proteins regulated by MAPK-dependent signaling pathway. Here, we introduce the quantitative phosphoproteomic analysis using a chemical labeling method. The isobaric tag for relative and absolute quantitation (iTRAQ) method is a MS-based technique to quantify protein expression among up to eight different samples in one experiment. In this technique, peptides were labeled by some stable isotope-coded covalent tags. We perform quantitative phosphoproteomics comparing Arabidopsis wild type and a stress-responsive mapkk mutant after phytotoxin treatment. To comprehensively identify the downstream phosphoproteins of MAPKK, total proteins were extracted from phytotoxin-treated wild-type and mapkk mutant plants. The phosphoproteins were purified by Pro-Q(®) Diamond Phosphoprotein Enrichment Kit and were digested with trypsin. Resulting peptides were labeled with iTRAQ reagents and were quantified and identified by MALDI TOF/TOF analyzer. We identified many phosphoproteins that were decreased in the mapkk mutant compared with wild type.

  1. Quantitative phosphoproteomics of proteasome inhibition in multiple myeloma cells.

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    Feng Ge

    Full Text Available BACKGROUND: The proteasome inhibitor bortezomib represents an important advance in the treatment of multiple myeloma (MM. Bortezomib inhibits the activity of the 26S proteasome and induces cell death in a variety of tumor cells; however, the mechanism of cytotoxicity is not well understood. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the differential phosphoproteome upon proteasome inhibition by using stable isotope labeling by amino acids in cell culture (SILAC in combination with phosphoprotein enrichment and LC-MS/MS analysis. In total 233 phosphoproteins were identified and 72 phosphoproteins showed a 1.5-fold or greater change upon bortezomib treatment. The phosphoproteins with expression alterations encompass all major protein classes, including a large number of nucleic acid binding proteins. Site-specific phosphopeptide quantitation revealed that Ser38 phosphorylation on stathmin increased upon bortezomib treatment, suggesting new mechanisms associated to bortezomib-induced apoptosis in MM cells. Further studies demonstrated that stathmin phosphorylation profile was modified in response to bortezomib treatment and the regulation of stathmin by phosphorylation at specific Ser/Thr residues participated in the cellular response induced by bortezomib. CONCLUSIONS/SIGNIFICANCE: Our systematic profiling of phosphorylation changes in response to bortezomib treatment not only advanced the global mechanistic understanding of the action of bortezomib on myeloma cells but also identified previously uncharacterized signaling proteins in myeloma cells.

  2. Characterization of early autophagy signaling by quantitative phosphoproteomics

    DEFF Research Database (Denmark)

    Rigbolt, Kristoffer Tg; Zarei, Mostafa; Sprenger, Adrian

    2014-01-01

    . To elucidate the regulation of early signaling events upon autophagy induction, we applied quantitative phosphoproteomics characterizing the temporal phosphorylation dynamics after starvation and rapamycin treatment. We obtained a comprehensive atlas of phosphorylation kinetics within the first 30 min upon...... induction of autophagy with both treatments affecting widely different cellular processes. The identification of dynamic phosphorylation already after 2 min demonstrates that the earliest events in autophagy signaling occur rapidly after induction. The data was subjected to extensive bioinformatics analysis...... of binding partners exhibiting dynamic phosphorylation patterns. The data presented here provide a valuable resource on phosphorylation events underlying early autophagy induction....

  3. Integrated quantitative analysis of the phosphoproteome and transcriptome in tamoxifen-resistant breast cancer.

    Science.gov (United States)

    Oyama, Masaaki; Nagashima, Takeshi; Suzuki, Takashi; Kozuka-Hata, Hiroko; Yumoto, Noriko; Shiraishi, Yuichi; Ikeda, Kazuhiro; Kuroki, Yoko; Gotoh, Noriko; Ishida, Takanori; Inoue, Satoshi; Kitano, Hiroaki; Okada-Hatakeyama, Mariko

    2011-01-07

    Quantitative phosphoproteome and transcriptome analysis of ligand-stimulated MCF-7 human breast cancer cells was performed to understand the mechanisms of tamoxifen resistance at a system level. Phosphoproteome data revealed that WT cells were more enriched with phospho-proteins than tamoxifen-resistant cells after stimulation with ligands. Surprisingly, decreased phosphorylation after ligand perturbation was more common than increased phosphorylation. In particular, 17β-estradiol induced down-regulation in WT cells at a very high rate. 17β-Estradiol and the ErbB ligand heregulin induced almost equal numbers of up-regulated phospho-proteins in WT cells. Pathway and motif activity analyses using transcriptome data additionally suggested that deregulated activation of GSK3β (glycogen-synthase kinase 3β) and MAPK1/3 signaling might be associated with altered activation of cAMP-responsive element-binding protein and AP-1 transcription factors in tamoxifen-resistant cells, and this hypothesis was validated by reporter assays. An examination of clinical samples revealed that inhibitory phosphorylation of GSK3β at serine 9 was significantly lower in tamoxifen-treated breast cancer patients that eventually had relapses, implying that activation of GSK3β may be associated with the tamoxifen-resistant phenotype. Thus, the combined phosphoproteome and transcriptome data set analyses revealed distinct signal transcription programs in tumor cells and provided a novel molecular target to understand tamoxifen resistance.

  4. Quantitative phosphoproteomics reveals the role of the AMPK plant ortholog SnRK1 as a metabolic master regulator under energy deprivation

    Science.gov (United States)

    Nukarinen, Ella; Nägele, Thomas; Pedrotti, Lorenzo; Wurzinger, Bernhard; Mair, Andrea; Landgraf, Ramona; Börnke, Frederik; Hanson, Johannes; Teige, Markus; Baena-Gonzalez, Elena; Dröge-Laser, Wolfgang; Weckwerth, Wolfram

    2016-01-01

    Since years, research on SnRK1, the major cellular energy sensor in plants, has tried to define its role in energy signalling. However, these attempts were notoriously hampered by the lethality of a complete knockout of SnRK1. Therefore, we generated an inducible amiRNA::SnRK1α2 in a snrk1α1 knock out background (snrk1α1/α2) to abolish SnRK1 activity to understand major systemic functions of SnRK1 signalling under energy deprivation triggered by extended night treatment. We analysed the in vivo phosphoproteome, proteome and metabolome and found that activation of SnRK1 is essential for repression of high energy demanding cell processes such as protein synthesis. The most abundant effect was the constitutively high phosphorylation of ribosomal protein S6 (RPS6) in the snrk1α1/α2 mutant. RPS6 is a major target of TOR signalling and its phosphorylation correlates with translation. Further evidence for an antagonistic SnRK1 and TOR crosstalk comparable to the animal system was demonstrated by the in vivo interaction of SnRK1α1 and RAPTOR1B in the cytosol and by phosphorylation of RAPTOR1B by SnRK1α1 in kinase assays. Moreover, changed levels of phosphorylation states of several chloroplastic proteins in the snrk1α1/α2 mutant indicated an unexpected link to regulation of photosynthesis, the main energy source in plants. PMID:27545962

  5. Urinary proteomic and non-prefractionation quantitative phosphoproteomic analysis during pregnancy and non-pregnancy

    National Research Council Canada - National Science Library

    Zheng, Jianhua; Liu, Liguo; Wang, Jin; Jin, Qi

    2013-01-01

    .... Furthermore, we also apply a non-prefractionation quantitative phosphoproteomic approach using mTRAQ labeling to evaluate the expression of specific phosphoproteins during pregnancy comparison with non-pregnancy...

  6. The current state of the art of quantitative phosphoproteomics and its applications to diabetes research.

    Science.gov (United States)

    Chan, Chi Yuet X'avia; Gritsenko, Marina A; Smith, Richard D; Qian, Wei-Jun

    2016-01-01

    Protein phosphorylation is a fundamental regulatory mechanism in many cellular processes and aberrant perturbation of phosphorylation has been implicated in various human diseases. Kinases and their cognate inhibitors have been considered as hotspots for drug development. Therefore, the emerging tools, which enable a system-wide quantitative profiling of phosphoproteome, would offer a powerful impetus in unveiling novel signaling pathways, drug targets and/or biomarkers for diseases of interest. This review highlights recent advances in phosphoproteomics, the current state of the art of the technologies and the challenges and future perspectives of this research area. Finally, some exemplary applications of phosphoproteomics in diabetes research are underscored.

  7. The calcium-dependent protein kinase 3 of toxoplasma influences basal calcium levels and functions beyond egress as revealed by quantitative phosphoproteome analysis.

    OpenAIRE

    Moritz Treeck; Sanders, John L.; Rajshekhar Y Gaji; Kacie A LaFavers; Child, Matthew A.; Gustavo Arrizabalaga; Elias, Joshua E.; John C Boothroyd

    2014-01-01

    Calcium-dependent protein kinases (CDPKs) are conserved in plants and apicomplexan parasites. In Toxoplasma gondii, TgCDPK3 regulates parasite egress from the host cell in the presence of a calcium-ionophore. The targets and the pathways that the kinase controls, however, are not known. To identify pathways regulated by TgCDPK3, we measured relative phosphorylation site usage in wild type and TgCDPK3 mutant and knock-out parasites by quantitative mass-spectrometry using stable isotope-labelin...

  8. In Vivo SILAC-Based Proteomics Reveals Phosphoproteome Changes during Mouse Skin Carcinogenesis

    Directory of Open Access Journals (Sweden)

    Sara Zanivan

    2013-02-01

    Full Text Available Cancer progresses through distinct stages, and mouse models recapitulating traits of this progression are frequently used to explore genetic, morphological, and pharmacological aspects of tumor development. To complement genomic investigations of this process, we here quantify phosphoproteomic changes in skin cancer development using the SILAC mouse technology coupled to high-resolution mass spectrometry. We distill protein expression signatures from our data that distinguish between skin cancer stages. A distinct phosphoproteome of the two stages of cancer progression is identified that correlates with perturbed cell growth and implicates cell adhesion as a major driver of malignancy. Importantly, integrated analysis of phosphoproteomic data and prediction of kinase activity revealed PAK4-PKC/SRC network to be highly deregulated in SCC but not in papilloma. This detailed molecular picture, both at the proteome and phosphoproteome level, will prove useful for the study of mechanisms of tumor progression.

  9. Phosphoproteome Profiling Reveals Circadian Clock Regulation of Posttranslational Modifications in the Murine Hippocampus

    Science.gov (United States)

    Chiang, Cheng-Kang; Xu, Bo; Mehta, Neel; Mayne, Janice; Sun, Warren Y. L.; Cheng, Kai; Ning, Zhibin; Dong, Jing; Zou, Hanfa; Cheng, Hai-Ying Mary; Figeys, Daniel

    2017-01-01

    The circadian clock is an endogenous oscillator that drives daily rhythms in physiology, behavior, and gene expression. The underlying mechanisms of circadian timekeeping are cell-autonomous and involve oscillatory expression of core clock genes that is driven by interconnecting transcription–translation feedback loops (TTFLs). Circadian clock TTFLs are further regulated by posttranslational modifications, in particular, phosphorylation. The hippocampus plays an important role in spatial memory and the conversion of short- to long-term memory. Several studies have reported the presence of a peripheral oscillator in the hippocampus and have highlighted the importance of circadian regulation in memory formation. Given the general importance of phosphorylation in circadian clock regulation, we performed global quantitative proteome and phosphoproteome analyses of the murine hippocampus across the circadian cycle, applying spiked-in labeled reference and high accuracy mass spectrometry (MS). Of the 3,052 proteins and 2,868 phosphosites on 1,368 proteins that were accurately quantified, 1.7% of proteins and 5.2% of phosphorylation events exhibited time-of-day-dependent expression profiles. The majority of circadian phosphopeptides displayed abrupt fluctuations at mid-to-late day without underlying rhythms of protein abundance. Bioinformatic analysis of cyclic phosphorylation events revealed their diverse distribution in different biological pathways, most notably, cytoskeletal organization and neuronal morphogenesis. This study provides the first large-scale, quantitative MS analysis of the circadian phosphoproteome and proteome of the murine hippocampus and highlights the significance of rhythmic regulation at the posttranslational level in this peripheral oscillator. In addition to providing molecular insights into the hippocampal circadian clock, our results will assist in the understanding of genetic factors that underlie rhythms-associated pathological states of

  10. 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.

  11. Quantitative phosphoproteomic analysis of prion-infected neuronal cells

    Directory of Open Access Journals (Sweden)

    Löwer Johannes

    2010-09-01

    Full Text Available Abstract Prion diseases or transmissible spongiform encephalopathies (TSEs are fatal diseases associated with the conversion of the cellular prion protein (PrPC to the abnormal prion protein (PrPSc. Since the molecular mechanisms in pathogenesis are widely unclear, we analyzed the global phospho-proteome and detected a differential pattern of tyrosine- and threonine phosphorylated proteins in PrPSc-replicating and pentosan polysulfate (PPS-rescued N2a cells in two-dimensional gel electrophoresis. To quantify phosphorylated proteins, we performed a SILAC (stable isotope labeling by amino acids in cell culture analysis and identified 105 proteins, which showed a regulated phosphorylation upon PrPSc infection. Among those proteins, we validated the dephosphorylation of stathmin and Cdc2 and the induced phosphorylation of cofilin in PrPSc-infected N2a cells in Western blot analyses. Our analysis showed for the first time a differentially regulated phospho-proteome in PrPSc infection, which could contribute to the establishment of novel protein markers and to the development of novel therapeutic intervention strategies in targeting prion-associated disease.

  12. Quantitative proteomics and phosphoproteomics on serial tumor biopsies from a sorafenib-treated HCC patient

    Science.gov (United States)

    Dazert, Eva; Colombi, Marco; Boldanova, Tujana; Moes, Suzette; Adametz, David; Quagliata, Luca; Roth, Volker; Terracciano, Luigi; Heim, Markus H.; Jenoe, Paul; Hall, Michael N.

    2016-01-01

    Compensatory signaling pathways in tumors confer resistance to targeted therapy, but the pathways and their mechanisms of activation remain largely unknown. We describe a procedure for quantitative proteomics and phosphoproteomics on snap-frozen biopsies of hepatocellular carcinoma (HCC) and matched nontumor liver tissue. We applied this procedure to monitor signaling pathways in serial biopsies taken from an HCC patient before and during treatment with the multikinase inhibitor sorafenib. At diagnosis, the patient had an advanced HCC. At the time of the second biopsy, abdominal imaging revealed progressive disease despite sorafenib treatment. Sorafenib was confirmed to inhibit MAPK signaling in the tumor, as measured by reduced ribosomal protein S6 kinase phosphorylation. Hierarchical clustering and enrichment analysis revealed pathways broadly implicated in tumor progression and resistance, such as epithelial-to-mesenchymal transition and cell adhesion pathways. Thus, we describe a protocol for quantitative analysis of oncogenic pathways in HCC biopsies and obtained first insights into the effect of sorafenib in vivo. This protocol will allow elucidation of mechanisms of resistance and enable precision medicine. PMID:26787912

  13. Interleukin-2 signaling pathway analysis by quantitative phosphoproteomics

    DEFF Research Database (Denmark)

    Osinalde, Nerea; Moss, Helle; Arrizabalaga, Onetsine

    2011-01-01

    in modulation of the immune response. The complete characterization of the IL-2 pathway is essential to understand how aberrant IL-2 signaling results in several diseases such as cancer or autoimmunity and also how IL-2 treatments affect cancer patients. To gain insights into the downstream machinery activated...... by IL-2, we aimed to define the global tyrosine-phosphoproteome of IL-2 pathway in human T cell line Kit225 using high resolution mass spectrometry combined with phosphotyrosine immunoprecipitation and SILAC. The molecular snapshot at 5min of IL-2 stimulation resulted in identification of 172 proteins...... with increased abundance in the tyrosine-phosphorylated complexes, of which 34 were not previously described. In addition, chemical inhibition of the identified IL-2-mediated JAK, PI3K and MAPK signaling pathways, resulted in distinct alteration on the IL-2 dependent proliferation....

  14. Quantitative Proteomic and Phosphoproteomic Approaches for Deciphering the Signaling Pathway for Tension Wood Formation in Poplar.

    Science.gov (United States)

    Mauriat, Mélanie; Leplé, Jean-Charles; Claverol, Stéphane; Bartholomé, Jérôme; Negroni, Luc; Richet, Nicolas; Lalanne, Céline; Bonneu, Marc; Coutand, Catherine; Plomion, Christophe

    2015-08-07

    Trees adjust their growth following forced changes in orientation to re-establish a vertical position. In angiosperms, this adjustment involves the differential regulation of vascular cambial activity between the lower (opposite wood) and upper (tension wood) sides of the leaning stem. We investigated the molecular mechanisms leading to the formation of differential wood types through a quantitative proteomic and phosphoproteomic analysis on poplar subjected to a gravitropic stimulus. We identified and quantified 675 phosphopeptides, corresponding to 468 phosphoproteins, and 3 763 nonphosphorylated peptides, corresponding to 1 155 proteins, in the differentiating xylem of straight-growing trees (control) and trees subjected to a gravitational stimulus during 8 weeks. About 1% of the peptides were specific to a wood type (straight, opposite, or tension wood). Proteins quantified in more than one type of wood were more numerous: a mixed linear model showed 389 phosphopeptides and 556 proteins to differ in abundance between tension wood and opposite wood. Twenty-one percent of the phosphoproteins identified here were described in their phosphorylated form for the first time. Our analyses revealed remarkable developmental molecular plasticity, with wood type-specific phosphorylation events, and highlighted the involvement of different proteins in the biosynthesis of cell wall components during the formation of the three types of wood.

  15. Quantitative Phosphoproteomic Analysis of Soybean Root Hairs Inoculated with Bradyrhizobium japonicum

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen, Tran H.; Brechenmacher, Laurent; Aldrich, Joshua T.; Clauss, Therese RW; Gritsenko, Marina A.; Hixson, Kim K.; Libault, Marc; Tanaka, Kiwamu; Yang, Feng; Yao, Qiuming; Pasa-Tolic, Ljiljana; Xu, Dong; Nguyen, Henry T.; Stacey, Gary

    2012-11-11

    Root hairs are single hair-forming cells on roots that function to increase root surface area, enhancing water and nutrient uptake. In leguminous plants, root hairs also play a critical role as the site of infection by symbiotic nitrogen fixing rhizobia, leading to the formation of a novel organ, the nodule. The initial steps in the rhizobia-root hair infection process are known to involve specific receptor kinases and subsequent kinase cascades. Here, we characterize the phosphoproteome of the root hairs and the corresponding stripped roots (i.e., roots from which root hairs were removed) during rhizobial colonization and infection to gain insight into the molecular mechanism of root hair cell biology. We chose soybean (Glycine max L.), one of the most important crop plants in the legume family, for this study because of its larger root size, which permits isolation of sufficient root hair material for phosphoproteomic analysis. Phosphopeptides derived from root hairs and stripped roots, mock inoculated or inoculated with the soybean-specific rhizobium Bradyrhizobium japonicum, were labeled with the isobaric tag 8-plex ITRAQ, enriched using Ni-NTA magnetic beads and subjected to nRPLC-MS/MS analysis using HCD and decision tree guided CID/ETD strategy. A total of 1,625 unique phosphopeptides, spanning 1,659 non-redundant phosphorylation sites, were detected from 1,126 soybean phosphoproteins. Among them, 273 phosphopeptides corresponding to 240 phosphoproteins were found to be significantly regulated (>1.5 fold abundance change) in response to inoculation with B. japonicum. The data reveal unique features of the soybean root hair phosphoproteome, including root hair and stripped root-specific phosphorylation suggesting a complex network of kinase-substrate and phosphatase-substrate interactions in response to rhizobial inoculation.

  16. Quantitative Phosphoproteomic Analysis of Soybean Root Hairs Inoculated with Bradyrhizobium japonicum*

    Science.gov (United States)

    Nguyen, Tran Hong Nha; Brechenmacher, Laurent; Aldrich, Joshua T.; Clauss, Therese R.; Gritsenko, Marina A.; Hixson, Kim K.; Libault, Marc; Tanaka, Kiwamu; Yang, Feng; Yao, Qiuming; Paša-Tolić, Ljiljana; Xu, Dong; Nguyen, Henry T.; Stacey, Gary

    2012-01-01

    Root hairs are single hair-forming cells on roots that function to increase root surface area, enhancing water and nutrient uptake. In leguminous plants, root hairs also play a critical role as the site of infection by symbiotic nitrogen fixing rhizobia, leading to the formation of a novel organ, the nodule. The initial steps in the rhizobia-root hair infection process are known to involve specific receptor kinases and subsequent kinase cascades. Here, we characterize the phosphoproteome of the root hairs and the corresponding stripped roots (i.e. roots from which root hairs were removed) during rhizobial colonization and infection to gain insight into the molecular mechanism of root hair cell biology. We chose soybean (Glycine max L.), one of the most important crop plants in the legume family, for this study because of its larger root size, which permits isolation of sufficient root hair material for phosphoproteomic analysis. Phosphopeptides derived from root hairs and stripped roots, mock inoculated or inoculated with the soybean-specific rhizobium Bradyrhizobium japonicum, were labeled with the isobaric tag eight-plex iTRAQ, enriched using Ni-NTA magnetic beads and subjected to nanoRPLC-MS/MS1 analysis using HCD and decision tree guided CID/ETD strategy. A total of 1625 unique phosphopeptides, spanning 1659 nonredundant phosphorylation sites, were detected from 1126 soybean phosphoproteins. Among them, 273 phosphopeptides corresponding to 240 phosphoproteins were found to be significantly regulated (>1.5-fold abundance change) in response to inoculation with B. japonicum. The data reveal unique features of the soybean root hair phosphoproteome, including root hair and stripped root-specific phosphorylation suggesting a complex network of kinase-substrate and phosphatase-substrate interactions in response to rhizobial inoculation. PMID:22843990

  17. Quantitative phosphoproteomic analysis of soybean root hairs inoculated with Bradyrhizobium japonicum.

    Science.gov (United States)

    Nguyen, Tran Hong Nha; Brechenmacher, Laurent; Aldrich, Joshua T; Clauss, Therese R; Gritsenko, Marina A; Hixson, Kim K; Libault, Marc; Tanaka, Kiwamu; Yang, Feng; Yao, Qiuming; Pasa-Tolić, Ljiljana; Xu, Dong; Nguyen, Henry T; Stacey, Gary

    2012-11-01

    Root hairs are single hair-forming cells on roots that function to increase root surface area, enhancing water and nutrient uptake. In leguminous plants, root hairs also play a critical role as the site of infection by symbiotic nitrogen fixing rhizobia, leading to the formation of a novel organ, the nodule. The initial steps in the rhizobia-root hair infection process are known to involve specific receptor kinases and subsequent kinase cascades. Here, we characterize the phosphoproteome of the root hairs and the corresponding stripped roots (i.e. roots from which root hairs were removed) during rhizobial colonization and infection to gain insight into the molecular mechanism of root hair cell biology. We chose soybean (Glycine max L.), one of the most important crop plants in the legume family, for this study because of its larger root size, which permits isolation of sufficient root hair material for phosphoproteomic analysis. Phosphopeptides derived from root hairs and stripped roots, mock inoculated or inoculated with the soybean-specific rhizobium Bradyrhizobium japonicum, were labeled with the isobaric tag eight-plex iTRAQ, enriched using Ni-NTA magnetic beads and subjected to nanoRPLC-MS/MS1 analysis using HCD and decision tree guided CID/ETD strategy. A total of 1625 unique phosphopeptides, spanning 1659 nonredundant phosphorylation sites, were detected from 1126 soybean phosphoproteins. Among them, 273 phosphopeptides corresponding to 240 phosphoproteins were found to be significantly regulated (>1.5-fold abundance change) in response to inoculation with B. japonicum. The data reveal unique features of the soybean root hair phosphoproteome, including root hair and stripped root-specific phosphorylation suggesting a complex network of kinase-substrate and phosphatase-substrate interactions in response to rhizobial inoculation.

  18. Quantitative phosphoproteomic analysis of early seed development in rice (Oryza sativa L.).

    Science.gov (United States)

    Qiu, Jiehua; Hou, Yuxuan; Tong, Xiaohong; Wang, Yifeng; Lin, Haiyan; Liu, Qing; Zhang, Wen; Li, Zhiyong; Nallamilli, Babi R; Zhang, Jian

    2016-02-01

    Rice (Oryza sativa L.) seed serves as a major food source for over half of the global population. Though it has been long recognized that phosphorylation plays an essential role in rice seed development, the phosphorylation events and dynamics in this process remain largely unknown so far. Here, we report the first large scale identification of rice seed phosphoproteins and phosphosites by using a quantitative phosphoproteomic approach. Thorough proteomic studies in pistils and seeds at 3, 7 days after pollination resulted in the successful identification of 3885, 4313 and 4135 phosphopeptides respectively. A total of 2487 proteins were differentially phosphorylated among the three stages, including Kip related protein 1, Rice basic leucine zipper factor 1, Rice prolamin box binding factor and numerous other master regulators of rice seed development. Moreover, differentially phosphorylated proteins may be extensively involved in the biosynthesis and signaling pathways of phytohormones such as auxin, gibberellin, abscisic acid and brassinosteroid. Our results strongly indicated that protein phosphorylation is a key mechanism regulating cell proliferation and enlargement, phytohormone biosynthesis and signaling, grain filling and grain quality during rice seed development. Overall, the current study enhanced our understanding of the rice phosphoproteome and shed novel insight into the regulatory mechanism of rice seed development.

  19. Temporal proteomic analysis of HIV infection reveals remodelling of the host phosphoproteome by lentiviral Vif variants

    Science.gov (United States)

    Greenwood, Edward JD; Matheson, Nicholas J; Wals, Kim; van den Boomen, Dick JH; Antrobus, Robin; Williamson, James C; Lehner, Paul J

    2016-01-01

    Viruses manipulate host factors to enhance their replication and evade cellular restriction. We used multiplex tandem mass tag (TMT)-based whole cell proteomics to perform a comprehensive time course analysis of >6500 viral and cellular proteins during HIV infection. To enable specific functional predictions, we categorized cellular proteins regulated by HIV according to their patterns of temporal expression. We focussed on proteins depleted with similar kinetics to APOBEC3C, and found the viral accessory protein Vif to be necessary and sufficient for CUL5-dependent proteasomal degradation of all members of the B56 family of regulatory subunits of the key cellular phosphatase PP2A (PPP2R5A-E). Quantitative phosphoproteomic analysis of HIV-infected cells confirmed Vif-dependent hyperphosphorylation of >200 cellular proteins, particularly substrates of the aurora kinases. The ability of Vif to target PPP2R5 subunits is found in primate and non-primate lentiviral lineages, and remodeling of the cellular phosphoproteome is therefore a second ancient and conserved Vif function. DOI: http://dx.doi.org/10.7554/eLife.18296.001 PMID:27690223

  20. Temporal proteomic analysis of HIV infection reveals remodelling of the host phosphoproteome by lentiviral Vif variants.

    Science.gov (United States)

    Greenwood, Edward Jd; Matheson, Nicholas J; Wals, Kim; van den Boomen, Dick Jh; Antrobus, Robin; Williamson, James C; Lehner, Paul J

    2016-09-30

    Viruses manipulate host factors to enhance their replication and evade cellular restriction. We used multiplex tandem mass tag (TMT)-based whole cell proteomics to perform a comprehensive time course analysis of >6500 viral and cellular proteins during HIV infection. To enable specific functional predictions, we categorized cellular proteins regulated by HIV according to their patterns of temporal expression. We focussed on proteins depleted with similar kinetics to APOBEC3C, and found the viral accessory protein Vif to be necessary and sufficient for CUL5-dependent proteasomal degradation of all members of the B56 family of regulatory subunits of the key cellular phosphatase PP2A (PPP2R5A-E). Quantitative phosphoproteomic analysis of HIV-infected cells confirmed Vif-dependent hyperphosphorylation of >200 cellular proteins, particularly substrates of the aurora kinases. The ability of Vif to target PPP2R5 subunits is found in primate and non-primate lentiviral lineages, and remodeling of the cellular phosphoproteome is therefore a second ancient and conserved Vif function.

  1. Phosphoproteomic analysis reveals compensatory effects in the piriform cortex of VX nerve agent exposed rats.

    Science.gov (United States)

    Nirujogi, Raja Sekhar; Wright, James D; Manda, Srikanth S; Zhong, Jun; Na, Chan Hyun; Meyerhoff, James; Benton, Bernard; Jabbour, Rabih; Willis, Kristen; Kim, Min-Sik; Pandey, Akhilesh; Sekowski, Jennifer W

    2015-01-01

    To gain insights into the toxicity induced by the nerve agent VX, an MS-based phosphoproteomic analysis was carried out on the piriform cortex region of brains from VX-treated rats. Using isobaric tag based TMT labeling followed by titanium dioxide enrichment strategy, we identified 9975 unique phosphosites derived from 3287 phosphoproteins. Temporal changes in the phosphorylation status of peptides were observed over a time period of 24 h in rats exposed to a 1× LD50, intravenous (i.v.) dose with the most notable changes occurring at the 1 h postexposure time point. Five major functional classes of proteins exhibited changes in their phosphorylation status: (i) ion channels/transporters, including ATPases, (ii) kinases/phosphatases, (iii) GTPases, (iv) structural proteins, and (v) transcriptional regulatory proteins. This study is the first quantitative phosphoproteomic analysis of VX toxicity in the brain. Understanding the toxicity and compensatory signaling mechanisms will improve the understanding of the complex toxicity of VX in the brain and aid in the elucidation of novel molecular targets that would be important for development of improved countermeasures. All MS data have been deposited in the ProteomeXchange with identifier PXD001184 (http://proteomecentral.proteomexchange.org/dataset/PXD001184).

  2. 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.

  3. Global phosphoproteomic analysis of human skeletal muscle reveals a network of exercise-regulated kinases and AMPK substrates

    DEFF Research Database (Denmark)

    Hoffman, Nolan J; Parker, Benjamin L; Chaudhuri, Rima

    2015-01-01

    the importance of AMPK in exercise-regulated metabolism, we performed a targeted in vitro AMPK screen and employed machine learning to predict exercise-regulated AMPK substrates. We validated eight predicted AMPK substrates, including AKAP1, using targeted phosphoproteomics. Functional characterization revealed...

  4. In Vivo SILAC-Based Proteomics Reveals Phosphoproteome Changes during Mouse Skin Carcinogenesis

    DEFF Research Database (Denmark)

    Zanivan, Sara; Meves, Alexander; Behrendt, Kristina

    2013-01-01

    SILAC technology in combination with high-resolution mass spectrometry (MS) can be successfully used to measure phosphoproteomes in vivo. Here, Zanivan, Mann, and colleagues have applied SILAC-based MS to investigate phosphoproteomic changes during skin carcinogenesis, using the DMBA/TPA two-stag...

  5. Quantitative Phosphoproteomics Identifies Filaggrin and other Targets of Ionizing Radiation in a Human Skin Model

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Feng; Waters, Katrina M.; Webb-Robertson, Bobbie-Jo M.; Sowa, Marianne B.; Freiin von Neubeck, Claere H.; Aldrich, Joshua T.; Markillie, Lye Meng; Wirgau, Rachel M.; Gristenko, Marina A.; Zhao, Rui; Camp, David G.; Smith, Richard D.; Stenoien, David L.

    2012-04-17

    Our objective here was to perform a quantitative phosphoproteomic study on a reconstituted human skin tissue to identify low and high dose ionizing radiation dependent signaling in a complex 3-dimensional setting. Application of an isobaric labeling strategy using sham and 3 radiation doses (3, 10, 200 cGy) resulted in the identification of 1113 unique phosphopeptides. Statistical analyses identified 151 phosphopeptides showing significant changes in response to radiation and radiation dose. Proteins responsible for maintaining skin structural integrity including keratins and desmosomal proteins (desmoglein, desmoplakin, plakophilin 1 and 2,) had altered phosphorylation levels following exposure to both low and high doses of radiation. A phosphorylation site present in multiple copies in the linker regions of human profilaggrin underwent the largest fold change. Increased phosphorylation of these sites coincided with altered profilaggrin processing suggesting a role for linker phosphorylation in human profilaggrin regulation. These studies demonstrate that the reconstituted human skin system undergoes a coordinated response to ionizing radiation involving multiple layers of the stratified epithelium that serve to maintain skin barrier functions and minimize the damaging consequences of radiation exposure.

  6. Quantitative cardiac phosphoproteomics profiling during ischemia-reperfusion in an immature swine model

    Energy Technology Data Exchange (ETDEWEB)

    Ledee, Dolena R.; Kang, Min A.; Kajimoto, Masaki; Purvine, Samuel O.; Brewer, Heather M.; Pasa Tolic, Ljiljana; Portman, Michael A.

    2017-07-01

    Ischemia-reperfusion (I/R) results in altered metabolic and molecular responses, and phosphorylation is one of the most noted regulatory mechanisms mediating signaling mechanisms during physiological stresses. To expand our knowledge of the potential phosphoproteomic changes in the myocardium during I/R, we used Isobaric Tags for Relative and Absolute Quantitation-based analyses in left ventricular samples obtained from porcine hearts under control or I/R conditions. The data are available via ProteomeXchange with identifier PXD006066. We identified 1,896 phosphopeptides within left ventricular control and I/R porcine samples. Significant differential phosphorylation between control and I/R groups was discovered in 111 phosphopeptides from 86 proteins. Analysis of the phosphopeptides using Motif-x identified five motifs: (..R..S..), (..SP..), (..S.S..), (..S…S..), and (..S.T..). Semiquantitative immunoblots confirmed site location and directional changes in phosphorylation for phospholamban and pyruvate dehydrogenase E1, two proteins known to be altered by I/R and identified by this study. Novel phosphorylation sites associated with I/R were also identified. Functional characterization of the phosphopeptides identified by our methodology could expand our understanding of the signaling mechanisms involved during I/R damage in the heart as well as identify new areas to target therapeutic strategies.

  7. Quantitative Phosphoproteomic Analysis of Arabidopsis in Response to Salt and Hydrogen Peroxide Stresses

    Institute of Scientific and Technical Information of China (English)

    Yanmei Chen

    2012-01-01

    Salinity and oxidative stresses are major factors in affecting and limiting the productivity of agricultural crops.The study of biochemical and molecular responses of plants in response to those stresses is important for crop genetics and breeding.Extensive evidence shows that reversible protein phosphorylation plays a central role in mediating stress-regulated physiological responses,but little is known about its extent and function.Mass spectrometry provides a powerful tool for the in-depth analysis of systems biology.In this study,we performed a global quantitative analysis of the Arabidopsis phosphoproteomics in response to a time course of stress treatments using 15N-metabolic labeling and subcellular fractionation approaches.In total,we found 176 phosphoproteins showed to be regulated under stresses.Nine SnRK2 kinases identified to be differentially phosphorylated at multiple serine/threonine residues in their kinase domains following stress treatments,demonstrating different temporal phosphorylation induction of the various isoforms.K+ and Na+ transporters showed coordinated phosphorylation regulation under salt stress.In particular,nuclear proteins and protein kinases have high phosphorylation site occupancy in response to stress treatment.This suggests that the wide range of signaling and cellular processes that are modulated in this study.

  8. Quantitative phosphoproteomics identifies filaggrin and other targets of ionizing radiation in a human skin model.

    Science.gov (United States)

    Yang, Feng; Waters, Katrina M; Webb-Robertson, Bobbie-Jo; Sowa, Marianne B; von Neubeck, Claere; Aldrich, Josh T; Markillie, Lye Meng; Wirgau, Rachel M; Gritsenko, Marina A; Zhao, Rui; Camp, David G; Smith, Richard D; Stenoien, David L

    2012-05-01

    Our objective here was to perform a quantitative phosphoproteomic study on a reconstituted human skin tissue to identify low- and high-dose ionizing radiation-dependent signalling in a complex three-dimensional setting. Application of an isobaric labelling strategy using sham and three radiation doses (3, 10, 200 cGy) resulted in the identification of 1052 unique phosphopeptides. Statistical analyses identified 176 phosphopeptides showing significant changes in response to radiation and radiation dose. Proteins responsible for maintaining skin structural integrity including keratins and desmosomal proteins (desmoglein, desmoplakin, plakophilin 1, 2 and 3) had altered phosphorylation levels following exposure to both low and high doses of radiation. Altered phosphorylation of multiple sites in profilaggrin linker domains coincided with altered profilaggrin processing suggesting a role for linker phosphorylation in human profilaggrin regulation. These studies demonstrate that the reconstituted human skin system undergoes a coordinated response to both low and high doses of ionizing radiation involving multiple layers of the stratified epithelium that serve to maintain tissue integrity and mitigate effects of radiation exposure.

  9. Phosphoproteome Analysis Reveals the Molecular Mechanisms Underlying Deoxynivalenol-Induced Intestinal Toxicity in IPEC-J2 Cells

    Science.gov (United States)

    Zhang, Zhi-Qi; Wang, Song-Bo; Wang, Rui-Guo; Zhang, Wei; Wang, Pei-Long; Su, Xiao-Ou

    2016-01-01

    Deoxynivalenol (DON) is a widespread trichothecene mycotoxin that commonly contaminates cereal crops and has various toxic effects in animals and humans. DON primarily targets the gastrointestinal tract, the first barrier against ingested food contaminants. In this study, an isobaric tag for relative and absolute quantitation (iTRAQ)-based phosphoproteomic approach was employed to elucidate the molecular mechanisms underlying DON-mediated intestinal toxicity in porcine epithelial cells (IPEC-J2) exposed to 20 μM DON for 60 min. There were 4153 unique phosphopeptides, representing 389 phosphorylation sites, detected in 1821 phosphoproteins. We found that 289 phosphopeptides corresponding to 255 phosphoproteins were differentially phosphorylated in response to DON. Comprehensive Gene Ontology (GO) analysis combined with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment revealed that, in addition to previously well-characterized mitogen-activated protein kinase (MAPK) signaling, DON exposure altered phosphatidylinositol 3-kinase/Akt (PI3K/Akt) and Janus kinase/signal transducer, and activator of transcription (JAK/STAT) pathways. These pathways are involved in a wide range of biological processes, including apoptosis, the intestinal barrier, intestinal inflammation, and the intestinal absorption of glucose. DON-induced changes are likely to contribute to the intestinal dysfunction. Overall, identification of relevant signaling pathways yielded new insights into the molecular mechanisms underlying DON-induced intestinal toxicity, and might help in the development of improved mechanism-based risk assessments in animals and humans. PMID:27669298

  10. Phosphoproteome Analysis Reveals the Molecular Mechanisms Underlying Deoxynivalenol-Induced Intestinal Toxicity in IPEC-J2 Cells

    Directory of Open Access Journals (Sweden)

    Zhi-Qi Zhang

    2016-09-01

    Full Text Available Deoxynivalenol (DON is a widespread trichothecene mycotoxin that commonly contaminates cereal crops and has various toxic effects in animals and humans. DON primarily targets the gastrointestinal tract, the first barrier against ingested food contaminants. In this study, an isobaric tag for relative and absolute quantitation (iTRAQ-based phosphoproteomic approach was employed to elucidate the molecular mechanisms underlying DON-mediated intestinal toxicity in porcine epithelial cells (IPEC-J2 exposed to 20 μM DON for 60 min. There were 4153 unique phosphopeptides, representing 389 phosphorylation sites, detected in 1821 phosphoproteins. We found that 289 phosphopeptides corresponding to 255 phosphoproteins were differentially phosphorylated in response to DON. Comprehensive Gene Ontology (GO analysis combined with Kyoto Encyclopedia of Genes and Genomes (KEGG pathway enrichment revealed that, in addition to previously well-characterized mitogen-activated protein kinase (MAPK signaling, DON exposure altered phosphatidylinositol 3-kinase/Akt (PI3K/Akt and Janus kinase/signal transducer, and activator of transcription (JAK/STAT pathways. These pathways are involved in a wide range of biological processes, including apoptosis, the intestinal barrier, intestinal inflammation, and the intestinal absorption of glucose. DON-induced changes are likely to contribute to the intestinal dysfunction. Overall, identification of relevant signaling pathways yielded new insights into the molecular mechanisms underlying DON-induced intestinal toxicity, and might help in the development of improved mechanism-based risk assessments in animals and humans.

  11. Phosphoproteomic analysis reveals interconnected system-wide responses to perturbations of kinases and phosphatases in yeast.

    Science.gov (United States)

    Bodenmiller, Bernd; Wanka, Stefanie; Kraft, Claudine; Urban, Jörg; Campbell, David; Pedrioli, Patrick G; Gerrits, Bertran; Picotti, Paola; Lam, Henry; Vitek, Olga; Brusniak, Mi-Youn; Roschitzki, Bernd; Zhang, Chao; Shokat, Kevan M; Schlapbach, Ralph; Colman-Lerner, Alejandro; Nolan, Garry P; Nesvizhskii, Alexey I; Peter, Matthias; Loewith, Robbie; von Mering, Christian; Aebersold, Ruedi

    2010-12-21

    The phosphorylation and dephosphorylation of proteins by kinases and phosphatases constitute an essential regulatory network in eukaryotic cells. This network supports the flow of information from sensors through signaling systems to effector molecules and ultimately drives the phenotype and function of cells, tissues, and organisms. Dysregulation of this process has severe consequences and is one of the main factors in the emergence and progression of diseases, including cancer. Thus, major efforts have been invested in developing specific inhibitors that modulate the activity of individual kinases or phosphatases; however, it has been difficult to assess how such pharmacological interventions would affect the cellular signaling network as a whole. Here, we used label-free, quantitative phosphoproteomics in a systematically perturbed model organism (Saccharomyces cerevisiae) to determine the relationships between 97 kinases, 27 phosphatases, and more than 1000 phosphoproteins. We identified 8814 regulated phosphorylation events, describing the first system-wide protein phosphorylation network in vivo. Our results show that, at steady state, inactivation of most kinases and phosphatases affected large parts of the phosphorylation-modulated signal transduction machinery-and not only the immediate downstream targets. The observed cellular growth phenotype was often well maintained despite the perturbations, arguing for considerable robustness in the system. Our results serve to constrain future models of cellular signaling and reinforce the idea that simple linear representations of signaling pathways might be insufficient for drug development and for describing organismal homeostasis.

  12. Phosphoproteomics Reveals Distinct Modes of Mec1/ATR Signaling During DNA Replication

    Science.gov (United States)

    de Oliveira, Francisco Meirelles Bastos; Kim, Dongsung; Cussiol, Jose Renato; Das, Jishnu; Jeong, Min Cheol; Doerfler, Lillian; Schmidt, Kristina Hildegard; Yu, Haiyuan; Smolka, Marcus Bustamante

    2015-01-01

    SUMMARY The Mec1/Tel1 kinases (human ATR/ATM) play numerous roles in the DNA replication stress response. Despite the multi-functionality of these kinases, studies of their in vivo action have mostly relied on a few well-established substrates. Here we employed a combined genetic-phosphoproteomic approach to monitor Mec1/Tel1 signaling in a systematic, unbiased and quantitative manner. Unexpectedly, we find that Mec1 is highly active during normal DNA replication, at levels comparable or higher than Mec1’s activation state induced by replication stress. This “replication-correlated” mode of Mec1 action requires the 9-1-1 clamp and the Dna2 lagging-strand factor, and is distinguishable from Mec1’s action in activating the downstream kinase Rad53. We propose that Mec1/ATR performs key functions during ongoing DNA synthesis that are distinct from their canonical checkpoint role during replication stress. PMID:25752575

  13. Ultradeep Human Phosphoproteome Reveals a Distinct Regulatory Nature of Tyr and Ser/Thr-Based Signaling

    Directory of Open Access Journals (Sweden)

    Kirti Sharma

    2014-09-01

    Full Text Available Regulatory protein phosphorylation controls normal and pathophysiological signaling in eukaryotic cells. Despite great advances in mass-spectrometry-based proteomics, the extent, localization, and site-specific stoichiometry of this posttranslational modification (PTM are unknown. Here, we develop a stringent experimental and computational workflow, capable of mapping more than 50,000 distinct phosphorylated peptides in a single human cancer cell line. We detected more than three-quarters of cellular proteins as phosphoproteins and determined very high stoichiometries in mitosis or growth factor signaling by label-free quantitation. The proportion of phospho-Tyr drastically decreases as coverage of the phosphoproteome increases, whereas Ser/Thr sites saturate only for technical reasons. Tyrosine phosphorylation is maintained at especially low stoichiometric levels in the absence of specific signaling events. Unexpectedly, it is enriched on higher-abundance proteins, and this correlates with the substrate KM values of tyrosine kinases. Our data suggest that P-Tyr should be considered a functionally separate PTM of eukaryotic proteomes.

  14. Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis

    DEFF Research Database (Denmark)

    Miller, Martin Lee; Brunak, Søren; Olsen, JV

    2010-01-01

    ) or CDK2 were almost fully phosphorylated in mitotic cells. In particular, nuclear proteins and proteins involved in regulating metabolic processes have high phosphorylation site occupancy in mitosis. This suggests that these proteins may be inactivated by phosphorylation in mitotic cells....

  15. Phosphoproteomic dynamics of chickpea (Cicer arietinum L.) reveals shared and distinct components of dehydration response.

    Science.gov (United States)

    Subba, Pratigya; Barua, Pragya; Kumar, Rajiv; Datta, Asis; Soni, Kamlesh Kumar; Chakraborty, Subhra; Chakraborty, Niranjan

    2013-11-01

    Reversible protein phosphorylation is a ubiquitous regulatory mechanism that plays critical roles in transducing stress signals to bring about coordinated intracellular responses. To gain better understanding of dehydration response in plants, we have developed a differential phosphoproteome in a food legume, chickpea (Cicer arietinum L.). Three-week-old chickpea seedlings were subjected to progressive dehydration by withdrawing water, and the changes in the phosphorylation status of a large repertoire of proteins were monitored. The proteins were resolved by 2-DE and stained with phosphospecific fluorescent Pro-Q Diamond dye. Mass spectrometric analysis led to the identification of 91 putative phosphoproteins, presumably involved in a variety of functions including cell defense and rescue, photosynthesis and photorespiration, molecular chaperones, and ion transport, among others. Multiple sites of phosphorylation were predicted on several key elements, which include both the regulatory as well as the functional proteins. A critical survey of the phosphorylome revealed a DREPP (developmentally regulated plasma membrane protein) plasma membrane polypeptide family protein, henceforth designated CaDREPP1. The transcripts of CaDREPP1 were found to be differentially regulated under dehydration stress, further corroborating the proteomic results. This work provides new insights into the possible phosphorylation events triggered by the conditions of progressive water-deficit in plants.

  16. Phosphoproteome analysis of E-coli reveals evolutionary conservation of bacterial Ser/Thr/Tyr phosphorylation

    DEFF Research Database (Denmark)

    Macek, B.; Gnad, F.; Soufi, Boumediene

    2008-01-01

    we use a recently developed proteomics approach based on phosphopeptide enrichment and high accuracy MS to analyze the phosphoproteome of the model Gram-negative bacterium Escherichia coli. We report 81 phosphorylation sites on 79 E. coli proteins, with distribution of Ser/Thr/Tyr phosphorylation...... sites 68%/23%/9%. Despite their phylogenetic distance, phosphoproteomes of E. coli and B. subtilis show striking similarity in size, classes of phosphorylated proteins, and distribution of Ser/Thr/Tyr phosphorylation sites. By combining the two datasets, we created the largest phosphorylation site...

  17. Global investigation of interleukin-1β signaling in primary β-cells using quantitative phosphoproteomics

    DEFF Research Database (Denmark)

    Engholm-Keller, Kasper; Størling, Joachim; Pociot, Flemming

    Novel Aspect: Global phosphoproteomic analysis of cytokine signaling in primary β-cells Introduction The insulin-producing β-cells of the pancreatic islets of Langerhans are targeted by aberrant immune system responses in diabetes mellitus involving cytokines, especially interleukin-1β (IL-1 β...

  18. Quantitative Phosphoproteomics Unravels Biased Phosphorylation of Serotonin 2A Receptor at Ser280 by Hallucinogenic versus Nonhallucinogenic Agonists*

    Science.gov (United States)

    Karaki, Samah; Becamel, Carine; Murat, Samy; Mannoury la Cour, Clotilde; Millan, Mark J.; Prézeau, Laurent; Bockaert, Joël; Marin, Philippe; Vandermoere, Franck

    2014-01-01

    The serotonin 5-HT2A receptor is a primary target of psychedelic hallucinogens such as lysergic acid diethylamine, mescaline, and psilocybin, which reproduce some of the core symptoms of schizophrenia. An incompletely resolved paradox is that only some 5-HT2A receptor agonists exhibit hallucinogenic activity, whereas structurally related agonists with comparable affinity and activity lack such a psychoactive activity. Using a strategy combining stable isotope labeling by amino acids in cell culture with enrichment in phosphorylated peptides by means of hydrophilic interaction liquid chromatography followed by immobilized metal affinity chromatography, we compared the phosphoproteome in HEK-293 cells transiently expressing the 5-HT2A receptor and exposed to either vehicle or the synthetic hallucinogen 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI) or the nonhallucinogenic 5-HT2A agonist lisuride. Among the 5995 identified phosphorylated peptides, 16 sites were differentially phosphorylated upon exposure of cells to DOI versus lisuride. These include a serine (Ser280) located in the third intracellular loop of the 5-HT2A receptor, a region important for its desensitization. The specific phosphorylation of Ser280 by hallucinogens was further validated by quantitative mass spectrometry analysis of immunopurified receptor digests and by Western blotting using a phosphosite specific antibody. The administration of DOI, but not of lisuride, to mice, enhanced the phosphorylation of 5-HT2A receptors at Ser280 in the prefrontal cortex. Moreover, hallucinogens induced a less pronounced desensitization of receptor-operated signaling in HEK-293 cells and neurons than did nonhallucinogenic agonists. The mutation of Ser280 to aspartic acid (to mimic phosphorylation) reduced receptor desensitization by nonhallucinogenic agonists, whereas its mutation to alanine increased the ability of hallucinogens to desensitize the receptor. This study reveals a biased phosphorylation of

  19. Quantitative phosphoproteomics in nuclei of vasopressin-sensitive renal collecting duct cells

    OpenAIRE

    Bolger, Steven J.; Hurtado, Patricia A. Gonzales; Hoffert, Jason D.; Saeed, Fahad; Pisitkun, Trairak; Knepper, Mark A.

    2012-01-01

    Vasopressin regulates transport across the collecting duct epithelium in part via effects on gene transcription. Transcriptional regulation occurs partially via changes in phosphorylation of transcription factors, transcriptional coactivators, and protein kinases in the nucleus. To test whether vasopressin alters the nuclear phosphoproteome of vasopressin-sensitive cultured mouse mpkCCD cells, we used stable isotope labeling and mass spectrometry to quantify thousands of phosphorylation sites...

  20. 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.

  1. Comparative Phosphoproteomics Reveals an Important Role of MKK2 in Banana (Musa spp.) Cold Signal Network

    Science.gov (United States)

    Gao, Jie; Zhang, Sheng; He, Wei-Di; Shao, Xiu-Hong; Li, Chun-Yu; Wei, Yue-Rong; Deng, Gui-Ming; Kuang, Rui-Bin; Hu, Chun-Hua; Yi, Gan-Jun; Yang, Qiao-Song

    2017-01-01

    Low temperature is one of the key environmental stresses, which greatly affects global banana production. However, little is known about the global phosphoproteomes in Musa spp. and their regulatory roles in response to cold stress. In this study, we conducted a comparative phosphoproteomic profiling of cold-sensitive Cavendish Banana and relatively cold tolerant Dajiao under cold stress. Phosphopeptide abundances of five phosphoproteins involved in MKK2 interaction network, including MKK2, HY5, CaSR, STN7 and kinesin-like protein, show a remarkable difference between Cavendish Banana and Dajiao in response to cold stress. Western blotting of MKK2 protein and its T31 phosphorylated peptide verified the phosphoproteomic results of increased T31 phosphopeptide abundance with decreased MKK2 abundance in Daojiao for a time course of cold stress. Meanwhile increased expression of MKK2 with no detectable T31 phosphorylation was found in Cavendish Banana. These results suggest that the MKK2 pathway in Dajiao, along with other cold-specific phosphoproteins, appears to be associated with the molecular mechanisms of high tolerance to cold stress in Dajiao. The results also provide new evidence that the signaling pathway of cellular MKK2 phosphorylation plays an important role in abiotic stress tolerance that likely serves as a universal plant cold tolerance mechanism. PMID:28106078

  2. Identification of novel protein functions and signaling mechanisms by genetics and quantitative phosphoproteomics in Caenorhabditis elegans

    DEFF Research Database (Denmark)

    Fredens, Julius; Engholm-Keller, Kasper; Møller-Jensen, Jakob;

    2014-01-01

    knockdown by feeding the nematode on pre-labeled lysine auxotroph Escherichia coli. In this chapter, we describe in details the generation of the E. coli strain, incorporation of heavy isotope-labeled lysine in C. elegans, and the procedure for a comprehensive global phosphoproteomic experiment.......Stable isotope labeling by amino acids combined with mass spectrometry is a widely used methodology for measuring relative changes in protein and phosphorylation levels at a global level. We have applied this method to the model organism Caenorhabditis elegans in combination with RNAi-mediated gene...

  3. Phosphoproteomics Reveals Regulatory T Cell-Mediated DEF6 Dephosphorylation That Affects Cytokine Expression in Human Conventional T Cells

    KAUST Repository

    Joshi, Rubin N.

    2017-09-25

    Regulatory T cells (Tregs) control key events of immune tolerance, primarily by suppression of effector T cells. We previously revealed that Tregs rapidly suppress T cell receptor (TCR)-induced calcium store depletion in conventional CD4CD25 T cells (Tcons) independently of IP levels, consequently inhibiting NFAT signaling and effector cytokine expression. Here, we study Treg suppression mechanisms through unbiased phosphoproteomics of primary human Tcons upon TCR stimulation and Treg-mediated suppression, respectively. Tregs induced a state of overall decreased phosphorylation as opposed to TCR stimulation. We discovered novel phosphosites (T595_S597) in the DEF6 (SLAT) protein that were phosphorylated upon TCR stimulation and conversely dephosphorylated upon coculture with Tregs. Mutation of these DEF6 phosphosites abrogated interaction of DEF6 with the IP receptor and affected NFAT activation and cytokine transcription in primary Tcons. This novel mechanism and phosphoproteomics data resource may aid in modifying sensitivity of Tcons to Treg-mediated suppression in autoimmune disease or cancer.

  4. Analysis of T4SS-induced signaling by H. pylori using quantitative phosphoproteomics

    Directory of Open Access Journals (Sweden)

    Frithjof eGlowinski

    2014-07-01

    Full Text Available Helicobacter pylori is a Gram-negative bacterial pathogen colonizing the human stomach. Infection with H. pylori causes chronic inflammation of the gastric mucosa and may lead to peptic ulceration and/or gastric cancer. A major virulence determinant of H. pylori is the type IV secretion system (T4SS, which is used to inject the virulence factor CagA into the host cell, triggering a wide range of cellular signaling events. Here, we used a phosphoproteomic approach to investigate tyrosine signaling in response to host-pathogen interaction, using stable isotope labeling in cell culture (SILAC of AGS cells to obtain a differential picture between multiple infection conditions. Cells were infected with wild type H. pylori P12, a P12ΔCagA deletion mutant, and a P12ΔT4SS deletion mutant to compare signaling changes over time and in the absence of CagA or the T4SS. Tryptic peptides were enriched for tyrosine (Tyr phosphopeptides and analysed by nano-LC-Orbitrap MS. In total, 58 different phosphosites were found to be regulated following infection. The majority of phosphosites identified were kinases of the MAPK familiy. CagA and the T4SS were found to be key regulators of Tyr phosphosites. Our findings indicate that CagA primarily induces activation of ERK1 and integrin linked factors, whereas the T4SS primarily modulates JNK and p38 activation.

  5. Quantitative phosphoproteomic analyses of the inferior parietal lobule from three different pathological stages of Alzheimer's disease.

    Science.gov (United States)

    Triplett, Judy C; Swomley, Aaron M; Cai, Jian; Klein, Jon B; Butterfield, D Allan

    2015-01-01

    Alzheimer's disease (AD), the most common age-related neurodegenerative disorder, is clinically characterized by progressive neuronal loss resulting in loss of memory and dementia. AD is histopathologically characterized by the extensive distribution of senile plaques and neurofibrillary tangles, and synapse loss. Amnestic mild cognitive impairment (MCI) is generally accepted to be an early stage of AD. MCI subjects have pathology and symptoms that fall on the scale intermediately between 'normal' cognition with little or no pathology and AD. A rare number of individuals, who exhibit normal cognition on psychometric tests but whose brains show widespread postmortem AD pathology, are classified as 'asymptomatic' or 'preclinical' AD (PCAD). In this study, we evaluated changes in protein phosphorylation states in the inferior parietal lobule of subjects with AD, MCI, PCAD, and control brain using a 2-D PAGE proteomics approach in conjunction with Pro-Q Diamond phosphoprotein staining. Statistically significant changes in phosphorylation levels were found in 19 proteins involved in energy metabolism, neuronal plasticity, signal transduction, and oxidative stress response. Changes in the disease state phosphoproteome may provide insights into underlying mechanisms for the preservation of memory with expansive AD pathology in PCAD and the progressive memory loss in amnestic MCI that escalates to the dementia and the characteristic pathology of AD brain.

  6. Analysis of EGFR signaling pathway in nasopharyngeal carcinoma cells by quantitative phosphoproteomics

    Directory of Open Access Journals (Sweden)

    He Qiu-Yan

    2011-06-01

    Full Text Available Abstract Background The epidermal growth factor receptor (EGFR is usually overexpressed in nasopharyngeal carcinoma (NPC and is associated with pathogenesis of NPC. However, the downstream signaling proteins of EGFR in NPC have not yet been completely understood at the system level. The aim of this study was identify novel downstream proteins of EGFR signaling pathway in NPC cells. Results We analyzed EGFR-regulated phosphoproteome in NPC CNE2 cells using 2D-DIGE and mass spectrometry analysis after phosphoprotein enrichment. As a result, 33 nonredundant phosphoproteins including five known EGFR-regulated proteins and twenty-eight novel EGFR-regulated proteins in CNE2 were identified, three differential phosphoproteins were selectively validated, and two differential phosphoproteins (GSTP1 and GRB2 were showed interacted with phospho-EGFR. Bioinformatics analysis showed that 32 of 33 identified proteins contain phosphorylation modification sites, and 17 identified proteins are signaling proteins. GSTP1, one of the EGFR-regulated proteins, associated with chemoresistance was analyzed. The results showed that GSTP1 could contribute to paclitaxel resistance in EGF-stimulated CNE2 cells. Furthermore, an EGFR signaling network based on the identified EGFR-regulated phosphoproteins were constructed using Pathway Studio 5.0 software, which includes canonical and novel EGFR-regulated proteins and implicates the possible biological roles for those proteins. Conclusion The data not only can extend our knowledge of canonical EGFR signaling, but also will be useful to understand the molecular mechanisms of EGFR in NPC pathogenesis and search therapeutic targets for NPC.

  7. Quantitative phosphoproteomics of murine Fmr1-KO cell lines provides new insights into FMRP-dependent signal transduction mechanisms.

    Science.gov (United States)

    Matic, Katarina; Eninger, Timo; Bardoni, Barbara; Davidovic, Laetitia; Macek, Boris

    2014-10-03

    Fragile X mental retardation protein (FMRP) is an RNA-binding protein that has a major effect on neuronal protein synthesis. Transcriptional silencing of the FMR1 gene leads to loss of FMRP and development of Fragile X syndrome (FXS), the most common known hereditary cause of intellectual impairment and autism. Here we utilize SILAC-based quantitative phosphoproteomics to analyze murine FMR1(-) and FMR1(+) fibroblastic cell lines derived from FMR1-KO embryos to identify proteins and phosphorylation sites dysregulated as a consequence of FMRP loss. We quantify FMRP-related changes in the levels of 5,023 proteins and 6,133 phosphorylation events and map them onto major signal transduction pathways. Our study confirms global downregulation of the MAPK/ERK pathway and decrease in phosphorylation level of ERK1/2 in the absence of FMRP, which is connected to attenuation of long-term potentiation. We detect differential expression of several key proteins from the p53 pathway, pointing to the involvement of p53 signaling in dysregulated cell cycle control in FXS. Finally, we detect differential expression and phosphorylation of proteins involved in pre-mRNA processing and nuclear transport, as well as Wnt and calcium signaling, such as PLC, PKC, NFAT, and cPLA2. We postulate that calcium homeostasis is likely affected in molecular pathogenesis of FXS.

  8. Phosphoproteomics Reveals HMGA1, a CK2 Substrate, as a Drug-Resistant Target in Non-Small Cell Lung Cancer

    Science.gov (United States)

    Wang, Yi-Ting; Pan, Szu-Hua; Tsai, Chia-Feng; Kuo, Ting-Chun; Hsu, Yuan-Ling; Yen, Hsin-Yung; Choong, Wai-Kok; Wu, Hsin-Yi; Liao, Yen-Chen; Hong, Tse-Ming; Sung, Ting-Yi; Yang, Pan-Chyr; Chen, Yu-Ju

    2017-01-01

    Although EGFR tyrosine kinase inhibitors (TKIs) have demonstrated good efficacy in non-small-cell lung cancer (NSCLC) patients harboring EGFR mutations, most patients develop intrinsic and acquired resistance. We quantitatively profiled the phosphoproteome and proteome of drug-sensitive and drug-resistant NSCLC cells under gefitinib treatment. The construction of a dose-dependent responsive kinase-substrate network of 1548 phosphoproteins and 3834 proteins revealed CK2-centric modules as the dominant core network for the potential gefitinib resistance-associated proteins. CK2 knockdown decreased cell survival in gefitinib-resistant NSCLCs. Using motif analysis to identify the CK2 core sub-network, we verified that elevated phosphorylation level of a CK2 substrate, HMGA1 was a critical node contributing to EGFR-TKI resistance in NSCLC cell. Both HMGA1 knockdown or mutation of the CK2 phosphorylation site, S102, of HMGA1 reinforced the efficacy of gefitinib in resistant NSCLC cells through reactivation of the downstream signaling of EGFR. Our results delineate the TKI resistance-associated kinase-substrate network, suggesting a potential therapeutic strategy for overcoming TKI-induced resistance in NSCLC. PMID:28290473

  9. Phosphoproteomics reveals the effect of ethylene in soybean root under flooding stress.

    Science.gov (United States)

    Yin, Xiaojian; Sakata, Katsumi; Komatsu, Setsuko

    2014-12-05

    Flooding has severe negative effects on soybean growth. To explore the flooding-responsive mechanisms in early-stage soybean, a phosphoproteomic approach was used. Two-day-old soybean plants were treated without or with flooding for 3, 6, 12, and 24 h, and root tip proteins were then extracted and analyzed at each time point. After 3 h of flooding exposure, the fresh weight of soybeans increased, whereas the ATP content of soybean root tips decreased. Using a gel-free proteomic technique, a total of 114 phosphoproteins were identified in the root tip samples, and 34 of the phosphoproteins were significantly changed with respect to phosphorylation status after 3 h of flooding stress. Among these phosphoproteins, eukaryotic translation initiation factors were dephosphorylated, whereas several protein synthesis-related proteins were phosphorylated. The mRNA expression levels of sucrose phosphate synthase 1F and eukaryotic translation initiation factor 4 G were down-regulated, whereas UDP-glucose 6-dehydrogenase mRNA expression was up-regulated during growth but down-regulated under flooding stress. Furthermore, bioinformatic protein interaction analysis of flooding-responsive proteins based on temporal phosphorylation patterns indicated that eukaryotic translation initiation factor 4 G was located in the center of the network during flooding. Soybean eukaryotic translation initiation factor 4 G has homology to programmed cell death 4 protein and is implicated in ethylene signaling. The weight of soybeans was increased with treatment by an ethylene-releasing agent under flooding condition, but it was decreased when plants were exposed to an ethylene receptor antagonist. These results suggest that the ethylene signaling pathway plays an important role, via the protein phosphorylation, in mechanisms of plant tolerance to the initial stages of flooding stress in soybean root tips.

  10. Comparative phosphoproteomics reveals components of host cell invasion and post-transcriptional regulation during Francisella infection

    Energy Technology Data Exchange (ETDEWEB)

    Nakayasu, Ernesto S.; Tempel, Rebecca; Cambronne, Xiaolu A.; Petyuk, Vladislav A.; Jones, Marcus B.; Gritsenko, Marina A.; Monroe, Matthew E.; Yang, Feng; Smith, Richard D.; Adkins, Joshua N.; Heffron, Fred

    2013-09-22

    Francisella tularensis is a facultative intracellular bacterium that causes the deadly disease tularemia. Most evidence suggests that Francisella is not well recognized by the innate immune system that normally leads to cytokine expression and cell death. In previous work, we identified new bacterial factors that were hyper-cytotoxic to macrophages. Four of the identified hyper-cytotoxic strains (lpcC, manB, manC and kdtA) had an impaired lipopolysaccharide (LPS) synthesis and produced an exposed lipid A lacking the O-antigen. These mutants were not only hyper-cytotoxic but also were phagocytosed at much higher rates compared to the wild type parent strain. To elucidate the cellular signaling underlying this enhanced phagocytosis and cell death, we performed a large-scale comparative phosphoproteomic analysis of cells infected with wild-type and delta-lpcC F. novicida. Our data suggest that not only actin but also intermediate filaments and microtubules are important for F. novicida entry into the host cells. In addition, we observed differential phosphorylation of tristetraprolin (TTP), a key component of the mRNA-degrading machinery that controls the expression of a variety of genes including many cytokines. Infection with the delta-lpcC mutant induced the hyper-phosphorylation and inhibition of TTP, leading to the production of cytokines such as IL-1beta and TNF-alpha which may kill the host cells by triggering apoptosis. Together, our data provide new insights for Francisella invasion and a post-transcriptional mechanism that prevents the expression of host immune response factors that controls infection by this pathogen.

  11. Comparative Phosphoproteomics Reveals Components of Host Cell Invasion and Post-transcriptional Regulation During Francisella Infection*

    Science.gov (United States)

    Nakayasu, Ernesto S.; Tempel, Rebecca; Cambronne, Xiaolu A.; Petyuk, Vladislav A.; Jones, Marcus B.; Gritsenko, Marina A.; Monroe, Matthew E.; Yang, Feng; Smith, Richard D.; Adkins, Joshua N.; Heffron, Fred

    2013-01-01

    Francisella tularensis is a facultative intracellular bacterium that causes the deadly disease tularemia. Most evidence suggests that Francisella is not well recognized by the innate immune system that normally leads to cytokine expression and cell death. In previous work, we identified new bacterial factors that were hyper-cytotoxic to macrophages. Four of the identified hyper-cytotoxic strains (lpcC, manB, manC, and kdtA) had an impaired lipopolysaccharide (LPS) synthesis and produced an exposed lipid A lacking the O-antigen. These mutants were not only hyper-cytotoxic but also were phagocytosed at much higher rates compared with the wild type parent strain. To elucidate the cellular signaling underlying this enhanced phagocytosis and cell death, we performed a large-scale comparative phosphoproteomic analysis of cells infected with wild-type and delta-lpcC F. novicida. Our data suggest that not only actin but also intermediate filaments and microtubules are important for F. novicida entry into the host cells. In addition, we observed differential phosphorylation of tristetraprolin, a key component of the mRNA-degrading machinery that controls the expression of a variety of genes including many cytokines. Infection with the delta-lpcC mutant induced the hyper-phosphorylation and inhibition of tristetraprolin, leading to the production of cytokines such as IL-1beta and TNF-alpha that may kill the host cells by triggering apoptosis. Together, our data provide new insights for Francisella invasion and a post-transcriptional mechanism that prevents the expression of host immune response factors that control infection by this pathogen. PMID:23970565

  12. Comparative phosphoproteomics reveals components of host cell invasion and post-transcriptional regulation during Francisella infection.

    Science.gov (United States)

    Nakayasu, Ernesto S; Tempel, Rebecca; Cambronne, Xiaolu A; Petyuk, Vladislav A; Jones, Marcus B; Gritsenko, Marina A; Monroe, Matthew E; Yang, Feng; Smith, Richard D; Adkins, Joshua N; Heffron, Fred

    2013-11-01

    Francisella tularensis is a facultative intracellular bacterium that causes the deadly disease tularemia. Most evidence suggests that Francisella is not well recognized by the innate immune system that normally leads to cytokine expression and cell death. In previous work, we identified new bacterial factors that were hyper-cytotoxic to macrophages. Four of the identified hyper-cytotoxic strains (lpcC, manB, manC, and kdtA) had an impaired lipopolysaccharide (LPS) synthesis and produced an exposed lipid A lacking the O-antigen. These mutants were not only hyper-cytotoxic but also were phagocytosed at much higher rates compared with the wild type parent strain. To elucidate the cellular signaling underlying this enhanced phagocytosis and cell death, we performed a large-scale comparative phosphoproteomic analysis of cells infected with wild-type and delta-lpcC F. novicida. Our data suggest that not only actin but also intermediate filaments and microtubules are important for F. novicida entry into the host cells. In addition, we observed differential phosphorylation of tristetraprolin, a key component of the mRNA-degrading machinery that controls the expression of a variety of genes including many cytokines. Infection with the delta-lpcC mutant induced the hyper-phosphorylation and inhibition of tristetraprolin, leading to the production of cytokines such as IL-1beta and TNF-alpha that may kill the host cells by triggering apoptosis. Together, our data provide new insights for Francisella invasion and a post-transcriptional mechanism that prevents the expression of host immune response factors that control infection by this pathogen.

  13. Quantitative phosphoproteomic analysis of neuronal intermediate filament proteins (NF-M/H) in Alzheimer's disease by iTRAQ.

    Science.gov (United States)

    Rudrabhatla, Parvathi; Grant, Philip; Jaffe, Howard; Strong, Michael J; Pant, Harish C

    2010-11-01

    Aberrant hyperphosphorylation of neuronal cytoskeletal proteins is one of the major pathological hallmarks of neurodegenerative disorders such as Alzheimer disease (AD), amyotrophic lateral sclerosis (ALS), and Parkinson's disease (PD). Human NF-M/H display a large number of multiple KSP repeats in the carboxy-terminal tail domain, which are phosphorylation sites of proline-directed serine/threonine (pSer/Thr-Pro, KS/T-P) kinases. The phosphorylation sites of NF-M/H have not been characterized in AD brain. Here, we use quantitative phosphoproteomic methodology, isobaric tag for relative and absolute quantitation (iTRAQ), for the characterization of NF-M/H phosphorylation sites in AD brain. We identified 13 hyperphosphorylated sites of NF-M; 9 Lys-Ser-Pro (KSP) sites; 2 variant motifs, Glu-Ser-Pro (ESP) Ser-736 and Leu-Ser-Pro (LSP) Ser-837; and 2 non-S/T-P motifs, Ser-783 and Ser-788. All the Ser/Thr residues are phosphorylated at significantly greater abundance in AD brain compared with control brain. Ten hyperphosphorylated KSP sites have been identified on the C-terminal tail domain of NF-H, with greater abundance of phosphorylation in AD brain compared with control brain. Our data provide the direct evidence that NF-M/H are hyperphosphorylated in AD compared with control brain and suggest the role of both proline-directed and non-proline-directed protein kinases in AD. This study represents the first comprehensive iTRAQ analyses and quantification of phosphorylation sites of human NF-M and NF-H from AD brain and suggests that aberrant hyperphosphorylation of neuronal intermediate filament proteins is involved in AD.

  14. Identification of Mediator Kinase Substrates in Human Cells using Cortistatin A and Quantitative Phosphoproteomics

    Directory of Open Access Journals (Sweden)

    Zachary C. Poss

    2016-04-01

    Full Text Available Cortistatin A (CA is a highly selective inhibitor of the Mediator kinases CDK8 and CDK19. Using CA, we now report a large-scale identification of Mediator kinase substrates in human cells (HCT116. We identified over 16,000 quantified phosphosites including 78 high-confidence Mediator kinase targets within 64 proteins, including DNA-binding transcription factors and proteins associated with chromatin, DNA repair, and RNA polymerase II. Although RNA-seq data correlated with Mediator kinase targets, the effects of CA on gene expression were limited and distinct from CDK8 or CDK19 knockdown. Quantitative proteome analyses, tracking around 7,000 proteins across six time points (0–24 hr, revealed that CA selectively affected pathways implicated in inflammation, growth, and metabolic regulation. Contrary to expectations, increased turnover of Mediator kinase targets was not generally observed. Collectively, these data support Mediator kinases as regulators of chromatin and RNA polymerase II activity and suggest their roles extend beyond transcription to metabolism and DNA repair.

  15. Quantitative Site-Specific Phosphoproteomics of Trichoderma reesei Signaling Pathways upon Induction of Hydrolytic Enzyme Production.

    Science.gov (United States)

    Nguyen, Elizabeth V; Imanishi, Susumu Y; Haapaniemi, Pekka; Yadav, Avinash; Saloheimo, Markku; Corthals, Garry L; Pakula, Tiina M

    2016-02-01

    The filamentous fungus Trichoderma reesei is used for industrial production of secreted enzymes including carbohydrate active enzymes, such as cellulases and hemicellulases. The production of many of these enzymes by T. reesei is influenced by the carbon source it grows on, where the regulation system controlling hydrolase genes involves various signaling pathways. T. reesei was cultivated in the presence of sorbitol, a carbon source that does not induce the production of cellulases and hemicellulases, and then exposed to either sophorose or spent-grain extract, which are efficient inducers of the enzyme production. Specific changes at phosphorylation sites were investigated in relation to the production of cellulases and hemicellulases using an MS-based framework. Proteome-wide phosphorylation following carbon source exchange was investigated in the early stages of induction: 0, 2, 5, and 10 min. The workflow involved sequential trypsin digestion, TiO2 enrichment, and MS analysis using a Q Exactive mass spectrometer. We report on the identification and quantitation of 1721 phosphorylation sites. Investigation of the data revealed a complex signaling network activated upon induction involving components related to light-mediated cellulase induction, osmoregulation, and carbon sensing. Changes in protein phosphorylation were detected in the glycolytic pathway, suggesting an inhibition of glucose catabolism at 10 min after the addition of sophorose and as early as 2 min after the addition of spent-grain extract. Differential phosphorylation of factors related to carbon storage, intracellular trafficking, cytoskeleton, and cellulase gene regulation were also observed.

  16. Phosphoproteomics Profiling of Human Skin Fibroblast Cells Reveals Pathways and Proteins Affected by Low Doses of Ionizing Radiation

    Science.gov (United States)

    Yang, Feng; Waters, Katrina M.; Miller, John H.; Gritsenko, Marina A.; Zhao, Rui; Du, Xiuxia; Livesay, Eric A.; Purvine, Samuel O.; Monroe, Matthew E.; Wang, Yingchun; Camp, David G.; Smith, Richard D.; Stenoien, David L.

    2010-01-01

    Background High doses of ionizing radiation result in biological damage; however, the precise relationships between long-term health effects, including cancer, and low-dose exposures remain poorly understood and are currently extrapolated using high-dose exposure data. Identifying the signaling pathways and individual proteins affected at the post-translational level by radiation should shed valuable insight into the molecular mechanisms that regulate dose-dependent responses to radiation. Principal Findings We have identified 7117 unique phosphopeptides (2566 phosphoproteins) from control and irradiated (2 and 50 cGy) primary human skin fibroblasts 1 h post-exposure. Semi-quantitative label-free analyses were performed to identify phosphopeptides that are apparently altered by radiation exposure. This screen identified phosphorylation sites on proteins with known roles in radiation responses including TP53BP1 as well as previously unidentified radiation-responsive proteins such as the candidate tumor suppressor SASH1. Bioinformatic analyses suggest that low and high doses of radiation affect both overlapping and unique biological processes and suggest a role for MAP kinase and protein kinase A (PKA) signaling in the radiation response as well as differential regulation of p53 networks at low and high doses of radiation. Conclusions Our results represent the most comprehensive analysis of the phosphoproteomes of human primary fibroblasts exposed to multiple doses of ionizing radiation published to date and provide a basis for the systems-level identification of biological processes, molecular pathways and individual proteins regulated in a dose dependent manner by ionizing radiation. Further study of these modified proteins and affected networks should help to define the molecular mechanisms that regulate biological responses to radiation at different radiation doses and elucidate the impact of low-dose radiation exposure on human health. PMID:21152398

  17. Phosphoproteomics profiling of human skin fibroblast cells reveals pathways and proteins affected by low doses of ionizing radiation.

    Directory of Open Access Journals (Sweden)

    Feng Yang

    Full Text Available BACKGROUND: High doses of ionizing radiation result in biological damage; however, the precise relationships between long-term health effects, including cancer, and low-dose exposures remain poorly understood and are currently extrapolated using high-dose exposure data. Identifying the signaling pathways and individual proteins affected at the post-translational level by radiation should shed valuable insight into the molecular mechanisms that regulate dose-dependent responses to radiation. PRINCIPAL FINDINGS: We have identified 7117 unique phosphopeptides (2566 phosphoproteins from control and irradiated (2 and 50 cGy primary human skin fibroblasts 1 h post-exposure. Semi-quantitative label-free analyses were performed to identify phosphopeptides that are apparently altered by radiation exposure. This screen identified phosphorylation sites on proteins with known roles in radiation responses including TP53BP1 as well as previously unidentified radiation-responsive proteins such as the candidate tumor suppressor SASH1. Bioinformatic analyses suggest that low and high doses of radiation affect both overlapping and unique biological processes and suggest a role for MAP kinase and protein kinase A (PKA signaling in the radiation response as well as differential regulation of p53 networks at low and high doses of radiation. CONCLUSIONS: Our results represent the most comprehensive analysis of the phosphoproteomes of human primary fibroblasts exposed to multiple doses of ionizing radiation published to date and provide a basis for the systems-level identification of biological processes, molecular pathways and individual proteins regulated in a dose dependent manner by ionizing radiation. Further study of these modified proteins and affected networks should help to define the molecular mechanisms that regulate biological responses to radiation at different radiation doses and elucidate the impact of low-dose radiation exposure on human health.

  18. Quantitative Phosphoproteome Analysis of Lysophosphatidic Acid Induced Chemotaxis applying Dual-step ¹⁸O Labeling Coupled with Immobilized Metal-ion Affinity Chromatography

    Energy Technology Data Exchange (ETDEWEB)

    Ding, Shi-Jian; Wang, Yingchun; Jacobs, Jon M.; Qian, Weijun; Yang, Feng; Tolmachev, Aleksey V.; Du, Xiuxia; Wang, Wei; Moore, Ronald J.; Monroe, Matthew E.; Purvine, Samuel O.; Waters, Katrina M.; Heibeck, Tyler H.; Adkins, Joshua N.; Camp, David G.; Klemke, Richard L.; Smith, Richard D.

    2008-10-01

    Reversible protein phosphorylation is a central cellular regulatory mechanism in modulating protein activity and propagating signals within cellular pathways and networks. Development of more effective methods for the simultaneous identification of phosphorylation sites and quantification of temporal changes in protein phosphorylation could provide important insights into molecular signaling mechanisms in a variety of different cellular processes. Here we present an integrated quantitative phosphoproteomics approach and its applications for comparative analysis of Cos-7 cells in response to lysophosphatidic acid (LPA) gradient stimulation. The approach combines trypsin-catalyzed 16O/18O labeling plus 16O/18O-methanol esterification labeling for quantitation, a macro- Immobilized Metal-ion Affinity Chromatography trap for phosphopeptide enrichment, and a monolithic capillary column with integrated electrospray emitter. LC separation and MS/MS is followed by neutral loss-dependent MS/MS/MS for phosphopeptide identification using a linear ion trap (LTQ)-FT mass spectrometer and complementary searching algorithms for interpreting MS/MS spectra. Protein phosphorylation involved in various signaling pathways of cell migration were identified and quantified, such as mitogen-activated protein kinase 1, dual-specificity mitogen-activated protein kinase kinase 2, and dual-specificity tyrosine-phosphorylation regulated kinase 1b, and a number of Rho GTPase-activating proteins. These results demonstrate the efficiency of this quantitative phosphoproteomics approach and its application for rapid discovery of phosphorylation events associated with gradient sensing and cell chemotaxis.

  19. Phosphoproteomics of collagen receptor networks reveals SHP-2 phosphorylation downstream of wild-type DDR2 and its lung cancer mutants.

    Science.gov (United States)

    Iwai, Leo K; Payne, Leo S; Luczynski, Maciej T; Chang, Francis; Xu, Huifang; Clinton, Ryan W; Paul, Angela; Esposito, Edward A; Gridley, Scott; Leitinger, Birgit; Naegle, Kristen M; Huang, Paul H

    2013-09-15

    Collagen is an important extracellular matrix component that directs many fundamental cellular processes including differentiation, proliferation and motility. The signalling networks driving these processes are propagated by collagen receptors such as the β1 integrins and the DDRs (discoidin domain receptors). To gain an insight into the molecular mechanisms of collagen receptor signalling, we have performed a quantitative analysis of the phosphorylation networks downstream of collagen activation of integrins and DDR2. Temporal analysis over seven time points identified 424 phosphorylated proteins. Distinct DDR2 tyrosine phosphorylation sites displayed unique temporal activation profiles in agreement with in vitro kinase data. Multiple clustering analysis of the phosphoproteomic data revealed several DDR2 candidate downstream signalling nodes, including SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2), NCK1 (non-catalytic region of tyrosine kinase adaptor protein 1), LYN, SHIP-2 [SH2 (Src homology 2)-domain-containing inositol phosphatase 2], PIK3C2A (phosphatidylinositol-4-phosphate 3-kinase, catalytic subunit type 2α) and PLCL2 (phospholipase C-like 2). Biochemical validation showed that SHP-2 tyrosine phosphorylation is dependent on DDR2 kinase activity. Targeted proteomic profiling of a panel of lung SCC (squamous cell carcinoma) DDR2 mutants demonstrated that SHP-2 is tyrosine-phosphorylated by the L63V and G505S mutants. In contrast, the I638F kinase domain mutant exhibited diminished DDR2 and SHP-2 tyrosine phosphorylation levels which have an inverse relationship with clonogenic potential. Taken together, the results of the present study indicate that SHP-2 is a key signalling node downstream of the DDR2 receptor which may have therapeutic implications in a subset of DDR2 mutations recently uncovered in genome-wide lung SCC sequencing screens.

  20. Phosphoproteomic Analysis of KSHV-Infected Cells Reveals Roles of ORF45-Activated RSK during Lytic Replication.

    Directory of Open Access Journals (Sweden)

    Denis Avey

    2015-07-01

    Full Text Available Kaposi's Sarcoma-Associated Herpesvirus (KSHV is an oncogenic virus which has adapted unique mechanisms to modulate the cellular microenvironment of its human host. The pathogenesis of KSHV is intimately linked to its manipulation of cellular signaling pathways, including the extracellular signal-regulated kinase (ERK mitogen-activated protein kinase (MAPK pathway. We have previously shown that KSHV ORF45 contributes to the sustained activation of both ERK and p90 ribosomal S6 kinase (RSK, a major functional mediator of ERK/MAPK signaling during KSHV lytic replication. ORF45-activated RSK is required for optimal KSHV lytic gene expression and progeny virion production, though the underlying mechanisms downstream of this activation are still unclear. We hypothesized that the activation of RSK by ORF45 causes differential phosphorylation of cellular and viral substrates, affecting biological processes essential for efficient KSHV lytic replication. Accordingly, we observed widespread and significant differences in protein phosphorylation upon induction of lytic replication. Mass-spectrometry-based phosphoproteomic screening identified putative substrates of ORF45-activated RSK in KSHV-infected cells. Bioinformatic analyses revealed that nuclear proteins, including several transcriptional regulators, were overrepresented among these candidates. We validated the ORF45/RSK-dependent phosphorylation of several putative substrates by employing KSHV BAC mutagenesis, kinase inhibitor treatments, and/or CRISPR-mediated knockout of RSK in KSHV-infected cells. Furthermore, we assessed the consequences of knocking out these substrates on ORF45/RSK-dependent regulation of gene expression and KSHV progeny virion production. Finally, we show data to support that ORF45 regulates the translational efficiency of a subset of viral/cellular genes with complex secondary structure in their 5' UTR. Altogether, these data shed light on the mechanisms by which KSHV ORF45

  1. Quantitative phosphoproteomics dissection of seven-transmembrane receptor signaling using full and biased agonists

    DEFF Research Database (Denmark)

    Christensen, Gitte L; Kelstrup, Christian D; Lyngsø, Christina

    2010-01-01

    (q)-dependent and -independent AT(1)R signaling. This study provides substantial novel insight into angiotensin II signal transduction and is the first study dissecting the differences between a full agonist and a biased agonist from a 7TMR on a systems-wide scale. Importantly, it reveals a previously unappreciated diversity...

  2. Quantitative phosphoproteomics dissection of 7TM receptor signaling using full and biased agonists

    DEFF Research Database (Denmark)

    Christensen, Gitte Lund; Kelstrup, Christian; Lyngsø, Christina

    2010-01-01

    into Angiotensin II signal transduction and is the first study dissecting the differences between a full agonist and a biased agonist from a 7TMR on a systems-wide scale. Importantly, it reveals a previously unappreciated diversity and quantity of Gaq protein-independent signaling and uncovers novel signaling...

  3. Targeted quantitative phosphoproteomic analysis of erythrocyte membranes during blood bank storage.

    Science.gov (United States)

    Rinalducci, Sara; Longo, Valentina; Ceci, Luigi R; Zolla, Lello

    2015-02-01

    One of the hallmarks of blood bank stored red blood cells (RBCs) is the irreversible transition from a discoid to a spherocyte-like morphology with membrane perturbation and cytoskeleton disorders. Therefore, identification of the storage-associated modifications in the protein-protein interactions between the cytoskeleton and the lipid bilayer may contribute to enlighten the molecular mechanisms involved in the alterations of mechanical properties of stored RBCs. Here we report the results obtained analyzing RBCs after 0, 21 and 35 days of storage under standard blood banking conditions by label free mass spectrometry (MS)-based experiments. We could quantitatively measure changes in the phosphorylation level of crucial phosphopeptides belonging to β-spectrin, ankyrin-1, α-adducin, dematin, glycophorin A and glycophorin C proteins. Data have been validated by both western blotting and pseudo-Multiple Reaction Monitoring (MRM). Although each phosphopeptide showed a distinctive trend, a sharp increase in the phosphorylation level during the storage duration was observed. Phosphopeptide mapping and structural modeling analysis indicated that the phosphorylated residues localize in protein functional domains fundamental for the maintenance of membrane structural integrity. Along with previous morphological evidence acquired by electron microscopy, our results seem to indicate that 21-day storage may represent a key point for the molecular processes leading to the erythrocyte deformability reduction observed during blood storage. These findings could therefore be helpful in understanding and preventing the morphology-linked mechanisms responsible for the post-transfusion survival of preserved RBCs.

  4. The Phosphoproteomes of Plasmodium falciparum and Toxoplasma gondii reveal unusual adaptations within and beyond the parasites’ boundaries

    OpenAIRE

    Treeck, Moritz; Sanders, John L.; Elias, Joshua E.; John C Boothroyd

    2011-01-01

    Plasmodium falciparum and Toxoplasma gondii are obligate intracellular apicomplexan parasites that rapidly invade and extensively modify host cells. Protein phosphorylation is one mechanism by which these parasites can control such processes. Here we present a phosphoproteome analysis of peptides enriched from schizont stage P. falciparum and T. gondii tachyzoites that are either “intracellular” or purified away from host material. Using liquid chromatography and tandem mass-spectrometry we i...

  5. Global phosphoproteomic profiling reveals perturbed signaling in a mouse model of dilated cardiomyopathy

    Science.gov (United States)

    Kuzmanov, Uros; Guo, Hongbo; Buchsbaum, Diana; Cosme, Jake; Abbasi, Cynthia; Isserlin, Ruth; Sharma, Parveen; Gramolini, Anthony O.; Emili, Andrew

    2016-01-01

    Phospholamban (PLN) plays a central role in Ca2+ homeostasis in cardiac myocytes through regulation of the sarco(endo)plasmic reticulum Ca2+-ATPase 2A (SERCA2A) Ca2+ pump. An inherited mutation converting arginine residue 9 in PLN to cysteine (R9C) results in dilated cardiomyopathy (DCM) in humans and transgenic mice, but the downstream signaling defects leading to decompensation and heart failure are poorly understood. Here we used precision mass spectrometry to study the global phosphorylation dynamics of 1,887 cardiac phosphoproteins in early affected heart tissue in a transgenic R9C mouse model of DCM compared with wild-type littermates. Dysregulated phosphorylation sites were quantified after affinity capture and identification of 3,908 phosphopeptides from fractionated whole-heart homogenates. Global statistical enrichment analysis of the differential phosphoprotein patterns revealed selective perturbation of signaling pathways regulating cardiovascular activity in early stages of DCM. Strikingly, dysregulated signaling through the Notch-1 receptor, recently linked to cardiomyogenesis and embryonic cardiac stem cell development and differentiation but never directly implicated in DCM before, was a prominently perturbed pathway. We verified alterations in Notch-1 downstream components in early symptomatic R9C transgenic mouse cardiomyocytes compared with wild type by immunoblot analysis and confocal immunofluorescence microscopy. These data reveal unexpected connections between stress-regulated cell signaling networks, specific protein kinases, and downstream effectors essential for proper cardiac function. PMID:27742792

  6. Phosphoproteomic analysis of protein kinase C signaling in Saccharomyces cerevisiae reveals Slt2 mitogen-activated protein kinase (MAPK)-dependent phosphorylation of eisosome core components.

    Science.gov (United States)

    Mascaraque, Victoria; Hernáez, María Luisa; Jiménez-Sánchez, María; Hansen, Rasmus; Gil, Concha; Martín, Humberto; Cid, Víctor J; Molina, María

    2013-03-01

    The cell wall integrity (CWI) pathway of the model organism Saccharomyces cerevisiae has been thoroughly studied as a paradigm of the mitogen-activated protein kinase (MAPK) pathway. It consists of a classic MAPK module comprising the Bck1 MAPK kinase kinase, two redundant MAPK kinases (Mkk1 and Mkk2), and the Slt2 MAPK. This module is activated under a variety of stimuli related to cell wall homeostasis by Pkc1, the only member of the protein kinase C family in budding yeast. Quantitative phosphoproteomics based on stable isotope labeling of amino acids in cell culture is a powerful tool for globally studying protein phosphorylation. Here we report an analysis of the yeast phosphoproteome upon overexpression of a PKC1 hyperactive allele that specifically activates CWI MAPK signaling in the absence of external stimuli. We found 82 phosphopeptides originating from 43 proteins that showed enhanced phosphorylation in these conditions. The MAPK S/T-P target motif was significantly overrepresented in these phosphopeptides. Hyperphosphorylated proteins provide putative novel targets of the Pkc1-cell wall integrity pathway involved in diverse functions such as the control of gene expression, protein synthesis, cytoskeleton maintenance, DNA repair, and metabolism. Remarkably, five components of the plasma-membrane-associated protein complex known as eisosomes were found among the up-regulated proteins. We show here that Pkc1-induced phosphorylation of the eisosome core components Pil1 and Lsp1 was not exerted directly by Pkc1, but involved signaling through the Slt2 MAPK module.

  7. Phosphoproteome reveals an atlas of protein signaling networks during osteoblast adhesion.

    Science.gov (United States)

    Milani, Renato; Ferreira, Carmen V; Granjeiro, José M; Paredes-Gamero, Edgar J; Silva, Rodrigo A; Justo, Giselle Z; Nader, Helena B; Galembeck, Eduardo; Peppelenbosch, Maikel P; Aoyama, Hiroshi; Zambuzzi, Willian F

    2010-04-01

    Cell adhesion on surfaces is a fundamental process in the emerging biomaterials field and developmental events as well. However, the mechanisms regulating this biological process in osteoblasts are not fully understood. Reversible phosphorylation catalyzed by kinases is probably the most important regulatory mechanism in eukaryotes. Therefore, the goal of this study is to assess osteoblast adhesion through a molecular prism under a peptide array technology, revealing essential signaling proteins governing adhesion-related events. First, we showed that there are main morphological changes on osteoblast shape during adhesion up to 3 h. Second, besides classical proteins activated upon integrin activation, our results showed a novel network involving signaling proteins such as Rap1A, PKA, PKC, and GSK3beta during osteoblast adhesion on polystyrene. Third, these proteins were grouped in different signaling cascades including focal adhesion establishment, cytoskeleton rearrangement, and cell-cycle arrest. We have thus provided evidence that a global phosphorylation screening is able to yield a systems-oriented look at osteoblast adhesion, providing new insights for understanding of bone formation and improvement of cell-substratum interactions. Altogether, these statements are necessary means for further intervention and development of new approaches for the progress of tissue engineering.

  8. Integrated phosphoproteomic and metabolomic profiling reveals NPM-ALK-mediated phosphorylation of PKM2 and metabolic reprogramming in anaplastic large cell lymphoma.

    Science.gov (United States)

    McDonnell, Scott R P; Hwang, Steven R; Rolland, Delphine; Murga-Zamalloa, Carlos; Basrur, Venkatesha; Conlon, Kevin P; Fermin, Damian; Wolfe, Thomas; Raskind, Alexander; Ruan, Chunhai; Jiang, Jian-Kang; Thomas, Craig J; Hogaboam, Cory M; Burant, Charles F; Elenitoba-Johnson, Kojo S J; Lim, Megan S

    2013-08-01

    The mechanisms underlying the pathogenesis of the constitutively active tyrosine kinase nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) expressing anaplastic large cell lymphoma are not completely understood. Here we show using an integrated phosphoproteomic and metabolomic strategy that NPM-ALK induces a metabolic shift toward aerobic glycolysis, increased lactate production, and biomass production. The metabolic shift is mediated through the anaplastic lymphoma kinase (ALK) phosphorylation of the tumor-specific isoform of pyruvate kinase (PKM2) at Y105, resulting in decreased enzymatic activity. Small molecule activation of PKM2 or expression of Y105F PKM2 mutant leads to reversal of the metabolic switch with increased oxidative phosphorylation and reduced lactate production coincident with increased cell death, decreased colony formation, and reduced tumor growth in an in vivo xenograft model. This study provides comprehensive profiling of the phosphoproteomic and metabolomic consequences of NPM-ALK expression and reveals a novel role of ALK in the regulation of multiple components of cellular metabolism. Our studies show that PKM2 is a novel substrate of ALK and plays a critical role in mediating the metabolic shift toward biomass production and tumorigenesis.

  9. Reconstruction of Insulin Signal Flow from Phosphoproteome and Metabolome Data

    Directory of Open Access Journals (Sweden)

    Katsuyuki Yugi

    2014-08-01

    Full Text Available Cellular homeostasis is regulated by signals through multiple molecular networks that include protein phosphorylation and metabolites. However, where and when the signal flows through a network and regulates homeostasis has not been explored. We have developed a reconstruction method for the signal flow based on time-course phosphoproteome and metabolome data, using multiple databases, and have applied it to acute action of insulin, an important hormone for metabolic homeostasis. An insulin signal flows through a network, through signaling pathways that involve 13 protein kinases, 26 phosphorylated metabolic enzymes, and 35 allosteric effectors, resulting in quantitative changes in 44 metabolites. Analysis of the network reveals that insulin induces phosphorylation and activation of liver-type phosphofructokinase 1, thereby controlling a key reaction in glycolysis. We thus provide a versatile method of reconstruction of signal flow through the network using phosphoproteome and metabolome data.

  10. Phosphoproteome analysis of functional mitochondria isolated from resting human muscle reveals extensive phosphorylation of inner membrane protein complexes and enzymes

    DEFF Research Database (Denmark)

    Zhao, Xiaolu; Leon, Ileana R; Bak, Steffen

    2011-01-01

    Mitochondria play a central role in energy metabolism and cellular survival, and consequently mitochondrial dysfunction is associated with a number of human pathologies. Reversible protein phosphorylation emerges as a central mechanism in the regulation of several mitochondrial processes. In skel......Mitochondria play a central role in energy metabolism and cellular survival, and consequently mitochondrial dysfunction is associated with a number of human pathologies. Reversible protein phosphorylation emerges as a central mechanism in the regulation of several mitochondrial processes....... In skeletal muscle, mitochondrial dysfunction is linked to insulin resistance in humans with obesity and type 2 diabetes. We performed a phosphoproteomic study of functional mitochondria isolated from human muscle biopsies with the aim to obtain a comprehensive overview of mitochondrial phosphoproteins...... for protein kinase A, protein kinase C, casein kinase II and DNA-dependent protein kinase. Our results demonstrate the feasibility of performing phosphoproteome analysis of organelles isolated from human tissue and provide novel targets for functional studies of reversible phosphorylation in mitochondria...

  11. Nuclear phosphoproteomics analysis reveals that CDK1/2 are involved in EGF-regulated constitutive pre-mRNA splicing in MDA-MB-468 cells.

    Science.gov (United States)

    Chen, Xianwei; Guo, Dan; Zhu, Yinghui; Xian, Feng; Liu, Siqi; Wu, Lin; Lou, Xiaomin

    2016-06-01

    The epidermal growth factor (EGF) receptor (EGFR) pathway is one of the most dysregulated and extensively investigated signaling pathways in human cancers and plays important roles in the regulation of nuclear functions through both cytoplasmic and nuclear EGFR pathways. However, the current understanding of the nuclear phosphorylation responses to activated EGFR pathways remains limited. In the present study, phosphoproteomics analysis revealed the increased phosphorylation of 90 nuclear proteins, primarily involved in RNA processing, pre-mRNA splicing and cell cycle regulation, upon EGF stimulation in MDA-MB-468 cells. Cellular splicing assays of the β-globin (HBB) minigene confirmed that EGF induced constitutive pre-mRNA splicing. Further analysis of phosphoproteomics data identified multiple CDK1/2 substrates in pre-mRNA splicing-related proteins, and both CDK1/2 inhibitors and CDK1/2 knockdowns reduced EGF-regulated pre-mRNA splicing. In conclusion, the results of the present study provide evidence that CDK1/2 participate in the regulation of constitutive pre-mRNA splicing by EGF stimulation in MDA-MB-468 cells. In this study, we successfully carried out a survey of nuclear phosphorylation changes in response to EGF stimulation. The results from the functional category analysis and pre-mRNA splicing assay strongly indicated that EGFR activation increased constitutive pre-mRNA splicing in MDA-MB-468 cells, revealing additional role of EGFR on regulation of mRNA maturation beyond alternative pre-mRNA splicing reported by previous studies. Furthermore, we found that CDK1/2 participated in constitutive pre-mRNA splicing regulation by EGF in MDA-MB-468 cells. Our study provides new knowledge for understanding the regulation of constitutive pre-mRNA splicing by EGF stimulation. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. In vivo Phosphoproteome of Human Skeletal Muscle Revealed by Phosphopeptide Enrichment and HPLC-ESI-MS/MS

    DEFF Research Database (Denmark)

    Højlund, Kurt; Bowen, Benjamin P; Hwang, Hyonson

    2009-01-01

    Protein phosphorylation plays an essential role in signal transduction pathways that regulate substrate and energy metabolism, contractile function, and muscle mass in human skeletal muscle. Abnormal phosphorylation of signaling enzymes has been identified in insulin resistant muscle using...... phosphoepitope-specific antibodies, but its role in other skeletal muscle disorders remains largely unknown. This may be in part due to insufficient knowledge of relevant targets. Here, we therefore present the first large-scale in vivo phosphoproteomic study of human skeletal muscle from 3 lean, healthy...... 240 phosphoserines, 53 phosphothreonines and 13 phosphotyrosines in at least 2 out of 3 subjects. In addition, 61 ambiguous phosphorylation sites were identified in at least 2 out of 3 subjects. The majority of phosphoproteins detected are involved in sarcomeric function, excitation...

  13. SILAC for global phosphoproteomic analysis.

    Science.gov (United States)

    Pimienta, Genaro; Chaerkady, Raghothama; Pandey, Akhilesh

    2009-01-01

    Establishing the phosphorylation pattern of proteins in a comprehensive fashion is an important goal of a majority of cell signaling projects. Phosphoproteomic strategies should be designed in such a manner as to identify sites of phosphorylation as well as to provide quantitative information about the extent of phosphorylation at the sites. In this chapter, we describe an experimental strategy that outlines such an approach using stable isotope labeling with amino acids in cell culture (SILAC) coupled to LC-MS/MS. We highlight the importance of quantitative strategies in signal transduction as a platform for a systematic and global elucidation of biological processes.

  14. Phosphoproteomics profiling of human skin fibroblast cells reveals pathways and proteins affected by low doses of ionizing radiation

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Feng; Waters, Katrina M.; Miller, John H.; Gritsenko, Marina A.; Zhao, Rui; Du, Xiuxia; Livesay, Eric A.; Purvine, Samuel O.; Monroe, Matthew E.; Wang, Yingchun; Camp, David G.; Smith, Richard D.; Stenoien, David L.

    2010-11-30

    Background: High doses of ionizing radiation result in biological damage, however the precise relationships between long term health effects, including cancer, and low dose exposures remain poorly understood and are currently extrapolated using high dose exposure data. Identifying the signaling pathways and individual proteins affected at the post-translational level by radiation should shed valuable insight into the molecular mechanisms that regulate dose dependent responses to radiation. Principle Findings: We have identified 6845 unique phosphopeptides (2566 phosphoproteins) from control and irradiated (2 and 50 cGy) primary human skin fibroblasts one hour post-exposure. Dual statistical analyses based on spectral counts and peak intensities identified 287 phosphopeptides (from 231 proteins) and 244 phosphopeptides (from 182 proteins) that varied significantly following exposure to 2 and 50 cGy respectively. This screen identified phosphorylation sites on proteins with known roles in radiation responses including TP53BP1 as well as previously unidentified radiation responsive proteins such as the candidate tumor suppressor SASH1. Bioinformatics analyses suggest that low and high doses of radiation affect both overlapping and unique biological processes and suggest a role of MAP kinase and protein kinase A (PKA) signaling in the radiation response as well as differential regulation of p53 networks at low and high doses of radiation. Conlcusions: Our results represent the most comprehensive analysis of the phosphoproteomes of human primary fibroblasts exposed to multiple doses of ionizing radiation published to date and provides a basis for the systems level identification of biological processes, molecular pathways and individual proteins regulated in a dose dependent manner by ionizing radiation. Further study of these modified proteins and affected networks should help to define the molecular mechanisms that regulate biological responses to radiation at

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

    Science.gov (United States)

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

    2013-06-07

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

  16. SILAC-Based Temporal Phosphoproteomics

    DEFF Research Database (Denmark)

    Francavilla, Chiara; Hekmat, Omid; Blagoev, Blagoy;

    2014-01-01

    signaling events. Here we provide an optimized SILAC-based proteomic workflow to analyze temporal changes in phosphoproteomes, which involve a generic three step enrichment protocol for phosphopeptides. SILAC-labeled peptides from digested whole cell lysates are as a first step enriched for phosphorylated...... pathways. Quantitative proteomics that combines stable isotope labeling by amino acid in cell culture (SILAC) with enrichment strategies for post-translational modification-bearing peptides and high-performance tandem mass spectrometry represents a powerful and unbiased approach to monitor dynamic...

  17. A high-resolution tissue-specific proteome and phosphoproteome atlas of maize primary roots reveals functional gradients along the root axes.

    Science.gov (United States)

    Marcon, Caroline; Malik, Waqas Ahmed; Walley, Justin W; Shen, Zhouxin; Paschold, Anja; Smith, Laurie G; Piepho, Hans-Peter; Briggs, Steven P; Hochholdinger, Frank

    2015-05-01

    A high-resolution proteome and phosphoproteome atlas of four maize (Zea mays) primary root tissues, the cortex, stele, meristematic zone, and elongation zone, was generated. High-performance liquid chromatography coupled with tandem mass spectrometry identified 11,552 distinct nonmodified and 2,852 phosphorylated proteins across the four root tissues. Two gradients reflecting the abundance of functional protein classes along the longitudinal root axis were observed. While the classes RNA, DNA, and protein peaked in the meristematic zone, cell wall, lipid metabolism, stress, transport, and secondary metabolism culminated in the differentiation zone. Functional specialization of tissues is underscored by six of 10 cortex-specific proteins involved in flavonoid biosynthesis. Comparison of this data set with high-resolution seed and leaf proteome studies revealed 13% (1,504/11,552) root-specific proteins. While only 23% of the 1,504 root-specific proteins accumulated in all four root tissues, 61% of all 11,552 identified proteins accumulated in all four root tissues. This suggests a much higher degree of tissue-specific functionalization of root-specific proteins. In summary, these data illustrate the remarkable plasticity of the proteomic landscape of maize primary roots and thus provide a starting point for gaining a better understanding of their tissue-specific functions.

  18. Quantitative phosphoproteomic analysis of early alterations in protein phosphorylation by 2,3,7,8-tetrachlorodibenzo-p-dioxin

    DEFF Research Database (Denmark)

    Schulz, Melanie; Brandner, Stefanie; Eberhagen, Carola;

    2013-01-01

    A comprehensive quantitative analysis of changes in protein phosphorylation preceding or accompanying transcriptional activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in 5L rat hepatoma cells was performed using the SILAC approach. Following exposure of the cells to DMSO or 1 nM TCDD for 0.......5 to 2 h, 5648 phosphorylated peptides corresponding to 2156 phosphoproteins were identified. Eight peptides exhibited a statistically significantly altered phosphorylation because of TCDD exposure and 22 showed a regulation factor of ≥ 1.5 in one of the experiments per time point. The vast majority...... of the TCCD-induced phosphorylation changes had not been reported before. The transcription factor ARNT, the obligate partner for gene activation by the TCDD-bound Ah receptor, exhibited an up-regulation of its Ser77 phosphorylation, a modification known to control the differential binding of ARNT homodimers...

  19. 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...

  20. Identifying differentially regulated subnetworks from phosphoproteomic data

    Directory of Open Access Journals (Sweden)

    Tebbe Andreas

    2010-06-01

    Full Text Available Abstract Background Various high throughput methods are available for detecting regulations at the level of transcription, translation or posttranslation (e.g. phosphorylation. Integrating these data with protein networks should make it possible to identify subnetworks that are significantly regulated. Furthermore, such integration can support identification of regulated entities from often noisy high throughput data. In particular, processing mass spectrometry-based phosphoproteomic data in this manner may expose signal transduction pathways and, in the case of experiments with drug-treated cells, reveal the drug's mode of action. Results Here, we introduce SubExtractor, an algorithm that combines phosphoproteomic data with protein network information from STRING to identify differentially regulated subnetworks and individual proteins. The method is based on a Bayesian probabilistic model combined with a genetic algorithm and rigorous significance testing. The Bayesian model accounts for information about both differential regulation and network topology. The method was tested with artificial data and subsequently applied to a comprehensive phosphoproteomics study investigating the mode of action of sorafenib, a small molecule kinase inhibitor. Conclusions SubExtractor reliably identifies differentially regulated subnetworks from phosphoproteomic data by integrating protein networks. The method can also be applied to gene or protein expression data.

  1. Battle through signaling between wheat and the fungal pathogen Septoria tritici revealed by proteomics and phosphoproteomics

    DEFF Research Database (Denmark)

    Yang, Fen; Braga, Marcella Nunes de Melo; Larsen, Martin Røssel

    2013-01-01

    compatible and incompatible interactions. However, differential regulation of the phosphorylation status of signaling proteins, transcription and translation regulators, and membrane-associated proteins was observed between two interactions. The proteomic data were correlated with a more rapid or stronger......-binding proteins, 14-3-3 proteins, and calcium-binding proteins. Quantitative PCR analysis showed the expression of fungal signaling genes and genes encoding a superoxide dismutase and cell-wall degrading enzymes. These results indicate roles of signaling, antioxidative stress mechanisms, and nutrient acquisition...... in facilitating the initial symptomless growth. Taken in its entirety, our dataset suggests interplay between the plant and S. tritici through complex signaling networks and downstream molecular events. Resistance is likely related to several rapidly and intensively triggered signal transduction cascades...

  2. Phosphoproteomics-based systems analysis of signal transduction networks

    Directory of Open Access Journals (Sweden)

    Hiroko eKozuka-Hata

    2012-01-01

    Full Text Available Signal transduction systems coordinate complex cellular information to regulate biological events such as cell proliferation and differentiation. Although the accumulating evidence on widespread association of signaling molecules has revealed essential contribution of phosphorylation-dependent interaction networks to cellular regulation, their dynamic behavior is mostly yet to be analyzed. Recent technological advances regarding mass spectrometry-based quantitative proteomics have enabled us to describe the comprehensive status of phosphorylated molecules in a time-resolved manner. Computational analyses based on the phosphoproteome dynamics accelerate generation of novel methodologies for mathematical analysis of cellular signaling. Phosphoproteomics-based numerical modeling can be used to evaluate regulatory network elements from a statistical point of view. Integration with transcriptome dynamics also uncovers regulatory hubs at the transcriptional level. These omics-based computational methodologies, which have firstly been applied to representative signaling systems such as the epidermal growth factor receptor pathway, have now opened up a gate for systems analysis of signaling networks involved in immune response and cancer.

  3. Analytical strategies for phosphoproteomics

    DEFF Research Database (Denmark)

    Thingholm, Tine E; Jensen, Ole N; Larsen, Martin R

    2009-01-01

    Protein phosphorylation is a key regulator of cellular signaling pathways. It is involved in most cellular events in which the complex interplay between protein kinases and protein phosphatases strictly controls biological processes such as proliferation, differentiation, and apoptosis. Defective...... sensitive and specific strategies. Today, most phosphoproteomic studies are conducted by mass spectrometric strategies in combination with phospho-specific enrichment methods. This review presents an overview of different analytical strategies for the characterization of phosphoproteins. Emphasis...

  4. Phosphoproteomics in cereals.

    Science.gov (United States)

    Yang, Pingfang

    2015-01-01

    Cereals are the most important crop plant supplying staple food throughout the world. The economic importance and continued breeding of crop plants such as rice, maize, wheat, or barley require a detailed scientific understanding of adaptive and developmental processes. Protein phosphorylation is one of the most important regulatory posttranslational modifications and its analysis allows deriving functional and regulatory principles in plants. This minireview summarizes the current knowledge of phosphoproteomic studies in cereals.

  5. Quantitative proteomics reveals cellular targets of celastrol.

    Directory of Open Access Journals (Sweden)

    Jakob Hansen

    Full Text Available Celastrol, a natural substance isolated from plant extracts used in traditional Chinese medicine, has been extensively investigated as a possible drug for treatment of cancer, autoimmune diseases, and protein misfolding disorders. Although studies focusing on celastrol's effects in specific cellular pathways have revealed a considerable number of targets in a diverse array of in vitro models there is an essential need for investigations that can provide a global view of its effects. To assess cellular effects of celastrol and to identify target proteins as biomarkers for monitoring treatment regimes, we performed large-scale quantitative proteomics in cultured human lymphoblastoid cells, a cell type that can be readily prepared from human blood samples. Celastrol substantially modified the proteome composition and 158 of the close to 1800 proteins with robust quantitation showed at least a 1.5 fold change in protein levels. Up-regulated proteins play key roles in cytoprotection with a prominent group involved in quality control and processing of proteins traversing the endoplasmic reticulum. Increased levels of proteins essential for the cellular protection against oxidative stress including heme oxygenase 1, several peroxiredoxins and thioredoxins as well as proteins involved in the control of iron homeostasis were also observed. Specific analysis of the mitochondrial proteome strongly indicated that the mitochondrial association of certain antioxidant defense and apoptosis-regulating proteins increased in cells exposed to celastrol. Analysis of selected mRNA transcripts showed that celastrol activated several different stress response pathways and dose response studies furthermore showed that continuous exposure to sub-micromolar concentrations of celastrol is associated with reduced cellular viability and proliferation. The extensive catalog of regulated proteins presented here identifies numerous cellular effects of celastrol and constitutes

  6. SILAC-based temporal phosphoproteomics.

    Science.gov (United States)

    Francavilla, Chiara; Hekmat, Omid; Blagoev, Blagoy; Olsen, Jesper V

    2014-01-01

    In recent years, thanks to advances in Mass Spectrometry (MS)-based quantitative proteomics, studies on signaling pathways have moved from a detailed description of individual components to system-wide analysis of entire signaling cascades, also providing spatio-temporal views of intracellular pathways. Quantitative proteomics that combines stable isotope labeling by amino acid in cell culture (SILAC) with enrichment strategies for post-translational modification-bearing peptides and high-performance tandem mass spectrometry represents a powerful and unbiased approach to monitor dynamic signaling events. Here we provide an optimized SILAC-based proteomic workflow to analyze temporal changes in phosphoproteomes, which involve a generic three step enrichment protocol for phosphopeptides. SILAC-labeled peptides from digested whole cell lysates are as a first step enriched for phosphorylated tyrosines by immunoaffinity and then further enriched for phosphorylated serine/threonine peptides by strong cation exchange in combination with titanium dioxide-beads chromatography. Analysis of enriched peptides on Orbitrap-based MS results in comprehensive and accurate reconstruction of temporal changes of signaling networks.

  7. The phosphoproteome of toll-like receptor-activated macrophages

    DEFF Research Database (Denmark)

    Weintz, Gabriele; Olsen, Jesper Velgaard; Frühauf, Katja;

    2010-01-01

    Recognition of microbial danger signals by toll-like receptors (TLR) causes re-programming of macrophages. To investigate kinase cascades triggered by the TLR4 ligand lipopolysaccharide (LPS) on systems level, we performed a global, quantitative and kinetic analysis of the phosphoproteome...

  8. Analytical strategies in mass spectrometry-based phosphoproteomics

    DEFF Research Database (Denmark)

    Rosenqvist, Heidi; Ye, Juanying; Jensen, Ole N

    2011-01-01

    to reveal key regulatory events and phosphorylation-mediated processes in the cell and in whole organisms. We present an overview of sensitive and robust analytical methods for phosphopeptide analysis, including calcium phosphate precipitation and affinity enrichment methods such as IMAC and TiO(2). We......Phosphoproteomics, the systematic study of protein phosphorylation events and cell signaling networks in cells and tissues, is a rapidly evolving branch of functional proteomics. Current phosphoproteomics research provides a large toolbox of strategies and protocols that may assist researchers...

  9. 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...

  10. Clinical and Technical Phosphoproteomic Research

    Directory of Open Access Journals (Sweden)

    Ferreira Antonio

    2011-06-01

    Full Text Available Abstract An encouraging approach for the diagnosis and effective therapy of immunological pathologies, which would include cancer, is the identification of proteins and phosphorylated proteins. Disease proteomics, in particular, is a potentially useful method for this purpose. A key role is played by protein phosphorylation in the regulation of normal immunology disorders and targets for several new cancer drugs and drug candidates are cancer cells and protein kinases. Protein phosphorylation is a highly dynamic process. The functioning of new drugs is of major importance as is the selection of those patients who would respond best to a specific treatment regime. In all major aspects of cellular life signalling networks are key elements which play a major role in inter- and intracellular communications. They are involved in diverse processes such as cell-cycle progression, cellular metabolism, cell-cell communication and appropriate response to the cellular environment. A whole range of networks that are involved in the regulation of cell development, differentiation, proliferation, apoptosis, and immunologic responses is contained in the latter. It is so necessary to understand and monitor kinase signalling pathways in order to understand many immunology pathologies. Enrichment of phosphorylated proteins or peptides from tissue or bodily fluid samples is required. The application of technologies such as immunoproteomic techniques, phosphoenrichments and mass spectrometry (MS is crucial for the identification and quantification of protein phosphorylation sites in order to advance in clinical research. Pharmacodynamic readouts of disease states and cellular drug responses in tumour samples will be provided as the field develops. We aim to detail the current and most useful techniques with research examples to isolate and carry out clinical phosphoproteomic studies which may be helpful for immunology and cancer research. Different phosphopeptide

  11. Phosphoproteomic analysis of apoptotic hematopoietic stem cells from hemoglobin E/β-thalassemia

    Directory of Open Access Journals (Sweden)

    Roytrakul Sittiruk

    2011-06-01

    Full Text Available Abstract Background Hemoglobin E/β-thalassemia is particularly common in Southeast Asia and has variable symptoms ranging from mild to severe anemia. Previous investigations demonstrated the remarkable symptoms of β-thalassemia in terms of the acceleration of apoptotic cell death. Ineffective erythropoiesis has been studied in human hematopoietic stem cells, however the distinct apoptotic mechanism was unclear. Methods The phosphoproteome of bone marrow HSCs/CD34+ cells from HbE/β-thalassemic patients was analyzed using IMAC phosphoprotein isolation followed by LC-MS/MS detection. Decyder MS software was used to quantitate differentially expressed proteins in 3 patients and 2 normal donors. The differentially expressed proteins from HSCs/CD34+ cells were compared with HbE/β-thalassemia and normal HSCs. Results A significant change in abundance of 229 phosphoproteins was demonstrated. Importantly, the analysis of the candidate proteins revealed a high abundance of proteins that are commonly found in apoptotic cells including cytochrome C, caspase 6 and apoptosis inducing factors. Moreover, in the HSCs patients a significant increase was observed in a specific type of phosphoserine/threonine binding protein, which is known to act as an important signal mediator for the regulation of cell survival and apoptosis in HbE/β-thalassemia. Conclusions Our study used a novel method to investigate proteins that influence a particular pathway in a given disease or physiological condition. Ultimately, phosphoproteome profiling in HbE/β-thalassemic stem cells is an effective method to further investigate the cell death mechanism of ineffective erythropoiesis in β-thalassemia. Our report provides a comprehensive phosphoproteome, an important resource for the study of ineffective erythropoiesis and developing therapies for HbE/β-thalassemia.

  12. Sperm phosphoproteome profiling by ultra performance liquid chromatography followed by data independent analysis (LC-MS(E)) reveals altered proteomic signatures in asthenozoospermia.

    Science.gov (United States)

    Parte, Priyanka P; Rao, Parimala; Redij, Shweta; Lobo, Vivian; D'Souza, Serena J; Gajbhiye, Rahul; Kulkarni, Vijay

    2012-10-22

    Sperm motility is an important prerequisite for successful fertilization and is regulated by cyclic AMP activated protein kinase A which phosphorylates flagella proteins like axonemal dynein and initiates motility. Increase in calcium influx reverses this process by dephosphorylation that is mediated by calcineurin. Analyzing the phosphoenriched fractions of spermatozoa lysates from eight normozoospermic-, and asthenozoospermic-samples, respectively, by Nano UPLC-MS(E), the present study investigates the phosphoproteins involved in sperm motility in an attempt to identify the key pathways regulating sperm motility and likely to be altered in spermatozoa of asthenozoospermic individuals. 66 phosphoproteins were differentially regulated in asthenozoospermia. The deregulated proteins comprised predominantly the HSPs, cytoskeletal proteins, proteins associated with the fibrous sheath, and those associated with energy metabolism. EM analysis of these spermatozoa demonstrated significant defects in mitochondria, and fibrous sheath and these defects could be correlated with the altered proteome. Pathway analysis revealed that carbohydrate and energy metabolism, cAMP mediated PKA signaling, PI3K/AKT signaling and pathway regulating actin based motility by Rho were significantly altered indicating that motility in spermatozoa is regulated through the concerted effort of these pathways. The data identified signature molecules that have the potential as biomarkers for diagnosing etiology of asthenozoospermia.

  13. Quantitative phosphoproteomic analysis of postmortem muscle development

    DEFF Research Database (Denmark)

    Huang, Honggang

    Meat quality development is highly dependent on postmortem (PM) metabolism and rigor mortis development in PM muscle. PM glycometabolism and rigor mortis fundamentally determine most of the important qualities of raw meat, such as ultimate pH, tenderness, color and water-holding capacity. Protein...

  14. Kinase activity ranking using phosphoproteomics data (KARP) quantifies the contribution of protein kinases to the regulation of cell viability.

    Science.gov (United States)

    Wilkes, Edmund H; Casado, Pedro; Rajeeve, Vinothini; Cutillas, Pedro R

    2017-09-01

    Cell survival is regulated by a signaling network driven by the activity of protein kinases; however, determining the contribution that each kinase in the network makes to such regulation remains challenging. Here, we report a computational approach that uses mass spectrometry-based phosphoproteomics data to rank protein kinases based on their contribution to cell regulation. We found that the scores returned by this algorithm, which we have termed kinase activity ranking using phosphoproteomics data (KARP), were a quantitative measure of the contribution that individual kinases make to the signaling output. Application of KARP to the analysis of eight hematological cell lines revealed that cyclin-dependent kinase (CDK) 1/2, casein kinase (CK) 2, extracellular signal-related kinase (ERK), and p21-activated kinase (PAK) were the most frequently highly ranked kinases in these cell models. The patterns of kinase activation were cell-line specific yet showed a significant association with cell viability as a function of kinase inhibitor treatment. Thus, our study exemplifies KARP as an untargeted approach to empirically and systematically identify regulatory kinases within signaling networks. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Machine learning of global phosphoproteomic profiles enables discrimination of direct versus indirect kinase substrates.

    Science.gov (United States)

    Kanshin, Evgeny; Giguere, Sebastien; Cheng, Jing; Tyers, Michael D; Thibault, Pierre

    2017-03-06

    Mass spectrometry allows quantification of tens of thousands of phosphorylation sites from minute amounts of cellular material. Despite this wealth of information, our understanding of phosphorylation-based signaling is limited, in part because it is not possible to deconvolute substrate phosphorylation that is directly mediated by a particular kinase versus phosphorylation that is mediated by downstream kinases. Here, we describe a framework for assignment of direct in-vivo kinase substrates using a combination of selective chemical inhibition, quantitative phosphoproteomics, and machine learning techniques. Our workflow allows classification of phosphorylation events following inhibition of an analog-sensitive kinase into kinase-independent effects of the inhibitor, direct effects on cognate substrates and indirect effects mediated by downstream kinases or phosphatases. We applied this method to identify many direct targets of Cdc28 and Snf1 kinases in the budding yeast S. cerevisiae. Global phosphoproteome analysis of acute time-series demonstrated that dephosphorylation of direct kinase substrates occurs more rapidly compared to indirect substrates, both after inhibitor treatment and under a physiological nutrient shift in wild-type cells. Mutagenesis experiments revealed a high proportion of functionally relevant phosphorylation sites on Snf1 targets. For example, Snf1 itself was inhibited through autophosphorylation on S391 and new phosphosites were discovered that modulate the activity of the Reg1 regulatory subunit of the Glc7 phosphatase and the Gal83 β-subunit of SNF1 complex. This methodology applies to any kinase for which a functional analog sensitive version can be constructed to facilitate the dissection of the global phosphorylation network.

  16. Comparative phosphoproteomics of zebrafish Fyn/Yes morpholino knockdown embryos.

    Science.gov (United States)

    Lemeer, Simone; Jopling, Chris; Gouw, Joost; Mohammed, Shabaz; Heck, Albert J R; Slijper, Monique; den Hertog, Jeroen

    2008-11-01

    The coordinated movement of cells is indispensable for normal vertebrate gastrulation. Several important players and signaling pathways have been identified in convergence and extension (CE) cell movements during gastrulation, including non-canonical Wnt signaling. Fyn and Yes, members of the Src family of kinases, are key regulators of CE movements as well. Here we investigated signaling pathways in early development by comparison of the phosphoproteome of wild type zebrafish embryos with Fyn/Yes knockdown embryos that display specific CE cell movement defects. For quantitation we used differential stable isotope labeling by reductive amination of peptides. Equal amounts of labeled peptides from wild type and Fyn/Yes knockdown embryos were mixed and analyzed by on-line reversed phase TiO(2)-reversed phase LC-MS/MS. Phosphorylated and non-phosphorylated peptides were quantified, and significant changes in protein expression and/or phosphorylation were detected. We identified 348 phosphoproteins of which 69 showed a decrease in phosphorylation in Fyn/Yes knockdown embryos and 72 showed an increase in phosphorylation. Among these phosphoproteins were known regulators of cell movements, including Adducin and PDLIM5. Our results indicate that quantitative phosphoproteomics combined with morpholino-mediated knockdowns can be used to identify novel signaling pathways that act in zebrafish development in vivo.

  17. Characterization of the Phosphoproteome in SLE Patients.

    Directory of Open Access Journals (Sweden)

    Xinzhou Zhang

    Full Text Available Protein phosphorylation is a complex regulatory event that is involved in the signaling networks that affect virtually every cellular process. The protein phosphorylation may be a novel source for discovering biomarkers and drug targets. However, a systematic analysis of the phosphoproteome in patients with SLE has not been performed. To clarify the pathogenesis of systemic lupus erythematosus (SLE, we compared phosphoprotein expression in PBMCs from SLE patients and normal subjects using proteomics analyses. Phosphopeptides were enriched using TiO₂ from PBMCs isolated from 15 SLE patients and 15 healthy subjects and then analyzed by automated LC-MS/MS analysis. Phosphorylation sites were identified and quantitated by MASCOT and MaxQuant. A total of 1035 phosphorylation sites corresponding to 618 NCBI-annotated genes were identified in SLE patients compared with normal subjects. Differentially expressed proteins, peptides and phosphorylation sites were then subjected to bioinformatics analyses. Gene ontology(GO and pathway analyses showed that nucleic acid metabolism, cellular component organization, transport and multicellular organismal development pathways made up the largest proportions of the differentially expressed genes. Pathway analyses showed that the mitogen-activated protein kinase (MAPK signaling pathway and actin cytoskeleton regulators made up the largest proportions of the metabolic pathways. Network analysis showed that rous sarcoma oncogene (SRC, v-rel reticuloendotheliosis viral oncogene homolog A (RELA, histone deacetylase (HDA1C and protein kinase C, delta (PRKCD play important roles in the stability of the network. These data suggest that aberrant protein phosphorylation may contribute to SLE pathogenesis.

  18. Phosphoproteomics analysis of a clinical Mycobacterium tuberculosis Beijing isolate: expanding the mycobacterial phosphoproteome catalog

    National Research Council Canada - National Science Library

    Fortuin, Suereta; Tomazella, Gisele G; Nagaraj, Nagarjuna; Sampson, Samantha L; Gey van Pittius, Nicolaas C; Soares, Nelson C; Wiker, Harald G; de Souza, Gustavo A; Warren, Robin M

    2015-01-01

    .... This study aimed to elucidate the phosphoproteomic landscape of a clinical isolate of M. tuberculosis. We performed a high throughput mass spectrometric analysis of proteins extracted from an early-logarithmic phase culture...

  19. Quantitative Phosphoproteomics of Tomato Mounting a Hypersensitive Response Reveals a Swift Suppression of Photosynthetic Activity and a Differential Role for Hsp90 Isoforms

    DEFF Research Database (Denmark)

    Stulemeijer, Iris J E; Joosten, Matthieu H A J; Jensen, Ole N

    2009-01-01

    secreted by the pathogenic fungus Cladosporium fulvum. Phosphopeptides were isolated from tomato seedlings expressing both Cf-4 and Avr4 using titanium dioxide columns and LC-MS/MS analysis led to the identification of 50 phosphoproteins, most of which have not been described in tomato before...

  20. Phosphoproteomics analysis of a clinical Mycobacterium tuberculosis Beijing isolate: expanding the mycobacterial phosphoproteome catalog.

    Science.gov (United States)

    Fortuin, Suereta; Tomazella, Gisele G; Nagaraj, Nagarjuna; Sampson, Samantha L; Gey van Pittius, Nicolaas C; Soares, Nelson C; Wiker, Harald G; de Souza, Gustavo A; Warren, Robin M

    2015-01-01

    Reversible protein phosphorylation, regulated by protein kinases and phosphatases, mediates a switch between protein activity and cellular pathways that contribute to a large number of cellular processes. The Mycobacterium tuberculosis genome encodes 11 Serine/Threonine kinases (STPKs) which show close homology to eukaryotic kinases. This study aimed to elucidate the phosphoproteomic landscape of a clinical isolate of M. tuberculosis. We performed a high throughput mass spectrometric analysis of proteins extracted from an early-logarithmic phase culture. Whole cell lysate proteins were processed using the filter-aided sample preparation method, followed by phosphopeptide enrichment of tryptic peptides by strong cation exchange (SCX) and Titanium dioxide (TiO2) chromatography. The MaxQuant quantitative proteomics software package was used for protein identification. Our analysis identified 414 serine/threonine/tyrosine phosphorylated sites, with a distribution of S/T/Y sites; 38% on serine, 59% on threonine and 3% on tyrosine; present on 303 unique peptides mapping to 214 M. tuberculosis proteins. Only 45 of the S/T/Y phosphorylated proteins identified in our study had been previously described in the laboratory strain H37Rv, confirming previous reports. The remaining 169 phosphorylated proteins were newly identified in this clinical M. tuberculosis Beijing strain. We identified 5 novel tyrosine phosphorylated proteins. These findings not only expand upon our current understanding of the protein phosphorylation network in clinical M. tuberculosis but the data set also further extends and complements previous knowledge regarding phosphorylated peptides and phosphorylation sites in M. tuberculosis.

  1. Phosphoproteome of the cyanobacterium Synechocystis sp. PCC 6803 and its dynamics during nitrogen starvation.

    Science.gov (United States)

    Spät, Philipp; Maček, Boris; Forchhammer, Karl

    2015-01-01

    Cyanobacteria have shaped the earth's biosphere as the first oxygenic photoautotrophs and still play an important role in many ecosystems. The ability to adapt to changing environmental conditions is an essential characteristic in order to ensure survival. To this end, numerous studies have shown that bacteria use protein post-translational modifications such as Ser/Thr/Tyr phosphorylation in cell signaling, adaptation, and regulation. Nevertheless, our knowledge of cyanobacterial phosphoproteomes and their dynamic response to environmental stimuli is relatively limited. In this study, we applied gel-free methods and high accuracy mass spectrometry toward the detection of Ser/Thr/Tyr phosphorylation events in the model cyanobacterium Synechocystis sp. PCC 6803. We could identify over 300 phosphorylation events in cultures grown on nitrate as exclusive nitrogen source. Chemical dimethylation labeling was applied to investigate proteome and phosphoproteome dynamics during nitrogen starvation. Our dataset describes the most comprehensive (phospho)proteome of Synechocystis to date, identifying 2382 proteins and 183 phosphorylation events and quantifying 2111 proteins and 148 phosphorylation events during nitrogen starvation. Global protein phosphorylation levels were increased in response to nitrogen depletion after 24 h. Among the proteins with increased phosphorylation, the PII signaling protein showed the highest fold-change, serving as positive control. Other proteins with increased phosphorylation levels comprised functions in photosynthesis and in carbon and nitrogen metabolism. This study reveals dynamics of Synechocystis phosphoproteome in response to environmental stimuli and suggests an important role of protein Ser/Thr/Tyr phosphorylation in fundamental mechanisms of homeostatic control in cyanobacteria.

  2. Enhanced Phosphoproteomic Profiling Workflow For Growth Factor Signaling Analysis

    DEFF Research Database (Denmark)

    Sylvester, Marc; Burbridge, Mike; Leclerc, Gregory

    2010-01-01

    understanding of pathological processes. In our study we create and integrate data on phosphorylations that are initiated by several growth factor receptors. We present an approach for quantitative, time-resolved phosphoproteomic profiling that integrates the important contributions by phosphotyrosines. Methods...... A549 lung carcinoma cells were used as a model and stimulated with hepatocyte growth factor, epidermal growth factor or fibroblast growth factor. We employed a quick protein digestion workflow with spin filters without using urea. Phosphopeptides in general were enriched by sequential elution from...... in order to maximize identification of peptides as well as localization of phosphorylation sites. Results and Conclusions The combination of SIMAC with phosphotyrosine enrichment leads to a significant increase in identification of potential signaling events in growth factor receptor signaling networks...

  3. Quantitative Genetic Interactions Reveal Layers of Biological Modularity

    Science.gov (United States)

    Beltrao, Pedro; Cagney, Gerard; Krogan, Nevan J.

    2010-01-01

    In the past, biomedical research has embraced a reductionist approach, primarily focused on characterizing the individual components that comprise a system of interest. Recent technical developments have significantly increased the size and scope of data describing biological systems. At the same time, advances in the field of systems biology have evoked a broader view of how the underlying components are interconnected. In this essay, we discuss how quantitative genetic interaction mapping has enhanced our view of biological systems, allowing a deeper functional interrogation at different biological scales. PMID:20510918

  4. Quantitative Proteomics of Intracellular Campylobacter jejuni Reveals Metabolic Reprogramming.

    Directory of Open Access Journals (Sweden)

    Xiaoyun Liu

    Full Text Available Campylobacter jejuni is the major cause of bacterial food-borne illness in the USA and Europe. An important virulence attribute of this bacterial pathogen is its ability to enter and survive within host cells. Here we show through a quantitative proteomic analysis that upon entry into host cells, C. jejuni undergoes a significant metabolic downshift. Furthermore, our results indicate that intracellular C. jejuni reprograms its respiration, favoring the respiration of fumarate. These results explain the poor ability of C. jejuni obtained from infected cells to grow under standard laboratory conditions and provide the bases for the development of novel anti microbial strategies that would target relevant metabolic pathways.

  5. System-wide temporal characterization of the proteome and phosphoproteome of human embryonic stem cell differentiation

    DEFF Research Database (Denmark)

    Rigbolt, Kristoffer T.G.; Prokhorova, Tatyana; Akimov, Vyacheslav

    2011-01-01

    To elucidate cellular events underlying the pluripotency of human embryonic stem cells (hESCs), we performed parallel quantitative proteomic and phosphoproteomic analyses of hESCs during differentiation initiated by a diacylglycerol analog or transfer to media that had not been conditioned...... by feeder cells. We profiled 6521 proteins and 23,522 phosphorylation sites, of which almost 50% displayed dynamic changes in phosphorylation status during 24 hours of differentiation. These data are a resource for studies of the events associated with the maintenance of hESC pluripotency and those...... accompanying their differentiation. From these data, we identified a core hESC phosphoproteome of sites with similar robust changes in response to the two distinct treatments. These sites exhibited distinct dynamic phosphorylation patterns, which were linked to known or predicted kinases on the basis...

  6. Quantitative analyses of the plant cytoskeleton reveal underlying organizational principles

    CERN Document Server

    Breuer, David; Sampathkumar, Arun; Hollandt, Florian; Persson, Staffan; Nikoloski, Zoran

    2015-01-01

    The actin and microtubule cytoskeletons are vital structures for cell growth and development across all species. While individual molecular mechanisms underpinning actin and microtubule dynamics have been intensively studied, principles that govern the cytoskeleton organization remain largely unexplored. Here, we captured biologically relevant characteristics of the plant cytoskeleton through a network-driven imaging-based approach allowing to quantitatively assess dynamic features of the cytoskeleton. By introducing suitable null models, we demonstrate that the plant cytoskeletal networks exhibit properties required for efficient transport, namely, short average path lengths and high robustness. We further show that these advantageous features are maintained during temporal cytoskeletal re-arrangements. Interestingly, man-made transportation networks exhibit similar properties, suggesting general laws of network organization supporting diverse transport processes. The proposed network-driven analysis can be ...

  7. Motif-specific sampling of phosphoproteomes.

    Science.gov (United States)

    Ruse, Cristian I; McClatchy, Daniel B; Lu, Bingwen; Cociorva, Daniel; Motoyama, Akira; Park, Sung Kyu; Yates, John R

    2008-05-01

    Phosphoproteomics, the targeted study of a subfraction of the proteome which is modified by phosphorylation, has become an indispensable tool to study cell signaling dynamics. We described a methodology that linked phosphoproteome and proteome analysis based on Ba2+ binding properties of amino acids. This technology selected motif-specific phosphopeptides independent of the system under analysis. MudPIT (Multidimensional Identification Technology) identified 1037 precipitated phosphopeptides from as little as 250 microg of proteins. To extend coverage of the phosphoproteome, we sampled the nuclear extract of HeLa cells with three values of Ba2+ ions molarity. The presence of more than 70% of identified phosphoproteins was further substantiated by their nonmodified peptides. Upon isoproterenol stimulation of HEK cells, we identified an increasing number of phosphoproteins from MAPK cascades and AKAP signaling hubs. We quantified changes in both protein and phosphorylation levels of 197 phosphoproteins including a critical kinase, MAPK1. Integration of differential phosphorylation of MAPK1 with knowledge bases constructed modules that correlated well with its role as node in cross-talk of canonical pathways.

  8. Quantitative metagenomics reveals unique gut microbiome biomarkers in ankylosing spondylitis.

    Science.gov (United States)

    Wen, Chengping; Zheng, Zhijun; Shao, Tiejuan; Liu, Lin; Xie, Zhijun; Le Chatelier, Emmanuelle; He, Zhixing; Zhong, Wendi; Fan, Yongsheng; Zhang, Linshuang; Li, Haichang; Wu, Chunyan; Hu, Changfeng; Xu, Qian; Zhou, Jia; Cai, Shunfeng; Wang, Dawei; Huang, Yun; Breban, Maxime; Qin, Nan; Ehrlich, Stanislav Dusko

    2017-07-27

    The assessment and characterization of the gut microbiome has become a focus of research in the area of human autoimmune diseases. Ankylosing spondylitis is an inflammatory autoimmune disease and evidence showed that ankylosing spondylitis may be a microbiome-driven disease. To investigate the relationship between the gut microbiome and ankylosing spondylitis, a quantitative metagenomics study based on deep shotgun sequencing was performed, using gut microbial DNA from 211 Chinese individuals. A total of 23,709 genes and 12 metagenomic species were shown to be differentially abundant between ankylosing spondylitis patients and healthy controls. Patients were characterized by a form of gut microbial dysbiosis that is more prominent than previously reported cases with inflammatory bowel disease. Specifically, the ankylosing spondylitis patients demonstrated increases in the abundance of Prevotella melaninogenica, Prevotella copri, and Prevotella sp. C561 and decreases in Bacteroides spp. It is noteworthy that the Bifidobacterium genus, which is commonly used in probiotics, accumulated in the ankylosing spondylitis patients. Diagnostic algorithms were established using a subset of these gut microbial biomarkers. Alterations of the gut microbiome are associated with development of ankylosing spondylitis. Our data suggest biomarkers identified in this study might participate in the pathogenesis or development process of ankylosing spondylitis, providing new leads for the development of new diagnostic tools and potential treatments.

  9. Quantitative flux analysis reveals folate-dependent NADPH production

    Science.gov (United States)

    Fan, Jing; Ye, Jiangbin; Kamphorst, Jurre J.; Shlomi, Tomer; Thompson, Craig B.; Rabinowitz, Joshua D.

    2014-06-01

    ATP is the dominant energy source in animals for mechanical and electrical work (for example, muscle contraction or neuronal firing). For chemical work, there is an equally important role for NADPH, which powers redox defence and reductive biosynthesis. The most direct route to produce NADPH from glucose is the oxidative pentose phosphate pathway, with malic enzyme sometimes also important. Although the relative contribution of glycolysis and oxidative phosphorylation to ATP production has been extensively analysed, similar analysis of NADPH metabolism has been lacking. Here we demonstrate the ability to directly track, by liquid chromatography-mass spectrometry, the passage of deuterium from labelled substrates into NADPH, and combine this approach with carbon labelling and mathematical modelling to measure NADPH fluxes. In proliferating cells, the largest contributor to cytosolic NADPH is the oxidative pentose phosphate pathway. Surprisingly, a nearly comparable contribution comes from serine-driven one-carbon metabolism, in which oxidation of methylene tetrahydrofolate to 10-formyl-tetrahydrofolate is coupled to reduction of NADP+ to NADPH. Moreover, tracing of mitochondrial one-carbon metabolism revealed complete oxidation of 10-formyl-tetrahydrofolate to make NADPH. As folate metabolism has not previously been considered an NADPH producer, confirmation of its functional significance was undertaken through knockdown of methylenetetrahydrofolate dehydrogenase (MTHFD) genes. Depletion of either the cytosolic or mitochondrial MTHFD isozyme resulted in decreased cellular NADPH/NADP+ and reduced/oxidized glutathione ratios (GSH/GSSG) and increased cell sensitivity to oxidative stress. Thus, although the importance of folate metabolism for proliferating cells has been long recognized and attributed to its function of producing one-carbon units for nucleic acid synthesis, another crucial function of this pathway is generating reducing power.

  10. Phosphoproteome of the cyanobacterium Synechocystis sp. PCC 6803 and its dynamics during nitrogen starvation.

    Directory of Open Access Journals (Sweden)

    Philipp eSpät

    2015-03-01

    Full Text Available Cyanobacteria have shaped the earth’s biosphere as the first oxygenic photoautotrophs and still play an important role in many ecosystems. The ability to adapt to changing environmental conditions is an essential characteristic in order to ensure survival. To this end, numerous studies have shown that bacteria use protein post-translational modifications such as Ser/Thr/Tyr phosphorylation in cell signalling, adaptation and regulation. Nevertheless, our knowledge of cyanobacterial phosphoproteomes and their dynamic response to environmental stimuli is relatively limited. In this study, we applied gel-free methods and high accuracy mass spectrometry towards the unbiased detection of Ser/Thr/Tyr phosphorylation events in the model cyanobacterium Synechocystis sp. PCC 6803. We could identify over 300 phosphorylation events in cultures grown on nitrate as exclusive nitrogen source. Chemical dimethylation labelling was applied to investigate proteome and phosphoproteome dynamics during nitrogen starvation. Our dataset describes the most comprehensive (phosphoproteome of Synechocystis to date, identifying 2,382 proteins and 183 phosphorylation events and quantifying 2,111 proteins and 148 phosphorylation events during nitrogen starvation. Global protein phosphorylation levels were increased in response to nitrogen depletion after 24 hours. Among the proteins with increased phosphorylation, the PII signalling protein showed the highest fold-change, serving as positive control. Other proteins with increased phosphorylation levels comprised functions in photosynthesis and in carbon and nitrogen metabolism. This study reveals dynamics of Synechocystis phosphoproteome in response to environmental stimuli and suggests an important role of protein Ser/Thr/Tyr phosphorylation in fundamental mechanisms of homeostatic control in cyanobacteria.

  11. Radiosensitization of Human Leukemic HL-60 Cells by ATR Kinase Inhibitor (VE-821: Phosphoproteomic Analysis

    Directory of Open Access Journals (Sweden)

    Barbora Šalovská

    2014-07-01

    Full Text Available DNA damaging agents such as ionizing radiation or chemotherapy are frequently used in oncology. DNA damage response (DDR—triggered by radiation-induced double strand breaks—is orchestrated mainly by three Phosphatidylinositol 3-kinase-related kinases (PIKKs: Ataxia teleangiectasia mutated (ATM, DNA-dependent protein kinase (DNA-PK and ATM and Rad3-related kinase (ATR. Their activation promotes cell-cycle arrest and facilitates DNA damage repair, resulting in radioresistance. Recently developed specific ATR inhibitor, VE-821 (3-amino-6-(4-(methylsulfonylphenyl-N-phenylpyrazine-2-carboxamide, has been reported to have a significant radio- and chemo-sensitizing effect delimited to cancer cells (largely p53-deficient without affecting normal cells. In this study, we employed SILAC-based quantitative phosphoproteomics to describe the mechanism of the radiosensitizing effect of VE-821 in human promyelocytic leukemic cells HL-60 (p53-negative. Hydrophilic interaction liquid chromatography (HILIC-prefractionation with TiO2-enrichment and nano-liquid chromatography—tandem mass spectrometry (LC-MS/MS analysis revealed 9834 phosphorylation sites. Proteins with differentially up-/down-regulated phosphorylation were mostly localized in the nucleus and were involved in cellular processes such as DDR, all phases of the cell cycle, and cell division. Moreover, sequence motif analysis revealed significant changes in the activities of kinases involved in these processes. Taken together, our data indicates that ATR kinase has multiple roles in response to DNA damage throughout the cell cycle and that its inhibitor VE-821 is a potent radiosensitizing agent for p53-negative HL-60 cells.

  12. Phosphoproteomic Analysis Identifies Focal Adhesion Kinase 2 (FAK2) as a Potential Therapeutic Target for Tamoxifen Resistance in Breast Cancer*

    Science.gov (United States)

    Wu, Xinyan; Zahari, Muhammad Saddiq; Renuse, Santosh; Nirujogi, Raja Sekhar; Kim, Min-Sik; Manda, Srikanth S.; Stearns, Vered; Gabrielson, Edward; Sukumar, Saraswati; Pandey, Akhilesh

    2015-01-01

    Tamoxifen, an estrogen receptor-α (ER) antagonist, is an important agent for the treatment of breast cancer. However, this therapy is complicated by the fact that a substantial number of patients exhibit either de novo or acquired resistance. To characterize the signaling mechanisms underlying this resistance, we treated the MCF7 breast cancer cell line with tamoxifen for over six months and showed that this cell line acquired resistance to tamoxifen in vitro and in vivo. We performed SILAC-based quantitative phosphoproteomic profiling on the tamoxifen resistant and vehicle-treated sensitive cell lines to quantify the phosphorylation alterations associated with tamoxifen resistance. From >5600 unique phosphopeptides identified, 1529 peptides exhibited hyperphosphorylation and 409 peptides showed hypophosphorylation in the tamoxifen resistant cells. Gene set enrichment analysis revealed that focal adhesion pathway was one of the most enriched signaling pathways activated in tamoxifen resistant cells. Significantly, we showed that the focal adhesion kinase FAK2 was not only hyperphosphorylated but also transcriptionally up-regulated in tamoxifen resistant cells. FAK2 suppression by specific siRNA knockdown or a small molecule inhibitor repressed cellular proliferation in vitro and tumor formation in vivo. More importantly, our survival analysis revealed that high expression of FAK2 is significantly associated with shorter metastasis-free survival in estrogen receptor-positive breast cancer patients treated with tamoxifen. Our studies suggest that FAK2 is a potential therapeutic target for the management of hormone-refractory breast cancers. PMID:26330541

  13. Dynamic changes of the phosphoproteome in postmortem mouse brains.

    Directory of Open Access Journals (Sweden)

    Tsutomu Oka

    Full Text Available Protein phosphorylation is deeply involved in the pathological mechanism of various neurodegenerative disorders. However, in human pathological samples, phosphorylation can be modified during preservation by postmortem factors such as time and temperature. Postmortem changes may also differ among proteins. Unfortunately, there is no comprehensive database that could support the analysis of protein phosphorylation in human brain samples from the standpoint of postmortem changes. As a first step toward addressing the issue, we performed phosphoproteome analysis with brain tissue dissected from mouse bodies preserved under different conditions. Quantitative whole proteome mass analysis showed surprisingly diverse postmortem changes in phosphoproteins that were dependent on temperature, time and protein species. Twelve hrs postmortem was a critical time point for preservation at room temperature. At 4°C, after the body was cooled down, most phosphoproteins were stable for 72 hrs. At either temperature, increase greater than 2-fold was exceptional during this interval. We found several standard proteins by which we can calculate the postmortem time at room temperature. The information obtained in this study will be indispensable for evaluating experimental data with human as well as mouse brain samples.

  14. Selection of an Appropriate Protein Extraction Method to Study the Phosphoproteome of Maize Photosynthetic Tissue

    Science.gov (United States)

    Luís, Inês M.; Alexandre, Bruno M.; Oliveira, M. Margarida

    2016-01-01

    Often plant tissues are recalcitrant and, due to that, methods relying on protein precipitation, such as TCA/acetone precipitation and phenol extraction, are usually the methods of choice for protein extraction in plant proteomic studies. However, the addition of precipitation steps to protein extraction methods may negatively impact protein recovery, due to problems associated with protein re-solubilization. Moreover, we show that when working with non-recalcitrant plant tissues, such as young maize leaves, protein extraction methods with precipitation steps compromise the maintenance of some labile post-translational modifications (PTMs), such as phosphorylation. Therefore, a critical issue when studying PTMs in plant proteins is to ensure that the protein extraction method is the most appropriate, both at qualitative and quantitative levels. In this work, we compared five methods for protein extraction of the C4-photosynthesis related proteins, in the tip of fully expanded third-leaves. These included: TCA/Acetone Precipitation; Phenol Extraction; TCA/Acetone Precipitation followed by Phenol Extraction; direct extraction in Lysis Buffer (a urea-based buffer); and direct extraction in Lysis Buffer followed by Cleanup with a commercial kit. Protein extraction in Lysis Buffer performed better in comparison to the other methods. It gave one of the highest protein yields, good coverage of the extracted proteome and phosphoproteome, high reproducibility, and little protein degradation. This was also the easiest and fastest method, warranting minimal sample handling. We also show that this method is adequate for the successful extraction of key enzymes of the C4-photosynthetic metabolism, such as PEPC, PPDK, PEPCK, and NADP-ME. This was confirmed by MALDI-TOF/TOF MS analysis of excised spots of 2DE analyses of the extracted protein pools. Staining for phosphorylated proteins in 2DE revealed the presence of several phosphorylated isoforms of PEPC, PPDK, and PEPCK. PMID

  15. Systems Analysis for Interpretation of Phosphoproteomics Data

    DEFF Research Database (Denmark)

    Munk, Stephanie; Refsgaard, Jan C; Olsen, Jesper V

    2016-01-01

    Global phosphoproteomics investigations yield overwhelming datasets with up to tens of thousands of quantified phosphosites. The main challenge after acquiring such large-scale data is to extract the biological meaning and relate this to the experimental question at hand. Systems level analysis...... provides the best means for extracting functional insights from such types of datasets, and this has primed a rapid development of bioinformatics tools and resources over the last decade. Many of these tools are specialized databases that can be mined for annotation and pathway enrichment, whereas others...... provide a platform to generate functional protein networks and explore the relations between proteins of interest. The use of these tools requires careful consideration with regard to the input data, and the interpretation demands a critical approach. This chapter provides a summary of the most...

  16. Posttranslational regulation of self-renewal capacity: insights from proteome and phosphoproteome analyses of stem cell leukemia

    Science.gov (United States)

    Trost, Matthias; Sauvageau, Martin; Hérault, Olivier; Deleris, Paul; Pomiès, Christelle; Chagraoui, Jalila; Mayotte, Nadine; Meloche, Sylvain; Sauvageau, Guy; Thibault, Pierre

    2017-01-01

    We recently generated 2 phenotypically similar Hoxa9+Meis1 overexpressing acute myeloid leukemias that differ by their in vivo biologic behavior. The first leukemia, named FLA2, shows a high frequency of leukemia stem cells (LSCs; 1 in 1.4 cells), whereas the second, FLB1, is more typical with a frequency of LSCs in the range of 1 per several hundred cells. To gain insights into possible mechanisms that determine LSC self-renewal, we profiled and compared the abundance of nuclear and cytoplasmic proteins and phosphoproteins from these leukemias using quantitative proteomics. These analyses revealed differences in proteins associated with stem cell fate, including a hyperactive p38 MAP kinase in FLB1 and a differentially localized Polycomb group protein Ezh2, which is mostly nuclear in FLA2 and predominantly cytoplasmic in FLB1. Together, these newly documented proteomes and phosphoproteomes represent a unique resource with more than 440 differentially expressed proteins and 11 543 unique phosphopeptides, of which 80% are novel and 7% preferentially phosphorylated in the stem cell–enriched leukemia. PMID:22802335

  17. TiSH--a robust and sensitive global phosphoproteomics strategy employing a combination of TiO2, SIMAC, and HILIC.

    Science.gov (United States)

    Engholm-Keller, Kasper; Birck, Pernille; Størling, Joachim; Pociot, Flemming; Mandrup-Poulsen, Thomas; Larsen, Martin R

    2012-10-22

    Large scale quantitative phosphoproteomics depends upon multidimensional strategies for peptide fractionation, phosphopeptide enrichment, and mass spectrometric analysis. Previously, most robust comprehensive large-scale phosphoproteomics strategies have relied on milligram amounts of protein. We have set up a multi-dimensional phosphoproteomics strategy combining a number of well-established enrichment and fraction methods: An initial TiO(2) phosphopeptide pre-enrichment step is followed by post-fractionation using sequential elution from IMAC (SIMAC) to separate multi- and mono-phosphorylated peptides, and hydrophilic interaction liquid chromatography (HILIC) of the mono-phosphorylated peptides (collectively abbreviated "TiSH"). The advantages of the strategy include a high specificity and sample preparation workload reduction due to the TiO(2) pre-enrichment step, as well as low adsorptive losses. We demonstrate the capability of this strategy by quantitative investigation of early interferon-γ signaling in low quantities of insulinoma cells. We identified ~6600 unique phosphopeptides from 300 μg of peptides/condition (22 unique phosphopeptides/μg) in a duplex dimethyl labeling experiment, with an enrichment specificity>94%. When doing network analysis of putative phosphorylation changes it could be noted that the identified protein interaction network centered upon proteins known to be affected by the interferon-γ pathway, thereby supporting the utility of this global phosphoproteomics strategy. This strategy thus shows great potential for interrogating signaling networks from low amounts of sample with high sensitivity and specificity.

  18. Phosphoproteomic analysis of the response of maize leaves to drought, heat and their combination stress

    Directory of Open Access Journals (Sweden)

    Xiuli eHu

    2015-05-01

    Full Text Available Drought and heat stress, especially their combination, greatly affect crop production. Many studies have described transcriptome, proteome and phosphoproteome changes in response of plants to drought or heat stress. However, the study about the phosphoproteomic changes in response of crops to the combination stress is scare. To understand the mechanism of maize responses to the drought and heat combination stress, phosphoproteomic analysis was performed on maize leaves by using multiplex iTRAQ-based quantitative proteomic and LC-MS/MS methods. Five-leaf-stage maize was subjected to drought, heat or their combination, and the leaves were collected. Globally, heat, drought and the combined stress significantly changed the phosphorylation levels of 172, 149 and 144 phosphopeptides, respectively. These phosphopeptides corresponded to 282 proteins. Among them, 23 only responded to the combined stress and could not be predicted from their responses to single stressors; 30 and 75 only responded to drought and heat, respectively. Notably, 19 proteins were phosphorylated on different sites in response to the single and combination stresses. Of the seven significantly enriched phosphorylation motifs identified, two were common for all stresses, two were common for heat and the combined stress, and one was specific to the combined stress. The signaling pathways in which the phosphoproteins were involved clearly differed among the three stresses. Functional characterization of the phosphoproteins and the pathways identified here could lead to new targets for the enhancement of crop stress tolerance, which will be particularly important in the face of climate change and the increasing prevalence of abiotic stressors.

  19. Reactive Oxygen Species (ROS)-Activated ATM-Dependent Phosphorylation of Cytoplasmic Substrates Identified by Large-Scale Phosphoproteomics Screen.

    Science.gov (United States)

    Kozlov, Sergei V; Waardenberg, Ashley J; Engholm-Keller, Kasper; Arthur, Jonathan W; Graham, Mark E; Lavin, Martin

    2016-03-01

    Ataxia-telangiectasia, mutated (ATM) protein plays a central role in phosphorylating a network of proteins in response to DNA damage. These proteins function in signaling pathways designed to maintain the stability of the genome and minimize the risk of disease by controlling cell cycle checkpoints, initiating DNA repair, and regulating gene expression. ATM kinase can be activated by a variety of stimuli, including oxidative stress. Here, we confirmed activation of cytoplasmic ATM by autophosphorylation at multiple sites. Then we employed a global quantitative phosphoproteomics approach to identify cytoplasmic proteins altered in their phosphorylation state in control and ataxia-telangiectasia (A-T) cells in response to oxidative damage. We demonstrated that ATM was activated by oxidative damage in the cytoplasm as well as in the nucleus and identified a total of 9,833 phosphorylation sites, including 6,686 high-confidence sites mapping to 2,536 unique proteins. A total of 62 differentially phosphorylated peptides were identified; of these, 43 were phosphorylated in control but not in A-T cells, and 19 varied in their level of phosphorylation. Motif enrichment analysis of phosphopeptides revealed that consensus ATM serine glutamine sites were overrepresented. When considering phosphorylation events, only observed in control cells (not observed in A-T cells), with predicted ATM sites phosphoSerine/phosphoThreonine glutamine, we narrowed this list to 11 candidate ATM-dependent cytoplasmic proteins. Two of these 11 were previously described as ATM substrates (HMGA1 and UIMCI/RAP80), another five were identified in a whole cell extract phosphoproteomic screens, and the remaining four proteins had not been identified previously in DNA damage response screens. We validated the phosphorylation of three of these proteins (oxidative stress responsive 1 (OSR1), HDGF, and ccdc82) as ATM dependent after H2O2 exposure, and another protein (S100A11) demonstrated ATM

  20. Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway

    DEFF Research Database (Denmark)

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

    2005-01-01

    . 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...

  1. Quantitative Proteomic and Phosphoproteomic Analysis of Trypanosoma cruzi Amastigogenesis

    DEFF Research Database (Denmark)

    Queiroz, Rayner M L; Charneau, Sebastien; Mandacaru, Samuel C;

    2014-01-01

    Chagas disease is a tropical neglected disease endemic in Latin America and it is caused by the protozoan Trypanosoma cruzi. The parasite has four major life stages: epimastigote, metacyclic trypomastigote, bloodstream trypomastigote and amastigote. The differentiation from infective trypomastigo......Chagas disease is a tropical neglected disease endemic in Latin America and it is caused by the protozoan Trypanosoma cruzi. The parasite has four major life stages: epimastigote, metacyclic trypomastigote, bloodstream trypomastigote and amastigote. The differentiation from infective...

  2. Quantitative phosphoproteomic analysis of porcine muscle within 24 h postmortem

    DEFF Research Database (Denmark)

    Huang, Honggang; Larsen, Martin Røssel; Palmisano, Giuseppe

    2014-01-01

    changes at the phosphorylation level but were relatively stable at the total protein level, suggesting that protein phosphorylation may have important roles in meat quality development through the regulation of proteins involved in glucose metabolism and muscle contraction, thereby affecting glycolysis...... proteins underwent significant changes at phosphorylation level but were relatively stable at the total protein level, suggesting that protein phosphorylation may have important roles in meat development through the regulation of proteins involved in metabolism and muscle contraction, thereby affecting......UNLABELLED: Protein phosphorylation can regulate most of the important processes in muscle, such as metabolism and contraction. The postmortem (PM) metabolism and rigor mortis have essential effects on meat quality. In order to identify and characterize the protein phosphorylation events involved...

  3. Phosphoproteomics Analysis of Endometrium in Women with or without Endometriosis

    Institute of Scientific and Technical Information of China (English)

    Hong-Mei Xu; Hai-Teng Deng; Chong-Dong Liu; Yu-Ling Chen; Zhen-Yu Zhang

    2015-01-01

    Background:The molecular mechanisms underlying the endometriosis are still not completely understood.In order to test the hypothesis that the approaches in phosphoproteomics might contribute to the identification of key biomarkers to assess disease pathogenesis and drug targets,we carried out a phosphoproteomics analysis of human endometrium.Methods:A large-scale differential phosphoproteome analysis,using peptide enrichment of titanium dioxide purify and sequential elution from immobilized metal affinity chromatography with linear trap quadrupole-tandem mass spectrometry,was performed in endometrium tissues from 8 women with or without endometriosis.Results:The phosphorylation profiling of endometrium from endometriosis patients had been obtained,and found that identified 516 proteins were modified at phosphorylation level during endometriosis.Gene ontology annotation analysis showed that these proteins were enriched in cellular processes of binding and catalytic activity.Further pathway analysis showed that ribosome pathway and focal adhesion pathway were the top two pathways,which might be deregulated during the development of endometriosis.Conclusions:That large-scale phosphoproteome quantification has been successfully identified in endometrium tissues of women with or without endometriosis will provide new insights to understand the molecular mechanisms of the development of endometriosis.

  4. Technologies and challenges in large-scale phosphoproteomics

    DEFF Research Database (Denmark)

    Engholm-Keller, Kasper; Larsen, Martin Røssel

    2013-01-01

    , and apoptosis, rely on phosphorylation. This PTM is thus involved in many diseases, rendering localization and assessment of extent of phosphorylation of major scientific interest. MS-based phosphoproteomics, which aims at describing all phosphorylation sites in a specific type of cell, tissue, or organism, has...... with focus on the various challenges and limitations this field currently faces....

  5. Phosphoproteomic Analysis Identifies Focal Adhesion Kinase 2 (FAK2) as a Potential Therapeutic Target for Tamoxifen Resistance in Breast Cancer.

    Science.gov (United States)

    Wu, Xinyan; Zahari, Muhammad Saddiq; Renuse, Santosh; Nirujogi, Raja Sekhar; Kim, Min-Sik; Manda, Srikanth S; Stearns, Vered; Gabrielson, Edward; Sukumar, Saraswati; Pandey, Akhilesh

    2015-11-01

    Tamoxifen, an estrogen receptor-α (ER) antagonist, is an important agent for the treatment of breast cancer. However, this therapy is complicated by the fact that a substantial number of patients exhibit either de novo or acquired resistance. To characterize the signaling mechanisms underlying this resistance, we treated the MCF7 breast cancer cell line with tamoxifen for over six months and showed that this cell line acquired resistance to tamoxifen in vitro and in vivo. We performed SILAC-based quantitative phosphoproteomic profiling on the tamoxifen resistant and vehicle-treated sensitive cell lines to quantify the phosphorylation alterations associated with tamoxifen resistance. From >5600 unique phosphopeptides identified, 1529 peptides exhibited hyperphosphorylation and 409 peptides showed hypophosphorylation in the tamoxifen resistant cells. Gene set enrichment analysis revealed that focal adhesion pathway was one of the most enriched signaling pathways activated in tamoxifen resistant cells. Significantly, we showed that the focal adhesion kinase FAK2 was not only hyperphosphorylated but also transcriptionally up-regulated in tamoxifen resistant cells. FAK2 suppression by specific siRNA knockdown or a small molecule inhibitor repressed cellular proliferation in vitro and tumor formation in vivo. More importantly, our survival analysis revealed that high expression of FAK2 is significantly associated with shorter metastasis-free survival in estrogen receptor-positive breast cancer patients treated with tamoxifen. Our studies suggest that FAK2 is a potential therapeutic target for the management of hormone-refractory breast cancers. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Molecular indexing enables quantitative targeted RNA sequencing and reveals poor efficiencies in standard library preparations.

    Science.gov (United States)

    Fu, Glenn K; Xu, Weihong; Wilhelmy, Julie; Mindrinos, Michael N; Davis, Ronald W; Xiao, Wenzhong; Fodor, Stephen P A

    2014-02-01

    We present a simple molecular indexing method for quantitative targeted RNA sequencing, in which mRNAs of interest are selectively captured from complex cDNA libraries and sequenced to determine their absolute concentrations. cDNA fragments are individually labeled so that each molecule can be tracked from the original sample through the library preparation and sequencing process. Multiple copies of cDNA fragments of identical sequence become distinct through labeling, and replicate clones created during PCR amplification steps can be identified and assigned to their distinct parent molecules. Selective capture enables efficient use of sequencing for deep sampling and for the absolute quantitation of rare or transient transcripts that would otherwise escape detection by standard sequencing methods. We have also constructed a set of synthetic barcoded RNA molecules, which can be introduced as controls into the sample preparation mix and used to monitor the efficiency of library construction. The quantitative targeted sequencing revealed extremely low efficiency in standard library preparations, which were further confirmed by using synthetic barcoded RNA molecules. This finding shows that standard library preparation methods result in the loss of rare transcripts and highlights the need for monitoring library efficiency and for developing more efficient sample preparation methods.

  7. HOPE-fixation of lung tissue allows retrospective proteome and phosphoproteome studies.

    Science.gov (United States)

    Shevchuk, Olga; Abidi, Nada; Klawonn, Frank; Wissing, Josef; Nimtz, Manfred; Kugler, Christian; Steinert, Michael; Goldmann, Torsten; Jänsch, Lothar

    2014-11-07

    Hepes-glutamic acid buffer-mediated organic solvent protection effect (HOPE)-fixation has been introduced as an alternative to formalin fixation of clinical samples. Beyond preservation of morphological structures for histology, HOPE-fixation was demonstrated to be compatible with recent methods for RNA and DNA sequencing. However, the suitability of HOPE-fixed materials for the inspection of proteomes by mass spectrometry so far remained undefined. This is of particular interest, since proteins constitute a prime resource for drug research and can give valuable insights into the activity status of signaling pathways. In this study, we extracted proteins from human lung tissue and tested HOPE-treated and snap-frozen tissues comparatively by proteome and phosphoproteome analyses. High confident data from accurate mass spectrometry allowed the identification of 2603 proteins and 3036 phosphorylation sites. HOPE-fixation did not hinder the representative extraction of proteins, and investigating their biochemical properties, covered subcellular localizations, and cellular processes revealed no bias caused by the type of fixation. In conclusion, proteome as well as phosphoproteome data of HOPE lung samples were qualitatively equivalent to results obtained from snap-frozen tissues. Thus, HOPE-treated tissues match clinical demands in both histology and retrospective proteome analyses of patient samples by proteomics.

  8. NeuCode Labeling in Nematodes: Proteomic and Phosphoproteomic Impact of Ascaroside Treatment in Caenorhabditis elegans*

    Science.gov (United States)

    Rhoads, Timothy W.; Prasad, Aman; Kwiecien, Nicholas W.; Merrill, Anna E.; Zawack, Kelson; Westphall, Michael S.; Schroeder, Frank C.; Kimble, Judith; Coon, Joshua J.

    2015-01-01

    The nematode Caenorhabditis elegans is an important model organism for biomedical research. We previously described NeuCode stable isotope labeling by amino acids in cell culture (SILAC), a method for accurate proteome quantification with potential for multiplexing beyond the limits of traditional stable isotope labeling by amino acids in cell culture. Here we apply NeuCode SILAC to profile the proteomic and phosphoproteomic response of C. elegans to two potent members of the ascaroside family of nematode pheromones. By consuming labeled E. coli as part of their diet, C. elegans nematodes quickly and easily incorporate the NeuCode heavy lysine isotopologues by the young adult stage. Using this approach, we report, at high confidence, one of the largest proteomic and phosphoproteomic data sets to date in C. elegans: 6596 proteins at a false discovery rate ≤ 1% and 6620 phosphorylation isoforms with localization probability ≥75%. Our data reveal a post-translational signature of pheromone sensing that includes many conserved proteins implicated in longevity and response to stress. PMID:26392051

  9. NeuCode Labeling in Nematodes: Proteomic and Phosphoproteomic Impact of Ascaroside Treatment in Caenorhabditis elegans.

    Science.gov (United States)

    Rhoads, Timothy W; Prasad, Aman; Kwiecien, Nicholas W; Merrill, Anna E; Zawack, Kelson; Westphall, Michael S; Schroeder, Frank C; Kimble, Judith; Coon, Joshua J

    2015-11-01

    The nematode Caenorhabditis elegans is an important model organism for biomedical research. We previously described NeuCode stable isotope labeling by amino acids in cell culture (SILAC), a method for accurate proteome quantification with potential for multiplexing beyond the limits of traditional stable isotope labeling by amino acids in cell culture. Here we apply NeuCode SILAC to profile the proteomic and phosphoproteomic response of C. elegans to two potent members of the ascaroside family of nematode pheromones. By consuming labeled E. coli as part of their diet, C. elegans nematodes quickly and easily incorporate the NeuCode heavy lysine isotopologues by the young adult stage. Using this approach, we report, at high confidence, one of the largest proteomic and phosphoproteomic data sets to date in C. elegans: 6596 proteins at a false discovery rate ≤ 1% and 6620 phosphorylation isoforms with localization probability ≥75%. Our data reveal a post-translational signature of pheromone sensing that includes many conserved proteins implicated in longevity and response to stress.

  10. Quantitative protein localization signatures reveal an association between spatial and functional divergences of proteins.

    Science.gov (United States)

    Loo, Lit-Hsin; Laksameethanasan, Danai; Tung, Yi-Ling

    2014-03-01

    Protein subcellular localization is a major determinant of protein function. However, this important protein feature is often described in terms of discrete and qualitative categories of subcellular compartments, and therefore it has limited applications in quantitative protein function analyses. Here, we present Protein Localization Analysis and Search Tools (PLAST), an automated analysis framework for constructing and comparing quantitative signatures of protein subcellular localization patterns based on microscopy images. PLAST produces human-interpretable protein localization maps that quantitatively describe the similarities in the localization patterns of proteins and major subcellular compartments, without requiring manual assignment or supervised learning of these compartments. Using the budding yeast Saccharomyces cerevisiae as a model system, we show that PLAST is more accurate than existing, qualitative protein localization annotations in identifying known co-localized proteins. Furthermore, we demonstrate that PLAST can reveal protein localization-function relationships that are not obvious from these annotations. First, we identified proteins that have similar localization patterns and participate in closely-related biological processes, but do not necessarily form stable complexes with each other or localize at the same organelles. Second, we found an association between spatial and functional divergences of proteins during evolution. Surprisingly, as proteins with common ancestors evolve, they tend to develop more diverged subcellular localization patterns, but still occupy similar numbers of compartments. This suggests that divergence of protein localization might be more frequently due to the development of more specific localization patterns over ancestral compartments than the occupation of new compartments. PLAST enables systematic and quantitative analyses of protein localization-function relationships, and will be useful to elucidate protein

  11. Quantitative protein localization signatures reveal an association between spatial and functional divergences of proteins.

    Directory of Open Access Journals (Sweden)

    Lit-Hsin Loo

    2014-03-01

    Full Text Available Protein subcellular localization is a major determinant of protein function. However, this important protein feature is often described in terms of discrete and qualitative categories of subcellular compartments, and therefore it has limited applications in quantitative protein function analyses. Here, we present Protein Localization Analysis and Search Tools (PLAST, an automated analysis framework for constructing and comparing quantitative signatures of protein subcellular localization patterns based on microscopy images. PLAST produces human-interpretable protein localization maps that quantitatively describe the similarities in the localization patterns of proteins and major subcellular compartments, without requiring manual assignment or supervised learning of these compartments. Using the budding yeast Saccharomyces cerevisiae as a model system, we show that PLAST is more accurate than existing, qualitative protein localization annotations in identifying known co-localized proteins. Furthermore, we demonstrate that PLAST can reveal protein localization-function relationships that are not obvious from these annotations. First, we identified proteins that have similar localization patterns and participate in closely-related biological processes, but do not necessarily form stable complexes with each other or localize at the same organelles. Second, we found an association between spatial and functional divergences of proteins during evolution. Surprisingly, as proteins with common ancestors evolve, they tend to develop more diverged subcellular localization patterns, but still occupy similar numbers of compartments. This suggests that divergence of protein localization might be more frequently due to the development of more specific localization patterns over ancestral compartments than the occupation of new compartments. PLAST enables systematic and quantitative analyses of protein localization-function relationships, and will be useful to

  12. Hippocampal phosphoproteomics of F344 rats exposed to 1-bromopropane

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Zhenlie [Guangdong Provincial Key Laboratory of Occupational Disease Prevention and Treatment, Guangdong Province Hospital for Occupational Disease Prevention and Treatment, Guangzhou 510-300 (China); Department of Occupational and Environmental Health, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Ichihara, Sahoko [Graduate School of Regional Innovation Studies, Mie University, Tsu 514-8507 (Japan); Oikawa, Shinji [Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Mie 514-8507 (Japan); Chang, Jie [Department of Occupational and Environmental Health, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Graduate School of Regional Innovation Studies, Mie University, Tsu 514-8507 (Japan); Zhang, Lingyi [Department of Occupational and Environmental Health, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Department of Occupational and Environmental Health, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Noda 278-8510 (Japan); Hu, Shijie [Guangdong Provincial Key Laboratory of Occupational Disease Prevention and Treatment, Guangdong Province Hospital for Occupational Disease Prevention and Treatment, Guangzhou 510-300 (China); Huang, Hanlin, E-mail: huanghl@gdoh.org [Guangdong Provincial Key Laboratory of Occupational Disease Prevention and Treatment, Guangdong Province Hospital for Occupational Disease Prevention and Treatment, Guangzhou 510-300 (China); Ichihara, Gaku, E-mail: gak@rs.tus.ac.jp [Department of Occupational and Environmental Health, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Department of Occupational and Environmental Health, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Noda 278-8510 (Japan)

    2015-01-15

    1-Bromopropane (1-BP) is neurotoxic in both experimental animals and human. To identify phosphorylated modification on the unrecognized post-translational modifications of proteins and investigate their role in 1-BP-induced neurotoxicity, changes in hippocampal phosphoprotein expression levels were analyzed quantitatively in male F344 rats exposed to 1-BP inhalation at 0, 400, or 1000 ppm for 8 h/day for 1 or 4 weeks. Hippocampal protein extracts were analyzed qualitatively and quantitatively by Pro-Q Diamond gel staining and SYPRO Ruby staining coupled with two-dimensional difference in gel electrophoresis (2D-DIGE), respectively, as well as by matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) to identify phosphoproteins. Changes in selected proteins were further confirmed by Manganese II (Mn{sup 2+})-Phos-tag SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Bax and cytochrome c protein levels were determined by western blotting. Pro-Q Diamond gel staining combined with 2D-DIGE identified 26 phosphoprotein spots (p < 0.05), and MALDI-TOF/MS identified 18 up-regulated proteins and 8 down-regulated proteins. These proteins are involved in the biological process of response to stimuli, metabolic processes, and apoptosis signaling. Changes in the expression of phosphorylated 14-3-3 θ were further confirmed by Mn{sup 2+}-Phos-tag SDS-PAGE. Western blotting showed overexpression of Bax protein in the mitochondria with down-regulation in the cytoplasm, whereas cytochrome c expression was high in the cytoplasm but low in the mitochondria after 1-BP exposure. Our results suggest that the pathogenesis of 1-BP-induced hippocampal damage involves inhibition of antiapoptosis process. Phosphoproteins identified in this study can potentially serve as biomarkers for 1-BP-induced neurotoxicity. - Highlights: • 1-BP modified hippocampal phosphoproteome in rat and 23 altered proteins were identified. • 1-BP changed phosphorylation

  13. Quantitative proteomics reveal proteins enriched in tubular endoplasmic reticulum of Saccharomyces cerevisiae

    Science.gov (United States)

    Wang, Xinbo; Li, Shanshan; Wang, Haicheng; Shui, Wenqing; Hu, Junjie

    2017-01-01

    The tubular network is a critical part of the endoplasmic reticulum (ER). The network is shaped by the reticulons and REEPs/Yop1p that generate tubules by inducing high membrane curvature, and the dynamin-like GTPases atlastin and Sey1p/RHD3 that connect tubules via membrane fusion. However, the specific functions of this ER domain are not clear. Here, we isolated tubule-based microsomes from Saccharomyces cerevisiae via classical cell fractionation and detergent-free immunoprecipitation of Flag-tagged Yop1p, which specifically localizes to ER tubules. In quantitative comparisons of tubule-derived and total microsomes, we identified a total of 79 proteins that were enriched in the ER tubules, including known proteins that organize the tubular ER network. Functional categorization of the list of proteins revealed that the tubular ER network may be involved in membrane trafficking, lipid metabolism, organelle contact, and stress sensing. We propose that affinity isolation coupled with quantitative proteomics is a useful tool for investigating ER functions. DOI: http://dx.doi.org/10.7554/eLife.23816.001 PMID:28287394

  14. Quantitative H2S-mediated protein sulfhydration reveals metabolic reprogramming during the integrated stress response

    Science.gov (United States)

    Gao, Xing-Huang; Krokowski, Dawid; Guan, Bo-Jhih; Bederman, Ilya; Majumder, Mithu; Parisien, Marc; Diatchenko, Luda; Kabil, Omer; Willard, Belinda; Banerjee, Ruma; Wang, Benlian; Bebek, Gurkan; Evans, Charles R.; Fox, Paul L.; Gerson, Stanton L.; Hoppel, Charles L.; Liu, Ming; Arvan, Peter; Hatzoglou, Maria

    2015-01-01

    The sulfhydration of cysteine residues in proteins is an important mechanism involved in diverse biological processes. We have developed a proteomics approach to quantitatively profile the changes of sulfhydrated cysteines in biological systems. Bioinformatics analysis revealed that sulfhydrated cysteines are part of a wide range of biological functions. In pancreatic β cells exposed to endoplasmic reticulum (ER) stress, elevated H2S promotes the sulfhydration of enzymes in energy metabolism and stimulates glycolytic flux. We propose that transcriptional and translational reprogramming by the integrated stress response (ISR) in pancreatic β cells is coupled to metabolic alternations triggered by sulfhydration of key enzymes in intermediary metabolism. DOI: http://dx.doi.org/10.7554/eLife.10067.001 PMID:26595448

  15. Ultra-deep and quantitative saliva proteome reveals dynamics of the oral microbiome

    DEFF Research Database (Denmark)

    Grassl, Niklas; Kulak, Nils Alexander; Pichler, Garwin

    2016-01-01

    , disruptions in saliva secretion and changes in the oral microbiome contribute to conditions such as tooth decay and respiratory tract infections. Here we set out to quantitatively map the saliva proteome in great depth with a rapid and in-depth mass spectrometry-based proteomics workflow. METHODS: We used...... with next-generation sequencing data from the Human Microbiome Project as well as a comparison to MALDI-TOF mass spectrometry on microbial cultures revealed strong agreement. The oral microbiome differs between individuals and changes drastically upon eating and tooth brushing. CONCLUSION: Rapid shotgun...... and robust technology can now simultaneously characterize the human and microbiome contributions to the proteome of a body fluid and is therefore a valuable complement to genomic studies. This opens new frontiers for the study of host-pathogen interactions and clinical saliva diagnostics....

  16. Towards single-cell LC-MS phosphoproteomics

    OpenAIRE

    Polat, Ayşe Nur; Özlü, Nurhan

    2014-01-01

    Protein phosphorylation is a ubiquitous posttranslational modification, which is heavily involved in signal transduction. Misregulation of protein phosphorylation is often associated with a decrease in cell viability and complex diseases such as cancer. The dynamic and low abundant nature of phosphorylated proteins makes studying phosphoproteome a challenging task. In this review, we summarize state of the art proteomic techniques to study and quantify peptide phosphorylation in biological sy...

  17. Phosphoproteomics analysis of postmortem porcine muscle with pH decline rate and time difference

    DEFF Research Database (Denmark)

    Huang, Honggang; Larsen, Martin R; Karlsson, Anders H

    2012-01-01

    The aim of this study was to characterize the protein phosphorylation in postmortem (PM) muscle and reveal the change during meat quality development. The gel-based phosphoproteomic analysis of PM porcine muscle was performed in three pig groups with different pH decline rates from PM 1h to 24 h...... the reverse case. The phosphorylation level of 12 bands in sarcoplasmic fraction and 3 bands in myofibrillar fraction were significantly affected by the synergy effects of pH and time (pproteins were identified. The phosphorylation patterns of pyruvate kinase, triosephosphate isomerase-1......, tropomyosin and myosin regulatory light chain 2 showed to be related to PM muscle pH decline rate and time. Our work sheds light on the potential role of protein phosphorylation on regulation of meat quality development....

  18. 哺乳动物精子磷酸化蛋白质组学研究进展%Resent Advances on Phosphoproteomics Researches of Mammalian Sperm Capacitation

    Institute of Scientific and Technical Information of China (English)

    张媛媛; 胡启蒙; 王亮亮; 李新红

    2012-01-01

    Studies on protein expression, modilicalion and interaction have become important in proteomics in the post-genomics era. Phosphorylation/dephosphorylation of proteins is the main mechanism of signal transduction and regulation of enzymes in sperm cells, what' s more, it plays a key role during the sperm-egg recognition and fertilization process. Researches about function of phosphorylated proteins contribute to the understanding of the molecular mechanism of sperm capacitation, hyperactive motility and acrosome reaction process. The progress in the researches of mammalian sperm phosphoproteomics was reviewed that include the phosphoproteomics technology, phosphorylated proteins identification and quantitation, function analysis benefits in finding new important fertilization related biological markers and revealing the sperm development, reproductive potential changes and molecular mechanism of fertilization.%蛋白质的表达、修饰及相互作用的研究已成为后基因组学时代蛋白质组学中的重要内容.蛋白质磷酸化和去磷酸化作为最普遍的翻译后修饰之一,是精子细胞信号转导和酶调控、表达的主要分子机制,亦是精子、卵细胞信号识别及完成受精作用的关键环节.对精子磷酸化蛋白功能的研究有助于深入理解精子的获能、超激活运动的维持、发生顶体反应及精卵结合等受精过程的分子调控机理.对哺乳动物精子磷酸化蛋白质组学的研究进展,包括动物精子磷酸化蛋白质组学研究的技术方法、磷酸化蛋白质种类的鉴定、定量及其功能分析进行了综述,为进一步发掘与受精相关的重要生物标志物,揭示精子发育、繁殖潜能变化及受精分子机理奠定基础.

  19. Quantitative trait locus mapping reveals complex genetic architecture of quantitative virulence in the wheat pathogen Zymoseptoria tritici.

    Science.gov (United States)

    Stewart, Ethan L; Croll, Daniel; Lendenmann, Mark H; Sanchez-Vallet, Andrea; Hartmann, Fanny E; Palma-Guerrero, Javier; Ma, Xin; McDonald, Bruce A

    2016-11-21

    We conducted a comprehensive analysis of virulence in the fungal wheat pathogen Zymoseptoria tritici using quantitative trait locus (QTL) mapping. High-throughput phenotyping based on automated image analysis allowed the measurement of pathogen virulence on a scale and with a precision that was not previously possible. Across two mapping populations encompassing more than 520 progeny, 540 710 pycnidia were counted and their sizes and grey values were measured. A significant correlation was found between pycnidia size and both spore size and number. Precise measurements of percentage leaf area covered by lesions provided a quantitative measure of host damage. Combining these large and accurate phenotypic datasets with a dense panel of restriction site-associated DNA sequencing (RADseq) genetic markers enabled us to genetically dissect pathogen virulence into components related to host damage and those related to pathogen reproduction. We showed that different components of virulence can be under separate genetic control. Large- and small-effect QTLs were identified for all traits, with some QTLs specific to mapping populations, cultivars and traits and other QTLs shared among traits within the same mapping population. We associated the presence of four accessory chromosomes with small, but significant, increases in several virulence traits, providing the first evidence for a meaningful function associated with accessory chromosomes in this organism. A large-effect QTL involved in host specialization was identified on chromosome 7, leading to the identification of candidate genes having a large effect on virulence.

  20. 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.

  1. Rotating Snakes Illusion—Quantitative Analysis Reveals a Region in Luminance Space With Opposite Illusory Rotation

    Science.gov (United States)

    Atala-Gérard, Lea

    2017-01-01

    The Rotating Snakes Illusion employs patterns with repetitive asymmetric luminance steps forming a “snake wheel.” In the underlying luminance sequence {black, dark grey, white, light grey}, coded as {0, g1, 100, g2}, we varied g1 and g2 and measured illusion strength via nulling: Saccades were performed next to a “snake wheel” that rotated physically; observers adjusted rotation until a stationary percept obtained. Observers performed the perceptual nulling of the seeming rotation reliably. Typical settings for (g1, g2), measured from images by Kitaoka, are around (20%, 60%). Indeed, we found a marked illusion in the region (g1≈{0%–25%}, g2≈{20%–75%}) with a rotation speed of ≈1°/s. Surprisingly, we detected a second “island” around (70%, 95%) with opposite direction of the illusory rotation and weaker illusion. Our quantitative measurements of illusion strength confirmed the optimal luminance choices of the standard snake wheel and, unexpectedly, revealed an opposite rotation illusion. PMID:28228928

  2. Rotating Snakes Illusion-Quantitative Analysis Reveals a Region in Luminance Space With Opposite Illusory Rotation.

    Science.gov (United States)

    Atala-Gérard, Lea; Bach, Michael

    2017-01-01

    The Rotating Snakes Illusion employs patterns with repetitive asymmetric luminance steps forming a "snake wheel." In the underlying luminance sequence {black, dark grey, white, light grey}, coded as {0, g1, 100, g2}, we varied g1 and g2 and measured illusion strength via nulling: Saccades were performed next to a "snake wheel" that rotated physically; observers adjusted rotation until a stationary percept obtained. Observers performed the perceptual nulling of the seeming rotation reliably. Typical settings for (g1, g2), measured from images by Kitaoka, are around (20%, 60%). Indeed, we found a marked illusion in the region (g1≈{0%-25%}, g2≈{20%-75%}) with a rotation speed of ≈1°/s. Surprisingly, we detected a second "island" around (70%, 95%) with opposite direction of the illusory rotation and weaker illusion. Our quantitative measurements of illusion strength confirmed the optimal luminance choices of the standard snake wheel and, unexpectedly, revealed an opposite rotation illusion.

  3. Quantitative proteomics analysis reveals the tolerance of Mirabilis jalapa L. to petroleum contamination.

    Science.gov (United States)

    Chen, Shuisen; Ma, Hui; Guo, Zhifu; Feng, Yaping; Lin, Jingwei; Zhang, Menghua; Zhong, Ming

    2017-03-01

    Petroleum is not only an important energy resource but is also a major soil pollutant. To gain better insight into the adaptability mechanism of Mirabilis jalapa to petroleum-contaminated soil, the protein profiles of M. jalapa root were investigated using label-free quantitative proteomics technique. After exposing to petroleum-contaminated soil for 24 h, 34 proteins significantly changed their protein abundance and most of the proteins increased in protein abundance (91.18%). Combined with gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses as well as data from previous studies, our results revealed that M. jalapa enhanced tolerance to petroleum by changing antioxidation and detoxification, cell wall organization, amino acid and carbohydrate metabolism, transportation and protein process, and so on. These metabolism alterations could result in the production and secretion of low molecular carbohydrate, amino acid, and functional protein, which enhanced the bioavailability of petroleum and reducing the toxicity of the petroleum. Taken together, these results provided novel information for better understanding of the tolerance of M. jalapa to petroleum stress.

  4. Dynamics of Natural Killer Cell Receptor Revealed by Quantitative Analysis of Photoswitchable Protein

    Science.gov (United States)

    Pageon, Sophie V.; Aquino, Gerardo; Lagrue, Kathryn; Köhler, Karsten; Endres, Robert G.; Davis, Daniel M.

    2013-11-01

    Natural Killer (NK) cell activation is dynamically regulated by numerous activating and inhibitory surface receptors that accumulate at the immune synapse. Quantitative analysis of receptor dynamics has been limited by methodologies which rely on indirect measurements such as fluorescence recovery after photobleaching. Here, we report a novel approach to study how proteins traffic to and from the immune synapse using NK cell receptors tagged with the photoswitchable fluorescent protein tdEosFP, which can be irreversibly photoswitched from a green to red fluorescent state by ultraviolet light. Thus, following a localized switching event, the movement of the photoswitched molecules can be temporally and spatially resolved by monitoring fluorescence in two regions of interest. By comparing images with mathematical models, we evaluated the diffusion coefficient of the receptor KIR2DL1 (0.23 +- 0.06 micron^2/s) and assessed how synapse formation affects receptor dynamics. Our data conclude that the inhibitory NK cell receptor KIR2DL1 is continually trafficked into the synapse and remains surprisingly stable there. Unexpectedly however, in NK cells forming synapses with multiple target cells simultaneously, KIR2DL1 at one synapse can relocate to another synapse. Thus, our results reveal a previously undetected inter-synaptic exchange of protein.

  5. Modeling development and quantitative trait mapping reveal independent genetic modules for leaf size and shape.

    Science.gov (United States)

    Baker, Robert L; Leong, Wen Fung; Brock, Marcus T; Markelz, R J Cody; Covington, Michael F; Devisetty, Upendra K; Edwards, Christine E; Maloof, Julin; Welch, Stephen; Weinig, Cynthia

    2015-10-01

    Improved predictions of fitness and yield may be obtained by characterizing the genetic controls and environmental dependencies of organismal ontogeny. Elucidating the shape of growth curves may reveal novel genetic controls that single-time-point (STP) analyses do not because, in theory, infinite numbers of growth curves can result in the same final measurement. We measured leaf lengths and widths in Brassica rapa recombinant inbred lines (RILs) throughout ontogeny. We modeled leaf growth and allometry as function valued traits (FVT), and examined genetic correlations between these traits and aspects of phenology, physiology, circadian rhythms and fitness. We used RNA-seq to construct a SNP linkage map and mapped trait quantitative trait loci (QTL). We found genetic trade-offs between leaf size and growth rate FVT and uncovered differences in genotypic and QTL correlations involving FVT vs STPs. We identified leaf shape (allometry) as a genetic module independent of length and width and identified selection on FVT parameters of development. Leaf shape is associated with venation features that affect desiccation resistance. The genetic independence of leaf shape from other leaf traits may therefore enable crop optimization in leaf shape without negative effects on traits such as size, growth rate, duration or gas exchange.

  6. Quantitative proteomics reveals distinct differences in the protein content of outer membrane vesicle vaccines.

    Science.gov (United States)

    van de Waterbeemd, Bas; Mommen, Geert P M; Pennings, Jeroen L A; Eppink, Michel H; Wijffels, René H; van der Pol, Leo A; de Jong, Ad P J M

    2013-04-05

    At present, only vaccines containing outer membrane vesicles (OMV) have successfully stopped Neisseria meningitidis serogroup B epidemics. These vaccines however require detergent-extraction to remove endotoxin, which changes immunogenicity and causes production difficulties. To investigate this in more detail, the protein content of detergent-extracted OMV is compared with two detergent-free alternatives. A novel proteomics strategy has been developed that allows quantitative analysis of many biological replicates despite inherent multiplex restrictions of dimethyl labeling. This enables robust statistical analysis of relative protein abundance. The comparison with detergent-extracted OMV reveales that detergent-free OMV are enriched with membrane (lipo)proteins and contain less cytoplasmic proteins due to a milder purification process. These distinct protein profiles are substantiated with serum blot proteomics, confirming enrichment with immunogenic proteins in both detergent-free alternatives. Therefore, the immunogenic protein content of OMV vaccines depends at least partially on the purification process. This study demonstrates that detergent-free OMV have a preferred composition.

  7. Optomechanical properties of cancer cells revealed by light-induced deformation and quantitative phase microscopy

    Science.gov (United States)

    Kastl, Lena; Budde, Björn; Isbach, Michael; Rommel, Christina; Kemper, Björn; Schnekenburger, Jürgen

    2015-05-01

    There is a growing interest in cell biology and clinical diagnostics in label-free, optical techniques as the interaction with the sample is minimized and substances like dyes or fixatives do not affect the investigated cells. Such techniques include digital holographic microscopy (DHM) and the optical stretching by fiber optical two beam traps. DHM enables quantitative phase contrast imaging and thereby the determination of the cellular refractive index, dry mass and the volume, whereas optical cell stretching reveals the deformability of cells. Since optical stretching strongly depends on the optical properties and the shape of the investigated material we combined the usage of fiber optical stretching and DHM for the characterization of pancreatic tumor cells. The risk of tumors is their potential to metastasize, spread through the bloodstream and build distal tumors/metastases. The grade of dedifferentiation in which the cells lose their cell type specific properties is a measure for this metastatic potential. The less differentiated the cells are, the higher is their risk to metastasize. Our results demonstrate that pancreatic tumor cells, which are from the same tumor but vary in their grade of differentiation, show significant differences in their deformability. The retrieved data show that differentiated cells have a higher stiffness than less differentiated cells of the same tumor. Even cells that differ only in the expression of a single tumor suppressor gene which is responsible for cell-cell adhesions can be distinguished by their mechanical properties. Additionally, results from DHM measurements yield that the refractive index shows only few variations, indicating that it does not significantly influence optical cell stretching. The obtained results show a promising new approach for the phenotyping of different cell types, especially in tumor cell characterization and cancer diagnostics.

  8. Quantitative mass spectrometry reveals plasticity of metabolic networks in Mycobacterium smegmatis.

    Science.gov (United States)

    Chopra, Tarun; Hamelin, Romain; Armand, Florence; Chiappe, Diego; Moniatte, Marc; McKinney, John D

    2014-11-01

    Mycobacterium tuberculosis has a remarkable ability to persist within the human host as a clinically inapparent or chronically active infection. Fatty acids are thought to be an important carbon source used by the bacteria during long term infection. Catabolism of fatty acids requires reprogramming of metabolic networks, and enzymes central to this reprogramming have been targeted for drug discovery. Mycobacterium smegmatis, a nonpathogenic relative of M. tuberculosis, is often used as a model system because of the similarity of basic cellular processes in these two species. Here, we take a quantitative proteomics-based approach to achieve a global view of how the M. smegmatis metabolic network adjusts to utilization of fatty acids as a carbon source. Two-dimensional liquid chromatography and mass spectrometry of isotopically labeled proteins identified a total of 3,067 proteins with high confidence. This number corresponds to 44% of the predicted M. smegmatis proteome and includes most of the predicted metabolic enzymes. Compared with glucose-grown cells, 162 proteins showed differential abundance in acetate- or propionate-grown cells. Among these, acetate-grown cells showed a higher abundance of proteins that could constitute a functional glycerate pathway. Gene inactivation experiments confirmed that both the glyoxylate shunt and the glycerate pathway are operational in M. smegmatis. In addition to proteins with annotated functions, we demonstrate carbon source-dependent differential abundance of proteins that have not been functionally characterized. These proteins might play as-yet-unidentified roles in mycobacterial carbon metabolism. This study reveals several novel features of carbon assimilation in M. smegmatis, which suggests significant functional plasticity of metabolic networks in this organism.

  9. Quantitative X-ray Diffraction (QXRD) analysis for revealing thermal transformations of red mud.

    Science.gov (United States)

    Liao, Chang-Zhong; Zeng, Lingmin; Shih, Kaimin

    2015-07-01

    Red mud is a worldwide environmental problem, and many authorities are trying to find an economic solution for its beneficial application or/and safe disposal. Ceramic production is one of the potential waste-to-resource strategies for using red mud as a raw material. Before implementing such a strategy, an unambiguous understanding of the reaction behavior of red mud under thermal conditions is essential. In this study, the phase compositions and transformation processes were revealed for the Pingguo red mud (PRM) heat-treated at different sintering temperatures. Hematite, perovskite, andradite, cancrinite, kaolinite, diaspore, gibbsite and calcite phases were observed in the samples. However, unlike those red mud samples from the other regions, no TiO2 (rutile or anatase) or quartz were observed. Titanium was found to exist mainly in perovskite and andradite while the iron mainly existed in hematite and andradite. A new silico-ferrite of calcium and aluminum (SFCA) phase was found in samples treated at temperatures above 1100°C, and two possible formation pathways for SFCA were suggested. This is the first SFCA phase to be reported in thermally treated red mud, and this finding may turn PRM waste into a material resource for the iron-making industry. Titanium was found to be enriched in the perovskite phase after 1200°C thermal treatment, and this observation indicated a potential strategy for the recovery of titanium from PRM. In addition to noting these various resource recovery opportunities, this is also the first study to quantitatively summarize the reaction details of PRM phase transformations at various temperatures.

  10. Quantitative trait loci mapping reveals candidate pathways regulating cell cycle duration in Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    Siwo Geoffrey

    2010-10-01

    Full Text Available Abstract Background Elevated parasite biomass in the human red blood cells can lead to increased malaria morbidity. The genes and mechanisms regulating growth and development of Plasmodium falciparum through its erythrocytic cycle are not well understood. We previously showed that strains HB3 and Dd2 diverge in their proliferation rates, and here use quantitative trait loci mapping in 34 progeny from a cross between these parent clones along with integrative bioinformatics to identify genetic loci and candidate genes that control divergences in cell cycle duration. Results Genetic mapping of cell cycle duration revealed a four-locus genetic model, including a major genetic effect on chromosome 12, which accounts for 75% of the inherited phenotype variation. These QTL span 165 genes, the majority of which have no predicted function based on homology. We present a method to systematically prioritize candidate genes using the extensive sequence and transcriptional information available for the parent lines. Putative functions were assigned to the prioritized genes based on protein interaction networks and expression eQTL from our earlier study. DNA metabolism or antigenic variation functional categories were enriched among our prioritized candidate genes. Genes were then analyzed to determine if they interact with cyclins or other proteins known to be involved in the regulation of cell cycle. Conclusions We show that the divergent proliferation rate between a drug resistant and drug sensitive parent clone is under genetic regulation and is segregating as a complex trait in 34 progeny. We map a major locus along with additional secondary effects, and use the wealth of genome data to identify key candidate genes. Of particular interest are a nucleosome assembly protein (PFL0185c, a Zinc finger transcription factor (PFL0465c both on chromosome 12 and a ribosomal protein L7Ae-related on chromosome 4 (PFD0960c.

  11. Multiplexed detection of O-GlcNAcome, phosphoproteome and whole proteome within the same gel

    Directory of Open Access Journals (Sweden)

    Caroline eCieniewski-Bernard

    2014-10-01

    Full Text Available The cellular diversity of proteins results in part from their post-translational modifications. Among all of them, the O-GlcNAcylation is an atypical glycosylation, more similar to phosphorylation than classical glycosylations. Highly dynamic, reversible, and exclusively localized on cytosolic, nuclear and mitochondrial proteins, O-GlcNAcylation is known to regulate almost all if not all cellular processes. Fundamental for the cell life, O-GlcNAcylation abnormalities are involved in the etiology of several inherited diseases. Assessing to O-GlcNAcylation pattern will permit to get relevant data about the role of O-GlcNAcylation in cell physiology. To get understanding about the role of O-GlcNAcylation, as also considering its interplay with phosphorylation, the O-GlcNAc profiling remains a real challenge for the community of proteomists/glycoproteomists. Due to development of multiplexed proteomics based on fluorescent detection of proteins, there is growing body of evidence about the proteome knowledge’s. We propose herein a multiplexed proteomic strategy to detect O-GlcNAcylated proteins, phosphoproteins, and the whole proteome within the same bidimensional gel. In particular, we investigated the phosphoproteome through the ProQ Diamond staining, while the whole proteome was visualized through Sypro Ruby staining, or after the labeling of proteins with a T-Dye fluorophore. The O-GlcNAcome was revealed by the way of the Click chemistry and the azide-alkyne cycloaddition of a fluorophore on GlcNAc moieties. This method permits, after sequential image acquisition, the direct in-gel detection of O-GlcNAcome, phosphoproteome and whole proteome.

  12. Phosphoproteomic Analysis of the Highly-Metastatic Hepatocellular Carcinoma Cell Line, MHCC97-H

    Directory of Open Access Journals (Sweden)

    Miaomiao Tian

    2015-02-01

    Full Text Available Invasion and metastasis of hepatocellular carcinoma (HCC is a major cause for lethal liver cancer. Signaling pathways associated with cancer progression are frequently reconfigured by aberrant phosphorylation of key proteins. To capture the key phosphorylation events in HCC metastasis, we established a methodology by an off-line high-pH HPLC separation strategy combined with multi-step IMAC and LC–MS/MS to study the phosphoproteome of a metastatic HCC cell line, MHCC97-H (high metastasis. In total, 6593 phosphopeptides with 6420 phosphorylation sites (p-sites of 2930 phosphoproteins were identified. Statistical analysis of gene ontology (GO categories for the identified phosphoproteins showed that several of the biological processes, such as transcriptional regulation, mRNA processing and RNA splicing, were over-represented. Further analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG annotations demonstrated that phosphoproteins in multiple pathways, such as spliceosome, the insulin signaling pathway and the cell cycle, were significantly enriched. In particular, we compared our dataset with a previously published phosphoproteome in a normal liver sample, and the results revealed that a number of proteins in the spliceosome pathway, such as U2 small nuclear RNA Auxiliary Factor 2 (U2AF2, Eukaryotic Initiation Factor 4A-III (EIF4A3, Cell Division Cycle 5-Like (CDC5L and Survival Motor Neuron Domain Containing 1 (SMNDC1, were exclusively identified as phosphoproteins only in the MHCC97-H cell line. These results indicated that the phosphorylation of spliceosome proteins may participate in the metastasis of HCC by regulating mRNA processing and RNA splicing.

  13. What Is "Good" Research? Revealing the Paradigmatic Tensions in Quantitative Criticalist Work

    Science.gov (United States)

    Hernández, Ebelia

    2014-01-01

    If quantitative criticalism is thought to be a bridge between positivist epistemologies prevalent in quantitative work and social constructionism often found in critical qualitative work, then this bridge is fraught with challenges and tensions. This chapter examines the methodological issues, questions, and tensions that emerged from a research…

  14. What Is "Good" Research? Revealing the Paradigmatic Tensions in Quantitative Criticalist Work

    Science.gov (United States)

    Hernández, Ebelia

    2014-01-01

    If quantitative criticalism is thought to be a bridge between positivist epistemologies prevalent in quantitative work and social constructionism often found in critical qualitative work, then this bridge is fraught with challenges and tensions. This chapter examines the methodological issues, questions, and tensions that emerged from a research…

  15. Phosphoproteomic analysis of chromoplasts from sweet orange during fruit ripening.

    Science.gov (United States)

    Zeng, Yunliu; Pan, Zhiyong; Wang, Lun; Ding, Yuduan; Xu, Qiang; Xiao, Shunyuan; Deng, Xiuxin

    2014-02-01

    Like other types of plastids, chromoplasts have essential biosynthetic and metabolic activities which may be regulated via post-translational modifications, such as phosphorylation, of their resident proteins. We here report a proteome-wide mapping of in vivo phosphorylation sites in chromoplast-enriched samples prepared from sweet orange [Citrus sinensis (L.) Osbeck] at different ripening stages by titanium dioxide-based affinity chromatography for phosphoprotein enrichment with LC-MS/MS. A total of 109 plastid-localized phosphoprotein candidates were identified that correspond to 179 unique phosphorylation sites in 135 phosphopeptides. On the basis of Motif-X analysis, two distinct types of phosphorylation sites, one as proline-directed phosphorylation motif and the other as casein kinase II motif, can be generalized from these identified phosphopeptides. While most identified phosphoproteins show high homology to those already identified in plastids, approximately 22% of them are novel based on BLAST search using the public databases PhosPhAt and P(3) DB. A close comparative analysis showed that approximately 50% of the phosphoproteins identified in citrus chromoplasts find obvious counterparts in the chloroplast phosphoproteome, suggesting a rather high-level of conservation in basic metabolic activities in these two types of plastids. Not surprisingly, the phosphoproteome of citrus chromoplasts is also characterized by the lack of phosphoproteins involved in photosynthesis and by the presence of more phosphoproteins implicated in stress/redox responses. This study presents the first comprehensive phosphoproteomic analysis of chromoplasts and may help to understand how phosphorylation regulates differentiation of citrus chromoplasts during fruit ripening.

  16. Fiber architecture in remodeled myocardium revealed with a quantitative diffusion CMR tractography framework and histological validation

    National Research Council Canada - National Science Library

    Choukri Mekkaoui; Shuning Huang; Howard H Chen; Guangping Dai; Timothy G Reese; William J Kostis; Aravinda Thiagalingam; Pal Maurovich-Horvat; Jeremy N Ruskin; Udo Hoffmann; Marcel P Jackowski; David E Sosnovik

    2012-01-01

    .... The aim of this study was therefore to develop a technique for quantitative 3D diffusion CMR tractography of the heart, and to apply this method to quantify fiber architecture in the remote zone of remodeled hearts. Methods...

  17. Search Databases and Statistics: Pitfalls and Best Practices in Phosphoproteomics.

    Science.gov (United States)

    Refsgaard, Jan C; Munk, Stephanie; Jensen, Lars J

    2016-01-01

    Advances in mass spectrometric instrumentation in the past 15 years have resulted in an explosion in the raw data yield from typical phosphoproteomics workflows. This poses the challenge of confidently identifying peptide sequences, localizing phosphosites to proteins and quantifying these from the vast amounts of raw data. This task is tackled by computational tools implementing algorithms that match the experimental data to databases, providing the user with lists for downstream analysis. Several platforms for such automated interpretation of mass spectrometric data have been developed, each having strengths and weaknesses that must be considered for the individual needs. These are reviewed in this chapter. Equally critical for generating highly confident output datasets is the application of sound statistical criteria to limit the inclusion of incorrect peptide identifications from database searches. Additionally, careful filtering and use of appropriate statistical tests on the output datasets affects the quality of all downstream analyses and interpretation of the data. Our considerations and general practices on these aspects of phosphoproteomics data processing are presented here.

  18. Phosphorylation of proteins during human myometrial contractions: A phosphoproteomic approach.

    Science.gov (United States)

    Hudson, Claire A; López Bernal, Andrés

    2017-01-22

    Phasic myometrial contractility is a key component of human parturition and the contractions are driven by reversible phosphorylation of myosin light chains catalyzed by the calcium (Ca(2+))-dependent enzyme myosin light chain kinase (MYLK). Other yet unknown phosphorylation or de-phosphorylation events may contribute to myometrial contraction and relaxation. In this study we have performed a global phosphoproteomic analysis of human myometrial tissue using tandem mass tagging to detect changes in the phosphorylation status of individual myometrial proteins during spontaneous and oxytocin-driven phasic contractions. We were able to detect 22 individual phosphopeptides whose relative ratio changed (fold > 2 or contraction. The most significant changes in phosphorylation were to MYLK on serine 1760, a site associated with reductions in calmodulin binding and subsequent kinase activity. Phosphorylated MYLK (ser1760) increased significantly during spontaneous (9.83 ± 3.27 fold, P contractions and we were able to validate these data using immunoblotting. Pathway analysis suggested additional proteins involved in calcium signalling, cGMP-PRKG signalling, adrenergic signalling and oxytocin signalling were also phosphorylated during contractions. This study demonstrates that a global phosphoproteomic analysis of myometrial tissue is a sensitive approach to detect changes in the phosphorylation of proteins during myometrial contractions, and provides a platform for further validation of these changes and for identification of their functional significance.

  19. A Methodological Self-Study of Quantitizing: Negotiating Meaning and Revealing Multiplicity

    Science.gov (United States)

    Seltzer-Kelly, Deborah; Westwood, Sean J.; Pena-Guzman, David M.

    2012-01-01

    This inquiry developed during the process of "quantitizing" qualitative data the authors had gathered for a mixed methods curriculum efficacy study. Rather than providing the intended rigor to their data coding process, their use of an intercoder reliability metric prompted their investigation of the multiplicity and messiness that, as they…

  20. Simple and Reproducible Sample Preparation for Single-Shot Phosphoproteomics with High Sensitivity

    DEFF Research Database (Denmark)

    Jersie-Christensen, Rosa R.; Sultan, Abida; Olsen, Jesper V

    2016-01-01

    The traditional sample preparation workflow for mass spectrometry (MS)-based phosphoproteomics is time consuming and usually requires multiple steps, e.g., lysis, protein precipitation, reduction, alkylation, digestion, fractionation, and phosphopeptide enrichment. Each step can introduce chemica...

  1. Quantitative PCR analysis of house dust can reveal abnormal mold conditions†

    OpenAIRE

    Meklin, Teija; Haugland, Richard A.; Reponen, Tiina; Varma, Manju; Lummus, Zana; Bernstein, David; Wymer, Larry J; Vesper, Stephen J.

    2004-01-01

    Indoor mold concentrations were measured in the dust of moldy homes (MH) and reference homes (RH) by quantitative PCR (QPCR) assays for 82 species or related groups of species (assay groups). About 70% of the species and groups were never or only rarely detected. The ratios (MH geometric mean : RH geometric mean) for 6 commonly detected species (Aspergillus ochraceus, A. penicillioides, A. unguis, A. versicolor, Eurotium group, and Cladosporium sphaerospermum) were > 1 (Group I). Logistic reg...

  2. Infrared spectroscopy reveals both qualitative and quantitative differences in equine subchondral bone during maturation

    Science.gov (United States)

    Kobrina, Yevgeniya; Isaksson, Hanna; Sinisaari, Miikka; Rieppo, Lassi; Brama, Pieter A.; van Weeren, René; Helminen, Heikki J.; Jurvelin, Jukka S.; Saarakkala, Simo

    2010-11-01

    The collagen phase in bone is known to undergo major changes during growth and maturation. The objective of this study is to clarify whether Fourier transform infrared (FTIR) microspectroscopy, coupled with cluster analysis, can detect quantitative and qualitative changes in the collagen matrix of subchondral bone in horses during maturation and growth. Equine subchondral bone samples (n = 29) from the proximal joint surface of the first phalanx are prepared from two sites subjected to different loading conditions. Three age groups are studied: newborn (0 days old), immature (5 to 11 months old), and adult (6 to 10 years old) horses. Spatial collagen content and collagen cross-link ratio are quantified from the spectra. Additionally, normalized second derivative spectra of samples are clustered using the k-means clustering algorithm. In quantitative analysis, collagen content in the subchondral bone increases rapidly between the newborn and immature horses. The collagen cross-link ratio increases significantly with age. In qualitative analysis, clustering is able to separate newborn and adult samples into two different groups. The immature samples display some nonhomogeneity. In conclusion, this is the first study showing that FTIR spectral imaging combined with clustering techniques can detect quantitative and qualitative changes in the collagen matrix of subchondral bone during growth and maturation.

  3. In-depth phosphoproteomic analysis of royal jelly derived from western and eastern honeybee species.

    Science.gov (United States)

    Han, Bin; Fang, Yu; Feng, Mao; Lu, Xiaoshan; Huo, Xinmei; Meng, Lifeng; Wu, Bin; Li, Jianke

    2014-12-01

    The proteins in royal jelly (RJ) play a pivotal role in the nutrition, immune defense, and cast determination of honeybee larvae and have a wide range of pharmacological and health-promoting functions for humans as well. Although the importance of post-translational modifications (PTMs) in protein function is known, investigation of protein phosphorylation of RJ proteins is still very limited. To this end, two complementary phosphopeptide enrichment materials (Ti(4+)-IMAC and TiO2) and high-sensitivity mass spectrometry were applied to establish a detailed phosphoproteome map and to qualitatively and quantitatively compare the phosphoproteomes of RJ produced by Apis mellifera ligustica (Aml) and Apis cerana cerana (Acc). In total, 16 phosphoproteins carrying 67 phosphorylation sites were identified in RJ derived from western bees, and nine proteins phosphorylated on 71 sites were found in RJ produced by eastern honeybees. Of which, eight phosphorylated proteins were common to both RJ samples, and the same motif ([S-x-E]) was extracted, suggesting that the function of major RJ proteins as nutrients and immune agents is evolutionary preserved in both of these honeybee species. All eight overlapping phosphoproteins showed significantly higher abundance in Acc-RJ than in Aml-RJ, and the phosphorylation of Jelleine-II (an antimicrobial peptide, TPFKLSLHL) at S(6) in Acc-RJ had stronger antimicrobial properties than that at T(1) in Aml-RJ even though the overall antimicrobial activity of Jelleine-II was found to decrease after phosphorylation. The differences in phosphosites, peptide abundance, and antimicrobial activity of the phosphorylated RJ proteins indicate that the two major honeybee species employ distinct phosphorylation strategies that align with their different biological characteristics shaped by evolution. The phosphorylation of RJ proteins are potentially driven by the activity of extracellular serine/threonine protein kinase FAM20C-like protein (FAM20C

  4. Quantitative trait variation is revealed in a novel hypomethylated population of woodland strawberry (Fragaria vesca).

    Science.gov (United States)

    Xu, Jihua; Tanino, Karen K; Horner, Kyla N; Robinson, Stephen J

    2016-11-04

    Phenotypic variation is determined by a combination of genotype, environment and their interactions. The realization that allelic diversity can be both genetic and epigenetic allows the environmental component to be further separated. Partitioning phenotypic variation observed among inbred lines with an altered epigenome can allow the epigenetic component controlling quantitative traits to be estimated. To assess the contribution of epialleles on phenotypic variation and determine the fidelity with which epialleles are inherited, we have developed a novel hypomethylated population of strawberry (2n = 2x = 14) using 5-azacytidine from which individuals with altered phenotypes can be identified, selected and characterized. The hypomethylated population was generated using an inbred strawberry population in the F. vesca ssp. vesca accession Hawaii 4. Analysis of whole genome sequence data from control and hypomethylated lines indicate that 5-azacytidine exposure does not increase SNP above background levels. The populations contained only Hawaii 4 alleles, removing introgression of alternate F. vesca alleles as a potential source of variation. Although genome sequencing and genetic marker data are unable to rule out 5-azacytidine induced chromosomal rearrangements as a potential source of the trait variation observed, none were detected in our survey. Quantitative trait variation focusing on flowering time and rosette diameter was scored in control and treated populations where expanded levels of variation were observed among the hypomethylated lines. Methylation sensitive molecular markers indicated that 5-azacytidine induced alterations in DNA methylation patterns and inheritance of methylation patterns were confirmed by bisulfite sequencing of targeted regions. It is possible that methylation polymorphisms might underlie or have induced genetic changes underlying the observable differences in quantitative phenotypes. This population developed in a uniform

  5. Quantitative iTRAQ Proteomics Revealed Possible Roles for Antioxidant Proteins in Sorghum Aluminum Tolerance

    Science.gov (United States)

    Zhou, Dangwei; Yang, Yong; Zhang, Jinbiao; Jiang, Fei; Craft, Eric; Thannhauser, Theodore W.; Kochian, Leon V.; Liu, Jiping

    2017-01-01

    Aluminum (Al) toxicity inhibits root growth and limits crop yields on acid soils worldwide. However, quantitative information is scarce on protein expression profiles under Al stress in crops. In this study, we report on the identification of potential Al responsive proteins from root tips of Al sensitive BR007 and Al tolerant SC566 sorghum lines using a strategy employing iTRAQ and 2D-liquid chromatography (LC) coupled to MS/MS (2D-LC-MS/MS). A total of 771 and 329 unique proteins with abundance changes of >1.5 or sorghum. PMID:28119720

  6. Phosphoproteomics Identifies CK2 as a Negative Regulator of Beige Adipocyte Thermogenesis and Energy Expenditure.

    Science.gov (United States)

    Shinoda, Kosaku; Ohyama, Kana; Hasegawa, Yutaka; Chang, Hsin-Yi; Ogura, Mayu; Sato, Ayaka; Hong, Haemin; Hosono, Takashi; Sharp, Louis Z; Scheel, David W; Graham, Mark; Ishihama, Yasushi; Kajimura, Shingo

    2015-12-01

    Catecholamines promote lipolysis both in brown and white adipocytes, whereas the same stimuli preferentially activate thermogenesis in brown adipocytes. Molecular mechanisms for the adipose-selective activation of thermogenesis remain poorly understood. Here, we employed quantitative phosphoproteomics to map global and temporal phosphorylation profiles in brown, beige, and white adipocytes under β3-adrenenoceptor activation and identified kinases responsible for the adipose-selective phosphorylation profiles. We found that casein kinase2 (CK2) activity is preferentially higher in white adipocytes than brown/beige adipocytes. Genetic or pharmacological blockade of CK2 in white adipocytes activates the thermogenic program in response to cAMP stimuli. Such activation is largely through reduced CK2-mediated phosphorylation of class I HDACs. Notably, inhibition of CK2 promotes beige adipocyte biogenesis and leads to an increase in whole-body energy expenditure and ameliorates diet-induced obesity and insulin resistance. These results indicate that CK2 is a plausible target to rewire the β3-adrenenoceptor signaling cascade that promotes thermogenesis in adipocytes.

  7. Phosphoproteomics Profiling of Tobacco Mature Pollen and Pollen Activated in vitro.

    Science.gov (United States)

    Fíla, Jan; Radau, Sonja; Matros, Andrea; Hartmann, Anja; Scholz, Uwe; Feciková, Jana; Mock, Hans-Peter; Čapková, Věra; Zahedi, René Peiman; Honys, David

    2016-04-01

    Tobacco mature pollen has extremely desiccated cytoplasm, and is metabolically quiescent. Upon re-hydration it becomes metabolically active and that results in later emergence of rapidly growing pollen tube. These changes in cytoplasm hydration and metabolic activity are accompanied by protein phosphorylation. In this study, we subjected mature pollen, 5-min-activated pollen, and 30-min-activated pollen to TCA/acetone protein extraction, trypsin digestion and phosphopeptide enrichment by titanium dioxide. The enriched fraction was subjected to nLC-MS/MS. We identified 471 phosphopeptides that carried 432 phosphorylation sites, position of which was exactly matched by mass spectrometry. These 471 phosphopeptides were assigned to 301 phosphoproteins, because some proteins carried more phosphorylation sites. Of the 13 functional groups, the majority of proteins were put into these categories: transcription, protein synthesis, protein destination and storage, and signal transduction. Many proteins were of unknown function, reflecting the fact that male gametophyte contains many specific proteins that have not been fully functionally annotated. The quantitative data highlighted the dynamics of protein phosphorylation during pollen activation; the identified phosphopeptides were divided into seven groups based on the regulatory trends. The major group comprised mature pollen-specific phosphopeptides that were dephosphorylated during pollen activation. Several phosphopeptides representing the same phosphoprotein had different regulation, which pinpointed the complexity of protein phosphorylation and its clear functional context. Collectively, we showed the first phosphoproteomics data on activated pollen where the position of phosphorylation sites was clearly demonstrated and regulatory kinetics was resolved.

  8. Quantitative Proteomics of Sleep-Deprived Mouse Brains Reveals Global Changes in Mitochondrial Proteins

    Science.gov (United States)

    Li, Tie-Mei; Zhang, Ju-en; Lin, Rui; Chen, She; Luo, Minmin; Dong, Meng-Qiu

    2016-01-01

    Sleep is a ubiquitous, tightly regulated, and evolutionarily conserved behavior observed in almost all animals. Prolonged sleep deprivation can be fatal, indicating that sleep is a physiological necessity. However, little is known about its core function. To gain insight into this mystery, we used advanced quantitative proteomics technology to survey the global changes in brain protein abundance. Aiming to gain a comprehensive profile, our proteomics workflow included filter-aided sample preparation (FASP), which increased the coverage of membrane proteins; tandem mass tag (TMT) labeling, for relative quantitation; and high resolution, high mass accuracy, high throughput mass spectrometry (MS). In total, we obtained the relative abundance ratios of 9888 proteins encoded by 6070 genes. Interestingly, we observed significant enrichment for mitochondrial proteins among the differentially expressed proteins. This finding suggests that sleep deprivation strongly affects signaling pathways that govern either energy metabolism or responses to mitochondrial stress. Additionally, the differentially-expressed proteins are enriched in pathways implicated in age-dependent neurodegenerative diseases, including Parkinson’s, Huntington’s, and Alzheimer’s, hinting at possible connections between sleep loss, mitochondrial stress, and neurodegeneration. PMID:27684481

  9. Quantitative metabolomics reveals an epigenetic blueprint for iron acquisition in uropathogenic Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Jeffrey P Henderson

    2009-02-01

    Full Text Available Bacterial pathogens are frequently distinguished by the presence of acquired genes associated with iron acquisition. The presence of specific siderophore receptor genes, however, does not reliably predict activity of the complex protein assemblies involved in synthesis and transport of these secondary metabolites. Here, we have developed a novel quantitative metabolomic approach based on stable isotope dilution to compare the complement of siderophores produced by Escherichia coli strains associated with intestinal colonization or urinary tract disease. Because uropathogenic E. coli are believed to reside in the gut microbiome prior to infection, we compared siderophore production between urinary and rectal isolates within individual patients with recurrent UTI. While all strains produced enterobactin, strong preferential expression of the siderophores yersiniabactin and salmochelin was observed among urinary strains. Conventional PCR genotyping of siderophore receptors was often insensitive to these differences. A linearized enterobactin siderophore was also identified as a product of strains with an active salmochelin gene cluster. These findings argue that qualitative and quantitative epi-genetic optimization occurs in the E. coli secondary metabolome among human uropathogens. Because the virulence-associated biosynthetic pathways are distinct from those associated with rectal colonization, these results suggest strategies for virulence-targeted therapies.

  10. Comparative mapping reveals quantitative trait loci that affect spawning time in coho salmon (Oncorhynchus kisutch

    Directory of Open Access Journals (Sweden)

    Cristian Araneda

    2012-01-01

    Full Text Available Spawning time in salmonids is a sex-limited quantitative trait that can be modified by selection. In rainbow trout (Oncorhynchus mykiss, various quantitative trait loci (QTL that affect the expression of this trait have been discovered. In this study, we describe four microsatellite loci associated with two possible spawning time QTL regions in coho salmon (Oncorhynchus kisutch. The four loci were identified in females from two populations (early and late spawners produced by divergent selection from the same base population. Three of the loci (OmyFGT34TUF, One2ASC and One19ASC that were strongly associated with spawning time in coho salmon (p < 0.0002 were previously associated with QTL for the same trait in rainbow trout; a fourth loci (Oki10 with a suggestive association (p = 0.00035 mapped 10 cM from locus OmyFGT34TUF in rainbow trout. The changes in allelic frequency observed after three generations of selection were greater than expected because of genetic drift. This work shows that comparing information from closely-related species is a valid strategy for identifying QTLs for marker-assisted selection in species whose genomes are poorly characterized or lack a saturated genetic map.

  11. Quantitative fluorescence imaging reveals point of release for lipoproteins during LDLR-dependent uptake[S

    Science.gov (United States)

    Pompey, Shanica; Zhao, Zhenze; Luby-Phelps, Kate; Michaely, Peter

    2013-01-01

    The LDL receptor (LDLR) supports efficient uptake of both LDL and VLDL remnants by binding lipoprotein at the cell surface, internalizing lipoprotein through coated pits, and releasing lipoprotein in endocytic compartments before returning to the surface for further rounds of uptake. While many aspects of lipoprotein binding and receptor entry are well understood, it is less clear where, when, and how the LDLR releases lipoprotein. To address these questions, the current study employed quantitative fluorescence imaging to visualize the uptake and endosomal processing of LDL and the VLDL remnant β-VLDL. We find that lipoprotein release is rapid, with most release occurring prior to entry of lipoprotein into early endosomes. Published biochemical studies have identified two mechanisms of lipoprotein release: one that involves the β-propeller module of the LDLR and a second that is independent of this module. Quantitative imaging comparing uptake supported by the normal LDLR or by an LDLR variant incapable of β-propeller-dependent release shows that the β-propeller-independent process is sufficient for release for both lipoproteins but that the β-propeller process accelerates both LDL and β-VLDL release. Together these findings define where, when, and how lipoprotein release occurs and provide a generalizable methodology for visualizing endocytic handling in situ. PMID:23296879

  12. Quantitative fluorescence imaging reveals point of release for lipoproteins during LDLR-dependent uptake.

    Science.gov (United States)

    Pompey, Shanica; Zhao, Zhenze; Luby-Phelps, Kate; Michaely, Peter

    2013-03-01

    The LDL receptor (LDLR) supports efficient uptake of both LDL and VLDL remnants by binding lipoprotein at the cell surface, internalizing lipoprotein through coated pits, and releasing lipoprotein in endocytic compartments before returning to the surface for further rounds of uptake. While many aspects of lipoprotein binding and receptor entry are well understood, it is less clear where, when, and how the LDLR releases lipoprotein. To address these questions, the current study employed quantitative fluorescence imaging to visualize the uptake and endosomal processing of LDL and the VLDL remnant β-VLDL. We find that lipoprotein release is rapid, with most release occurring prior to entry of lipoprotein into early endosomes. Published biochemical studies have identified two mechanisms of lipoprotein release: one that involves the β-propeller module of the LDLR and a second that is independent of this module. Quantitative imaging comparing uptake supported by the normal LDLR or by an LDLR variant incapable of β-propeller-dependent release shows that the β-propeller-independent process is sufficient for release for both lipoproteins but that the β-propeller process accelerates both LDL and β-VLDL release. Together these findings define where, when, and how lipoprotein release occurs and provide a generalizable methodology for visualizing endocytic handling in situ.

  13. Quantitative proteomic analysis of mice corneal tissues reveals angiogenesis-related proteins involved in corneal neovascularization.

    Science.gov (United States)

    Shen, Minqian; Tao, Yimin; Feng, Yifan; Liu, Xing; Yuan, Fei; Zhou, Hu

    2016-07-01

    Corneal neovascularization (CNV) was induced in Balb/c mice by alkali burns in the central area of the cornea with a diameter of 2.5mm. After fourteen days, the cornea from one eye was collected for histological staining for CNV examination, while the cornea from the other eye of the same mouse was harvested for proteomic analysis. The label-free quantitative proteomic approach was applied to analyze five normal corneal tissues (normal group mice n=5) and five corresponding neovascularized corneal tissues (model group mice n=5). A total of 2124 proteins were identified, and 1682 proteins were quantified from these corneal tissues. Among these quantified proteins, 290 proteins were significantly changed between normal and alkali burned corneal tissues. Of these significantly changed proteins, 35 were reported or predicted as angiogenesis-related proteins. Then, these 35 proteins were analyzed using Ingenuity Pathway Analysis Software, resulting in 26 proteins enriched and connected to each other in the protein-protein interaction network, such as Lcn-2, αB-crystallin and Serpinf1 (PEDF). These three significantly changed proteins were selected for further Western blotting validation. Consistent with the quantitative proteomic results, Western blotting showed that Lcn-2 and αB-crystallin were significantly up-regulated in CNV model, while PEDF was down-regulated. This study provided increased understanding of angiogenesis-related proteins involved in corneal vascular development, which will be useful in the ophthalmic clinic of specifically target angiogenesis.

  14. Phosphoproteome analysis of Lotus japonicus seeds.

    Science.gov (United States)

    Ino, Yoko; Ishikawa, Akiyo; Nomura, Ayako; Kajiwara, Hideyuki; Harada, Kyuya; Hirano, Hisashi

    2014-01-01

    In this study, we report the first dataset of phosphoproteins of the seeds of a model plant, Lotus japonicus. This dataset might be useful in studying the regulatory mechanisms of seed germination in legume plants. By proteomic analysis of seeds following water absorption, we identified a total of 721 phosphopeptides derived from 343 phosphoproteins in cotyledons, and 931 phosphopeptides from 473 phosphoproteins in hypocotyls. Kinase-specific prediction analyses revealed that different kinases were activated in cotyledons and hypocotyls. In particular, many peptides containing ATM-kinase target motifs, X-X-pS/pT-Q-X-X, were detected in cotyledons. Moreover, by real-time RT-PCR analysis, we found that expression of a homolog of ATM kinase is upregulated specifically in cotyledons, suggesting that this ATM-kinase homolog plays a significant role in cell proliferation in the cotyledons of L. japonicus seeds. The data have been deposited to the ProteomeXchange with identifier PXD000053 (http://proteomecentral.proteomexchange.org/dataset/PXD000053).

  15. Quantitative Lipoproteomics in Clostridium difficile Reveals a Role for Lipoproteins in Sporulation.

    Science.gov (United States)

    Charlton, Thomas M; Kovacs-Simon, Andrea; Michell, Stephen L; Fairweather, Neil F; Tate, Edward W

    2015-11-19

    Bacterial lipoproteins are surface exposed, anchored to the membrane by S-diacylglyceryl modification of the N-terminal cysteine thiol. They play important roles in many essential cellular processes and in bacterial pathogenesis. For example, Clostridium difficile is a Gram-positive anaerobe that causes severe gastrointestinal disease; however, its lipoproteome remains poorly characterized. Here we describe the application of metabolic tagging with alkyne-tagged lipid analogs, in combination with quantitative proteomics, to profile protein lipidation across diverse C. difficile strains and on inactivation of specific components of the lipoprotein biogenesis pathway. These studies provide the first comprehensive map of the C. difficile lipoproteome, demonstrate the existence of two active lipoprotein signal peptidases, and provide insights into lipoprotein function, implicating the lipoproteome in transmission of this pathogen.

  16. 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.

  17. iTRAQ quantitative proteomic analysis reveals the pathways for methanation of propionate facilitated by magnetite

    DEFF Research Database (Denmark)

    Jing, Yuhang; Wan, Jingjing; Angelidaki, Irini

    2017-01-01

    by around 44% in batch experiments, and both direct interspecies electron transfer and interspecies H2 transfer were thermodynamically feasible with the addition of magnetite. The methanation of propionate facilitated by magnetite was also demonstrated in a long-term operated continuous reactor. The methane...... enriched with the addition of magnetite. iTRAQ quantitative proteomic analysis, which was used in mixed culture for the first time, showed that magnetite induced the changes of protein expression levels involved in various pathways during the methanation of propionate. The up-regulation of proteins...... electron transfer considering its up-regulation with the addition of magnetite and origination from Thauera. Most of the up-regulated proteins in methane metabolism were originated from Methanosaeta, while most of the enzymes with down-regulated proteins were originated from Methanosarcina. However, the up-regulated...

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

    Science.gov (United States)

    Beisser, Daniela; Kaschani, Farnusch; Graupner, Nadine; Grossmann, Lars; Jensen, Manfred; Ninck, Sabrina; Schulz, Florian; Rahmann, Sven; Boenigk, Jens; Kaiser, Markus

    2017-01-01

    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 a direct

  19. Quantitative MS-based enzymology of caspases reveals distinct protein substrate specificities, hierarchies, and cellular roles.

    Science.gov (United States)

    Julien, Olivier; Zhuang, Min; Wiita, Arun P; O'Donoghue, Anthony J; Knudsen, Giselle M; Craik, Charles S; Wells, James A

    2016-04-05

    Proteases constitute the largest enzyme family, yet their biological roles are obscured by our rudimentary understanding of their cellular substrates. There are 12 human caspases that play crucial roles in inflammation and cell differentiation and drive the terminal stages of cell death. Recent N-terminomics technologies have begun to enumerate the diverse substrates individual caspases can cleave in complex cell lysates. It is clear that many caspases have shared substrates; however, few data exist about the catalytic efficiencies (kcat/KM) of these substrates, which is critical to understanding their true substrate preferences. In this study, we use quantitative MS to determine the catalytic efficiencies for hundreds of natural protease substrates in cellular lysate for two understudied members: caspase-2 and caspase-6. Most substrates are new, and the cleavage rates vary up to 500-fold. We compare the cleavage rates for common substrates with those found for caspase-3, caspase-7, and caspase-8, involved in apoptosis. There is little correlation in catalytic efficiencies among the five caspases, suggesting each has a unique set of preferred substrates, and thus more specialized roles than previously understood. We synthesized peptide substrates on the basis of protein cleavage sites and found similar catalytic efficiencies between the protein and peptide substrates. These data suggest the rates of proteolysis are dominated more by local primary sequence, and less by the tertiary protein fold. Our studies highlight that global quantitative rate analysis for posttranslational modification enzymes in complex milieus for native substrates is critical to better define their functions and relative sequence of events.

  20. Quantitative mass spectrometry measurements reveal stoichiometry of principal postsynaptic density proteins.

    Science.gov (United States)

    Lowenthal, Mark S; Markey, Sanford P; Dosemeci, Ayse

    2015-06-05

    Quantitative studies are presented of postsynaptic density (PSD) fractions from rat cerebral cortex with the ultimate goal of defining the average copy numbers of proteins in the PSD complex. Highly specific and selective isotope dilution mass spectrometry assays were developed using isotopically labeled polypeptide concatemer internal standards. Interpretation of PSD protein stoichiometry was achieved as a molar ratio with respect to PSD-95 (SAP-90, DLG4), and subsequently, copy numbers were estimated using a consensus literature value for PSD-95. Average copy numbers for several proteins at the PSD were estimated for the first time, including those for AIDA-1, BRAGs, and densin. Major findings include evidence for the high copy number of AIDA-1 in the PSD (144 ± 30)-equivalent to that of the total GKAP family of proteins (150 ± 27)-suggesting that AIDA-1 is an element of the PSD scaffold. The average copy numbers for NMDA receptor sub-units were estimated to be 66 ± 18, 27 ± 9, and 45 ± 15, respectively, for GluN1, GluN2A, and GluN2B, yielding a total of 34 ± 10 NMDA channels. Estimated average copy numbers for AMPA channels and their auxiliary sub-units TARPs were 68 ± 36 and 144 ± 38, respectively, with a stoichiometry of ∼1:2, supporting the assertion that most AMPA receptors anchor to the PSD via TARP sub-units. This robust, quantitative analysis of PSD proteins improves upon and extends the list of major PSD components with assigned average copy numbers in the ongoing effort to unravel the complex molecular architecture of the PSD.

  1. Quantitative Multiplex Immunohistochemistry Reveals Myeloid-Inflamed Tumor-Immune Complexity Associated with Poor Prognosis

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    Takahiro Tsujikawa

    2017-04-01

    Full Text Available Here, we describe a multiplexed immunohistochemical platform with computational image processing workflows, including image cytometry, enabling simultaneous evaluation of 12 biomarkers in one formalin-fixed paraffin-embedded tissue section. To validate this platform, we used tissue microarrays containing 38 archival head and neck squamous cell carcinomas and revealed differential immune profiles based on lymphoid and myeloid cell densities, correlating with human papilloma virus status and prognosis. Based on these results, we investigated 24 pancreatic ductal adenocarcinomas from patients who received neoadjuvant GVAX vaccination and revealed that response to therapy correlated with degree of mono-myelocytic cell density and percentages of CD8+ T cells expressing T cell exhaustion markers. These data highlight the utility of in situ immune monitoring for patient stratification and provide digital image processing pipelines to the community for examining immune complexity in precious tissue sections, where phenotype and tissue architecture are preserved to improve biomarker discovery and assessment.

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

    DEFF Research Database (Denmark)

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

    2015-01-01

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

  3. 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.

  4. Quantitative immunohistochemical analysis reveals association between sodium iodide symporter and estrogen receptor expression in breast cancer.

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    Sushmita Chatterjee

    Full Text Available BACKGROUND: Human sodium iodide symporter (hNIS gene over-expression is under active consideration worldwide as an alternative target molecule for breast cancer (BC diagnosis and targeted radio-iodine treatment. However, the field demands better stratified analysis of endogenous hNIS expression across major BC subtypes. Therefore, we have analyzed subtype-specific variation of hNIS overexpression in breast tumor tissue samples by immunohistochemistry (IHC and also report the development of a homogeneous, quantitative analysis method of digital IHC images. METHODS: hNIS expression was analyzed from 108 BC tissue samples by IHC. Sub-cellular localization of hNIS protein was analyzed by dual immunofluorescence (IF staining method using hNIS and HER2 antibodies. An ImageJ based two-step digital analysis method was developed and applied for the bias-free analysis of the images. RESULTS: Staining of the tumor samples show 70% cases are hNIS positive indicating high incidence of hNIS positive cases in BC. More importantly, a subtype specific analysis done for the first time shows that hNIS expression is overly dominated in estrogen receptor (ER positive cases than the receptor negative cases. Further, 56% of the ER+ve, PgR+ve, HER2-ve and 36% of ER+ve, PgR+ve, HER2+ve cases show highest intensity staining equivalent to the thyroid tissue. A significant positive correlation is also observed between hNIS and estrogen receptor expression (p = 0.0033, CI = 95% suggesting hNIS mediated targeted radio-iodine therapy procedures may benefit both ER+ve, PgR+ve, HER2-ve as well as HER2+ve cases. Further, in a few cases, hNIS and HER2 protein localization is demonstrated by overlapping membrane co-expression. ImageJ based image analysis method shows over 70% match with manual pathological scoring method. CONCLUSION: The study indicates a positive link between hNIS and ER expression in BC. The quantitative IHC image analysis method reported here will

  5. Quantitative transcription dynamic analysis reveals candidate genes and key regulators for ethanol tolerance in Saccharomyces cerevisiae.

    Science.gov (United States)

    Ma, Menggen; Liu, Lewis Z

    2010-06-10

    Derived from our lignocellulosic conversion inhibitor-tolerant yeast, we generated an ethanol-tolerant strain Saccharomyces cerevisiae NRRL Y-50316 by enforced evolutionary adaptation. Using a newly developed robust mRNA reference and a master equation unifying gene expression data analyses, we investigated comparative quantitative transcription dynamics of 175 genes selected from previous studies for an ethanol-tolerant yeast and its closely related parental strain. A highly fitted master equation was established and applied for quantitative gene expression analyses using pathway-based qRT-PCR array assays. The ethanol-tolerant Y-50316 displayed significantly enriched background of mRNA abundance for at least 35 genes without ethanol challenge compared with its parental strain Y-50049. Under the ethanol challenge, the tolerant Y-50316 responded in consistent expressions over time for numerous genes belonging to groups of heat shock proteins, trehalose metabolism, glycolysis, pentose phosphate pathway, fatty acid metabolism, amino acid biosynthesis, pleiotropic drug resistance gene family and transcription factors. The parental strain showed repressed expressions for many genes and was unable to withstand the ethanol stress and establish a viable culture and fermentation. The distinct expression dynamics between the two strains and their close association with cell growth, viability and ethanol fermentation profiles distinguished the tolerance-response from the stress-response in yeast under the ethanol challenge. At least 82 genes were identified as candidate and key genes for ethanol-tolerance and subsequent fermentation under the stress. Among which, 36 genes were newly recognized by the present study. Most of the ethanol-tolerance candidate genes were found to share protein binding motifs of transcription factors Msn4p/Msn2p, Yap1p, Hsf1p and Pdr1p/Pdr3p. Enriched background of transcription abundance and enhanced expressions of ethanol-tolerance genes

  6. Quantitative transcription dynamic analysis reveals candidate genes and key regulators for ethanol tolerance in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Ma Menggen

    2010-06-01

    Full Text Available Abstract Background Derived from our lignocellulosic conversion inhibitor-tolerant yeast, we generated an ethanol-tolerant strain Saccharomyces cerevisiae NRRL Y-50316 by enforced evolutionary adaptation. Using a newly developed robust mRNA reference and a master equation unifying gene expression data analyses, we investigated comparative quantitative transcription dynamics of 175 genes selected from previous studies for an ethanol-tolerant yeast and its closely related parental strain. Results A highly fitted master equation was established and applied for quantitative gene expression analyses using pathway-based qRT-PCR array assays. The ethanol-tolerant Y-50316 displayed significantly enriched background of mRNA abundance for at least 35 genes without ethanol challenge compared with its parental strain Y-50049. Under the ethanol challenge, the tolerant Y-50316 responded in consistent expressions over time for numerous genes belonging to groups of heat shock proteins, trehalose metabolism, glycolysis, pentose phosphate pathway, fatty acid metabolism, amino acid biosynthesis, pleiotropic drug resistance gene family and transcription factors. The parental strain showed repressed expressions for many genes and was unable to withstand the ethanol stress and establish a viable culture and fermentation. The distinct expression dynamics between the two strains and their close association with cell growth, viability and ethanol fermentation profiles distinguished the tolerance-response from the stress-response in yeast under the ethanol challenge. At least 82 genes were identified as candidate and key genes for ethanol-tolerance and subsequent fermentation under the stress. Among which, 36 genes were newly recognized by the present study. Most of the ethanol-tolerance candidate genes were found to share protein binding motifs of transcription factors Msn4p/Msn2p, Yap1p, Hsf1p and Pdr1p/Pdr3p. Conclusion Enriched background of transcription abundance

  7. Nuclear phosphoproteome of developing chickpea seedlings (Cicer arietinum L.) and protein-kinase interaction network.

    Science.gov (United States)

    Kumar, Rajiv; Kumar, Amit; Subba, Pratigya; Gayali, Saurabh; Barua, Pragya; Chakraborty, Subhra; Chakraborty, Niranjan

    2014-06-13

    Nucleus, the control centre of eukaryotic cell, houses most of the genetic machineries required for gene expression and their regulation. Post translational modifications of proteins, particularly phosphorylation control a wide variety of cellular processes but its functional connectivity, in plants, is still elusive. This study profiled the nuclear phosphoproteome of a grain legume, chickpea, to gain better understanding of such event. Intact nuclei were isolated from 3-week-old seedlings using two independent methods, and nuclear proteins were resolved by 2-DE. In a separate set of experiments, phosphoproteins were enriched using IMAC method and resolved by 1-DE. The separated proteins were stained with phosphospecific Pro-Q Diamond stain. Proteomic analyses led to the identification of 107 putative phosphoproteins, of which 86 were non-redundant. Multiple sites of phosphorylation were predicted on several key elements, which included both regulatory and functional proteins. The analysis revealed an array of phosphoproteins, presumably involved in a variety of cellular functions, viz., protein folding (24%), signalling and gene regulation (22%), DNA replication, repair and modification (16%), and metabolism (13%), among others. These results represent the first nucleus-specific phosphoproteome map of a non-model legume, which would provide insights into the possible function of protein phosphorylation in plants. Chickpea is grown over 10 million hectares of land worldwide, and global production hovers around 8.5 million metric tons annually. Despite its nutritional merits, it is often referred to as 'orphan' legume and has remained outside the realm of large-scale functional genomics studies. While current chickpea genome initiative has primarily focused on sequence information and functional annotation, proteomics analyses are limited. It is thus important to study the proteome of the cell organelle particularly the nucleus, which harbors most of the genetic

  8. Mass Spectrometry-Based Quantitative Metabolomics Revealed a Distinct Lipid Profile in Breast Cancer Patients

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    Yun Yen

    2013-04-01

    Full Text Available Breast cancer accounts for the largest number of newly diagnosed cases in female cancer patients. Although mammography is a powerful screening tool, about 20% of breast cancer cases cannot be detected by this method. New diagnostic biomarkers for breast cancer are necessary. Here, we used a mass spectrometry-based quantitative metabolomics method to analyze plasma samples from 55 breast cancer patients and 25 healthy controls. A number of 30 patients and 20 age-matched healthy controls were used as a training dataset to establish a diagnostic model and to identify potential biomarkers. The remaining samples were used as a validation dataset to evaluate the predictive accuracy for the established model. Distinct separation was obtained from an orthogonal partial least squares-discriminant analysis (OPLS-DA model with good prediction accuracy. Based on this analysis, 39 differentiating metabolites were identified, including significantly lower levels of lysophosphatidylcholines and higher levels of sphingomyelins in the plasma samples obtained from breast cancer patients compared with healthy controls. Using logical regression, a diagnostic equation based on three metabolites (lysoPC a C16:0, PC ae C42:5 and PC aa C34:2 successfully differentiated breast cancer patients from healthy controls, with a sensitivity of 98.1% and a specificity of 96.0%.

  9. iTRAQ-based quantitative proteomic analysis reveals potential virulence factors of Erysipelothrix rhusiopathiae.

    Science.gov (United States)

    Wang, Ya; Li, Jingtao; Zhang, Anding; Zhu, Weifeng; Zhang, Qiang; Xu, Zhongmin; Yan, Shuxian; Sun, Xiaomei; Chen, Huanchun; Jin, Meilin

    2017-03-08

    Erysipelothrix rhusiopathiae is a ubiquitous pathogen that has caused considerable economic losses to pig farmers. However, the mechanisms of E. rhusiopathiae pathogenesis remain unclear. To identify new virulence-associated factors, the differentially abundant cell wall-associated proteins (CWPs) between high- and low-virulence strains were investigated through isobaric Tags for Relative and Absolute Quantitation (iTRAQ) combined with liquid chromatography-quadrupole mass spectrometry (LC-MS/MS). In total, 100 CWPs showed significant differences in abundance. Selected differences were verified by western blotting to support the iTRAQ data. Among the differential proteins, the proteins with higher abundance in the high-virulence strain were mostly ABC transporter proteins and adhesion proteins, and the proteins with lower abundance in the high-virulence strain were mainly stress-response proteins. The more abundant proteins in the high-virulence strain may be related to bacterial virulence. The iTRAQ results showed that the abundance of the sugar ABC transporter substrate-binding protein Sbp (No. 5) was higher by 1.73-fold. We further constructed an sbp-deletion mutant. Experiments in animal models showed that the sbp-deletion mutant caused decreased mortality. Together, our data indicated that transporter proteins and adhesion proteins may play important roles in E. rhusiopathiae virulence and confirmed that sbp contributed to the virulence of E. rhusiopathiae.

  10. Quantitative PCR analysis of house dust can reveal abnormal mold conditions.

    Science.gov (United States)

    Meklin, Teija; Haugland, Richard A; Reponen, Tiina; Varma, Manju; Lummus, Zana; Bernstein, David; Wymer, Larry J; Vesper, Stephen J

    2004-07-01

    Indoor mold concentrations were measured in the dust of moldy homes (MH) and reference homes (RH) by quantitative PCR (QPCR) assays for 82 species or related groups of species (assay groups). About 70% of the species and groups were never or only rarely detected. The ratios (MH geometric mean : RH geometric mean) for 6 commonly detected species (Aspergillus ochraceus, A. penicillioides, A. unguis, A. versicolor, Eurotium group, and Cladosporium sphaerospermum) were >1 (Group I). Logistic regression analysis of the sum of the logs of the concentrations of Group I species resulted in a 95% probability for separating MH from RH. These results suggest that it may be possible to evaluate whether a home has an abnormal mold condition by quantifying a limited number of mold species in a dust sample. Also, four common species of Aspergillus were quantified by standard culturing procedures and their concentrations compared to QPCR results. Culturing underestimated the concentrations of these four species by 2 to 3 orders of magnitude compared to QPCR.

  11. Quantitative PCR analysis of house dust can reveal abnormal mold conditions†

    Science.gov (United States)

    Meklin, Teija; Haugland, Richard A.; Reponen, Tiina; Varma, Manju; Lummus, Zana; Bernstein, David; Wymer, Larry J.; Vesper, Stephen J.

    2007-01-01

    Indoor mold concentrations were measured in the dust of moldy homes (MH) and reference homes (RH) by quantitative PCR (QPCR) assays for 82 species or related groups of species (assay groups). About 70% of the species and groups were never or only rarely detected. The ratios (MH geometric mean : RH geometric mean) for 6 commonly detected species (Aspergillus ochraceus, A. penicillioides, A. unguis, A. versicolor, Eurotium group, and Cladosporium sphaerospermum) were > 1 (Group I). Logistic regression analysis of the sum of the logs of the concentrations of Group I species resulted in a 95% probability for separating MH from RH. These results suggest that it may be possible to evaluate whether a home has an abnormal mold condition by quantifying a limited number of mold species in a dust sample. Also, four common species of Aspergillus were quantified by standard culturing procedures and their concentrations compared to QPCR results. Culturing underestimated the concentrations of these four species by 2 to 3 orders of magnitude compared to QPCR. PMID:15237292

  12. Quantitative ChIP-Seq Normalization Reveals Global Modulation of the Epigenome

    Directory of Open Access Journals (Sweden)

    David A. Orlando

    2014-11-01

    Full Text Available Epigenomic profiling by chromatin immunoprecipitation coupled with massively parallel DNA sequencing (ChIP-seq is a prevailing methodology used to investigate chromatin-based regulation in biological systems such as human disease, but the lack of an empirical methodology to enable normalization among experiments has limited the precision and usefulness of this technique. Here, we describe a method called ChIP with reference exogenous genome (ChIP-Rx that allows one to perform genome-wide quantitative comparisons of histone modification status across cell populations using defined quantities of a reference epigenome. ChIP-Rx enables the discovery and quantification of dynamic epigenomic profiles across mammalian cells that would otherwise remain hidden using traditional normalization methods. We demonstrate the utility of this method for measuring epigenomic changes following chemical perturbations and show how reference normalization of ChIP-seq experiments enables the discovery of disease-relevant changes in histone modification occupancy.

  13. Quantitative proteomic profiling reveals photosynthesis responsible for inoculum size dependent variation in Chlorella sorokiniana.

    Science.gov (United States)

    Ma, Qian; Wang, Jiangxin; Lu, Shuhuan; Lv, Yajin; Yuan, Yingjin

    2013-03-01

    High density cultivation is essential to industrial production of biodiesel from microalgae, which involves in variations of micro-environment around individual cells, including light intensity, nutrition distribution, other abiotic stress and so on. To figure out the main limit factor in high inoculum cultivation, a quantitative proteomic analysis (iTRAQ-on-line 2-D nano-LC/MS) in a non-model green microalga, Chlorella sorokiniana, under different inoculum sizes was conducted. The resulting high-quality proteomic dataset consisted of 695 proteins. Using a cutoff of P photosynthesis (light reaction) and Calvin cycle (carbon reaction pathway) had highest expression levels under inoculum size of 1 × 10(6) cells mL(-1), and lowest levels under 1 × 10(7) cells mL(-1). Canonical correlation analysis of the photosynthesis related proteins and metabolites biomarkers showed that a good correlation existed between them (canonical coefficient was 0.987), suggesting photosynthesis process greatly affected microalgae biodiesel productivity and quality. Proteomic study of C. sorokiniana under different illuminations was also conducted to confirm light intensity as a potential limit factor of high inoculum size. Nearly two thirds of proteins showed up-regulation under the illumination of 70-110 µmol m(-2) s(-1), compared to those of 40 µmol m(-2) s(-1). This result suggested that by elegantly adjusting light conditions, high cell density cultivation and high biodiesel production might be achieved. Copyright © 2012 Wiley Periodicals, Inc.

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

    Science.gov (United States)

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

    2015-08-01

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

  15. Quantitative Susceptibility Mapping Reveals an Association between Brain Iron Load and Depression Severity

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    Shun Yao

    2017-08-01

    Full Text Available Previous studies have detected abnormal serum ferritin levels in patients with depression; however, the results have been inconsistent. This study used quantitative susceptibility mapping (QSM for the first time to examine brain iron concentration in depressed patients and evaluated whether it is related to severity. We included three groups of age- and gender-matched participants: 30 patients with mild-moderate depression (MD, 14 patients with major depression disorder (MDD and 20 control subjects. All participants underwent MR scans with a 3D gradient-echo sequence reconstructing for QSM and performed the 17-item Hamilton Depression Rating Scale (HDRS test. In MDD, the susceptibility value in the bilateral putamen was significantly increased compared with MD or control subjects. In addition, a significant difference was also observed in the left thalamus in MDD patients compared with controls. However, the susceptibility values did not differ between MD patients and controls. The susceptibility values positively correlated with the severity of depression as indicated by the HDRS scores. Our results provide evidence that brain iron deposition may be associated with depression and may even be a biomarker for investigating the pathophysiological mechanism of depression.

  16. Context influences on TALE-DNA binding revealed by quantitative profiling.

    Science.gov (United States)

    Rogers, Julia M; Barrera, Luis A; Reyon, Deepak; Sander, Jeffry D; Kellis, Manolis; Joung, J Keith; Bulyk, Martha L

    2015-06-11

    Transcription activator-like effector (TALE) proteins recognize DNA using a seemingly simple DNA-binding code, which makes them attractive for use in genome engineering technologies that require precise targeting. Although this code is used successfully to design TALEs to target specific sequences, off-target binding has been observed and is difficult to predict. Here we explore TALE-DNA interactions comprehensively by quantitatively assaying the DNA-binding specificities of 21 representative TALEs to ∼5,000-20,000 unique DNA sequences per protein using custom-designed protein-binding microarrays (PBMs). We find that protein context features exert significant influences on binding. Thus, the canonical recognition code does not fully capture the complexity of TALE-DNA binding. We used the PBM data to develop a computational model, Specificity Inference For TAL-Effector Design (SIFTED), to predict the DNA-binding specificity of any TALE. We provide SIFTED as a publicly available web tool that predicts potential genomic off-target sites for improved TALE design.

  17. Quantitative Analysis of Fundus-Image Sequences Reveals Phase of Spontaneous Venous Pulsations

    Science.gov (United States)

    Moret, Fabrice; Reiff, Charlotte M.; Lagrèze, Wolf A.; Bach, Michael

    2015-01-01

    Purpose Spontaneous venous pulsation correlates negatively with elevated intracranial pressure and papilledema, and it relates to glaucoma. Yet, its etiology remains unclear. A key element to elucidate its underlying mechanism is the time at which collapse occurs with respect to the heart cycle, but previous reports are contradictory. We assessed this question in healthy subjects using quantitative measurements of both vein diameters and artery lateral displacements; the latter being used as the marker of the ocular systole time. Methods We recorded 5-second fundus sequences with a near-infrared scanning laser ophthalmoscope in 12 young healthy subjects. The image sequences were coregistered, cleaned from microsaccades, and filtered via a principal component analysis to remove nonpulsatile dynamic features. Time courses of arterial lateral displacement and of diameter at sites of spontaneous venous pulsation or proximal to the disk were retrieved from those image sequences and compared. Results Four subjects displayed both arterial and venous pulsatile waveforms. On those, we observed venous diameter waveforms differing markedly among the subjects, ranging from a waveform matching the typical intraocular pressure waveform to a close replica of the arterial waveform. Conclusions The heterogeneity in waveforms and arteriovenous phases suggests that the mechanism governing the venous outflow resistance differs among healthy subjects. Translational relevance Further characterizations are necessary to understand the heterogeneous mechanisms governing the venous outflow resistance as this resistance is altered in glaucoma and is instrumental when monitoring intracranial hypertension based on fundus observations. PMID:26396929

  18. Mechanisms of cell cycle control revealed by a systematic and quantitative overexpression screen in S. cerevisiae.

    Directory of Open Access Journals (Sweden)

    Wei Niu

    2008-07-01

    Full Text Available Regulation of cell cycle progression is fundamental to cell health and reproduction, and failures in this process are associated with many human diseases. Much of our knowledge of cell cycle regulators derives from loss-of-function studies. To reveal new cell cycle regulatory genes that are difficult to identify in loss-of-function studies, we performed a near-genome-wide flow cytometry assay of yeast gene overexpression-induced cell cycle delay phenotypes. We identified 108 genes whose overexpression significantly delayed the progression of the yeast cell cycle at a specific stage. Many of the genes are newly implicated in cell cycle progression, for example SKO1, RFA1, and YPR015C. The overexpression of RFA1 or YPR015C delayed the cell cycle at G2/M phases by disrupting spindle attachment to chromosomes and activating the DNA damage checkpoint, respectively. In contrast, overexpression of the transcription factor SKO1 arrests cells at G1 phase by activating the pheromone response pathway, revealing new cross-talk between osmotic sensing and mating. More generally, 92%-94% of the genes exhibit distinct phenotypes when overexpressed as compared to their corresponding deletion mutants, supporting the notion that many genes may gain functions upon overexpression. This work thus implicates new genes in cell cycle progression, complements previous screens, and lays the foundation for future experiments to define more precisely roles for these genes in cell cycle progression.

  19. Quantitative proteomics analysis by iTRAQ revealed underlying changes in thermotolerance of Arthrospira platensis.

    Science.gov (United States)

    Chang, Rong; Lv, Bingxin; Li, Bosheng

    2017-08-08

    Growth temperature is a critical factor that affects cultivation of Arthrospira platensis which is a type of cyanobacterium widely known as Spirulina that has significant commercial value. To investigate the molecular mechanism underlying the thermotolerance of Spirulina, differential protein expression profiling was carried out using iTRAQ-based quantitative proteomic analysis. This study only analyzed changes in thylakoids. Among the 2085 proteins quantified, 43 differentially expressed proteins were selected based on the fold change cutoff scores of ≥2 or ≤0.5 for up-regulation or down-regulation, respectively. An analysis of these 43 proteins found that 23% of them are photosynthetic system proteins which include photosynthetic enzymes and pigment proteins. The dynamic change of these proteins indicates that photosynthetic system functions were profoundly affected under heat stress and the light-dependent reactions were probably the most sensitive to temperature changes. Meanwhile, to cope with the low energy production due to impaired photosynthesis there was a considerable down-shift in protein synthesis which is a very energy demanding process. The impaired photosynthesis led to low energy generation that was compensated by a down-shift in translation (the most energy-demanding process) and an up-shift of glycolysis. The reduction of many ribosome proteins may lead to a loss in translation efficiency; therefore, Spirulina may adopted a different mechanism to increase translational elongation under heat stress to compensate for this loss, such as elevate L7/L12 proteins. Changes were also found in the classical heat shock proteins, the ROS scavenging system, DNA-binding proteins, and some membrane proteins. In conclusion, this research demonstrate that heat stress induces profound changes in cellular physiology and shed light on the mechanism of the heat stress response and thermotolerance of Arthrospira platensis. Arthrospira platensis, widely known as

  20. Quantitative trait locus (QTL mapping reveals a role for unstudied genes in Aspergillus virulence.

    Directory of Open Access Journals (Sweden)

    Julian K Christians

    Full Text Available Infections caused by the fungus Aspergillus are a major cause of morbidity and mortality in immunocompromised populations. To identify genes required for virulence that could be used as targets for novel treatments, we mapped quantitative trait loci (QTL affecting virulence in the progeny of a cross between two strains of A. nidulans (FGSC strains A4 and A91. We genotyped 61 progeny at 739 single nucleotide polymorphisms (SNP spread throughout the genome, and constructed a linkage map that was largely consistent with the genomic sequence, with the exception of one potential inversion of ∼527 kb on Chromosome V. The estimated genome size was 3705 cM and the average intermarker spacing was 5.0 cM. The average ratio of physical distance to genetic distance was 8.1 kb/cM, which is similar to previous estimates, and variation in recombination rate was significantly positively correlated with GC content, a pattern seen in other taxa. To map QTL affecting virulence, we measured the ability of each progeny strain to kill model hosts, larvae of the wax moth Galleria mellonella. We detected three QTL affecting in vivo virulence that were distinct from QTL affecting in vitro growth, and mapped the virulence QTL to regions containing 7-24 genes, excluding genes with no sequence variation between the parental strains and genes with only synonymous SNPs. None of the genes in our QTL target regions have been previously associated with virulence in Aspergillus, and almost half of these genes are currently annotated as "hypothetical". This study is the first to map QTL affecting the virulence of a fungal pathogen in an animal host, and our results illustrate the power of this approach to identify a short list of unknown genes for further investigation.

  1. Fiber architecture in remodeled myocardium revealed with a quantitative diffusion CMR tractography framework and histological validation

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    Mekkaoui Choukri

    2012-10-01

    Full Text Available Abstract Background The study of myofiber reorganization in the remote zone after myocardial infarction has been performed in 2D. Microstructural reorganization in remodeled hearts, however, can only be fully appreciated by considering myofibers as continuous 3D entities. The aim of this study was therefore to develop a technique for quantitative 3D diffusion CMR tractography of the heart, and to apply this method to quantify fiber architecture in the remote zone of remodeled hearts. Methods Diffusion Tensor CMR of normal human, sheep, and rat hearts, as well as infarcted sheep hearts was performed ex vivo. Fiber tracts were generated with a fourth-order Runge-Kutta integration technique and classified statistically by the median, mean, maximum, or minimum helix angle (HA along the tract. An index of tract coherence was derived from the relationship between these HA statistics. Histological validation was performed using phase-contrast microscopy. Results In normal hearts, the subendocardial and subepicardial myofibers had a positive and negative HA, respectively, forming a symmetric distribution around the midmyocardium. However, in the remote zone of the infarcted hearts, a significant positive shift in HA was observed. The ratio between negative and positive HA variance was reduced from 0.96 ± 0.16 in normal hearts to 0.22 ± 0.08 in the remote zone of the remodeled hearts (p Conclusions A significant reorganization of the 3D fiber continuum is observed in the remote zone of remodeled hearts. The positive (rightward shift in HA in the remote zone is greatest in the subepicardium, but involves all layers of the myocardium. Tractography-based quantification, performed here for the first time in remodeled hearts, may provide a framework for assessing regional changes in the left ventricle following infarction.

  2. Resting-state quantitative electroencephalography reveals increased neurophysiologic connectivity in depression.

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    Andrew F Leuchter

    Full Text Available Symptoms of Major Depressive Disorder (MDD are hypothesized to arise from dysfunction in brain networks linking the limbic system and cortical regions. Alterations in brain functional cortical connectivity in resting-state networks have been detected with functional imaging techniques, but neurophysiologic connectivity measures have not been systematically examined. We used weighted network analysis to examine resting state functional connectivity as measured by quantitative electroencephalographic (qEEG coherence in 121 unmedicated subjects with MDD and 37 healthy controls. Subjects with MDD had significantly higher overall coherence as compared to controls in the delta (0.5-4 Hz, theta (4-8 Hz, alpha (8-12 Hz, and beta (12-20 Hz frequency bands. The frontopolar region contained the greatest number of "hub nodes" (surface recording locations with high connectivity. MDD subjects expressed higher theta and alpha coherence primarily in longer distance connections between frontopolar and temporal or parietooccipital regions, and higher beta coherence primarily in connections within and between electrodes overlying the dorsolateral prefrontal cortical (DLPFC or temporal regions. Nearest centroid analysis indicated that MDD subjects were best characterized by six alpha band connections primarily involving the prefrontal region. The present findings indicate a loss of selectivity in resting functional connectivity in MDD. The overall greater coherence observed in depressed subjects establishes a new context for the interpretation of previous studies showing differences in frontal alpha power and synchrony between subjects with MDD and normal controls. These results can inform the development of qEEG state and trait biomarkers for MDD.

  3. Quantitative MRI reveals decelerated fatty infiltration in muscles of active FSHD patients.

    Science.gov (United States)

    Janssen, Barbara; Voet, Nicoline; Geurts, Alexander; van Engelen, Baziel; Heerschap, Arend

    2016-05-03

    To investigate the effects of aerobic exercise training (AET) and cognitive-behavioral therapy (CBT), directed towards an increase in daily physical activity, on the progression of fatty infiltration and edema in skeletal muscles of patients with facioscapulohumeral muscular dystrophy (FSHD) type 1 by T2 MRI. Quantitative T2 MRI (qT2 MRI) and fat-suppressed T2 MRI of the thigh were performed at 3T on 31 patients, 13 of whom received usual care (UC), 9 AET, and 9 CBT. Muscle-specific fat fractions (%), derived from qT2 MRI, were recorded pretreatment and posttreatment. Intervention effects were analyzed by comparing fat fraction progression rates of the UC with the treated groups using Mann-Whitney tests, and intermuscle differences by a linear mixed model. Edematous hyperintense lesions were identified on the fat-suppressed T2 MRI. The intraclass correlation coefficient for reproducibility of qT2 MRI fat assessment was 0.99. In the UC group, the fat fraction increased by 6.7/year (95% confidence interval [CI] 4.3 to 9.1). This rate decreased to 2.9/year (95% CI 0.7 to 5.2) in the AET (p = 0.03) and 1.7/year (95% CI -0.2 to 3.6) in the CBT group (p = 0.00015). The treatment effect differed among individual muscles. Fewer new edematous lesions occurred after therapy. Fat fraction derived from qT2 MRI is a reproducible and sensitive biomarker to monitor the effects of increased physical activity in individual muscles. This biomarker reports a favorable effect of AET and CBT on the rate of muscular deterioration in FSHD as reflected in decelerated fat replacement. This study provides Class II evidence that for patients with FSHD type 1, both AET and CBT decrease the rate of fatty infiltration in muscles. © 2016 American Academy of Neurology.

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

    Science.gov (United States)

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

    2015-03-19

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

  5. Quantitative expression profiling guided by common retroviral insertion sites reveals novel and cell type–specific cancer genes in leukemia

    Science.gov (United States)

    Sauvageau, Martin; Miller, Michelle; Lemieux, Sébastien; Lessard, Julie; Hébert, Josée; Sauvageau, Guy

    2017-01-01

    Proviral insertional mutagenesis is a powerful tool for the discovery of cancer-associated genes. The ability of integrated proviruses to affect gene expression over long distances combined with the lack of methods to determine the expression levels of large numbers of genes in a systematic and truly quantitative manner have limited the identification of cancer genes by proviral insertional mutagenesis. Here, we have characterized a new model of proviral insertional mutagenesis-induced lymphoid tumors derived from Eed Polycomb group gene mutant mice and quantitatively determined the expression levels of all genes within 100 kb of 20 different retroviral common insertion sites (CISs) identified in these tumors. Using high-throughput quantitative reverse transcription–polymerase chain reaction (Q-RT-PCR), we document an average of 13 CIS-associated genes deregulated per tumor, half of which are leukemia subtype–specific, while the others are coordinately deregulated in the majority of tumors analyzed. Interestingly, we find that genes located distantly from common proviral integration sites are as frequently deregulated as proximal genes, with multiple genes affected per integration. Our studies reveal an unsuspected conservation in the group of genes deregulated among phenotypically similar subtypes of lymphoid leukemias, and suggest that identification of common molecular determinants of this disease is within reach. PMID:17906077

  6. Comparison of fecal and cecal microbiotas reveals qualitative similarities but quantitative differences.

    Science.gov (United States)

    Stanley, Dragana; Geier, Mark S; Chen, Honglei; Hughes, Robert J; Moore, Robert J

    2015-02-27

    quantitatively different. Fecal samples can be effectively used to detect some shifts and responses of cecal microbiota.

  7. Cis-Expression Quantitative Trait Loci Mapping Reveals Replicable Associations with Heroin Addiction in OPRM1

    Science.gov (United States)

    Hancock, Dana B.; Levy, Joshua L.; Gaddis, Nathan C.; Glasheen, Cristie; Saccone, Nancy L.; Page, Grier P.; Hulse, Gary; Wildenauer, Dieter; Kelty, Erin; Schwab, Sibylle; Degenhardt, Louisa; Martin, Nicholas G.; Montgomery, Grant W.; Attia, John; Holliday, Elizabeth G.; McEvoy, Mark; Scott, Rodney J.; Bierut, Laura J.; Nelson, Elliot C.; Kral, Alex; Johnson, Eric O.

    2015-01-01

    Background No opioid receptor, mu 1 (OPRM1) gene polymorphisms, including the functional single nucleotide polymorphism (SNP) rs1799971, have been conclusively associated with heroin/other opioid addiction, despite their biological plausibility. We used evidence of polymorphisms altering OPRM1 expression in normal human brain tissue to nominate and then test associations with heroin addiction. Methods We tested 103 OPRM1 SNPs for association with OPRM1 mRNA expression in prefrontal cortex from 224 European Americans and African Americans of the BrainCloud cohort. We then tested the 16 putative cis-quantitative trait loci (cis-eQTL) SNPs for association with heroin addiction in the Urban Health Study and two replication cohorts, totaling 16,729 European Americans, African Americans, and Australians of European ancestry. Results Four putative cis-eQTL SNPs were significantly associated with heroin addiction in the Urban Health Study (smallest P=8.9×10−5): rs9478495, rs3778150, rs9384169, and rs562859. Rs3778150, located in OPRM1 intron 1, was significantly replicated (P=6.3×10−5). Meta-analysis across all case-control cohorts resulted in P=4.3×10−8: the rs3778150-C allele (frequency=16%-19%) being associated with increased heroin addiction risk. Importantly, the functional SNP allele rs1799971-A was associated with heroin addiction only in the presence of rs3778150-C (P=1.48×10−6 for rs1799971-A/rs3778150-C and P=0.79 for rs1799971-A/rs3778150-T haplotypes). Lastly, replication was observed for six other intron 1 SNPs which had prior suggestive associations with heroin addiction (smallest P=2.7×10−8 for rs3823010). Conclusions Our findings show that common OPRM1 intron 1 SNPs have replicable associations with heroin addiction. The haplotype structure of rs3778150 and nearby SNPs may underlie the inconsistent associations between rs1799971 and heroin addiction. PMID:25744370

  8. Effects of poly-ether B on proteome and phosphoproteome expression in biofouling Balanus amphitrite cyprids

    KAUST Repository

    Dash, Swagatika

    2012-04-01

    Biofouling is ubiquitous in marine environments, and the barnacle Balanus amphitrite is one of the most recalcitrant and aggressive biofoulers in tropical waters. Several natural antifoulants that were claimed to be non-toxic have been isolated in recent years, although the mechanism by which they inhibit fouling is yet to be investigated. Poly-ether B has shown promise in the non-toxic inhibition of larval barnacle attachment. Hence, in this study, multiplex two-dimensional electrophoresis (2-DE) was applied in conjunction with mass spectrometry to investigate the effects of poly-ether B on barnacle larvae at the molecular level. The cyprid proteome response to poly-ether B treatment was analyzed at the total proteome and phosphoproteome levels, with 65 protein and 19 phosphoprotein spots found to be up- or down-regulated. The proteins were found to be related to energy-metabolism, oxidative stress, and molecular chaperones, thus indicating that poly-ether B may interfere with the redox-regulatory mechanisms governing the settlement of barnacle larvae. The results of this study demonstrate the usefulness of the proteomic technique in revealing the working mechanisms of antifouling compounds. © 2012 Copyright Taylor and Francis Group, LLC.

  9. The beginnings of crop phosphoproteomics: exploring early warning systems of stress.

    Directory of Open Access Journals (Sweden)

    Christof eRampitsch

    2012-07-01

    Full Text Available This review examines why a knowledge of plant protein phosphorylation events is important in devising strategies to protect crops from both biotic and abiotic stresses, and why proteomics should be included when studying stress pathways. Most of the achievements in elucidating phospho-signalling pathways in biotic and abiotic stress are reported from model systems: while these are discussed, this review attempts mainly to focus on work done with crops, with examples of achievements reported from rice, maize, wheat, grape, Brassica, tomato and soy bean after cold acclimation, hormonal and oxidative H2O2 treatment, salt stress, mechanical wounding or pathogen challenge. The challenges that remain to transfer this information into a format that can be used to protect crops against biotic and abiotic stresses are enormous. The tremendous increase in the speed and ease of DNA sequencing is poised to reveal the whole genomes of many crop species in the near future, which will facilitate phosphoproteomics and phosphogenomics research.

  10. The differential hippocampal phosphoproteome of Apodemus sylvaticus paralleling spatial memory retrieval in the Barnes maze.

    Science.gov (United States)

    Li, Lin; Csaszar, Edina; Szodorai, Edit; Patil, Sudarshan; Pollak, Arnold; Lubec, Gert

    2014-05-01

    Protein phosphorylation is a well-known and well-documented mechanism in memory processes. Although a large series of protein kinases involved in memory processes have been reported, information on phosphoproteins is limited. It was therefore the aim of the study to determine a partial and differential phosphoproteome along with the corresponding network in hippocampus of a wild caught mouse strain with excellent performance in several paradigms of spatial memory. Apodemus sylvaticus mice were trained in the Barnes maze, a non-invasive test system for spatial memory and untrained mice served as controls. Animals were sacrificed 6h following memory retrieval, hippocampi were taken, proteins extracted and in-solution digestion was carried out with subsequent iTRAQ double labelling. Phosphopeptides were enriched by a TiO2-based method and semi-quantified using two fragmentation principles on the LTQ-orbitrap Velos. In hippocampi of trained animals phosphopeptide levels representing signalling, neuronal, synaptosomal, cytoskeletal and metabolism proteins were at least twofold reduced or increased. Furthermore, a network revealing a link to pathways of ubiquitination, the androgen receptor, small GTPase Rab5 and MAPK signaling as well as synucleins was constructed. This work is relevant for interpretation of previous work and the design of future studies on protein phosphorylation in spatial memory.

  11. Phosphoproteomics and bioinformatics analyses of spinal cord proteins in rats with morphine tolerance.

    Directory of Open Access Journals (Sweden)

    Wen-Jinn Liaw

    Full Text Available INTRODUCTION: Morphine is the most effective pain-relieving drug, but it can cause unwanted side effects. Direct neuraxial administration of morphine to spinal cord not only can provide effective, reliable pain relief but also can prevent the development of supraspinal side effects. However, repeated neuraxial administration of morphine may still lead to morphine tolerance. METHODS: To better understand the mechanism that causes morphine tolerance, we induced tolerance in rats at the spinal cord level by giving them twice-daily injections of morphine (20 µg/10 µL for 4 days. We confirmed tolerance by measuring paw withdrawal latencies and maximal possible analgesic effect of morphine on day 5. We then carried out phosphoproteomic analysis to investigate the global phosphorylation of spinal proteins associated with morphine tolerance. Finally, pull-down assays were used to identify phosphorylated types and sites of 14-3-3 proteins, and bioinformatics was applied to predict biological networks impacted by the morphine-regulated proteins. RESULTS: Our proteomics data showed that repeated morphine treatment altered phosphorylation of 10 proteins in the spinal cord. Pull-down assays identified 2 serine/threonine phosphorylated sites in 14-3-3 proteins. Bioinformatics further revealed that morphine impacted on cytoskeletal reorganization, neuroplasticity, protein folding and modulation, signal transduction and biomolecular metabolism. CONCLUSIONS: Repeated morphine administration may affect multiple biological networks by altering protein phosphorylation. These data may provide insight into the mechanism that underlies the development of morphine tolerance.

  12. TiO2-Based Phosphoproteomic Analysis of the Plasma Membrane and the Effects of Phosphatase Inhibitor Treatment

    DEFF Research Database (Denmark)

    Thingholm, Tine; Larsen, Martin Røssel; Ingrell, Christian

    2008-01-01

    of the plasma membrane phosphoproteome are challenging. We present an optimized analytical strategy for plasma membrane phosphoproteomics that combines efficient plasma membrane protein preparation with TiO 2-based phosphopeptide enrichment and high-performance mass spectrometry for phosphopeptide sequencing...

  13. A quorum-sensing factor in vegetative Dictyostelium discoideum cells revealed by quantitative migration analysis.

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    Laurent Golé

    Full Text Available BACKGROUND: Many cells communicate through the production of diffusible signaling molecules that accumulate and once a critical concentration has been reached, can activate or repress a number of target genes in a process termed quorum sensing (QS. In the social amoeba Dictyostelium discoideum, QS plays an important role during development. However little is known about its effect on cell migration especially in the growth phase. METHODS AND FINDINGS: To investigate the role of cell density on cell migration in the growth phase, we use multisite timelapse microscopy and automated cell tracking. This analysis reveals a high heterogeneity within a given cell population, and the necessity to use large data sets to draw reliable conclusions on cell motion. In average, motion is persistent for short periods of time (t ≤ 5 min, but normal diffusive behavior is recovered over longer time periods. The persistence times are positively correlated with the migrated distances. Interestingly, the migrated distance decreases as well with cell density. The adaptation of cell migration to cell density highlights the role of a secreted quorum sensing factor (QSF on cell migration. Using a simple model describing the balance between the rate of QSF generation and the rate of QSF dilution, we were able to gather all experimental results into a single master curve, showing a sharp cell transition between high and low motile behaviors with increasing QSF. CONCLUSION: This study unambiguously demonstrates the central role played by QSF on amoeboid motion in the growth phase.

  14. Deficiencies in jasmonate-mediated plant defense reveal quantitative variation in Botrytis cinerea pathogenesis.

    Directory of Open Access Journals (Sweden)

    Heather C Rowe

    2010-04-01

    Full Text Available Despite the described central role of jasmonate signaling in plant defense against necrotrophic pathogens, the existence of intraspecific variation in pathogen capacity to activate or evade plant jasmonate-mediated defenses is rarely considered. Experimental infection of jasmonate-deficient and jasmonate-insensitive Arabidopsis thaliana with diverse isolates of the necrotrophic fungal pathogen Botrytis cinerea revealed pathogen variation for virulence inhibition by jasmonate-mediated plant defenses and induction of plant defense metabolites. Comparison of the transcriptional effects of infection by two distinct B. cinerea isolates showed only minor differences in transcriptional responses of wild-type plants, but notable isolate-specific transcript differences in jasmonate-insensitive plants. These transcriptional differences suggest B. cinerea activation of plant defenses that require plant jasmonate signaling for activity in response to only one of the two B. cinerea isolates tested. Thus, similar infection phenotypes observed in wild-type plants result from different signaling interactions with the plant that are likely integrated by jasmonate signaling.

  15. Isotopic Zonation Within Sulfate Evaporite Mineral Crystals Reveal Quantitative Paleoenvironment Details

    Science.gov (United States)

    Coleman, M.; Rhorssen, M.; Mielke, R. E.

    2008-12-01

    developed a new analytical method [2]. We use a modification of the standard TC/EA continuous-flow protocol to measure both hydrogen and oxygen of water of hydration from the same small sample. We have proved the concept of this new approach by analyzing zones within crystals and individual grains, growing epsomite (magnesium sulfate heptahydrate) in the laboratory and by analysis of natural gypsum evaporites. We are now exploring the effects of varying the controlling parameters. Eventual application to Martian sulfates will reveal amount of water involved in sulfate formation, its isotopic composition(s) and details of the paleo-atmospheric humidity. [1] Gat JR and Gonfiantini R, (Eds) (1981) IAEA Technical Report Series. [2] Rohrssen MK, Brunner B Mielke RE and Coleman M (2008) Analyt. Chem. (in press).

  16. 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.

  17. A Quantitative Profiling Tool for Diverse Genomic Data Types Reveals Potential Associations between Chromatin and Pre-mRNA Processing.

    Science.gov (United States)

    Kremsky, Isaac; Bellora, Nicolás; Eyras, Eduardo

    2015-01-01

    High-throughput sequencing, and genome-based datasets in general, are often represented as profiles centered at reference points to study the association of protein binding and other signals to particular regulatory mechanisms. Although these profiles often provide compelling evidence of these associations, they do not provide a quantitative assessment of the enrichment, which makes the comparison between signals and conditions difficult. In addition, a number of biases can confound profiles, but are rarely accounted for in the tools currently available. We present a novel computational method, ProfileSeq, for the quantitative assessment of biological profiles to provide an exact, nonparametric test that specific regions of the test profile have higher or lower signal densities than a control set. The method is applicable to high-throughput sequencing data (ChIP-Seq, GRO-Seq, CLIP-Seq, etc.) and to genome-based datasets (motifs, etc.). We validate ProfileSeq by recovering and providing a quantitative assessment of several results reported before in the literature using independent datasets. We show that input signal and mappability have confounding effects on the profile results, but that normalizing the signal by input reads can eliminate these biases while preserving the biological signal. Moreover, we apply ProfileSeq to ChIP-Seq data for transcription factors, as well as for motif and CLIP-Seq data for splicing factors. In all examples considered, the profiles were robust to biases in mappability of sequencing reads. Furthermore, analyses performed with ProfileSeq reveal a number of putative relationships between transcription factor binding to DNA and splicing factor binding to pre-mRNA, adding to the growing body of evidence relating chromatin and pre-mRNA processing. ProfileSeq provides a robust way to quantify genome-wide coordinate-based signal. Software and documentation are freely available for academic use at https://bitbucket.org/regulatorygenomicsupf/profileseq/.

  18. A Quantitative Profiling Tool for Diverse Genomic Data Types Reveals Potential Associations between Chromatin and Pre-mRNA Processing.

    Directory of Open Access Journals (Sweden)

    Isaac Kremsky

    Full Text Available High-throughput sequencing, and genome-based datasets in general, are often represented as profiles centered at reference points to study the association of protein binding and other signals to particular regulatory mechanisms. Although these profiles often provide compelling evidence of these associations, they do not provide a quantitative assessment of the enrichment, which makes the comparison between signals and conditions difficult. In addition, a number of biases can confound profiles, but are rarely accounted for in the tools currently available. We present a novel computational method, ProfileSeq, for the quantitative assessment of biological profiles to provide an exact, nonparametric test that specific regions of the test profile have higher or lower signal densities than a control set. The method is applicable to high-throughput sequencing data (ChIP-Seq, GRO-Seq, CLIP-Seq, etc. and to genome-based datasets (motifs, etc.. We validate ProfileSeq by recovering and providing a quantitative assessment of several results reported before in the literature using independent datasets. We show that input signal and mappability have confounding effects on the profile results, but that normalizing the signal by input reads can eliminate these biases while preserving the biological signal. Moreover, we apply ProfileSeq to ChIP-Seq data for transcription factors, as well as for motif and CLIP-Seq data for splicing factors. In all examples considered, the profiles were robust to biases in mappability of sequencing reads. Furthermore, analyses performed with ProfileSeq reveal a number of putative relationships between transcription factor binding to DNA and splicing factor binding to pre-mRNA, adding to the growing body of evidence relating chromatin and pre-mRNA processing. ProfileSeq provides a robust way to quantify genome-wide coordinate-based signal. Software and documentation are freely available for academic use at https://bitbucket.org/regulatorygenomicsupf/profileseq/.

  19. Involvement of GABA transporters in atropine-treated myopic retina as revealed by iTRAQ quantitative proteomics.

    Science.gov (United States)

    Barathi, Veluchamy A; Chaurasia, Shyam S; Poidinger, Michael; Koh, Siew Kwan; Tian, Dechao; Ho, Candice; Iuvone, P Michael; Beuerman, Roger W; Zhou, Lei

    2014-11-07

    Atropine, a muscarinic antagonist, is known to inhibit myopia progression in several animal models and humans. However, the mode of action is not established yet. In this study, we compared quantitative iTRAQ proteomic analysis in the retinas collected from control and lens-induced myopic (LIM) mouse eyes treated with atropine. The myopic group received a (-15D) spectacle lens over the right eye on postnatal day 10 with or without atropine eye drops starting on postnatal day 24. Axial length was measured by optical low coherence interferometry (OLCI), AC-Master, and refraction was measured by automated infrared photorefractor at postnatal 24, 38, and 52 days. Retinal tissue samples were pooled from six eyes for each group. The experiments were repeated twice, and technical replicates were also performed for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. MetaCore was used to perform protein profiling for pathway analysis. We identified a total of 3882 unique proteins with retina proteome reported to date. Thirty proteins were found to be up-regulated (ratio for myopia/control > global mean ratio + 1 standard deviation), and 28 proteins were down-regulated (ratio for myopia/control retinas. Pathway analysis using MetaCore revealed regulation of γ-aminobutyric acid (GABA) levels in the myopic eyes. Detailed analysis of the quantitative proteomics data showed that the levels of GABA transporter 1 (GAT-1) were elevated in myopic retina and significantly reduced after atropine treatment. These results were further validated with immunohistochemistry and Western blot analysis. In conclusion, this study provides a comprehensive quantitative proteomic analysis of atropine-treated mouse retina and suggests the involvement of GABAergic signaling in the antimyopic effects of atropine in mouse eyes. The GABAergic transmission in the neural retina plays a pivotal role in the maintenance of axial eye growth in mammals.

  20. Quantitative proteomics reveals regulatory differences in the chondrocyte secretome from human medial and lateral femoral condyles in osteoarthritic patients.

    Science.gov (United States)

    Stenberg, Johan; Rüetschi, Ulla; Skiöldebrand, Eva; Kärrholm, Johan; Lindahl, Anders

    2013-10-04

    Osteoarthritis (OA) is a destructive joint disease and there are no known biomarkers available for an early diagnosis. To identify potential disease biomarkers and gain further insight into the disease mechanisms of OA we applied quantitative proteomics with SILAC technology on the secretomes from chondrocytes of OA knees, designated as high Mankin (HM) scored secretome. A quantitative comparison was made between the secretomes of the medial and lateral femur condyle chondrocytes in the same knee since the medial femur condyle is usually more affected in OA than the lateral condyle, which was confirmed by Mankin scoring. The medial/lateral comparison was also made on the secretomes from chondrocytes taken from one individual with no clinically apparent joint-disease, designated as low Mankin (LM) scored secretome. We identified 825 proteins in the HM secretome and 69 of these showed differential expression when comparing the medial and lateral femoral compartment. The LM scored femoral condyle showed early signs of OA in the medial compartment as assessed by Mankin score. We here report the identification and relative quantification of several proteins of interest for the OA disease mechanism e.g. CYTL1, DMD and STAB1 together with putative early disease markers e.g. TIMP1, PPP2CA and B2M. The present study reveals differences in protein abundance between medial/lateral femur condyles in OA patients. These regulatory differences expand the knowledge regarding OA disease markers and mechanisms.

  1. A Quantitative Model of Motility Reveals Low-Dimensional Variation in Exploratory Behavior Across Multiple Nematode Species

    Science.gov (United States)

    Helms, Stephen; Avery, Leon; Stephens, Greg; Shimizu, Tom

    2014-03-01

    Animal behavior emerges from many layers of biological organization--from molecular signaling pathways and neuronal networks to mechanical outputs of muscles. In principle, the large number of interconnected variables at each of these layers could imply dynamics that are complex and hard to control or even tinker with. Yet, for organisms to survive in a competitive, ever-changing environment, behavior must readily adapt. We applied quantitative modeling to identify important aspects of behavior in chromadorean nematodes ranging from the lab strain C. elegans N2 to wild strains and distant species. We revealed subtle yet important features such as speed control and heavy-tailed directional changes. We found that the parameters describing this behavioral model varied among individuals and across species in a correlated way that is consistent with a trade-off between exploratory and exploitative behavior.

  2. Phosphoproteomic profiling of in vivo signaling in liver by the mammalian target of rapamycin complex 1 (mTORC1.

    Directory of Open Access Journals (Sweden)

    Gokhan Demirkan

    Full Text Available Our understanding of signal transduction networks in the physiological context of an organism remains limited, partly due to the technical challenge of identifying serine/threonine phosphorylated peptides from complex tissue samples. In the present study, we focused on signaling through the mammalian target of rapamycin (mTOR complex 1 (mTORC1, which is at the center of a nutrient- and growth factor-responsive cell signaling network. Though studied extensively, the mechanisms involved in many mTORC1 biological functions remain poorly understood.We developed a phosphoproteomic strategy to purify, enrich and identify phosphopeptides from rat liver homogenates. Using the anticancer drug rapamycin, the only known target of which is mTORC1, we characterized signaling in liver from rats in which the complex was maximally activated by refeeding following 48 hr of starvation. Using protein and peptide fractionation methods, TiO(2 affinity purification of phosphopeptides and mass spectrometry, we reproducibly identified and quantified over four thousand phosphopeptides. Along with 5 known rapamycin-sensitive phosphorylation events, we identified 62 new rapamycin-responsive candidate phosphorylation sites. Among these were PRAS40, gephyrin, and AMP kinase 2. We observed similar proportions of increased and reduced phosphorylation in response to rapamycin. Gene ontology analysis revealed over-representation of mTOR pathway components among rapamycin-sensitive phosphopeptide candidates.In addition to identifying potential new mTORC1-mediated phosphorylation events, and providing information relevant to the biology of this signaling network, our experimental and analytical approaches indicate the feasibility of large-scale phosphoproteomic profiling of tissue samples to study physiological signaling events in vivo.

  3. Rules of RNA specificity of hnRNP A1 revealed by global and quantitative analysis of its affinity distribution

    Science.gov (United States)

    Jain, Niyati; Lin, Hsuan-Chun; Morgan, Christopher E.; Harris, Michael E.; Tolbert, Blanton S.

    2017-01-01

    Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a multipurpose RNA-binding protein (RBP) involved in normal and pathological RNA metabolism. Transcriptome-wide mapping and in vitro evolution identify consensus hnRNP A1 binding motifs; however, such data do not reveal how surrounding RNA sequence and structural context modulate affinity. We determined the affinity of hnRNP A1 for all possible sequence variants (n = 16,384) of the HIV exon splicing silencer 3 (ESS3) 7-nt apical loop. Analysis of the affinity distribution identifies the optimal motif 5′-YAG-3′ and shows how its copy number, position in the loop, and loop structure modulate affinity. For a subset of ESS3 variants, we show that specificity is determined by association rate constants and that variants lacking the minimal sequence motif bind competitively with consensus RNA. Thus, the results reveal general rules of specificity of hnRNP A1 and provide a quantitative framework for understanding how it discriminates between alternative competing RNA ligands in vivo. PMID:28193894

  4. Quantitative Proteomic Analysis Reveals Populus cathayana Females Are More Sensitive and Respond More Sophisticatedly to Iron Deficiency than Males.

    Science.gov (United States)

    Zhang, Sheng; Zhang, Yunxiang; Cao, Yanchun; Lei, Yanbao; Jiang, Hao

    2016-03-04

    Previous studies have shown that there are significant sexual differences in the morphological and physiological responses of Populus cathayana Rehder to nitrogen and phosphorus deficiencies, but little is known about the sex-specific differences in responses to iron deficiency. In this study, the effects of iron deficiency on the morphology, physiology, and proteome of P. cathayana males and females were investigated. The results showed that iron deficiency (25 days) significantly decreased height growth, photosynthetic rate, chlorophyll content, and tissue iron concentration in both sexes. A comparison between the sexes indicated that iron-deficient males had less height inhibition and photosynthesis system II or chloroplast ultrastructural damage than iron-deficient females. iTRAQ-based quantitative proteomic analysis revealed that 144 and 68 proteins were decreased in abundance (e.g., proteins involved in photosynthesis, carbohydrate and energy metabolism, and gene expression regulation) and 78 and 39 proteins were increased in abundance (e.g., proteins involved in amino acid metabolism and stress response) according to the criterion of ratio ≥1.5 in females and males, respectively. A comparison between the sexes indicated that iron-deficient females exhibited a greater change in the proteins involved in photosynthesis, carbon and energy metabolism, the redox system, and stress responsive proteins. This study reveals females are more sensitive and have a more sophisticated response to iron deficiency compared with males and provides new insights into differential sexual responses to nutrient deficiency.

  5. Salt-induced changes in cardiac phosphoproteome in a rat model of chronic renal failure.

    Science.gov (United States)

    Su, Zhengxiu; Zhu, Hongguo; Zhang, Menghuan; Wang, Liangliang; He, Hanchang; Jiang, Shaoling; Hou, Fan Fan; Li, Aiqing

    2014-01-01

    Heart damage is widely present in patients with chronic kidney disease. Salt diet is the most important environmental factor affecting development of chronic renal failure and cardiovascular diseases. The proteins involved in chronic kidney disease -induced heart damage, especially their posttranslational modifications, remain largely unknown to date. Sprague-Dawley rats underwent 5/6 nephrectomy (chronic renal failure model) or sham operation were treated for 2 weeks with a normal-(0.4% NaCl), or high-salt (4% NaCl) diet. We employed TiO2 enrichment, iTRAQ labeling and liquid-chromatography tandem mass spectrometry strategy for phosphoproteomic profiling of left ventricular free walls in these animals. A total of 1724 unique phosphopeptides representing 2551 non-redundant phosphorylation sites corresponding to 763 phosphoproteins were identified. During normal salt feeding, 89 (54%) phosphopeptides upregulated and 76 (46%) phosphopeptides downregulated in chronic renal failure rats relative to sham rats. In chronic renal failure rats, high salt intake induced upregulation of 84 (49%) phosphopeptides and downregulation of 88 (51%) phosphopeptides. Database searches revealed that most of the identified phospholproteins were important signaling molecules such as protein kinases, receptors and phosphatases. These phospholproteins were involved in energy metabolism, cell communication, cell differentiation, cell death and other biological processes. The Search Tool for the Retrieval of Interacting Genes analysis revealed functional links among 15 significantly regulated phosphoproteins in chronic renal failure rats compared to sham group, and 23 altered phosphoproteins induced by high salt intake. The altered phosphorylation levels of two proteins involved in heart damage, lamin A and phospholamban were validated. Expression of the downstream genes of these two proteins, desmin and SERCA2a, were also analyzed.

  6. Salt-induced changes in cardiac phosphoproteome in a rat model of chronic renal failure.

    Directory of Open Access Journals (Sweden)

    Zhengxiu Su

    Full Text Available Heart damage is widely present in patients with chronic kidney disease. Salt diet is the most important environmental factor affecting development of chronic renal failure and cardiovascular diseases. The proteins involved in chronic kidney disease -induced heart damage, especially their posttranslational modifications, remain largely unknown to date. Sprague-Dawley rats underwent 5/6 nephrectomy (chronic renal failure model or sham operation were treated for 2 weeks with a normal-(0.4% NaCl, or high-salt (4% NaCl diet. We employed TiO2 enrichment, iTRAQ labeling and liquid-chromatography tandem mass spectrometry strategy for phosphoproteomic profiling of left ventricular free walls in these animals. A total of 1724 unique phosphopeptides representing 2551 non-redundant phosphorylation sites corresponding to 763 phosphoproteins were identified. During normal salt feeding, 89 (54% phosphopeptides upregulated and 76 (46% phosphopeptides downregulated in chronic renal failure rats relative to sham rats. In chronic renal failure rats, high salt intake induced upregulation of 84 (49% phosphopeptides and downregulation of 88 (51% phosphopeptides. Database searches revealed that most of the identified phospholproteins were important signaling molecules such as protein kinases, receptors and phosphatases. These phospholproteins were involved in energy metabolism, cell communication, cell differentiation, cell death and other biological processes. The Search Tool for the Retrieval of Interacting Genes analysis revealed functional links among 15 significantly regulated phosphoproteins in chronic renal failure rats compared to sham group, and 23 altered phosphoproteins induced by high salt intake. The altered phosphorylation levels of two proteins involved in heart damage, lamin A and phospholamban were validated. Expression of the downstream genes of these two proteins, desmin and SERCA2a, were also analyzed.

  7. Transcriptome- Assisted Label-Free Quantitative Proteomics Analysis Reveals Novel Insights into Piper nigrum-Phytophthora capsici Phytopathosystem.

    Science.gov (United States)

    Mahadevan, Chidambareswaren; Krishnan, Anu; Saraswathy, Gayathri G; Surendran, Arun; Jaleel, Abdul; Sakuntala, Manjula

    2016-01-01

    Black pepper (Piper nigrum L.), a tropical spice crop of global acclaim, is susceptible to Phytophthora capsici, an oomycete pathogen which causes the highly destructive foot rot disease. A systematic understanding of this phytopathosystem has not been possible owing to lack of genome or proteome information. In this study, we explain an integrated transcriptome-assisted label-free quantitative proteomics pipeline to study the basal immune components of black pepper when challenged with P. capsici. We report a global identification of 532 novel leaf proteins from black pepper, of which 518 proteins were functionally annotated using BLAST2GO tool. A label-free quantitation of the protein datasets revealed 194 proteins common to diseased and control protein datasets of which 22 proteins showed significant up-regulation and 134 showed significant down-regulation. Ninety-three proteins were identified exclusively on P. capsici infected leaf tissues and 245 were expressed only in mock (control) infected samples. In-depth analysis of our data gives novel insights into the regulatory pathways of black pepper which are compromised during the infection. Differential down-regulation was observed in a number of critical pathways like carbon fixation in photosynthetic organism, cyano-amino acid metabolism, fructose, and mannose metabolism, glutathione metabolism, and phenylpropanoid biosynthesis. The proteomics results were validated with real-time qRT-PCR analysis. We were also able to identify the complete coding sequences for all the proteins of which few selected genes were cloned and sequence characterized for further confirmation. Our study is the first report of a quantitative proteomics dataset in black pepper which provides convincing evidence on the effectiveness of a transcriptome-based label-free proteomics approach for elucidating the host response to biotic stress in a non-model spice crop like P. nigrum, for which genome information is unavailable. Our dataset

  8. Transcriptome- Assisted Label-Free Quantitative Proteomics Analysis Reveals Novel Insights into Piper nigrum—Phytophthora capsici Phytopathosystem

    Science.gov (United States)

    Mahadevan, Chidambareswaren; Krishnan, Anu; Saraswathy, Gayathri G.; Surendran, Arun; Jaleel, Abdul; Sakuntala, Manjula

    2016-01-01

    Black pepper (Piper nigrum L.), a tropical spice crop of global acclaim, is susceptible to Phytophthora capsici, an oomycete pathogen which causes the highly destructive foot rot disease. A systematic understanding of this phytopathosystem has not been possible owing to lack of genome or proteome information. In this study, we explain an integrated transcriptome-assisted label-free quantitative proteomics pipeline to study the basal immune components of black pepper when challenged with P. capsici. We report a global identification of 532 novel leaf proteins from black pepper, of which 518 proteins were functionally annotated using BLAST2GO tool. A label-free quantitation of the protein datasets revealed 194 proteins common to diseased and control protein datasets of which 22 proteins showed significant up-regulation and 134 showed significant down-regulation. Ninety-three proteins were identified exclusively on P. capsici infected leaf tissues and 245 were expressed only in mock (control) infected samples. In-depth analysis of our data gives novel insights into the regulatory pathways of black pepper which are compromised during the infection. Differential down-regulation was observed in a number of critical pathways like carbon fixation in photosynthetic organism, cyano-amino acid metabolism, fructose, and mannose metabolism, glutathione metabolism, and phenylpropanoid biosynthesis. The proteomics results were validated with real-time qRT-PCR analysis. We were also able to identify the complete coding sequences for all the proteins of which few selected genes were cloned and sequence characterized for further confirmation. Our study is the first report of a quantitative proteomics dataset in black pepper which provides convincing evidence on the effectiveness of a transcriptome-based label-free proteomics approach for elucidating the host response to biotic stress in a non-model spice crop like P. nigrum, for which genome information is unavailable. Our dataset

  9. Phosphoproteomic profiling of selenate-treated Alzheimer's disease model cells.

    Directory of Open Access Journals (Sweden)

    Ping Chen

    Full Text Available The reversible phosphorylation of proteins regulates most biological processes, while abnormal phosphorylation is a cause or consequence of many diseases including Alzheimer's disease (AD. One of the hallmarks of AD is the formation of neurofibrillary tangles (NFTs, which is composed of hyperphosphorylated tau proteins. Sodium selenate has been recently found to reduce tau hyperphosphorylation and NFTs formation, and to improve spatial learning and motor performance in AD mice. In the current study, the phosphoproteomics of N2aSW cells treated with selenate were investigated. To avoid missing low-abundance phosphoproteins, both the total proteins of cells and the phosphor-enriched proteins were extracted and subjected to the two-dimensional gel electrophoresis with Pro-Q diamond staining and then LC-MS/MS analysis. A total of 65 proteins were altered in phosphorylation level, of which 39 were up-regulated and 26 were down-regulated. All identified phosphoproteins were bioinformatically annotated according to their physiochemical features, subcellular location, and biological function. Most of these significantly changed phosphoproteins are involved in crucial neural processes such as protesome activity, oxidative stress, cysteine and methionine metabolism, and energy metabolism. Furthermore, decreases were found in homocysteine, phosphor-tau and amyloid β upon selenate treatment. Our results suggest that selenate may intervene in the pathological process of AD by altering the phosphorylation of some key proteins involved in oxidative stress, energy metabolism and protein degradation, thus play important roles in maintaining redox homeostasis, generating ATP, and clearing misfolded proteins and aggregates. The present paper provides some new clues to the mechanism of selenate in AD prevention.

  10. ROS-activated ATM-dependent phosphorylation of cytoplasmic substrates identified by large scale phosphoproteomics screen

    DEFF Research Database (Denmark)

    Kozlov, Sergei V; Waardenberg, Ashley J; Engholm-Keller, Kasper

    2016-01-01

    substrates (HMGA1 and UIMCI/RAP80), another five were identified in a whole cell extract phosphoproteomic screens and the remaining four proteins had not been identified previously in DNA damage response screens. We validated the phosphorylation of three of these proteins (OSR1, HDGF and ccdc82) as ATM...

  11. Quantitative Analysis of MicroRNAs in Vaccinia virus Infection Reveals Diversity in Their Susceptibility to Modification and Suppression.

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    Amy H Buck

    Full Text Available Vaccinia virus (VACV is a large cytoplasmic DNA virus that causes dramatic alterations to many cellular pathways including microRNA biogenesis. The virus encodes a poly(A polymerase which was previously shown to add poly(A tails to the 3' end of cellular miRNAs, resulting in their degradation by 24 hours post infection (hpi. Here we used small RNA sequencing to quantify the impact of VACV infection on cellular miRNAs in human cells at both early (6 h and late (24 h times post infection. A detailed quantitative analysis of individual miRNAs revealed marked diversity in the extent of their modification and relative change in abundance during infection. Some miRNAs became highly modified (e.g. miR-29a-3p, miR-27b-3p whereas others appeared resistant (e.g. miR-16-5p. Furthermore, miRNAs that were highly tailed at 6 hpi were not necessarily among the most reduced at 24 hpi. These results suggest that intrinsic features of human cellular miRNAs cause them to be differentially polyadenylated and altered in abundance during VACV infection. We also demonstrate that intermediate and late VACV gene expression are required for optimal repression of some miRNAs including miR-27-3p. Overall this work reveals complex and varied consequences of VACV infection on host miRNAs and identifies miRNAs which are largely resistant to VACV-induced polyadenylation and are therefore present at functional levels during the initial stages of infection and replication.

  12. SWATH-MS Quantitative Proteomic Investigation Reveals a Role of Jasmonic Acid during Lead Response in Arabidopsis.

    Science.gov (United States)

    Zhu, Fu-Yuan; Chan, Wai-Lung; Chen, Mo-Xian; Kong, Ricky P W; Cai, Congxi; Wang, Qiaomei; Zhang, Jian-Hua; Lo, Clive

    2016-10-07

    Lead (Pb) pollution is a growing environment problem that continuously threatens the productivity of crops. To understand the molecular mechanisms of plant adaptation to Pb toxicity, we examined proteome changes in Arabidopsis seedlings following Pb treatment by SWATH-MS, a label-free quantitative proteomic platform. We identified and quantified the expression of 1719 proteins in water- and Pb-treated plants. Among them, 231 proteins showed significant abundance changes (151 elevated and 80 reduced) upon Pb exposure. Functional categorization revealed that most of the Pb-responsive proteins are involved in different metabolic processes. For example, down-regulation of photosynthesis and biosynthesis of isoprenoids and tetrapyrroles in chloroplasts were observed. On the contrary, pathways leading to glutathione, jasmonic acid (JA), glucosinolate (GSL), and phenylpropanoid production are up-regulated. Experimental characterizations demonstrated a rapid elevation of endogenic JA production in Pb-treated Arabidopsis seedlings, while a JA-deficient mutant and a JA-insensitive mutant showed hypersensitivity to root inhibition by Pb, implicating an essential role of JA during Pb responses. Consistently, methyl jasmonate supplementation alleviated Pb toxicity in the wild-type and JA-deficient mutant. Furthermore, GSL levels were substantially enhanced following Pb treatment, while such induction was not detected in the JA mutant, suggesting that the Pb-induced GSL accumulation is JA-dependent. Overall, our work represents the first SWATH-MS analysis in Arabidopsis and highlights a potential mediating role of JA during Pb stress.

  13. Label-free quantitative proteomics reveals differentially regulated proteins in the latex of sticky diseased Carica papaya L. plants.

    Science.gov (United States)

    Rodrigues, Silas P; Ventura, José A; Aguilar, Clemente; Nakayasu, Ernesto S; Choi, HyungWon; Sobreira, Tiago J P; Nohara, Lilian L; Wermelinger, Luciana S; Almeida, Igor C; Zingali, Russolina B; Fernandes, Patricia M B

    2012-06-18

    Papaya meleira virus (PMeV) is so far the only described laticifer-infecting virus, the causal agent of papaya (Carica papaya L.) sticky disease. The effects of PMeV on the laticifers' regulatory network were addressed here through the proteomic analysis of papaya latex. Using both 1-DE- and 1D-LC-ESI-MS/MS, 160 unique papaya latex proteins were identified, representing 122 new proteins in the latex of this plant. Quantitative analysis by normalized spectral counting revealed 10 down-regulated proteins in the latex of diseased plants, 9 cysteine proteases (chymopapain) and 1 latex serine proteinase inhibitor. A repression of papaya latex proteolytic activity during PMeV infection was hypothesized. This was further confirmed by enzymatic assays that showed a reduction of cysteine-protease-associated proteolytic activity in the diseased papaya latex. These findings are discussed in the context of plant responses against pathogens and may greatly contribute to understand the roles of laticifers in plant stress responses.

  14. Quantitative genome-wide genetic interaction screens reveal global epistatic relationships of protein complexes in Escherichia coli.

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    Mohan Babu

    2014-02-01

    Full Text Available Large-scale proteomic analyses in Escherichia coli have documented the composition and physical relationships of multiprotein complexes, but not their functional organization into biological pathways and processes. Conversely, genetic interaction (GI screens can provide insights into the biological role(s of individual gene and higher order associations. Combining the information from both approaches should elucidate how complexes and pathways intersect functionally at a systems level. However, such integrative analysis has been hindered due to the lack of relevant GI data. Here we present a systematic, unbiased, and quantitative synthetic genetic array screen in E. coli describing the genetic dependencies and functional cross-talk among over 600,000 digenic mutant combinations. Combining this epistasis information with putative functional modules derived from previous proteomic data and genomic context-based methods revealed unexpected associations, including new components required for the biogenesis of iron-sulphur and ribosome integrity, and the interplay between molecular chaperones and proteases. We find that functionally-linked genes co-conserved among γ-proteobacteria are far more likely to have correlated GI profiles than genes with divergent patterns of evolution. Overall, examining bacterial GIs in the context of protein complexes provides avenues for a deeper mechanistic understanding of core microbial systems.

  15. Quantitative proteome analysis of an antibiotic resistant Escherichia coli exposed to tetracycline reveals multiple affected metabolic and peptidoglycan processes.

    Science.gov (United States)

    Jones-Dias, Daniela; Carvalho, Ana Sofia; Moura, Inês Barata; Manageiro, Vera; Igrejas, Gilberto; Caniça, Manuela; Matthiesen, Rune

    2017-03-06

    Tetracyclines are among the most commonly used antibiotics administrated to farm animals for disease treatment and prevention, contributing to the worldwide increase in antibiotic resistance in animal and human pathogens. Although tetracycline mechanisms of resistance are well known, the role of metabolism in bacterial reaction to antibiotic stress is still an important assignment and could contribute to the understanding of tetracycline related stress response. In this study, spectral counts-based label free quantitative proteomics has been applied to study the response to tetracycline of the environmental-borne Escherichia coli EcAmb278 isolate soluble proteome. A total of 1484 proteins were identified by high resolution mass spectrometry at a false discovery rate threshold of 1%, of which 108 were uniquely identified under absence of tetracycline whereas 126 were uniquely identified in presence of tetracycline. These proteins revealed interesting difference in e.g. proteins involved in peptidoglycan-based cell wall proteins and energy metabolism. Upon treatment, 12 proteins were differentially regulated showing more than 2-fold change and presistant E. coli provides novel insight into tetracycline related stress. The lack of new antibiotics to fight infections caused by multidrug resistant microorganisms has motivated the use of old antibiotics, and the search for new drug targets. The evolution of antibiotic resistance is complex, but it is known that agroecosystems play an important part in the selection of antibiotic resistance bacteria. Tetracyclines are still used as phytopharmaceutical agents in crops, selecting resistant bacteria and changing the ecology of farm soil. Little is known about the metabolic response of genetically resistant populations to antibiotic exposure. Indeed, to date there are no quantitative tetracycline resistance studies performed with the latest generation of high resolution mass spectrometers allowing high mass accuracy in both

  16. Plasma proteome response to severe burn injury revealed by 18O-labeled "universal" reference-based quantitative proteomics.

    Science.gov (United States)

    Qian, Wei-Jun; Petritis, Brianne O; Kaushal, Amit; Finnerty, Celeste C; Jeschke, Marc G; Monroe, Matthew E; Moore, Ronald J; Schepmoes, Athena A; Xiao, Wenzhong; Moldawer, Lyle L; Davis, Ronald W; Tompkins, Ronald G; Herndon, David N; Camp, David G; Smith, Richard D

    2010-09-01

    A burn injury represents one of the most severe forms of human trauma and is responsible for significant mortality worldwide. Here, we present the first quantitative proteomics investigation of the blood plasma proteome response to severe burn injury by comparing the plasma protein concentrations of 10 healthy control subjects with those of 15 severe burn patients at two time-points following the injury. The overall analytical strategy for this work integrated immunoaffinity depletion of the 12 most abundant plasma proteins with cysteinyl-peptide enrichment-based fractionation prior to LC-MS analyses of individual patient samples. Incorporation of an 18O-labeled "universal" reference among the sample sets enabled precise relative quantification across samples. In total, 313 plasma proteins confidently identified with two or more unique peptides were quantified. Following statistical analysis, 110 proteins exhibited significant abundance changes in response to the burn injury. The observed changes in protein concentrations suggest significant inflammatory and hypermetabolic response to the injury, which is supported by the fact that many of the identified proteins are associated with acute phase response signaling, the complement system, and coagulation system pathways. The regulation of approximately 35 proteins observed in this study is in agreement with previous results reported for inflammatory or burn response, but approximately 50 potentially novel proteins previously not known to be associated with burn response or inflammation are also found. Elucidating proteins involved in the response to severe burn injury may reveal novel targets for therapeutic interventions as well as potential predictive biomarkers for patient outcomes such as multiple organ failure.

  17. A quantitative validated model reveals two phases of transcriptional regulation for the gap gene giant in Drosophila.

    Science.gov (United States)

    Hoermann, Astrid; Cicin-Sain, Damjan; Jaeger, Johannes

    2016-03-15

    Understanding eukaryotic transcriptional regulation and its role in development and pattern formation is one of the big challenges in biology today. Most attempts at tackling this problem either focus on the molecular details of transcription factor binding, or aim at genome-wide prediction of expression patterns from sequence through bioinformatics and mathematical modelling. Here we bridge the gap between these two complementary approaches by providing an integrative model of cis-regulatory elements governing the expression of the gap gene giant (gt) in the blastoderm embryo of Drosophila melanogaster. We use a reverse-engineering method, where mathematical models are fit to quantitative spatio-temporal reporter gene expression data to infer the regulatory mechanisms underlying gt expression in its anterior and posterior domains. These models are validated through prediction of gene expression in mutant backgrounds. A detailed analysis of our data and models reveals that gt is regulated by domain-specific CREs at early stages, while a late element drives expression in both the anterior and the posterior domains. Initial gt expression depends exclusively on inputs from maternal factors. Later, gap gene cross-repression and gt auto-activation become increasingly important. We show that auto-regulation creates a positive feedback, which mediates the transition from early to late stages of regulation. We confirm the existence and role of gt auto-activation through targeted mutagenesis of Gt transcription factor binding sites. In summary, our analysis provides a comprehensive picture of spatio-temporal gene regulation by different interacting enhancer elements for an important developmental regulator.

  18. Novel X-linked genes revealed by quantitative polymerase chain reaction in the green anole, Anolis carolinensis.

    Science.gov (United States)

    Rovatsos, Michail; Altmanová, Marie; Pokorná, Martina Johnson; Kratochvíl, Lukáš

    2014-08-28

    The green anole, Anolis carolinensis (ACA), is the model reptile for a vast array of biological disciplines. It was the first nonavian reptile to have its genome fully sequenced. During the genome project, the XX/XY system of sex chromosomes homologous to chicken chromosome 15 (GGA15) was revealed, and 106 X-linked genes were identified. We selected 38 genes located on eight scaffolds in ACA and having orthologs located on GGA15, then tested their linkage to ACA X chromosome by using comparative quantitative fluorescent real-time polymerase chain reaction applied to male and female genomic DNA. All tested genes appeared to be X-specific and not present on the Y chromosome. Assuming that all genes located on these scaffolds should be localized to the ACA X chromosome, we more than doubled the number of known X-linked genes in ACA, from 106 to 250. While demonstrating that the gene content of chromosome X in ACA and GGA15 is largely conserved, we nevertheless showed that numerous interchromosomal rearrangements had occurred since the splitting of the chicken and anole evolutionary lineages. The presence of many ACA X-specific genes localized to distinct contigs indicates that the ACA Y chromosome should be highly degenerated, having lost a large amount of its original gene content during evolution. The identification of novel genes linked to the X chromosome and absent on the Y chromosome in the model lizard species contributes to ongoing research as to the evolution of sex determination in reptiles and provides important information for future comparative and functional genomics.

  19. Phosphoproteome analysis demonstrates the potential role of THRAP3 phosphorylation in androgen-independent prostate cancer cell growth.

    Science.gov (United States)

    Ino, Yoko; Arakawa, Noriaki; Ishiguro, Hitoshi; Uemura, Hiroji; Kubota, Yoshinobu; Hirano, Hisashi; Toda, Tosifusa

    2016-04-01

    Elucidating the androgen-independent growth mechanism is critical for developing effective treatment strategies to combat androgen-independent prostate cancer. We performed a comparative phosphoproteome analysis using a prostate cancer cell line, LNCaP, and an LNCaP-derived androgen-independent cell line, LNCaP-AI, to identify phosphoproteins involved in this mechanism. We performed quantitative comparisons of the phosphopeptide levels in tryptic digests of protein extracts from these cell lines using MS. We found that the levels of 69 phosphopeptides in 66 proteins significantly differed between LNCaP and LNCaP-AI. In particular, we focused on thyroid hormone receptor associated protein 3 (THRAP3), which is a known transcriptional coactivator of the androgen receptor. The phosphorylation level of THRAP3 was significantly lower at S248 and S253 in LNCaP-AI cells. Furthermore, pull-down assays showed that 32 proteins uniquely bound to the nonphosphorylatable mutant form of THRAP3, whereas 31 other proteins uniquely bound to the phosphorylation-mimic form. Many of the differentially interacting proteins were identified as being involved with RNA splicing and processing. These results suggest that the phosphorylation state of THRAP3 at S248 and S253 might be involved in the mechanism of androgen-independent prostate cancer cell growth by changing the interaction partners.

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

    Directory of Open Access Journals (Sweden)

    Linghua Chen

    Full Text Available 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

  1. Investigating temporal changes in the yeast phosphoproteome upon fatty acid starvation

    DEFF Research Database (Denmark)

    Pultz, Dennis; Bennetzen, Martin; Andersen, Jens S.

    2011-01-01

    Investigating stemporal changes in the yeast phosphoproteome upon fatty acid starvation Dennis Pultz*, Martin Bennetzen*, Jens S. Andersen and Nils J.Færgeman. Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark, 5230 Reducing food intake to induce...... that the nutrient sensor TOR1 is central in the regulation and orchestration of the downstream cellular response upon fatty acid starvation. By use of mass spectrometry and a SILAC (stable isotope labelling by amino acids in cell culture) based approach we wish to unravel the temporal changes in the phosphoproteome...... and the physiological changes DR induces, only little is known about the genetics and signalling networks which regulate the DR response. We have recently shown that inhibition of fatty acid synthesis in Saccharomyces cerevisiae results in a dependency on autophagy in maintaining normal life span. We further believe...

  2. Dataset of the Botrytis cinerea phosphoproteome induced by different plant-based elicitors

    Directory of Open Access Journals (Sweden)

    Eva Liñeiro

    2016-06-01

    Full Text Available Phosphorylation is one of the main post-translational modification (PTM involved in signaling network in the ascomycete Botrytis cinerea, one of the most relevant phytopathogenic fungus. The data presented in this article provided a differential mass spectrometry-based analysis of the phosphoproteome of B. cinerea under two different phenotypical conditions induced by the use of two different elicitors: glucose and deproteinized Tomate Cell Walls (TCW. A total 1138 and 733 phosphoproteins were identified for glucose and TCW culture conditions respectively. Raw data are deposited at the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier (PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD003099. Further interpretation and discussion of these data are provided in our research article entitled “Phosphoproteome analysis of B.cinerea in response to different plant-based elicitors” (Liñeiro et al., 2016 [1].

  3. Predicting Kinase Activity in Angiotensin Receptor Phosphoproteomes Based on Sequence-Motifs and Interactions

    DEFF Research Database (Denmark)

    Bøgebo, Rikke; Horn, Heiko; Olsen, Jesper V;

    2014-01-01

    -arrestin dependent signalling. Two complimentary global phosphoproteomics studies have analyzed the complex signalling induced by the AT1aR. Here we integrate the data sets from these studies and perform a joint analysis using a novel method for prediction of differential kinase activity from phosphoproteomics data......Recent progress in the understanding of seven-transmembrane receptor (7TMR) signalling has promoted the development of a new generation of pathway selective ligands. The angiotensin II type I receptor (AT1aR) is one of the most studied 7TMRs with respect to selective activation of the β...... likely activated kinases. This suggested that AT1aR-dependent signalling activates 48 of the 285 kinases detected in HEK293 cells. Of these, Aurora B, CLK3 and PKG1 have not previously been described in the pathway whereas others, such as PKA, PKB and PKC, are well known. In summary, we have developed...

  4. Recent findings and technological advances in phosphoproteomics for cells and tissues

    DEFF Research Database (Denmark)

    von Stechow, Louise; Francavilla, Chiara; Olsen, Jesper V

    2015-01-01

    in different diseases, including cancer. Large-scale studies of phosphoproteins - termed phosphoproteomics - strongly rely on the use of high-performance mass spectrometric instrumentation. This powerful technology has been applied to study a great number of phosphorylation-based phenotypes. Nevertheless, many...... technical and biological challenges have to be overcome to identify biologically relevant phosphorylation sites in cells and tissues. This review describes different technological strategies to identify and quantify phosphorylation sites with high accuracy, without significant loss of analysis speed...

  5. SIMAC - A phosphoproteomic strategy for the rapid separation of mono-phosphorylated from multiply phosphorylated peptides

    DEFF Research Database (Denmark)

    Thingholm, Tine E; Jensen, Ole N; Robinson, Phillip J

    2008-01-01

    spectrometric analysis, such as immobilized metal affinity chromatography or titanium dioxide the coverage of the phosphoproteome of a given sample is limited. Here we report a simple and rapid strategy - SIMAC - for sequential separation of mono-phosphorylated peptides and multiply phosphorylated peptides from...... and an optimized titanium dioxide chromatographic method. More than double the total number of identified phosphorylation sites was obtained with SIMAC, primarily from a three-fold increase in recovery of multiply phosphorylated peptides....

  6. Refined phosphopeptide enrichment by phosphate additive and the analysis of human brain phosphoproteome.

    Science.gov (United States)

    Tan, Haiyan; Wu, Zhiping; Wang, Hong; Bai, Bing; Li, Yuxin; Wang, Xusheng; Zhai, Bo; Beach, Thomas G; Peng, Junmin

    2015-01-01

    Alzheimer's disease (AD) is the most common form of dementia, characterized by progressive loss of cognitive function. One of the pathological hallmarks of AD is the formation of neurofibrillary tangles composed of abnormally hyperphosphorylated tau protein, but global deregulation of protein phosphorylation in AD is not well analyzed. Here, we report a pilot investigation of AD phosphoproteome by titanium dioxide enrichment coupled with high resolution LC-MS/MS. During the optimization of the enrichment method, we found that phosphate ion at a low concentration (e.g. 1 mM) worked efficiently as a nonphosphopeptide competitor to reduce background. The procedure was further tuned with respect to peptide-to-bead ratio, phosphopeptide recovery, and purity. Using this refined method and 9 h LC-MS/MS, we analyzed phosphoproteome in one milligram of digested AD brain lysate, identifying 5243 phosphopeptides containing 3715 nonredundant phosphosites on 1455 proteins, including 31 phosphosites on the tau protein. This modified enrichment method is simple and highly efficient. The AD case study demonstrates its feasibility of dissecting phosphoproteome in a limited amount of postmortem human brain. All MS data have been deposited in the ProteomeXchange with identifier PXD001180 (http://proteomecentral.proteomexchange.org/dataset/PXD001180).

  7. The proteome and phosphoproteome of maize pollen uncovers fertility candidate proteins.

    Science.gov (United States)

    Chao, Qing; Gao, Zhi-Fang; Wang, Yue-Feng; Li, Zhe; Huang, Xia-He; Wang, Ying-Chun; Mei, Ying-Chang; Zhao, Biligen-Gaowa; Li, Liang; Jiang, Yu-Bo; Wang, Bai-Chen

    2016-06-01

    Maize is unique since it is both monoecious and diclinous (separate male and female flowers on the same plant). We investigated the proteome and phosphoproteome of maize pollen containing modified proteins and here we provide a comprehensive pollen proteome and phosphoproteome which contain 100,990 peptides from 6750 proteins and 5292 phosphorylated sites corresponding to 2257 maize phosphoproteins, respectively. Interestingly, among the total 27 overrepresented phosphosite motifs we identified here, 11 were novel motifs, which suggested different modification mechanisms in plants compared to those of animals. Enrichment analysis of pollen phosphoproteins showed that pathways including DNA synthesis/chromatin structure, regulation of RNA transcription, protein modification, cell organization, signal transduction, cell cycle, vesicle transport, transport of ions and metabolisms, which were involved in pollen development, the following germination and pollen tube growth, were regulated by phosphorylation. In this study, we also found 430 kinases and 105 phosphatases in the maize pollen phosphoproteome, among which calcium dependent protein kinases (CDPKs), leucine rich repeat kinase, SNF1 related protein kinases and MAPK family proteins were heavily enriched and further analyzed. From our research, we also uncovered hundreds of male sterility-associated proteins and phosphoproteins that might influence maize productivity and serve as targets for hybrid maize seed production. At last, a putative complex signaling pathway involving CDPKs, MAPKs, ubiquitin ligases and multiple fertility proteins was constructed. Overall, our data provides new insight for further investigation of protein phosphorylation status in mature maize pollen and construction of maize male sterile mutants in the future.

  8. Novel aspects of grapevine response to phytoplasma infection investigated by a proteomic and phospho-proteomic approach with data integration into functional networks

    Directory of Open Access Journals (Sweden)

    Margaria Paolo

    2013-01-01

    Full Text Available Abstract Background Translational and post-translational protein modifications play a key role in the response of plants to pathogen infection. Among the latter, phosphorylation is critical in modulating protein structure, localization and interaction with other partners. In this work, we used a multiplex staining approach with 2D gels to study quantitative changes in the proteome and phosphoproteome of Flavescence dorée-affected and recovered ‘Barbera’ grapevines, compared to healthy plants. Results We identified 48 proteins that differentially changed in abundance, phosphorylation, or both in response to Flavescence dorée phytoplasma infection. Most of them did not show any significant difference in recovered plants, which, by contrast, were characterized by changes in abundance, phosphorylation, or both for 17 proteins not detected in infected plants. Some enzymes involved in the antioxidant response that were up-regulated in infected plants, such as isocitrate dehydrogenase and glutathione S-transferase, returned to healthy-state levels in recovered plants. Others belonging to the same functional category were even down-regulated in recovered plants (oxidoreductase GLYR1 and ascorbate peroxidase. Our proteomic approach thus agreed with previously published biochemical and RT-qPCR data which reported down-regulation of scavenging enzymes and accumulation of H2O2 in recovered plants, possibly suggesting a role for this molecule in remission from infection. Fifteen differentially phosphorylated proteins (| ratio | > 2, p  Conclusions Proteomic data were integrated into biological networks and their interactions were represented through a hypothetical model, showing the effects of protein modulation on primary metabolic ways and related secondary pathways. By following a multiplex-staining approach, we obtained new data on grapevine proteome pathways that specifically change at the phosphorylation level during phytoplasma infection

  9. Phosphoproteome profiles of the phytopathogenic fungi Alternaria brassicicola and Botrytis cinerea during exponential growth in axenic cultures.

    Science.gov (United States)

    Davanture, Marlène; Dumur, Jérôme; Bataillé-Simoneau, Nelly; Campion, Claire; Valot, Benoît; Zivy, Michel; Simoneau, Philippe; Fillinger, Sabine

    2014-07-01

    This study describes the gel-free phosphoproteomic analysis of the phytopathogenic fungi Alternaria brassicicola and Botrytis cinerea grown in vitro under nonlimiting conditions. Using a combination of strong cation exchange and IMAC prior to LC-MS, we identified over 1350 phosphopeptides per fungus representing over 800 phosphoproteins. The preferred phosphorylation sites were found on serine (>80%) and threonine (>15%), whereas phosphorylated tyrosine residues were found at less than 1% in A. brassicicola and at a slightly higher ratio in B. cinerea (1.5%). Biological processes represented principally among the phoshoproteins were those involved in response and transduction of stimuli as well as in regulation of cellular and metabolic processes. Most known elements of signal transduction were found in the datasets of both fungi. This study also revealed unexpected phosphorylation sites in histidine kinases, a category overrepresented in filamentous ascomycetes compared to yeast. The data have been deposited to the ProteomeXchange database with identifier PXD000817 (http://proteomecentral.proteomexchange.org/dataset/PXD000817).

  10. Quantitative proteomic analysis of wheat grain proteins reveals differential effects of silencing of omega-5 gliadin genes in transgenic lines

    Science.gov (United States)

    Novel wheat lines with altered flour compositions can be used to decipher the roles of specific gluten proteins in flour quality. Grain proteins from transgenic wheat lines in which genes encoding the omega-5 gliadins were silenced by RNA interference (RNAi) were analyzed in detail by quantitative 2...

  11. Searching for novel Cdk5 substrates in brain by comparative phosphoproteomics of wild type and Cdk5-/- mice.

    Directory of Open Access Journals (Sweden)

    Erick Contreras-Vallejos

    Full Text Available Protein phosphorylation is the most common post-translational modification that regulates several pivotal functions in cells. Cyclin-dependent kinase 5 (Cdk5 is a proline-directed serine/threonine kinase which is mostly active in the nervous system. It regulates several biological processes such as neuronal migration, cytoskeletal dynamics, axonal guidance and synaptic plasticity among others. In search for novel substrates of Cdk5 in the brain we performed quantitative phosphoproteomics analysis, isolating phosphoproteins from whole brain derived from E18.5 Cdk5+/+ and Cdk5-/- embryos, using an Immobilized Metal-Ion Affinity Chromatography (IMAC, which specifically binds to phosphorylated proteins. The isolated phosphoproteins were eluted and isotopically labeled for relative and absolute quantitation (iTRAQ and mass spectrometry identification. We found 40 proteins that showed decreased phosphorylation at Cdk5-/- brains. In addition, out of these 40 hypophosphorylated proteins we characterized two proteins, :MARCKS (Myristoylated Alanine-Rich protein Kinase C substrate and Grin1 (G protein regulated inducer of neurite outgrowth 1. MARCKS is known to be phosphorylated by Cdk5 in chick neural cells while Grin1 has not been reported to be phosphorylated by Cdk5. When these proteins were overexpressed in N2A neuroblastoma cell line along with p35, serine phosphorylation in their Cdk5 motifs was found to be increased. In contrast, treatments with roscovitine, the Cdk5 inhibitor, resulted in an opposite effect on serine phosphorylation in N2A cells and primary hippocampal neurons transfected with MARCKS. In summary, the results presented here identify Grin 1 as novel Cdk5 substrate and confirm previously identified MARCKS as a a bona fide Cdk5 substrate.

  12. Analysis of T4SS-induced signaling by H. pylori using quantitative phosphoproteomics

    National Research Council Canada - National Science Library

    Glowinski, Frithjof; Holland, Carsten; Thiede, Bernd; Jungblut, Peter R; Meyer, Thomas F

    2014-01-01

    .... A major virulence determinant of H. pylori is the type IV secretion system (T4SS), which is used to inject the virulence factor CagA into the host cell, triggering a wide range of cellular signaling events...

  13. iTRAQ labeling is superior to mTRAQ for quantitative global proteomics and phosphoproteomics

    National Research Council Canada - National Science Library

    Mertins, Philipp; Udeshi, Namrata D; Clauser, Karl R; Mani, D R; Patel, Jinal; Ong, Shao-en; Jaffe, Jacob D; Carr, Steven A

    2012-01-01

    .... Isobaric labeling techniques such as iTRAQ™ or TMT™ allow for relative quantification of peptides based on ratios of reporter ions in the low m/z region of spectra produced by precursor ion fragmentation...

  14. Quantitative site-specific phosphoproteomics of Trichoderma reesei signaling pathways upon induction of hydrolytic enzyme production

    NARCIS (Netherlands)

    E.V. Nguyen; S.Y. Imanishi; P. Haapaniemi; A. Yadav; M. Saloheimo; G.L. Corthals; T.M. Pakula

    2016-01-01

    The filamentous fungus Trichoderma reesei is used for industrial production of secreted enzymes including carbohydrate active enzymes, such as cellulases and hemicellulases. The production of many of these enzymes by T. reesei is influenced by the carbon source it grows on, where the regulation syst

  15. Global investigation of interleukin-1β signaling in primary β-cells using quantitative phosphoproteomics

    DEFF Research Database (Denmark)

    Engholm-Keller, Kasper; Størling, Joachim; Pociot, Flemming

    to culture medium with or without IL-1 β for 10 min. After cellular lysis in 8M urea, the proteome was digested using Lys-C and trypsin, and stable isotope-labeled by reductive dimethylation for subsequent mass spectrometry-based quantification. Phosphopeptides were enriched by TiO2 chromatography...... in the HCD-cell. Preliminary Data We performed three biological replicates of control versus 10 min. IL-1β stimulation of 600, 500, and 800 randomly picked rat islets of Langerhans (approximately 110, 90, and 150 µg of protein per condition, respectively) using quantification by stable isotope labeling via...... and separated into monophosphorylated and multiphosphorylated peptide pools using Sequential elution from IMAC (SIMAC). The monophosphorylated sample was fractionated by microscale HILIC HPLC and all fractions analyzed by nanoLC-ESI-MS/MS on an LTQ-Orbitrap Velos using beam-type collision-induced dissociation...

  16. Triple quad ICPMS (ICPQQQ) as a new tool for absolute quantitative proteomics and phosphoproteomics.

    Science.gov (United States)

    Diez Fernández, Silvia; Sugishama, Naoki; Ruiz Encinar, Jorge; Sanz-Medel, Alfredo

    2012-07-17

    It is clear that sensitive and interference-free quantification of ICP-detectable elements naturally present in proteins will boost the role of ICPMS in proteomics. In this study, a completely new way of polyatomic interference removal in ICPMS for detection of sulfur (present in the majority of proteins as methionine or cysteine) and phosphorus (present in phosphorylated proteins) is presented. It is based on the concept of tandem mass spectrometry (QQQ) typically used in molecular MS. Briefly, the first quadrupole can be operated as 1 amu window band-pass mass filter to select target analyte ions ((31)P, (32)S, and their on-mass polyatomic interferences). In this way, only selected ions enter the cell and react with O(2), reducing the interferences produced by matrix ions as well as background noise. After optimization of the cell conditions, product ions formed for the targets, (47)PO(+) and (48)SO(+), could be detected with enhanced sensitivity and selectivity. The coupling to capillary HPLC allowed analysis of S- and P-containing species with the lowest detection limits ever published (11 and 6.6 fmol, respectively). The potential of the approach for proteomics studies was demonstrated for the highly sensitive simultaneous absolute quantification of different S-containing peptides and phosphopeptides.

  17. Quantitative secretome analysis reveals the interactions between epithelia and tumor cells by in vitro modulating colon cancer microenvironment.

    Science.gov (United States)

    Zeng, Xiao; Yang, Pengbo; Chen, Bing; Jin, Xuewen; Liu, Yuling; Zhao, Xia; Liang, Shufang

    2013-08-26

    In tumor microenvironment, interactions among multiple cell types are critical for cancer progression. Secreted proteins are responsible for crosstalk among these cells within tumor microenvironment. To elucidate the interactions of tumor and epithelia, we co-cultured colon cancer cell line HT29 with normal human colon mucosal epithelial cell line NCM460 to mimic tumor microenvironment in vitro and investigated the differential expression pattern of secretome. A quantitative proteomics approach based on stable isotope labeling by amino acids in cell culture (SILAC) and LC-mass spectrometry was used for secretome analysis. Totally 45 proteins were altered over 2-fold in co-cultured cellular supernatants between equal amounts of NCM460 and HT29 cells, compared with mono-cultured conditions. These differential secreted proteins involve in multiple tumor-associated biological functions. The secretion level and acting pattern of acrogranin, IGFBP6 and vimentin were changed along with different co-cultured cell number ratios between NCM460 and HT29 cells, simulating early, middle or advanced stage of colon cancer. Therefore, a quantitative secretome profiling based on a co-culture system can track secreted protein changes and their associated biological roles between tumor and epithelia, which gives a new insight on communications between tumor and epithelia as well as cancer biotherapy by inhibiting cell interactions. Tumor microenvironment is a complex system and comprised of cancer cells and host stromal cells. The growth and progression of tumor have been recognized were affected by multidirectional interactions of secreted proteins (secretome), which were produced by the cells within tumor microenvironment. Focus on general secreted molecules of living cells via proteomic tools, is promising for investigating cell communication. Stable isotope labeling by amino acids in cell culture (SILAC) is a metabolic labeling strategy for quantitative analysis, which is gaining

  18. Quantitative immunologic analysis of the methanogenic flora of digestors reveals a considerable diversity. [Methanobacterium formicicum; Methanobrevibacter arboriphilus

    Energy Technology Data Exchange (ETDEWEB)

    Macario, A.J.L.; de Macario, E.C.

    1988-01-01

    To determine which methanogens occur in digestors, we performed a quantitative immunologic analysis of a variety of samples. A comprehensive panel of calibrated polyclonal antibody probes of predefined specificity spectra was used. This allowed precise identification of bacteria by antigenic fingerprinting. A considerable diversity of methanogens was uncovered, much larger than previously reported, encompassing at least 14 strains of 11 species. Strategies were developed to measure the load of any given methanogen in a sample and to compare samples quantitatively. Two methanogens were found to predominate which were antigenically closely related with either Methanobacterium formicicum MF or Methanobrevibacter arboriphilus AZ. Fundamental data, probes, and methods are now available to monitor methanogenic subpopulations during digestor operation and thus learn about their respective roles and predictive and predictive significance.

  19. Identification and quantitation of signal molecule-dependent protein phosphorylation

    KAUST Repository

    Groen, Arnoud J.

    2013-09-03

    Phosphoproteomics is a fast-growing field that aims at characterizing phosphorylated proteins in a cell or a tissue at a given time. Phosphorylation of proteins is an important regulatory mechanism in many cellular processes. Gel-free phosphoproteome technique involving enrichment of phosphopeptide coupled with mass spectrometry has proven to be invaluable to detect and characterize phosphorylated proteins. In this chapter, a gel-free quantitative approach involving 15N metabolic labelling in combination with phosphopeptide enrichment by titanium dioxide (TiO2) and their identification by MS is described. This workflow can be used to gain insights into the role of signalling molecules such as cyclic nucleotides on regulatory networks through the identification and quantification of responsive phospho(proteins). © Springer Science+Business Media New York 2013.

  20. In Vivo Phosphoproteomics Analysis Reveals the Cardiac Targets of β-Adrenergic Receptor Signaling

    DEFF Research Database (Denmark)

    Lundby, Alicia; Andersen, Martin N; Steffensen, Annette B;

    2013-01-01

    -X-X-pS/T), and integrative analysis of sequence motifs and interaction networks suggested that the kinases AMPK (adenosine 5'-monophosphate-activated protein kinase), Akt, and mTOR (mammalian target of rapamycin) mediate βAR signaling, in addition to the well-established pathways mediated by PKA (cyclic adenosine...

  1. Global phosphoproteome profiling reveals unanticipated networks responsive to cisplatin treatment of embryonic stem cells

    DEFF Research Database (Denmark)

    Pines, Alex; Kelstrup, Christian D; Vrouwe, Mischa G

    2011-01-01

    (stable isotope labeling by amino acids in cell culture)-labeled murine embryonic stem cells with the anticancer drug cisplatin. Network and pathway analyses indicated that processes related to the DNA damage response and cytoskeleton organization were significantly affected. Although the ATM (ataxia...

  2. Phosphoproteome Reveals an Atlas of Protein Signaling Networks During Osteoblast Adhesion

    NARCIS (Netherlands)

    Milani, Renato; Ferreira, Carmen V.; Granjeiro, Jose M.; Paredes-Gamero, Edgar J.; Silva, Rodrigo A.; Justo, Giselle Z.; Nader, Helena B.; Galembeck, Eduardo; Peppelenbosch, Maikel P.; Aoyama, Hiroshi; Zambuzzi, Willian F.

    2010-01-01

    Cell adhesion on surfaces is a fundamental process in the emerging biomaterials field and developmental events as well. However, the mechanisms regulating this biological process in osteoblasts are not fully understood. Reversible phosphorylation catalyzed by kinases is probably the most important r

  3. Phosphoproteome Reveals an Atlas of Protein Signaling Networks During Osteoblast Adhesion

    NARCIS (Netherlands)

    Milani, Renato; Ferreira, Carmen V.; Granjeiro, Jose M.; Paredes-Gamero, Edgar J.; Silva, Rodrigo A.; Justo, Giselle Z.; Nader, Helena B.; Galembeck, Eduardo; Peppelenbosch, Maikel P.; Aoyama, Hiroshi; Zambuzzi, Willian F.

    2010-01-01

    Cell adhesion on surfaces is a fundamental process in the emerging biomaterials field and developmental events as well. However, the mechanisms regulating this biological process in osteoblasts are not fully understood. Reversible phosphorylation catalyzed by kinases is probably the most important

  4. Phosphoproteome analysis of streptomyces development reveals extensive protein phosphorylation accompanying bacterial differentiation

    DEFF Research Database (Denmark)

    Manteca, Angel; Ye, Juanying; Sánchez, Jesús

    2011-01-01

    Streptomycetes are bacterial species that undergo a complex developmental cycle that includes programmed cell death (PCD) events and sporulation. They are widely used in biotechnology because they produce most clinically relevant secondary metabolites. Although Streptomyces coelicolor is one...... events were detected during the presporulation and sporulation stages (80%). Most of these phosphorylations were not reported before in Streptomyces, and included sporulation factors, transcriptional regulators, protein kinases and other regulatory proteins. Several of the identified phosphorylated...

  5. Phosphoproteome dynamics reveal heat-shock protein complexes specific to the Leishmania donovani infectious stage

    OpenAIRE

    Morales, M. A.; R. WATANABE; Dacher, M.; Chafey, P.; Osorio y Fortea, J.; Scott, D A; Beverley, S. M.; van Ommen, G.; CLOS, J.; Hem, S.; Lenormand, P.; Rousselle, J.-C.; Namane, A.; Spath, G. F.

    2010-01-01

    Leishmania is exposed to a sudden increase in environmental temperature during the infectious cycle that triggers stage differentiation and adapts the parasite phenotype to intracellular survival in the mammalian host. The absence of classical promoter-dependent mechanisms of gene regulation and constitutive expression of most of the heat-shock proteins (HSPs) in these human pathogens raise important unresolved questions as to regulation of the heat-shock response and stage-specific functions...

  6. Phosphoproteomic analysis of differentiating Leishmania parasites reveals a unique stage-specific phosphorylation motif

    OpenAIRE

    Tsigankov, Polina; Gherardini, Pier Federico; Helmer-Citterich, Manuela; Späth, Gerald F; Zilberstein, Dan

    2013-01-01

    International audience; Protists of the genus Leishmania are obligatory intracellular parasites that cause a wide range of cutaneous, mucocutaneous, and visceral diseases in humans. They cycle between phagolysosomes of mammalian macrophages and the sand fly midgut, proliferating as intracellular amastigotes and extracellular promastigotes, respectively. Exposure to a lysosomal environment, i.e. acidic pH and body temperature, signals promastigotes to differentiate into amastigotes. Time cours...

  7. 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.

  8. Membrane Phosphoproteomics of Yeast Early Response to Acetic Acid: Role of Hrk1 Kinase and Lipid Biosynthetic Pathways, in Particular Sphingolipids.

    Science.gov (United States)

    Guerreiro, Joana F; Mira, Nuno P; Santos, Aline X S; Riezman, Howard; Sá-Correia, Isabel

    2017-01-01

    Saccharomyces cerevisiae response and tolerance to acetic acid is critical in industrial biotechnology and in acidic food and beverages preservation. The HRK1 gene, encoding a protein kinase of unknown function belonging to the "Npr1-family" of kinases known to be involved in the regulation of plasma membrane transporters, is an important determinant of acetic acid tolerance. This study was performed to identify the alterations occurring in yeast membrane phosphoproteome profile during the adaptive early response to acetic acid stress (following 1 h of exposure to a sub-lethal inhibitory concentration; 50 mM at pH 4.0) and the effect of HRK1 expression on the phosphoproteome. Results from mass spectrometry analysis following the prefractionation and specific enrichment of phosphorylated peptides using TiO2 beads highlight the contribution of processes related with translation, protein folding and processing, transport, and cellular homeostasis in yeast response to acetic acid stress, with particular relevance for changes in phosphorylation of transport-related proteins, found to be highly dependent on the Hrk1 kinase. Twenty different phosphoproteins known to be involved in lipid and sterol metabolism were found to be differently phosphorylated in response to acetic acid stress, including several phosphopeptides that had not previously been described as being phosphorylated. The suggested occurrence of cellular lipid composition remodeling during the short term yeast response to acetic acid was confirmed: Hrk1 kinase-independent reduction in phytoceramide levels and a reduction in phosphatidylcholine and phosphatidylinositol levels under acetic acid stress in the more susceptible hrk1Δ strain were revealed by a lipidomic analysis.

  9. 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).

  10. Parsing ERK Activation Reveals Quantitatively Equivalent Contributions From Epidermal Growth Factor Receptor and HER2 In Human Mammary Epithelial Cells

    Energy Technology Data Exchange (ETDEWEB)

    Hendriks, Bart S.; Orr, Galya; Wells, Alan H.; Wiley, H. S.; Lauffenburger, Douglas A.

    2005-02-18

    HER2, a member of the EGFR tyrosine kinase family, functions as an accessory EGFR signaling component and alters EGFR trafficking by heterodimerization. HER2 overexpression leads to aberrant cell behavior including enhanced proliferation and motility. Here we apply a combination of computational modeling and quantitative experimental studies of the dynamic interactions between EGFR and HER2, and their downstream activation of extracellular signal-related kinase (ERK) to understand this complex signaling system. Using cells expressing different levels of HER2 relative to the EGFR, we can separate relative contributions of EGFR and HER2 to signaling amplitude and duration. Based on our model calculations, we demonstrate that, in contrast with previous suggestions in the literature, the intrinsic capabilities of EGFR and HER2 to activated ERK are quantitatively equivalent . We find that HER2-mediated effects on EGFR dimerization and trafficking are sufficient to explain the detected HER2-mediated amplification of EGF-induced ERK signaling. Our model suggests that transient amplification of ERK activity by HER2 arises predominantly from the 2-to-1 stoichiometry of receptor kinase to bound ligand in EGFR/HER2 heterodimers compared to the 1-to-1 stoichiometry of the EGFR homodimer, but alterations in receptor trafficking, with resultant EGFR sparing, cause the sustained HER2-mediated enhancement of ERK signaling.

  11. Correlative and quantitative 1H NMR-based metabolomics reveals specific metabolic pathway disturbances in diabetic rats.

    Science.gov (United States)

    Zhang, Shucha; Nagana Gowda, G A; Asiago, Vincent; Shanaiah, Narasimhamurthy; Barbas, Coral; Raftery, Daniel

    2008-12-01

    Type 1 diabetes was induced in Sprague-Dawley rats using streptozotocin. Rat urine samples (8 diabetic and 10 control) were analyzed by 1H nuclear magnetic resonance (NMR) spectroscopy. The derived metabolites using univariate and multivariate statistical analysis were subjected to correlative analysis. Plasma metabolites were measured by a series of bioassays. A total of 17 urinary metabolites were identified in the 1H NMR spectra and the loadings plots after principal components analysis. Diabetic rats showed significantly increased levels of glucose (P cycle and a contribution from gut microbial metabolism. Specific perturbed metabolic pathways include the glucose-alanine and Cori cycles, the acetate switch, and choline metabolism. Detection of the altered metabolic pathways and bacterial metabolites using this correlative and quantitative NMR-based metabolomics approach should help to further the understanding of diabetes-related mechanisms.

  12. Behaviour of pathogenic and indicator bacteria during urban wastewater treatment and sludge composting, as revealed by quantitative PCR.

    Science.gov (United States)

    Wéry, Nathalie; Lhoutellier, Claire; Ducray, Florence; Delgenès, Jean-Philippe; Godon, Jean-Jacques

    2008-01-01

    Two enteric pathogens, Salmonella spp. and Campylobacter jejuni, and two bacteria commonly used as indicators, Escherichia coli and Clostridium perfringens, were monitored using quantitative real-time PCR during municipal wastewater treatment and sludge composting. The results were compared with those obtained using standard culture methods. A reduction of all bacteria was observed during wastewater treatment and during the thermophilic phase of composting. However, the bacterial groups studied behaved differently during the process, and the main differences were observed during biological treatment in activated sludge basins. In particular, Salmonella spp. and C. jejuni survived better during activated sludge treatment than E. coli. C. jejuni was the most resistant to wastewater treatment among the four bacterial groups. Overall, differences in survival were observed for all bacteria studied, when submitted to the same environmental pressure. This holds both for differences between indicators and pathogenic bacteria and between pathogenic bacteria. These results show the difficulty in defining reliable indicators.

  13. 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

  14. Quantitative Live Imaging of Human Embryonic Stem Cell Derived Neural Rosettes Reveals Structure-Function Dynamics Coupled to Cortical Development.

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    Omer Ziv

    2015-10-01

    Full Text Available Neural stem cells (NSCs are progenitor cells for brain development, where cellular spatial composition (cytoarchitecture and dynamics are hypothesized to be linked to critical NSC capabilities. However, understanding cytoarchitectural dynamics of this process has been limited by the difficulty to quantitatively image brain development in vivo. Here, we study NSC dynamics within Neural Rosettes--highly organized multicellular structures derived from human pluripotent stem cells. Neural rosettes contain NSCs with strong epithelial polarity and are expected to perform apical-basal interkinetic nuclear migration (INM--a hallmark of cortical radial glial cell development. We developed a quantitative live imaging framework to characterize INM dynamics within rosettes. We first show that the tendency of cells to follow the INM orientation--a phenomenon we referred to as radial organization, is associated with rosette size, presumably via mechanical constraints of the confining structure. Second, early forming rosettes, which are abundant with founder NSCs and correspond to the early proliferative developing cortex, show fast motions and enhanced radial organization. In contrast, later derived rosettes, which are characterized by reduced NSC capacity and elevated numbers of differentiated neurons, and thus correspond to neurogenesis mode in the developing cortex, exhibit slower motions and decreased radial organization. Third, later derived rosettes are characterized by temporal instability in INM measures, in agreement with progressive loss in rosette integrity at later developmental stages. Finally, molecular perturbations of INM by inhibition of actin or non-muscle myosin-II (NMII reduced INM measures. Our framework enables quantification of cytoarchitecture NSC dynamics and may have implications in functional molecular studies, drug screening, and iPS cell-based platforms for disease modeling.

  15. Quantitative Live Imaging of Human Embryonic Stem Cell Derived Neural Rosettes Reveals Structure-Function Dynamics Coupled to Cortical Development.

    Science.gov (United States)

    Ziv, Omer; Zaritsky, Assaf; Yaffe, Yakey; Mutukula, Naresh; Edri, Reuven; Elkabetz, Yechiel

    2015-10-01

    Neural stem cells (NSCs) are progenitor cells for brain development, where cellular spatial composition (cytoarchitecture) and dynamics are hypothesized to be linked to critical NSC capabilities. However, understanding cytoarchitectural dynamics of this process has been limited by the difficulty to quantitatively image brain development in vivo. Here, we study NSC dynamics within Neural Rosettes--highly organized multicellular structures derived from human pluripotent stem cells. Neural rosettes contain NSCs with strong epithelial polarity and are expected to perform apical-basal interkinetic nuclear migration (INM)--a hallmark of cortical radial glial cell development. We developed a quantitative live imaging framework to characterize INM dynamics within rosettes. We first show that the tendency of cells to follow the INM orientation--a phenomenon we referred to as radial organization, is associated with rosette size, presumably via mechanical constraints of the confining structure. Second, early forming rosettes, which are abundant with founder NSCs and correspond to the early proliferative developing cortex, show fast motions and enhanced radial organization. In contrast, later derived rosettes, which are characterized by reduced NSC capacity and elevated numbers of differentiated neurons, and thus correspond to neurogenesis mode in the developing cortex, exhibit slower motions and decreased radial organization. Third, later derived rosettes are characterized by temporal instability in INM measures, in agreement with progressive loss in rosette integrity at later developmental stages. Finally, molecular perturbations of INM by inhibition of actin or non-muscle myosin-II (NMII) reduced INM measures. Our framework enables quantification of cytoarchitecture NSC dynamics and may have implications in functional molecular studies, drug screening, and iPS cell-based platforms for disease modeling.

  16. Quantitative Proteomic and Transcriptomic Study on Autotetraploid Paulownia and Its Diploid Parent Reveal Key Metabolic Processes Associated with Paulownia Autotetraploidization.

    Science.gov (United States)

    Dong, Yanpeng; Deng, Minjie; Zhao, Zhenli; Fan, Guoqiang

    2016-01-01

    Polyploidy plays a very important role in speciation and plant evolution by way of genomic merging and doubling. In the process of polyploidy, rapid genomic, and transcriptomic changes have been observed and researched. However, proteomic divergence caused by the effects of polyploidization is still poorly understood. In the present study, we used iTRAQ coupled with mass spectrometry to quantitatively analyze proteomic changes in the leaves of autotetraploid Paulownia and its diploid parent. A total of 2963 proteins were identified and quantified. Among them, 463 differentially abundant proteins were detected between autotetraploid Paulownia and its diploid parent, and 198 proteins were found to be non-additively abundant in autotetraploid Paulownia, suggesting the presence of non-additive protein regulation during genomic merger and doubling. We also detected 1808 protein-encoding genes in previously published RNA sequencing data. We found that 59 of the genes that showed remarkable changes at mRNA level encoded proteins with consistant changes in their abundance levels, while a further 48 genes that showed noteworthy changes in their expression levels encoded proteins with opposite changes in their abundance levels. Proteins involved in posttranslational modification, protein turnover, and response to stimulus, were significantly enriched among the non-additive proteins, which may provide some of the driving power for variation and adaptation in autopolyploids. Quantitative real-time PCR analysis verified the expression patterns of related protein-coding genes. In addition, we found that the percentage of differentially abundant proteins that matched previously reported differentially expressed genes was relatively low.

  17. Ex vivo measures of muscle mitochondrial capacity reveal quantitative limits of oxygen delivery by the circulation during exercise

    DEFF Research Database (Denmark)

    Boushel, Robert; Saltin, Bengt

    2013-01-01

    of the body mass will be discussed in relation to mitochondrial capacity measured ex vivo. These analyses reveal that as the mass of muscle engaged in exercise increases from one-leg knee extension, to 2-arm cranking, to 2-leg cycling and x-country skiing, the magnitude of blood flow and oxygen delivery...... decrease. Accordingly, a 2-fold higher oxygen delivery and oxygen uptake per unit muscle mass are seen in vivo during 1-leg exercise compared to 2-leg cycling indicating a significant limitation of the circulation during exercise with a large muscle mass. This analysis also reveals that mitochondrial......Muscle mitochondrial respiratory capacity measured ex vivo provides a physiological reference to assess cellular oxidative capacity as a component in the oxygen cascade in vivo. In this article, the magnitude of muscle blood flow and oxygen uptake during exercise involving a small-to-large fraction...

  18. Proteome Differences between Hepatitis B Virus Genotype-B- and Genotype-C-Induced Hepatocellular Carcinoma Revealed by iTRAQ-Based Quantitative Proteomics.

    Science.gov (United States)

    Wei, Dahai; Zeng, Yongyi; Xing, Xiaohua; Liu, Hongzhi; Lin, Minjie; Han, Xiao; Liu, Xiaolong; Liu, Jingfeng

    2016-02-05

    Hepatitis B virus (HBV) is the main cause of hepatocellular carcinoma (HCC) in southeast Asia where HBV genotype B and genotype C are the most prevalent. Viral genotypes have been reported to significantly affect the clinical outcomes of HCC. However, the underlying molecular differences among different genotypes of HBV virus infected HCC have not been revealed. Here, we applied isobaric tags for relative and absolute quantitation (iTRAQ) technology integrated with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to identify the proteome differences between the HBV genotypes B- and C-induced HCC. In brief, a total of 83 proteins in the surrounding noncancerous tissues and 136 proteins in the cancerous tissues between HBV genotype-B- and genotype-C-induced HCC were identified, respectively. This information revealed that there might be different molecular mechanisms of the tumorigenesis and development of HBV genotypes B- and C-induced HCC. Furthermore, our results indicate that the two proteins ARFIP2 and ANXA1 might be potential biomarkers for distinguishing the HBV genotypes B- and C-induced HCC. Thus, the quantitative proteomic analysis revealed molecular differences between the HBV genotypes B- and C-induced HCC, and might provide fundamental information for further deep study.

  19. Phosphoproteomic analysis of the Chlamydia caviae elementary body and reticulate body forms.

    Science.gov (United States)

    Fisher, Derek J; Adams, Nancy E; Maurelli, Anthony T

    2015-08-01

    Chlamydia are Gram-negative, obligate intracellular bacteria responsible for significant diseases in humans and economically important domestic animals. These pathogens undergo a unique biphasic developmental cycle transitioning between the environmentally stable elementary body (EB) and the replicative intracellular reticulate body (RB), a conversion that appears to require extensive regulation of protein synthesis and function. However, Chlamydia possess a limited number of canonical mechanisms of transcriptional regulation. Ser/Thr/Tyr phosphorylation of proteins in bacteria has been increasingly recognized as an important mechanism of post-translational control of protein function. We utilized 2D gel electrophoresis coupled with phosphoprotein staining and MALDI-TOF/TOF analysis to map the phosphoproteome of the EB and RB forms of Chlamydia caviae. Forty-two non-redundant phosphorylated proteins were identified (some proteins were present in multiple locations within the gels). Thirty-four phosphorylated proteins were identified in EBs, including proteins found in central metabolism and protein synthesis, Chlamydia-specific hypothetical proteins and virulence-related proteins. Eleven phosphorylated proteins were identified in RBs, mostly involved in protein synthesis and folding and a single virulence-related protein. Only three phosphoproteins were found in both EB and RB phosphoproteomes. Collectively, 41 of 42 C. caviae phosphoproteins were present across Chlamydia species, consistent with the existence of a conserved chlamydial phosphoproteome. The abundance of stage-specific phosphoproteins suggests that protein phosphorylation may play a role in regulating the function of developmental-stage-specific proteins and/or may function in concert with other factors in directing EB-RB transitions.

  20. 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

  1. Picking the right tool for the job--Phosphoproteomics of egg activation.

    Science.gov (United States)

    Wessel, Gary M

    2015-12-01

    Eggs are the rarest cell in the human body, yet their study is essential for the fields of fertility, reproduction, and fetal health. Guo et al. (Proteomics 2015, 15, 4080-4095) use a "surrogate" animal to discover the phosphoproteomic pathways involved in egg activation. With datasets of several thousand phosphosites on 2500 different proteins, these investigators have defined new pathways, connections to pathways, and priorities in searches for how eggs are activated at fertilization. These results in a sea urchin are now transposable to mammals for testing on a per candidate strategy.

  2. Quantitative Proteomic Analysis of Mitochondrial Proteins Reveals Pro-Survival Mechanisms in the Perpetuation of Radiation-Induced Genomic Instability

    Energy Technology Data Exchange (ETDEWEB)

    Thomas, Stefani N.; Waters, Katrina M.; Morgan, William F.; Yang, Austin; Baulch, Janet E.

    2012-07-26

    Radiation induced genomic instability is a well-studied phenomenon that is measured as mitotically heritable genetic alterations observed in the progeny of an irradiated cell. The mechanisms that perpetuate this instability are unclear, however, a role for chronic oxidative stress has consistently been demonstrated. In the chromosomally unstable LS12 cell line, oxidative stress and genomic instability were correlated with mitochondrial dysfunction. To clarify this mitochondrial dysfunction and gain insight into the mechanisms underlying radiation induced genomic instability we have evaluated the mitochondrial sub-proteome and performed quantitative mass spectrometry (MS) analysis of LS12 cells. Of 98 quantified mitochondrial proteins, 17 met criteria for fold changes and reproducibility; and 11 were statistically significant in comparison with the stable parental GM10115 cell line. Previous observations implicated defects in the electron transport chain (ETC) in the LS12 cell mitochondrial dysfunction. Proteomic analysis supports these observations, demonstrating significantly reduced levels of mitochondrial cytochrome c, the intermediary between complexes III and IV of the ETC. Results also suggest that LS12 cells compensate for ETC dysfunction and oxidative stress through increased levels of tricarboxylic acid cycle enzymes and up-regulation of proteins that protect against oxidative stress and apoptosis. More than one cellular defect is likely to contribute to the genomic instability phenotype. These data suggest that LS12 cells have adapted mechanisms that allow survival under sub-optimal conditions of oxidative stress and compromised mitochondrial function to perpetuate genomic instability.

  3. Quantitative imaging reveals real-time Pou5f3–Nanog complexes driving dorsoventral mesendoderm patterning in zebrafish

    Science.gov (United States)

    Perez-Camps, Mireia; Tian, Jing; Chng, Serene C; Sem, Kai Pin; Sudhaharan, Thankiah; Teh, Cathleen; Wachsmuth, Malte; Korzh, Vladimir; Ahmed, Sohail; Reversade, Bruno

    2016-01-01

    Formation of the three embryonic germ layers is a fundamental developmental process that initiates differentiation. How the zebrafish pluripotency factor Pou5f3 (homologous to mammalian Oct4) drives lineage commitment is unclear. Here, we introduce fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy to assess the formation of Pou5f3 complexes with other transcription factors in real-time in gastrulating zebrafish embryos. We show, at single-cell resolution in vivo, that Pou5f3 complexes with Nanog to pattern mesendoderm differentiation at the blastula stage. Later, during gastrulation, Sox32 restricts Pou5f3–Nanog complexes to the ventrolateral mesendoderm by binding Pou5f3 or Nanog in prospective dorsal endoderm. In the ventrolateral endoderm, the Elabela / Aplnr pathway limits Sox32 levels, allowing the formation of Pou5f3–Nanog complexes and the activation of downstream BMP signaling. This quantitative model shows that a balance in the spatiotemporal distribution of Pou5f3–Nanog complexes, modulated by Sox32, regulates mesendoderm specification along the dorsoventral axis. DOI: http://dx.doi.org/10.7554/eLife.11475.001 PMID:27684073

  4. Cancer-associated p53 tetramerization domain mutants: quantitative analysis reveals a low threshold for tumor suppressor inactivation

    Energy Technology Data Exchange (ETDEWEB)

    Kamada, R.; Anderson, C.; Nomura, T.; Sakaguchi, K.

    2011-01-07

    The tumor suppressor p53, a 393-amino acid transcription factor, induces cell cycle arrest and apoptosis in response to genotoxic stress. Its inactivation via the mutation of its gene is a key step in tumor progression, and tetramer formation is critical for p53 post-translational modification and its ability to activate or repress the transcription of target genes vital in inhibiting tumor growth. About 50% of human tumors have TP53 gene mutations; most are missense ones that presumably lower the tumor suppressor activity of p53. In this study, we explored the effects of known tumor-derived missense mutations on the stability and oligomeric structure of p53; our comprehensive, quantitative analyses encompassed the tetramerization domain peptides representing 49 such substitutions in humans. Their effects on tetrameric structure were broad, and the stability of the mutant peptides varied widely ({Delta}T{sub m} = 4.8 {approx} -46.8 C). Because formation of a tetrameric structure is critical for protein-protein interactions, DNA binding, and the post-translational modification of p53, a small destabilization of the tetrameric structure could result in dysfunction of tumor suppressor activity. We suggest that the threshold for loss of tumor suppressor activity in terms of the disruption of the tetrameric structure of p53 could be extremely low. However, other properties of the tetramerization domain, such as electrostatic surface potential and its ability to bind partner proteins, also may be important.

  5. Quantitative FLIM-FRET Microscopy to Monitor Nanoscale Chromatin Compaction In Vivo Reveals Structural Roles of Condensin Complexes

    Directory of Open Access Journals (Sweden)

    David Llères

    2017-02-01

    Full Text Available How metazoan genomes are structured at the nanoscale in living cells and tissues remains unknown. Here, we adapted a quantitative FRET (Förster resonance energy transfer-based fluorescence lifetime imaging microscopy (FLIM approach to assay nanoscale chromatin compaction in living organisms. Caenorhabditis elegans was chosen as a model system. By measuring FRET between histone-tagged fluorescent proteins, we visualized distinct chromosomal regions and quantified the different levels of nanoscale compaction in meiotic cells. Using RNAi and repetitive extrachromosomal array approaches, we defined the heterochromatin state and showed that its architecture presents a nanoscale-compacted organization controlled by Heterochromatin Protein-1 (HP1 and SETDB1 H3-lysine-9 methyltransferase homologs in vivo. Next, we functionally explored condensin complexes. We found that condensin I and condensin II are essential for heterochromatin compaction and that condensin I additionally controls lowly compacted regions. Our data show that, in living animals, nanoscale chromatin compaction is controlled not only by histone modifiers and readers but also by condensin complexes.

  6. Quantitative Trait Locus Analysis of Seed Germination and Seedling Vigor in Brassica rapa Reveals QTL Hotspots and Epistatic Interactions.

    Science.gov (United States)

    Basnet, Ram K; Duwal, Anita; Tiwari, Dev N; Xiao, Dong; Monakhos, Sokrat; Bucher, Johan; Visser, Richard G F; Groot, Steven P C; Bonnema, Guusje; Maliepaard, Chris

    2015-01-01

    The genetic basis of seed germination and seedling vigor is largely unknown in Brassica species. We performed a study to evaluate the genetic basis of these important traits in a B. rapa doubled haploid population from a cross of a yellow-seeded oil-type yellow sarson and a black-seeded vegetable-type pak choi. We identified 26 QTL regions across all 10 linkage groups for traits related to seed weight, seed germination and seedling vigor under non-stress and salt stress conditions illustrating the polygenic nature of these traits. QTLs for multiple traits co-localized and we identified eight hotspots for quantitative trait loci (QTL) of seed weight, seed germination, and root and shoot lengths. A QTL hotspot for seed germination on A02 mapped at the B. rapa Flowering Locus C (BrFLC2). Another hotspot on A05 with salt stress specific QTLs co-located with the B. rapa Fatty acid desaturase 2 (BrFAD2) locus. Epistatic interactions were observed between QTL hotspots for seed germination on A02 and A10 and with a salt tolerance QTL on A05. These results contribute to the understanding of the genetics of seed quality and seeding vigor in B. rapa and can offer tools for Brassica breeding.

  7. Quantitative mass spectrometry analysis reveals similar substrate consensus motif for human Mps1 kinase and Plk1.

    Directory of Open Access Journals (Sweden)

    Zhen Dou

    Full Text Available BACKGROUND: Members of the Mps1 kinase family play an essential and evolutionarily conserved role in the spindle assembly checkpoint (SAC, a surveillance mechanism that ensures accurate chromosome segregation during mitosis. Human Mps1 (hMps1 is highly phosphorylated during mitosis and many phosphorylation sites have been identified. However, the upstream kinases responsible for these phosphorylations are not presently known. METHODOLOGY/PRINCIPAL FINDINGS: Here, we identify 29 in vivo phosphorylation sites in hMps1. While in vivo analyses indicate that Aurora B and hMps1 activity are required for mitotic hyper-phosphorylation of hMps1, in vitro kinase assays show that Cdk1, MAPK, Plk1 and hMps1 itself can directly phosphorylate hMps1. Although Aurora B poorly phosphorylates hMps1 in vitro, it positively regulates the localization of Mps1 to kinetochores in vivo. Most importantly, quantitative mass spectrometry analysis demonstrates that at least 12 sites within hMps1 can be attributed to autophosphorylation. Remarkably, these hMps1 autophosphorylation sites closely resemble the consensus motif of Plk1, demonstrating that these two mitotic kinases share a similar substrate consensus. CONCLUSIONS/SIGNIFICANCE: hMps1 kinase is regulated by Aurora B kinase and its autophosphorylation. Analysis on hMps1 autophosphorylation sites demonstrates that hMps1 has a substrate preference similar to Plk1 kinase.

  8. Fusion-related host proteins are actively regulated by NA during influenza infection as revealed by quantitative proteomics analysis.

    Directory of Open Access Journals (Sweden)

    Zhiwei Sui

    Full Text Available Three recombinant influenza A viruses with different neuraminidases (NAs in the background of A/PR/8/34 (PR8, named rPR8-H5N1NA, rPR8-H9N2NA, and rPR8-H1N1NA, derived from H5N1, H9N2, H1N1 (swine viruses, respectively, were constructed. We performed a quantitative proteomics analysis to investigate differential protein expression in Madin-Darby canine kidney (MDCK cells infected with recombinant and wild-type influenza viruses to determine whether NA replacement would alter host cell gene expression. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-TOF MS and two-dimensional gel electrophoresis (2-DE, we identified 12 up-regulated and 49 down-regulated protein spots, including cytoskeletal proteins, molecular biosynthesis proteins, ubiquitin-proteasome pathway proteins, and heat shock proteins. The most significant changes in infected cells were observed for molecular biosynthesis proteins. We found more differentially expressed protein spots in cells infected with rPR8-H5N1NA or rPR8-H9N2NA viruses than cells infected with wild-type virus. Many of those proteins are postulated to be involved in cell-cell fusion, but the full mechanism remains to be explored. Meanwhile, our data demonstrate that the wild-type virus has evolutionary advantages over recombinant viruses.

  9. Quantitative analysis reveals asynchronous and more than DSB-associated histone H2AX phosphorylation after exposure to ionizing radiation.

    Science.gov (United States)

    Han, Jianxun; Hendzel, Michael J; Allalunis-Turner, Joan

    2006-03-01

    Rapid phosphorylation of histone H2AX after exposure of cells to ionizing radiation occurs at DSB sites and extends to a region including as much as 30 Mbp of chromatin to form visible microscopic structures called gamma-H2AX foci. Although the kinetics of total cellular histone H2AX phosphorylation after irradiation has been characterized, we still know little about the phosphorylation kinetics of individual gamma-H2AX foci. In addition, there are hundreds of smaller gamma-H2AX foci that are not associated with DNA double-strand breaks. We refer to these sites as DSB-unrelated gamma-H2AX foci. By using indirect immunofluorescence microscopy, deconvolution and three-dimensional image analysis, we established an objective method to quantitatively analyze each gamma-H2AX focus as well as to discriminate DSB-related gamma-H2AX foci from DSB-unrelated gamma-H2AX foci. Using this method, we found that histone H2AX phosphorylation at different DSB sites was asynchronous after exposure to ionizing radiation. This may reflect the heterogeneous characteristic of free DNA ends that are generated under these conditions. In addition, we found that increased histone H2AX phosphorylation also occurred outside of DSB sites after exposure to ionizing radiation. The function of this DSB-unassociated phosphorylation is not known.

  10. Quantitative mapping of zinc fluxes in the mammalian egg reveals the origin of fertilization-induced zinc sparks

    Energy Technology Data Exchange (ETDEWEB)

    Que, Emily L.; Bleher, Reiner; Duncan, Francesca E.; Kong, Betty Y.; Gleber, Sophie C.; Vogt, Stefan; Chen, Si; Garwin, Seth A.; Bayer, Amanda R.; Dravid, Vinayak P.; Woodruff, Teresa K.; O' Halloran, Thomas V.

    2014-12-15

    Fertilization of a mammalian egg initiates a series of 'zinc sparks' that are necessary to induce the egg-to-embryo transition. Despite the importance of these zinc-efflux events little is known about their origin. To understand the molecular mechanism of the zinc spark we combined four physical approaches that resolve zinc distributions in single cells: a chemical probe for dynamic live-cell fluorescence imaging and a combination of scanning transmission electron microscopy with energy-dispersive spectroscopy, X-ray fluorescence microscopy and three-dimensional elemental tomography for high-resolution elemental mapping. We show that the zinc spark arises from a system of thousands of zinc-loaded vesicles, each of which contains, on average, 10(6) zinc atoms. These vesicles undergo dynamic movement during oocyte maturation and exocytosis at the time of fertilization. The discovery of these vesicles and the demonstration that zinc sparks originate from them provides a quantitative framework for understanding how zinc fluxes regulate cellular processes

  11. New insights into the diets of harbor seals in the Salish Sea revealed by quantitative fatty acid signature analysis

    Science.gov (United States)

    Bromaghin, Jeffrey F.; Lance, Monique M.; Elliott, Elizabeth W.; Jeffries, Steven J.; Acevedo-Gutiérrez, Alejandro; Kennish, John M.

    2012-01-01

    Harbor seals (Phoca vitulina) are an abundant predator along the west coast of North America, and there is considerable interest in their diet composition, especially in regard to predation on valued fish stocks. Available information on harbor seal diets, primarily derived from scat analysis, suggests that adult salmon (Oncorhynchus spp.), Pacific Herring (Clupea pallasii), and gadids predominate. Because diet assessments based on scat analysis may be biased, we investigated diet composition through quantitative analysis of fatty acid signatures. Blubber samples from 49 harbor seals captured in western North America from haul-outs within the area of the San Juan Islands and southern Strait of Georgia in the Salish Sea were analyzed for fatty acid composition, along with 269 fish and squid specimens representing 27 potential prey classes. Diet estimates varied spatially, demographically, and among individual harbor seals. Findings confirmed the prevalence of previously identified prey species in harbor seal diets, but other species also contributed significantly. In particular, Black (Sebastes melanops) and Yellowtail (S. flavidus) Rockfish were estimated to compose up to 50% of some individual seal diets. Specialization and high predation rates on Black and Yellowtail Rockfish by a subset of harbor seals may play a role in the population dynamics of these regional rockfish stocks that is greater than previously realized.

  12. The Pseudomonas community in metal-contaminated sediments as revealed by quantitative PCR: a link with metal bioavailability.

    Science.gov (United States)

    Roosa, Stéphanie; Wauven, Corinne Vander; Billon, Gabriel; Matthijs, Sandra; Wattiez, Ruddy; Gillan, David C

    2014-10-01

    Pseudomonas bacteria are ubiquitous Gram-negative and aerobic microorganisms that are known to harbor metal resistance mechanisms such as efflux pumps and intracellular redox enzymes. Specific Pseudomonas bacteria have been quantified in some metal-contaminated environments, but the entire Pseudomonas population has been poorly investigated under these conditions, and the link with metal bioavailability was not previously examined. In the present study, quantitative PCR and cell cultivation were used to monitor and characterize the Pseudomonas population at 4 different sediment sites contaminated with various levels of metals. At the same time, total metals and metal bioavailability (as estimated using an HCl 1 m extraction) were measured. It was found that the total level of Pseudomonas, as determined by qPCR using two different genes (oprI and the 16S rRNA gene), was positively and significantly correlated with total and HCl-extractable Cu, Co, Ni, Pb and Zn, with high correlation coefficients (>0.8). Metal-contaminated sediments featured isolates of the Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas lutea and Pseudomonas aeruginosa groups, with other bacterial genera such as Mycobacterium, Klebsiella and Methylobacterium. It is concluded that Pseudomonas bacteria do proliferate in metal-contaminated sediments, but are still part of a complex community.

  13. Microdialysis Sampling from Wound Fluids Enables Quantitative Assessment of Cytokines, Proteins, and Metabolites Reveals Bone Defect-Specific Molecular Profiles.

    Directory of Open Access Journals (Sweden)

    Yvonne Förster

    Full Text Available Bone healing involves a variety of different cell types and biological processes. Although certain key molecules have been identified, the molecular interactions of the healing progress are not completely understood. Moreover, a clinical routine for predicting the quality of bone healing after a fracture in an early phase is missing. This is mainly due to a lack of techniques to comprehensively screen for cytokines, growth factors and metabolites at their local site of action. Since all soluble molecules of interest are present in the fracture hematoma, its in-depth assessment could reveal potential markers for the monitoring of bone healing. Here, we describe an approach for sampling and quantification of cytokines and metabolites by using microdialysis, combined with solid phase extractions of proteins from wound fluids. By using a control group with an isolated soft tissue wound, we could reveal several bone defect-specific molecular features. In bone defect dialysates the neutrophil chemoattractants CXCL1, CXCL2 and CXCL3 were quantified with either a higher or earlier response compared to dialysate from soft tissue wound. Moreover, by analyzing downstream adaptions of the cells on protein level and focusing on early immune response, several proteins involved in the immune cell migration and activity could be identified to be specific for the bone defect group, e.g. immune modulators, proteases and their corresponding inhibitors. Additionally, the metabolite screening revealed different profiles between the bone defect group and the control group. In summary, we identified potential biomarkers to indicate imbalanced healing progress on all levels of analysis.

  14. 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

  15. Asynchrony of the early maturation of white matter bundles in healthy infants: Quantitative landmarks revealed noninvasively by diffusion tensor imaging

    Energy Technology Data Exchange (ETDEWEB)

    Dubois, J.; Perrin, M.; Mangin, J.F.; Cointepas, Y.; Duchesnay, E.; Le Bihan, D.; Hertz-Pannier, L. [CEA, Serv Hosp Frederic Joliot, UNAF, F-91406 Orsay (France); Dehaene-Lambertz, G. [INSERM, U562, Orsay (France); Dubois, J.; Dehaene-Lambertz, G.; Perrin, M.; Mangin, J.F.; Cointepas, Y.; Duchesnay, E.; Le Bihan, D.; Hertz-Pannier, L. [IFR49, Paris (France)

    2008-07-01

    Normal cognitive development in infants follows a well-known temporal sequence, which is assumed to be correlated with the structural maturation of underlying functional networks. Postmortem studies and, more recently, structural MR imaging studies have described qualitatively the heterogeneous spatio-temporal progression of white matter myelination. However, in vivo quantification of the maturation phases of fiber bundles is still lacking. We used noninvasive diffusion tensor MR imaging and tractography in twenty-three 1-4-month-old healthy infants to quantify the early maturation of the main cerebral fascicles. A specific maturation model, based on the respective roles of different maturational processes on the diffusion phenomena, was designed to highlight asynchronous maturation across bundles by evaluating the time-course of mean diffusivity and anisotropy changes over the considered developmental period. Using an original approach, a progression of maturation in four relative stages was determined in each tract by estimating the maturation state and speed, from the diffusion indices over the infants group compared with an adults group on one hand, and in each tract compared with the average over bundles on the other hand. Results were coherent with, and extended previous findings in 8 of 11 bundles, showing the anterior limb of the internal capsule and cingulum as the most immature, followed by the optic radiations, arcuate and inferior longitudinal fascicles, then the spino-thalamic tract and fornix, and finally the cortico-spinal tract as the most mature bundle. Thus, this approach provides new quantitative landmarks for further noninvasive research on brain-behavior relationships during normal and abnormal development. (authors)

  16. The CesA gene family of barley. Quantitative analysis of transcripts reveals two groups of co-expressed genes.

    Science.gov (United States)

    Burton, Rachel A; Shirley, Neil J; King, Brendon J; Harvey, Andrew J; Fincher, Geoffrey B

    2004-01-01

    Sequence data from cDNA and genomic clones, coupled with analyses of expressed sequence tag databases, indicate that the CesA (cellulose synthase) gene family from barley (Hordeum vulgare) has at least eight members, which are distributed across the genome. Quantitative polymerase chain reaction has been used to determine the relative abundance of mRNA transcripts for individual HvCesA genes in vegetative and floral tissues, at different stages of development. To ensure accurate expression profiling, geometric averaging of multiple internal control gene transcripts has been applied for the normalization of transcript abundance. Total HvCesA mRNA levels are highest in coleoptiles, roots, and stems and much lower in floral tissues, early developing grain, and in the elongation zone of leaves. In most tissues, HvCesA1, HvCesA2, and HvCesA6 predominate, and their relative abundance is very similar; these genes appear to be coordinately transcribed. A second group, comprising HvCesA4, HvCesA7, and HvCesA8, also appears to be coordinately transcribed, most obviously in maturing stem and root tissues. The HvCesA3 expression pattern does not fall into either of these two groups, and HvCesA5 transcript levels are extremely low in all tissues. Thus, the HvCesA genes fall into two general groups of three genes with respect to mRNA abundance, and the co-expression of the groups identifies their products as candidates for the rosettes that are involved in cellulose biosynthesis at the plasma membrane. Phylogenetic analysis allows the two groups of genes to be linked with orthologous Arabidopsis CesA genes that have been implicated in primary and secondary wall synthesis.

  17. Quantitative magnetic resonance analysis and a morphometric predictive model reveal lean body mass changes in migrating Nearctic-Neotropical passerines.

    Science.gov (United States)

    Seewagen, Chad L; Guglielmo, Christopher G

    2011-04-01

    Most studies of lean mass dynamics in free-living passerine birds have focused on Old World species at geographical barriers where they are challenged to make the longest non-stop flight of their migration. We examined lean mass variation in New World passerines in an area where the distribution of stopover habitat does not require flights to exceed more than a few hours and most migrants stop flying well before fat stores near exhaustion. We used either quantitative magnetic resonance (QMR) analysis or a morphometric model to measure or estimate, respectively, the fat and lean body mass of migrants during stopovers in New York, USA. With these data, we examined (1) variance in total body mass explained by lean body mass, (2) hourly rates of fat and lean body mass change in single-capture birds, and (3) net changes in fat and lean mass in recaptured birds. Lean mass contributed to 50% of the variation in total body mass among white-throated sparrows Zonotrichia albicollis and hermit thrushes Catharus guttatus. Lean mass of refueling gray catbirds Dumetella carolinensis and white-throated sparrows, respectively, increased 1.123 and 0.320 g h(-1). Lean mass of ovenbirds Seiurus aurocapillus accounted for an estimated 33-40% of hourly gains in total body mass. On average 35% of the total mass gained among recaptured birds was lean mass. Substantial changes in passerine lean mass are not limited to times when birds are forced to make long, non-stop flights across barriers. Protein usage during migration is common across broad taxonomic groups, migration systems, and migration strategies.

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

    Institute of Scientific and Technical Information of China (English)

    Qiaosong Yang; Junhua Wu; Chunyu Li; Yuerong Wei; Ou Sheng; Chunhua Hu; Ruibin Kuang

    2012-01-01

    Banana and its close relative,plantain are globally important crops and there is of considerable interest in optimizing their cultivation.Plantain has superior cold tolerance compared to 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℃ and subsequently allowed to recover for 24 h at 28℃.A total of 3,477 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,while 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 7 differentially expressed,cold-response candidate plantain proteins in order to validate the proteomics data.Similar analyses of the 7 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 higher 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 proteomic analysis.

  19. iTRAQ-based quantitative proteomics of stratum corneum of dandruff scalp reveals new insights into its aetiology and similarities with atopic dermatitis.

    Science.gov (United States)

    Cavusoglu, Nükhet; Delattre, Caroline; Donovan, Mark; Bourassa, Sylvie; Droit, Arnaud; El Rawadi, Charles; Jourdain, Roland; Bernard, Dominique

    2016-11-01

    The study aimed at detecting differentially expressed proteins in the stratum corneum of dandruff versus non-dandruff scalps to better understand dandruff aetiology. iTRAQ-based quantitative proteomic analysis revealed a total of 68 differentially expressed biomarkers. A detailed analysis of their known physiological functions provided new insights into the affected metabolic pathways of a dandruff scalp. Dandruff scalp showed (1) profound changes in the expression and maturation of structural and epidermal differentiation related proteins, that are responsible for the integrity of the skin, (2) altered relevant factors that regulate skin hydration, and (3) an imbalanced physiological protease-protease inhibitor ratio. Stratum corneum proteins with antimicrobial activity, mainly those derived from sweat and sebaceous glands were also found modified. Comparing our data with those reported for atopic dermatitis revealed that about 50 % of the differentially expressed proteins in the superficial layers of the stratum corneum from dandruff and atopic dermatitis are identical.

  20. Quantitative lipidomics reveals age-dependent perturbations of whole-body lipid metabolism in ACBP deficient mice

    DEFF Research Database (Denmark)

    Gallego, Sandra F; Sprenger, Richard R; Neess, Ditte

    2017-01-01

    and delayed weaning, the physiological process where newborns transit from a fat-based milk diet to a carbohydrate-rich diet. To gain insights into how ACBP impinges on weaning and the concomitant remodeling of whole-body lipid metabolism we performed a comparative lipidomics analysis charting the absolute......The acyl-CoA binding protein (ACBP) plays a key role in chaperoning long-chain acyl-CoAs into lipid metabolic processes and acts as an important regulatory hub in mammalian physiology. This is highlighted by the recent finding that mice devoid of ACBP suffer from a compromised epidermal barrier...... abundance of 613 lipid molecules in liver, muscle and plasma from weaning and adult Acbp knockout and wild type mice. Our results reveal that ACBP deficiency affects primarily lipid metabolism of liver and plasma during weaning. Specifically, we show that ACBP deficient mice have elevated levels of hepatic...

  1. Quantitative lipidomics reveals age-dependent perturbations of whole-body lipid metabolism in ACBP deficient mice

    DEFF Research Database (Denmark)

    Gallego, Sandra Fernandez; Sprenger, Richard; Neess, Ditte;

    2016-01-01

    The acyl-CoA binding protein (ACBP) plays a key role in chaperoning long-chain acyl-CoAs into lipid metabolic processes and acts as an important regulatory hub in mammalian physiology. This is highlighted by the recent finding that mice devoid of ACBP suffer from a compromised epidermal barrier...... and delayed weaning, the physiological process where newborns transit from a fat-based milk diet to a carbohydrate-rich diet. To gain insights into how ACBP impinges on weaning and the concomitant remodeling of whole-body lipid metabolism we performed a comparative lipidomics analysis charting the absolute...... abundance of 613 lipid molecules in liver, muscle and plasma from weaning and adult Acbp knockout and wild type mice. Our results reveal that ACBP deficiency affects primarily lipid metabolism of liver and plasma during weaning. Specifically, we show that ACBP deficient mice have elevated levels of hepatic...

  2. Quantitative analysis of Nipah virus proteins released as virus-like particles reveals central role for the matrix protein

    Directory of Open Access Journals (Sweden)

    Eaton Bryan T

    2007-01-01

    Full Text Available Abstract Background Nipah virus (NiV is an emerging paramyxovirus distinguished by its ability to cause fatal disease in both animal and human hosts. Together with Hendra virus (HeV, they comprise the genus Henipavirus in the Paramyxoviridae family. NiV and HeV are also restricted to Biosafety Level-4 containment and this has hampered progress towards examining details of their replication and morphogenesis. Here, we have established recombinant expression systems to study NiV particle assembly and budding through the formation of virus-like particles (VLPs. Results When expressed by recombinant Modified Vaccinia virus Ankara (rMVA or plasmid transfection, individual NiV matrix (M, fusion (F and attachment (G proteins were all released into culture supernatants in a membrane-associated state as determined by sucrose density gradient flotation and immunoprecipitation. However, co-expression of F and G along with M revealed a shift in their distribution across the gradient, indicating association with M in VLPs. Protein release was also altered depending on the context of viral proteins being expressed, with F, G and nucleocapsid (N protein reducing M release, and N release dependent on the co-expression of M. Immunoelectron microscopy and density analysis revealed VLPs that were similar to authentic virus. Differences in the budding dynamics of NiV proteins were also noted between rMVA and plasmid based strategies, suggesting that over-expression by poxvirus may not be appropriate for studying the details of recombinant virus particle assembly and release. Conclusion Taken together, the results indicate that NiV M, F, and G each possess some ability to bud from expressing cells, and that co-expression of these viral proteins results in a more organized budding process with M playing a central role. These findings will aid our understanding of paramyxovirus particle assembly in general and could help facilitate the development of a novel vaccine

  3. A quantitative multiplex nuclease protection assay reveals immunotoxicity gene expression profiles in the rabbit model for vaginal drug safety evaluation

    Energy Technology Data Exchange (ETDEWEB)

    Fichorova, Raina N., E-mail: rfichorova@rics.bwh.harvard.edu [Laboratory of Genital Tract Biology, Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women' s Hospital and Harvard Medical School, Boston, MA (United States); Mendonca, Kevin; Yamamoto, Hidemi S.; Murray, Ryan [Laboratory of Genital Tract Biology, Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women' s Hospital and Harvard Medical School, Boston, MA (United States); Chandra, Neelima; Doncel, Gustavo F. [CONRAD, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, VA (United States)

    2015-06-15

    Any vaginal product that alters the mucosal environment and impairs the immune barrier increases the risk of sexually transmitted infections, especially HIV infection, which thrives on mucosal damage and inflammation. The FDA-recommended rabbit vaginal irritation (RVI) model serves as a first line selection tool for vaginal products; however, for decades it has been limited to histopathology scoring, insufficient to select safe anti-HIV microbicides. In this study we incorporate to the RVI model a novel quantitative nuclease protection assay (qNPA) to quantify mRNA levels of 25 genes representing leukocyte differentiation markers, toll-like receptors (TLR), cytokines, chemokines, epithelial repair, microbicidal and vascular markers, by designing two multiplex arrays. Tissue sections were obtained from 36 rabbits (6 per treatment arm) after 14 daily applications of a placebo gel, saline, 4% nonoxynol-9 (N-9), and three combinations of the anti-HIV microbicides tenofovir (TFV) and UC781 in escalating concentrations (highest: 10% TFV + 2.5%UC781). Results showed that increased expression levels of toll-like receptor (TLR)-4, interleukin (IL)-1β, CXCL8, epithelial membrane protein (EMP)-1 (P < 0.05), and decreased levels of TLR2 (P < 0.05), TLR3 and bactericidal permeability increasing protein (BPI) (P < 0.001) were associated with cervicovaginal mucosal alteration (histopathology). Seven markers showed a significant linear trend predicting epithelial damage (up with CD4, IL-1β, CXCL8, CCL2, CCL21, EMP1 and down with BPI). Despite the low tissue damage RVI scores, the high-dose microbicide combination gel caused activation of HIV host cells (SLC and CD4) while N-9 caused proinflammatory gene upregulation (IL-8 and TLR4) suggesting a potential for increasing risk of HIV via different mechanisms depending on the chemical nature of the test product. - Highlights: • A transcriptome nuclease protection assay assessed microbicides for vaginal safety. • Biomarkers were

  4. Quantitative analysis of DNA methylation at all human imprinted regions reveals preservation of epigenetic stability in adult somatic tissue

    Directory of Open Access Journals (Sweden)

    Woodfine Kathryn

    2011-01-01

    Full Text Available Abstract Background Genes subject to genomic imprinting are mono-allelically expressed in a parent-of-origin dependent manner. Each imprinted locus has at least one differentially methylated region (DMR which has allele specific DNA methylation and contributes to imprinted gene expression. Once DMRs are established, they are potentially able to withstand normal genome reprogramming events that occur during cell differentiation and germ-line DMRs are stably maintained throughout development. These DMRs, in addition to being either maternally or paternally methylated, have differences in whether methylation was acquired in the germ-line or post fertilization and are present in a variety of genomic locations with different Cytosine-phosphate guanine (CpG densities and CTCF binding capacities. We therefore examined the stability of maintenance of DNA methylation imprints and determined the normal baseline DNA methylation levels in several adult tissues for all imprinted genes. In order to do this, we first developed and validated 50 highly specific, quantitative DNA methylation pyrosequencing assays for the known DMRs associated with human imprinted genes. Results Remarkable stability of the DNA methylation imprint was observed in all germ-line DMRs and paternally methylated somatic DMRs (which maintained average methylation levels of between 35% - 65% in all somatic tissues, independent of gene expression. Maternally methylated somatic DMRs were found to have more variation with tissue specific methylation patterns. Most DMRs, however, showed some intra-individual variability for DNA methylation levels in peripheral blood, suggesting that more than one DMR needs to be examined in order to get an overall impression of the epigenetic stability in a tissue. The plasticity of DNA methylation at imprinted genes was examined in a panel of normal and cancer cell lines. All cell lines showed changes in DNA methylation, especially at the paternal germ

  5. Time-resolved dissection of early phosphoproteome and ensuing proteome changes in response to TGF-β

    DEFF Research Database (Denmark)

    J D'Souza, Rochelle C; Knittle, Anna M; Nagaraj, Nagarjuna

    2014-01-01

    Transforming growth factor-β (TGF-β) signaling promotes cell motility by inducing epithelial-to-mesenchymal transitions (EMTs) in normal physiology and development, as well as in pathological conditions, such as cancer. We performed a time-resolved analysis of the proteomic and phosphoproteomic c...

  6. Quantitative lipidomics reveals age-dependent perturbations of whole-body lipid metabolism in ACBP deficient mice.

    Science.gov (United States)

    Gallego, Sandra F; Sprenger, Richard R; Neess, Ditte; Pauling, Josch K; Færgeman, Nils J; Ejsing, Christer S

    2017-02-01

    The acyl-CoA binding protein (ACBP) plays a key role in chaperoning long-chain acyl-CoAs into lipid metabolic processes and acts as an important regulatory hub in mammalian physiology. This is highlighted by the recent finding that mice devoid of ACBP suffer from a compromised epidermal barrier and delayed weaning, the physiological process where newborns transit from a fat-based milk diet to a carbohydrate-rich diet. To gain insights into how ACBP impinges on weaning and the concomitant remodeling of whole-body lipid metabolism we performed a comparative lipidomics analysis charting the absolute abundance of 613 lipid molecules in liver, muscle and plasma from weaning and adult Acbp knockout and wild type mice. Our results reveal that ACBP deficiency affects primarily lipid metabolism of liver and plasma during weaning. Specifically, we show that ACBP deficient mice have elevated levels of hepatic cholesteryl esters, and that lipids featuring an 18:1 fatty acid moiety are increased in Acbp depleted mice across all tissues investigated. Our results also show that the perturbation of systemic lipid metabolism in Acbp knockout mice is transient and becomes normalized and similar to that of wild type as mice grow older. These findings demonstrate that ACBP serves crucial functions in maintaining lipid metabolic homeostasis in mice during weaning.

  7. Quantitative superresolution microscopy reveals differences in nuclear DNA organization of multiple myeloma and monoclonal gammopathy of undetermined significance.

    Science.gov (United States)

    Sathitruangsak, Chirawadee; Righolt, Christiaan H; Klewes, Ludger; Tammur, Pille; Ilus, Tiiu; Tamm, Anu; Punab, Mari; Olujohungbe, Adebayo; Mai, Sabine

    2015-05-01

    The mammalian nucleus has a distinct substructure that cannot be visualized directly by conventional microscopy. In this study, the organization of the DNA within the nucleus of multiple myeloma (MM) cells, their precursor cells (monoclonal gammopathy of undetermined significance; MGUS) and control lymphocytes of the representative patients is visualized and quantified by superresolution microscopy. Three-dimensional structured illumination microscopy (3D-SIM) increases the spatial resolution beyond the limits of conventional widefield fluorescence microscopy. 3D-SIM reveals new insights into the nuclear architecture of cancer as we show for the first time that it resolves organizational differences in intranuclear DNA organization of myeloma cells in MGUS and in MM patients. In addition, we report a significant increase in nuclear submicron DNA structure and structure of the DNA-free space in myeloma nuclei compared to normal lymphocyte nuclei. Our study provides previously unknown details of the nanoscopic DNA architecture of interphase nuclei of the normal lymphocytes, MGUS and MM cells. This study opens new avenues to understanding the disease progression from MGUS to MM. © 2014 Wiley Periodicals, Inc.

  8. Proteomic and phosphoproteomic analyses of chromatin-associated proteins from Arabidopsis thaliana

    KAUST Repository

    Bigeard, Jean

    2014-07-10

    The nucleus is the organelle where basically all DNA-related processes take place in eukaryotes, such as replication, transcription, and splicing as well as epigenetic regulation. The identification and description of the nuclear proteins is one of the requisites toward a comprehensive understanding of the biological functions accomplished in the nucleus. Many of the regulatory mechanisms of protein functions rely on their PTMs among which phosphorylation is probably one of the most important properties affecting enzymatic activity, interaction with other molecules, localization, or stability. So far, the nuclear and subnuclear proteome and phosphoproteome of the model plant Arabidopsis thaliana have been the subject of very few studies. In this work, we developed a purification protocol of Arabidopsis chromatin-associated proteins and performed proteomic and phosphoproteomic analyses identifying a total of 879 proteins of which 198 were phosphoproteins that were mainly involved in chromatin remodeling, transcriptional regulation, and RNA processing. From 230 precisely localized phosphorylation sites (phosphosites), 52 correspond to hitherto unidentified sites. This protocol and data thereby obtained should be a valuable resource for many domains of plant research.

  9. Phosphoproteomic profiling of human myocardial tissues distinguishes ischemic from non-ischemic end stage heart failure.

    Science.gov (United States)

    Schechter, Matthew A; Hsieh, Michael K H; Njoroge, Linda W; Thompson, J Will; Soderblom, Erik J; Feger, Bryan J; Troupes, Constantine D; Hershberger, Kathleen A; Ilkayeva, Olga R; Nagel, Whitney L; Landinez, Gina P; Shah, Kishan M; Burns, Virginia A; Santacruz, Lucia; Hirschey, Matthew D; Foster, Matthew W; Milano, Carmelo A; Moseley, M Arthur; Piacentino, Valentino; Bowles, Dawn E

    2014-01-01

    The molecular differences between ischemic (IF) and non-ischemic (NIF) heart failure are poorly defined. A better understanding of the molecular differences between these two heart failure etiologies may lead to the development of more effective heart failure therapeutics. In this study extensive proteomic and phosphoproteomic profiles of myocardial tissue from patients diagnosed with IF or NIF were assembled and compared. Proteins extracted from left ventricular sections were proteolyzed and phosphopeptides were enriched using titanium dioxide resin. Gel- and label-free nanoscale capillary liquid chromatography coupled to high resolution accuracy mass tandem mass spectrometry allowed for the quantification of 4,436 peptides (corresponding to 450 proteins) and 823 phosphopeptides (corresponding to 400 proteins) from the unenriched and phospho-enriched fractions, respectively. Protein abundance did not distinguish NIF from IF. In contrast, 37 peptides (corresponding to 26 proteins) exhibited a ≥ 2-fold alteration in phosphorylation state (pfailure etiology examined. Proteins exhibiting phosphorylation alterations were grouped into functional categories: transcriptional activation/RNA processing; cytoskeleton structure/function; molecular chaperones; cell adhesion/signaling; apoptosis; and energetic/metabolism. Phosphoproteomic analysis demonstrated profound post-translational differences in proteins that are involved in multiple cellular processes between different heart failure phenotypes. Understanding the roles these phosphorylation alterations play in the development of NIF and IF has the potential to generate etiology-specific heart failure therapeutics, which could be more effective than current therapeutics in addressing the growing concern of heart failure.

  10. Quantitative analysis of commensal Escherichia coli populations reveals host-specific enterotypes at the intra-species level.

    Science.gov (United States)

    Smati, Mounira; Clermont, Olivier; Bleibtreu, Alexandre; Fourreau, Frédéric; David, Anthony; Daubié, Anne-Sophie; Hignard, Cécile; Loison, Odile; Picard, Bertrand; Denamur, Erick

    2015-08-01

    The primary habitat of the Escherichia coli species is the gut of warm-blooded vertebrates. The E. coli species is structured into four main phylogenetic groups A, B1, B2, and D. We estimated the relative proportions of these phylogroups in the feces of 137 wild and domesticated animals with various diets living in the Ile de France (Paris) region by real-time PCR. We distinguished three main clusters characterized by a particular abundance of two or more phylogroups within the E. coli animal commensal populations, which we called "enterocolitypes" by analogy with the enterotypes defined in the human gut microbiota at the genus level. These enterocolitypes were characterized by a dominant (>50%) B2, B1, or A phylogroup and were associated with different host species, diets, and habitats: wild and herbivorous species (wild rabbits and deer), domesticated herbivorous species (domesticated rabbits, horses, sheep, and cows), and omnivorous species (boar, pigs, and chickens), respectively. By analyzing retrospectively the data obtained using the same approach from 98 healthy humans living in Ile de France (Smati et al. 2013, Appl. Environ. Microbiol. 79, 5005-5012), we identified a specific human enterocolitype characterized by the dominant and/or exclusive (>90%) presence of phylogroup B2. We then compared B2 strains isolated from animals and humans, and revealed that human and animal strains differ regarding O-type and B2 subgroup. Moreover, two genes, sfa/foc and clbQ, were associated with the exclusive character of strains, observed only in humans. In conclusion, a complex network of interactions exists at several levels (genus and intra-species) within the intestinal microbiota. © 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  11. Real-time PCR for quantitative analysis of human commensal Escherichia coli populations reveals a high frequency of subdominant phylogroups.

    Science.gov (United States)

    Smati, Mounira; Clermont, Olivier; Le Gal, Frédéric; Schichmanoff, Olivier; Jauréguy, Françoise; Eddi, Alain; Denamur, Erick; Picard, Bertrand

    2013-08-01

    Escherichia coli is divided into four main phylogenetic groups, which each exhibit ecological specialization. To understand the population structure of E. coli in its primary habitat, we directly assessed the relative proportions of these phylogroups from the stools of 100 healthy human subjects using a new real-time PCR method, which allows a large number of samples to be studied. The detection threshold for our technique was 0.1% of the E. coli population, i.e., 10(5) CFU/g of feces; in other methods based on individual colony analysis, the threshold is 10%. One, two, three, or four phylogenetic groups were simultaneously found in 21%, 48%, 21%, and 8% of the subjects, respectively. Phylogroups present at a threshold of less than 10% of the population were found in 40% of the subjects, revealing high within-individual diversity. Phylogroups A and B2 were detected in 74% and 70% of the subjects, respectively; phylogroups B1 and D were detected in 36% and 32%, respectively. When phylogroup B2 was dominant, it tended not to cooccur with other phylogroups. In contrast, other phylogroups were present when phylogroup A was dominant. These data indicate a complex pattern of interactions between the members of a single species within the human gut and identify a reservoir of clones that are present at a low frequency. The presence of these minor clones could explain the fluctuation in the composition of the E. coli microbiota within single individuals that may be seen over time. They could also constitute reservoirs of virulent and/or resistant strains.

  12. CeleST: computer vision software for quantitative analysis of C. elegans swim behavior reveals novel features of locomotion.

    Science.gov (United States)

    Restif, Christophe; Ibáñez-Ventoso, Carolina; Vora, Mehul M; Guo, Suzhen; Metaxas, Dimitris; Driscoll, Monica

    2014-07-01

    In the effort to define genes and specific neuronal circuits that control behavior and plasticity, the capacity for high-precision automated analysis of behavior is essential. We report on comprehensive computer vision software for analysis of swimming locomotion of C. elegans, a simple animal model initially developed to facilitate elaboration of genetic influences on behavior. C. elegans swim test software CeleST tracks swimming of multiple animals, measures 10 novel parameters of swim behavior that can fully report dynamic changes in posture and speed, and generates data in several analysis formats, complete with statistics. Our measures of swim locomotion utilize a deformable model approach and a novel mathematical analysis of curvature maps that enable even irregular patterns and dynamic changes to be scored without need for thresholding or dropping outlier swimmers from study. Operation of CeleST is mostly automated and only requires minimal investigator interventions, such as the selection of videotaped swim trials and choice of data output format. Data can be analyzed from the level of the single animal to populations of thousands. We document how the CeleST program reveals unexpected preferences for specific swim "gaits" in wild-type C. elegans, uncovers previously unknown mutant phenotypes, efficiently tracks changes in aging populations, and distinguishes "graceful" from poor aging. The sensitivity, dynamic range, and comprehensive nature of CeleST measures elevate swim locomotion analysis to a new level of ease, economy, and detail that enables behavioral plasticity resulting from genetic, cellular, or experience manipulation to be analyzed in ways not previously possible.

  13. Quantitative morphometry of electrophysiologically identified CA3b interneurons reveals robust local geometry and distinct cell classes.

    Science.gov (United States)

    Ascoli, Giorgio A; Brown, Kerry M; Calixto, Eduardo; Card, J Patrick; Galván, E J; Perez-Rosello, T; Barrionuevo, Germán

    2009-08-20

    The morphological and electrophysiological diversity of inhibitory cells in hippocampal area CA3 may underlie specific computational roles and is not yet fully elucidated. In particular, interneurons with somata in strata radiatum (R) and lacunosum-moleculare (L-M) receive converging stimulation from the dentate gyrus and entorhinal cortex as well as within CA3. Although these cells express different forms of synaptic plasticity, their axonal trees and connectivity are still largely unknown. We investigated the branching and spatial patterns, plus the membrane and synaptic properties, of rat CA3b R and L-M interneurons digitally reconstructed after intracellular labeling. We found considerable variability within but no difference between the two layers, and no correlation between morphological and biophysical properties. Nevertheless, two cell types were identified based on the number of dendritic bifurcations, with significantly different anatomical and electrophysiological features. Axons generally branched an order of magnitude more than dendrites. However, interneurons on both sides of the R/L-M boundary revealed surprisingly modular axodendritic arborizations with consistently uniform local branch geometry. Both axons and dendrites followed a lamellar organization, and axons displayed a spatial preference toward the fissure. Moreover, only a small fraction of the axonal arbor extended to the outer portion of the invaded volume, and tended to return toward the proximal region. In contrast, dendritic trees demonstrated more limited but isotropic volume occupancy. These results suggest a role of predominantly local feedforward and lateral inhibitory control for both R and L-M interneurons. Such a role may be essential to balance the extensive recurrent excitation of area CA3 underlying hippocampal autoassociative memory function.

  14. Enhanced Phosphoproteomic Profiling Workflow For Growth Factor Signaling Analysis

    DEFF Research Database (Denmark)

    Sylvester, Marc; Burbridge, Mike; Leclerc, Gregory;

    2010-01-01

    Background Our understanding of complex signaling networks is still fragmentary. Isolated processes have been studied extensively but cross-talk is omnipresent and precludes intuitive predictions of signaling outcomes. The need for quantitative data on dynamic systems is apparent especially for our...... A549 lung carcinoma cells were used as a model and stimulated with hepatocyte growth factor, epidermal growth factor or fibroblast growth factor. We employed a quick protein digestion workflow with spin filters without using urea. Phosphopeptides in general were enriched by sequential elution from...... transfer dissociation adds confidence in modification site assignment. The workflow is relatively simple but the integration of complementary techniques leads to a deeper insight into cellular signaling networks and the potential pharmacological intervention thereof....

  15. Hippocampal phosphoproteomics of F344 rats exposed to 1-bromopropane.

    Science.gov (United States)

    Huang, Zhenlie; Ichihara, Sahoko; Oikawa, Shinji; Chang, Jie; Zhang, Lingyi; Hu, Shijie; Huang, Hanlin; Ichihara, Gaku

    2015-01-15

    1-Bromopropane (1-BP) is neurotoxic in both experimental animals and human. To identify phosphorylated modification on the unrecognized post-translational modifications of proteins and investigate their role in 1-BP-induced neurotoxicity, changes in hippocampal phosphoprotein expression levels were analyzed quantitatively in male F344 rats exposed to 1-BP inhalation at 0, 400, or 1000 ppm for 8 h/day for 1 or 4 weeks. Hippocampal protein extracts were analyzed qualitatively and quantitatively by Pro-Q Diamond gel staining and SYPRO Ruby staining coupled with two-dimensional difference in gel electrophoresis (2D-DIGE), respectively, as well as by matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) to identify phosphoproteins. Changes in selected proteins were further confirmed by Manganese II (Mn(2+))-Phos-tag SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Bax and cytochrome c protein levels were determined by western blotting. Pro-Q Diamond gel staining combined with 2D-DIGE identified 26 phosphoprotein spots (p<0.05), and MALDI-TOF/MS identified 18 up-regulated proteins and 8 down-regulated proteins. These proteins are involved in the biological process of response to stimuli, metabolic processes, and apoptosis signaling. Changes in the expression of phosphorylated 14-3-3 θ were further confirmed by Mn(2+)-Phos-tag SDS-PAGE. Western blotting showed overexpression of Bax protein in the mitochondria with down-regulation in the cytoplasm, whereas cytochrome c expression was high in the cytoplasm but low in the mitochondria after 1-BP exposure. Our results suggest that the pathogenesis of 1-BP-induced hippocampal damage involves inhibition of antiapoptosis process. Phosphoproteins identified in this study can potentially serve as biomarkers for 1-BP-induced neurotoxicity.

  16. Improving the Phosphoproteome Coverage for Limited Sample Amounts Using TiO2-SIMAC-HILIC (TiSH) Phosphopeptide Enrichment and Fractionation

    DEFF Research Database (Denmark)

    Engholm-Keller, Kasper; Larsen, Martin R

    2016-01-01

    Obtaining high phosphoproteome coverage requires specific enrichment of phosphorylated peptides from the often extremely complex peptide mixtures generated by proteolytic digestion of biological samples, as well as extensive chromatographic fractionation prior to liquid chromatography-tandem mass...

  17. Quantitative proteomic analysis reveals that anti-cancer effects of selenium-binding protein 1 in vivo are associated with metabolic pathways.

    Science.gov (United States)

    Ying, Qi; Ansong, Emmanuel; Diamond, Alan M; Lu, Zhaoxin; Yang, Wancai; Bie, Xiaomei

    2015-01-01

    Previous studies have shown the tumor-suppressive role of selenium-binding protein 1 (SBP1), but the underlying mechanisms are unclear. In this study, we found that induction of SBP1 showed significant inhibition of colorectal cancer cell growth and metastasis in mice. We further employed isobaric tags for relative and absolute quantitation (iTRAQ) to identify proteins that were involved in SBP1-mediated anti-cancer effects in tumor tissues. We identified 132 differentially expressed proteins, among them, 53 proteins were upregulated and 79 proteins were downregulated. Importantly, many of the differentially altered proteins were associated with lipid/glucose metabolism, which were also linked to Glycolysis, MAPK, Wnt, NF-kB, NOTCH and epithelial-mesenchymal transition (EMT) signaling pathways. These results have revealed a novel mechanism that SBP1-mediated cancer inhibition is through altering lipid/glucose metabolic signaling pathways.

  18. Genome-wide analysis of intracellular pH reveals quantitative control of cell division rate by pH(c) in Saccharomyces cerevisiae.

    Science.gov (United States)

    Orij, Rick; Urbanus, Malene L; Vizeacoumar, Franco J; Giaever, Guri; Boone, Charles; Nislow, Corey; Brul, Stanley; Smits, Gertien J

    2012-09-10

    Because protonation affects the properties of almost all molecules in cells, cytosolic pH (pH(c)) is usually assumed to be constant. In the model organism yeast, however, pH(c) changes in response to the presence of nutrients and varies during growth. Since small changes in pH(c) can lead to major changes in metabolism, signal transduction, and phenotype, we decided to analyze pH(c) control. Introducing a pH-sensitive reporter protein into the yeast deletion collection allowed quantitative genome-wide analysis of pH(c) in live, growing yeast cultures. pH(c) is robust towards gene deletion; no single gene mutation led to a pH(c) of more than 0.3 units lower than that of wild type. Correct pH(c) control required not only vacuolar proton pumps, but also strongly relied on mitochondrial function. Additionally, we identified a striking relationship between pH(c) and growth rate. Careful dissection of cause and consequence revealed that pH(c) quantitatively controls growth rate. Detailed analysis of the genetic basis of this control revealed that the adequate signaling of pH(c) depended on inositol polyphosphates, a set of relatively unknown signaling molecules with exquisitely pH sensitive properties. While pH(c) is a very dynamic parameter in the normal life of yeast, genetically it is a tightly controlled cellular parameter. The coupling of pH(c) to growth rate is even more robust to genetic alteration. Changes in pH(c) control cell division rate in yeast, possibly as a signal. Such a signaling role of pH(c) is probable, and may be central in development and tumorigenesis.

  19. Modulation of the Chromatin Phosphoproteome by the Haspin Protein Kinase

    DEFF Research Database (Denmark)

    Maiolica, Alessio; de Medina-Redondo, Maria; Schoof, Erwin

    2014-01-01

    protein- protein interaction network. We determined the Haspin consensus motif and the co-crystal structure of the kinase with the histone H3 tail. The structure revealed a unique bent substrate binding mode positioning the histone H3 residues Arg2 and Lys4 adjacent to the Haspin phosphorylated threonine...... into acidic binding pockets. This unique conformation of the kinase-substrate complex explains the reported modulation of Haspin activity by methylation of Lys4 of the histone H3. In addition, the identification of the structural basis of substrate recognition and the amino acid sequence preferences of Haspin......Recent discoveries have highlighted the importance of Haspin kinase activity for the correct positioning of the kinase Aurora B at the centromere. Haspin phosphorylates Thr3 of the histone H3 (H3), which provides a signal for Aurora B to localize to the centromere of mitotic chromosomes. To date...

  20. Quantitative Transcriptomics Reveals the Growth- and Nutrient-Dependent Response of a Streamlined Marine Methylotroph to Methanol and Naturally Occurring Dissolved Organic Matter.

    Science.gov (United States)

    Gifford, Scott M; Becker, Jamie W; Sosa, Oscar A; Repeta, Daniel J; DeLong, Edward F

    2016-11-22

    substantial influence on marine organic matter flux, yet the carbon components targeted by specific bacterial groups, as well as how those groups' metabolic activities change under different conditions, are not well understood. Gene expression studies of model organisms can identify these responses under defined conditions, which can then be compared to environmental transcriptomes to elucidate in situ activities. This integration, however, is limited by the data's relative nature. Here, we report the fully quantitative transcriptome of a marine bacterium, providing a genome-wide survey of cellular transcript abundances and how they change with different states of growth, nutrient conditions, and carbon substrates. The results revealed the dynamic metabolic strategies this methylotroph has for processing both simple one-carbon compounds and the complex multicarbon substrates of naturally derived marine organic matter and provide baseline quantitative data for identifying their in situ activities and impact on the marine carbon cycle.

  1. Recent findings and technological advances in phosphoproteomics for cells and tissues.

    Science.gov (United States)

    von Stechow, Louise; Francavilla, Chiara; Olsen, Jesper V

    2015-01-01

    Site-specific phosphorylation is a fast and reversible covalent post-translational modification that is tightly regulated in cells. The cellular machinery of enzymes that write, erase and read these modifications (kinases, phosphatases and phospho-binding proteins) is frequently deregulated in different diseases, including cancer. Large-scale studies of phosphoproteins - termed phosphoproteomics - strongly rely on the use of high-performance mass spectrometric instrumentation. This powerful technology has been applied to study a great number of phosphorylation-based phenotypes. Nevertheless, many technical and biological challenges have to be overcome to identify biologically relevant phosphorylation sites in cells and tissues. This review describes different technological strategies to identify and quantify phosphorylation sites with high accuracy, without significant loss of analysis speed and reproducibility in tissues and cells. Moreover, computational tools for analysis, integration and biological interpretation of phosphorylation events are discussed.

  2. Offline High pH Reversed-Phase Peptide Fractionation for Deep Phosphoproteome Coverage

    DEFF Research Database (Denmark)

    Batth, Tanveer S; Olsen, Jesper V

    2016-01-01

    Protein phosphorylation, a process in which kinases modify serines, threonines, and tyrosines with phosphoryl groups is of major importance in eukaryotic biology. Protein phosphorylation events are key initiators of signaling responses which determine cellular outcomes after environmental...... and metabolic stimuli, and are thus highly regulated. Therefore, studying the mechanism of regulation by phosphorylation, and pinpointing the exact site of phosphorylation on proteins is of high importance. This protocol describes in detail a phosphoproteomics workflow for ultra-deep coverage by fractionating...... peptide mixtures based on high pH (basic) reversed-phase chromatography prior to phosphopeptide enrichment and mass spectrometric analysis. Peptides are separated on a C18 reversed-phase column under basic conditions and fractions collected in timed intervals followed by concatenation of the fractions...

  3. Characterization of the human plasma phosphoproteome using linear ion trap mass spectrometry and multiple search engines.

    Science.gov (United States)

    Carrascal, Montserrat; Gay, Marina; Ovelleiro, David; Casas, Vanessa; Gelpí, Emilio; Abian, Joaquin

    2010-02-05

    Major plasma protein families play different roles in blood physiology and hemostasis and in immunodefense. Other proteins in plasma can be involved in signaling as chemical messengers or constitute biological markers of the status of distant tissues. In this respect, the plasma phosphoproteome holds potentially relevant information on the mechanisms modulating these processes through the regulation of protein activity. In this work we describe for the first time a collection of phosphopeptides identified in human plasma using immunoaffinity separation of the seven major serum protein families from other plasma proteins, SCX fractionation, and TiO(2) purification prior to LC-MS/MS analysis. One-hundred and twenty-seven phosphosites in 138 phosphopeptides mapping 70 phosphoproteins were identified with FDR < 1%. A high-confidence collection of phosphosites was obtained using a combined search with the OMSSA, SEQUEST, and Phenyx search engines.

  4. Automated Immobilized Metal Affinity Chromatography System for Enrichment of Escherichia coli Phosphoproteome

    Energy Technology Data Exchange (ETDEWEB)

    Qu, Yi; Wu, Si; Zhao, Rui; Zink, Erika M.; Orton, Daniel J.; Moore, Ronald J.; Meng, Da; Clauss, Therese RW; Aldrich, Joshua T.; Lipton, Mary S.; Pasa-Tolic, Ljiljana

    2013-06-05

    Enrichment of bacterial phosphopeptides is an essential step prior to bottom-up mass spectrometry-based analysis of the phosphoproteome, which is fundamental to understanding the role of phosphoproteins in cell signaling and regulation of protein activity. We developed an automated IMAC system to enrich strong cation exchange-fractionated phosphopeptides from the soluble proteome of Escherichia coli MG1655 grown on minimal medium. Initial demonstration of the system resulted in identification of 75 phosphopeptides covering 52 phosphoproteins. Consistent with previous studies, many of these phosphoproteins are involved in the carbohydrate portion of central metabolism. The automated system utilizes a large capacity IMAC column that can effectively enrich phosphopeptides from a bacterial sample by increasing peptide loading and reducing the wash time. An additional benefit of the automated IMAC system is reduced labor and associated costs.

  5. Phosphoproteomic profiling of human myocardial tissues distinguishes ischemic from non-ischemic end stage heart failure.

    Directory of Open Access Journals (Sweden)

    Matthew A Schechter

    Full Text Available The molecular differences between ischemic (IF and non-ischemic (NIF heart failure are poorly defined. A better understanding of the molecular differences between these two heart failure etiologies may lead to the development of more effective heart failure therapeutics. In this study extensive proteomic and phosphoproteomic profiles of myocardial tissue from patients diagnosed with IF or NIF were assembled and compared. Proteins extracted from left ventricular sections were proteolyzed and phosphopeptides were enriched using titanium dioxide resin. Gel- and label-free nanoscale capillary liquid chromatography coupled to high resolution accuracy mass tandem mass spectrometry allowed for the quantification of 4,436 peptides (corresponding to 450 proteins and 823 phosphopeptides (corresponding to 400 proteins from the unenriched and phospho-enriched fractions, respectively. Protein abundance did not distinguish NIF from IF. In contrast, 37 peptides (corresponding to 26 proteins exhibited a ≥ 2-fold alteration in phosphorylation state (p<0.05 when comparing IF and NIF. The degree of protein phosphorylation at these 37 sites was specifically dependent upon the heart failure etiology examined. Proteins exhibiting phosphorylation alterations were grouped into functional categories: transcriptional activation/RNA processing; cytoskeleton structure/function; molecular chaperones; cell adhesion/signaling; apoptosis; and energetic/metabolism. Phosphoproteomic analysis demonstrated profound post-translational differences in proteins that are involved in multiple cellular processes between different heart failure phenotypes. Understanding the roles these phosphorylation alterations play in the development of NIF and IF has the potential to generate etiology-specific heart failure therapeutics, which could be more effective than current therapeutics in addressing the growing concern of heart failure.

  6. Celiac Anti-Type 2 Transglutaminase Antibodies Induce Phosphoproteome Modification in Intestinal Epithelial Caco-2 Cells

    Science.gov (United States)

    Marabotti, Anna; Lepretti, Marilena; Salzano, Anna Maria; Scaloni, Andrea; Vitale, Monica; Zambrano, Nicola; Sblattero, Daniele; Esposito, Carla

    2013-01-01

    Background Celiac disease is an inflammatory condition of the small intestine that affects genetically predisposed individuals after dietary wheat gliadin ingestion. Type 2-transglutaminase (TG2) activity seems to be responsible for a strong autoimmune response in celiac disease, TG2 being the main autoantigen. Several studies support the concept that celiac anti-TG2 antibodies may contribute to disease pathogenesis. Our recent findings on the ability of anti-TG2 antibodies to induce a rapid intracellular mobilization of calcium ions, as well as extracellular signal-regulated kinase phosphorylation, suggest that they potentially act as signaling molecules. In line with this concept, we have investigated whether anti-TG2 antibodies can induce phosphoproteome modification in an intestinal epithelial cell line. Methods and Principal Findings We studied phosphoproteome modification in Caco-2 cells treated with recombinant celiac anti-TG2 antibodies. We performed a two-dimensional electrophoresis followed by specific staining of phosphoproteins and mass spectrometry analysis of differentially phosphorylated proteins. Of 14 identified proteins (excluding two uncharacterized proteins), three were hypophosphorylated and nine were hyperphosphorylated. Bioinformatics analyses confirmed the presence of phosphorylation sites in all the identified proteins and highlighted their involvement in several fundamental biological processes, such as cell cycle progression, cell stress response, cytoskeletal organization and apoptosis. Conclusions Identification of differentially phosphorylated proteins downstream of TG2-antibody stimulation suggests that in Caco-2 cells these antibodies perturb cell homeostasis by behaving as signaling molecules. We hypothesize that anti-TG2 autoantibodies may destabilize the integrity of the intestinal mucosa in celiac individuals, thus contributing to celiac disease establishment and progression. Since several proteins here identified in this study

  7. Celiac anti-type 2 transglutaminase antibodies induce phosphoproteome modification in intestinal epithelial Caco-2 cells.

    Directory of Open Access Journals (Sweden)

    Gaetana Paolella

    Full Text Available BACKGROUND: Celiac disease is an inflammatory condition of the small intestine that affects genetically predisposed individuals after dietary wheat gliadin ingestion. Type 2-transglutaminase (TG2 activity seems to be responsible for a strong autoimmune response in celiac disease, TG2 being the main autoantigen. Several studies support the concept that celiac anti-TG2 antibodies may contribute to disease pathogenesis. Our recent findings on the ability of anti-TG2 antibodies to induce a rapid intracellular mobilization of calcium ions, as well as extracellular signal-regulated kinase phosphorylation, suggest that they potentially act as signaling molecules. In line with this concept, we have investigated whether anti-TG2 antibodies can induce phosphoproteome modification in an intestinal epithelial cell line. METHODS AND PRINCIPAL FINDINGS: We studied phosphoproteome modification in Caco-2 cells treated with recombinant celiac anti-TG2 antibodies. We performed a two-dimensional electrophoresis followed by specific staining of phosphoproteins and mass spectrometry analysis of differentially phosphorylated proteins. Of 14 identified proteins (excluding two uncharacterized proteins, three were hypophosphorylated and nine were hyperphosphorylated. Bioinformatics analyses confirmed the presence of phosphorylation sites in all the identified proteins and highlighted their involvement in several fundamental biological processes, such as cell cycle progression, cell stress response, cytoskeletal organization and apoptosis. CONCLUSIONS: Identification of differentially phosphorylated proteins downstream of TG2-antibody stimulation suggests that in Caco-2 cells these antibodies perturb cell homeostasis by behaving as signaling molecules. We hypothesize that anti-TG2 autoantibodies may destabilize the integrity of the intestinal mucosa in celiac individuals, thus contributing to celiac disease establishment and progression. Since several proteins here

  8. Quantitative Analysis of the Association Angle between T-cell Receptor Vα/Vβ Domains Reveals Important Features for Epitope Recognition.

    Science.gov (United States)

    Hoffmann, Thomas; Krackhardt, Angela M; Antes, Iris

    2015-07-01

    T-cell receptors (TCR) play an important role in the adaptive immune system as they recognize pathogen- or cancer-based epitopes and thus initiate the cell-mediated immune response. Therefore there exists a growing interest in the optimization of TCRs for medical purposes like adoptive T-cell therapy. However, the molecular mechanisms behind T-cell signaling are still predominantly unknown. For small sets of TCRs it was observed that the angle between their Vα- and Vβ-domains, which bind the epitope, can vary and might be important for epitope recognition. Here we present a comprehensive, quantitative study of the variation in the Vα/Vβ interdomain-angle and its influence on epitope recognition, performing a systematic bioinformatics analysis based on a representative set of experimental TCR structures. For this purpose we developed a new, cuboid-based superpositioning method, which allows a unique, quantitative analysis of the Vα/Vβ-angles. Angle-based clustering led to six significantly different clusters. Analysis of these clusters revealed the unexpected result that the angle is predominantly influenced by the TCR-clonotype, whereas the bound epitope has only a minor influence. Furthermore we could identify a previously unknown center of rotation (CoR), which is shared by all TCRs. All TCR geometries can be obtained by rotation around this center, rendering it a new, common TCR feature with the potential of improving the accuracy of TCR structure prediction considerably. The importance of Vα/Vβ rotation for signaling was confirmed as we observed larger variances in the Vα/Vβ-angles in unbound TCRs compared to epitope-bound TCRs. Our results strongly support a two-step mechanism for TCR-epitope: First, preformation of a flexible TCR geometry in the unbound state and second, locking of the Vα/Vβ-angle in a TCR-type specific geometry upon epitope-MHC association, the latter being driven by rotation around the unique center of rotation.

  9. The phosphoproteome in regenerating protoplasts from Physcomitrella patens protonemata shows changes paralleling postembryonic development in higher plants.

    Science.gov (United States)

    Wang, Xiaoqin; Qi, Meiyan; Li, Jingyun; Ji, Zhongzhong; Hu, Yong; Bao, Fang; Mahalingam, Ramamurthy; He, Yikun

    2014-05-01

    The moss Physcomitrella patens is an ideal model plant to study plant developmental processes. To better understand the mechanism of protoplast regeneration, a phosphoproteome analysis was performed. Protoplasts were prepared from protonemata. By 4 d of protoplast regeneration, the first cell divisions had ensued. Through a highly selective titanium dioxide (TiO2)-based phosphopeptide enrichment method and mass spectrometric technology, more than 300 phosphoproteins were identified as protoplast regeneration responsive. Of these, 108 phosphoproteins were present on day 4 but not in fresh protoplasts or those cultured for 2 d. These proteins are catalogued here. They were involved in cell-wall metabolism, transcription, signal transduction, cell growth/division, and cell structure. These protein functions are related to cell morphogenesis, organogenesis, and development adjustment. This study presents a comprehensive analysis of phosphoproteome involved in protoplast regeneration and indicates that the mechanism of plant protoplast regeneration is similar to that of postembryonic development.

  10. Chronic low-dose-rate ionising radiation affects the hippocampal phosphoproteome in the ApoE-/- Alzheimer's mouse model

    DEFF Research Database (Denmark)

    Kempf, S. J.; Janik, Dirk; Barjaktarovic, Zarko

    2016-01-01

    Accruing data indicate that radiation-induced consequences resemble pathologies of neurodegenerative diseases such as Alzheimer's. The aim of this study was to elucidate the effect on hippocampus of chronic low-dose-rate radiation exposure (1 mGy/day or 20 mGy/day) given over 300 days...... that several molecular targets induced by chronic low-dose-rate radiation overlap with those of Alzheimer's pathology. It may suggest that ionising radiation functions as a contributing risk factor to this neurodegenerative disease....... with cumulative doses of 0.3 Gy and 6.0 Gy, respectively. ApoE deficient mutant C57Bl/6 mouse was used as an Alzheimer's model. Using mass spectrometry, a marked alteration in the phosphoproteome was found at both dose rates. The radiation-induced changes in the phosphoproteome were associated with the control...

  11. Coupling functionalized cobalt ferrite nanoparticle enrichment with online LC/MS/MS for top-down phosphoproteomics.

    Science.gov (United States)

    Chen, Bifan; Hwang, Leekyoung; Ochowicz, William; Lin, Ziqing; Guardado-Alvarez, Tania M; Cai, Wenxuan; Xiu, Lichen; Dani, Kunal; Colah, Cyrus; Jin, Song; Ge, Ying

    2017-06-01

    Phosphorylation plays pivotal roles in cellular processes and dysregulated phosphorylation is considered as an underlying mechanism in many human diseases. Top-down mass spectrometry (MS) analyzes intact proteins and provides a comprehensive analysis of protein phosphorylation. However, top-down MS-based phosphoproteomics is challenging due to the difficulty in enriching low abundance intact phosphoproteins as well as separating and detecting the enriched phosphoproteins from complex mixtures. Herein, we have designed and synthesized the next generation functionalized superparamagnetic cobalt ferrite (CoFe2O4) nanoparticles (NPs), and have further developed a top-down phosphoproteomics strategy coupling phosphoprotein enrichment enabled by the functionalized CoFe2O4 NPs with online liquid chromatography (LC)/MS/MS for comprehensive characterization of phosphoproteins. We have demonstrated the highly specific enrichment of a minimal amount of spike-in β-casein from a complex tissue lysate as well as effective separation and quantification of its phosphorylated genetic variants. More importantly, this integrated top-down phosphoproteomics strategy allows for enrichment, identification, quantification, and comprehensive characterization of low abundance endogenous phosphoproteins from complex tissue extracts on a chromatographic time scale.

  12. Selecting Sample Preparation Workflows for Mass Spectrometry-Based Proteomic and Phosphoproteomic Analysis of Patient Samples with Acute Myeloid Leukemia

    Directory of Open Access Journals (Sweden)

    Maria Hernandez-Valladares

    2016-08-01

    Full Text Available Global mass spectrometry (MS-based proteomic and phosphoproteomic studies of acute myeloid leukemia (AML biomarkers represent a powerful strategy to identify and confirm proteins and their phosphorylated modifications that could be applied in diagnosis and prognosis, as a support for individual treatment regimens and selection of patients for bone marrow transplant. MS-based studies require optimal and reproducible workflows that allow a satisfactory coverage of the proteome and its modifications. Preparation of samples for global MS analysis is a crucial step and it usually requires method testing, tuning and optimization. Different proteomic workflows that have been used to prepare AML patient samples for global MS analysis usually include a standard protein in-solution digestion procedure with a urea-based lysis buffer. The enrichment of phosphopeptides from AML patient samples has previously been carried out either with immobilized metal affinity chromatography (IMAC or metal oxide affinity chromatography (MOAC. We have recently tested several methods of sample preparation for MS analysis of the AML proteome and phosphoproteome and introduced filter-aided sample preparation (FASP as a superior methodology for the sensitive and reproducible generation of peptides from patient samples. FASP-prepared peptides can be further fractionated or IMAC-enriched for proteome or phosphoproteome analyses. Herein, we will review both in-solution and FASP-based sample preparation workflows and encourage the use of the latter for the highest protein and phosphorylation coverage and reproducibility.

  13. In-vivo quantitative proteomics reveals a key contribution of post-transcriptional mechanisms to the circadian regulation of liver metabolism.

    Directory of Open Access Journals (Sweden)

    Maria S Robles

    2014-01-01

    Full Text Available Circadian clocks are endogenous oscillators that drive the rhythmic expression of a broad array of genes, orchestrating metabolism and physiology. Recent evidence indicates that post-transcriptional and post-translational mechanisms play essential roles in modulating temporal gene expression for proper circadian function, particularly for the molecular mechanism of the clock. Due to technical limitations in large-scale, quantitative protein measurements, it remains unresolved to what extent the circadian clock regulates metabolism by driving rhythms of protein abundance. Therefore, we aimed to identify global circadian oscillations of the proteome in the mouse liver by applying in vivo SILAC mouse technology in combination with state of the art mass spectrometry. Among the 3000 proteins accurately quantified across two consecutive cycles, 6% showed circadian oscillations with a defined phase of expression. Interestingly, daily rhythms of one fifth of the liver proteins were not accompanied by changes at the transcript level. The oscillations of almost half of the cycling proteome were delayed by more than six hours with respect to the corresponding, rhythmic mRNA. Strikingly we observed that the length of the time lag between mRNA and protein cycles varies across the day. Our analysis revealed a high temporal coordination in the abundance of proteins involved in the same metabolic process, such as xenobiotic detoxification. Apart from liver specific metabolic pathways, we identified many other essential cellular processes in which protein levels are under circadian control, for instance vesicle trafficking and protein folding. Our large-scale proteomic analysis reveals thus that circadian post-transcriptional and post-translational mechanisms play a key role in the temporal orchestration of liver metabolism and physiology.

  14. iTRAQ-based quantitative proteomic analysis reveals potential factors associated with the enhancement of phenazine-1-carboxamide production in Pseudomonas chlororaphis P3.

    Science.gov (United States)

    Jin, Xue-Jie; Peng, Hua-Song; Hu, Hong-Bo; Huang, Xian-Qing; Wang, Wei; Zhang, Xue-Hong

    2016-06-07

    Phenazine-1-carboxamide (PCN), a phenazine derivative, is strongly antagonistic to fungal phytopathogens. Pseudomonas chlororaphis HT66 is a PCN-producing, non-pathogenic biocontrol strain, and we obtained the mutant P. chlororaphis P3, which produces 4.7 times more PCN than the wild-type HT66 strain. To reveal the cause of PCN production enhancement in P3 and find potential factors related to PCN biosynthesis, an iTRAQ-based quantitative proteomic analysis was used to study the expression changes between the two strains. Of the 452 differentially expressed proteins, most were functionally mapped into PCN biosynthesis pathway or other related metabolisms. The upregulation of proteins, including PhzA/B, PhzD, PhzF, PhzG, and PhzH, involved in PCN biosynthesis was in agreement with the efficient production of PCN in P3. A number of proteins that function primarily in energy production, amino acid metabolism, and secondary metabolism played important roles in PCN biosynthesis. Notably, proteins involved in the uptake and conversion of phosphate, inorganic nitrogen sources, and iron improved the PCN production. Furthermore, the type VI secretion system may participate in the secretion or/and indirect biosynthetic regulation of PCN in P. chlororaphis. This study provides valuable clues to better understand the biosynthesis, excretion and regulation of PCN in Pseudomonas and also provides potential gene targets for further engineering high-yield strains.

  15. Quantitative Analysis of the Human Milk Whey Proteome Reveals Developing Milk and Mammary-Gland Functions across the First Year of Lactation

    Directory of Open Access Journals (Sweden)

    Qiang Zhang

    2013-09-01

    Full Text Available In-depth understanding of the changing functions of human milk (HM proteins and the corresponding physiological adaptions of the lactating mammary gland has been inhibited by incomplete knowledge of the HM proteome. We analyzed the HM whey proteome (n = 10 women with samples at 1 week and 1, 3, 6, 9 and 12 months using a quantitative proteomic approach. One thousand three hundred and thirty three proteins were identified with 615 being quantified. Principal component analysis revealed a transition in the HM whey proteome-throughout the first year of lactation. Abundance changes in IgG, sIgA and sIgM display distinct features during the first year. Complement components and other acute-phase proteins are generally at higher levels in early lactation. Proteomic analysis further suggests that the sources of milk fatty acids (FA shift from more direct blood influx to more de novo mammary synthesis over lactation. The abundances of the majority of glycoproteins decline over lactation, which is consistent with increased enzyme expression in glycoprotein degradation and decreased enzyme expression in glycoprotein synthesis. Cellular detoxification machinery may be transformed as well, thereby accommodating increased metabolic activities in late lactation. The multiple developing functions of HM proteins and the corresponding mammary adaption become more apparent from this study.

  16. Quantitative Analysis of the Human Milk Whey Proteome Reveals Developing Milk and Mammary-Gland Functions across the First Year of Lactation.

    Science.gov (United States)

    Zhang, Qiang; Cundiff, Judy K; Maria, Sarah D; McMahon, Robert J; Woo, Jessica G; Davidson, Barbara S; Morrow, Ardythe L

    2013-09-03

    In-depth understanding of the changing functions of human milk (HM) proteins and the corresponding physiological adaptions of the lactating mammary gland has been inhibited by incomplete knowledge of the HM proteome. We analyzed the HM whey proteome (n = 10 women with samples at 1 week and 1, 3, 6, 9 and 12 months) using a quantitative proteomic approach. One thousand three hundred and thirty three proteins were identified with 615 being quantified. Principal component analysis revealed a transition in the HM whey proteome-throughout the first year of lactation. Abundance changes in IgG, sIgA and sIgM display distinct features during the first year. Complement components and other acute-phase proteins are generally at higher levels in early lactation. Proteomic analysis further suggests that the sources of milk fatty acids (FA) shift from more direct blood influx to more de novo mammary synthesis over lactation. The abundances of the majority of glycoproteins decline over lactation, which is consistent with increased enzyme expression in glycoprotein degradation and decreased enzyme expression in glycoprotein synthesis. Cellular detoxification machinery may be transformed as well, thereby accommodating increased metabolic activities in late lactation. The multiple developing functions of HM proteins and the corresponding mammary adaption become more apparent from this study.

  17. Coordinating Role of RXRα in Downregulating Hepatic Detoxification during Inflammation Revealed by Fuzzy-Logic Modeling.

    Science.gov (United States)

    Keller, Roland; Klein, Marcus; Thomas, Maria; Dräger, Andreas; Metzger, Ute; Templin, Markus F; Joos, Thomas O; Thasler, Wolfgang E; Zell, Andreas; Zanger, Ulrich M

    2016-01-01

    During various inflammatory processes circulating cytokines including IL-6, IL-1β, and TNFα elicit a broad and clinically relevant impairment of hepatic detoxification that is based on the simultaneous downregulation of many drug metabolizing enzymes and transporter genes. To address the question whether a common mechanism is involved we treated human primary hepatocytes with IL-6, the major mediator of the acute phase response in liver, and characterized acute phase and detoxification responses in quantitative gene expression and (phospho-)proteomics data sets. Selective inhibitors were used to disentangle the roles of JAK/STAT, MAPK, and PI3K signaling pathways. A prior knowledge-based fuzzy logic model comprising signal transduction and gene regulation was established and trained with perturbation-derived gene expression data from five hepatocyte donors. Our model suggests a greater role of MAPK/PI3K compared to JAK/STAT with the orphan nuclear receptor RXRα playing a central role in mediating transcriptional downregulation. Validation experiments revealed a striking similarity of RXRα gene silencing versus IL-6 induced negative gene regulation (rs = 0.79; P<0.0001). These results concur with RXRα functioning as obligatory heterodimerization partner for several nuclear receptors that regulate drug and lipid metabolism.

  18. Searching for Novel Cdk5 Substrates in Brain by Comparative Phosphoproteomics of Wild Type and Cdk5−/− Mice

    Science.gov (United States)

    Contreras-Vallejos, Erick; Utreras, Elías; Bórquez, Daniel A.; Prochazkova, Michaela; Terse, Anita; Jaffe, Howard; Toledo, Andrea; Arruti, Cristina; Pant, Harish C.; Kulkarni, Ashok B.; González-Billault, Christian

    2014-01-01

    Protein phosphorylation is the most common post-translational modification that regulates several pivotal functions in cells. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase which is mostly active in the nervous system. It regulates several biological processes such as neuronal migration, cytoskeletal dynamics, axonal guidance and synaptic plasticity among others. In search for novel substrates of Cdk5 in the brain we performed quantitative phosphoproteomics analysis, isolating phosphoproteins from whole brain derived from E18.5 Cdk5+/+ and Cdk5−/− embryos, using an Immobilized Metal-Ion Affinity Chromatography (IMAC), which specifically binds to phosphorylated proteins. The isolated phosphoproteins were eluted and isotopically labeled for relative and absolute quantitation (iTRAQ) and mass spectrometry identification. We found 40 proteins that showed decreased phosphorylation at Cdk5−/− brains. In addition, out of these 40 hypophosphorylated proteins we characterized two proteins, :MARCKS (Myristoylated Alanine-Rich protein Kinase C substrate) and Grin1 (G protein regulated inducer of neurite outgrowth 1). MARCKS is known to be phosphorylated by Cdk5 in chick neural cells while Grin1 has not been reported to be phosphorylated by Cdk5. When these proteins were overexpressed in N2A neuroblastoma cell line along with p35, serine phosphorylation in their Cdk5 motifs was found to be increased. In contrast, treatments with roscovitine, the Cdk5 inhibitor, resulted in an opposite effect on serine phosphorylation in N2A cells and primary hippocampal neurons transfected with MARCKS. In summary, the results presented here identify Grin 1 as novel Cdk5 substrate and confirm previously identified MARCKS as a a bona fide Cdk5 substrate. PMID:24658276

  19. Quantitative Analysis of the Association Angle between T-cell Receptor Vα/Vβ Domains Reveals Important Features for Epitope Recognition.

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    Thomas Hoffmann

    2015-07-01

    Full Text Available T-cell receptors (TCR play an important role in the adaptive immune system as they recognize pathogen- or cancer-based epitopes and thus initiate the cell-mediated immune response. Therefore there exists a growing interest in the optimization of TCRs for medical purposes like adoptive T-cell therapy. However, the molecular mechanisms behind T-cell signaling are still predominantly unknown. For small sets of TCRs it was observed that the angle between their Vα- and Vβ-domains, which bind the epitope, can vary and might be important for epitope recognition. Here we present a comprehensive, quantitative study of the variation in the Vα/Vβ interdomain-angle and its influence on epitope recognition, performing a systematic bioinformatics analysis based on a representative set of experimental TCR structures. For this purpose we developed a new, cuboid-based superpositioning method, which allows a unique, quantitative analysis of the Vα/Vβ-angles. Angle-based clustering led to six significantly different clusters. Analysis of these clusters revealed the unexpected result that the angle is predominantly influenced by the TCR-clonotype, whereas the bound epitope has only a minor influence. Furthermore we could identify a previously unknown center of rotation (CoR, which is shared by all TCRs. All TCR geometries can be obtained by rotation around this center, rendering it a new, common TCR feature with the potential of improving the accuracy of TCR structure prediction considerably. The importance of Vα/Vβ rotation for signaling was confirmed as we observed larger variances in the Vα/Vβ-angles in unbound TCRs compared to epitope-bound TCRs. Our results strongly support a two-step mechanism for TCR-epitope: First, preformation of a flexible TCR geometry in the unbound state and second, locking of the Vα/Vβ-angle in a TCR-type specific geometry upon epitope-MHC association, the latter being driven by rotation around the unique center of rotation.

  20. In-depth Qualitative and Quantitative Profiling of Tyrosine Phosphorylation Using a Combination of Phosphopeptide Immunoaffinity Purification and Stable Isotope Dimethyl Labeling*

    Science.gov (United States)

    Boersema, Paul J.; Foong, Leong Yan; Ding, Vanessa M. Y.; Lemeer, Simone; van Breukelen, Bas; Philp, Robin; Boekhorst, Jos; Snel, Berend; den Hertog, Jeroen; Choo, Andre B. H.; Heck, Albert J. R.

    2010-01-01

    Several mass spectrometry-based assays have emerged for the quantitative profiling of cellular tyrosine phosphorylation. Ideally, these methods should reveal the exact sites of tyrosine phosphorylation, be quantitative, and not be cost-prohibitive. The latter is often an issue as typically several milligrams of (stable isotope-labeled) starting protein material are required to enable the detection of low abundance phosphotyrosine peptides. Here, we adopted and refined a peptidecentric immunoaffinity purification approach for the quantitative analysis of tyrosine phosphorylation by combining it with a cost-effective stable isotope dimethyl labeling method. We were able to identify by mass spectrometry, using just two LC-MS/MS runs, more than 1100 unique non-redundant phosphopeptides in HeLa cells from about 4 mg of starting material without requiring any further affinity enrichment as close to 80% of the identified peptides were tyrosine phosphorylated peptides. Stable isotope dimethyl labeling could be incorporated prior to the immunoaffinity purification, even for the large quantities (mg) of peptide material used, enabling the quantification of differences in tyrosine phosphorylation upon pervanadate treatment or epidermal growth factor stimulation. Analysis of the epidermal growth factor-stimulated HeLa cells, a frequently used model system for tyrosine phosphorylation, resulted in the quantification of 73 regulated unique phosphotyrosine peptides. The quantitative data were found to be exceptionally consistent with the literature, evidencing that such a targeted quantitative phosphoproteomics approach can provide reproducible results. In general, the combination of immunoaffinity purification of tyrosine phosphorylated peptides with large scale stable isotope dimethyl labeling provides a cost-effective approach that can alleviate variation in sample preparation and analysis as samples can be combined early on. Using this approach, a rather complete qualitative

  1. Elucidating the CXCL12/CXCR4 signaling network in chronic lymphocytic leukemia through phosphoproteomics analysis.

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    Morgan O'Hayre

    Full Text Available BACKGROUND: Chronic Lymphocytic Leukemia (CLL pathogenesis has been linked to the prolonged survival and/or apoptotic resistance of leukemic B cells in vivo, and is thought to be due to enhanced survival signaling responses to environmental factors that protect CLL cells from spontaneous and chemotherapy-induced death. Although normally associated with cell migration, the chemokine, CXCL12, is one of the factors known to support the survival of CLL cells. Thus, the signaling pathways activated by CXCL12 and its receptor, CXCR4, were investigated as components of these pathways and may represent targets that if inhibited, could render resistant CLL cells more susceptible to chemotherapy. METHODOLOGY/PRINCIPAL FINDINGS: To determine the downstream signaling targets that contribute to the survival effects of CXCL12 in CLL, we took a phosphoproteomics approach to identify and compare phosphopeptides in unstimulated and CXCL12-stimulated primary CLL cells. While some of the survival pathways activated by CXCL12 in CLL are known, including Akt and ERK1/2, this approach enabled the identification of additional signaling targets and novel phosphoproteins that could have implications in CLL disease and therapy. In addition to the phosphoproteomics results, we provide evidence from western blot validation that the tumor suppressor, programmed cell death factor 4 (PDCD4, is a previously unidentified phosphorylation target of CXCL12 signaling in all CLL cells probed. Additionally, heat shock protein 27 (HSP27, which mediates anti-apoptotic signaling and has previously been linked to chemotherapeutic resistance, was detected in a subset (approximately 25% of CLL patients cells examined. CONCLUSIONS/SIGNIFICANCE: Since PDCD4 and HSP27 have previously been associated with cancer and regulation of cell growth and apoptosis, these proteins may have novel implications in CLL cell survival and represent potential therapeutic targets. PDCD4 also represents a

  2. Reverse transcription quantitative PCR revealed persistency of thermophilic lactic acid bacteria metabolic activity until the end of the ripening of Emmental cheese.

    Science.gov (United States)

    Falentin, Hélène; Henaff, Nadine; Le Bivic, Pierre; Deutsch, Stéphanie-Marie; Parayre, Sandrine; Richoux, Romain; Sohier, Daniele; Thierry, Anne; Lortal, Sylvie; Postollec, Florence

    2012-02-01

    For Emmental manufacture two kinds of adjunct culture are added: (i) thermophilic lactic acid bacteria (starters) such as Lactobacillus helveticus (LH), and Streptococcus thermophilus (ST) growing the first day of the manufacture and (ii) ripening culture. ST and LH have a key role in curd acidification and proteolysis at the beginning of the manufacture but are considered to be lyzed for a great part of them at the ripening step. The aim of this work was to assess the metabolic activity of these bacteria throughout manufacture and ripening. During Emmental cheesemaking, LH and ST were subjected to i) population quantification by numerations and by quantitative PCR (qPCR) ii) reverse transcription (RT) Temporal Temperature Gel Electrophoresis (TTGE) iii) transcript quantification by RT-qPCR targeting 16S rRNA, tuf and groL mRNAs to evaluate bacterial metabolic activity. During ripening, ST and LH numerations showed a 2.5 log(10) loss of culturability whereas qPCR on pelleted cells revealed only one log(10) of decrease for both of these species. 10(9) ST and 10(8) LH cells/g of cheese still remained. They contained a stable number of 16S transcript and at least 10(6) copies of mRNAs per 10(9) cells until the end of ripening. These results prove the unexpected persistency of thermophilic lactic acid bacteria starters (ST and LH) metabolic activity until the end of ripening and open new perspectives in term of their involvement in the quality of cheeses during ripening.

  3. In vivo organization of the FtsZ-ring by ZapA and ZapB revealed by quantitative super-resolution microscopy.

    Science.gov (United States)

    Buss, Jackson; Coltharp, Carla; Huang, Tao; Pohlmeyer, Chris; Wang, Shih-Chin; Hatem, Christine; Xiao, Jie

    2013-09-01

    In most bacterial cells, cell division is dependent on the polymerization of the FtsZ protein to form a ring-like structure (Z-ring) at the midcell. Despite its essential role, the molecular architecture of the Z-ring remains elusive. In this work we examine the roles of two FtsZ-associated proteins, ZapA and ZapB, in the assembly dynamics and structure of the Z-ring in Escherichia coli cells. In cells deleted of zapA or zapB, we observed abnormal septa and highly dynamic FtsZ structures. While details of these FtsZ structures are difficult to discern under conventional fluorescence microscopy, single-molecule-based super-resolution imaging method Photoactivated Localization Microscopy (PALM) reveals that these FtsZ structures arise from disordered arrangements of FtsZ clusters. Quantitative analysis finds these clusters are larger and comprise more molecules than a single FtsZ protofilament, and likely represent a distinct polymeric species that is inherent to the assembly pathway of the Z-ring. Furthermore, we find these clusters are not due to the loss of ZapB-MatP interaction in ΔzapA and ΔzapB cells. Our results suggest that the main function of ZapA and ZapB in vivo may not be to promote the association of individual protofilaments but to align FtsZ clusters that consist of multiple FtsZ protofilaments. © 2013 John Wiley & Sons Ltd.

  4. Clusters of Insomnia Disorder: An Exploratory Cluster Analysis of Objective Sleep Parameters Reveals Differences in Neurocognitive Functioning, Quantitative EEG, and Heart Rate Variability.

    Science.gov (United States)

    Miller, Christopher B; Bartlett, Delwyn J; Mullins, Anna E; Dodds, Kirsty L; Gordon, Christopher J; Kyle, Simon D; Kim, Jong Won; D'Rozario, Angela L; Lee, Rico S C; Comas, Maria; Marshall, Nathaniel S; Yee, Brendon J; Espie, Colin A; Grunstein, Ronald R

    2016-11-01

    To empirically derive and evaluate potential clusters of Insomnia Disorder through cluster analysis from polysomnography (PSG). We hypothesized that clusters would differ on neurocognitive performance, sleep-onset measures of quantitative (q)-EEG and heart rate variability (HRV). Research volunteers with Insomnia Disorder (DSM-5) completed a neurocognitive assessment and overnight PSG measures of total sleep time (TST), wake time after sleep onset (WASO), and sleep onset latency (SOL) were used to determine clusters. From 96 volunteers with Insomnia Disorder, cluster analysis derived at least two clusters from objective sleep parameters: Insomnia with normal objective sleep duration (I-NSD: n = 53) and Insomnia with short sleep duration (I-SSD: n = 43). At sleep onset, differences in HRV between I-NSD and I-SSD clusters suggest attenuated parasympathetic activity in I-SSD (P clusters by retaining the I-NSD and splitting the I-SSD cluster into two: I-SSD A (n = 29): defined by high WASO and I-SSD B (n = 14): a second I-SSD cluster with high SOL and medium WASO. The I-SSD B cluster performed worse than I-SSD A and I-NSD for sustained attention (P ≤ 0.05). In an exploratory analysis, q-EEG revealed reduced spectral power also in I-SSD B before (Delta, Alpha, Beta-1) and after sleep-onset (Beta-2) compared to I-SSD A and I-NSD (P ≤ 0.05). Two insomnia clusters derived from cluster analysis differ in sleep onset HRV. Preliminary data suggest evidence for three clusters in insomnia with differences for sustained attention and sleep-onset q-EEG. Insomnia 100 sleep study: Australia New Zealand Clinical Trials Registry (ANZCTR) identification number 12612000049875. URL: https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=347742.

  5. Quantitative meta-analysis of fMRI and PET studies reveals consistent activation in fronto-striatal-parietal regions and cerebellum during antisaccades and prosaccades

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    Sharna eJamadar

    2013-10-01

    Full Text Available The antisaccade task is a classic task of oculomotor control that requires participants to inhibit a saccade to a target and instead make a voluntary saccade to the mirror opposite location. By comparison, the prosaccade task requires participants to make a visually-guided saccade to the target. These tasks have been studied extensively using behavioural oculomotor, electrophysiological and neuroimaging in both non-human primates and humans. In humans, the antisaccade task is under active investigation as a potential endophenotype or biomarker for multiple psychiatric and neurological disorders. A large and growing body of literature has used functional magnetic resonance imaging (fMRI and positron emission tomography (PET to study the neural correlates of the antisaccade and prosaccade tasks. We present a quantitative meta-analysis of all published voxel-wise fMRI and PET studies (18 of the antisaccade task and show that consistent activation for antisaccades and prosaccades is obtained in a fronto-subcortical-parietal network encompassing frontal and supplementary eye fields, thalamus, striatum and intraparietal cortex. This network is strongly linked to oculomotor control and was activated to a greater extent for antisaccade than prosaccade trials. Antisaccade but not prosaccade trials additionally activated dorsolateral and ventrolateral prefrontal cortices. We also found that a number of additional regions not classically linked to oculomotor control were activated to a greater extent for antisaccade versus prosaccade trials; these regions are often reported in antisaccade studies but rarely commented upon. While the number of studies eligible to be included in this meta-analysis was small, the results of this systematic review reveal that antisaccade and prosaccade trials consistently activate a distributed network of regions both within and outside the classic definition of the oculomotor network.

  6. Improving the Phosphoproteome Coverage for Limited Sample Amounts Using TiO2-SIMAC-HILIC (TiSH) Phosphopeptide Enrichment and Fractionation.

    Science.gov (United States)

    Engholm-Keller, Kasper; Larsen, Martin R

    2016-01-01

    Obtaining high phosphoproteome coverage requires specific enrichment of phosphorylated peptides from the often extremely complex peptide mixtures generated by proteolytic digestion of biological samples, as well as extensive chromatographic fractionation prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Due to the sample loss resulting from fractionation, this procedure is mainly performed when large quantities of sample are available. To make large-scale phosphoproteomics applicable to smaller amounts of protein we have recently combined highly specific TiO2-based phosphopeptide enrichment with sequential elution from immobilized metal affinity chromatography (SIMAC) for fractionation of mono- and multi-phosphorylated peptides prior to capillary scale hydrophilic interaction liquid chromatography (HILIC) based fractionation of monophosphorylated peptides. In the following protocol we describe the procedure step by step to allow for comprehensive coverage of the phosphoproteome utilizing only a few hundred micrograms of protein.

  7. An automated platform for analysis of phosphoproteomic datasets: application to kidney collecting duct phosphoproteins.

    Science.gov (United States)

    Hoffert, Jason D; Wang, Guanghui; Pisitkun, Trairak; Shen, Rong-Fong; Knepper, Mark A

    2007-09-01

    Large-scale phosphoproteomic analysis employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) often requires a significant amount of manual manipulation of phosphopeptide datasets in the post-acquisition phase. To assist in this process, we have created software, PhosphoPIC (PhosphoPeptide Identification and Compilation), which can perform a variety of useful functions including automated selection and compilation of phosphopeptide identifications from multiple MS levels, estimation of dataset false discovery rate, and application of appropriate cross-correlation (XCorr) filters. In addition, the output files generated by this program are compatible with downstream phosphorylation site assignment using the Ascore algorithm, as well as phosphopeptide quantification via QUOIL. In this report, we utilized this software to analyze phosphoproteins from short-term vasopressin-treated rat kidney inner medullary collecting duct (IMCD). A total of 925 phosphopeptides representing 173 unique proteins were identified from membrane-enriched fractions of IMCD with a false discovery rate of 1.5%. Of these proteins, 106 were found only in the membrane-enriched fraction of IMCD cells and not in whole IMCD cell lysates. These identifications included a number of well-studied ion and solute transporters including ClC-1, LAT4, MCT2, NBC3, and NHE1, all of which contained novel phosphorylation sites. Using a label-free quantification approach, we identified phosphoproteins that changed in abundance with vasopressin exposure including aquaporin-2 (AQP2), Hnrpa3, IP3 receptor 3, and pur-beta.

  8. Comparative Proteome and Phosphoproteome Analyses during Cyprid Development of the Barnacle Balanus ( =Amphibalanus ) amphitrite

    KAUST Repository

    Zhang, Yu

    2010-06-04

    The barnacle Balanus amphitrite (=Amphibalanus amphitrite) is a major marine biofouling invertebrate worldwide. It has a complex life cycle during which the larva (called a nauplius) molts six times before transforming into the cyprid stage. The cyprid stage in B. amphitrite is the critical stage for the larval decision to attach and metamorphose. In this study, proteome and phosphoproteome alterations during cyprid development/aging and upon treatment with the antifouling agent butenolide were examined with a two-dimensional electrophoresis (2-DE) multiplexed fluorescent staining approach. Optimized protein separation strategies, including solution-phase isoelectric fractionation and narrow-pH-range 2-DE, were used in a proteomic analysis. Our results show that the differential regulation of the target proteins is highly dynamic on the levels of both protein expression and posttranslational modification. Two groups of proteins, stress-associated and energy metabolism-related proteins, are differentially expressed during cyprid development. Comparison of the control and treatment groups suggests that butenolide exerts its effects by sustaining the expression levels of these proteins. Altogether, our data suggest that proteins involved in stress regulation and energy metabolism play crucial roles in regulating larval attachment and metamorphosis of B. amphitrite. © 2010 American Chemical Society.

  9. Site-Specific Ser/Thr/Tyr Phosphoproteome of Sinorhizobium meliloti at Stationary Phase.

    Science.gov (United States)

    Liu, Tao; Tian, Chang Fu; Chen, Wen Xin

    2015-01-01

    Sinorhizobium meliloti, a facultative microsymbiont of alfalfa, should fine-tune its cellular processes to live saprophytically in soils characterized with limited nutrients and diverse stresses. In this study, TiO2 enrichment and LC-MS/MS were used to uncover the site-specific Ser/Thr/Tyr phosphoproteome of S. meliloti in minimum medium at stationary phase. There are a total of 96 unique phosphorylated sites, with a Ser/Thr/Tyr distribution of 63:28:5, in 77 proteins. Phosphoproteins identified in S. meliloti showed a wide distribution pattern regarding to functional categories, such as replication, transcription, translation, posttranslational modification, transport and metabolism of amino acids, carbohydrate, inorganic ion, succinoglycan etc. Ser/Thr/Tyr phosphosites identified within the conserved motif in proteins of key cellular function indicate a crucial role of phosphorylation in modulating cellular physiology. Moreover, phosphorylation in proteins involved in processes related to rhizobial adaptation was also discussed, such as those identified in SMa0114 and PhaP2 (polyhydroxybutyrate synthesis), ActR (pH stress and microaerobic adaption), SupA (potassium stress), chaperonin GroEL2 (viability and potentially symbiosis), and ExoP (succinoglycan synthesis and secretion). These Ser/Thr/Tyr phosphosites identified herein would be helpful for our further investigation and understanding of the role of phosphorylation in rhizobial physiology.

  10. Site-Specific Ser/Thr/Tyr Phosphoproteome of Sinorhizobium meliloti at Stationary Phase.

    Directory of Open Access Journals (Sweden)

    Tao Liu

    Full Text Available Sinorhizobium meliloti, a facultative microsymbiont of alfalfa, should fine-tune its cellular processes to live saprophytically in soils characterized with limited nutrients and diverse stresses. In this study, TiO2 enrichment and LC-MS/MS were used to uncover the site-specific Ser/Thr/Tyr phosphoproteome of S. meliloti in minimum medium at stationary phase. There are a total of 96 unique phosphorylated sites, with a Ser/Thr/Tyr distribution of 63:28:5, in 77 proteins. Phosphoproteins identified in S. meliloti showed a wide distribution pattern regarding to functional categories, such as replication, transcription, translation, posttranslational modification, transport and metabolism of amino acids, carbohydrate, inorganic ion, succinoglycan etc. Ser/Thr/Tyr phosphosites identified within the conserved motif in proteins of key cellular function indicate a crucial role of phosphorylation in modulating cellular physiology. Moreover, phosphorylation in proteins involved in processes related to rhizobial adaptation was also discussed, such as those identified in SMa0114 and PhaP2 (polyhydroxybutyrate synthesis, ActR (pH stress and microaerobic adaption, SupA (potassium stress, chaperonin GroEL2 (viability and potentially symbiosis, and ExoP (succinoglycan synthesis and secretion. These Ser/Thr/Tyr phosphosites identified herein would be helpful for our further investigation and understanding of the role of phosphorylation in rhizobial physiology.

  11. The proteome and phosphoproteome of Neurospora crassa in response to cellulose, sucrose and carbon starvation.

    Science.gov (United States)

    Xiong, Yi; Coradetti, Samuel T; Li, Xin; Gritsenko, Marina A; Clauss, Therese; Petyuk, Vlad; Camp, David; Smith, Richard; Cate, Jamie H D; Yang, Feng; Glass, N Louise

    2014-11-01

    Improving cellulolytic enzyme production by plant biomass degrading fungi holds great potential in reducing costs associated with production of next-generation biofuels generated from lignocellulose. How fungi sense cellulosic materials and respond by secreting enzymes has mainly been examined by assessing function of transcriptional regulators and via transcriptional profiling. Here, we obtained global proteomic and phosphoproteomic profiles of the plant biomass degrading filamentous fungus Neurospora crassa grown on different carbon sources, i.e. sucrose, no carbon, and cellulose, by performing isobaric tags for relative and absolute quantification (iTRAQ)-based LC-MS/MS analyses. A comparison between proteomes and transcriptomes under identical carbon conditions suggests that extensive post-transcriptional regulation occurs in N. crassa in response to exposure to cellulosic material. Several hundred amino acid residues with differential phosphorylation levels on crystalline cellulose (Avicel) or carbon-free medium vs sucrose medium were identified, including phosphorylation sites in a major transcriptional activator for cellulase genes, CLR1, as well as a cellobionic acid transporter, CBT1. Mutation of phosphorylation sites on CLR1 did not have a major effect on transactivation of cellulase production, while mutation of phosphorylation sites in CBT1 increased its transporting capacity. Our data provides rich information at both the protein and phosphorylation levels of the early cellular responses to carbon starvation and cellulosic induction and aids in a greater understanding of the underlying post-transcriptional regulatory mechanisms in filamentous fungi.

  12. Newly fabricated magnetic lanthanide oxides core-shell nanoparticles in phosphoproteomics.

    Science.gov (United States)

    Jabeen, Fahmida; Najam-Ul-Haq, Muhammad; Rainer, Matthias; Güzel, Yüksel; Huck, Christian W; Bonn, Guenther K

    2015-01-01

    Metal oxides show high selectivity and sensitivity toward mass spectrometry based enrichment strategies. Phosphopeptides/phosphoproteins enrichment from biological samples is cumbersome because of their low abundance. Phosphopeptides are of interest in enzymes and phosphorylation pathways which lead to the clinical links of a disease. Magnetic core-shell lanthanide oxide nanoparticles (Fe3O4@SiO2-La2O3 and Fe3O4@SiO2-Sm2O3) are fabricated, characterized by SEM, FTIR, and EDX and employed in the enrichment of phosphopeptides. The nanoparticles enrich phosphopeptides from casein variants, nonfat milk, egg yolk, human serum and HeLa cell extract. The materials and enrichment protocols are designed in a way that there are almost no nonspecific bindings. The selectivity is achieved up to 1:8500 using β-casein/BSA mixture and sensitivity down to 1 atto-mole. Batch-to-batch reproducibility is high with the reuse of core-shell nanoparticles up to four cycles. The enrichment followed by MALDI-MS analyses is carried out for the identification of phosphopeptides from serum digest and HeLa cell extract. Characteristic phosphopeptides of phosphoproteins are identified from human serum after the enrichment, which have the diagnostic potential toward prostate cancer. Thus, the lanthanide based magnetic core-shell materials offer a highly selective and sensitive workflow in phosphoproteomics.

  13. Deoxygenation affects tyrosine phosphoproteome of red cell membrane from patients with sickle cell disease.

    Science.gov (United States)

    Siciliano, Angela; Turrini, Franco; Bertoldi, Mariarita; Matte, Alessandro; Pantaleo, Antonella; Olivieri, Oliviero; De Franceschi, Lucia

    2010-04-15

    Sickle cell disease (SCD) is a worldwide distributed hereditary red cell disorder related to the production of a defective form of hemoglobin, hemoglobin S (HbS). One of the hallmarks of SCD is the presence of dense, dehydrate highly adhesive sickle red blood cells (RBCs) that result from persistent membrane damage associated with HbS polymerization, abnormal activation of membrane cation transports and generation of distorted and rigid red cells with membrane perturbation and cytoskeleton dysfunction. Although modulation of phosphorylation state of the proteins from membrane and cytoskeleton networks has been proposed to participate in red cell homeostasis, much still remains to be investigated in normal and diseased red cells. Here, we report that tyrosine (Tyr-) phosphoproteome of sickle red cells was different from normal controls and was affected by deoxygenation. We found proteins, p55 and band 4.1, from the junctional complex, differently Tyr-phosphorylated in SCD RBCs compared to normal RBCs under normoxia and modulated by deoxygenation, while band 4.2 was similarly Tyr-phosphorylated in both conditions. In SCD RBCs we identified the phosphopeptides for protein 4.1R located in the protein FERM domain (Tyr-13) and for alpha-spectrin located near or in a linker region (Tyr-422 and Tyr-1498) involving protein areas crucial for their functions in the context of red cell membrane properties, suggesting that Tyr-phosphorylation may be part of the events involved in maintaining membrane mechanical stability in SCD red cells.

  14. Identification of BCAP-{sub L} as a negative regulator of the TLR signaling-induced production of IL-6 and IL-10 in macrophages by tyrosine phosphoproteomics

    Energy Technology Data Exchange (ETDEWEB)

    Matsumura, Takayuki [Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, Shinjuku-ku, Tokyo 162-0041 (Japan); Department of Life Science and Medical Bio-Science, Waseda University, Shinjuku-ku, Tokyo 162-8480 (Japan); Oyama, Masaaki; Kozuka-Hata, Hiroko [Medical Proteomics Laboratory, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639 (Japan); Ishikawa, Kosuke; Inoue, Takafumi [Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, Shinjuku-ku, Tokyo 162-0041 (Japan); Department of Life Science and Medical Bio-Science, Waseda University, Shinjuku-ku, Tokyo 162-8480 (Japan); Muta, Tatsushi [Laboratory of Cell Recognition and Response, Graduate School of Life Sciences, Tohoku University, Sendai 980-8578 (Japan); Semba, Kentaro, E-mail: ksemba@waseda.jp [Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, Shinjuku-ku, Tokyo 162-0041 (Japan); Department of Life Science and Medical Bio-Science, Waseda University, Shinjuku-ku, Tokyo 162-8480 (Japan); Inoue, Jun-ichiro, E-mail: jun-i@ims.u-tokyo.ac.jp [Medical Proteomics Laboratory, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639 (Japan); Division of Cellular and Molecular Biology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639 (Japan)

    2010-09-17

    Research highlights: {yields} Twenty five tyrosine-phosphorylated proteins in LPS-stimulated macrophages were determined. {yields} BCAP is a novel tyrosine-phosphorylated protein in LPS-stimulated macrophages. {yields} BCAP-{sub L} inhibits IL-6 and IL-10 production in LPS-stimulated macrophages. -- Abstract: Toll-like receptor (TLR) signaling in macrophages is essential for anti-pathogen responses such as cytokine production and antigen presentation. Although numerous reports suggest that protein tyrosine kinases (PTKs) are involved in cytokine induction in response to lipopolysaccharides (LPS; TLR4 ligand) in macrophages, the PTK-mediated signal transduction pathway has yet to be analyzed in detail. Here, we carried out a comprehensive and quantitative dynamic tyrosine phosphoproteomic analysis on the TLR4-mediated host defense system in RAW264.7 macrophages using stable isotope labeling by amino acids in cell culture (SILAC). We determined the temporal profiles of 25 proteins based on SILAC-encoded peptide(s). Of these, we focused on the tyrosine phosphorylation of B-cell adaptor for phosphatidylinositol 3-kinase (BCAP) because the function of BCAP remains unknown in TLR signaling in macrophages. Furthermore, Bcap has two distinct transcripts, a full-length (Bcap-{sub L}) and an alternatively initiated or spliced (Bcap-{sub S}) mRNA, and little is known about the differential functions of the BCAP-{sub L} and BCAP-{sub S} proteins. Our study showed, for the first time, that RNAi-mediated selective depletion of BCAP-{sub L} enhanced IL-6 and IL-10 production but not TNF-{alpha} production in TLR ligand-stimulated macrophages. We propose that BCAP-{sub L} (but not BCAP-{sub S}) is a negative regulator of the TLR-mediated host defense system in macrophages.

  15. Male fertility versus sterility, cytotype, and DNA quantitative variation in seed production in diploid and tetraploid sea lavenders (Limonium sp., Plumbaginaceae) reveal diversity in reproduction modes.

    Science.gov (United States)

    Róis, Ana Sofia; Teixeira, Generosa; Sharbel, Timothy F; Fuchs, Jörg; Martins, Sérgio; Espírito-Santo, Dalila; Caperta, Ana D

    2012-12-01

    The genus Limonium Miller, a complex taxonomic group, comprises annuals and perennials that can produce sexual and/or asexual seeds (apomixis). In this study, we used diverse cytogenetic and cytometric approaches to analyze male sporogenesis and gametogenesis for characterizing male reproductive output on seed production in Limonium ovalifolium and Limonium multiflorum. We showed here that the first species is mostly composed of diploid cytotypes with 2n = 16 chromosomes and the latter species by tetraploid cytotypes with 2n = 32, 34, 35, 36 chromosomes and had a genome roughly twice as big as the former one. In both species, euploid and aneuploid cytotypes with large metacentric chromosomes having decondensed interstitial sites were found within and among populations, possibly involved in chromosomal reconstructions. L. ovalifolium diploids showed regular meiosis resulting in normal tetrads, while diverse chromosome pairing and segregation irregularities leading to the formation of abnormal meiotic products are found in balanced and non-balanced L. multiflorum tetraploids. Before anther dehiscence, the characteristic unicellular, bicellular, or tricellular pollen grains showing the typical Limonium micro- or macro-reticulate exine ornamentation patterns were observed in L. ovalifolium using scanning electron microscopy. Most of these grains were viable and able to produce pollen tubes in vitro. In both balanced and unbalanced L. multiflorum tetraploids, microspores only developed until the "ring-vacuolate stage" with a collapsed morphology without the typical exine patterns, pointing to a sporophytic defect. These microspores were unviable and therefore never germinated in vitro. L. ovalifolium individuals presented larger pollen grains than those of L. multiflorum, indicating that pollen size and ploidy levels are not correlated in the Limonium system. Cytohistological studies in mature seeds from both species revealed that an embryo and a residual endosperm

  16. In vivo quantitative phosphoproteomic profiling identifies novel regulators of castration-resistant prostate cancer growth

    DEFF Research Database (Denmark)

    Jiang, Nan; Hjorth-Jensen, Kim; Hekmat, Omid

    2015-01-01

    pathways in castration-resistant tumors, a notion that was confirmed by tumor transcriptome analysis. Further analysis demonstrated that the activation of mTORC1, PAK2 and the increased levels of YAP1 in castration-resistant tumors can be explained by the loss of androgen inhibitory actions. The analysis...

  17. Identification of novel PAMP-triggered phosphorylation and dephosphorylation events in arabidopsis thaliana by quantitative phosphoproteomic analysis

    KAUST Repository

    Rayapuram, Naganand

    2014-04-04

    Signaling cascades rely strongly on protein kinase-mediated substrate phosphorylation. Currently a major challenge in signal transduction research is to obtain high confidence substrate phosphorylation sites and assign them to specific kinases. In response to bacterial flagellin, a pathogen-associated molecular pattern (PAMP), we searched for rapidly phosphorylated proteins in Arabidopsis thaliana by combining multistage activation (MSA) and electron transfer dissociation (ETD) fragmentation modes, which generate complementary spectra and identify phosphopeptide sites with increased reliability. Of a total of 825 phosphopeptides, we identified 58 to be differentially phosphorylated. These peptides harbor kinase motifs of mitogen-activated protein kinases (MAPKs) and calcium-dependent protein kinases (CDPKs), as well as yet unknown protein kinases. Importantly, 12 of the phosphopeptides show reduced phosphorylation upon flagellin treatment. Since protein abundance levels did not change, these results indicate that flagellin induces not only various protein kinases but also protein phosphatases, even though a scenario of inhibited kinase activity may also be possible. © 2014 American Chemical Society.

  18. Quantitative Detection of Chlamydia psittaci and C. pecorum by High-Sensitivity Real-Time PCR Reveals High Prevalence of Vaginal Infection in Cattle

    OpenAIRE

    DeGraves, Fred J.; Gao, Dongya; Hehnen, Hans-Robert; Schlapp, Tobias; Kaltenboeck, Bernhard

    2003-01-01

    Bovine vaginal cytobrush specimens were analyzed for the presence of Chlamydia spp. by a high-sensitivity, high-specificity quantitative PCR. The 53% prevalence of low-level Chlamydia psittaci and C. pecorum genital infection detected in virgin heifers suggests predominantely extragenital transmission of Chlamydia in cattle and conforms to the high seroprevalence of anti-Chlamydia antibodies.

  19. Simple preparation of magnetic metal-organic frameworks composite as a "bait" for phosphoproteome research.

    Science.gov (United States)

    Han, Guobin; Zeng, Qiaoling; Jiang, Zhongwei; Deng, Wenchan; Huang, Chengzhi; Li, Yuanfang

    2017-08-15

    Phosphospecific enrichment techniques and mass spectrometry (MS) are primary tools for comprehending the cellular phosphoproteome. In this work, a rational and extremely facile route to synthesize the magnetic metal-organic frameworks (mMOFs) was employed and the prepared composite was first utilized as a "bait" for selective enrichment of phosphopeptides. Typically, the mMOFs was synthesized via electrostatic self-assembly between the negatively charged Fe3O4 magnetic nanoparticles (MNPs) and positively charged MIL-101(Fe). The obtained Fe3O4/MIL-101(Fe) composite possessed well-defined structures, rough surface, highly specific surface area and excellent magnetic property. To demonstrate their ability for enrichment of phosphopeptides, we applied Fe3O4/MIL-101(Fe) as a "bait" to capture the phosphopeptides from standard protein digestion and practical samples. The enriched phosphopeptides were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The MS results show that the Fe3O4/MIL-101(Fe) exhibits superior enrichment performance for phosphopeptides with low detectable concentration assessed to be 8 fmol, selectivity investigated to be 1:1000 using β-casein/bovine serum albumin mixture and enrichment recovery evaluated to be 89.8%. Based on these excellent properties, the prepared composite was used to enrich the phosphopeptides from tilapia eggs biological samples for the first time. A total number of 51 phosphorylation sites were identified from the digest of tilapia eggs proteins, suggesting the excellent potential of Fe3O4/MIL-101(Fe) composite in the practical application. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. The design and synthesis of a hydrophilic core-shell-shell structured magnetic metal-organic framework as a novel immobilized metal ion affinity platform for phosphoproteome research.

    Science.gov (United States)

    Zhao, Man; Deng, Chunhui; Zhang, Xiangmin

    2014-06-14

    In this work, polydopamine (PDA)-coated magnetic microspheres with surface modification of zirconium-based MOFs were synthesized for the first time. The as-synthesized Fe3O4@PDA@Zr-MOF composites were successfully applied as a novel immobilized metal ion affinity platform for phosphoproteome research.

  1. Proteomic and phosphoproteomic comparison of human ES and iPS cells.

    Science.gov (United States)

    Phanstiel, Douglas H; Brumbaugh, Justin; Wenger, Craig D; Tian, Shulan; Probasco, Mitchell D; Bailey, Derek J; Swaney, Danielle L; Tervo, Mark A; Bolin, Jennifer M; Ruotti, Victor; Stewart, Ron; Thomson, James A; Coon, Joshua J

    2011-01-01

    Combining high-mass-accuracy mass spectrometry, isobaric tagging and software for multiplexed, large-scale protein quantification, we report deep proteomic coverage of four human embryonic stem cell and four induced pluripotent stem cell lines in biological triplicate. This 24-sample comparison resulted in a very large set of identified proteins and phosphorylation sites in pluripotent cells. The statistical analysis afforded by our approach revealed subtle but reproducible differences in protein expression and protein phosphorylation between embryonic stem cells and induced pluripotent cells. Merging these results with RNA-seq analysis data, we found functionally related differences across each tier of regulation. We also introduce the Stem Cell-Omics Repository (SCOR), a resource to collate and display quantitative information across multiple planes of measurement, including mRNA, protein and post-translational modifications.

  2. Phosphoproteomics of Klebsiella pneumoniae NTUH-K2044 Reveals a Tight Link between Tyrosine Phosphorylation and Virulence

    National Research Council Canada - National Science Library

    Miao-Hsia Lin; Tung-Li Hsu; Shu-Yu Lin; Yi-Jiun Pan; Jia-Tsrong Jan; Jin-Town Wang; Kay-Hooi Khoo; Shih-Hsiung Wu

    2009-01-01

    .... The capsular polysaccharide on K. pneumoniae surface was determined as the key to virulence. Although the regulation of capsular polysaccharide biosynthesis is largely unclear, it was found that protein-tyrosine kinases and phosphatases are involved...

  3. Nuclear phosphoproteome analysis of 3T3-L1 preadipocyte differentiation reveals system-wide phosphorylation of transcriptional regulators

    DEFF Research Database (Denmark)

    Rabiee, Atefeh; Schwämmle, Veit; Sidoli, Simone

    2017-01-01

    are associated with adverse metabolic function. Adipogenesis is the process whereby preadipocyte precursor cells differentiate into lipid laden mature adipocytes. This process is driven by a network of transcriptional regulators (TRs). We hypothesized that protein post-translational modifications (PTMs...... of which were assigned as regulators of gene expression. Among 288 identified transcriptional regulators, 49 were regulated within four hours of adipogenic stimulation including several known and many novel potential adipogenic regulators. A kinase-substrate database for 3T3-L1 preadipocytes established....... New insights into phosphorylation-dependent signaling networks that impact on nuclear proteins and controls adipocyte differentiation and cell fate. Adipocytes (fat cells) are important endocrine and metabolic cells critical for systemic insulin sensitivity. Both adipose excess and insufficiency...

  4. Proteomic and Phosphoproteomic Insights into a Signaling Hub Role for Cdc14 in Asexual Development and Multiple Stress Responses in Beauveria bassiana.

    Science.gov (United States)

    Wang, Zhi-Kang; Wang, Jie; Liu, Jing; Ying, Sheng-Hua; Peng, Xiao-Jun; Feng, Ming-Guang

    2016-01-01

    Cdc14 is a dual-specificity phosphatase that regulates nuclear behavior by dephosphorylating phosphotyrosine and phosphoserine/phosphothreonine in fungi. Previously, Cdc14 was shown to act as a positive regulator of cytokinesis, asexual development and multiple stress responses in Beauveria bassiana, a fungal insect pathogen. This study seeks to gain deep insight into a pivotal role of Cdc14 in the signaling network of B. bassiana by analyzing the Cdc14-specific proteome and phosphoproteome generated by the 8-plex iTRAQ labeling and MS/MS analysis of peptides and phosphopeptides. Under normal conditions, 154 proteins and 86 phosphorylation sites in 67 phosphoproteins were upregulated in Δcdc14 versus wild-type, whereas 117 proteins and 85 phosphorylation sites in 58 phosphoproteins were significantly downregulated. Co-cultivation of Δcdc14 with NaCl (1 M), H2O2 (3 mM) and Congo red (0.15 mg/ml) resulted in the upregulation / downregulation of 23/63, 41/39 and 79/79 proteins and of 127/112, 52/47 and 105/226 phosphorylation sites in 85/92, 45/36 and 79/146 phosphoproteins, respectively. Bioinformatic analyses revealed that Cdc14 could participate in many biological and cellular processes, such as carbohydrate metabolism, glycerophospholipid metabolism, the MAP Kinase signaling pathway, and DNA conformation, by regulating protein expression and key kinase phosphorylation in response to different environmental cues. These indicate that in B. bassiana, Cdc14 is a vital regulator of not only protein expression but also many phosphorylation events involved in developmental and stress-responsive pathways. Fourteen conserved and novel motifs were identified in the fungal phosphorylation events.

  5. Up-to-Date Workflow for Plant (Phospho)proteomics Identifies Differential Drought-Responsive Phosphorylation Events in Maize Leaves.

    Science.gov (United States)

    Vu, Lam Dai; Stes, Elisabeth; Van Bel, Michiel; Nelissen, Hilde; Maddelein, Davy; Inzé, Dirk; Coppens, Frederik; Martens, Lennart; Gevaert, Kris; De Smet, Ive

    2016-12-02

    Protein phosphorylation is one of the most common post-translational modifications (PTMs), which can regulate protein activity and localization as well as protein-protein interactions in numerous cellular processes. Phosphopeptide enrichment techniques enable plant researchers to acquire insight into phosphorylation-controlled signaling networks in various plant species. Most phosphoproteome analyses of plant samples still involve stable isotope labeling, peptide fractionation, and demand a lot of mass spectrometry (MS) time. Here, we present a simple workflow to probe, map, and catalogue plant phosphoproteomes, requiring relatively low amounts of starting material, no labeling, no fractionation, and no excessive analysis time. Following optimization of the different experimental steps on Arabidopsis thaliana samples, we transferred our workflow to maize, a major monocot crop, to study signaling upon drought stress. In addition, we included normalization to protein abundance to identify true phosphorylation changes. Overall, we identified a set of new phosphosites in both Arabidopsis thaliana and maize, some of which are differentially phosphorylated upon drought. All data are available via ProteomeXchange with identifier PXD003634, but to provide easy access to our model plant and crop data sets, we created an online database, Plant PTM Viewer ( bioinformatics.psb.ugent.be/webtools/ptm_viewer/ ), where all phosphosites identified in our study can be consulted.

  6. Quantitative Assessment of Chromatin Immunoprecipitation Grade Antibodies Directed against Histone Modifications Reveals Patterns of Co-occurring Marks on Histone Protein Molecules*

    OpenAIRE

    Peach, Sally E.; Rudomin, Emily L.; Udeshi, Namrata D.; Carr, Steven A.; Jaffe, Jacob D.

    2012-01-01

    The defining step in most chromatin immunoprecipitation (ChIP) assays is the use of an antibody to enrich for a particular protein or histone modification state associated with segments of chromatin. The specificity of the antibody is critical to the interpretation of the experiment, yet this property is rarely reported. Here, we present a quantitative method using mass spectrometry to characterize the specificity of key histone H3 modification-targeting antibodies that have previously been u...

  7. Quantitative weaknesses of the Marcus-Hush theory of electrode kinetics revealed by Reverse Scan Square Wave Voltammetry: The reduction of 2-methyl-2-nitropropane at mercury microelectrodes

    Science.gov (United States)

    Laborda, Eduardo; Wang, Yijun; Henstridge, Martin C.; Martínez-Ortiz, Francisco; Molina, Angela; Compton, Richard G.

    2011-08-01

    The Marcus-Hush and Butler-Volmer kinetic electrode models are compared experimentally by studying the reduction of 2-methyl-2-nitropropane in acetonitrile at mercury microelectrodes using Reverse Scan Square Wave Voltammetry. This technique is found to be very sensitive to the electrode kinetics and to permit critical comparison of the two models. The Butler-Volmer model satisfactorily fits the experimental data whereas Marcus-Hush does not quantitatively describe this redox system.

  8. Dominant Microbial Composition and Its Vertical Distribution in Saline Meromictic Lake Kaiike (Japan) as Revealed by Quantitative Oligonucleotide Probe Membrane Hybridization

    OpenAIRE

    Koizumi, Yoshikazu; Kojima, Hisaya; Fukui, Manabu

    2004-01-01

    Vertical distributions of dominant bacterial populations in saline meromictic Lake Kaiike were investigated throughout the water column and sediment by quantitative oligonucleotide probe membrane hybridization. Three oligonucleotide probes specific for the small-subunit (SSU) rRNA of three groups of Chlorobiaceae were newly designed. In addition, three general domain (Bacteria, Archaea, and Eukarya)-specific probes, two δ-Proteobacteria-specific probes, a Chlorobiaceae-specific probe, and a C...

  9. Population average T2 MRI maps reveal quantitative regional transformations in the degenerating rabbit intervertebral disc that vary by lumbar level.

    Science.gov (United States)

    Martin, John T; Collins, Christopher M; Ikuta, Kensuke; Mauck, Robert L; Elliott, Dawn M; Zhang, Yeija; Anderson, D Greg; Vaccaro, Alexander R; Albert, Todd J; Arlet, Vincent; Smith, Harvey E

    2015-01-01

    Magnetic resonance imaging (MRI) with T2-weighting is routinely performed to assess intervertebral disc degeneration. Standard clinical evaluations of MR images are qualitative, however, and do not focus on region-specific alterations in the disc. Utilizing a rabbit needle puncture model, T2 mapping was performed on injured discs to develop a quantitative description of the degenerative process following puncture. To do so, an 18G needle was inserted into four discs per rabbit (L3/L4 to L6/L7) and T2 maps were generated pre- and 4 weeks post-injury. Individual T2 maps were normalized to a disc-specific coordinate system and then averaged for pre- and post-injury population composite T2 maps. We also developed a method to automatically segment the nucleus pulposus by fitting the NP region of the T2 maps with modified 2-D and 3-D Gaussian distribution functions. Puncture injury produced alterations in MR signal intensity in a region-specific manner mirroring human degeneration. Population average T2 maps provided a quantitative representation of the injury response, and identified deviations of individual degenerate discs from the pre-injury population. We found that the response to standardized injury was modest at lower lumbar levels, likely as a result of increased disc dimensions. These tools will be valuable for the quantitative characterization of disc degeneration in future clinical and pre-clinical studies. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  10. Bigger Is Fitter? Quantitative Genetic Decomposition of Selection Reveals an Adaptive Evolutionary Decline of Body Mass in a Wild Rodent Population.

    Science.gov (United States)

    Bonnet, Timothée; Wandeler, Peter; Camenisch, Glauco; Postma, Erik

    2017-01-01

    In natural populations, quantitative trait dynamics often do not appear to follow evolutionary predictions. Despite abundant examples of natural selection acting on heritable traits, conclusive evidence for contemporary adaptive evolution remains rare for wild vertebrate populations, and phenotypic stasis seems to be the norm. This so-called "stasis paradox" highlights our inability to predict evolutionary change, which is especially concerning within the context of rapid anthropogenic environmental change. While the causes underlying the stasis paradox are hotly debated, comprehensive attempts aiming at a resolution are lacking. Here, we apply a quantitative genetic framework to individual-based long-term data for a wild rodent population and show that despite a positive association between body mass and fitness, there has been a genetic change towards lower body mass. The latter represents an adaptive response to viability selection favouring juveniles growing up to become relatively small adults, i.e., with a low potential adult mass, which presumably complete their development earlier. This selection is particularly strong towards the end of the snow-free season, and it has intensified in recent years, coinciding which a change in snowfall patterns. Importantly, neither the negative evolutionary change, nor the selective pressures that drive it, are apparent on the phenotypic level, where they are masked by phenotypic plasticity and a non causal (i.e., non genetic) positive association between body mass and fitness, respectively. Estimating selection at the genetic level enabled us to uncover adaptive evolution in action and to identify the corresponding phenotypic selective pressure. We thereby demonstrate that natural populations can show a rapid and adaptive evolutionary response to a novel selective pressure, and that explicitly (quantitative) genetic models are able to provide us with an understanding of the causes and consequences of selection that is

  11. TRI12 based quantitative real-time PCR assays reveal the distribution of trichothecene genotype of F. graminearum and F. culmorum isolates in Danish small grain cereals

    DEFF Research Database (Denmark)

    Nielsen, L. K.; Jensen, J. D.; Rodríguez, A.

    2012-01-01

    Quantitative real-time PCR assays, based on polymorphisms in the TRI12 gene of the trichothecene pathway, were developed to identify and quantify the trichothecene genotypes producing 3-acetyl-deoxynivalenol (3ADON), 15-acetyl-deoxynivalenol (15ADON) or nivalenol (NIV) in the Fusarium graminearum...... in the sample representing the years from 1997 to 2000. Detection of low amounts of the 15ADON genotype in these historical samples and the relatively high amounts of 15ADON genotype in 2003 and following years correspond well with the occurrence of F. graminearum and indicates that the 15ADON genotype...

  12. Quantitative proteome profiling of dystrophic dog skeletal muscle reveals a stabilized muscular architecture and protection against oxidative stress after systemic delivery of MuStem cells.

    Science.gov (United States)

    Lardenois, Aurélie; Jagot, Sabrina; Lagarrigue, Mélanie; Guével, Blandine; Ledevin, Mireille; Larcher, Thibaut; Dubreil, Laurence; Pineau, Charles; Rouger, Karl; Guével, Laëtitia

    2016-07-01

    Proteomic profiling plays a decisive role in the elucidation of molecular signatures representative of a specific clinical context. MuStem cell based therapy represents a promising approach for clinical applications to cure Duchenne muscular dystrophy (DMD). To expand our previous studies collected in the clinically relevant DMD animal model, we decided to investigate the skeletal muscle proteome 4 months after systemic delivery of allogenic MuStem cells. Quantitative proteomics with isotope-coded protein labeling was used to compile quantitative changes in the protein expression profiles of muscle in transplanted Golden Retriever muscular dystrophy (GRMD) dogs as compared to Golden Retriever muscular dystrophy dogs. A total of 492 proteins were quantified, including 25 that were overrepresented and 46 that were underrepresented after MuStem cell transplantation. Interestingly, this study demonstrates that somatic stem cell therapy impacts on the structural integrity of the muscle fascicle by acting on fibers and its connections with the extracellular matrix. We also show that cell infusion promotes protective mechanisms against oxidative stress and favors the initial phase of muscle repair. This study allows us to identify putative candidates for tissue markers that might be of great value in objectively exploring the clinical benefits resulting from our cell-based therapy for DMD. All MS data have been deposited in the ProteomeXchange with identifier PXD001768 (http://proteomecentral.proteomexchange.org/dataset/PXD001768).

  13. Quantitative Metaproteomics and Activity-Based Probe Enrichment Reveals Significant Alterations in Protein Expression from a Mouse Model of Inflammatory Bowel Disease.

    Science.gov (United States)

    Mayers, Michael D; Moon, Clara; Stupp, Gregory S; Su, Andrew I; Wolan, Dennis W

    2017-02-03

    Tandem mass spectrometry based shotgun proteomics of distal gut microbiomes is exceedingly difficult due to the inherent complexity and taxonomic diversity of the samples. We introduce two new methodologies to improve metaproteomic studies of microbiome samples. These methods include the stable isotope labeling in mammals to permit protein quantitation across two mouse cohorts as well as the application of activity-based probes to enrich and analyze both host and microbial proteins with specific functionalities. We used these technologies to study the microbiota from the adoptive T cell transfer mouse model of inflammatory bowel disease (IBD) and compare these samples to an isogenic control, thereby limiting genetic and environmental variables that influence microbiome composition. The data generated highlight quantitative alterations in both host and microbial proteins due to intestinal inflammation and corroborates the observed phylogenetic changes in bacteria that accompany IBD in humans and mouse models. The combination of isotope labeling with shotgun proteomics resulted in the total identification of 4434 protein clusters expressed in the microbial proteomic environment, 276 of which demonstrated differential abundance between control and IBD mice. Notably, application of a novel cysteine-reactive probe uncovered several microbial proteases and hydrolases overrepresented in the IBD mice. Implementation of these methods demonstrated that substantial insights into the identity and dysregulation of host and microbial proteins altered in IBD can be accomplished and can be used in the interrogation of other microbiome-related diseases.

  14. Quantitative measurements of alternating finger tapping in Parkinson's disease correlate with UPDRS motor disability and reveal the improvement in fine motor control from medication and deep brain stimulation.

    Science.gov (United States)

    Taylor Tavares, Ana Lisa; Jefferis, Gregory S X E; Koop, Mandy; Hill, Bruce C; Hastie, Trevor; Heit, Gary; Bronte-Stewart, Helen M

    2005-10-01

    The Unified Parkinson's Disease Rating Scale (UPDRS) is the primary outcome measure in most clinical trials of Parkinson's disease (PD) therapeutics. Each subscore of the motor section (UPDRS III) compresses a wide range of motor performance into a coarse-grained scale from 0 to 4; the assessment of performance can also be subjective. Quantitative digitography (QDG) is an objective, quantitative assessment of digital motor control using a computer-interfaced musical keyboard. In this study, we show that the kinematics of a repetitive alternating finger-tapping (RAFT) task using QDG correlate with the UPDRS motor score, particularly with the bradykinesia subscore, in 33 patients with PD. We show that dopaminergic medication and an average of 9.5 months of bilateral subthalamic nucleus deep brain stimulation (B-STN DBS) significantly improve UPDRS and QDG scores but may have different effects on certain kinematic parameters. This study substantiates the use of QDG to measure motor outcome in trials of PD therapeutics and shows that medication and B-STN DBS both improve fine motor control.

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

    Science.gov (United States)

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

    2014-03-01

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

  16. Quantitative profiling of glucosinolates by LC-MS analysis reveals several cultivars of cabbage and kale as promising sources of sulforaphane.

    Science.gov (United States)

    Sasaki, Katsunori; Neyazaki, Makiko; Shindo, Kazutoshi; Ogawa, Toshiya; Momose, Masaki

    2012-08-15

    Sulforaphane is an isothiocyanate well known for its potential health benefits. With the aim of finding sulforaphane supply sources, its precursor, glucoraphanin, was widely searched for among Brassica oleracea varieties. Quantitative profiling of seven glucosinolates by LC-MS analysis was performed on 6 cultivars of broccoli, 32 of cabbage and 24 cultivars of kale. The glucoraphanin levels found in three cultivars of cabbage and six cultivars of kale were comparable with, or even higher than, the highest of broccoli (119.4 mg/100g FW). The most promising group belonged to the black kale, Cavolo nero. Use of a C30 column and an ammonium formate buffer in LC-MS and a micro plate solid phase extraction technique was highly effective.

  17. An anti-phospholipase A2 receptor quantitative immunoassay and epitope analysis in membranous nephropathy reveals different antigenic domains of the receptor.

    Science.gov (United States)

    Behnert, Astrid; Fritzler, Marvin J; Teng, Beina; Zhang, Meifeng; Bollig, Frank; Haller, Hermann; Skoberne, Andrej; Mahler, Michael; Schiffer, Mario

    2013-01-01

    The phospholipase A2 receptor (PLA2R) was recently discovered as a target autoantigen in patients with idiopathic membranous nephropathy (IMN). Published evidence suggests that the autoantibodies directed towards a conformation dependent epitope are currently effectively detected by a cell based assay (CBA) utilizing indirect immunofluorescence (IIF) on tissue culture cells transfected with the PLA2R cDNA. Limitations of such IIF-CBA assays include observer dependent subjective evaluation of semi-quantitative test results and the protocols are not amenable to high throughput diagnostic testing. We developed a quantitative, observer independent, high throughput capture immunoassay for detecting PLA2R autoantibodies on an addressable laser bead immunoassay (ALBIA) platform. Since reactive domains of PLA2R (i.e. epitopes) could be used to improve diagnostic tests by using small peptides in various high throughput diagnostic platforms, we identified PLA2R epitopes that bound autoantibodies of IMN patients. These studies confirmed that inter-molecular epitope spreading occurs in IMN but use of the cognate synthetic peptides in immunoassays was unable to conclusively distinguish between IMN patients and normal controls. However, combinations of these peptides were able to effectively absorb anti-PLA2R reactivity in IIF-CBA and an immunoassay that employed a lysate derived from HEK cells tranfected with and overexpressing PLA2R. While we provide evidence of intermolecular epitope spreading, our data indicates that in addition to conformational epitopes, human anti-PLA2R reactivity in a commercially available CBA and an addressable laser bead immunoassay is significantly absorbed by peptides representing epitopes of PLA2R.

  18. An anti-phospholipase A2 receptor quantitative immunoassay and epitope analysis in membranous nephropathy reveals different antigenic domains of the receptor.

    Directory of Open Access Journals (Sweden)

    Astrid Behnert

    Full Text Available The phospholipase A2 receptor (PLA2R was recently discovered as a target autoantigen in patients with idiopathic membranous nephropathy (IMN. Published evidence suggests that the autoantibodies directed towards a conformation dependent epitope are currently effectively detected by a cell based assay (CBA utilizing indirect immunofluorescence (IIF on tissue culture cells transfected with the PLA2R cDNA. Limitations of such IIF-CBA assays include observer dependent subjective evaluation of semi-quantitative test results and the protocols are not amenable to high throughput diagnostic testing. We developed a quantitative, observer independent, high throughput capture immunoassay for detecting PLA2R autoantibodies on an addressable laser bead immunoassay (ALBIA platform. Since reactive domains of PLA2R (i.e. epitopes could be used to improve diagnostic tests by using small peptides in various high throughput diagnostic platforms, we identified PLA2R epitopes that bound autoantibodies of IMN patients. These studies confirmed that inter-molecular epitope spreading occurs in IMN but use of the cognate synthetic peptides in immunoassays was unable to conclusively distinguish between IMN patients and normal controls. However, combinations of these peptides were able to effectively absorb anti-PLA2R reactivity in IIF-CBA and an immunoassay that employed a lysate derived from HEK cells tranfected with and overexpressing PLA2R. While we provide evidence of intermolecular epitope spreading, our data indicates that in addition to conformational epitopes, human anti-PLA2R reactivity in a commercially available CBA and an addressable laser bead immunoassay is significantly absorbed by peptides representing epitopes of PLA2R.

  19. Quantitative mass spectrometry of histones H3.2 and H3.3 in Suz12-deficient mouse embryonic stem cells reveals distinct, dynamic post-translational modifications at Lys-27 and Lys-36

    DEFF Research Database (Denmark)

    Jung, Hye Ryung; Pasini, Diego; Helin, Kristian

    2010-01-01

    SUZ12 is a core component of the polycomb repressive complex 2 (PRC2) and is required for the differentiation of mouse embryonic stem cells (ESCs). PRC2 is associated with transcriptional repression via methylation of H3 Lys-27. We applied quantitative mass spectrometry to investigate the effects....... The combined use of ETD and CID MS/MS increased the total number of identified modified peptides. Comparative quantitative analysis of histones from wild type and Suz12-deficient ESCs using stable isotope labeling with amino acids in cell culture and LC-MS/MS revealed a dramatic reduction of H3K27me2 and H3K27...... analysis of the dynamics of coexisting post-translational modifications in proteins....

  20. Data set from the phosphoproteomic analysis of Magnaporthe oryzae-responsive proteins in susceptible and resistant rice cultivars

    Directory of Open Access Journals (Sweden)

    Yunfeng Li

    2015-06-01

    Full Text Available Rice blast, caused by the fungal pathogen Magnaporthe oryzae, is the most destructive disease of rice and causes tremendous losses of rice yield worldwide. To explore the molecular mechanisms involved in the rice–M. oryzae interaction, we conducted a time-course phosphoproteomic analysis of leaf samples from resistant and susceptible rice cultivars infected with M. oryzae. This data article contains additional results and analysis of M. oryzae-regulated phosphoproteins in rice leaves [1]. We report the analysis of M. oryzae-regulated phosphoproteins at all time points, including Venn diagram analysis, close-up views, relative intensities, and functional category, and the MS spectra of representative phosphoprotein and representative phosphorylated peptides.

  1. Quantitative proteomics reveals significant changes in cell shape and an energy shift after IPTG induction via an optimized SILAC approach for Escherichia coli.

    Science.gov (United States)

    Ping, Lingyan; Zhang, Heng; Zhai, Linhui; Dammer, Eric B; Duong, Duc M; Li, Ning; Yan, Zili; Wu, Junzhu; Xu, Ping

    2013-12-01

    Stable isotope labeling by amino acids in cell culture (SILAC) has been widely used in yeast, mammalian cells, and even some multicellular organisms. However, the lack of optimized SILAC media limits its application in Escherichia coli, the most commonly used model organism. We optimized SILACE medium (SILAC medium created in this study for E. coli) for nonauxotrophic E. coli with high growth speed and complete labeling efficiency of the whole proteome in 12 generations. We applied a swapped SILAC workflow and pure null experiment with the SILACE medium using E. coli BL21 (DE3) cells hosting a recombinant plasmid coding for glutathione-S-transferase (GST) and ubiquitin binding domain before and after isopropyl thiogalactoside (IPTG) induction. Finally, we identified 1251 proteins with a significant change in abundance. Pathway analysis suggested that cell growth and fissiparism were inhibited accompanied by the down-regulation of proteins related to energy and metabolism, cell division, and the cell cycle, resulting in the size and shape change of the induced cells. Taken together, the results confirm the development of SILACE medium suitable for efficient and complete labeling of E. coli cells and a data filtering strategy for SILAC-based quantitative proteomics studies of E. coli.

  2. Quantitative shotgun proteomics reveals extensive changes to the proteome of the orbitofrontal cortex in rats that are hyperactive following withdrawal from a high sugar diet.

    Science.gov (United States)

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

    2016-02-01

    In most Westernized societies, there has been an alarming increase in the consumption of sugar-sweetened drinks. For many adults these drinks represent a substantial proportion of their total daily caloric intake. Here we investigated whether extended exposure to sugar changes behavior and protein expression in the orbitofrontal cortex (OFC). Male adult Sprague-Dawley rats (n = 8 per group) were treated for 26 days with either water or a 10% sucrose solution. Locomotor behavior was measured on the first and last day of treatment, then 1 week after treatment. Following the 1-week period free from treatment, sucrose treated rats were significantly more active than the control. Two hours following final behavioral testing, brains were rapidly removed and prepared for proteomic analysis of the OFC. Label free quantitative shotgun proteomic analyses of three rats from each group found 290 proteins were differentially expressed in the sucrose treated group when compared to the control group. Major changes in the proteome were seen in proteins related to energy metabolism, mitochondrial function and the cellular response to stress. This research does not seek to suggest that sugar will cause specific neurological disorders, however similar changes in proteins have been seen in neurological disorders such as Alzheimer's disease, Parkinson's disease and schizophrenia.

  3. A quantitative label-free analysis of the extracellular proteome of human supraspinatus tendon reveals damage to the pericellular and elastic fibre niches in torn and aged tissue.

    Science.gov (United States)

    Hakimi, Osnat; Ternette, Nicola; Murphy, Richard; Kessler, Benedikt M; Carr, Andrew

    2017-01-01

    Tears of the human supraspinatus tendon are common and often cause painful and debilitating loss of function. Progressive failure of the tendon leading to structural abnormality and tearing is accompanied by numerous cellular and extra-cellular matrix (ECM) changes in the tendon tissue. This proteomics study aimed to compare torn and aged rotator cuff tissue to young and healthy tissue, and provide the first ECM inventory of human supraspinatus tendon generated using label-free quantitative LC-MS/MS. Employing two digestion protocols (trypsin and elastase), we analysed grain-sized tendon supraspinatus biopsies from older patients with torn tendons and from healthy, young controls. Our findings confirm measurable degradation of collagen fibrils and associated proteins in old and torn tendons, suggesting a significant loss of tissue organisation. A particularly marked reduction of cartilage oligomeric matrix protein (COMP) raises the possibility of using changes in levels of this glycoprotein as a marker of abnormal tissue, as previously suggested in horse models. Surprisingly, and despite using an elastase digestion for validation, elastin was not detected, suggesting that it is not highly abundant in human supraspinatus tendon as previously thought. Finally, we identified marked changes to the elastic fibre, fibrillin-rich niche and the pericellular matrix. Further investigation of these regions may yield other potential biomarkers and help to explain detrimental cellular processes associated with tendon ageing and tendinopathy.

  4. 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.

  5. A genome-wide association study reveals a quantitative trait locus for days open on chromosome 2 in Japanese Black cattle.

    Science.gov (United States)

    Sasaki, Shinji; Ibi, Takayuki; Kojima, Takatoshi; Sugimoto, Yoshikazu

    2016-02-01

    Days open (DO), which is the interval from calving to conception, is an important trait related to reproductive performance in cattle. To identify quantitative trait loci for DO in Japanese Black cattle, we conducted a genome-wide association study with 33,303 single nucleotide polymorphisms (SNPs) using 459 animals with extreme DO values selected from a larger group of 15,488 animals. We identified a SNP on bovine chromosome 2 (BTA2) that was associated with DO. After imputation using phased haplotype data inferred from 586 812 SNPs of 1041 Japanese Black cattle, six SNPs associated with DO were located in an 8.5-kb region of high linkage disequilibrium on BTA2. These SNPs were located on the telomeric side at a distance of 177 kb from the parathyroid hormone 2 receptor (PTH2R) gene. The association was replicated in a sample of 1778 animals. In the replicated population, the frequency of the reduced-DO allele (Q) was 0.63, and it accounted for 1.72% of the total genetic variance. The effect of a Q-to-q allele substitution on DO was a decrease of 3.74 days. The results suggest that the Q allele could serve as a marker in Japanese Black cattle to select animals with superior DO performance.

  6. The Microbiome and Metabolites in Fermented Pu-erh Tea as Revealed by High-Throughput Sequencing and Quantitative Multiplex Metabolite Analysis.

    Science.gov (United States)

    Zhang, Yongjie; Skaar, Ida; Sulyok, Michael; Liu, Xingzhong; Rao, Mingyong; Taylor, John W

    2016-01-01

    Pu-erh is a tea produced in Yunnan, China by microbial fermentation of fresh Camellia sinensis leaves by two processes, the traditional raw fermentation and the faster, ripened fermentation. We characterized fungal and bacterial communities in leaves and both Pu-erhs by high-throughput, rDNA-amplicon sequencing and we characterized the profile of bioactive extrolite mycotoxins in Pu-erh teas by quantitative liquid chromatography-tandem mass spectrometry. We identified 390 fungal and 629 bacterial OTUs from leaves and both Pu-erhs. Major findings are: 1) fungal diversity drops and bacterial diversity rises due to raw or ripened fermentation, 2) fungal and bacterial community composition changes significantly between fresh leaves and both raw and ripened Pu-erh, 3) aging causes significant changes in the microbial community of raw, but not ripened, Pu-erh, and, 4) ripened and well-aged raw Pu-erh have similar microbial communities that are distinct from those of young, raw Ph-erh tea. Twenty-five toxic metabolites, mainly of fungal origin, were detected, with patulin and asperglaucide dominating and at levels supporting the Chinese custom of discarding the first preparation of Pu-erh and using the wet tea to then brew a pot for consumption.

  7. From beavis to beak color: a simulation study to examine how much qtl mapping can reveal about the genetic architecture of quantitative traits.

    Science.gov (United States)

    Slate, Jon

    2013-05-01

    Quantitative trait locus (QTL) mapping is frequently used in evolutionary studies to understand the genetic architecture of continuously varying traits. The majority of studies have been conducted in specially created crosses, in which genetic differences between parental lines are identified by linkage analysis. Detecting QTL segregating within populations is more problematic, especially in wild populations, because these populations typically have complicated and unbalanced multigenerational pedigrees. However, QTL mapping can still be conducted in such populations using a variance components mixed model approach, and the advent of appropriate statistical frameworks and better genotyping methods mean that the approach is gaining popularity. In this study it is shown that all studies described to date report evidence of QTL of major effect on trait variation, but that these findings are probably caused by inflated estimates of QTL effect sizes due to the Beavis effect. Using simulations I show that even the most powerful studies conducted to date are likely to give misleading descriptions of the genetic architecture of a trait. I show that an interpretation of a mapping study of beak color in the zebra finch (Taeniopygia guttata), that suggested genetic variation was determined by a small number of loci of large effect, which are possibly maintained by antagonistic pleiotropy, is likely to be incorrect. More generally, recommendations are made to how QTL mapping can be combined with other approaches to provide more accurate descriptions of a trait's genetic architecture.

  8. Quantitative in vivo Analyses Reveal Calcium-dependent Phosphorylation Sites and Identifies a Novel Component of the Toxoplasma Invasion Motor Complex

    Science.gov (United States)

    Nebl, Thomas; Prieto, Judith Helena; Kapp, Eugene; Smith, Brian J.; Williams, Melanie J.; Yates, John R.; Cowman, Alan F.; Tonkin, Christopher J.

    2011-01-01

    Apicomplexan parasites depend on the invasion of host cells for survival and proliferation. Calcium-dependent signaling pathways appear to be essential for micronemal release and gliding motility, yet the target of activated kinases remains largely unknown. We have characterized calcium-dependent phosphorylation events during Toxoplasma host cell invasion. Stimulation of live tachyzoites with Ca2+-mobilizing drugs leads to phosphorylation of numerous parasite proteins, as shown by differential 2-DE display of 32[P]-labeled protein extracts. Multi-dimensional Protein Identification Technology (MudPIT) identified ∼546 phosphorylation sites on over 300 Toxoplasma proteins, including 10 sites on the actomyosin invasion motor. Using a Stable Isotope of Amino Acids in Culture (SILAC)-based quantitative LC-MS/MS analyses we monitored changes in the abundance and phosphorylation of the invasion motor complex and defined Ca2+-dependent phosphorylation patterns on three of its components - GAP45, MLC1 and MyoA. Furthermore, calcium-dependent phosphorylation of six residues across GAP45, MLC1 and MyoA is correlated with invasion motor activity. By analyzing proteins that appear to associate more strongly with the invasion motor upon calcium stimulation we have also identified a novel 15-kDa Calmodulin-like protein that likely represents the MyoA Essential Light Chain of the Toxoplasma invasion motor. This suggests that invasion motor activity could be regulated not only by phosphorylation but also by the direct binding of calcium ions to this new component. PMID:21980283

  9. Quantitative trait mapping reveals a regulatory axis involving peroxisome proliferator-activated receptors, PRDM16, transforming growth factor-β2 and FLT3 in hematopoiesis.

    Science.gov (United States)

    Avagyan, Serine; Aguilo, Francesca; Kamezaki, Kenjiro; Snoeck, Hans-Willem

    2011-12-01

    Hematopoiesis is the process whereby BM HSCs renew to maintain their number or to differentiate into committed progenitors to generate all blood cells. One approach to gain mechanistic insight into this complex process is the investigation of quantitative genetic variation in hematopoietic function among inbred mouse strains. We previously showed that TGF-β2 is a genetically determined positive regulator of hematopoiesis. In the presence of unknown nonprotein serum factors TGF-β2, but not TGF-β1 or -β3, enhances progenitor proliferation in vitro, an effect that is subject to mouse strain-dependent variation mapping to a locus on chr.4, Tb2r1. TGF-β2-deficient mice show hematopoietic defects, demonstrating the physiologic role of this cytokine. Here, we show that TGF-β2 specifically and predominantly cell autonomously enhances signaling by FLT3 in vitro and in vivo. A coding polymorphism in Prdm16 (PR-domain-containing 16) underlies Tb2r1 and differentially regulates transcriptional activity of peroxisome proliferator-activated receptor-γ (PPARγ), identifying lipid PPAR ligands as the serum factors required for regulation of FLT3 signaling by TGF-β2. We furthermore show that PPARγ agonists play a FLT3-dependent role in stress responses of progenitor cells. These observations identify a novel regulatory axis that includes PPARs, Prdm16, and TGF-β2 in hematopoiesis.

  10. Quantitative assessment of chromatin immunoprecipitation grade antibodies directed against histone modifications reveals patterns of co-occurring marks on histone protein molecules.

    Science.gov (United States)

    Peach, Sally E; Rudomin, Emily L; Udeshi, Namrata D; Carr, Steven A; Jaffe, Jacob D

    2012-05-01

    The defining step in most chromatin immunoprecipitation (ChIP) assays is the use of an antibody to enrich for a particular protein or histone modification state associated with segments of chromatin. The specificity of the antibody is critical to the interpretation of the experiment, yet this property is rarely reported. Here, we present a quantitative method using mass spectrometry to characterize the specificity of key histone H3 modification-targeting antibodies that have previously been used to characterize the "histone code." We further extend the use of these antibody reagents to the observation of long range correlations among disparate histone modifications. Using purified human histones representing the mixture of chromatin states present in living cells, we were able to quantify the degree of target enrichment and the specificity of several commonly used, commercially available ChIP grade antibodies. We found significant differences in enrichment efficiency among various reagents directed against four frequently studied chromatin marks: H3K4me2, H3K4me3, H3K9me3, and H3K27me3. For some antibodies, we also detected significant off target enrichment of alternate modifications at the same site (i.e., enrichment of H3K4me2 by an antibody directed against H3K4me3). Through cluster analysis, we were able to recognize patterns of co-enrichment of marks at different sites on the same histone protein. Surprisingly, these co-enrichments corresponded well to "canonical" chromatin states that are exemplary of activated and repressed regions of chromatin. Altogether, our findings suggest that 1) the results of ChIP experiments need to be evaluated with caution given the potential for cross-reactivity of the commonly used histone modification recognizing antibodies, 2) multiple marks with consistent biological interpretation exist on the same histone protein molecule, and 3) some components of the histone code may be transduced on single proteins in living cells.

  11. Field-Based High-Throughput Plant Phenotyping Reveals the Temporal Patterns of Quantitative Trait Loci Associated with Stress-Responsive Traits in Cotton.

    Science.gov (United States)

    Pauli, Duke; Andrade-Sanchez, Pedro; Carmo-Silva, A Elizabete; Gazave, Elodie; French, Andrew N; Heun, John; Hunsaker, Douglas J; Lipka, Alexander E; Setter, Tim L; Strand, Robert J; Thorp, Kelly R; Wang, Sam; White, Jeffrey W; Gore, Michael A

    2016-04-07

    The application of high-throughput plant phenotyping (HTPP) to continuously study plant populations under relevant growing conditions creates the possibility to more efficiently dissect the genetic basis of dynamic adaptive traits. Toward this end, we employed a field-based HTPP system that deployed sets of sensors to simultaneously measure canopy temperature, reflectance, and height on a cotton (Gossypium hirsutum L.) recombinant inbred line mapping population. The evaluation trials were conducted under well-watered and water-limited conditions in a replicated field experiment at a hot, arid location in central Arizona, with trait measurements taken at different times on multiple days across 2010-2012. Canopy temperature, normalized difference vegetation index (NDVI), height, and leaf area index (LAI) displayed moderate-to-high broad-sense heritabilities, as well as varied interactions among genotypes with water regime and time of day. Distinct temporal patterns of quantitative trait loci (QTL) expression were mostly observed for canopy temperature and NDVI, and varied across plant developmental stages. In addition, the strength of correlation between HTPP canopy traits and agronomic traits, such as lint yield, displayed a time-dependent relationship. We also found that the genomic position of some QTL controlling HTPP canopy traits were shared with those of QTL identified for agronomic and physiological traits. This work demonstrates the novel use of a field-based HTPP system to study the genetic basis of stress-adaptive traits in cotton, and these results have the potential to facilitate the development of stress-resilient cotton cultivars. Copyright © 2016 Pauli et al.

  12. Field-Based High-Throughput Plant Phenotyping Reveals the Temporal Patterns of Quantitative Trait Loci Associated with Stress-Responsive Traits in Cotton

    Directory of Open Access Journals (Sweden)

    Duke Pauli

    2016-04-01

    Full Text Available The application of high-throughput plant phenotyping (HTPP to continuously study plant populations under relevant growing conditions creates the possibility to more efficiently dissect the genetic basis of dynamic adaptive traits. Toward this end, we employed a field-based HTPP system that deployed sets of sensors to simultaneously measure canopy temperature, reflectance, and height on a cotton (Gossypium hirsutum L. recombinant inbred line mapping population. The evaluation trials were conducted under well-watered and water-limited conditions in a replicated field experiment at a hot, arid location in central Arizona, with trait measurements taken at different times on multiple days across 2010–2012. Canopy temperature, normalized difference vegetation index (NDVI, height, and leaf area index (LAI displayed moderate-to-high broad-sense heritabilities, as well as varied interactions among genotypes with water regime and time of day. Distinct temporal patterns of quantitative trait loci (QTL expression were mostly observed for canopy temperature and NDVI, and varied across plant developmental stages. In addition, the strength of correlation between HTPP canopy traits and agronomic traits, such as lint yield, displayed a time-dependent relationship. We also found that the genomic position of some QTL controlling HTPP canopy traits were shared with those of QTL identified for agronomic and physiological traits. This work demonstrates the novel use of a field-based HTPP system to study the genetic basis of stress-adaptive traits in cotton, and these results have the potential to facilitate the development of stress-resilient cotton cultivars.

  13. iTRAQ-based quantitative proteomic analysis reveals Bai-Hu-Tang enhances phagocytosis and cross-presentation against LPS fever in rabbit.

    Science.gov (United States)

    Zhang, Shidong; Wang, Dongsheng; Dong, Shuwei; Yang, Zhiqiang; Yan, Zuoting

    2017-07-31

    Bai-Hu-Tang (BHT), a classical anti-febrile Chinese formula comprising of liquorice, anemarrhena rhizome, gypsum and rice, has been traditionally used to anti-febrile treatment and promote the production of body fluid to relieve thirst. In this paper, we aim to explore anti-febrile mechanism of BHT at protein level through analyzing alteration of differentially expressed proteins (DEPs) both lipopolysaccharide (LPS) fever syndrome and that was treated with BHT in rabbits. Febrile model was induced by LPS injection (i.v.) in rabbits, and BHT (750mg dry extract/kg body weight) was gavaged to another group of LPS fever rabbits. After sacrifice of animals, total protein of liver tissue was isolated, and two-dimensional liquid chromatography (LC) - tandem mass spectrometry (MS) coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling analysis was employed to quantitatively identify differentially expressed proteins in two group animals, which were compared with control group. Then bioinformatic analysis of DEPs was conducted through hierarchical Clustering, Venn analysis, gene ontology (GO) annotation enrichment, and kyoto encyclopedia of genes and genomes (KEGG) pathways enrichment. The results demonstrated there were 63 and 109 DEPs in LPS fever group and BHT-treated group, respectively. Enrichment analysis of GO annotations indicated that BHT mainly regulated expression of some extracellular structural proteins for response to stimulus and stress. KEGG analysis showed that ribosome and phagosome were the most significant pathways. Thereinto, several proteins in phagosome pathway were significantly up-regulated by BHT, including F-actin, coronin, Rac, and major histocompatibility complex class I (MHC I), which work in phagocytosis and cross-presentation CONCLUSION: BHT may contribute to pyrogen clearance by boosting antigenic phagocytosis, degradation, and cross presentation in the liver. Copyright © 2017. Published by Elsevier B.V.

  14. Dominant microbial composition and its vertical distribution in saline meromictic Lake Kaiike (Japan) as revealed by quantitative oligonucleotide probe membrane hybridization.

    Science.gov (United States)

    Koizumi, Yoshikazu; Kojima, Hisaya; Fukui, Manabu

    2004-08-01

    Vertical distributions of dominant bacterial populations in saline meromictic Lake Kaiike were investigated throughout the water column and sediment by quantitative oligonucleotide probe membrane hybridization. Three oligonucleotide probes specific for the small-subunit (SSU) rRNA of three groups of Chlorobiaceae were newly designed. In addition, three general domain (Bacteria, Archaea, and Eukarya)-specific probes, two delta-Proteobacteria-specific probes, a Chlorobiaceae-specific probe, and a Chloroflexi-specific probe were used after optimization of their washing conditions. The abundance of the sum of SSU rRNAs hybridizing with probes specific for three groups of Chlorobiaceae relative to total SSU rRNA peaked in the chemocline, accounting for up to 68%. The abundance of the delta-proteobacterial SSU rRNA relative to total SSU rRNA rapidly increased just below the chemocline up to 29% in anoxic water and peaked at the 2- to 3-cm sediment depth at ca. 34%. The abundance of SSU rRNAs hybridizing with the probe specific for the phylum Chloroflexi relative to total SSU rRNA was highest (31 to 54%) in the top of the sediment but then steeply declined with depth and became stable at 11 to 19%, indicating the robust coexistence of sulfate-reducing bacteria and Chloroflexi in the top of the sediment. Any SSU rRNA of Chloroflexi in the water column was under the detection limit. The summation of the signals of group-specific probes used in this study accounted for up to 89% of total SSU rRNA, suggesting that the DGGE-oligonucleotide probe hybridization approach, in contrast to conventional culture-dependent approaches, was very effective in covering dominant populations.

  15. Human mesenchymal stem cell expression program upon extended ex-vivo cultivation, as revealed by 2-DE-based quantitative proteomics.

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    Andreia Madeira

    Full Text Available Human mesenchymal stem cells (MSC have been on the focus of intense clinical-oriented research due to their multilineage differentiation potential and immunomodulatory properties. However, to reach the clinically meaningful cell numbers for cellular therapy and tissue engineering applications, MSC ex-vivo expansion is mandatory but sequential cell passaging results in loss of proliferative, clonogenic and differentiation potential. To get clues into the molecular mechanisms underlying cellular senescence resulting from extended ex-vivo cultivation of bone marrow (BM MSC, we explored a two-dimensional gel electrophoresis (2-DE based quantitative proteomics to compare the expression programs of Passage 3 cells (P3, commonly used in clinical studies with expanded MSC, and Passage 7 (P7 cells, which already demonstrated significant signs of culture-induced senescence. Proteins of the functional categories "Structural components and cellular cytoskeleton" and "Folding and stress response proteins" are less abundant in P7 cells, compared to P3, while proteins involved in "Energy metabolism", "Cell cycle regulation and aging" and "Apoptosis" are more abundant. The large number of multiple size and charge isoforms with an altered content that were identified in this study in P7 versus P3, namely the cytoskeleton components β-actin (7 forms and vimentin (24 forms, also emphasizes the importance of post-transcriptional modification upon long-term cultivation. The differential protein expression registered suggests that cellular senescence occurring during ex-vivo expansion of BM MSC is associated with the impairment of cytoskeleton remodeling and/or organization and the repair of damaged proteins resulting from cell exposure to culture stress. The genome-wide expression approach used in this study has proven useful for getting mechanistic insights into the observed decrease on the proliferative and clonogenic potential of P7 versus P3 cells and paves the

  16. Human mesenchymal stem cell expression program upon extended ex-vivo cultivation, as revealed by 2-DE-based quantitative proteomics.

    Science.gov (United States)

    Madeira, Andreia; da Silva, Cláudia L; dos Santos, Francisco; Camafeita, Emilio; Cabral, Joaquim M S; Sá-Correia, Isabel

    2012-01-01

    Human mesenchymal stem cells (MSC) have been on the focus of intense clinical-oriented research due to their multilineage differentiation potential and immunomodulatory properties. However, to reach the clinically meaningful cell numbers for cellular therapy and tissue engineering applications, MSC ex-vivo expansion is mandatory but sequential cell passaging results in loss of proliferative, clonogenic and differentiation potential. To get clues into the molecular mechanisms underlying cellular senescence resulting from extended ex-vivo cultivation of bone marrow (BM) MSC, we explored a two-dimensional gel electrophoresis (2-DE) based quantitative proteomics to compare the expression programs of Passage 3 cells (P3), commonly used in clinical studies with expanded MSC, and Passage 7 (P7) cells, which already demonstrated significant signs of culture-induced senescence. Proteins of the functional categories "Structural components and cellular cytoskeleton" and "Folding and stress response proteins" are less abundant in P7 cells, compared to P3, while proteins involved in "Energy metabolism", "Cell cycle regulation and aging" and "Apoptosis" are more abundant. The large number of multiple size and charge isoforms with an altered content that were identified in this study in P7 versus P3, namely the cytoskeleton components β-actin (7 forms) and vimentin (24 forms), also emphasizes the importance of post-transcriptional modification upon long-term cultivation. The differential protein expression registered suggests that cellular senescence occurring during ex-vivo expansion of BM MSC is associated with the impairment of cytoskeleton remodeling and/or organization and the repair of damaged proteins resulting from cell exposure to culture stress. The genome-wide expression approach used in this study has proven useful for getting mechanistic insights into the observed decrease on the proliferative and clonogenic potential of P7 versus P3 cells and paves the way to set

  17. Quantitative real-time reverse transcription-PCR analysis reveals stable and prolonged neurotoxin cluster gene activity in a Clostridium botulinum type E strain at refrigeration temperature.

    Science.gov (United States)

    Chen, Ying; Korkeala, Hannu; Lindén, Jere; Lindström, Miia

    2008-10-01

    The relative expression levels of six botulinum neurotoxin cluster genes in a group II Clostridium botulinum type E strain grown at 10 or 30 degrees C were investigated using quantitative real-time reverse transcription-PCR. An enzyme-linked immunosorbent assay was used to confirm neurotoxin expression. Distinct mRNA and toxin production patterns were observed at the two temperatures. The average relative mRNA levels at 10 degrees C were higher than (ntnh and p47), similar to (botE), or lower than (orfx1, orfx2, orfx3) those at 30 degrees C. The maximum botE expression levels and average neurotoxin levels at 10 degrees C were 45 to 65% of those at 30 degrees C. The relative mRNA levels at 10 degrees C declined generally slowly within 8 days, as opposed to the rapid decline observed at 30 degrees C within 24 h. Distinct expression patterns of the six genes at the two temperatures suggest that the type E neurotoxin cluster genes are transcribed as two tricistronic operons at 30 degrees C, whereas at 10 degrees C monocistronic (botE or orfx1 alone) and bicistronic (ntnh-p47 and orfx2-orfx3) transcription may dominate. Thus, type E botulinum neurotoxin production may be involved with various temperature-dependent regulatory events. In light of group II C. botulinum type E being a dangerous food-borne pathogen, these findings may be important in terms of the safety of refrigerated packaged foods of extended durability.

  18. Quantitative PCR reveals strong spatial and temporal variation of the wasting disease pathogen, Labyrinthula zosterae in northern European eelgrass (Zostera marina beds.

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    Anna-Christina Bockelmann

    Full Text Available Seagrass beds are the foundation species of functionally important coastal ecosystems worldwide. The world's largest losses of the widespread seagrass Zostera marina (eelgrass have been reported as a consequence of wasting disease, an infection with the endophytic protist Labyrinthula zosterae. During one of the most extended epidemics in the marine realm, ∼90% of East and Western Atlantic eelgrass beds died-off between 1932 and 1934. Today, small outbreaks continue to be reported, but the current extent of L. zosterae in European meadows is completely unknown. In this study we quantify the abundance and prevalence of the wasting disease pathogen among 19 Z. marina populations in northern European coastal waters, using quantitative PCR (QPCR with primers targeting a species specific portion of the internally transcribed spacer (ITS1 of L. zosterae. Spatially, we found marked variation among sites with abundances varying between 0 and 126 cells mg(-1 Z. marina dry weight (mean: 5.7 L. zosterae cells mg(-1 Z. marina dry weight ±1.9 SE and prevalences ranged from 0-88.9%. Temporarily, abundances varied between 0 and 271 cells mg(-1 Z. marina dry weight (mean: 8.5±2.6 SE, while prevalences ranged from zero in winter and early spring to 96% in summer. Field concentrations accessed via bulk DNA extraction and subsequent QPCR correlated well with prevalence data estimated via isolation and cultivation from live plant tissue. L. zosterae was not only detectable in black lesions, a sign of Labyrinthula-induced necrosis, but also occurred in green, apparently healthy tissue. We conclude that L. zosterae infection is common (84% infected populations in (northern European eelgrass populations with highest abundances during the summer months. In the light of global climate change and increasing rate of marine diseases our data provide a baseline for further studies on the causes of pathogenic outbreaks of L. zosterae.

  19. Quantitative profiling of the shedding rate of the three Marek's disease virus (MDV) serotypes reveals that challenge with virulent MDV markedly increases shedding of vaccinal viruses.

    Science.gov (United States)

    Islam, Aminul; Walkden-Brown, Stephen W

    2007-08-01

    The shedding profile of Marek's disease virus serotype 1 (MDV1, virulent), serotype 2 (MDV2, vaccinal) and herpesvirus of turkeys (HVT, vaccinal) in commercial broiler chickens was determined by measuring the daily rate of production of feather dander from chickens housed in isolators and by quantifying the viral load of each of these serotypes in the dander using quantitative real-time PCR (qPCR). MDV1 and HVT viruses were detectable in dander filtered from isolator exhaust air from day 7 and MDV2 from day 12 after infection and thereafter until the end of the experiment at 61 days of age of the chickens. There was no difference in shedding rate among the three MDV1 isolates. Daily shedding of MDV1 increased sharply between days 7 and 28 and stabilized thereafter at about 10(9) virus copies per chicken per day, irrespective of vaccination status. Challenge with the three different MDV1 isolates markedly increased shedding of the vaccinal viruses HVT and MDV2 in dander by 38- and 75-fold, respectively. These results demonstrate the utility of qPCR for the differentiation and quantification of different MDV serotypes in feather dander and have significant implications for the routine monitoring of Marek's disease using qPCR assays of dust, for epidemiological modelling of the behaviour and spread of MDVs in chicken populations and for studies into the evolution of virulence in MDV1 in the face of blanket vaccination with imperfect vaccines that ameliorate disease but do not prevent infection and replication of virulent virus.

  20. 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.

  1. A Novel Function for Arabidopsis CYCLASE1 in Programmed Cell Death Revealed by Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) Analysis of Extracellular Matrix Proteins.

    Science.gov (United States)

    Smith, Sarah J; Kroon, Johan T M; Simon, William J; Slabas, Antoni R; Chivasa, Stephen

    2015-06-01

    Programmed cell death is essential for plant development and stress adaptation. A detailed understanding of the signal transduction pathways that regulate plant programmed cell death requires identification of the underpinning protein networks. Here, we have used a protagonist and antagonist of programmed cell death triggered by fumonisin B1 as probes to identify key cell death regulatory proteins in Arabidopsis. Our hypothesis was that changes in the abundance of cell death-regulatory proteins induced by the protagonist should be blocked or attenuated by concurrent treatment with the antagonist. We focused on proteins present in the mobile phase of the extracellular matrix on the basis that they are important for cell-cell communications during growth and stress-adaptive responses. Salicylic acid, a plant hormone that promotes programmed cell death, and exogenous ATP, which can block fumonisin B1-induced cell death, were used to treat Arabidopsis cell suspension cultures prior to isobaric-tagged relative and absolute quantitation analysis of secreted proteins. A total of 33 proteins, whose response to salicylic acid was suppressed by ATP, were identified as putative cell death-regulatory proteins. Among these was CYCLASE1, which was selected for further analysis using reverse genetics. Plants in which CYCLASE1 gene expression was knocked out by insertion of a transfer-DNA sequence manifested dramatically increased cell death when exposed to fumonisin B1 or a bacterial pathogen that triggers the defensive hypersensitive cell death. Although pathogen inoculation altered CYCLASE1 gene expression, multiplication of bacterial pathogens was indistinguishable between wild type and CYCLASE1 knockout plants. However, remarkably severe chlorosis symptoms developed on gene knockout plants in response to inoculation with either a virulent bacterial pathogen or a disabled mutant that is incapable of causing disease in wild type plants. These results show that CYCLASE1, which

  2. Quantitative analysis of viral load per haploid genome revealed the different biological features of Merkel cell polyomavirus infection in skin tumor.

    Directory of Open Access Journals (Sweden)

    Satoshi Ota

    Full Text Available Merkel cell polyomavirus (MCPyV has recently been identified in Merkel cell carcinoma (MCC, an aggressive cancer that occurs in sun-exposed skin. Conventional technologies, such as polymerase chain reaction (PCR and immunohistochemistry, have produced conflicting results for MCPyV infections in non-MCC tumors. Therefore, we performed quantitative analyses of the MCPyV copy number in various skin tumor tissues, including MCC (n = 9 and other sun exposure-related skin tumors (basal cell carcinoma [BCC, n = 45], actinic keratosis [AK, n = 52], Bowen's disease [n = 34], seborrheic keratosis [n = 5], primary cutaneous anaplastic large-cell lymphoma [n = 5], malignant melanoma [n = 5], and melanocytic nevus [n = 6]. In a conventional PCR analysis, MCPyV DNA was detected in MCC (9 cases; 100%, BCC (1 case; 2%, and AK (3 cases; 6%. We then used digital PCR technology to estimate the absolute viral copy number per haploid human genome in these tissues. The viral copy number per haploid genome was estimated to be around 1 in most MCC tissues, and there were marked differences between the MCC (0.119-42.8 and AK (0.02-0.07 groups. PCR-positive BCC tissue showed a similar viral load as MCC tissue (0.662. Immunohistochemistry with a monoclonal antibody against the MCPyV T antigen (CM2B4 demonstrated positive nuclear localization in most of the high-viral-load tumor groups (8 of 9 MCC and 1 BCC, but not in the low-viral-load or PCR-negative tumor groups. These results demonstrated that MCPyV infection is possibly involved in a minority of sun-exposed skin tumors, including BCC and AK, and that these tumors display different modes of infection.

  3. Quantitative in vivo analyses reveal calcium-dependent phosphorylation sites and identifies a novel component of the Toxoplasma invasion motor complex.

    Directory of Open Access Journals (Sweden)

    Thomas Nebl

    2011-09-01

    Full Text Available Apicomplexan parasites depend on the invasion of host cells for survival and proliferation. Calcium-dependent signaling pathways appear to be essential for micronemal release and gliding motility, yet the target of activated kinases remains largely unknown. We have characterized calcium-dependent phosphorylation events during Toxoplasma host cell invasion. Stimulation of live tachyzoites with Ca²⁺-mobilizing drugs leads to phosphorylation of numerous parasite proteins, as shown by differential 2-DE display of ³²[P]-labeled protein extracts. Multi-dimensional Protein Identification Technology (MudPIT identified ∼546 phosphorylation sites on over 300 Toxoplasma proteins, including 10 sites on the actomyosin invasion motor. Using a Stable Isotope of Amino Acids in Culture (SILAC-based quantitative LC-MS/MS analyses we monitored changes in the abundance and phosphorylation of the invasion motor complex and defined Ca²⁺-dependent phosphorylation patterns on three of its components--GAP45, MLC1 and MyoA. Furthermore, calcium-dependent phosphorylation of six residues across GAP45, MLC1 and MyoA is correlated with invasion motor activity. By analyzing proteins that appear to associate more strongly with the invasion motor upon calcium stimulation we have also identified a novel 15-kDa Calmodulin-like protein that likely represents the MyoA Essential Light Chain of the Toxoplasma invasion motor. This suggests that invasion motor activity could be regulated not only by phosphorylation but also by the direct binding of calcium ions to this new component.

  4. Quantitative Assessment of Chromatin Immunoprecipitation Grade Antibodies Directed against Histone Modifications Reveals Patterns of Co-occurring Marks on Histone Protein Molecules*

    Science.gov (United States)

    Peach, Sally E.; Rudomin, Emily L.; Udeshi, Namrata D.; Carr, Steven A.; Jaffe, Jacob D.

    2012-01-01

    The defining step in most chromatin immunoprecipitation (ChIP) assays is the use of an antibody to enrich for a particular protein or histone modification state associated with segments of chromatin. The specificity of the antibody is critical to the interpretation of the experiment, yet this property is rarely reported. Here, we present a quantitative method using mass spectrometry to characterize the specificity of key histone H3 modification-targeting antibodies that have previously been used to characterize the “histone code.” We further extend the use of these antibody reagents to the observation of long range correlations among disparate histone modifications. Using purified human histones representing the mixture of chromatin states present in living cells, we were able to quantify the degree of target enrichment and the specificity of several commonly used, commercially available ChIP grade antibodies. We found significant differences in enrichment efficiency among various reagents directed against four frequently studied chromatin marks: H3K4me2, H3K4me3, H3K9me3, and H3K27me3. For some antibodies, we also detected significant off target enrichment of alternate modifications at the same site (i.e., enrichment of H3K4me2 by an antibody directed against H3K4me3). Through cluster analysis, we were able to recognize patterns of co-enrichment of marks at different sites on the same histone protein. Surprisingly, these co-enrichments corresponded well to “canonical” chromatin states that are exemplary of activated and repressed regions of chromatin. Altogether, our findings suggest that 1) the results of ChIP experiments need to be evaluated with caution given the potential for cross-reactivity of the commonly used histone modification recognizing antibodies, 2) multiple marks with consistent biological interpretation exist on the same histone protein molecule, and 3) some components of the histone code may be transduced on single proteins in living

  5. A Quantitative Model of the GIRK1/2 Channel Reveals That Its Basal and Evoked Activities Are Controlled by Unequal Stoichiometry of Gα and Gβγ

    Science.gov (United States)

    Rubinstein, Moran; Farhy-Tselnicker, Isabella; Styr, Boaz; Keren-Raifman, Tal; Dessauer, Carmen W.; Dascal, Nathan

    2015-01-01

    G protein-gated K+ channels (GIRK; Kir3), activated by Gβγ subunits derived from Gi/o proteins, regulate heartbeat and neuronal excitability and plasticity. Both neurotransmitter-evoked (Ievoked) and neurotransmitter-independent basal (Ibasal) GIRK activities are physiologically important, but mechanisms of Ibasal and its relation to Ievoked are unclear. We have previously shown for heterologously expressed neuronal GIRK1/2, and now show for native GIRK in hippocampal neurons, that Ibasal and Ievoked are interrelated: the extent of activation by neurotransmitter (activation index, Ra) is inversely related to Ibasal. To unveil the underlying mechanisms, we have developed a quantitative model of GIRK1/2 function. We characterized single-channel and macroscopic GIRK1/2 currents, and surface densities of GIRK1/2 and Gβγ expressed in Xenopus oocytes. Based on experimental results, we constructed a mathematical model of GIRK1/2 activity under steady-state conditions before and after activation by neurotransmitter. Our model accurately recapitulates Ibasal and Ievoked in Xenopus oocytes, HEK293 cells and hippocampal neurons; correctly predicts the dose-dependent activation of GIRK1/2 by coexpressed Gβγ and fully accounts for the inverse Ibasal-Ra correlation. Modeling indicates that, under all conditions and at different channel expression levels, between 3 and 4 Gβγ dimers are available for each GIRK1/2 channel. In contrast, available Gαi/o decreases from ~2 to less than one Gα per channel as GIRK1/2's density increases. The persistent Gβγ/channel (but not Gα/channel) ratio support a strong association of GIRK1/2 with Gβγ, consistent with recruitment to the cell surface of Gβγ, but not Gα, by GIRK1/2. Our analysis suggests a maximal stoichiometry of 4 Gβγ but only 2 Gαi/o per one GIRK1/2 channel. The unique, unequal association of GIRK1/2 with G protein subunits, and the cooperative nature of GIRK gating by Gβγ, underlie the complex pattern of

  6. A quantitative, high-throughput reverse genetic screen reveals novel connections between Pre-mRNA splicing and 5' and 3' end transcript determinants.

    Directory of Open Access Journals (Sweden)

    Laura-Oana Albulescu

    Full Text Available Here we present the development and implementation of a genome-wide reverse genetic screen in the budding yeast, Saccharomyces cerevisiae, that couples high-throughput strain growth, robotic RNA isolation and cDNA synthesis, and quantitative PCR to allow for a robust determination of the level of nearly any cellular RNA in the background of ~5,500 different mutants. As an initial test of this approach, we sought to identify the full complement of factors that impact pre-mRNA splicing. Increasing lines of evidence suggest a relationship between pre-mRNA splicing and other cellular pathways including chromatin remodeling, transcription, and 3' end processing, yet in many cases the specific proteins responsible for functionally connecting these pathways remain unclear. Moreover, it is unclear whether all pathways that are coupled to splicing have been identified. As expected, our approach sensitively detects pre-mRNA accumulation in the vast majority of strains containing mutations in known splicing factors. Remarkably, however, several additional candidates were found to cause increases in pre-mRNA levels similar to that seen for canonical splicing mutants, none of which had previously been implicated in the splicing pathway. Instead, several of these factors have been previously implicated to play roles in chromatin remodeling, 3' end processing, and other novel categories. Further analysis of these factors using splicing-sensitive microarrays confirms that deletion of Bdf1, a factor that links transcription initiation and chromatin remodeling, leads to a global splicing defect, providing evidence for a novel connection between pre-mRNA splicing and this component of the SWR1 complex. By contrast, mutations in 3' end processing factors such as Cft2 and Yth1 also result in pre-mRNA splicing defects, although only for a subset of transcripts, suggesting that spliceosome assembly in S. cerevisiae may more closely resemble mammalian models of exon

  7. Parallel β-sheet vibrational couplings revealed by 2D IR spectroscopy of an isotopically labeled macrocycle: quantitative benchmark for the interpretation of amyloid and protein infrared spectra.

    Science.gov (United States)

    Woys, Ann Marie; Almeida, Aaron M; Wang, Lu; Chiu, Chi-Cheng; McGovern, Michael; de Pablo, Juan J; Skinner, James L; Gellman, Samuel H; Zanni, Martin T

    2012-11-21

    Infrared spectroscopy is playing an important role in the elucidation of amyloid fiber formation, but the coupling models that link spectra to structure are not well tested for parallel β-sheets. Using a synthetic macrocycle that enforces a two stranded parallel β-sheet conformation, we measured the lifetimes and frequency for six combinations of doubly (13)C═(18)O labeled amide I modes using 2D IR spectroscopy. The average vibrational lifetime of the isotope labeled residues was 550 fs. The frequencies of the labels ranged from 1585 to 1595 cm(-1), with the largest frequency shift occurring for in-register amino acids. The 2D IR spectra of the coupled isotope labels were calculated from molecular dynamics simulations of a series of macrocycle structures generated from replica exchange dynamics to fully sample the conformational distribution. The models used to simulate the spectra include through-space coupling, through-bond coupling, and local frequency shifts caused by environment electrostatics and hydrogen bonding. The calculated spectra predict the line widths and frequencies nearly quantitatively. Historically, the characteristic features of β-sheet infrared spectra have been attributed to through-space couplings such as transition dipole coupling. We find that frequency shifts of the local carbonyl groups due to nearest neighbor couplings and environmental factors are more important, while the through-space couplings dictate the spectral intensities. As a result, the characteristic absorption spectra empirically used for decades to assign parallel β-sheet secondary structure arises because of a redistribution of oscillator strength, but the through-space couplings do not themselves dramatically alter the frequency distribution of eigenstates much more than already exists in random coil structures. Moreover, solvent exposed residues have amide I bands with >20 cm(-1) line width. Narrower line widths indicate that the amide I backbone is solvent

  8. Quantitative Persulfide Site Identification (qPerS-SID) Reveals Protein Targets of H2S Releasing Donors in Mammalian Cells.

    Science.gov (United States)

    Longen, Sebastian; Richter, Florian; Köhler, Yvette; Wittig, Ilka; Beck, Karl-Friedrich; Pfeilschifter, Josef

    2016-07-14

    H2S is an important signalling molecule involved in diverse biological processes. It mediates the formation of cysteine persulfides (R-S-SH), which affect the activity of target proteins. Like thiols, persulfides show reactivity towards electrophiles and behave similarly to other cysteine modifications in a biotin switch assay. In this manuscript, we report on qPerS-SID a mass spectrometry-based method allowing the isolation of persulfide containing peptides in the mammalian proteome. With this method, we demonstrated that H2S donors differ in their efficacy to induce persulfides in HEK293 cells. Furthermore, data analysis revealed that persulfide formation affects all subcellular compartments and various cellular processes. Negatively charged amino acids appeared more frequently adjacent to cysteines forming persulfides. We confirmed our proteomic data using pyruvate kinase M2 as a model protein and showed that several cysteine residues are prone to persulfide formation finally leading to its inactivation. Taken together, the site-specific identification of persulfides on a proteome scale can help to identify target proteins involved in H2S signalling and enlightens the biology of H2S and its releasing agents.

  9. MiRNA Analysis by Quantitative PCR in Preterm Human Breast Milk Reveals Daily Fluctuations of hsa-miR-16-5p.

    Directory of Open Access Journals (Sweden)

    Ilaria Floris

    Full Text Available Human breast milk is an extremely dynamic fluid containing many biologically-active components which change throughout the feeding period and throughout the day. We designed a miRNA assay on minimized amounts of raw milk obtained from mothers of preterm infants. We investigated changes in miRNA expression within month 2 of lactation and then over the course of 24 hours.Analyses were performed on pooled breast milk, made by combining samples collected at different clock times from the same mother donor, along with time series collected over 24 hours from four unsynchronized mothers. Whole milk, lipids or skim milk fractions were processed and analyzed by qPCR. We measured hsa-miR-16-5p, hsa-miR-21-5p, hsa-miR-146-5p, and hsa-let-7a, d and g (all -5p. Stability of miRNA endogenous controls was evaluated using RefFinder, a web tool integrating geNorm, Normfinder, BestKeeper and the comparative ΔΔCt method.MiR-21 and miR-16 were stably expressed in whole milk collected within month 2 of lactation from four mothers. Analysis of lipids and skim milk revealed that miR-146b and let-7d were better references in both fractions. Time series (5H-23H allowed the identification of a set of three endogenous reference genes (hsa-let-7d, hsa-let-7g and miR-146b to normalize raw quantification cycle (Cq data. We identified a daily oscillation of miR-16-5p.Our assay allows exploring miRNA levels of breast milk from mother with preterm baby collected in time series over 48-72 hours.

  10. A comparison of two common sample preparation techniques for lipid and fatty acid analysis in three different coral morphotypes reveals quantitative and qualitative differences.

    Science.gov (United States)

    Conlan, Jessica A; Rocker, Melissa M; Francis, David S

    2017-01-01

    Lipids are involved in a host of biochemical and physiological processes in corals. Therefore, changes in lipid composition reflect changes in the ecology, nutrition, and health of corals. As such, accurate lipid extraction, quantification, and identification is critical to obtain comprehensive insight into a coral's condition. However, discrepancies exist in sample preparation methodology globally, and it is currently unknown whether these techniques generate analogous results. This study compared the two most common sample preparation techniques for lipid analysis in corals: (1) tissue isolation by air-spraying and (2) crushing the coral in toto. Samples derived from each preparation technique were subsequently analysed to quantify lipids and their constituent classes and fatty acids in four common, scleractinian coral species representing three distinct morphotypes (Acropora millepora, Montipora crassotuberculata, Porites cylindrica, and Pocillopora damicornis). Results revealed substantial amounts of organic material, including lipids, retained in the skeletons of all species following air-spraying, causing a marked underestimation of total lipid concentration using this method. Moreover, lipid class and fatty acid compositions between the denuded skeleton and sprayed tissue were substantially different. In particular, the majority of the total triacylglycerol and total fatty acid concentrations were retained in the skeleton (55-69% and 56-64%, respectively). As such, the isolated, sprayed tissue cannot serve as a reliable proxy for lipid quantification or identification in the coral holobiont. The in toto crushing method is therefore recommended for coral sample preparation prior to lipid analysis to capture the lipid profile of the entire holobiont, permitting accurate diagnoses of coral condition.

  11. 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.

  12. Nuclear Phosphoproteomic Screen Uncovers ACLY as Mediator of IL-2-induced Proliferation of CD4+ T lymphocytes.

    Science.gov (United States)

    Osinalde, Nerea; Mitxelena, Jone; Sánchez-Quiles, Virginia; Akimov, Vyacheslav; Aloria, Kerman; Arizmendi, Jesus M; Zubiaga, Ana M; Blagoev, Blagoy; Kratchmarova, Irina

    2016-06-01

    Anti-cancer immunotherapies commonly rely on the use of interleukin-2 (IL-2) to promote the expansion of T lymphocytes. IL-2- dependent proliferation is the culmination of a complex network of phosphorylation-driven signaling events that impact on gene transcription through mechanisms that are not clearly understood. To study the role of IL-2 in the regulation of nuclear protein function we have performed an unbiased mass spectrometry-based study of the nuclear phosphoproteome of resting and IL-2-treated CD4(+) T lymphocytes. We detected 8521distinct phosphosites including many that are not yet reported in curated phosphorylation databases. Although most phosphorylation sites remained unaffected upon IL-2 treatment, 391 sites corresponding to 288 gene products showed robust IL-2-dependent regulation. Importantly, we show that ATP-citrate lyase (ACLY) is a key phosphoprotein effector of IL-2-mediated T-cell responses. ACLY becomes phosphorylated on serine 455 in T lymphocytes upon IL-2-driven activation of AKT, and depletion or inactivation of ACLY compromises IL-2-promoted T-cell growth. Mechanistically, we demonstrate that ACLY is required for enhancing histone acetylation levels and inducing the expression of cell cycle regulating genes in response to IL-2. Thus, the metabolic enzyme ACLY emerges as a bridge between cytokine signaling and proliferation of T lymphocytes, and may be an attractive candidate target for the development of more efficient anti-cancer immunotherapies.

  13. A novel double-component MOAC honeycomb composite with pollen grains as a template for phosphoproteomics research.

    Science.gov (United States)

    Wang, Jiaxi; Li, Jie; Wang, Yanan; Gao, Mingxia; Zhang, Xiangmin; Deng, Chunhui

    2016-07-01

    The enrichment and separation of phosphopeptides from mixed biological samples is a technologically very significance, but highly challenging work. Current designed materials are mainly based on the broad and effective adsorptive character of metal oxide affinity chromatography (MOAC). Though significant progress has been made in the enrichment of phosphopeptides with MOAC material, there are chances for further development. In this study, a novel pollen-based MOAC honeycomb material was firstly explored in which the suitable hydrophilic channels preferentially enrich much more endogenous phosphopeptides than nonphosphopeptides or proteins while doping binary metal oxides at the atomic level and the ultra-high specific surface area have further allowed it to possess more effective active sites. Based on these unique features, the pollen-based material exhibited high selectivity for β-casein (mass ratio of β-casein/BSA, 1:1500), ultra-low detection limit (0.1fmol), desirable reusability. Moreover, the bionics MOAC composites were investigated in the enrichment of phosphopeptides from nonfat milk, human serum (male and female at the same age) and mice liver, results of which indicate the great potential of the composite for the phosphoproteome analysis of complex biological samples through the cheap and environmentally friendly process.

  14. Changes in the proteome and phosphoproteome expression in the bryozoan Bugula neritina larvae in response to the antifouling agent butenolide

    KAUST Repository

    Qian, Pei Yuan

    2010-09-08

    Larval attachment and metamorphosis, commonly referred to as larval settlement, of marine sessile invertebrates can be triggered or blocked by chemical cues and affected by changes in overall protein expression pattern and phosphorylation dynamics. This study focuses on the effects of butenolide, an effective larval settlement inhibitor, on larval settlement at the proteome level in the bryozoan Bugula neritina. Liquid-phase IEF sample prefractionation combined with 2-DE and MALDI-TOF MS was used to identify the differentially expressed proteins. Substantial changes occurred both in protein abundance and in phosphorylation status during larval settlement and when settling larvae were challenged with butenolide. The proteins that responded to treatment were identified as structural proteins, molecular chaperones, mitochondrial peptidases and calcium-binding proteins. Compared with our earlier results, both genistein and butenolide inhibited larval settlement of B. neritina primarily by changes in protein abundance and the phosphorylation status of proteins but have different protein targets in the same species. Clearly, to design potent antifouling compounds and to understand the mode of action of compounds, more studies on the effects of different compounds on proteome and phosphoproteome of different larval species are required. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.

  15. The hsp 16 Gene of the Probiotic Lactobacillus acidophilus Is Differently Regulated by Salt, High Temperature and Acidic Stresses, as Revealed by Reverse Transcription Quantitative PCR (qRT-PCR Analysis

    Directory of Open Access Journals (Sweden)

    Daniela Fiocco

    2011-08-01

    Full Text Available Small heat shock proteins (sHsps are ubiquitous conserved chaperone-like proteins involved in cellular proteins protection under stressful conditions. In this study, a reverse transcription quantitative PCR (RT-qPCR procedure was developed and used to quantify the transcript level of a small heat shock gene (shs in the probiotic bacterium Lactobacillus acidophilus NCFM, under stress conditions such as heat (45 °C and 53 °C, bile (0.3% w/v, hyperosmosis (1 M and 2.5 M NaCl, and low pH value (pH 4. The shs gene of L. acidophilus NCFM was induced by salt, high temperature and acidic stress, while repression was observed upon bile stress. Analysis of the 5' noncoding region of the hsp16 gene reveals the presence of an inverted repeat (IR sequence (TTAGCACTC-N9-GAGTGCTAA homologue to the controlling IR of chaperone expression (CIRCE elements found in the upstream regulatory region of Gram-positive heat shock operons, suggesting that the hsp16 gene of L. acidophilus might be transcriptionally controlled by HrcA. In addition, the alignment of several small heat shock proteins identified so far in lactic acid bacteria, reveals that the Hsp16 of L. acidophilus exhibits a strong evolutionary relationship with members of the Lactobacillus acidophilus group.

  16. The hsp 16 gene of the probiotic Lactobacillus acidophilus is differently regulated by salt, high temperature and acidic stresses, as revealed by reverse transcription quantitative PCR (qRT-PCR) analysis.

    Science.gov (United States)

    Capozzi, Vittorio; Arena, Mattia Pia; Crisetti, Elisabetta; Spano, Giuseppe; Fiocco, Daniela

    2011-01-01

    Small heat shock proteins (sHsps) are ubiquitous conserved chaperone-like proteins involved in cellular proteins protection under stressful conditions. In this study, a reverse transcription quantitative PCR (RT-qPCR) procedure was developed and used to quantify the transcript level of a small heat shock gene (shs) in the probiotic bacterium Lactobacillus acidophilus NCFM, under stress conditions such as heat (45 °C and 53 °C), bile (0.3% w/v), hyperosmosis (1 M and 2.5 M NaCl), and low pH value (pH 4). The shs gene of L. acidophilus NCFM was induced by salt, high temperature and acidic stress, while repression was observed upon bile stress. Analysis of the 5' noncoding region of the hsp16 gene reveals the presence of an inverted repeat (IR) sequence (TTAGCACTC-N9-GAGTGCTAA) homologue to the controlling IR of chaperone expression (CIRCE) elements found in the upstream regulatory region of Gram-positive heat shock operons, suggesting that the hsp16 gene of L. acidophilus might be transcriptionally controlled by HrcA. In addition, the alignment of several small heat shock proteins identified so far in lactic acid bacteria, reveals that the Hsp16 of L. acidophilus exhibits a strong evolutionary relationship with members of the Lactobacillus acidophilus group.

  17. TiSH--a robust and sensitive global phosphoproteomics strategy employing a combination of TiO2, SIMAC, and HILIC

    DEFF Research Database (Denmark)

    Engholm-Keller, Kasper; Birck, Pernille; Størling, Joachim

    2012-01-01

    have set up a multi-dimensional phosphoproteomics strategy combining a number of well-established enrichment and fraction methods: An initial TiO(2) phosphopeptide pre-enrichment step is followed by post-fractionation using sequential elution from IMAC (SIMAC) to separate multi- and mono......-phosphorylated peptides, and hydrophilic interaction liquid chromatography (HILIC) of the mono-phosphorylated peptides (collectively abbreviated "TiSH"). The advantages of the strategy include a high specificity and sample preparation workload reduction due to the TiO(2) pre-enrichment step, as well as low adsorptive...

  18. Differences in Beef Quality between Angus (Bos taurus taurus) and Nellore (Bos taurus indicus) Cattle through a Proteomic and Phosphoproteomic Approach.

    Science.gov (United States)

    Rodrigues, Rafael Torres de Souza; Chizzotti, Mario Luiz; Vital, Camilo Elber; Baracat-Pereira, Maria Cristina; Barros, Edvaldo; Busato, Karina Costa; Gomes, Rafael Aparecido; Ladeira, Márcio Machado; Martins, Taiane da Silva

    2017-01-01

    Proteins are the major constituents of muscle and are key molecules regulating the metabolic changes during conversion of muscle to meat. Brazil is one of the largest exporters of beef and most Brazilian cattle are composed by zebu (Nellore) genotype. Bos indicus beef is generally leaner and tougher than Bos taurus such as Angus. The aim of this study was to compare the muscle proteomic and phosphoproteomic profile of Angus and Nellore. Seven animals of each breed previously subjected the same growth management were confined for 84 days. Proteins were extracted from Longissimus lumborum samples collected immediately after slaughter and separated by two-dimensional electrophoresis. Pro-Q Diamond stain was used in phosphoproteomics. Proteins identification was performed using matrix assisted laser desorption/ionization time-of-flight mass spectrometry. Tropomyosin alpha-1 chain, troponin-T, myosin light chain-1 fragment, cytoplasmic malate dehydrogenase, alpha-enolase and 78 kDa glucose-regulated protein were more abundant in Nellore, while myosin light chain 3, prohibitin, mitochondrial stress-70 protein and heat shock 70 kDa protein 6 were more abundant in Angus (Pbeef quality between Angus and Nellore. Furthermore, prohibitin appears to be a potential biomarker of intramuscular fat in cattle. Additionally, differences in phosphorylation of myofilaments and glycolytic enzymes could be involved with differences in muscle contraction force, susceptibility to calpain, apoptosis and postmortem glycolysis, which might also be related to differences in beef quality among Angus and Nellore.

  19. A comparison of the chicken and turkey proteomes and phosphoproteomes in the development of poultry-specific immuno-metabolism kinome peptide arrays

    Directory of Open Access Journals (Sweden)

    Ryan J Arsenault

    2014-11-01

    Full Text Available The use of species-specific peptide arrays for the study of animal kinomes has a proven track record of success. This technique has been used in a variety of species for the study of host-pathogen interactions and metabolism. Species-specific peptide arrays have been designed previously for use with chicken, but a turkey array has never been attempted. In addition, arrays designed around individual cellular functions have been designed and utilized, but cross-function immuno-metabolic arrays have not been considered previously. Antecedent to designing separate chicken and turkey immuno-metabolic kinome peptide arrays, we show that while the chicken and turkey genomes are quite similar, the two species are much more distinct at the proteome and phosphoproteome levels. Despite a genome identity of approximately 90%, we observe that only 83% of chicken and turkey orthologous proteins display sequence matches between the two species. Further, less than 70% of kinase recognition target sequences are exact matches between chicken and turkey. Thus, our analysis shows that, at the proteome and kinome level, these two species must be considered separately in the design of novel peptide arrays. Our ultimate array design covers numerous immune and metabolic processes including innate and adaptive immunity, inflammatory responses, carbohydrate, protein and fat metabolism, and response to hormones. We have shown the proteomic and phosphoproteomic diversity of chicken and turkey and have designed a valuable research tool for the study of immuno-metabolism within these two species.

  20. Signal transduction in cerebral arteries after subarachnoid hemorrhage-a phosphoproteomic approach

    DEFF Research Database (Denmark)

    Parker, Benjamin; Larsen, Martin Røssel; Povlsen, Gro Klitgaard

    2013-01-01

    After subarachnoid hemorrhage (SAH), pathologic changes in cerebral arteries contribute to delayed cerebral ischemia and poor outcome. We hypothesize such changes are triggered by early intracellular signals, targeting of which may prevent SAH-induced vasculopathy. We performed an unbiased quanti......-induced signaling components downstream and upstream of ERK1/2.Journal of Cerebral Blood Flow & Metabolism advance online publication, 29 May 2013; doi:10.1038/jcbfm.2013.78....... quantitative analysis of early SAH-induced phosphorylations in cerebral arteries and evaluated identified signaling components as targets for prevention of delayed vasculopathy and ischemia. Labeled phosphopeptides from rat cerebral arteries were quantified by high-resolution tandem mass spectrometry. Selected...

  1. Kinomic and phospho-proteomic analysis of breast cancer stem-like cells

    DEFF Research Database (Denmark)

    Leth-Larsen, Rikke; Christensen, Anne Geske Lindhard; Ehmsen, Sidse

    cell death, while the bulk of a tumor lacks these capacities. The resistance mechanisms may cause these cells to survive and become the source of later tumor recurrence, highlighting the need for therapeutic strategies that specifically target pathways central to these cancer stem cells. The CD44hi....../CD24-/low compartment of human breast cancer is enriched in tumor-initiating cells; however the functional heterogeneity within this subpopulation remains poorly defined. From a triple-negative breast cancer cell line we isolated and cloned CD44hi single-cells that exhibited functional heterogeneity....... Transcriptomic analysis using Affymetrix Human Gene 1.0 ST Arrays revealed similar alterations at the gene expression level. We used these single cell clones to further investigate the molecular mechanisms underlying the association between selected proteins, some of them phosphorylated, and cancer stem cell...

  2. Quantitative research.

    Science.gov (United States)

    Watson, Roger

    2015-04-01

    This article describes the basic tenets of quantitative research. The concepts of dependent and independent variables are addressed and the concept of measurement and its associated issues, such as error, reliability and validity, are explored. Experiments and surveys – the principal research designs in quantitative research – are described and key features explained. The importance of the double-blind randomised controlled trial is emphasised, alongside the importance of longitudinal surveys, as opposed to cross-sectional surveys. Essential features of data storage are covered, with an emphasis on safe, anonymous storage. Finally, the article explores the analysis of quantitative data, considering what may be analysed and the main uses of statistics in analysis.

  3. Gel-based phosphoproteomics analysis of sarcoplasmic proteins in postmortem porcine muscle with pH decline rate and time differences

    DEFF Research Database (Denmark)

    Huang, Honggang; Larsen, Martin Røssel; Karlsson, Anders H

    2011-01-01

    Meat quality development is highly influenced by the pH decline caused by the postmortem (PM) glycolysis. Protein phosphorylation is an important mechanism in regulating the activity of glycometabolic enzymes. Here, a gel-based phosphoproteomic study was performed to analyze the protein...... phosphorylation in sarcoplasmic proteins from three groups of pigs with different pH decline rates from PM 1 to 24¿h. Globally, the fast pH decline group had the highest phosphorylation level at PM 1¿h, but lowest at 24¿h, whereas the slow pH decline group showed the reverse case. The same pattern was also...... observed in most individual bands in 1-DE. The protein phosphorylation levels of 12 bands were significantly affected by the synergy effects of pH and time (p...

  4. Differences in Beef Quality between Angus (Bos taurus taurus) and Nellore (Bos taurus indicus) Cattle through a Proteomic and Phosphoproteomic Approach

    Science.gov (United States)

    Chizzotti, Mario Luiz; Vital, Camilo Elber; Baracat-Pereira, Maria Cristina; Barros, Edvaldo; Busato, Karina Costa; Gomes, Rafael Aparecido; Ladeira, Márcio Machado; Martins, Taiane da Silva

    2017-01-01

    Proteins are the major constituents of muscle and are key molecules regulating the metabolic changes during conversion of muscle to meat. Brazil is one of the largest exporters of beef and most Brazilian cattle are composed by zebu (Nellore) genotype. Bos indicus beef is generally leaner and tougher than Bos taurus such as Angus. The aim of this study was to compare the muscle proteomic and phosphoproteomic profile of Angus and Nellore. Seven animals of each breed previously subjected the same growth management were confined for 84 days. Proteins were extracted from Longissimus lumborum samples collected immediately after slaughter and separated by two-dimensional electrophoresis. Pro-Q Diamond stain was used in phosphoproteomics. Proteins identification was performed using matrix assisted laser desorption/ionization time-of-flight mass spectrometry. Tropomyosin alpha-1 chain, troponin-T, myosin light chain-1 fragment, cytoplasmic malate dehydrogenase, alpha-enolase and 78 kDa glucose-regulated protein were more abundant in Nellore, while myosin light chain 3, prohibitin, mitochondrial stress-70 protein and heat shock 70 kDa protein 6 were more abundant in Angus (P<0.05). Nellore had higher phosphorylation of myosin regulatory light chain-2, alpha actin-1, triosephosphate isomerase and 14-3-3 protein epsilon. However, Angus had greater phosphorylation of phosphoglucomutase-1 and troponin-T (P<0.05). Therefore, proteins involved in contraction and muscle organization, myofilaments expressed in fast or slow-twitch fibers and heat shock proteins localized in mitochondria or sarcoplasmic reticulum and involved in cell flux of calcium and apoptosis might be associated with differences in beef quality between Angus and Nellore. Furthermore, prohibitin appears to be a potential biomarker of intramuscular fat in cattle. Additionally, differences in phosphorylation of myofilaments and glycolytic enzymes could be involved with differences in muscle contraction force

  5. Phosphoproteome analysis during larval development and metamorphosis in the spionid polychaete Pseudopolydora vexillosa

    KAUST Repository

    Chandramouli, Kondethimmanahalli

    2011-05-25

    Background: The metamorphosis of the spionid polychaete Pseudopolydora vexillosa includes spontaneous settlement onto soft-bottom habitats and morphogenesis that can be completed in a very short time. A previous study on the total changes to the proteome during the various developmental stages of P. vexillosa suggested that little or no de novo protein synthesis occurs during metamorphosis. In this study, we used multicolor fluorescence detection of proteins in 2-D gels for differential analysis of proteins and phosphoproteins to reveal the dynamics of post-translational modification proteins in this species. A combination of affinity chromatography, 2D-PAGE, and mass spectrometry was used to identify the phosphoproteins in pre-competent larvae, competent larvae, and newly metamorphosed juveniles. Results: We reproducibly detected 210, 492, and 172 phosphoproteins in pre-competent larvae, competent larvae, and newly metamorphosed juveniles, respectively. The highest percentage of phosphorylation was observed during the competent larval stage. About 64 stage-specific phosphoprotein spots were detected in the competent stage, and 32 phosphoproteins were found to be significantly differentially expressed in the three stages. We identified 38 phosphoproteins, 10 of which were differentially expressed during metamorphosis. These phosphoproteins belonged to six categories of biological processes: (1) development, (2) cell differentiation and integrity, (3) transcription and translation, (4) metabolism, (5) protein-protein interaction and proteolysis, and (6) receptors and enzymes. Conclusion: This is the first study to report changes in phosphoprotein expression patterns during the metamorphosis of the marine polychaete P. vexillosa. The higher degree of phosphorylation during the process of attaining competence to settle and metamorphose may be due to fast morphological transitions regulated by various mechanisms. Our data are consistent with previous studies showing a

  6. Exploring the human leukocyte phosphoproteome using a microfluidic reversed-phase-TiO2-reversed-phase high-performance liquid chromatography phosphochip coupled to a quadrupole time-of-flight mass spectrometer.

    Science.gov (United States)

    Raijmakers, Reinout; Kraiczek, Karsten; de Jong, Ad P; Mohammed, Shabaz; Heck, Albert J R

    2010-02-01

    The study of protein phosphorylation events is one of the most important challenges in proteome analysis. Despite the importance of phosphorylation for many regulatory processes in cells and many years of phosphoprotein and phosphopeptide research, the identification and characterization of phosphorylation by mass spectrometry is still a challenging task. Recently, we introduced an approach that facilitates the analysis of phosphopeptides by performing automated, online, TiO(2) enrichment of phosphopeptides prior to mass spectrometry (MS) analysis. The implementation of that method on a "plug-and-play" microfluidic high-performance liquid chromatography (HPLC) chip design will potentially open up efficient phosphopeptide enrichment methods enabling phosphoproteomics analyses by a broader research community. Following our initial proof of principle, whereby the device was coupled to an ion trap, we now show that this so-called phosphochip is capable of the enrichment of large numbers of phosphopeptides from complex cellular lysates, which can be more readily identified when coupled to a higher resolution quadrupole time-of-flight (Q-TOF) mass spectrometer. We use the phosphochip-Q-TOF setup to explore the phosphoproteome of nonstimulated primary human leukocytes where we identify 1012 unique phosphopeptides corresponding to 960 different phosphorylation sites providing for the first time an overview of the phosphoproteome of these important circulating white blood cells.

  7. Deep Proteomics of Breast Cancer Cells Reveals that Metformin Rewires Signaling Networks Away from a Pro-growth State.

    Science.gov (United States)

    Sacco, Francesca; Silvestri, Alessandra; Posca, Daniela; Pirrò, Stefano; Gherardini, Pier Federico; Castagnoli, Luisa; Mann, Matthias; Cesareni, Gianni

    2016-03-23

    Metformin is the most frequently prescribed drug for type 2 diabetes. In addition to its hypoglycemic effects, metformin also lowers cancer incidence. This anti-cancer activity is incompletely understood. Here, we profiled the metformin-dependent changes in the proteome and phosphoproteome of breast cancer cells using high-resolution mass spectrometry. In total, we quantified changes of 7,875 proteins and 15,813 phosphosites after metformin changes. To interpret these datasets, we developed a generally applicable strategy that overlays metformin-dependent changes in the proteome and phosphoproteome onto a literature-derived network. This approach suggested that metformin treatment makes cancer cells more sensitive to apoptotic stimuli and less sensitive to pro-growth stimuli. These hypotheses were tested in vivo; as a proof-of-principle, we demonstrated that metformin inhibits the p70S6K-rpS6 axis in a PP2A-phosphatase dependent manner. In conclusion, analysis of deep proteomics reveals both detailed and global mechanisms that contribute to the anti-cancer activity of metformin.

  8. Quantitative Proteomics Reveals That the Specific Methyltransferases Txr1p and Ezl2p Differentially Affect the Mono-, Di- and Trimethylation States of Histone H3 Lysine 27 (H3K27)*

    OpenAIRE

    Zhang, Chunchao; Molascon, Anthony J.; Gao, Shan; Liu, Yifan; Andrews, Philip C.

    2012-01-01

    Nuclear DNA in eukaryotic cells is assembled into the hierarchical chromatin structure via a process that is dynamically affected by the combinatorial set of post-translational modifications (PTMs) of histones in a dynamic manner responsive to physiological and environmental changes. The precise quantification of these complex modifications is challenging. Here we present a robust MS-based quantitative proteomics method for studying histone PTMs using 15N metabolically labeled histones as the...

  9. The quantitative Morse theorem

    OpenAIRE

    Loi, Ta Le; Phien, Phan

    2013-01-01

    In this paper, we give a proof of the quantitative Morse theorem stated by {Y. Yomdin} in \\cite{Y1}. The proof is based on the quantitative Sard theorem, the quantitative inverse function theorem and the quantitative Morse lemma.

  10. Different binding motifs of the celiac disease-associated HLA molecules DQ2.5, DQ2.2, and DQ7.5 revealed by relative quantitative proteomics of endogenous peptide repertoires

    DEFF Research Database (Denmark)

    Bergseng, Elin; Dørum, Siri; Arntzen, Magnus Ø.

    2014-01-01

    . It was recently demonstrated that T cells of DQ2.5 and DQ2.2 patients recognize distinct sets of gluten epitopes, suggesting that these two DQ2 variants select different peptides for display. To explore whether this is the case, we performed a comprehensive comparison of the endogenous self-peptides bound to HLA......-DQ molecules of B-lymphoblastoid cell lines. Peptides were eluted from affinity-purified HLA molecules of nine cell lines and subjected to quadrupole orbitrap mass spectrometry and MaxQuant software analysis. Altogether, 12,712 endogenous peptides were identified at very different relative abundances...... at position P3. This study demonstrates that relative quantitative comparison of endogenous peptides sampled from our protein metabolism by HLA molecules provides clues to understand HLA association with disease....

  11. Sequential Enrichment with Titania-coated Magnetic Mesoporous Hollow Silica Microspheres and Zirconium Arsenate-modified Magnetic Nanoparticles for the Study of Phosphoproteome of HL60 Cells

    Science.gov (United States)

    Yu, Qiong-Wei; Li, Xiao-Shui; Xiao, Yongsheng; Guo, Lei; Zhang, Fan; Cai, Qian; Feng, Yu-Qi; Yuan, Bi-Feng; Wang, Yinsheng

    2014-01-01

    As one of the most important types of post-translational modifications, reversible phosphorylation of proteins plays crucial roles in a large number of biological processes. However, owing to the relatively low abundance and dynamic nature of phosphorylation and the presence of the unphosphorylated peptides in large excess, phosphopeptide enrichment is indispensable in large-scale phosphoproteomic analysis. Metal oxides including titanium dioxide have become prominent affinity materials to enrich phosphopeptides prior to their analysis using liquid chromatography-mass spectrometry (LC-MS). In the current study, we established a novel strategy, which encompassed strong cation exchange chromatography, sequential enrichment of phosphopeptides using titania-coated magnetic mesoporous hollow silica microspheres (TiO2/MHMSS) and zirconium arsenate-modified magnetic nanoparticles (ZrAs-Fe3O4@SiO2), and LC-MS/MS analysis, for the proteome-wide identification of phosphosites of proteins in HL60 cells. In total, we were able to identify 11579 unique phosphorylation sites in 3432 unique proteins. Additionally, our results suggested that TiO2/MHMSS and ZrAs-Fe3O4@SiO2 are complementary in phosphopeptide enrichment, where the two types of materials displayed preferential binding of peptides carrying multiple and single phosphorylation sites, respectively. PMID:25262027

  12. Sequential enrichment with titania-coated magnetic mesoporous hollow silica microspheres and zirconium arsenate-modified magnetic nanoparticles for the study of phosphoproteome of HL60 cells.

    Science.gov (United States)

    Yu, Qiong-Wei; Li, Xiao-Shui; Xiao, Yongsheng; Guo, Lei; Zhang, Fan; Cai, Qian; Feng, Yu-Qi; Yuan, Bi-Feng; Wang, Yinsheng

    2014-10-24

    As one of the most important types of post-translational modifications, reversible phosphorylation of proteins plays crucial roles in a large number of biological processes. However, owing to the relatively low abundance and dynamic nature of phosphorylation and the presence of the unphosphorylated peptides in large excess, phosphopeptide enrichment is indispensable in large-scale phosphoproteomic analysis. Metal oxides including titanium dioxide have become prominent affinity materials to enrich phosphopeptides prior to their analysis using liquid chromatography-mass spectrometry (LC-MS). In the current study, we established a novel strategy, which encompassed strong cation exchange chromatography, sequential enrichment of phosphopeptides using titania-coated magnetic mesoporous hollow silica microspheres (TiO2/MHMSS) and zirconium arsenate-modified magnetic nanoparticles (ZrAs-Fe3O4@SiO2), and LC-MS/MS analysis, for the proteome-wide identification of phosphosites of proteins in HL60 cells. In total, we were able to identify 11,579 unique phosphorylation sites in 3432 unique proteins. Additionally, our results suggested that TiO2/MHMSS and ZrAs-Fe3O4@SiO2 are complementary in phosphopeptide enrichment, where the two types of materials displayed preferential binding of peptides carrying multiple and single phosphorylation sites, respectively.

  13. 王浆高产蜜蜂咽下腺磷酸化蛋白质组分析%Phosphoproteome Analysis of Hypopharyngeal Glands of High Royal Jelly Producing Bee (Apis mellifera L.)

    Institute of Scientific and Technical Information of China (English)

    鲁小山; 韩宾; 张兰; 冯毛; 房宇; 李荣丽; 周天娥; 李建科

    2013-01-01

    [Objective]High royal jelly producing bee (Apis mellifera L.) is the unique honeybee resource in China. However, the mechanism of high royal jelly producing has not been clearly addressed. In order to reveal the significance of protein phosphorylation in hypopharyngeal gland for royal jelly synthesis and secretion, the phosphoproteome of hypopharyngeal gland of nurse bee (6-12 day) was analyzed.[Method]IMAC (immobilized metal-affinity chromatography) phosphoprotein enrichment, SCX (strong cation exchange) separation, LC-MS/MS (liquid chromatography-mass/mass) identification and bioinformatics analysis were applied to analyze phosphoproteome of hypopharyngeal gland of nurse bee.[Result]Of the identified 117 proteins in the hypopharyngeal gland, 6 of them were phosphorylated on 6 phosphopeptides and assigned 8 phosphorylated sites. They were related to protein translation and synthesis, such as 60S acidic ribosomal proteins P0, P1, P2 and 60S ribosomal protein L15, and major royal jelly proteins 1 and 7 precursor.[Conclusion]The phosphorylation modifications occurred on ribosome proteins of hypopharyngeal gland mainly contribute to the high efficiency of synthesizing and secreting of royal jelly protein. The phosphorylation of royal jelly proteins 1 and 7 maintain the reasonable ratio of calcium to phosphorus of royal jelly with the increasing yield, thus meeting the nutrition demand of fertile egg-laying queens and developing larvae. Hence, the data obtained in this study will provide new knowledge to deeply understand the mechanism how high royal jelly producing bees could produce higher amount of royal jelly at the level of protein phosphorylation.%[目的]通过对王浆高产蜜蜂(Apis mellifera L.)(浙江浆蜂)哺育蜂(6-12 d)咽下腺的磷酸化蛋白质组分析,以期探明蛋白质磷酸化修饰对王浆分泌的生物学意义。[方法]将哺育蜂咽下腺蛋白质液内酶切后,用固相金属离子亲和层析色谱法(IMAC

  14. Quantitative proteomic analysis of HIV-1 infected CD4+ T cells reveals an early host response in important biological pathways: Protein synthesis, cell proliferation, and T-cell activation

    Energy Technology Data Exchange (ETDEWEB)

    Navare, Arti T.; Sova, Pavel; Purdy, David E.; Weiss, Jeffrey M. [Department of Microbiology, University of Washington, Seattle, WA (United States); Wolf-Yadlin, Alejandro [Department of Genome Sciences, University of Washington, Seattle, WA (United States); Korth, Marcus J.; Chang, Stewart T.; Proll, Sean C. [Department of Microbiology, University of Washington, Seattle, WA (United States); Jahan, Tahmina A. [Proteomics Resource, UW Medicine at South Lake Union, Seattle, WA (United States); Krasnoselsky, Alexei L.; Palermo, Robert E. [Department of Microbiology, University of Washington, Seattle, WA (United States); Katze, Michael G., E-mail: honey@uw.edu [Department of Microbiology, University of Washington, Seattle, WA (United States); Washington National Primate Research Center, University of Washington, Seattle, WA (United States)

    2012-07-20

    Human immunodeficiency virus (HIV-1) depends upon host-encoded proteins to facilitate its replication while at the same time inhibiting critical components of innate and/or intrinsic immune response pathways. To characterize the host cell response on protein levels in CD4+ lymphoblastoid SUP-T1 cells after infection with HIV-1 strain LAI, we used mass spectrometry (MS)-based global quantitation with iTRAQ (isobaric tag for relative and absolute quantification). We found 266, 60 and 22 proteins differentially expressed (DE) (P-value{<=}0.05) at 4, 8, and 20 hours post-infection (hpi), respectively, compared to time-matched mock-infected samples. The majority of changes in protein abundance occurred at an early stage of infection well before the de novo production of viral proteins. Functional analyses of these DE proteins showed enrichment in several biological pathways including protein synthesis, cell proliferation, and T-cell activation. Importantly, these early changes before the time of robust viral production have not been described before.

  15. Ambiguity Revealed

    OpenAIRE

    Subir Bose; Matthew Polisson; Ludovic Renou

    2012-01-01

    We derive necessary and suffcient conditions for data sets composed of state-contingent prices and consumption to be consistent with two prominent models of decision making under ambiguity: variational preferences and smooth ambiguity. The revealed preference conditions for the maxmin expected utility and subjective expected utility models are characterized as special cases.

  16. Ambiguity revealed

    OpenAIRE

    Bayer, Ralph-C; Bose, Subir; Polisson, Matthew; Renou, Ludovic

    2013-01-01

    We derive necessary and sufficient conditions for data sets composed of state-contingent prices and consumption to be consistent with two prominent models of decision making under uncertainty: variational preferences and smooth ambiguity. The revealed preference conditions for subjective expected utility, maxmin expected utility, and multiplier preferences are characterised as special cases. We implement our tests on data from a portfolio choice experiment.

  17. Global expression profiling of rice microRNAs by one-tube stem-loop reverse transcription quantitative PCR revealed important roles of microRNAs in abiotic stress responses.

    Science.gov (United States)

    Shen, Jianqiang; Xie, Kabin; Xiong, Lizhong

    2010-12-01

    MicroRNAs are a class of endogenous small RNA molecules (20-24 nucleotides) that have pivotal roles in regulating gene expression mostly at posttranscriptional levels in plants. Plant microRNAs have been implicated in the regulation of diverse biological processes including growth and stress responses. However, the information about microRNAs in regulating abiotic stress responses in rice is limited. We optimized a one-tube stem-loop reverse transcription quantitative PCR (ST-RT qPCR) for high-throughput expression profiling analysis of microRNAs in rice under normal and stress conditions. The optimized ST-RT qPCR method was as accurate as small RNA gel blotting and was more convenient and time-saving than other methods in quantifying microRNAs. With this method, 41 rice microRNAs were quantified for their relative expression levels after drought, salt, cold, and abscisic acid (ABA) treatments. Thirty-two microRNAs showed induced or suppressed expression after stress or ABA treatment. Further analysis suggested that stress-responsive cis-elements were enriched in the promoters of stress-responsive microRNA genes. The expressions of five and seven microRNAs were significantly affected in the rice plant with defects in stress tolerance regulatory genes OsSKIPa and OsbZIP23, respectively. Some of the predicted target genes of these microRNAs were also related to abiotic stresses. We conclude that ST-RT qPCR is an efficient and reliable method for expression profiling of microRNAs and a significant portion of rice microRNAs participate in abiotic stress response and regulation.

  18. TRI12 based quantitative real-time PCR assays reveal the distribution of trichothecene genotype of F. graminearum and F. culmorum isolates in Danish small grain cereals

    DEFF Research Database (Denmark)

    Nielsen, L. K.; Jensen, J. D.; Rodríguez, A.;

    2012-01-01

    species complex, Fusarium culmorum, Fusarium cerealis and Fusarium pseudograminearum. These assays were applied on a total of 378 field samples of cereal grain of wheat, barley, triticale, rye and oats collected from 2003 to 2007 to study the trichothecene genotype composition in Danish cereals. The three...... in wheat. The NIV genotype was found at low levels in most samples. Study of genotype composition within the Danish F. graminearum and F. culmorum population was based on principal component analysis (PCA). PCA revealed that the dominating genotype of F. graminearum in wheat is 15ADON. For barley, the PCA...... analysis indicated that the F. graminearum population consisted of all three genotypes, and in triticale, the F. graminearum population consisted mainly of 15ADON genotype. F. culmorum/F. cerealis showed correlation to the NIV genotype in wheat and triticale but not in barley. F. culmorum/F. cerealis also...

  19. Mathematics revealed

    CERN Document Server

    Berman, Elizabeth

    1979-01-01

    Mathematics Revealed focuses on the principles, processes, operations, and exercises in mathematics.The book first offers information on whole numbers, fractions, and decimals and percents. Discussions focus on measuring length, percent, decimals, numbers as products, addition and subtraction of fractions, mixed numbers and ratios, division of fractions, addition, subtraction, multiplication, and division. The text then examines positive and negative numbers and powers and computation. Topics include division and averages, multiplication, ratios, and measurements, scientific notation and estim

  20. Major Quantitative Trait Loci and Putative Candidate Genes for Powdery Mildew Resistance and Fruit-Related Traits Revealed by an Intraspecific Genetic Map for Watermelon (Citrullus lanatus var. lanatus).

    Science.gov (United States)

    Kim, Kwang-Hwan; Hwang, Ji-Hyun; Han, Dong-Yeup; Park, Minkyu; Kim, Seungill; Choi, Doil; Kim, Yongjae; Lee, Gung Pyo; Kim, Sun-Tae; Park, Young-Hoon

    2015-01-01

    An intraspecific genetic map for watermelon was constructed using an F2 population derived from 'Arka Manik' × 'TS34' and transcript sequence variants and quantitative trait loci (QTL) for resistance to powdery mildew (PMR), seed size (SS), and fruit shape (FS) were analyzed. The map consists of 14 linkage groups (LGs) defined by 174 cleaved amplified polymorphic sequences (CAPS), 2 derived-cleaved amplified polymorphic sequence markers, 20 sequence-characterized amplified regions, and 8 expressed sequence tag-simple sequence repeat markers spanning 1,404.3 cM, with a mean marker interval of 6.9 cM and an average of 14.6 markers per LG. Genetic inheritance and QTL analyses indicated that each of the PMR, SS, and FS traits is controlled by an incompletely dominant effect of major QTLs designated as pmr2.1, ss2.1, and fsi3.1, respectively. The pmr2.1, detected on chromosome 2 (Chr02), explained 80.0% of the phenotypic variation (LOD = 30.76). This QTL was flanked by two CAPS markers, wsb2-24 (4.00 cM) and wsb2-39 (13.97 cM). The ss2.1, located close to pmr2.1 and CAPS marker wsb2-13 (1.00 cM) on Chr02, explained 92.3% of the phenotypic variation (LOD = 68.78). The fsi3.1, detected on Chr03, explained 79.7% of the phenotypic variation (LOD = 31.37) and was flanked by two CAPS, wsb3-24 (1.91 cM) and wsb3-9 (7.00 cM). Candidate gene-based CAPS markers were developed from the disease resistance and fruit shape gene homologs located on Chr.02 and Chr03 and were mapped on the intraspecific map. Colocalization of these markers with the major QTLs indicated that watermelon orthologs of a nucleotide-binding site-leucine-rich repeat class gene containing an RPW8 domain and a member of SUN containing the IQ67 domain are candidate genes for pmr2.1 and fsi3.1, respectively. The results presented herein provide useful information for marker-assisted breeding and gene cloning for PMR and fruit-related traits.

  1. The quantitative analysis by stem-loop real-time PCR revealed the microRNA-34a, microRNA-155 and microRNA-200c overexpression in human colorectal cancer.

    Science.gov (United States)

    Wang, Mojin; Zhang, Peng; Li, Yuan; Liu, Guanghui; Zhou, Bin; Zhan, Lan; Zhou, Zongguang; Sun, Xiaofeng

    2012-12-01

    The recently identified class of microRNAs (miRNAs) provided a new insight in cancer research. As the member of miRNAs family, miR-34a, miR-155 and miR-200c abnormalities have been found in various types of cancer. However, the relationship between these three miRNAs (miR-34a, miR-155 and miR-200c) and colorectal cancer is unclear. In this study, we applied stem-loop real-time PCR to quantitatively detect miR-34a, miR-155 and miR-200c expression in 109 pair-matched human colorectal cancers and the corresponding normal mucosa. MiR-34a (2.2-fold), miR-155 (2.3-fold) and miR-200c (3.1-fold) were all expressed at higher levels in colorectal cancer (P = 0.001, 0.005 and 0.001, respectively). In rectum, miR-34a and miR-200c were significantly upregulated (P = 0.006 and 0.007), while the miR-155 overexpression was not statistically significant (P = 0.083). In colon, the higher expression of three miRNAs was seen, however, without significant difference (P > 0.05). We also found that the miR-34a expression was higher in rectal cancer having more advanced TNM stage (III + IV, P = 0.03). Then miR-200c expression was positively correlated with and sera CEA level of rectal cancer patients (P = 0.04). In conclusion, our results thus suggest that the overexpression of miR-34a, miR-155 and miR-200c be associated with the development of colorectal cancer, meanwhile miR-34a may be involved in the development and progression of rectal cancer. The more deeply and larger scale research are required to prove the correlation.

  2. REVEALED ALTRUISM

    OpenAIRE

    Cox, James C; Friedman, Daniel; Sadiraj, Vjollca

    2009-01-01

    This pap er develops a theory of revealed preferences over oneís own and othersímonetary payo§s. We intro duce ìmore altruistic thanî(MAT), a partial ordering over preferences, and interpret it with known parametric mo dels. We also intro duce and illustrate ìmore generous thanî (MGT), a partial ordering over opp ortunity sets. Several recent discussions of altruism fo cus on two player extensive form games of complete information in which the Örst mover (FM) cho oses a more or less gen...

  3. Insights into chemoselectivity principles in metal oxide affinity chromatography using tailored nanocast metal oxide microspheres and mass spectrometry-based phosphoproteomics.

    Science.gov (United States)

    Leitner, Alexander; Sakeye, Motolani; Zimmerli, Christian Eugen; Smått, Jan-Henrik

    2017-05-30

    The ability to comprehensively characterize biological samples, including tissues and body fluids, opens up new possibilities to diagnose and treat diseases and to better understand fundamental biological processes. For this purpose, suitable experimental workflows need to be designed. In this context, materials with particular chemoselective properties are used for the enrichment of certain classes of (bio)molecules. Metal oxides such as titanium dioxide have become the materials of choice for the large-scale study of protein phosphorylation in phosphoproteomics. Despite their widespread use, the main factors influencing their performance (for example, affinity and specificity) are not completely understood. This understanding is, however, crucial to develop improved materials and methods. Here, we used the nanocasting method to prepare microspheres of seven metal oxides with comparable textural properties, allowing an objective comparison of the materials and their binding properties. We evaluated these materials with samples of different complexity, ranging from synthetic peptides to whole cell lysates, using liquid chromatography-tandem mass spectrometry as a readout. A set of more than 7000 identified phosphopeptides allowed us to study differences between the metal oxide sorbents in detail. Importantly, the performance of the affinity materials was found to be mainly correlated with the oxides' isoelectric points (IEPs), with the materials that enriched the highest number of phosphopeptides having an IEP of around 6. This included the widely used TiO2 and ZrO2, but also In2O3 that was not previously known to possess affinity to phosphates. This finding supports the conclusion that the IEP has a stronger influence than the particular type of metal oxide and contrasts earlier reports that compared a limited number of materials with often unknown textural properties. Taken together, we introduce new metal oxides suitable for phosphopeptide enrichment, provide

  4. Phosphoproteome and transcription factor activity profiling identify actions of the anti-inflammatory agent UTL-5g in LPS stimulated RAW 264.7 cells including disrupting actin remodeling and STAT-3 activation.

    Science.gov (United States)

    Carruthers, Nicholas J; Stemmer, Paul M; Chen, Ben; Valeriote, Frederick; Gao, Xiaohua; Guatam, Subhash C; Shaw, Jiajiu

    2017-09-15

    UTL-5g is a novel small-molecule TNF-alpha modulator. It reduces cisplatin-induced side effects by protecting kidney, liver, and platelets, thereby increasing tolerance for cisplatin. UTL-5g also reduces radiation-induced acute liver toxicity. The mechanism of action for UTL-5g is not clear at the present time. A phosphoproteomic analysis to a depth of 4943 phosphopeptides and a luminescence-based transcription factor activity assay were used to provide complementary analyses of signaling events that were disrupted by UTL-5g in RAW 264.7 cells. Transcriptional activity downstream of the interferon gamma, IL-6, type 1 Interferon, TGF-β, PKC/Ca(2+) and the glucocorticoid receptor pathways were disrupted by UTL-5g. Phosphoproteomic analysis indicated that hyperphosphorylation of proteins involved in actin remodeling was suppressed by UTL-5g (gene set analysis, FDR 5g. This global characterization of UTL-5g activity in a macrophage cell line discovered that it disrupts selected aspects of LPS signaling including Stat3 activation and actin remodeling providing new insight on how UTL-5g acts to reduce cisplatin-induced side effects. Copyright © 2017 Elsevier B.V. All rights reserved.