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Sample records for enables quantitative phosphoproteomics

  1. Sample Collection Method Bias Effects in Quantitative Phosphoproteomics.

    PubMed

    Kanshin, Evgeny; Tyers, Michael; Thibault, Pierre

    2015-07-01

    Current advances in selective enrichment, fractionation, and MS detection of phosphorylated peptides allowed identification and quantitation of tens of thousands phosphosites from minute amounts of biological material. One of the major challenges in the field is preserving the in vivo phosphorylation state of the proteins throughout the sample preparation workflow. This is typically achieved by using phosphatase inhibitors and denaturing conditions during cell lysis. Here we determine if the upstream cell collection techniques could introduce changes in protein phosphorylation. To evaluate the effect of sample collection protocols on the global phosphorylation status of the cell, we compared different sample workflows by metabolic labeling and quantitative mass spectrometry on Saccharomyces cerevisiae cell cultures. We identified highly similar phosphopeptides for cells harvested in ice cold isotonic phosphate buffer, cold ethanol, trichloroacetic acid, and liquid nitrogen. However, quantitative analyses revealed that the commonly used phosphate buffer unexpectedly activated signaling events. Such effects may introduce systematic bias in phosphoproteomics measurements and biochemical analysis. PMID:26040406

  2. Quantitative Phosphoproteomics Analysis of ERBB3/ERBB4 Signaling

    PubMed Central

    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. PMID:26745281

  3. 15N labeled brain enables quantification of proteome and phosphoproteome in cultured primary neurons

    PubMed Central

    Liao, Lujian; Sando, Richard C.; Farnum, John B.; Vanderklish, Peter W.; Maximov, Anton; Yates, John R.

    2011-01-01

    Terminally differentiated primary cells represent a valuable in vitro model to study signaling events associated within a specific tissue. Quantitative proteomic methods using metabolic labeling in primary cells encounter labeling efficiency issues hindering the use of these cells. Here we developed a method to quantify the proteome and phosphoproteome of cultured neurons using 15N labeled brain tissue as an internal standard, and applied this method to determine how an inhibitor of an excitatory neural transmitter receptor, phencyclidine (PCP), affects the global phosphoproteome of cortical neurons. We identified over 10,000 phosphopeptides and made accurate quantitative measurements of the neuronal phosphoproteome after neuronal inhibition. We show that short PCP treatments lead to changes in phosphorylation for 7% of neuronal phosphopeptides and that prolonged PCP treatment alters the total levels of several proteins essential for synaptic transmission and plasticity and leads to a massive reduction in the synaptic strength of inhibitory synapses. The results provide valuable insights into the dynamics of molecular networks implicated in PCP-mediated NMDA receptor inhibition and sensorimotor deficits. PMID:22070516

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

    PubMed

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

    2016-02-01

    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

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

    PubMed Central

    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

  6. Quantitative Phosphoproteomics Revealed Glucose-Stimulated Responses of Islet Associated with Insulin Secretion.

    PubMed

    Li, Jiaming; Li, Qingrun; Tang, Jiashu; Xia, Fangying; Wu, Jiarui; Zeng, Rong

    2015-11-01

    As central tissue of glucose homeostasis, islet has been an important focus of diabetes research. Phosphorylation plays pivotal roles in islet function, especially in islet glucose-stimulated insulin secretion. A systematic view on how phosphorylation networks were coordinately regulated in this process remains lacking, partially due to the limited amount of islets from an individual for a phosphoproteomic analysis. Here we optimized the in-tip and best-ratio phosphopeptide enrichment strategy and a SILAC-based workflow for processing rat islet samples. With limited islet lysates from each individual rat (20-47 μg), we identified 8539 phosphosites on 2487 proteins. Subsequent quantitative analyses uncovered that short-term (30 min) high glucose stimulation induced coordinate responses of islet phosphoproteome on multiple biological levels, including insulin secretion related pathways, cytoskeleton dynamics, protein processing in ER and Golgi, transcription and translation, and so on. Furthermore, three glucose-responsive phosphosites (Prkar1a pT75pS77 and Tagln2 pS163) from the data set were proved to be correlated with insulin secretion. Overall, we initially gave an in-depth map of islet phosphoproteome regulated by glucose on individual rat level. This was a significant addition to our knowledge about how phosphorylation networks responded in insulin secretion. Also, the list of changed phosphosites was a valuable resource for molecular researchers in diabetes field. PMID:26437020

  7. Quantitative phospho-proteomics to investigate the polo-like kinase 1-dependent phospho-proteome.

    PubMed

    Grosstessner-Hain, Karin; Hegemann, Björn; Novatchkova, Maria; Rameseder, Jonathan; Joughin, Brian A; Hudecz, Otto; Roitinger, Elisabeth; Pichler, Peter; Kraut, Norbert; Yaffe, Michael B; Peters, Jan-Michael; Mechtler, Karl

    2011-11-01

    Polo-like kinase 1 (PLK1) is a key regulator of mitotic progression and cell division, and small molecule inhibitors of PLK1 are undergoing clinical trials to evaluate their utility in cancer therapy. Despite this importance, current knowledge about the identity of PLK1 substrates is limited. Here we present the results of a proteome-wide analysis of PLK1-regulated phosphorylation sites in mitotic human cells. We compared phosphorylation sites in HeLa cells that were or were not treated with the PLK1-inhibitor BI 4834, by labeling peptides via methyl esterification, fractionation of peptides by strong cation exchange chromatography, and phosphopeptide enrichment via immobilized metal affinity chromatography. Analysis by quantitative mass spectrometry identified 4070 unique mitotic phosphorylation sites on 2069 proteins. Of these, 401 proteins contained one or multiple phosphorylation sites whose abundance was decreased by PLK1 inhibition. These include proteins implicated in PLK1-regulated processes such as DNA damage, mitotic spindle formation, spindle assembly checkpoint signaling, and chromosome segregation, but also numerous proteins that were not suspected to be regulated by PLK1. Analysis of amino acid sequence motifs among phosphorylation sites down-regulated under PLK1 inhibition in this data set identified two potential novel variants of the PLK1 consensus motif. PMID:21857030

  8. Quantitative Phosphoproteomics Analysis of Nitric Oxide–Responsive Phosphoproteins in Cotton Leaf

    PubMed Central

    Song, Meizhen; Pang, Chaoyou; Wei, Hengling; Liu, Ji; Zhan, Xianjin; Lan, Jiayang; Feng, Changhui; Zhang, Shengxi; Yu, Shuxun

    2014-01-01

    Knowledge of phosphorylation events and their regulation is crucial to understanding the functional biology of plant proteins, but very little is currently known about nitric oxide–responsive phosphorylation in plants. Here, we report the first large-scale, quantitative phosphoproteome analysis of cotton (Gossypium hirsutum) treated with sodium nitroprusside (nitric oxide donor) by utilizing the isobaric tag for relative and absolute quantitation (iTRAQ) method. A total of 1315 unique phosphopeptides, spanning 1528 non-redundant phosphorylation sites, were detected from 1020 cotton phosphoproteins. Among them, 183 phosphopeptides corresponding to 167 phosphoproteins were found to be differentially phosphorylated in response to sodium nitroprusside. Several of the phosphorylation sites that we identified, including RQxS, DSxE, TxxxxSP and SPxT, have not, to our knowledge, been reported to be protein kinase sites in other species. The phosphoproteins identified are involved in a wide range of cellular processes, including signal transduction, RNA metabolism, intracellular transport and so on. This study reveals unique features of the cotton phosphoproteome and provides new insight into the biochemical pathways that are regulated by nitric oxide. PMID:24714030

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

    PubMed

    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. PMID:26613898

  10. Systems-wide Analysis of a Phosphatase Knock-down by Quantitative Proteomics and Phosphoproteomics

    PubMed Central

    Hilger, Maximiliane; Bonaldi, Tiziana; Gnad, Florian; Mann, Matthias

    2009-01-01

    Signal transduction in metazoans regulates almost all aspects of biological function, and aberrant signaling is involved in many diseases. Perturbations in phosphorylation-based signaling networks are typically studied in a hypothesis-driven approach, using phospho-specific antibodies. Here we apply quantitative, high-resolution mass spectrometry to determine the systems response to the depletion of one signaling component. Drosophila cells were metabolically labeled using stable isotope labeling by amino acids in cell culture (SILAC) and the phosphatase Ptp61F, the ortholog of mammalian PTB1B, a drug target for diabetes, was knocked down by RNAi. In total we detected more than 10,000 phosphorylation sites in the phosphoproteome of Drosophila Schneider cells and trained a phosphorylation site predictor with this data. SILAC-based quantitation after phosphatase knock-down showed that apart from the phosphatase, the proteome was minimally affected whereas 288 of 6,478 high-confidence phosphorylation sites changed significantly. Responses at the phosphotyrosine level included the already described Ptp61F substrates Stat92E and Abi. Our analysis highlights a connection of Ptp61F to cytoskeletal regulation through GTPase regulating proteins and focal adhesion components. PMID:19429919

  11. Quantitative Circadian Phosphoproteomic Analysis of Arabidopsis Reveals Extensive Clock Control of Key Components in Physiological, Metabolic, and Signaling Pathways*

    PubMed Central

    Choudhary, Mani Kant; Nomura, Yuko; Wang, Lei; Nakagami, Hirofumi; Somers, David E.

    2015-01-01

    The circadian clock provides adaptive advantages to an organism, resulting in increased fitness and survival. The phosphorylation events that regulate circadian-dependent signaling and the processes which post-translationally respond to clock-gated signals are largely unknown. To better elucidate post-translational events tied to the circadian system we carried out a survey of circadian-regulated protein phosphorylation events in Arabidopsis seedlings. A large-scale mass spectrometry-based quantitative phosphoproteomics approach employing TiO2-based phosphopeptide enrichment techniques identified and quantified 1586 phosphopeptides on 1080 protein groups. A total of 102 phosphopeptides displayed significant changes in abundance, enabling the identification of specific patterns of response to circadian rhythms. Our approach was sensitive enough to quantitate oscillations in the phosphorylation of low abundance clock proteins (EARLY FLOWERING4; ELF4 and PSEUDORESPONSE REGULATOR3; PRR3) as well as other transcription factors and kinases. During constant light, extensive cyclic changes in phosphorylation status occurred in critical regulators, implicating direct or indirect regulation by the circadian system. These included proteins influencing transcriptional regulation, translation, metabolism, stress and phytohormones-mediated responses. We validated our analysis using the elf4–211 allele, in which an S45L transition removes the phosphorylation herein identified. We show that removal of this phosphorylatable site diminishes interaction with EARLY FLOWERING3 (ELF3), a key partner in a tripartite evening complex required for circadian cycling. elf4–211 lengthens period, which increases with increasing temperature, relative to the wild type, resulting in a more stable temperature compensation of circadian period over a wider temperature range. PMID:26091701

  12. Research Resource: Identification of Novel Growth Hormone-Regulated Phosphorylation Sites by Quantitative Phosphoproteomics

    PubMed Central

    Ray, Bridgette N.; Kweon, Hye Kyong; Argetsinger, Lawrence S.; Fingar, Diane C.; Andrews, Philip C.

    2012-01-01

    GH and GH receptors are expressed throughout life, and GH elicits a diverse range of responses, including growth and altered metabolism. It is therefore important to understand the full spectrum of GH signaling pathways and cellular responses. We applied mass spectrometry-based phosphoproteomics combined with stable isotope labeling with amino acids in cell culture to identify proteins rapidly phosphorylated in response to GH in 3T3-F442A preadipocytes. We identified 132 phosphosites in 95 proteins that exhibited rapid (5 or 15 min) GH-dependent statistically significant increases in phosphorylation by more than or equal to 50% and 96 phosphosites in 46 proteins that were down-regulated by GH by more than or equal to 30%. Several of the GH-stimulated phosphorylation sites were known (e.g. regulatory Thr/Tyr in Erks 1 and 2, Tyr in signal transducers and activators of transcription (Stat) 5a and 5b, Ser939 in tuberous sclerosis protein (TSC) 2 or tuberin). The remaining 126 GH-stimulated sites were not previously associated with GH. Kyoto Encyclopedia of Genes and Genomes pathway analysis of GH-stimulated sites indicated enrichment in proteins associated with the insulin and mammalian target of rapamycin (mTOR) pathways, regulation of the actin cytoskeleton, and focal adhesions. Akt/protein kinase A consensus sites (RXRXXS/T) were the most commonly phosphorylated consensus sites. Immunoblotting confirmed GH-stimulated phosphorylation of all seven novel GH-dependent sites tested [regulatory sites in proline-rich Akt substrate, 40 kDA (PRAS40), regulatory associated protein of mTOR, ATP-citrate lyase, Na+/H+ exchanger-1, N-myc downstream regulated gene 1, and Shc]). The immunoblot results suggest that many, if not most, of the GH-stimulated phosphosites identified in this large-scale quantitative phosphoproteomics analysis, including sites in multiple proteins in the Akt/ mTOR complex 1 pathway, are phosphorylated in response to GH. Their identification significantly

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

    SciTech Connect

    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.

  14. Quantitative Phosphoproteomic Profiling of Human Non-Small Cell Lung Cancer Tumors

    PubMed Central

    Schweppe, Devin K.; Rigas, James R.; Gerber, Scott A.

    2013-01-01

    Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths worldwide. Within the molecular scope of NCSLC, a complex landscape of dysregulated cellular signaling has emerged, defined largely by mutations in select mediators of signal transduction, including the epidermal growth factor receptor (EGFR) and anaplastic lymphoma (ALK) kinases. Consequently, these mutant kinases become constitutively activated and targets for chemotherapeutic intervention. Encouragingly, small molecule inhibitors of these pathways have shown promise in clinical trials or are approved for clinical use. However, many protein kinases are dysregulated in NSCLC without genetic mutations. To quantify differences in tumor cell signaling that are transparent to genomic methods, we established a super-SILAC internal standard derived from NSCLC cell lines grown in vitro and labeled with heavy lysine and arginine, and deployed them in a phosphoproteomics workflow. We identified 9019 and 8753 phosphorylation sites in two separate tumors. Relative quantification of phosphopeptide abundance between tumor samples allowed for the determination of specific hubs and pathways differing between each tumor. Sites downstream of Ras showed decreased inhibitory phosphorylation (Raf/Mek) and increased activating phosphorylation (Erk1/2) in one tumor versus another. In this way, we were able to quantitatively access oncogenic kinase signaling in primary human tumors. PMID:23911959

  15. Automated, Reproducible, Titania-Based Phosphopeptide Enrichment Strategy for Label-Free Quantitative Phosphoproteomics

    PubMed Central

    Richardson, Brenna McJury; Soderblom, Erik J.; Thompson, J. Will; Moseley, M. Arthur

    2013-01-01

    An automated phosphopeptide enrichment strategy is described using titanium dioxide (TiO2)-packed, fused silica capillaries for use with liquid chromatography (LC)-mass spectrometry (MS)/MS-based, label-free proteomics workflows. To correlate an optimum peptide:TiO2 loading ratio between different particle types, the ratio of phenyl phosphate-binding capacities was used. The optimum loading for the column was then verified through replicate enrichments of a range of quantities of digested rat brain tissue cell lysate. Fractions were taken during sample loading, multiple wash steps, and the elution steps and analyzed by LC-MS/MS to gauge the efficiency and reproducibility of the enrichment. Greater than 96% of the total phosphopeptides were detected in the elution fractions, indicating efficient trapping of the phosphopeptides on the first pass of enrichment. The quantitative reproducibility of the automated setup was also improved greatly with phosphopeptide intensities from replicate enrichments exhibiting a median coefficient of variation (CV) of 5.8%, and 80% of the identified phosphopeptides had CVs below 11.1%, while maintaining >85% specificity. By providing this high degree of analytical reproducibility, this method allows for label-free phosphoproteomics over large sample sets with complex experimental designs (multiple biological conditions, multiple biological replicates, multiple time-points, etc.), including large-scale clinical cohorts. PMID:23542237

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

    PubMed

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

    2015-08-01

    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. PMID:26112267

  17. Quantitative Phosphoproteomics Reveals the Role of Protein Arginine Phosphorylation in the Bacterial Stress Response*

    PubMed Central

    Schmidt, Andreas; Trentini, Débora Broch; Spiess, Silvia; Fuhrmann, Jakob; Ammerer, Gustav; Mechtler, Karl; Clausen, Tim

    2014-01-01

    Arginine phosphorylation is an emerging protein modification implicated in the general stress response of Gram-positive bacteria. The modification is mediated by the arginine kinase McsB, which phosphorylates and inactivates the heat shock repressor CtsR. In this study, we developed a mass spectrometric approach accounting for the peculiar chemical properties of phosphoarginine. The improved methodology was used to analyze the dynamic changes in the Bacillus subtilis arginine phosphoproteome in response to different stress situations. Quantitative analysis showed that a B. subtilis mutant lacking the YwlE arginine phosphatase accumulated a strikingly large number of arginine phosphorylations (217 sites in 134 proteins), however only a minor fraction of these sites was increasingly modified during heat shock or oxidative stress. The main targets of McsB-mediated arginine phosphorylation comprise central factors of the stress response system including the CtsR and HrcA heat shock repressors, as well as major components of the protein quality control system such as the ClpCP protease and the GroEL chaperonine. These findings highlight the impact of arginine phosphorylation in orchestrating the bacterial stress response. PMID:24263382

  18. Quantitative Phosphoproteome Profiling of Iron-Deficient Arabidopsis Roots1[C][W

    PubMed Central

    Lan, Ping; Li, Wenfeng; Wen, Tuan-Nan; Schmidt, Wolfgang

    2012-01-01

    Iron (Fe) is an essential mineral nutrient for plants, but often it is not available in sufficient quantities to sustain optimal growth. To gain insights into adaptive processes to low Fe availability at the posttranslational level, we conducted a quantitative analysis of Fe deficiency-induced changes in the phosphoproteome profile of Arabidopsis (Arabidopsis thaliana) roots. Isobaric tags for relative and absolute quantitation-labeled phosphopeptides were analyzed by liquid chromatography-tandem mass spectrometry on an LTQ-Orbitrap with collision-induced dissociation and high-energy collision dissociation capabilities. Using a combination of titanium dioxide and immobilized metal affinity chromatography to enrich phosphopeptides, we extracted 849 uniquely identified phosphopeptides corresponding to 425 proteins and identified several not previously described phosphorylation motifs. A subset of 45 phosphoproteins was defined as being significantly changed in abundance upon Fe deficiency. Kinase motifs in Fe-responsive proteins matched to protein kinase A/calcium calmodulin-dependent kinase II, casein kinase II, and proline-directed kinase, indicating a possible critical function of these kinase classes in Fe homeostasis. To validate our analysis, we conducted site-directed mutagenesis on IAA-CONJUGATE-RESISTANT4 (IAR4), a protein putatively functioning in auxin homeostasis. iar4 mutants showed compromised root hair formation and developed shorter primary roots. Changing serine-296 in IAR4 to alanine resulted in a phenotype intermediate between mutant and wild type, whereas acidic substitution to aspartate to mimic phosphorylation was either lethal or caused an extreme dwarf phenotype, supporting the critical importance of this residue in Fe homeostasis. Our analyses further disclose substantial changes in the abundance of phosphoproteins involved in primary carbohydrate metabolism upon Fe deficiency, complementing the picture derived from previous proteomic and

  19. Identification of Candidate Cyclin-dependent kinase 1 (Cdk1) Substrates in Mitosis by Quantitative Phosphoproteomics.

    PubMed

    Petrone, Adam; Adamo, Mark E; Cheng, Chao; Kettenbach, Arminja N

    2016-07-01

    Cyclin-dependent kinase 1 (Cdk1) is an essential regulator of many mitotic processes including the reorganization of the cytoskeleton, chromosome segregation, and formation and separation of daughter cells. Deregulation of Cdk1 activity results in severe defects in these processes. Although the role of Cdk1 in mitosis is well established, only a limited number of Cdk1 substrates have been identified in mammalian cells. To increase our understanding of Cdk1-dependent phosphorylation pathways in mitosis, we conducted a quantitative phosphoproteomics analysis in mitotic HeLa cells using two small molecule inhibitors of Cdk1, Flavopiridol and RO-3306. In these analyses, we identified a total of 24,840 phosphopeptides on 4,273 proteins, of which 1,215 phosphopeptides on 551 proteins were significantly reduced by 2.5-fold or more upon Cdk1 inhibitor addition. Comparison of phosphopeptide quantification upon either inhibitor treatment revealed a high degree of correlation (R(2) value of 0.87) between the different datasets. Motif enrichment analysis of significantly regulated phosphopeptides revealed enrichment of canonical Cdk1 kinase motifs. Interestingly, the majority of proteins identified in this analysis contained two or more Cdk1 inhibitor-sensitive phosphorylation sites, were highly connected with other candidate Cdk1 substrates, were enriched at specific subcellular structures, or were part of protein complexes as identified by the CORUM database. Furthermore, candidate Cdk1 substrates were enriched in G2 and M phase-specific genes. Finally, we validated a subset of candidate Cdk1 substrates by in vitro kinase assays. Our findings provide a valuable resource for the cell signaling and mitosis research communities and greatly increase our knowledge of Cdk1 substrates and Cdk1-dependent signaling pathways. PMID:27134283

  20. Quantitative phosphoproteomics of protein kinase SnRK1 regulated protein phosphorylation in Arabidopsis under submergence.

    PubMed

    Cho, Hsing-Yi; Wen, Tuan-Nan; Wang, Ying-Tsui; Shih, Ming-Che

    2016-04-01

    SNF1 RELATED PROTEIN KINASE 1 (SnRK1) is proposed to be a central integrator of the plant stress and energy starvation signalling pathways. We observed that the Arabidopsis SnRK1.1 dominant negative mutant (SnRK1.1 (K48M) ) had lower tolerance to submergence than the wild type, suggesting that SnRK1.1-dependent phosphorylation of target proteins is important in signalling pathways triggered by submergence. We conducted quantitative phosphoproteomics and found that the phosphorylation levels of 57 proteins increased and the levels of 27 proteins decreased in Col-0 within 0.5-3h of submergence. Among the 57 proteins with increased phosphorylation in Col-0, 38 did not show increased phosphorylation levels in SnRK1.1 (K48M) under submergence. These proteins are involved mainly in sugar and protein synthesis. In particular, the phosphorylation of MPK6, which is involved in regulating ROS responses under abiotic stresses, was disrupted in the SnRK1.1 (K48M) mutant. In addition, PTP1, a negative regulator of MPK6 activity that directly dephosphorylates MPK6, was also regulated by SnRK1.1. We also showed that energy conservation was disrupted in SnRK1.1 (K48M) , mpk6, and PTP1 (S7AS8A) under submergence. These results reveal insights into the function of SnRK1 and the downstream signalling factors related to submergence. PMID:27029354

  1. Quantitative Phosphoproteomics Reveals Wee1 Kinase as a Therapeutic Target in a Model of Proneural Glioblastoma.

    PubMed

    Lescarbeau, Rebecca S; Lei, Liang; Bakken, Katrina K; Sims, Peter A; Sarkaria, Jann N; Canoll, Peter; White, Forest M

    2016-06-01

    Glioblastoma (GBM) is the most common malignant primary brain cancer. With a median survival of about a year, new approaches to treating this disease are necessary. To identify signaling molecules regulating GBM progression in a genetically engineered murine model of proneural GBM, we quantified phosphotyrosine-mediated signaling using mass spectrometry. Oncogenic signals, including phosphorylated ERK MAPK, PI3K, and PDGFR, were found to be increased in the murine tumors relative to brain. Phosphorylation of CDK1 pY15, associated with the G2 arrest checkpoint, was identified as the most differentially phosphorylated site, with a 14-fold increase in phosphorylation in the tumors. To assess the role of this checkpoint as a potential therapeutic target, syngeneic primary cell lines derived from these tumors were treated with MK-1775, an inhibitor of Wee1, the kinase responsible for CDK1 Y15 phosphorylation. MK-1775 treatment led to mitotic catastrophe, as defined by increased DNA damage and cell death by apoptosis. To assess the extensibility of targeting Wee1/CDK1 in GBM, patient-derived xenograft (PDX) cell lines were also treated with MK-1775. Although the response was more heterogeneous, on-target Wee1 inhibition led to decreased CDK1 Y15 phosphorylation and increased DNA damage and apoptosis in each line. These results were also validated in vivo, where single-agent MK-1775 demonstrated an antitumor effect on a flank PDX tumor model, increasing mouse survival by 1.74-fold. This study highlights the ability of unbiased quantitative phosphoproteomics to reveal therapeutic targets in tumor models, and the potential for Wee1 inhibition as a treatment approach in preclinical models of GBM. Mol Cancer Ther; 15(6); 1332-43. ©2016 AACR. PMID:27196784

  2. Quantitative phosphoproteomics of protein kinase SnRK1 regulated protein phosphorylation in Arabidopsis under submergence

    PubMed Central

    Cho, Hsing-Yi; Wen, Tuan-Nan; Wang, Ying-Tsui; Shih, Ming-Che

    2016-01-01

    SNF1 RELATED PROTEIN KINASE 1 (SnRK1) is proposed to be a central integrator of the plant stress and energy starvation signalling pathways. We observed that the Arabidopsis SnRK1.1 dominant negative mutant (SnRK1.1 K48M) had lower tolerance to submergence than the wild type, suggesting that SnRK1.1-dependent phosphorylation of target proteins is important in signalling pathways triggered by submergence. We conducted quantitative phosphoproteomics and found that the phosphorylation levels of 57 proteins increased and the levels of 27 proteins decreased in Col-0 within 0.5–3h of submergence. Among the 57 proteins with increased phosphorylation in Col-0, 38 did not show increased phosphorylation levels in SnRK1.1 K48M under submergence. These proteins are involved mainly in sugar and protein synthesis. In particular, the phosphorylation of MPK6, which is involved in regulating ROS responses under abiotic stresses, was disrupted in the SnRK1.1 K48M mutant. In addition, PTP1, a negative regulator of MPK6 activity that directly dephosphorylates MPK6, was also regulated by SnRK1.1. We also showed that energy conservation was disrupted in SnRK1.1 K48M, mpk6, and PTP1 S7AS8A under submergence. These results reveal insights into the function of SnRK1 and the downstream signalling factors related to submergence. PMID:27029354

  3. Quantitative Phosphoproteomics Reveals Crosstalk Between Phosphorylation and O-GlcNAc in the DNA Damage Response Pathway

    PubMed Central

    Zhong, Jun; Martinez, Marissa; Sengupta, Srona; Lee, Albert; Wu, Xinyan; Chaerkady, Raghothama; Chatterjee, Aditi; O’Meally, Robert N.; Cole, Robert N.; Pandey, Akhilesh; Zachara, Natasha E.

    2015-01-01

    The modification of intracellular proteins by monosaccharides of O-linked β-N-acetylglucosamine (O-GlcNAc) is an essential and dynamic post-translational modification of metazoans. The addition and removal of O-GlcNAc is catalyzed by the O-GlcNAc transferase (OGT) and O-GlcNAcase, respectively. One mechanism by which O-GlcNAc is thought to mediate proteins is by regulating phosphorylation. To provide insight into the pathways regulated by O-GlcNAc, we have utilized stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative proteomics to carry out comparisons of site-specific phosphorylation in OGT wild-type (WT) and Null cells. Quantitation of the phosphoproteome demonstrated that out of 5,529 phosphoserine, phosphothreonine and phosphotyrosine sites, 232 phosphosites were upregulated and 133 downregulated in the absence of O-GlcNAc. Collectively, these data suggest that deletion of OGT has a profound effect on the phosphorylation of cell cycle and DNA damage response proteins. Key events were confirmed by biochemical analyses and demonstrate a increase in the activating autophosphorylation event on ATM (Ser1987) and on ATM’s downstream targets p53, H2AX and Chk2. Together, these data support widespread changes in the phosphoproteome upon removal of O-GlcNAc, suggesting that O-GlcNAc regulates processes such as the cell cycle, genomic stability, and lysosomal biogenesis. PMID:25263469

  4. Label-free quantitative phosphoproteomic analysis reveals differentially regulated proteins and pathway in PRRSV-infected pulmonary alveolar macrophages.

    PubMed

    Luo, Rui; Fang, Liurong; Jin, Hui; Wang, Dang; An, Kang; Xu, Ningzhi; Chen, Huanchun; Xiao, Shaobo

    2014-03-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen of swine worldwide and causes significant economic losses. Through regulating the host proteins phosphorylation, PRRSV was found to manipulate the activities of several signaling molecules to regulate innate immune responses. However, the role of protein phosphorylation during PRRSV infection and the signal pathways responsible for it are relatively unknown. Here liquid chromatography-tandem mass spectrometry for label-free quantitative phosphoproteomics was applied to systematically investigate the global phosphorylation events in PRRSV-infected pulmonary alveolar macrophages. In total, we identified 2125 unique phosphosites, of which the phosphorylation level of 292 phosphosites on 242 proteins and 373 phosphosites on 249 proteins was significantly altered at 12 and 36 h pi, respectively. The phosphoproteomics data were analyzed using ingenuity pathways analysis to identify defined canonical pathways and functional networks. Pathway analysis revealed that PRRSV-induced inflammatory cytokines production was probably due to the activation of mitogen-activated protein kinase and NF-κB signal pathway, which were regulated by several protein kinases during virus infection. Interacting network analysis indicated that altered phosphoproteins were involved in cellular assembly and organization, protein synthesis, molecular transport, and signal transduction in PRRSV infected cells. These pathways and functional networks analysis could provide direct insights into the biological significance of phosphorylation events modulated by PRRSV and may help us elucidate the pathogenic mechanisms of PRRSV infection. PMID:24533505

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

    SciTech Connect

    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.

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

    PubMed

    Fredens, Julius; Engholm-Keller, Kasper; Møller-Jensen, Jakob; Larsen, Martin Røssel; Færgeman, Nils J

    2014-01-01

    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 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. PMID:25059608

  7. Discovery of Mouse Spleen Signaling Responses to Anthrax using Label-Free Quantitative Phosphoproteomics via Mass Spectrometry*

    PubMed Central

    Manes, Nathan P.; Dong, Li; Zhou, Weidong; Du, Xiuxia; Reghu, Nikitha; Kool, Arjan C.; Choi, Dahan; Bailey, Charles L.; Petricoin, Emanuel F.; Liotta, Lance A.; Popov, Serguei G.

    2011-01-01

    Inhalational anthrax is caused by spores of the bacterium Bacillus anthracis (B. anthracis), and is an extremely dangerous disease that can kill unvaccinated victims within 2 weeks. Modern antibiotic-based therapy can increase the survival rate to ∼50%, but only if administered presymptomatically (within 24–48 h of exposure). To discover host signaling responses to presymptomatic anthrax, label-free quantitative phosphoproteomics via liquid chromatography coupled to mass spectrometry was used to compare spleens from uninfected and spore-challenged mice over a 72 h time-course. Spleen proteins were denatured using urea, reduced using dithiothreitol, alkylated using iodoacetamide, and digested into peptides using trypsin, and the resulting phosphopeptides were enriched using titanium dioxide solid-phase extraction and analyzed by nano-liquid chromatography-Linear Trap Quadrupole-Orbitrap-MS(/MS). The fragment ion spectra were processed using DeconMSn and searched using both Mascot and SEQUEST resulting in 252,626 confident identifications of 6248 phosphopeptides (corresponding to 5782 phosphorylation sites). The precursor ion spectra were deisotoped using Decon2LS and aligned using MultiAlign resulting in the confident quantitation of 3265 of the identified phosphopeptides. ANOVAs were used to produce a q-value ranked list of host signaling responses. Late-stage (48–72 h postchallenge) Sterne strain (lethal) infections resulted in global alterations to the spleen phosphoproteome. In contrast, ΔSterne strain (asymptomatic; missing the anthrax toxin) infections resulted in 188 (5.8%) significantly altered (q<0.05) phosphopeptides. Twenty-six highly tentative phosphorylation responses to early-stage (24 h postchallenge) anthrax were discovered (q<0.5), and ten of these originated from eight proteins that have known roles in the host immune response. These tentative early-anthrax host response signaling events within mouse spleens may translate into presymptomatic

  8. Quantitative Phosphoproteomics Reveals Signaling Mechanisms Associated with Rapid Cold Hardening in a Chill-Tolerant Fly.

    PubMed

    Teets, Nicholas M; Denlinger, David L

    2016-08-01

    Rapid cold hardening (RCH) is a physiological adaptation in which brief chilling (minutes to hours) significantly enhances the cold tolerance of insects. RCH allows insects to cope with sudden cold snaps and diurnal variation in temperature, but the mechanistic basis of this rapid stress response is poorly understood. Here, we used phosphoproteomics to identify phosphorylation-mediated signaling events that are regulated by chilling that induces RCH. Phosphoproteomic changes were measured in both brain and fat bodies, two tissues that are essential for sensing cold and coordinating RCH at the organismal level. Tissues were chilled ex vivo, and changes in phosphoprotein abundance were measured using 2D electrophoresis coupled with Pro-Q diamond labeling of phosphoproteins followed by protein identification via LC-MS/MS. In both tissues, we observed an abundance of protein phosphorylation events in response to chilling. Some of the proteins regulated by RCH-inducing chilling include proteins involved in cytoskeletal reorganization, heat shock proteins, and proteins involved in the degradation of damaged cellular components via the proteasome and autophagosome. Our results suggest that phosphorylation-mediated signaling cascades are major drivers of RCH and enhance our mechanistic understanding of this complex phenotype. PMID:27362561

  9. Identification of Putative Mek1 Substrates during Meiosis in Saccharomyces cerevisiae Using Quantitative Phosphoproteomics

    PubMed Central

    Suhandynata, Raymond T.; Wan, Lihong; Zhou, Huilin; Hollingsworth, Nancy M.

    2016-01-01

    Meiotic recombination plays a key role in sexual reproduction as it generates crossovers that, in combination with sister chromatid cohesion, physically connect homologous chromosomes, thereby promoting their proper segregation at the first meiotic division. Meiotic recombination is initiated by programmed double strand breaks (DSBs) catalyzed by the evolutionarily conserved, topoisomerase-like protein Spo11. Repair of these DSBs is highly regulated to create crossovers between homologs that are distributed throughout the genome. This repair requires the presence of the mitotic recombinase, Rad51, as well as the strand exchange activity of the meiosis-specific recombinase, Dmc1. A key regulator of meiotic DSB repair in Saccharomyces cerevisiae is the meiosis-specific kinase Mek1, which promotes interhomolog strand invasion and is required for the meiotic recombination checkpoint and the crossover/noncrossover decision. Understanding how Mek1 regulates meiotic recombination requires the identification of its substrates. Towards that end, an unbiased phosphoproteomic approach utilizing Stable Isotope Labeling by Amino Acids in Cells (SILAC) was utilized to generate a list of potential Mek1 substrates, as well as proteins containing consensus phosphorylation sites for cyclin-dependent kinase, the checkpoint kinases, Mec1/Tel1, and the polo-like kinase, Cdc5. These experiments represent the first global phosphoproteomic dataset for proteins in meiotic budding yeast. PMID:27214570

  10. Quantitative analysis of changes in the phosphoproteome of maize induced by the plant hormone salicylic acid

    PubMed Central

    Wu, Liuji; Hu, Xiuli; Wang, Shunxi; Tian, Lei; Pang, Yanjie; Han, Zanping; Wu, Liancheng; Chen, Yanhui

    2015-01-01

    Phytohormone salicylic acid (SA) plays an important role in regulating various physiological and biochemical processes. Our previous study identified several protein kinases responsive to SA, suggesting that phosphorylation events play an important role in the plant response to SA. In this study, we characterized the phosphoproteome of maize in response to SA using isotope tags for relative and absolute quantification (iTRAQ) technology and TiO2 enrichment method. Based on LC-MS/MS analysis, we found a total of 858 phosphoproteins among 1495 phosphopeptides. Among them, 291 phosphopeptides corresponding to 244 phosphoproteins were found to be significantly changed after SA treatment. The phosphoproteins identified are involved in a wide range of biological processes, which indicate that the response to SA encompasses a reformatting of major cellular processes. Furthermore, some of the phosphoproteins which were not previously known to be involved with SA were found to have significantly changed phosphorylation levels. Many of these changes are phosphorylation decreases, indicating that other currently unknown SA signaling pathways that result in decreased phosphorylation of downstream targets must be involved. Our study represents the first attempt at global phosphoproteome profiling in response to SA, and provides a better understanding of the molecular mechanisms regulated by SA. PMID:26659305

  11. Identification of Putative Mek1 Substrates during Meiosis in Saccharomyces cerevisiae Using Quantitative Phosphoproteomics.

    PubMed

    Suhandynata, Raymond T; Wan, Lihong; Zhou, Huilin; Hollingsworth, Nancy M

    2016-01-01

    Meiotic recombination plays a key role in sexual reproduction as it generates crossovers that, in combination with sister chromatid cohesion, physically connect homologous chromosomes, thereby promoting their proper segregation at the first meiotic division. Meiotic recombination is initiated by programmed double strand breaks (DSBs) catalyzed by the evolutionarily conserved, topoisomerase-like protein Spo11. Repair of these DSBs is highly regulated to create crossovers between homologs that are distributed throughout the genome. This repair requires the presence of the mitotic recombinase, Rad51, as well as the strand exchange activity of the meiosis-specific recombinase, Dmc1. A key regulator of meiotic DSB repair in Saccharomyces cerevisiae is the meiosis-specific kinase Mek1, which promotes interhomolog strand invasion and is required for the meiotic recombination checkpoint and the crossover/noncrossover decision. Understanding how Mek1 regulates meiotic recombination requires the identification of its substrates. Towards that end, an unbiased phosphoproteomic approach utilizing Stable Isotope Labeling by Amino Acids in Cells (SILAC) was utilized to generate a list of potential Mek1 substrates, as well as proteins containing consensus phosphorylation sites for cyclin-dependent kinase, the checkpoint kinases, Mec1/Tel1, and the polo-like kinase, Cdc5. These experiments represent the first global phosphoproteomic dataset for proteins in meiotic budding yeast. PMID:27214570

  12. Quantitative phosphoproteomic analysis of the PI3K-regulated signaling network.

    PubMed

    Gnad, Florian; Wallin, Jeffrey; Edgar, Kyle; Doll, Sophia; Arnott, David; Robillard, Liliane; Kirkpatrick, Donald S; Stokes, Matthew P; Vijapurkar, Ulka; Hatzivassiliou, Georgia; Friedman, Lori S; Belvin, Marcia

    2016-07-01

    The PI3K pathway is commonly activated in cancer. Only a few studies have attempted to explore the spectrum of phosphorylation signaling downstream of the PI3K cascade. Such insight, however, is imperative to understand the mechanisms responsible for oncogenic phenotypes. By applying MS-based phosphoproteomics, we mapped 2509 phosphorylation sites on 1096 proteins, and quantified their responses to activation or inhibition of PIK3CA using isogenic knock-in derivatives and a series of targeted inhibitors. We uncovered phosphorylation changes in a wide variety of proteins involved in cell growth and proliferation, many of which have not been previously associated with PI3K signaling. A significant update of the posttranslational modification database PHOSIDA (http://www.phosida.com) allows efficient use of the data. All MS data have been deposited in the ProteomeXchange with identifier PXD003899 (http://proteomecentral.proteomexchange.org/dataset/PXD003899). PMID:27282143

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

    PubMed Central

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

    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 in nuclear extracts and nuclear pellet fractions. Measurements were made in the presence and absence of the vasopressin analog dDAVP. Of the 1,251 sites quantified, 39 changed significantly in response to dDAVP. Network analysis of the regulated proteins revealed two major clusters (“cell-cell adhesion” and “transcriptional regulation”) that were connected to known elements of the vasopressin signaling pathway. The hub proteins for these two clusters were the transcriptional coactivator β-catenin and the transcription factor c-Jun. Phosphorylation of β-catenin at Ser552 was increased by dDAVP [log2(dDAVP/vehicle) = 1.79], and phosphorylation of c-Jun at Ser73 was decreased [log2(dDAVP/vehicle) = −0.53]. The β-catenin site is known to be targeted by either protein kinase A or Akt, both of which are activated in response to vasopressin. The c-Jun site is a canonical target for the MAP kinase Jnk2, which is downregulated in response to vasopressin in the collecting duct. The data support the idea that vasopressin-mediated control of transcription in collecting duct cells involves selective changes in the nuclear phosphoproteome. All data are available to users at http://helixweb.nih.gov/ESBL/Database/mNPPD/. PMID:22992673

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

    PubMed Central

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

    2014-01-01

    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 P12Δ CagA deletion mutant, and a P12Δ PAI 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 analyzed by nano-LC-Orbitrap MS. In total, 85 different phosphosites were found to be regulated following infection. The majority of phosphosites identified were kinases of the MAPK family. 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. PMID:25101063

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

    PubMed

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

    2014-10-01

    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. PMID:25168779

  16. Effects of MEK inhibitors GSK1120212 and PD0325901 in vivo using 10-plex quantitative proteomics and phosphoproteomics

    PubMed Central

    Paulo, Joao A.; McAllister, Fiona E.; Everley, Robert A.; Beausoleil, Sean A.; Banks, Alexander S.; Gygi, Steven P.

    2015-01-01

    Multiplexed isobaric tag-based quantitative proteomics and phosphoproteomics strategies can comprehensively analyze drug treatments effects on biological systems. Given the role of MEK signaling in cancer and MAPK-dependent diseases, we sought to determine if this pathway could be inhibited safely by examining the downstream molecular consequences. We used a series of TMT10-plex experiments to analyze the effect of two MEK inhibitors (GSK1120212 and PD0325901) on three tissues (kidney, liver, and pancreas) from nine mice. We quantified ~6000 proteins in each tissue, but significant protein level alterations were minimal with inhibitor treatment. Of particular interest was kidney tissue, as edema is an adverse effect of these inhibitors. From kidney tissue, we enriched phosphopeptides using titanium dioxide (TiO2) and quantified 10,562 phosphorylation events. Further analysis by phosphotyrosine (pY) peptide immunoprecipitation quantified an additional 592 phosphorylation events. Phosphorylation motif analysis revealed that the inhibitors decreased phosphorylation levels of PxSP and SP sites, consistent with ERK inhibition. The MEK inhibitors had the greatest decrease on the phosphorylation of two proteins, Barttin and Slc12a3, which have roles in ion transport and fluid balance. Further studies will provide insight into the effect of these MEK inhibitors with respect to edema and other adverse events in mouse models and human patients. PMID:25195567

  17. Quantitative phosphoproteomics revealed interplay between Syk and Lyn in the resistance to nilotinib in chronic myeloid leukemia cells.

    PubMed

    Gioia, Romain; Leroy, Cédric; Drullion, Claire; Lagarde, Valérie; Etienne, Gabriel; Dulucq, Stéphanie; Lippert, Eric; Roche, Serge; Mahon, François-Xavier; Pasquet, Jean-Max

    2011-08-25

    In this study, we have addressed how Lyn kinase signaling mediates nilotinib-resistance by quantitative phospho-proteomics using Stable Isotope Labeling with Amino acid in Cell culture. We have found an increased tyrosine phosphorylation of 2 additional tyrosine kinases in nilotinib-resistant cells: the spleen tyrosine kinase Syk and the UFO family receptor tyrosine kinase Axl. This increased tyrosine phosphorylation involved an interaction of these tyrosine kinases with Lyn. Inhibition of Syk by the inhibitors R406 or BAY 61-3606 or by RNA interference restored the capacity of nilotinib to inhibit cell proliferation. Conversely, coexpression of Lyn and Syk were required to fully induce resistance to nilotinib in drug-sensitive cells. Surprisingly, the knockdown of Syk also strongly decreased tyrosine phosphorylation of Lyn and Axl, thus uncovering interplay between Syk and Lyn. We have shown the involvement of the adaptor protein CDCP-1 in resistance to nilotinib. Interestingly, the expression of Axl and CDCP1 were found increased both in a nilotinib-resistant cell line and in nilotinib-resistant CML patients. We conclude that an oncogenic signaling mediated by Lyn and Syk can bypass the need of Bcr-Abl in CML cells. Thus, targeting these kinases may be of therapeutic value to override imatinib or nilotinib resistance in CML. PMID:21730355

  18. Wide-scale quantitative phosphoproteomic analysis reveals that cold treatment of T cells closely mimics soluble antibody stimulation

    PubMed Central

    Ji, Qinqin; Salomon, Arthur R.

    2015-01-01

    The activation of T-lymphocytes through antigen-mediated T-cell receptor (TCR) clustering is vital in regulating the adaptive-immune response. Although T cell receptor signaling has been extensively studied, the fundamental mechanisms for signal initiation are not fully understood. Reduced temperature initiated some of the hallmarks of TCR signaling such as increased phosphorylation and activation on ERK and calcium release from the endoplasmic reticulum as well as coalesce T-cell membrane microdomains. The precise mechanism of TCR signaling initiation due to temperature change remains obscure. One critical question is whether signaling initiated by cold treatment of T cells differs from signaling initiated by crosslinking of the T cell receptor. To address this uncertainty, a wide-scale, quantitative mass spectrometry-based phosphoproteomic analysis was performed on T cells stimulated either by temperature shift or through crosslinking of the TCR. Careful statistical comparison between the two stimulations revealed a striking level of identity between the subset of 339 sites that changed significantly with both stimulations. This study demonstrates for the first time, at unprecedented detail, that T cell cold treatment was sufficient to initiate signaling patterns nearly identical to soluble antibody stimulation, shedding new light on the mechanism of activation of these critically important immune cells. PMID:25839225

  19. Quantitative analysis of the TNF-α-induced phosphoproteome reveals AEG-1/MTDH/LYRIC as an IKKβ substrate

    PubMed Central

    Krishnan, Ramesh K.; Nolte, Hendrik; Sun, Tianliang; Kaur, Harmandeep; Sreenivasan, Krishnamoorthy; Looso, Mario; Offermanns, Stefan; Krüger, Marcus; Swiercz, Jakub M.

    2015-01-01

    The inhibitor of the nuclear factor-κB (IκB) kinase (IKK) complex is a key regulator of the canonical NF-κB signalling cascade and is crucial for fundamental cellular functions, including stress and immune responses. The majority of IKK complex functions are attributed to NF-κB activation; however, there is increasing evidence for NF-κB pathway-independent signalling. Here we combine quantitative mass spectrometry with random forest bioinformatics to dissect the TNF-α-IKKβ-induced phosphoproteome in MCF-7 breast cancer cells. In total, we identify over 20,000 phosphorylation sites, of which ∼1% are regulated up on TNF-α stimulation. We identify various potential novel IKKβ substrates including kinases and regulators of cellular trafficking. Moreover, we show that one of the candidates, AEG-1/MTDH/LYRIC, is directly phosphorylated by IKKβ on serine 298. We provide evidence that IKKβ-mediated AEG-1 phosphorylation is essential for IκBα degradation as well as NF-κB-dependent gene expression and cell proliferation, which correlate with cancer patient survival in vivo. PMID:25849741

  20. Quantitative phospho-proteomics reveals the Plasmodium merozoite triggers pre-invasion host kinase modification of the red cell cytoskeleton

    PubMed Central

    Zuccala, Elizabeth S.; Satchwell, Timothy J.; Angrisano, Fiona; Tan, Yan Hong; Wilson, Marieangela C.; Heesom, Kate J.; Baum, Jake

    2016-01-01

    The invasive blood-stage malaria parasite – the merozoite – induces rapid morphological changes to the target erythrocyte during entry. However, evidence for active molecular changes in the host cell that accompany merozoite invasion is lacking. Here, we use invasion inhibition assays, erythrocyte resealing and high-definition imaging to explore red cell responses during invasion. We show that although merozoite entry does not involve erythrocyte actin reorganisation, it does require ATP to complete the process. Towards dissecting the ATP requirement, we present an in depth quantitative phospho-proteomic analysis of the erythrocyte during each stage of invasion. Specifically, we demonstrate extensive increased phosphorylation of erythrocyte proteins on merozoite attachment, including modification of the cytoskeletal proteins beta-spectrin and PIEZO1. The association with merozoite contact but not active entry demonstrates that parasite-dependent phosphorylation is mediated by host-cell kinase activity. This provides the first evidence that the erythrocyte is stimulated to respond to early invasion events through molecular changes in its membrane architecture. PMID:26830761

  1. Quantitative phospho-proteomics reveals the Plasmodium merozoite triggers pre-invasion host kinase modification of the red cell cytoskeleton.

    PubMed

    Zuccala, Elizabeth S; Satchwell, Timothy J; Angrisano, Fiona; Tan, Yan Hong; Wilson, Marieangela C; Heesom, Kate J; Baum, Jake

    2016-01-01

    The invasive blood-stage malaria parasite - the merozoite - induces rapid morphological changes to the target erythrocyte during entry. However, evidence for active molecular changes in the host cell that accompany merozoite invasion is lacking. Here, we use invasion inhibition assays, erythrocyte resealing and high-definition imaging to explore red cell responses during invasion. We show that although merozoite entry does not involve erythrocyte actin reorganisation, it does require ATP to complete the process. Towards dissecting the ATP requirement, we present an in depth quantitative phospho-proteomic analysis of the erythrocyte during each stage of invasion. Specifically, we demonstrate extensive increased phosphorylation of erythrocyte proteins on merozoite attachment, including modification of the cytoskeletal proteins beta-spectrin and PIEZO1. The association with merozoite contact but not active entry demonstrates that parasite-dependent phosphorylation is mediated by host-cell kinase activity. This provides the first evidence that the erythrocyte is stimulated to respond to early invasion events through molecular changes in its membrane architecture. PMID:26830761

  2. Integrative Network Analysis Combined with Quantitative Phosphoproteomics Reveals Transforming Growth Factor-beta Receptor type-2 (TGFBR2) as a Novel Regulator of Glioblastoma Stem Cell Properties.

    PubMed

    Narushima, Yuta; Kozuka-Hata, Hiroko; Koyama-Nasu, Ryo; Tsumoto, Kouhei; Inoue, Jun-ichiro; Akiyama, Tetsu; Oyama, Masaaki

    2016-03-01

    Glioblastoma is one of the most malignant brain tumors with poor prognosis and their development and progression are known to be driven by glioblastoma stem cells. Although glioblastoma stem cells lose their cancer stem cell properties during cultivation in serum-containing medium, little is known about the molecular mechanisms regulating signaling alteration in relation to reduction of stem cell-like characteristics. To elucidate the global phosphorylation-related signaling events, we performed a SILAC-based quantitative phosphoproteome analysis of serum-induced dynamics in glioblastoma stem cells established from the tumor tissues of the patient. Among a total of 2876 phosphorylation sites on 1584 proteins identified in our analysis, 732 phosphorylation sites on 419 proteins were regulated through the alteration of stem cell-like characteristics. The integrative computational analyses based on the quantified phosphoproteome data revealed the relevant changes of phosphorylation levels regarding the proteins associated with cytoskeleton reorganization such as Rho family GTPase and Intermediate filament signaling, in addition to transforming growth factor-β receptor type-2 (TGFBR2) as a prominent upstream regulator involved in the serum-induced phosphoproteome regulation. The functional association of transforming growth factor-β receptor type-2 with stem cell-like properties was experimentally validated through signaling perturbation using the corresponding inhibitors, which indicated that transforming growth factor-β receptor type-2 could play an important role as a novel cell fate determinant in glioblastoma stem cell regulation. PMID:26670566

  3. Phosphoproteomics of the Dopamine Pathway Enables Discovery of Rap1 Activation as a Reward Signal In Vivo.

    PubMed

    Nagai, Taku; Nakamuta, Shinichi; Kuroda, Keisuke; Nakauchi, Sakura; Nishioka, Tomoki; Takano, Tetsuya; Zhang, Xinjian; Tsuboi, Daisuke; Funahashi, Yasuhiro; Nakano, Takashi; Yoshimoto, Junichiro; Kobayashi, Kenta; Uchigashima, Motokazu; Watanabe, Masahiko; Miura, Masami; Nishi, Akinori; Kobayashi, Kazuto; Yamada, Kiyofumi; Amano, Mutsuki; Kaibuchi, Kozo

    2016-02-01

    Dopamine (DA) type 1 receptor (D1R) signaling in the striatum presumably regulates neuronal excitability and reward-related behaviors through PKA. However, whether and how D1Rs and PKA regulate neuronal excitability and behavior remain largely unknown. Here, we developed a phosphoproteomic analysis method to identify known and novel PKA substrates downstream of the D1R and obtained more than 100 candidate substrates, including Rap1 GEF (Rasgrp2). We found that PKA phosphorylation of Rasgrp2 activated its guanine nucleotide-exchange activity on Rap1. Cocaine exposure activated Rap1 in the nucleus accumbens in mice. The expression of constitutively active PKA or Rap1 in accumbal D1R-expressing medium spiny neurons (D1R-MSNs) enhanced neuronal firing rates and behavioral responses to cocaine exposure through MAPK. Knockout of Rap1 in the accumbal D1R-MSNs was sufficient to decrease these phenotypes. These findings demonstrate a novel DA-PKA-Rap1-MAPK intracellular signaling mechanism in D1R-MSNs that increases neuronal excitability to enhance reward-related behaviors. PMID:26804993

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

    PubMed Central

    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

  5. Quantitative phosphoproteomics unravels biased phosphorylation of serotonin 2A receptor at Ser280 by hallucinogenic versus nonhallucinogenic agonists.

    PubMed

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

    2014-05-01

    The serotonin 5-HT(2A) 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-HT(2A) 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-HT(2A) receptor and exposed to either vehicle or the synthetic hallucinogen 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI) or the nonhallucinogenic 5-HT(2A) 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 (Ser(280)) located in the third intracellular loop of the 5-HT(2A) receptor, a region important for its desensitization. The specific phosphorylation of Ser(280) 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-HT(2A) receptors at Ser(280) 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 Ser(280) 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

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

    SciTech Connect

    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.

  7. Phosphoproteomics in Cancer

    PubMed Central

    Harsha, H. C.; Pandey, Akhilesh

    2010-01-01

    Reversible protein phosphorylation serves as a basis for regulating a number of cellular processes. Aberrant activation of kinase signaling pathways is commonly associated with several cancers. Recent developments in phosphoprotein/phosphopeptide enrichment strategies and quantitative mass spectrometry have resulted in robust pipelines for high-throughput characterization of phosphorylation in a global fashion. Today, it is possible to profile site-specific phosphorylation events on thousands of proteins in a single experiment. The potential of this approach is already being realized to characterize signaling pathways that govern oncogenesis. In addition, chemical proteomic strategies have been used to unravel targets of kinase inhibitors, which are otherwise difficult to characterize. This review summarizes various approaches used for analysis of the phosphoproteome in general, and protein kinases in particular, highlighting key cancer phosphoproteomic studies. PMID:20937571

  8. Quantitative Phosphoproteomic Analysis Reveals a Role for Serine and Threonine Kinases in the Cytoskeletal Reorganization in Early T Cell Receptor Activation in Human Primary T Cells*

    PubMed Central

    Ruperez, Patricia; Gago-Martinez, Ana; Burlingame, A. L.; Oses-Prieto, Juan A.

    2012-01-01

    Protein phosphorylation-dephosphorylation events play a primary role in regulation of almost all aspects of cell function including signal transduction, cell cycle, or apoptosis. Thus far, T cell phosphoproteomics have focused on analysis of phosphotyrosine residues, and little is known about the role of serine/threonine phosphorylation in early activation of the T cell receptor (TCR). Therefore, we performed a quantitative mass spectrometry-based analysis of the global phosphoproteome of human primary T cells in response to 5 min of TCR activation with anti-CD3 antibody. Combining immunoprecipitation with an antiphosphotyrosine antibody, titanium dioxide phosphopeptide enrichment, isobaric tag for the relative and absolute quantitation methodology, and strong cation exchange separation, we were able to identify 2814 phosphopeptides. These unique sites were employed to investigate the site-specific phosphorylation dynamics. Five hundred and seventeen phosphorylation sites showed TCR-responsive changes. We found that upon 5 min of stimulation of the TCR, specific serine and threonine kinase motifs are overrepresented in the set of responsive phosphorylation sites. These phosphorylation events targeted proteins with many different activities and are present in different subcellular locations. Many of these proteins are involved in intracellular signaling cascades related mainly to cytoskeletal reorganization and regulation of small GTPase-mediated signal transduction, probably involved in the formation of the immune synapse. PMID:22499768

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

    PubMed

    Poss, Zachary C; Ebmeier, Christopher C; Odell, Aaron T; Tangpeerachaikul, Anupong; Lee, Thomas; Pelish, Henry E; Shair, Matthew D; Dowell, Robin D; Old, William M; Taatjes, Dylan J

    2016-04-12

    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. PMID:27050516

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

    PubMed

    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. PMID:26689635

  11. Global Effects of Kinase Inhibitors on Signaling Networks Revealed by Quantitative Phosphoproteomics*

    PubMed Central

    Pan, Cuiping; Olsen, Jesper V.; Daub, Henrik; Mann, Matthias

    2009-01-01

    Aberrant signaling causes many diseases, and manipulating signaling pathways with kinase inhibitors has emerged as a promising area of drug research. Most kinase inhibitors target the conserved ATP-binding pocket; therefore specificity is a major concern. Proteomics has previously been used 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-ABL, which is the cause of chronic myelogenous leukemia, affected nearly 1,000 phosphopeptides. In addition to the proximal effects on ABL and its immediate targets, dasatinib broadly affected the downstream MAPK pathways. Pathway mapping of regulated sites implicated a variety of cellular functions, such as chromosome remodeling, RNA splicing, and cytoskeletal organization, some of which have been described in the literature before. Our assay is streamlined and generic and could become a useful tool in kinase drug development. PMID:19651622

  12. Informatics Methods to Enable Sharing of Quantitative Imaging Research Data

    PubMed Central

    Levy, Mia A.; Freymann, John B.; Kirby, Justin S.; Fedorov, Andriy; Fennessy, Fiona M.; Eschrich, Steven A.; Berglund, Anders E.; Fenstermacher, David A.; Tan, Yongqiang; Guo, Xiaotao; Casavant, Thomas L.; Brown, Bartley J.; Braun, Terry A.; Dekker, Andre; Roelofs, Erik; Mountz, James M.; Boada, Fernando; Laymon, Charles; Oborski, Matt; Rubin, Daniel L

    2012-01-01

    Introduction The National Cancer Institute (NCI) Quantitative Research Network (QIN) is a collaborative research network whose goal is to share data, algorithms and research tools to accelerate quantitative imaging research. A challenge is the variability in tools and analysis platforms used in quantitative imaging. Our goal was to understand the extent of this variation and to develop an approach to enable sharing data and to promote reuse of quantitative imaging data in the community. Methods We performed a survey of the current tools in use by the QIN member sites for representation and storage of their QIN research data including images, image meta-data and clinical data. We identified existing systems and standards for data sharing and their gaps for the QIN use case. We then proposed a system architecture to enable data sharing and collaborative experimentation within the QIN. Results There area variety of tools currently used by each QIN institution. We developed a general information system architecture to support the QIN goals. We also describe the remaining architecture gaps we are developing to enable members to share research images and image meta-data across the network. Conclusions As a research network, the QIN will stimulate quantitative imaging research by pooling data, algorithms and research tools. However, there are gaps in current functional requirements that will need to be met by future informatics development. Special attention must be given to the technical requirements needed to translate these methods into the clinical research workflow to enable validation and qualification of these novel imaging biomarkers. PMID:22770688

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

    PubMed Central

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

    2015-01-01

    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. PMID:26278961

  14. Quantitative Phosphoproteomics of the Ataxia Telangiectasia-Mutated (ATM) and Ataxia Telangiectasia-Mutated and Rad3-related (ATR) Dependent DNA Damage Response in Arabidopsis thaliana*

    PubMed Central

    Roitinger, Elisabeth; Hofer, Manuel; Köcher, Thomas; Pichler, Peter; Novatchkova, Maria; Yang, Jianhua; Schlögelhofer, Peter; Mechtler, Karl

    2015-01-01

    The reversible phosphorylation of proteins on serine, threonine, and tyrosine residues is an important biological regulatory mechanism. In the context of genome integrity, signaling cascades driven by phosphorylation are crucial for the coordination and regulation of DNA repair. The two serine/threonine protein kinases ataxia telangiectasia-mutated (ATM) and Ataxia telangiectasia-mutated and Rad3-related (ATR) are key factors in this process, each specific for different kinds of DNA lesions. They are conserved across eukaryotes, mediating the activation of cell-cycle checkpoints, chromatin modifications, and regulation of DNA repair proteins. We designed a novel mass spectrometry-based phosphoproteomics approach to study DNA damage repair in Arabidopsis thaliana. The protocol combines filter aided sample preparation, immobilized metal affinity chromatography, metal oxide affinity chromatography, and strong cation exchange chromatography for phosphopeptide generation, enrichment, and separation. Isobaric labeling employing iTRAQ (isobaric tags for relative and absolute quantitation) was used for profiling the phosphoproteome of atm atr double mutants and wild type plants under either regular growth conditions or challenged by irradiation. A total of 10,831 proteins were identified and 15,445 unique phosphopeptides were quantified, containing 134 up- and 38 down-regulated ATM/ATR dependent phosphopeptides. We identified known and novel ATM/ATR targets such as LIG4 and MRE11 (needed for resistance against ionizing radiation), PIE1 and SDG26 (implicated in chromatin remodeling), PCNA1, WAPL, and PDS5 (implicated in DNA replication), and ASK1 and HTA10 (involved in meiosis). PMID:25561503

  15. Label-free quantitative analysis of the casein kinase 2-responsive phosphoproteome of the marine minimal model species Ostreococcus tauri.

    PubMed

    Le Bihan, Thierry; Hindle, Matthew; Martin, Sarah F; Barrios-Llerena, Martin E; Krahmer, Johanna; Kis, Katalin; Millar, Andrew J; van Ooijen, Gerben

    2015-12-01

    Casein kinase 2 (CK2) is a protein kinase that phosphorylates a plethora of cellular target proteins involved in processes including DNA repair, cell cycle control, and circadian timekeeping. CK2 is functionally conserved across eukaryotes, although the substrate proteins identified in a range of complex tissues are often different. The marine alga Ostreococcus tauri is a unicellular eukaryotic model organism ideally suited to efficiently study generic roles of CK2 in the cellular circadian clock. Overexpression of CK2 leads to a slow circadian rhythm, verifying functional conservation of CK2 in timekeeping. The proteome was analysed in wild-type and CK2-overexpressing algae at dawn and dusk, revealing that differential abundance of the global proteome across the day is largely unaffected by overexpression. However, CK2 activity contributed more strongly to timekeeping at dusk than at dawn. The phosphoproteome of a CK2 overexpression line and cells treated with CK2 inhibitor was therefore analysed and compared to control cells at dusk. We report an extensive catalogue of 447 unique CK2-responsive differential phosphopeptide motifs to inform future studies into CK2 activity in the circadian clock of more complex tissues. All MS data have been deposited in the ProteomeXchange with identifier PXD000975 (http://proteomecentral.proteomexchange.org/dataset/PXD000975). PMID:25930153

  16. Enrichment Strategies in Phosphoproteomics.

    PubMed

    Leitner, Alexander

    2016-01-01

    The comprehensive study of the phosphoproteome is heavily dependent on appropriate enrichment strategies that are most often, but not exclusively, carried out on the peptide level. In this chapter, I give an overview of the most widely used techniques. In addition to dedicated antibodies, phosphopeptides are enriched by their selective interaction with metals in the form of chelated metal ions or metal oxides. The negative charge of the phosphate group is also exploited in a variety of chromatographic fractionation methods that include different types of ion exchange chromatography, hydrophilic interaction chromatography (HILIC), and electrostatic repulsion HILIC (ERLIC) chromatography. Selected examples from the literature will demonstrate how a combination of these techniques with current high-performance mass spectrometry enables the identification of thousands of phosphorylation sites from various sample types. PMID:26584921

  17. Quantitative Phosphoproteomics Analysis Reveals a Key Role of Insulin Growth Factor 1 Receptor (IGF1R) Tyrosine Kinase in Human Sperm Capacitation*

    PubMed Central

    Wang, Jing; Qi, Lin; Huang, Shaoping; Zhou, Tao; Guo, Yueshuai; Wang, Gaigai; Guo, Xuejiang; Zhou, Zuomin; Sha, Jiahao

    2015-01-01

    One of the most important changes during sperm capacitation is the enhancement of tyrosine phosphorylation. However, the mechanisms of protein tyrosine phosphorylation during sperm capacitation are not well studied. We used label-free quantitative phosphoproteomics to investigate the overall phosphorylation events during sperm capacitation in humans and identified 231 sites with increased phosphorylation levels. Motif analysis using the NetworKIN algorithm revealed that the activity of tyrosine phosphorylation kinases insulin growth factor 1 receptor (IGF1R)/insulin receptor is significantly enriched among the up-regulated phosphorylation substrates during capacitation. Western blotting further confirmed inhibition of IGF1R with inhibitors GSK1904529A and NVP-AEW541, which inhibited the increase in tyrosine phosphorylation levels during sperm capacitation. Additionally, sperm hyperactivated motility was also inhibited by GSK1904529A and NVP-AEW541 but could be up-regulated by insulin growth factor 1, the ligand of IGF1R. Thus, the IGF1R-mediated tyrosine phosphorylation pathway may play important roles in the regulation of sperm capacitation in humans and could be a target for improvement in sperm functions in infertile men. PMID:25693802

  18. Quantitative phosphoproteomics reveals genistein as a modulator of cell cycle and DNA damage response pathways in triple-negative breast cancer cells

    PubMed Central

    FANG, YI; ZHANG, QIAN; WANG, XIN; YANG, XUE; WANG, XIANGYU; HUANG, ZHEN; JIAO, YUCHEN; WANG, JING

    2016-01-01

    Around one sixth of breast cancer cases are classified as triple-negative breast cancer (TNBC), named after the absence of the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2); however, patients with TNBC suffer from poor clinical outcome and shortage of targeted therapy. Genistein, an estrogenic soy isoflavone, shows anticancer effects in TNBC cells such as inducing G2/M cell cycle arrest and apoptosis. However, the underlying mechanism of its anticancer effects is poorly understood and its elucidation can help the development of novel therapeutic strategies for TNBC. In this study, by combining isobaric tag-based TMT labeling with titanium dioxide-based phosphopeptide enrichment, we quantitated 5,445 phosphorylation sites on 2,008 phosphoproteins in the TNBC cell line MDA-MB-231, upon genistein treatment. Our analysis revealed 332 genistein-regulated phosphorylation sites on 226 proteins. Our data show that genistein can regulate several biological processes during the cell cycle, including DNA replication, cohesin complex cleavage, and kinetochore formation. Furthermore, genistein can also activate DNA damage response, including activation of ATR and BRCA1 complex. Overall, our study presents evidence at a phosphoproteomic level that genistein is able to inhibit TNBC cell growth by regulating the cell cycle and DNA damage response in a more complex manner. Our findings help elucidate the mechanisms through which genistein exerts its anticancer effects in TNBC cells. PMID:26783066

  19. Regulation of Platelet Derived Growth Factor Signaling by Leukocyte Common Antigen-related (LAR) Protein Tyrosine Phosphatase: A Quantitative Phosphoproteomics Study.

    PubMed

    Sarhan, Adil R; Patel, Trushar R; Creese, Andrew J; Tomlinson, Michael G; Hellberg, Carina; Heath, John K; Hotchin, Neil A; Cunningham, Debbie L

    2016-06-01

    Intracellular signaling pathways are reliant on protein phosphorylation events that are controlled by a balance of kinase and phosphatase activity. Although kinases have been extensively studied, the role of phosphatases in controlling specific cell signaling pathways has been less so. Leukocyte common antigen-related protein (LAR) is a member of the LAR subfamily of receptor-like protein tyrosine phosphatases (RPTPs). LAR is known to regulate the activity of a number of receptor tyrosine kinases, including platelet-derived growth factor receptor (PDGFR). To gain insight into the signaling pathways regulated by LAR, including those that are PDGF-dependent, we have carried out the first systematic analysis of LAR-regulated signal transduction using SILAC-based quantitative proteomic and phosphoproteomic techniques. We haveanalyzed differential phosphorylation between wild-type mouse embryo fibroblasts (MEFs) and MEFs in which the LAR cytoplasmic phosphatase domains had been deleted (LARΔP), and found a significant change in abundance of phosphorylation on 270 phosphosites from 205 proteins because of the absence of the phosphatase domains of LAR. Further investigation of specific LAR-dependent phosphorylation sites and enriched biological processes reveal that LAR phosphatase activity impacts on a variety of cellular processes, most notably regulation of the actin cytoskeleton. Analysis of putative upstream kinases that may play an intermediary role between LAR and the identified LAR-dependent phosphorylation events has revealed a role for LAR in regulating mTOR and JNK signaling. PMID:27074791

  20. Quantitative phosphoproteomics reveals genistein as a modulator of cell cycle and DNA damage response pathways in triple-negative breast cancer cells.

    PubMed

    Fang, Yi; Zhang, Qian; Wang, Xin; Yang, Xue; Wang, Xiangyu; Huang, Zhen; Jiao, Yuchen; Wang, Jing

    2016-03-01

    Around one sixth of breast cancer cases are classified as triple-negative breast cancer (TNBC), named after the absence of the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2); however, patients with TNBC suffer from poor clinical outcome and shortage of targeted therapy. Genistein, an estrogenic soy isoflavone, shows anticancer effects in TNBC cells such as inducing G2/M cell cycle arrest and apoptosis. However, the underlying mechanism of its anticancer effects is poorly understood and its elucidation can help the development of novel therapeutic strategies for TNBC. In this study, by combining isobaric tag-based TMT labeling with titanium dioxide-based phosphopeptide enrichment, we quantitated 5,445 phosphorylation sites on 2,008 phosphoproteins in the TNBC cell line MDA-MB-231, upon genistein treatment. Our analysis revealed 332 genistein-regulated phosphorylation sites on 226 proteins. Our data show that genistein can regulate several biological processes during the cell cycle, including DNA replication, cohesin complex cleavage, and kinetochore formation. Furthermore, genistein can also activate DNA damage response, including activation of ATR and BRCA1 complex. Overall, our study presents evidence at a phosphoproteomic level that genistein is able to inhibit TNBC cell growth by regulating the cell cycle and DNA damage response in a more complex manner. Our findings help elucidate the mechanisms through which genistein exerts its anticancer effects in TNBC cells. PMID:26783066

  1. Quantitative phosphoproteomic analysis of signaling downstream of the prostaglandin e2/g-protein coupled receptor in human synovial fibroblasts: potential antifibrotic networks.

    PubMed

    Gerarduzzi, Casimiro; He, QingWen; Antoniou, John; Di Battista, John A

    2014-11-01

    The Prostaglandin E2 (PGE2) signaling mechanism within fibroblasts is of growing interest as it has been shown to prevent numerous fibrotic features of fibroblast activation with limited evidence of downstream pathways. To understand the mechanisms of fibroblasts producing tremendous amounts of PGE2 with autocrine effects, we apply a strategy of combining a wide-screening of PGE2-induced kinases with quantitative phosphoproteomics. Our large-scale proteomic approach identified a PKA signal transmitted through phosphorylation of its substrates harboring the R(R/X)X(S*/T*) motif. We documented 115 substrates, of which 72 had 89 sites with a 2.5-fold phosphorylation difference in PGE2-treated cells than in untreated cells, where approximately half of such sites were defined as being novel. They were compiled by networking software to focus on highlighted activities and to associate them with a functional readout of fibroblasts. The substrates were associated with a variety of cellular functions including cytoskeletal structures (migration/motility), regulators of G-protein coupled receptor function, protein kinases, and transcriptional/translational regulators. For the first time, we extended the PGE2 pathway into an elaborate network of interconnecting phosphoproteins, providing vital information to a once restricted signalosome. These data provide new insights into eicosanoid-initiated cell signaling with regards to the regulation of fibroblast activation and the identification of new targets for evidenced-based pharmacotherapy against fibrosis. PMID:25223752

  2. Targeted Phosphoproteome Analysis Using Selected/Multiple Reaction Monitoring (SRM/MRM).

    PubMed

    Adachi, Jun; Narumi, Ryohei; Tomonaga, Takeshi

    2016-01-01

    Mass spectrometry-based phosphoproteomics has been rapidly spread based on the advancement of mass spectrometry and development of efficient enrichment techniques for phosphorylated proteins or peptides. Non-targeted approach has been employed in most of the studies for phosphoproteome analysis. However, targeted approach using selected/multiple reaction monitoring (SRM/MRM) is an indispensible technique used for the quantitation of known targets especially when we have many samples to quantitate phosphorylation events on proteins in biological or clinical samples. We herein describe the application of a large-scale phosphoproteome analysis and SRM-based quantitation for the systematic discovery and validation of biomarkers. PMID:26700043

  3. Quantitative phosphoproteomic analysis reveals γ-bisabolene inducing p53-mediated apoptosis of human oral squamous cell carcinoma via HDAC2 inhibition and ERK1/2 activation.

    PubMed

    Jou, Yu-Jen; Chen, Chao-Jung; Liu, Yu-Ching; Way, Tzong-Der; Lai, Chih-Ho; Hua, Chun-Hung; Wang, Ching-Ying; Huang, Su-Hua; Kao, Jung-Yie; Lin, Cheng-Wen

    2015-10-01

    γ-Bisabolene, one of main components in cardamom, showed potent in vitro and in vivo anti-proliferative activities against human oral squamous cell carcinoma (OSCC). γ-Bisabolene activated caspases-3/9 and decreased mitochondrial memebrane potential, leading to apoptosis of OSCC cell lines (Ca9-22 and SAS), but not normal oral fibroblast cells. Phosphoproteome profiling of OSCC cells treated with γ-bisabolene was identified using TiO2-PDMS plate and LC-MS/MS, then confirmed using Western blotting and real-time RT-PCR assays. Phosphoproteome profiling revealed that γ-bisabolene increased the phosphorylation of ERK1/2, protein phosphatases 1 (PP1), and p53, as well as decreased the phosphorylation of histone deacetylase 2 (HDAC2) in the process of apoptosis induction. Protein-protein interaction network analysis proposed the involvement of PP1-HDAC2-p53 and ERK1/2-p53 pathways in γ-bisabolene-induced apoptosis. Subsequent assays indicated γ-bisabolene eliciting p53 acetylation that enhanced the expression of p53-regulated apoptotic genes. PP1 inhibitor-2 restored the status of HDAC2 phosphorylation, reducing p53 acetylation and PUMA mRNA expression in γ-bisabolene-treated Ca9-22 and SAS cells. Meanwhile, MEK and ERK inhibitors significantly decreased γ-bisabolene-induced PUMA expression in both cancer cell lines. Notably, the results ascertained the involvement of PP1-HDAC2-p53 and ERK1/2-p53 pathways in mitochondria-mediated apoptosis of γ-bisabolene-treated cells. This study demonstrated γ-bisabolene displaying potent anti-proliferative and apoptosis-inducing activities against OSCC in vitro and in vivo, elucidating molecular mechanisms of γ-bisabolene-induced apoptosis. The novel insight could be useful for developing anti-cancer drugs. PMID:26194454

  4. Quantitative Phosphoproteomic Study Reveals that Protein Kinase A Regulates Neural Stem Cell Differentiation Through Phosphorylation of Catenin Beta-1 and Glycogen Synthase Kinase 3β.

    PubMed

    Wang, Shuxin; Li, Zheyi; Shen, Hongyan; Zhang, Zhong; Yin, Yuxin; Wang, Qingsong; Zhao, Xuyang; Ji, Jianguo

    2016-08-01

    Protein phosphorylation is central to the understanding of multiple cellular signaling pathways responsible for regulating the self-renewal and differentiation of neural stem cells (NSCs). Here we performed a large-scale phosphoproteomic analysis of rat fetal NSCs using strong cation exchange chromatography prefractionation and citric acid-assisted two-step enrichment with TiO2 strategy followed by nanoLC-MS/MS analysis. Totally we identified 32,546 phosphosites on 5,091 phosphoproteins, among which 23,945 were class I phosphosites, and quantified 16,000 sites during NSC differentiation. More than 65% of class I phosphosites were novel when compared with PhosphoSitePlus database. Quantification results showed that the early and late stage of NSC differentiation differ greatly. We mapped 69 changed phosphosites on 20 proteins involved in Wnt signaling pathway, including S552 on catenin beta-1 (Ctnnb1) and S9 on glycogen synthase kinase 3β (Gsk3β). Western blotting and real-time PCR results proved that Wnt signaling pathway plays critical roles in NSC fate determination. Furthermore, inhibition and activation of PKA dramatically affected the phosphorylation state of Ctnnb1 and Gsk3β, which regulates the differentiation of NSCs. Our data provides a valuable resource for studying the self-renewal and differentiation of NSCs. Stem Cells 2016;34:2090-2101. PMID:27097102

  5. Stable Isotope Metabolic Labeling-based Quantitative Phosphoproteomic Analysis of Arabidopsis Mutants Reveals Ethylene-regulated Time-dependent Phosphoproteins and Putative Substrates of Constitutive Triple Response 1 Kinase*

    PubMed Central

    Yang, Zhu; Guo, Guangyu; Zhang, Manyu; Liu, Claire Y.; Hu, Qin; Lam, Henry; Cheng, Han; Xue, Yu; Li, Jiayang; Li, Ning

    2013-01-01

    Ethylene is an important plant hormone that regulates numerous cellular processes and stress responses. The mode of action of ethylene is both dose- and time-dependent. Protein phosphorylation plays a key role in ethylene signaling, which is mediated by the activities of ethylene receptors, constitutive triple response 1 (CTR1) kinase, and phosphatase. To address how ethylene alters the cellular protein phosphorylation profile in a time-dependent manner, differential and quantitative phosphoproteomics based on 15N stable isotope labeling in Arabidopsis was performed on both one-minute ethylene-treated Arabidopsis ethylene-overly-sensitive loss-of-function mutant rcn1-1, deficient in PP2A phosphatase activity, and a pair of long-term ethylene-treated wild-type and loss-of-function ethylene signaling ctr1-1 mutants, deficient in mitogen-activated kinase kinase kinase activity. In total, 1079 phosphopeptides were identified, among which 44 were novel. Several one-minute ethylene-regulated phosphoproteins were found from the rcn1-1. Bioinformatic analysis of the rcn1-1 phosphoproteome predicted nine phosphoproteins as the putative substrates for PP2A phosphatase. In addition, from CTR1 kinase-enhanced phosphosites, we also found putative CTR1 kinase substrates including plastid transcriptionally active protein and calcium-sensing receptor. These regulatory proteins are phosphorylated in the presence of ethylene. Analysis of ethylene-regulated phosphosites using the group-based prediction system with a protein–protein interaction filter revealed a total of 14 kinase–substrate relationships that may function in both CTR1 kinase- and PP2A phosphatase-mediated phosphor-relay pathways. Finally, several ethylene-regulated post-translational modification network models have been built using molecular systems biology tools. It is proposed that ethylene regulates the phosphorylation of arginine/serine-rich splicing factor 41, plasma membrane intrinsic protein 2A, light

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

    PubMed

    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

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

    PubMed Central

    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

  8. Novel Host Proteins and Signaling Pathways in Enteropathogenic E. coli Pathogenesis Identified by Global Phosphoproteome Analysis.

    PubMed

    Scholz, Roland; Imami, Koshi; Scott, Nichollas E; Trimble, William S; Foster, Leonard J; Finlay, B Brett

    2015-07-01

    Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system (T3SS) to directly translocate effector proteins into host cells where they play a pivotal role in subverting host cell signaling needed for disease. However, our knowledge of how EPEC affects host protein phosphorylation is limited to a few individual protein studies. We employed a quantitative proteomics approach to globally map alterations in the host phosphoproteome during EPEC infection. By characterizing host phosphorylation events at various time points throughout infection, we examined how EPEC dynamically impacts the host phosphoproteome over time. This experimental setup also enabled identification of T3SS-dependent and -independent changes in host phosphorylation. Specifically, T3SS-regulated events affected various cellular processes that are known EPEC targets, including cytoskeletal organization, immune signaling, and intracellular trafficking. However, the involvement of phosphorylation in these events has thus far been poorly studied. We confirmed the MAPK family as an established key host player, showed its central role in signal transduction during EPEC infection, and extended the repertoire of known signaling hubs with previously unrecognized proteins, including TPD52, CIN85, EPHA2, and HSP27. We identified altered phosphorylation of known EPEC targets, such as cofilin, where the involvement of phosphorylation has so far been undefined, thus providing novel mechanistic insights into the roles of these proteins in EPEC infection. An overlap of regulated proteins, especially those that are cytoskeleton-associated, was observed when compared with the phosphoproteome of Shigella-infected cells. We determined the biological relevance of the phosphorylation of a novel protein in EPEC pathogenesis, septin-9 (SEPT9). Both siRNA knockdown and a phosphorylation-impaired SEPT9 mutant decreased bacterial adherence and EPEC-mediated cell death. In contrast, a phosphorylation

  9. Predicting outbreak detection in public health surveillance: quantitative analysis to enable evidence-based method selection.

    PubMed

    Buckeridge, David L; Okhmatovskaia, Anna; Tu, Samson; O'Connor, Martin; Nyulas, Csongor; Musen, Mark A

    2008-01-01

    Public health surveillance is critical for accurate and timely outbreak detection and effective epidemic control. A wide range of statistical algorithms is used for surveillance, and important differences have been noted in the ability of these algorithms to detect outbreaks. The evidence about the relative performance of these algorithms, however, remains limited and mainly qualitative. Using simulated outbreak data, we developed and validated quantitative models for predicting the ability of commonly used surveillance algorithms to detect different types of outbreaks. The developed models accurately predict the ability of different algorithms to detect different types of outbreaks. These models enable evidence-based algorithm selection and can guide research into algorithm development. PMID:18999264

  10. Predicting Outbreak Detection in Public Health Surveillance: Quantitative Analysis to Enable Evidence-Based Method Selection

    PubMed Central

    Buckeridge, David L.; Okhmatovskaia, Anna; Tu, Samson; O’Connor, Martin; Nyulas, Csongor; Musen, Mark A.

    2008-01-01

    Public health surveillance is critical for accurate and timely outbreak detection and effective epidemic control. A wide range of statistical algorithms is used for surveillance, and important differences have been noted in the ability of these algorithms to detect outbreaks. The evidence about the relative performance of these algorithms, however, remains limited and mainly qualitative. Using simulated outbreak data, we developed and validated quantitative models for predicting the ability of commonly used surveillance algorithms to detect different types of outbreaks. The developed models accurately predict the ability of different algorithms to detect different types of outbreaks. These models enable evidence-based algorithm selection and can guide research into algorithm development. PMID:18999264

  11. Portable SERS-enabled micropipettes for microarea sampling and reliably quantitative detection of surface organic residues.

    PubMed

    Fang, Wei; Zhang, Xinwei; Chen, Yong; Wan, Liang; Huang, Weihua; Shen, Aiguo; Hu, Jiming

    2015-09-15

    We report the first microsampling device for reliably quantitative, label-free and separation-free detection of multicomponents of surface organic residues (SORs) by means of a quality controllable surface-enhanced Raman scattering (SERS)-enabled micropipette. The micropipette is comprised of a drawn glass capillary with a tiny orifice (∼50 μm) at the distal tip, where the specially designed nanorattles (NRs) are compactly coated on the inner wall surface. SERS signals of 4-mercapto benzoic acid (MBA) anchored inside the internal gap of NRs could be used to evaluate and control the quality of micropipettes and, therefore, allow us to overcome the limitations of a reliably quantitative SERS assay using traditional substrates without an internal standard. By dropping a trace extraction agent on targeting SORs located on a narrow surface, the capillary and SERS functionalities of these micropipettes allow on-site microsampling via capillary action and subsequent multiplex distinction/detection due to their molecularly narrow Raman peaks. For example, 8 nM thiram (TMTD), 8 nM malachite green (MG), and 1.5 μM (400 ppb) methyl parathion (MPT) on pepper and cucumber peels have been simultaneously detected in a wide detection range. The portable SERS-enabled device could potentially be facilely incorporated with liquid-liquid or solid phase micro-extracting devices for a broader range of applications in rapid and field analysis of food/public/environment security related SORs. PMID:26274894

  12. Improvement of phosphoproteome analyses using FAIMS and decision tree fragmentation. application to the insulin signaling pathway in Drosophila melanogaster S2 cells.

    PubMed

    Bridon, Gaëlle; Bonneil, Eric; Muratore-Schroeder, Tara; Caron-Lizotte, Olivier; Thibault, Pierre

    2012-02-01

    This report examines the analytical benefits of high-field asymmetric waveform ion mobility spectrometry (FAIMS) coupled to liquid chromatography mass spectrometry (LC-MS) for phosphoproteomics analyses. The ability of FAIMS to separate multiply charged peptide ions from chemical interferences confers a unique advantage in phosphoproteomics by enhancing the detection of low abundance phosphopeptides. LC-FAIMS-MS experiments performed on TiO(2)-enriched tryptic digests from Drosophila melanogaster provided a 50% increase in phosphopeptide identification compared to conventional LC-MS analysis. Also, FAIMS can be used to select different population of multiply charged phosphopeptide ions prior to their activation with either collision activated dissociation (CAD) or electron transfer dissociation (ETD). Importantly, FAIMS enabled the resolution of coeluting phosphoisomers of different abundances to facilitate their unambiguous identification using conventional database search engines. The benefits of FAIMS in large-scale phosphoproteomics of D. melanogaster are further investigated using label-free quantitation to identify differentially regulated phosphoproteins in response to insulin stimulation. PMID:22059388

  13. Challenges in plasma membrane phosphoproteomics

    PubMed Central

    Orsburn, Benjamin C; Stockwin, Luke H; Newton, Dianne L

    2011-01-01

    The response to extracellular stimuli often alters the phosphorylation state of plasma membrane-associated proteins. In this regard, generation of a comprehensive membrane phosphoproteome can significantly enhance signal transduction and drug mechanism studies. However, analysis of this subproteome is regarded as technically challenging, given the low abundance and insolubility of integral membrane proteins, combined with difficulties in isolating, ionizing and fragmenting phosphopeptides. In this article, we highlight recent advances in membrane and phosphoprotein enrichment techniques resulting in improved identification of these elusive peptides. We also describe the use of alternative fragmentation techniques, and assess their current and future value to the field of membrane phosphoproteomics. PMID:21819303

  14. Phosphoproteomics and Lung Cancer Research

    PubMed Central

    López, Elena; Cho, William C. S.

    2012-01-01

    Massive evidence suggests that genetic abnormalities contribute to the development of lung cancer. These molecular abnormalities may serve as diagnostic, prognostic and predictive biomarkers for this deadly disease. It is imperative to search these biomarkers in different tumorigenesis pathways so as to provide the most appropriate therapy for each individual patient with lung malignancy. Phosphoproteomics is a promising technology for the identification of biomarkers and novel therapeutic targets for cancer. Thousands of proteins interact via physical and chemical association. Moreover, some proteins can covalently modify other proteins post-translationally. These post-translational modifications ultimately give rise to the emergent functions of cells in sequence, space and time. Phosphoproteomics clinical researches imply the comprehensive analysis of the proteins that are expressed in cells or tissues and can be employed at different stages. In addition, understanding the functions of phosphorylated proteins requires the study of proteomes as linked systems rather than collections of individual protein molecules. In fact, proteomics approaches coupled with affinity chromatography strategies followed by mass spectrometry have been used to elucidate relevant biological questions. This article will discuss the relevant clues of post-translational modifications, phosphorylated proteins, and useful proteomics approaches to identify molecular cancer signatures. The recent progress in phosphoproteomics research in lung cancer will be also discussed. PMID:23202899

  15. Reduced-representation Phosphosignatures Measured by Quantitative Targeted MS Capture Cellular States and Enable Large-scale Comparison of Drug-induced Phenotypes.

    PubMed

    Abelin, Jennifer G; Patel, Jinal; Lu, Xiaodong; Feeney, Caitlin M; Fagbami, Lola; Creech, Amanda L; Hu, Roger; Lam, Daniel; Davison, Desiree; Pino, Lindsay; Qiao, Jana W; Kuhn, Eric; Officer, Adam; Li, Jianxue; Abbatiello, Susan; Subramanian, Aravind; Sidman, Richard; Snyder, Evan; Carr, Steven A; Jaffe, Jacob D

    2016-05-01

    Profiling post-translational modifications represents an alternative dimension to gene expression data in characterizing cellular processes. Many cellular responses to drugs are mediated by changes in cellular phosphosignaling. We sought to develop a common platform on which phosphosignaling responses could be profiled across thousands of samples, and created a targeted MS assay that profiles a reduced-representation set of phosphopeptides that we show to be strong indicators of responses to chemical perturbagens.To develop the assay, we investigated the coordinate regulation of phosphosites in samples derived from three cell lines treated with 26 different bioactive small molecules. Phosphopeptide analytes were selected from these discovery studies by clustering and picking 1 to 2 proxy members from each cluster. A quantitative, targeted parallel reaction monitoring assay was developed to directly measure 96 reduced-representation probes. Sample processing for proteolytic digestion, protein quantification, peptide desalting, and phosphopeptide enrichment have been fully automated, making possible the simultaneous processing of 96 samples in only 3 days, with a plate phosphopeptide enrichment variance of 12%. This highly reproducible process allowed ∼95% of the reduced-representation phosphopeptide probes to be detected in ∼200 samples.The performance of the assay was evaluated by measuring the probes in new samples generated under treatment conditions from discovery experiments, recapitulating the observations of deeper experiments using a fraction of the analytical effort. We measured these probes in new experiments varying the treatments, cell types, and timepoints to demonstrate generalizability. We demonstrated that the assay is sensitive to disruptions in common signaling pathways (e.g. MAPK, PI3K/mTOR, and CDK). The high-throughput, reduced-representation phosphoproteomics assay provides a platform for the comparison of perturbations across a range of

  16. Recent advances in enrichment and separation strategies for mass spectrometry-based phosphoproteomics

    PubMed Central

    Yang, Chenxi; Zhong, Xuefei; Li, Lingjun

    2016-01-01

    Due to the significance of protein phosphorylation in various biological processes and signaling events, new analytical techniques for enhanced phosphoproteomics have been rapidly introduced in recent years. The combinatorial use of the phospho-specific enrichment techniques and prefractionation methods prior to MS analysis enables comprehensive profiling of the phosphoproteome and facilitates deciphering the critical roles that phosphorylation plays in signaling pathways in various biological systems. This review places special emphasis on the recent five-year (2009–2013) advances for enrichment and separation techniques that have been utilized for phosphopeptides prior to MS analysis. PMID:24687451

  17. Optical digital coherent detection technology enabled flexible and ultra-fast quantitative phase imaging.

    PubMed

    Feng, Yuan-Hua; Lu, Xing; Song, Lu; Guo, Xiaojie; Wang, Yawei; Zhu, Linyan; Sui, Qi; Li, Jianping; Shi, Kebin; Li, Zhaohui

    2016-07-25

    Quantitative phase imaging has been an important labeling-free microscopy modality for many biomedical and material science applications. In which, ultra-fast quantitative phase imaging is indispensable for dynamic or transient characteristics analysis. Conventional wide field optical interferometry is a common scheme for quantitative phase imaging, while its data acquisition rate is usually hindered by the frame rate of arrayed detector. By utilizing novel balanced-photo-detector based digital optics coherent detection techniques, we report on a method of constructing ultra-fast quantitative phase microscopy at the line-scan rate of 100 MHz with ~2 μm spatial resolution. PMID:27464166

  18. Sperm phosphoproteomics: historical perspectives and current methodologies

    PubMed Central

    Porambo, James R; Salicioni, Ana M; Visconti, Pablo E; Platt, Mark D

    2013-01-01

    Mammalian sperm are differentiated germ cells that transfer genetic material from the male to the female. Owing to this essential role in the reproductive process, an understanding of the complex mechanisms that underlie sperm function has implications ranging from the development of novel contraceptives to the treatment of male infertility. While the importance of phosphorylation in sperm differentiation, maturation and fertilization has been well established, the ability to directly determine the sites of phosphorylation within sperm proteins and to quantitate the extent of phosphorylation at these sites is a recent development that has relied almost exclusively on advances in the field of proteomics. This review will summarize the work that has been carried out to date on sperm phosphoproteomics and discuss how the resulting qualitative and quantitative information has been used to provide insight into the manner in which protein phosphorylation events modulate sperm function. The authors also present the proteomics process as it is most often utilized for the elucidation of protein expression, with a particular emphasis on the way in which the process has been modified for the analysis of protein phosphorylation in sperm. PMID:23194270

  19. Phosphoproteomics for the masses.

    PubMed

    Grimsrud, Paul A; Swaney, Danielle L; Wenger, Craig D; Beauchene, Nicole A; Coon, Joshua J

    2010-01-15

    Protein phosphorylation serves as a primary mechanism of signal transduction in the cells of biological organisms. Technical advancements over the last several years in mass spectrometry now allow for the large-scale identification and quantitation of in vivo phosphorylation at unprecedented levels. These developments have occurred in the areas of sample preparation, instrumentation, quantitative methodology, and informatics so that today, 10 000-20 000 phosphorylation sites can be identified and quantified within a few weeks. With the rapid development and widespread availability of such data, its translation into biological insight and knowledge is a current obstacle. Here we present an overview of how this technology came to be and is currently applied, as well as future challenges for the field. PMID:20047291

  20. Phosphoproteomics for the masses

    PubMed Central

    Grimsrud, Paul A.; Swaney, Danielle L.; Wenger, Craig D.; Beauchene, Nicole A.; Coon, Joshua J.

    2010-01-01

    Protein phosphorylation serves as a primary mechanism of signal transduction in the cells of biological organisms. Technical advancements over the last several years in mass spectrometry now allow for the large-scale identification and quantitation of in vivo phosphorylation at unprecedented levels. These developments have occurred in the areas of sample preparation, instrumentation, quantitative methodology, and informatics so that today, ten to twenty thousand phosphorylation sites can be identified and quantified within a few weeks. With the rapid development and widespread availability of such data, its translation into biological insight and knowledge is a current obstacle. Here we present an overview of how this technology came to be and is currently applied, as well as future challenges for the field. PMID:20047291

  1. Nanotag-enabled photonic crystal fiber as quantitative surface-enhanced Raman scattering optofluidic platform

    SciTech Connect

    Pinkhasova, Polina; Chen, Hui; Du, Henry; Kanka, Jiri; Mergo, Pawel

    2015-02-16

    Core-shell nanotags that are active in surface-enhanced Raman scattering (SERS) and entrapped with thiocyanate (SCN) label molecules were immobilized in the air channels of suspended-core photonic crystal fiber (PCF) to impart quantitative capacity to SERS-based PCF optofluidic sensing platform. The Raman intensity of Rhodamine 6G increases with concentration, whereas the intensity of SCN remains constant when measured using this platform. The signal from the SCN label can be used as an internal reference to establish calibration for quantitative measurements of analytes of unknown concentrations. The long optical path-length PCF optofluidic platform integrated with SERS-active core-shell nanotags holds significant promise for sensitive quantitative chem/bio measurements with the added benefit of small sampling volume. The dependence of SERS intensity on the nanotag coverage density and PCF length was interpreted based on numerical-analytical simulations.

  2. Nanotag-enabled photonic crystal fiber as quantitative surface-enhanced Raman scattering optofluidic platform

    NASA Astrophysics Data System (ADS)

    Pinkhasova, Polina; Chen, Hui; Kanka, Jiri; Mergo, Pawel; Du, Henry

    2015-02-01

    Core-shell nanotags that are active in surface-enhanced Raman scattering (SERS) and entrapped with thiocyanate (SCN) label molecules were immobilized in the air channels of suspended-core photonic crystal fiber (PCF) to impart quantitative capacity to SERS-based PCF optofluidic sensing platform. The Raman intensity of Rhodamine 6G increases with concentration, whereas the intensity of SCN remains constant when measured using this platform. The signal from the SCN label can be used as an internal reference to establish calibration for quantitative measurements of analytes of unknown concentrations. The long optical path-length PCF optofluidic platform integrated with SERS-active core-shell nanotags holds significant promise for sensitive quantitative chem/bio measurements with the added benefit of small sampling volume. The dependence of SERS intensity on the nanotag coverage density and PCF length was interpreted based on numerical-analytical simulations.

  3. Nanoparticle-mediated photothermal effect enables a new method for quantitative biochemical analysis using a thermometer

    NASA Astrophysics Data System (ADS)

    Fu, Guanglei; Sanjay, Sharma T.; Dou, Maowei; Li, Xiujun

    2016-03-01

    A new biomolecular quantitation method, nanoparticle-mediated photothermal bioassay, using a common thermometer as the signal reader was developed. Using an immunoassay as a proof of concept, iron oxide nanoparticles (NPs) captured in the sandwich-type assay system were transformed into a near-infrared (NIR) laser-driven photothermal agent, Prussian blue (PB) NPs, which acted as a photothermal probe to convert the assay signal into heat through the photothermal effect, thus allowing sensitive biomolecular quantitation using a thermometer. This is the first report of biomolecular quantitation using a thermometer and also serves as the first attempt to introduce the nanoparticle-mediated photothermal effect for bioassays.A new biomolecular quantitation method, nanoparticle-mediated photothermal bioassay, using a common thermometer as the signal reader was developed. Using an immunoassay as a proof of concept, iron oxide nanoparticles (NPs) captured in the sandwich-type assay system were transformed into a near-infrared (NIR) laser-driven photothermal agent, Prussian blue (PB) NPs, which acted as a photothermal probe to convert the assay signal into heat through the photothermal effect, thus allowing sensitive biomolecular quantitation using a thermometer. This is the first report of biomolecular quantitation using a thermometer and also serves as the first attempt to introduce the nanoparticle-mediated photothermal effect for bioassays. Electronic supplementary information (ESI) available: Additional information on FTIR characterization (Fig. S1), photothermal immunoassay of PSA in human serum samples (Table S1), and the Experimental section, including preparation of antibody-conjugated iron oxide NPs, sandwich-type immunoassay, characterization, and photothermal detection protocol. See DOI: 10.1039/c5nr09051b

  4. High-Resolution Enabled 12-Plex DiLeu Isobaric Tags for Quantitative Proteomics

    PubMed Central

    2015-01-01

    Multiplex isobaric tags (e.g., tandem mass tags (TMT) and isobaric tags for relative and absolute quantification (iTRAQ)) are a valuable tool for high-throughput mass spectrometry based quantitative proteomics. We have developed our own multiplex isobaric tags, DiLeu, that feature quantitative performance on par with commercial offerings but can be readily synthesized in-house as a cost-effective alternative. In this work, we achieve a 3-fold increase in the multiplexing capacity of the DiLeu reagent without increasing structural complexity by exploiting mass defects that arise from selective incorporation of 13C, 15N, and 2H stable isotopes in the reporter group. The inclusion of eight new reporter isotopologues that differ in mass from the existing four reporters by intervals of 6 mDa yields a 12-plex isobaric set that preserves the synthetic simplicity and quantitative performance of the original implementation. We show that the new reporter variants can be baseline-resolved in high-resolution higher-energy C-trap dissociation (HCD) spectra, and we demonstrate accurate 12-plex quantitation of a DiLeu-labeled Saccharomyces cerevisiae lysate digest via high-resolution nano liquid chromatography–tandem mass spectrometry (nanoLC–MS2) analysis on an Orbitrap Elite mass spectrometer. PMID:25405479

  5. Advances in surface plasmon resonance imaging enable quantitative tracking of nanoscale changes in thickness and roughness.

    PubMed

    Raegen, Adam N; Reiter, Kyle; Dion, Alexander; Clarke, Anthony J; Lipkowski, Jacek; Dutcher, John R

    2014-04-01

    To date, detailed studies of the thickness of coatings using surface plasmon resonance have been limited to samples that are very uniform in thickness, and this technique has not been applied quantitatively to samples that are inherently rough or undergo instabilities with time. Our manuscript describes a significant improvement to surface plasmon resonance imaging (SPRi) that allows this sensitive technique to be used for quantitative tracking of the thickness and roughness of surface coatings that are rough on the scale of tens of nanometers. We tested this approach by studying samples with an idealized, one-dimensional roughness: patterned channels in a thin polymer film. We find that a novel analysis of the SPRi data collected with the plane of incidence parallel to the patterned channels allows the determination of the thickness profile of the channels in the polymer film, which is in agreement with that measured using atomic force microscopy. We have further validated our approach by performing SPRi measurements perpendicular to the patterned channels, for which the measured SPR curve agrees well with the single SPR curve calculated using the average thickness determined from the thickness profile as determined using AFM. We applied this analysis technique to track the average thickness and RMS roughness of cellulose microfibrils upon exposure to cellulolytic enzymes, providing quantitative determinations of the times of action of the enzymes that are of direct interest to the cellulosic ethanol industry. PMID:24605881

  6. Phosphoproteome Dynamics Upon Changes in Plant Water Status Reveal Early Events Associated With Rapid Growth Adjustment in Maize Leaves*

    PubMed Central

    Bonhomme, Ludovic; Valot, Benoît; Tardieu, François; Zivy, Michel

    2012-01-01

    Plant growth adjustment during water deficit is a crucial adaptive response. The rapid fine-tuned control achieved at the post-translational level is believed to be of considerable importance for regulating early changes in plant growth reprogramming. Aiming at a better understanding of early responses to contrasting plant water statuses, we carried out a survey of the protein phosphorylation events in the growing zone of maize leaves upon a range of water regimes. In this study, the impact of mild and severe water deficits were evaluated in comparison with constant optimal watering and with recovery periods lasting 5, 10, 20, 30, 45, and 60 min. Using four biological replicates per treatment and a robust quantitative phosphoproteomic methodology based on stable-isotope labeling, we identified 3664 unique phosphorylation sites on 2496 proteins. The abundance of nearly 1250 phosphorylated peptides was reproducibly quantified and profiled with high confidence among treatments. A total of 138 phosphopeptides displayed highly significant changes according to water regimes and enabled to identify specific patterns of response to changing plant water statuses. Further quantification of protein amounts emphasized that most phosphorylation changes did not reflect protein abundance variation. During water deficit and recovery, extensive changes in phosphorylation status occurred in critical regulators directly or indirectly involved in plant growth and development. These included proteins influencing epigenetic control, gene expression, cell cycle-dependent processes and phytohormone-mediated responses. Some of the changes depended on stress intensity whereas others depended on rehydration duration, including rapid recoveries that occurred as early as 5 or 10 mins after rewatering. By combining a physiological approach and a quantitative phosphoproteomic analysis, this work provides new insights into the in vivo early phosphorylation events triggered by rapid changes in

  7. Phosphoproteomic Analyses Reveal Signaling Pathways That Facilitate Lytic Gammaherpesvirus Replication

    PubMed Central

    Stahl, James A.; Chavan, Shweta S.; Sifford, Jeffrey M.; MacLeod, Veronica; Voth, Daniel E.; Edmondson, Ricky D.; Forrest, J. Craig

    2013-01-01

    Lytic gammaherpesvirus (GHV) replication facilitates the establishment of lifelong latent infection, which places the infected host at risk for numerous cancers. As obligate intracellular parasites, GHVs must control and usurp cellular signaling pathways in order to successfully replicate, disseminate to stable latency reservoirs in the host, and prevent immune-mediated clearance. To facilitate a systems-level understanding of phosphorylation-dependent signaling events directed by GHVs during lytic replication, we utilized label-free quantitative mass spectrometry to interrogate the lytic replication cycle of murine gammaherpesvirus-68 (MHV68). Compared to controls, MHV68 infection regulated by 2-fold or greater ca. 86% of identified phosphopeptides – a regulatory scale not previously observed in phosphoproteomic evaluations of discrete signal-inducing stimuli. Network analyses demonstrated that the infection-associated induction or repression of specific cellular proteins globally altered the flow of information through the host phosphoprotein network, yielding major changes to functional protein clusters and ontologically associated proteins. A series of orthogonal bioinformatics analyses revealed that MAPK and CDK-related signaling events were overrepresented in the infection-associated phosphoproteome and identified 155 host proteins, such as the transcription factor c-Jun, as putative downstream targets. Importantly, functional tests of bioinformatics-based predictions confirmed ERK1/2 and CDK1/2 as kinases that facilitate MHV68 replication and also demonstrated the importance of c-Jun. Finally, a transposon-mutant virus screen identified the MHV68 cyclin D ortholog as a viral protein that contributes to the prominent MAPK/CDK signature of the infection-associated phosphoproteome. Together, these analyses enhance an understanding of how GHVs reorganize and usurp intracellular signaling networks to facilitate infection and replication. PMID:24068923

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

    PubMed Central

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

    2015-01-01

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

  9. Quantitative Analyses of Core Promoters Enable Precise Engineering of Regulated Gene Expression in Mammalian Cells.

    PubMed

    Ede, Christopher; Chen, Ximin; Lin, Meng-Yin; Chen, Yvonne Y

    2016-05-20

    Inducible transcription systems play a crucial role in a wide array of synthetic biology circuits. However, the majority of inducible promoters are constructed from a limited set of tried-and-true promoter parts, which are susceptible to common shortcomings such as high basal expression levels (i.e., leakiness). To expand the toolbox for regulated mammalian gene expression and facilitate the construction of mammalian genetic circuits with precise functionality, we quantitatively characterized a panel of eight core promoters, including sequences with mammalian, viral, and synthetic origins. We demonstrate that this selection of core promoters can provide a wide range of basal gene expression levels and achieve a gradient of fold-inductions spanning 2 orders of magnitude. Furthermore, commonly used parts such as minimal CMV and minimal SV40 promoters were shown to achieve robust gene expression upon induction, but also suffer from high levels of leakiness. In contrast, a synthetic promoter, YB_TATA, was shown to combine low basal expression with high transcription rate in the induced state to achieve significantly higher fold-induction ratios compared to all other promoters tested. These behaviors remain consistent when the promoters are coupled to different genetic outputs and different response elements, as well as across different host-cell types and DNA copy numbers. We apply this quantitative understanding of core promoter properties to the successful engineering of human T cells that respond to antigen stimulation via chimeric antigen receptor signaling specifically under hypoxic environments. Results presented in this study can facilitate the design and calibration of future mammalian synthetic biology systems capable of precisely programmed functionality. PMID:26883397

  10. KinasePA: Phosphoproteomics data annotation using hypothesis driven kinase perturbation analysis.

    PubMed

    Yang, Pengyi; Patrick, Ellis; Humphrey, Sean J; Ghazanfar, Shila; James, David E; Jothi, Raja; Yang, Jean Yee Hwa

    2016-07-01

    Mass spectrometry (MS)-based quantitative phosphoproteomics has become a key approach for proteome-wide profiling of phosphorylation in tissues and cells. Traditional experimental design often compares a single treatment with a control, whereas increasingly more experiments are designed to compare multiple treatments with respect to a control. To this end, the development of bioinformatic tools that can integrate multiple treatments and visualise kinases and substrates under combinatorial perturbations is vital for dissecting concordant and/or independent effects of each treatment. Here, we propose a hypothesis driven kinase perturbation analysis (KinasePA) to annotate and visualise kinases and their substrates that are perturbed by various combinatorial effects of treatments in phosphoproteomics experiments. We demonstrate the utility of KinasePA through its application to two large-scale phosphoproteomics datasets and show its effectiveness in dissecting kinases and substrates within signalling pathways driven by unique combinations of cellular stimuli and inhibitors. We implemented and incorporated KinasePA as part of the "directPA" R package available from the comprehensive R archive network (CRAN). Furthermore, KinasePA also has an interactive web interface that can be readily applied to annotate user provided phosphoproteomics data (http://kinasepa.pengyiyang.org). PMID:27145998

  11. Phosphoproteomics in translational research: a sarcoma perspective.

    PubMed

    Noujaim, J; Payne, L S; Judson, I; Jones, R L; Huang, P H

    2016-05-01

    Phosphoproteomics has been extensively used as a preclinical research tool to characterize the phosphorylated components of the cancer proteome. Advances in the field have yielded insights into new drug targets, mechanisms of disease progression and drug resistance, and biomarker discovery. However, application of this technology to clinical research has been challenging because of practical issues relating to specimen integrity and tumour heterogeneity. Beyond these limitations, phosphoproteomics has the potential to play a pivotal role in translational studies and contribute to advances in different tumour groups, including rare disease sites like sarcoma. In this review, we propose that deploying phosphoproteomic technologies in translational research may facilitate the identification of better defined predictive biomarkers for patient stratification, inform drug selection in umbrella trials and identify new combinations to overcome drug resistance. We provide an overview of current phosphoproteomic technologies, such as affinity-based assays and mass spectrometry-based approaches, and discuss their advantages and limitations. We use sarcoma as an example to illustrate the current challenges in evaluating targeted kinase therapies in clinical trials. We then highlight useful lessons from preclinical studies in sarcoma biology to demonstrate how phosphoproteomics may address some of these challenges. Finally, we conclude by offering a perspective and list the key measures required to translate and benchmark a largely preclinical technology into a useful tool for translational research. PMID:26802162

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

    PubMed Central

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

    2015-01-01

    Yeast cells are protected by a cell wall that plays an important role in the exchange of substances with the environment. The cell wall structure is dynamic and can adapt to different physiological states or environmental conditions. For the investigation of morphological changes, selective staining with fluorescent dyes is a valuable tool. Furthermore, cell wall staining is used to facilitate sub-cellular localization experiments with fluorescently-labeled proteins and the detection of yeast cells in non-fungal host tissues. Here, we report staining of Saccharomyces cerevisiae cell wall with Trypan Blue, which emits strong red fluorescence upon binding to chitin and yeast glucan; thereby, it facilitates cell wall analysis by confocal and super-resolution microscopy. The staining pattern of Trypan Blue was similar to that of the widely used UV-excitable, blue fluorescent cell wall stain Calcofluor White. Trypan Blue staining facilitated quantification of cell size and cell wall volume when utilizing the optical sectioning capacity of a confocal microscope. This enabled the quantification of morphological changes during growth under anaerobic conditions and in the presence of chemicals, demonstrating the potential of this approach for morphological investigations or screening assays. PMID:25717323

  13. Quantitative hopanoid analysis enables robust pattern detection and comparison between laboratories.

    PubMed

    Wu, C-H; Kong, L; Bialecka-Fornal, M; Park, S; Thompson, A L; Kulkarni, G; Conway, S J; Newman, D K

    2015-07-01

    external diplopterol standards. These new quantitative approaches permit meaningful comparisons between studies, allowing more accurate hopanoid pattern detection in both laboratory and environmental samples. PMID:25865768

  14. Quantitative hopanoid analysis enables robust pattern detection and comparison between laboratories

    PubMed Central

    Wu, C-H; Kong, L; Bialecka-Fornal, M; Park, S; Thompson, A L; Kulkarni, G; Conway, S J; Newman, D K

    2015-01-01

    external diplopterol standards. These new quantitative approaches permit meaningful comparisons between studies, allowing more accurate hopanoid pattern detection in both laboratory and environmental samples. PMID:25865768

  15. Technical phosphoproteomic and bioinformatic tools useful in cancer research.

    PubMed

    López, Elena; Wesselink, Jan-Jaap; López, Isabel; Mendieta, Jesús; Gómez-Puertas, Paulino; Muñoz, Sarbelio Rodríguez

    2011-01-01

    Reversible protein phosphorylation is one of the most important forms of cellular regulation. Thus, phosphoproteomic analysis of protein phosphorylation in cells is a powerful tool to evaluate cell functional status. The importance of protein kinase-regulated signal transduction pathways in human cancer has led to the development of drugs that inhibit protein kinases at the apex or intermediary levels of these pathways. Phosphoproteomic analysis of these signalling pathways will provide important insights for operation and connectivity of these pathways to facilitate identification of the best targets for cancer therapies. Enrichment of phosphorylated proteins or peptides from tissue or bodily fluid samples is required. The application of technologies such as phosphoenrichments, mass spectrometry (MS) coupled to bioinformatics tools is crucial for the identification and quantification of protein phosphorylation sites for advancing in such relevant clinical research. A combination of different phosphopeptide enrichments, quantitative techniques and bioinformatic tools is necessary to achieve good phospho-regulation data and good structural analysis of protein studies. The current and most useful proteomics and bioinformatics techniques will be explained with research examples. Our aim in this article is to be helpful for cancer research via detailing proteomics and bioinformatic tools. PMID:21967744

  16. Technical phosphoproteomic and bioinformatic tools useful in cancer research

    PubMed Central

    2011-01-01

    Reversible protein phosphorylation is one of the most important forms of cellular regulation. Thus, phosphoproteomic analysis of protein phosphorylation in cells is a powerful tool to evaluate cell functional status. The importance of protein kinase-regulated signal transduction pathways in human cancer has led to the development of drugs that inhibit protein kinases at the apex or intermediary levels of these pathways. Phosphoproteomic analysis of these signalling pathways will provide important insights for operation and connectivity of these pathways to facilitate identification of the best targets for cancer therapies. Enrichment of phosphorylated proteins or peptides from tissue or bodily fluid samples is required. The application of technologies such as phosphoenrichments, mass spectrometry (MS) coupled to bioinformatics tools is crucial for the identification and quantification of protein phosphorylation sites for advancing in such relevant clinical research. A combination of different phosphopeptide enrichments, quantitative techniques and bioinformatic tools is necessary to achieve good phospho-regulation data and good structural analysis of protein studies. The current and most useful proteomics and bioinformatics techniques will be explained with research examples. Our aim in this article is to be helpful for cancer research via detailing proteomics and bioinformatic tools. PMID:21967744

  17. Pump-free gradient-based micro-device enables quantitative and high-throughput bacterial growth inhibition analysis.

    PubMed

    Ran, Min; Wang, Ying; Wang, Sida; Luo, Chunxiong

    2015-08-01

    Antibiotic susceptibility testing is very important in antibiotic therapy. Traditional methods to determine antibiotic susceptibility include disk diffusion and broth dilution. However, these tests are always labor intensive, time-consuming, and need large amounts of reagents. In this paper, we demonstrated a novel pump-free micro-device that enables quantitative and high-throughput bacterial growth inhibition analysis. This device consists of a series of wells and diffusion-based antibiotic gradient channels. The wells serve as antibiotic sources and buffer sinks, and we could easily observe the bacterial growth in the gradient channels .The design of the multi-wells is adapted to the commercialized multi-channel pipette, which makes it very convenient for loading reagents into the wells. For each assay, only about 20 μL antibiotic solution is needed. As a demonstration, we used both fluorescence images and dark-field images to quantify the bacterial growth inhibition effect under different antibiotics. The quantitative data of bacterial growth inhibition under different antibiotics can be obtained within 3 to 4 h. Considering the simple operation process and the high-throughput and quantitative result this device can offer, it has great potential to be widely used in clinics and may be useful for the study of the kinetics of bacterial growth. PMID:26044203

  18. Characterization of the Phosphoproteome in SLE Patients

    PubMed Central

    Huang, Jianrong; Dai, Yong

    2012-01-01

    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. PMID:23285258

  19. Phosphoproteomic Analysis of Cell-Based Resistance to BRAF Inhibitor Therapy in Melanoma

    PubMed Central

    Parker, Robert; Vella, Laura J.; Xavier, Dylan; Amirkhani, Ardeshir; Parker, Jimmy; Cebon, Jonathan; Molloy, Mark P.

    2015-01-01

    The treatment of melanoma by targeted inhibition of the mutated kinase BRAF with small molecules only temporarily suppresses metastatic disease. In the face of chemical inhibition tumor plasticity, both innate and adaptive, promotes survival through the biochemical and genetic reconfiguration of cellular pathways that can engage proliferative and migratory systems. To investigate this process, high-resolution mass spectrometry was used to characterize the phosphoproteome of this transition in vitro. A simple and accurate, label-free quantitative method was used to localize and quantitate thousands of phosphorylation events. We also correlated changes in the phosphoproteome with the proteome to more accurately determine changes in the activity of regulatory kinases determined by kinase landscape profiling. The abundance of phosphopeptides with sites that function in cytoskeletal regulation, GTP/GDP exchange, protein kinase C, IGF signaling, and melanosome maturation were highly divergent after transition to a drug resistant phenotype. PMID:26029660

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

    PubMed Central

    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. PMID:25713560

  1. Laboratory reflectance spectra of clay minerals mixed with Mars analog materials: Toward enabling quantitative clay abundances from Mars spectra

    NASA Astrophysics Data System (ADS)

    Roush, Ted L.; Bishop, Janice L.; Brown, Adrian J.; Blake, David F.; Bristow, Thomas F.

    2015-09-01

    Quantitative estimates of clay minerals on the martian surface, via remote sensing observations, provide constraints on activity, timing, duration, and extent of aqueous processes and the geochemical environment in martian history. We describe an analytical study to begin enabling quantitative estimates of phyllosilicates when mixed with martian analog materials. We characterize the chemistry, mineralogy, particle size distribution, and reflectance spectra of the end-member materials: saponite, montmorillonite, pyroxene, and palagonitic soil. Reflectance spectra were obtained for physical mixtures of saponite and montmorillonite with pyroxene, and saponite with palagonitic soil. We analyzed the diagnostic phyllosilicate spectral signatures in the 2.2-2.4 μm wavelength region in detail for the mixtures. This involved fitting the observed ∼2.3 or ∼2.2 μm band depth, associated with the presence of saponite and montmorillonite, respectively, as a function of the abundance of these materials in the mixtures. Based upon the band depth of the spectral features we find that 3-5 wt.% of the clay minerals in the mixture with pyroxene can be recognized and at 25 wt.% their presence is indisputable in the mixtures. When the saponite is mixed with the lower albedo palagonitic soil, its presence is clearly distinguishable via the 1.4 and 2.3 μm features at 25 wt.% abundance. These relationships, between abundance and band depth, provide an ability to quantitatively address the amount of these materials in mixtures. The trends described here provide guidance for estimating the presence of phyllosilicates in matrices on the martian surface.

  2. Improved Selection of Internal Transcribed Spacer-Specific Primers Enables Quantitative, Ultra-High-Throughput Profiling of Fungal Communities

    PubMed Central

    Bokulich, Nicholas A.

    2013-01-01

    Ultra-high-throughput sequencing (HTS) of fungal communities has been restricted by short read lengths and primer amplification bias, slowing the adoption of newer sequencing technologies to fungal community profiling. To address these issues, we evaluated the performance of several common internal transcribed spacer (ITS) primers and designed a novel primer set and work flow for simultaneous quantification and species-level interrogation of fungal consortia. Primer comparison and validation were predicted in silico and by sequencing a “mock community” of mixed yeast species to explore the challenges of amplicon length and amplification bias for reconstructing defined yeast community structures. The amplicon size and distribution of this primer set are smaller than for all preexisting ITS primer sets, maximizing sequencing coverage of hypervariable ITS domains by very-short-amplicon, high-throughput sequencing platforms. This feature also enables the optional integration of quantitative PCR (qPCR) directly into the HTS preparatory work flow by substituting qPCR with these primers for standard PCR, yielding quantification of individual community members. The complete work flow described here, utilizing any of the qualified primer sets evaluated, can rapidly profile mixed fungal communities and capably reconstructed well-characterized beer and wine fermentation fungal communities. PMID:23377949

  3. Computational phosphoproteomics: From identification to localization

    PubMed Central

    Lee, Dave C H; Jones, Andrew R; Hubbard, Simon J

    2015-01-01

    Analysis of the phosphoproteome by MS has become a key technology for the characterization of dynamic regulatory processes in the cell, since kinase and phosphatase action underlie many major biological functions. However, the addition of a phosphate group to a suitable side chain often confounds informatic analysis by generating product ion spectra that are more difficult to interpret (and consequently identify) relative to unmodified peptides. Collectively, these challenges have motivated bioinformaticians to create novel software tools and pipelines to assist in the identification of phosphopeptides in proteomic mixtures, and help pinpoint or “localize” the most likely site of modification in cases where there is ambiguity. Here we review the challenges to be met and the informatics solutions available to address them for phosphoproteomic analysis, as well as highlighting the difficulties associated with using them and the implications for data standards. PMID:25475148

  4. Phosphoproteomics data classify hematological cancer cell lines according to tumor type and sensitivity to kinase inhibitors

    PubMed Central

    2013-01-01

    Background Tumor classification based on their predicted responses to kinase inhibitors is a major goal for advancing targeted personalized therapies. Here, we used a phosphoproteomic approach to investigate biological heterogeneity across hematological cancer cell lines including acute myeloid leukemia, lymphoma, and multiple myeloma. Results Mass spectrometry was used to quantify 2,000 phosphorylation sites across three acute myeloid leukemia, three lymphoma, and three multiple myeloma cell lines in six biological replicates. The intensities of the phosphorylation sites grouped these cancer cell lines according to their tumor type. In addition, a phosphoproteomic analysis of seven acute myeloid leukemia cell lines revealed a battery of phosphorylation sites whose combined intensities correlated with the growth-inhibitory responses to three kinase inhibitors with remarkable correlation coefficients and fold changes (> 100 between the most resistant and sensitive cells). Modeling based on regression analysis indicated that a subset of phosphorylation sites could be used to predict response to the tested drugs. Quantitative analysis of phosphorylation motifs indicated that resistant and sensitive cells differed in their patterns of kinase activities, but, interestingly, phosphorylations correlating with responses were not on members of the pathway being targeted; instead, these mainly were on parallel kinase pathways. Conclusion This study reveals that the information on kinase activation encoded in phosphoproteomics data correlates remarkably well with the phenotypic responses of cancer cells to compounds that target kinase signaling and could be useful for the identification of novel markers of resistance or sensitivity to drugs that target the signaling network. PMID:23628362

  5. Sample Preparation for Phosphoproteomic Analysis of Circadian Time Series in Arabidopsis thaliana

    PubMed Central

    Krahmer, Johanna; Hindle, Matthew M.; Martin, Sarah F.; Le Bihan, Thierry; Millar, Andrew J.

    2015-01-01

    Systems biological approaches to study the Arabidopsis thaliana circadian clock have mainly focused on transcriptomics while little is known about the proteome, and even less about posttranslational modifications. Evidence has emerged that posttranslational protein modifications, in particular phosphorylation, play an important role for the clock and its output. Phosphoproteomics is the method of choice for a large-scale approach to gain more knowledge about rhythmic protein phosphorylation. Recent plant phosphoproteomics publications have identified several thousand phosphopeptides. However, the methods used in these studies are very labor-intensive and therefore not suitable to apply to a well-replicated circadian time series. To address this issue, we present and compare different strategies for sample preparation for phosphoproteomics that are compatible with large numbers of samples. Methods are compared regarding number of identifications, variability of quantitation, and functional categorization. We focus on the type of detergent used for protein extraction as well as methods for its removal. We also test a simple two-fraction separation of the protein extract. PMID:25662467

  6. Validation of reference genes in Penicillium echinulatum to enable gene expression study using real-time quantitative RT-PCR.

    PubMed

    Zampieri, Denise; Nora, Luísa C; Basso, Vanessa; Camassola, Marli; Dillon, Aldo J P

    2014-08-01

    Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is a methodology that facilitates the quantification of mRNA expression in a given sample. Analysis of relative gene expression by qRT-PCR requires normalization of the data using a reference gene that is expressed at a similar level in all evaluated conditions. Determining an internal control gene is essential for gene expression studies. Gene expression studies in filamentous fungi frequently use the β-actin gene (actb), β-tubulin, and glyceraldehyde-3-phosphate dehydrogenase as reference genes because they are known to have consistent expression levels. Until now, no study has been performed to select an internal control gene for the filamentous fungal species Penicillium echinulatum. The aim of this study was to evaluate and validate internal control genes to enable the study of gene expression in P. echinulatum using qRT-PCR. P. echinulatum strain S1M29 was grown in conditions to either induce (cellulose and sugar cane bagasse) or repress (glucose) gene expression to analyze 23 candidate normalization genes for stable expression. Two software programs, BestKeeper and geNorm, were used to assess the expression of the candidate normalization genes. The results indicate that the actb reference gene is more stably expressed in P. echinulatum. This is the first report in the literature that determines a normalization gene for this fungus. From the results obtained, we recommend the use of the P. echinulatum actb gene as an endogenous control for gene expression studies of cellulases and hemicellulases by qRT-PCR. PMID:24509829

  7. Assessment of beating parameters in human induced pluripotent stem cells enables quantitative in vitro screening for cardiotoxicity

    SciTech Connect

    Sirenko, Oksana; Cromwell, Evan F.; Crittenden, Carole; Wignall, Jessica A.; Wright, Fred A.; Rusyn, Ivan

    2013-12-15

    Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes show promise for screening during early drug development. Here, we tested a hypothesis that in vitro assessment of multiple cardiomyocyte physiological parameters enables predictive and mechanistically-interpretable evaluation of cardiotoxicity in a high-throughput format. Human iPSC-derived cardiomyocytes were exposed for 30 min or 24 h to 131 drugs, positive (107) and negative (24) for in vivo cardiotoxicity, in up to 6 concentrations (3 nM to 30 uM) in 384-well plates. Fast kinetic imaging was used to monitor changes in cardiomyocyte function using intracellular Ca{sup 2+} flux readouts synchronous with beating, and cell viability. A number of physiological parameters of cardiomyocyte beating, such as beat rate, peak shape (amplitude, width, raise, decay, etc.) and regularity were collected using automated data analysis. Concentration–response profiles were evaluated using logistic modeling to derive a benchmark concentration (BMC) point-of-departure value, based on one standard deviation departure from the estimated baseline in vehicle (0.3% dimethyl sulfoxide)-treated cells. BMC values were used for cardiotoxicity classification and ranking of compounds. Beat rate and several peak shape parameters were found to be good predictors, while cell viability had poor classification accuracy. In addition, we applied the Toxicological Prioritization Index (ToxPi) approach to integrate and display data across many collected parameters, to derive “cardiosafety” ranking of tested compounds. Multi-parameter screening of beating profiles allows for cardiotoxicity risk assessment and identification of specific patterns defining mechanism-specific effects. These data and analysis methods may be used widely for compound screening and early safety evaluation in drug development. - Highlights: • Induced pluripotent stem cell-derived cardiomyocytes are promising in vitro models. • We tested if evaluation

  8. DNA Replication Stress Phosphoproteome Profiles Reveal Novel Functional Phosphorylation Sites on Xrs2 in Saccharomyces cerevisiae.

    PubMed

    Huang, Dongqing; Piening, Brian D; Kennedy, Jacob J; Lin, Chenwei; Jones-Weinert, Corey W; Yan, Ping; Paulovich, Amanda G

    2016-05-01

    In response to replication stress, a phospho-signaling cascade is activated and required for coordination of DNA repair and replication of damaged templates (intra-S-phase checkpoint) . How phospho-signaling coordinates the DNA replication stress response is largely unknown. We employed state-of-the-art liquid chromatography tandem-mass spectrometry (LC-MS/MS) approaches to generate high-coverage and quantitative proteomic and phospho-proteomic profiles during replication stress in yeast, induced by continuous exposure to the DNA alkylating agent methyl methanesulfonate (MMS) . We identified 32,057 unique peptides representing the products of 4296 genes and 22,061 unique phosphopeptides representing the products of 3183 genes. A total of 542 phosphopeptides (mapping to 339 genes) demonstrated an abundance change of greater than or equal to twofold in response to MMS. The screen enabled detection of nearly all of the proteins known to be involved in the DNA damage response, as well as many novel MMS-induced phosphorylations. We assessed the functional importance of a subset of key phosphosites by engineering a panel of phosphosite mutants in which an amino acid substitution prevents phosphorylation. In total, we successfully mutated 15 MMS-responsive phosphorylation sites in seven representative genes including APN1 (base excision repair); CTF4 and TOF1 (checkpoint and sister-chromatid cohesion); MPH1 (resolution of homologous recombination intermediates); RAD50 and XRS2 (MRX complex); and RAD18 (PRR). All of these phosphorylation site mutants exhibited MMS sensitivity, indicating an important role in protecting cells from DNA damage. In particular, we identified MMS-induced phosphorylation sites on Xrs2 that are required for MMS resistance in the absence of the MRX activator, Sae2, and that affect telomere maintenance. PMID:27017623

  9. Phosphoproteomics technologies and applications in plant biology research

    PubMed Central

    Li, Jinna; Silva-Sanchez, Cecilia; Zhang, Tong; Chen, Sixue; Li, Haiying

    2015-01-01

    Protein phosphorylation has long been recognized as an essential mechanism to regulate many important processes of plant life. However, studies on phosphorylation mediated signaling events in plants are challenged with low stoichiometry and dynamic nature of phosphorylated proteins. Significant advances in mass spectrometry based phosphoproteomics have taken place in recent decade, including phosphoprotein/phosphopeptide enrichment, detection and quantification, and phosphorylation site localization. This review describes a variety of separation and enrichment methods for phosphoproteins and phosphopeptides, the applications of technological innovations in plant phosphoproteomics, and highlights significant achievement of phosphoproteomics in the areas of plant signal transduction, growth and development. PMID:26136758

  10. Large-Scale Multiplexed Quantitative Discovery Proteomics Enabled by the Use of an O-18-Labeled “Universal” Reference Sample

    SciTech Connect

    Qian, Weijun; Liu, Tao; Petyuk, Vladislav A.; Gritsenko, Marina A.; Petritis, Brianne O.; Polpitiya, Ashoka D.; Kaushal, Amit; Xiao, Wenzhong; Finnerty, Celeste C.; Jescheke, Marc G.; Jaitly, Navdeep; Monroe, Matthew E.; Moore, Ronald J.; Moldawer, Lyle L.; Davis, Ronald W.; Tompkins, Ronald G.; Hemdon, David N.; Camp, David G.; Smith, Richard D.

    2009-01-01

    Quantitative comparison of protein abundances across a relatively large number of patient samples is an important challenge for clinical proteomic applications. Herein we describe a dual-quantitation strategy that allows the simultaneous integration of complementary label-free and stable isotope labeling based approaches without increasing the number of LC-MS analyses. The approach utilizes a stable isotope 18O-labeled “universal” reference sample as a comprehensive set of internal standards spiked into each individually processed unlabeled patient sample. The quantitative data are based on both the direct 16O-MS intensities for label-free quantitation and the 16O/18O isotopic peptide pair ratios that compare each patient sample to the identical labeled reference. The effectiveness of this dual-quantitation approach for large scale quantitative proteomics is demonstrated by the application to a set of 38 clinical plasma samples from surviving and non-surviving severe burn patients. With the coupling of immunoaffinity depletion, cysteinyl-peptide enrichment based fractionation, high resolution LC-MS measurements, and the dual-quantitation approach, a total of 318 proteins were confidently quantified with at least two peptides and 263 proteins were quantified by both approaches. The strategy also enabled a direct comparison between the two approaches with the labeling approach showing significantly better precision in quantitation while the label-free approach resulted in more protein identifications. The relative abundance differences determined by the two approaches also show strong correlation. Finally, the dual-quantitation strategy allowed us to identify more candidate protein biomarkers, illustrating the complementary nature of the two quantitative methods.

  11. Toward defining the phosphoproteome of Xenopus laevis embryos

    PubMed Central

    McGivern, Jered V.; Swaney, Danielle L.; Coon, Joshua J.; Sheets, Michael D.

    2010-01-01

    Phosphorylation is universally used for controlling protein function, but knowledge of the phosphoproteome in vertebrate embryos has been limited. However, recent technical advances make it possible to define an organism's phosphoproteome at a more comprehensive level. Xenopus laevis offers established advantages for analyzing the regulation of protein function by phosphorylation. Functionally unbiased, comprehensive information about the Xenopus phosphoproteome would provide a powerful guide for future studies of phosphorylation in a developmental context. To this end, we performed a phosphoproteomic analysis of Xenopus oocytes, eggs, and embryos using recently developed mass spectrometry methods. We identified 1,441 phosphorylation sites present on 654 different Xenopus proteins, including hundreds of previously unknown phosphorylation sites. This approach identified several phosphorylation sites described in the literature and/or evolutionarily conserved in other organisms, validating the data's quality. These data will serve as a powerful resource for the exploration of phosphorylation and protein function within a developmental context. PMID:19384857

  12. Integrating Phosphoproteome and Transcriptome Reveals New Determinants of Macrophage Multinucleation*

    PubMed Central

    Rotival, Maxime; Ko, Jeong-Hun; Srivastava, Prashant K.; Kerloc'h, Audrey; Montoya, Alex; Mauro, Claudio; Faull, Peter; Cutillas, Pedro R.; Petretto, Enrico; Behmoaras, Jacques

    2015-01-01

    Macrophage multinucleation (MM) is essential for various biological processes such as osteoclast-mediated bone resorption and multinucleated giant cell-associated inflammatory reactions. Here we study the molecular pathways underlying multinucleation in the rat through an integrative approach combining MS-based quantitative phosphoproteomics (LC-MS/MS) and transcriptome (high-throughput RNA-sequencing) to identify new regulators of MM. We show that a strong metabolic shift toward HIF1-mediated glycolysis occurs at transcriptomic level during MM, together with modifications in phosphorylation of over 50 proteins including several ARF GTPase activators and polyphosphate inositol phosphatases. We use shortest-path analysis to link differential phosphorylation with the transcriptomic reprogramming of macrophages and identify LRRFIP1, SMARCA4, and DNMT1 as novel regulators of MM. We experimentally validate these predictions by showing that knock-down of these latter reduce macrophage multinucleation. These results provide a new framework for the combined analysis of transcriptional and post-translational changes during macrophage multinucleation, prioritizing essential genes, and revealing the sequential events leading to the multinucleation of macrophages. PMID:25532521

  13. Virtual-'Light-Sheet' Single-Molecule Localisation Microscopy Enables Quantitative Optical Sectioning for Super-Resolution Imaging

    PubMed Central

    Palayret, Matthieu; Armes, Helen; Basu, Srinjan; Watson, Adam T.; Herbert, Alex; Lando, David; Etheridge, Thomas J.; Endesfelder, Ulrike; Heilemann, Mike; Laue, Ernest; Carr, Antony M.; Klenerman, David; Lee, Steven F.

    2015-01-01

    Single-molecule super-resolution microscopy allows imaging of fluorescently-tagged proteins in live cells with a precision well below that of the diffraction limit. Here, we demonstrate 3D sectioning with single-molecule super-resolution microscopy by making use of the fitting information that is usually discarded to reject fluorophores that emit from above or below a virtual-'light-sheet', a thin volume centred on the focal plane of the microscope. We describe an easy-to-use routine (implemented as an open-source ImageJ plug-in) to quickly analyse a calibration sample to define and use such a virtual light-sheet. In addition, the plug-in is easily usable on almost any existing 2D super-resolution instrumentation. This optical sectioning of super-resolution images is achieved by applying well-characterised width and amplitude thresholds to diffraction-limited spots that can be used to tune the thickness of the virtual light-sheet. This allows qualitative and quantitative imaging improvements: by rejecting out-of-focus fluorophores, the super-resolution image gains contrast and local features may be revealed; by retaining only fluorophores close to the focal plane, virtual-'light-sheet' single-molecule localisation microscopy improves the probability that all emitting fluorophores will be detected, fitted and quantitatively evaluated. PMID:25884495

  14. Virtual-'light-sheet' single-molecule localisation microscopy enables quantitative optical sectioning for super-resolution imaging.

    PubMed

    Palayret, Matthieu; Armes, Helen; Basu, Srinjan; Watson, Adam T; Herbert, Alex; Lando, David; Etheridge, Thomas J; Endesfelder, Ulrike; Heilemann, Mike; Laue, Ernest; Carr, Antony M; Klenerman, David; Lee, Steven F

    2015-01-01

    Single-molecule super-resolution microscopy allows imaging of fluorescently-tagged proteins in live cells with a precision well below that of the diffraction limit. Here, we demonstrate 3D sectioning with single-molecule super-resolution microscopy by making use of the fitting information that is usually discarded to reject fluorophores that emit from above or below a virtual-'light-sheet', a thin volume centred on the focal plane of the microscope. We describe an easy-to-use routine (implemented as an open-source ImageJ plug-in) to quickly analyse a calibration sample to define and use such a virtual light-sheet. In addition, the plug-in is easily usable on almost any existing 2D super-resolution instrumentation. This optical sectioning of super-resolution images is achieved by applying well-characterised width and amplitude thresholds to diffraction-limited spots that can be used to tune the thickness of the virtual light-sheet. This allows qualitative and quantitative imaging improvements: by rejecting out-of-focus fluorophores, the super-resolution image gains contrast and local features may be revealed; by retaining only fluorophores close to the focal plane, virtual-'light-sheet' single-molecule localisation microscopy improves the probability that all emitting fluorophores will be detected, fitted and quantitatively evaluated. PMID:25884495

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

    PubMed Central

    Hu, Xiuli; Wu, Liuji; Zhao, Feiyun; Zhang, Dayong; Li, Nana; Zhu, Guohui; Li, Chaohao; Wang, Wei

    2015-01-01

    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. PMID:25999967

  16. Noninvasive device readouts validated by immunohistochemical analysis enable objective quantitative assessment of acute wound healing in human skin.

    PubMed

    Ud-Din, Sara; Greaves, Nicholas S; Sebastian, Anil; Baguneid, Mohamed; Bayat, Ardeshir

    2015-01-01

    Objective evaluation of cutaneous wounds through use of noninvasive devices has important implications for diagnosis, monitoring treatment efficacy, progression and may lead to development of improved theranostic treatment strategies. However, there is a lack of validation in the use of certain devices in wound repair, where objective measurements taken by noninvasive devices have been corroborated by immunohistochemical analysis. Thus, data from three acute wound-healing studies in healthy volunteers using three noninvasive objective devices were further evaluated by immunohistochemistry. One hundred ten participants had 5-mm diameter skin biopsies to their arms. Spectrophotometric intracutaneous analysis (SIAscopy), full-field laser perfusion imaging, and three-dimensional imaging provided quantitative measurements of melanin, hemoglobin, collagen, blood flow, and wound size; all of which were validated by immunohistochemistry. Full-field laser perfusion imaging showed blood flow increased to D7 and decreased by 40% to D14. SIAscopy showed that hemoglobin increased to D7 and reduced to D14. CD31 analysis corroborated this by showing a 76% increase in blood vessel density to D7 and a reduction by 14% to D14. Three-dimensional imaging showed that wound surface area reduced by 50% from day 7 to day 14. Alpha-smooth muscle Actin (Alpha-SMA) staining supported these trends by showing increased levels by 72% from D0 to D14 (corresponding to wound contraction). Collagen, measured by SIAscopy, decreased to D7 and increased to D14, which was validated by collagen III analysis. Additionally, collagen I increased by 14% from D0 to D14. SIAscopy measurements for melanin showed an increase at D7 and a slight reduction to D14, while melanogenesis increased by 46.7% from D0 to D14. These findings show the utility of noninvasive objective devices in the quantitative evaluation of wound-healing parameters in human skin as corroborated by immunohistochemistry. This may contribute

  17. Quantitative phosphoproteome analysis of embryonic stem cell differentiation toward blood

    PubMed Central

    Piazzi, Manuela; Williamson, Andrew; Lee, Chia-Fang; Pearson, Stella; Lacaud, Georges; Kouskoff, Valerie; McCubrey, James A.; Cocco, Lucio; Whetton, Anthony D.

    2015-01-01

    Murine embryonic stem (ES) cells can differentiate in vitro into three germ layers (endodermic, mesodermic, ectodermic). Studies on the differentiation of these cells to specific early differentiation stages has been aided by an ES cell line carrying the Green Fluorescent Protein (GFP) targeted to the Brachyury (Bry) locus which marks mesoderm commitment. Furthermore, expression of the Vascular Endothelial Growth Factor receptor 2 (Flk1) along with Bry defines hemangioblast commitment. Isobaric-tag for relative and absolute quantification (iTRAQTM) and phosphopeptide enrichment coupled to liquid chromatography separation and mass spectrometry allow the study of phosphorylation changes occurring at different stages of ES cell development using Bry and Flk1 expression respectively. We identified and relatively quantified 37 phosphoentities which are modulated during mesoderm-induced ES cells differentiation, comparing epiblast-like, early mesoderm and hemangioblast-enriched cells. Among the proteins differentially phosphorylated toward mesoderm differentiation were: the epigenetic regulator Dnmt3b, the protein kinase GSK3b, the chromatin remodeling factor Smarcc1, the transcription factor Utf1; as well as protein specifically related to stem cell differentiation, as Eomes, Hmga2, Ints1 and Rif1. As most key factors regulating early hematopoietic development have also been implicated in various types of leukemia, understanding the post-translational modifications driving their regulation during normal development could result in a better comprehension of their roles during abnormal hematopoiesis in leukemia. PMID:25890499

  18. In vivo Monitoring of Transcriptional Dynamics After Lower-Limb Muscle Injury Enables Quantitative Classification of Healing

    PubMed Central

    Aguilar, Carlos A.; Shcherbina, Anna; Ricke, Darrell O.; Pop, Ramona; Carrigan, Christopher T.; Gifford, Casey A.; Urso, Maria L.; Kottke, Melissa A.; Meissner, Alexander

    2015-01-01

    Traumatic lower-limb musculoskeletal injuries are pervasive amongst athletes and the military and typically an individual returns to activity prior to fully healing, increasing a predisposition for additional injuries and chronic pain. Monitoring healing progression after a musculoskeletal injury typically involves different types of imaging but these approaches suffer from several disadvantages. Isolating and profiling transcripts from the injured site would abrogate these shortcomings and provide enumerative insights into the regenerative potential of an individual’s muscle after injury. In this study, a traumatic injury was administered to a mouse model and healing progression was examined from 3 hours to 1 month using high-throughput RNA-Sequencing (RNA-Seq). Comprehensive dissection of the genome-wide datasets revealed the injured site to be a dynamic, heterogeneous environment composed of multiple cell types and thousands of genes undergoing significant expression changes in highly regulated networks. Four independent approaches were used to determine the set of genes, isoforms, and genetic pathways most characteristic of different time points post-injury and two novel approaches were developed to classify injured tissues at different time points. These results highlight the possibility to quantitatively track healing progression in situ via transcript profiling using high- throughput sequencing. PMID:26381351

  19. A Novel Image-Analysis Toolbox Enabling Quantitative Analysis of Root System Architecture1[W][OA

    PubMed Central

    Lobet, Guillaume; Pagès, Loïc; Draye, Xavier

    2011-01-01

    We present in this paper a novel, semiautomated image-analysis software to streamline the quantitative analysis of root growth and architecture of complex root systems. The software combines a vectorial representation of root objects with a powerful tracing algorithm that accommodates a wide range of image sources and quality. The root system is treated as a collection of roots (possibly connected) that are individually represented as parsimonious sets of connected segments. Pixel coordinates and gray level are therefore turned into intuitive biological attributes such as segment diameter and orientation as well as distance to any other segment or topological position. As a consequence, user interaction and data analysis directly operate on biological entities (roots) and are not hampered by the spatially discrete, pixel-based nature of the original image. The software supports a sampling-based analysis of root system images, in which detailed information is collected on a limited number of roots selected by the user according to specific research requirements. The use of the software is illustrated with a time-lapse analysis of cluster root formation in lupin (Lupinus albus) and an architectural analysis of the maize (Zea mays) root system. The software, SmartRoot, is an operating system-independent freeware based on ImageJ and relies on cross-platform standards for communication with data-analysis software. PMID:21771915

  20. Quantitative Proteomics Reveals That Hsp90 Inhibition Preferentially Targets Kinases and the DNA Damage Response*

    PubMed Central

    Sharma, Kirti; Vabulas, R. Martin; Macek, Boris; Pinkert, Stefan; Cox, Jürgen; Mann, Matthias; Hartl, F. Ulrich

    2012-01-01

    Despite the increasing importance of heat shock protein 90 (Hsp90) inhibitors as chemotherapeutic agents in diseases such as cancer, their global effects on the proteome remain largely unknown. Here we use high resolution, quantitative mass spectrometry to map protein expression changes associated with the application of the Hsp90 inhibitor, 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG). In depth data obtained from five replicate SILAC experiments enabled accurate quantification of about 6,000 proteins in HeLa cells. As expected, we observed activation of a heat shock response with induced expression of molecular chaperones, which refold misfolded proteins, and proteases, which degrade irreparably damaged polypeptides. Despite the broad range of known Hsp90 substrates, bioinformatics analysis revealed that particular protein classes were preferentially affected. These prominently included proteins involved in the DNA damage response, as well as protein kinases and especially tyrosine kinases. We followed up on this observation with a quantitative phosphoproteomic analysis of about 4,000 sites, which revealed that Hsp90 inhibition leads to much more down- than up-regulation of the phosphoproteome (34% down versus 6% up). This study defines the cellular response to Hsp90 inhibition at the proteome level and sheds light on the mechanisms by which it can be used to target cancer cells. PMID:22167270

  1. Quantitative radiography enabled by slot collimation and a novel scatter correction technique on a large-area flat panel x-ray detector

    NASA Astrophysics Data System (ADS)

    Yue, Meghan L.; Boden, Adam E.; Sabol, John M.

    2009-02-01

    In addition to causing loss of contrast and blurring in an image, scatter also makes quantitative measurements of xray attenuation impossible. Many devices, methods, and models have been developed to eliminate, estimate, and correct for the effects of scatter. Although these techniques can reduce the impact of scatter in a large-area image, no methods have proven to be practical and sufficient to enable quantitative analysis of image data in a routine clinical setting. This paper describes a method of scatter correction which uses moderate x-ray collimation in combination with a correction algorithm operating on data obtained from large-area flat panel detector images. The method involves acquiring slot collimated images of the object, and utilizing information from outside of the collimated region, in addition to a priori data, to estimate the scatter within the collimated region. This method requires no increase dose to the patient while providing high image quality and accurate estimates of the primary x-ray data. This scatter correction technique was validated through beam stop experiments and comparison of theoretically calculated and measured contrast of thin aluminum and polymethylmethacrelate objects. Measurements taken with various background material thicknesses, both with and without a grid, showed that the slot-scatter corrected contrast and the theoretical contrast were not significantly different given a 99% confidence interval. However, the uncorrected contrast was found to be significantly different from the corrected and theoretical contrasts. These findings indicate that this method of scatter correction can eliminate the effect of scatter on contrast and potentially enable quantitative x-ray imaging.

  2. Dynamic Adipocyte Phosphoproteome Reveals that Akt Directly Regulates mTORC2

    PubMed Central

    Humphrey, Sean J.; Yang, Guang; Yang, Pengyi; Fazakerley, Daniel J.; Stöckli, Jacqueline; Yang, Jean Y.; James, David E.

    2013-01-01

    Summary A major challenge of the post-genomics era is to define the connectivity of protein phosphorylation networks. Here, we quantitatively delineate the insulin signaling network in adipocytes by high-resolution mass spectrometry-based proteomics. These data reveal the complexity of intracellular protein phosphorylation. We identified 37,248 phosphorylation sites on 5,705 proteins in this single-cell type, with approximately 15% responding to insulin. We integrated these large-scale phosphoproteomics data using a machine learning approach to predict physiological substrates of several diverse insulin-regulated kinases. This led to the identification of an Akt substrate, SIN1, a core component of the mTORC2 complex. The phosphorylation of SIN1 by Akt was found to regulate mTORC2 activity in response to growth factors, revealing topological insights into the Akt/mTOR signaling network. The dynamic phosphoproteome described here contains numerous phosphorylation sites on proteins involved in diverse molecular functions and should serve as a useful functional resource for cell biologists. PMID:23684622

  3. Comprehensive Analysis of the Membrane Phosphoproteome Regulated by Oligogalacturonides in Arabidopsis thaliana.

    PubMed

    Mattei, Benedetta; Spinelli, Francesco; Pontiggia, Daniela; De Lorenzo, Giulia

    2016-01-01

    Early changes in the Arabidopsis thaliana membrane phosphoproteome in response to oligogalacturonides (OGs), a class of plant damage-associated molecular patterns (DAMPs), were analyzed by two complementary proteomic approaches. Differentially phosphorylated sites were determined through phosphopeptide enrichment followed by LC-MS/MS using label-free quantification; differentially phosphorylated proteins were identified by 2D-DIGE combined with phospho-specific fluorescent staining (phospho-DIGE). This large-scale phosphoproteome analysis of early OG-signaling enabled us to determine 100 regulated phosphosites using LC-MS/MS and 46 differential spots corresponding to 34 pdhosphoproteins using phospho-DIGE. Functional classification showed that the OG-responsive phosphoproteins include kinases, phosphatases and receptor-like kinases, heat shock proteins (HSPs), reactive oxygen species (ROS) scavenging enzymes, proteins related to cellular trafficking, transport, defense and signaling as well as novel candidates for a role in immunity, for which elicitor-induced phosphorylation changes have not been shown before. A comparison with previously identified elicitor-regulated phosphosites shows only a very limited overlap, uncovering the immune-related regulation of 70 phosphorylation sites and revealing novel potential players in the regulation of elicitor-dependent immunity. PMID:27532006

  4. Comprehensive Analysis of the Membrane Phosphoproteome Regulated by Oligogalacturonides in Arabidopsis thaliana

    PubMed Central

    Mattei, Benedetta; Spinelli, Francesco; Pontiggia, Daniela; De Lorenzo, Giulia

    2016-01-01

    Early changes in the Arabidopsis thaliana membrane phosphoproteome in response to oligogalacturonides (OGs), a class of plant damage-associated molecular patterns (DAMPs), were analyzed by two complementary proteomic approaches. Differentially phosphorylated sites were determined through phosphopeptide enrichment followed by LC-MS/MS using label-free quantification; differentially phosphorylated proteins were identified by 2D-DIGE combined with phospho-specific fluorescent staining (phospho-DIGE). This large-scale phosphoproteome analysis of early OG-signaling enabled us to determine 100 regulated phosphosites using LC-MS/MS and 46 differential spots corresponding to 34 pdhosphoproteins using phospho-DIGE. Functional classification showed that the OG-responsive phosphoproteins include kinases, phosphatases and receptor-like kinases, heat shock proteins (HSPs), reactive oxygen species (ROS) scavenging enzymes, proteins related to cellular trafficking, transport, defense and signaling as well as novel candidates for a role in immunity, for which elicitor-induced phosphorylation changes have not been shown before. A comparison with previously identified elicitor-regulated phosphosites shows only a very limited overlap, uncovering the immune-related regulation of 70 phosphorylation sites and revealing novel potential players in the regulation of elicitor-dependent immunity. PMID:27532006

  5. The use of elemental mass spectrometry in phosphoproteomic applications.

    PubMed

    Maes, Evelyne; Tirez, Kristof; Baggerman, Geert; Valkenborg, Dirk; Schoofs, Liliane; Encinar, Jorge Ruiz; Mertens, Inge

    2016-01-01

    Reversible phosphorylation is one of the most important post-translational modifications in mammalian cells. Because this molecular switch is an important mechanism that diversifies and regulates proteins in cellular processes, knowledge about the extent and quantity of phosphorylation is very important to understand the complex cellular interplay. Although phosphoproteomics strategies are applied worldwide, they mainly include only molecular mass spectrometry (like MALDI or ESI)-based experiments. Although identification and relative quantification of phosphopeptides is straightforward with these techniques, absolute quantification is more complex and usually requires for specific isotopically phosphopeptide standards. However, the use of elemental mass spectrometry, and in particular inductively coupled plasma mass spectrometry (ICP-MS), in phosphoproteomics-based experiments, allow one to absolutely quantify phosphopeptides. Here, these phosphoproteomic applications with ICP-MS as elemental detector are reviewed. Pioneering work and recent developments in the field are both described. Additionally, the advantage of the parallel use of molecular and elemental mass spectrometry is stressed. PMID:25139451

  6. An Initial Characterization of the Serum Phosphoproteome

    PubMed Central

    Zhou, Weidong; Ross, Mark M.; Tessitore, Alessandra; Ornstein, David; VanMeter, Amy; Liotta, Lance A.; Petricoin, Emanuel F.

    2009-01-01

    Phosphorylation is a dynamic post-translational protein modification that is the basis of a general mechanism for maintaining and regulating protein structure and function, and of course underpins key cellular processes through signal transduction. In the last several years, many studies of large-scale profiling of phosphoproteins and mapping phosphorylation sites from cultured human cells or tissues by mass spectrometry technique have been published; however, there is little information on general (or global) phosphoproteomic characterization and description of the content of phosphoprotein analytes within the circulation. Circulating phosphoproteins and phosphopeptides could represent important disease biomarkers because of their well-known importance in cellular function, and these analytes frequently are mutated and activated in human diseases such as cancer. Here we report an initial attempt to characterize the phosphoprotein content of serum. To accomplish this, we developed a method in which phosphopeptides are enriched from digested serum proteins and analyzed by LC-MS/MS using LTQ-Orbitrap (CID) and LTQ-ETD mass spectrometers. Using this approach we identified ~100 unique phosphopeptides with stringent filtering criteria and a lower than 1% false discovery rate. PMID:19824718

  7. A Novel Quantitative Hemolytic Assay Coupled with Restriction Fragment Length Polymorphisms Analysis Enabled Early Diagnosis of Atypical Hemolytic Uremic Syndrome and Identified Unique Predisposing Mutations in Japan

    PubMed Central

    Yoshida, Yoko; Miyata, Toshiyuki; Matsumoto, Masanori; Shirotani-Ikejima, Hiroko; Uchida, Yumiko; Ohyama, Yoshifumi; Kokubo, Tetsuro; Fujimura, Yoshihiro

    2015-01-01

    For thrombotic microangiopathies (TMAs), the diagnosis of atypical hemolytic uremic syndrome (aHUS) is made by ruling out Shiga toxin-producing Escherichia coli (STEC)-associated HUS and ADAMTS13 activity-deficient thrombotic thrombocytopenic purpura (TTP), often using the exclusion criteria for secondary TMAs. Nowadays, assays for ADAMTS13 activity and evaluation for STEC infection can be performed within a few hours. However, a confident diagnosis of aHUS often requires comprehensive gene analysis of the alternative complement activation pathway, which usually takes at least several weeks. However, predisposing genetic abnormalities are only identified in approximately 70% of aHUS. To facilitate the diagnosis of complement-mediated aHUS, we describe a quantitative hemolytic assay using sheep red blood cells (RBCs) and human citrated plasma, spiked with or without a novel inhibitory anti-complement factor H (CFH) monoclonal antibody. Among 45 aHUS patients in Japan, 24% (11/45) had moderate-to-severe (≥50%) hemolysis, whereas the remaining 76% (34/45) patients had mild or no hemolysis (<50%). The former group is largely attributed to CFH-related abnormalities, and the latter group has C3-p.I1157T mutations (16/34), which were identified by restriction fragment length polymorphism (RFLP) analysis. Thus, a quantitative hemolytic assay coupled with RFLP analysis enabled the early diagnosis of complement-mediated aHUS in 60% (27/45) of patients in Japan within a week of presentation. We hypothesize that this novel quantitative hemolytic assay would be more useful in a Caucasian population, who may have a higher proportion of CFH mutations than Japanese patients. PMID:25951460

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

    PubMed Central

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

    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. PMID:26415800

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

    SciTech Connect

    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{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

  10. Spatial proteomic and phospho-proteomic organization in three prototypical cell migration modes

    PubMed Central

    2014-01-01

    Background Tight spatio-temporal signaling of cytoskeletal and adhesion dynamics is required for localized membrane protrusion that drives directed cell migration. Different ensembles of proteins are therefore likely to get recruited and phosphorylated in membrane protrusions in response to specific cues. Results Here, we use an assay that allows to biochemically purify extending protrusions of cells migrating in response to three prototypical receptors: integrins, recepor tyrosine kinases and G-coupled protein receptors. Using quantitative proteomics and phospho-proteomics approaches, we provide evidence for the existence of cue-specific, spatially distinct protein networks in the different cell migration modes. Conclusions The integrated analysis of the large-scale experimental data with protein information from databases allows us to understand some emergent properties of spatial regulation of signaling during cell migration. This provides the cell migration community with a large-scale view of the distribution of proteins and phospho-proteins regulating directed cell migration. PMID:24987309

  11. Systematic profiling of the bacterial phosphoproteome reveals bacterium-specific features of phosphorylation.

    PubMed

    Lin, Miao-Hsia; Sugiyama, Naoyuki; Ishihama, Yasushi

    2015-09-15

    Protein phosphorylation is a crucial posttranslational modification for regulating cellular processes in bacteria; however, it has not been extensively studied because of technical difficulties in the enrichment of phosphopeptides. We devised an enrichment protocol that enabled the identification of >1000 phosphopeptides from a single bacterial sample. We discovered three high-confidence serine and threonine phosphorylation motifs, as well as 29 other motifs at various levels of confidence, from three distinct bacterial phosphoproteomes. We found that the proline-directed and basophilic phosphorylation motifs that are commonly enriched in eukaryotes were not observed in bacteria. Unlike eukaryotes, bacteria had a low occurrence of both phosphorylation and acetylation in N-terminal phosphopeptides. Because infection of host cells by bacterial pathogens is often accompanied by kinase-mediated phosphorylation events, the differences in phosphorylation preferences between bacteria and eukaryotes revealed by this study could be useful in identifying bacterial-specific targets for future therapies. PMID:26373674

  12. Brief Isoflurane Anesthesia Produces Prominent Phosphoproteomic Changes in the Adult Mouse Hippocampus.

    PubMed

    Kohtala, Samuel; Theilmann, Wiebke; Suomi, Tomi; Wigren, Henna-Kaisa; Porkka-Heiskanen, Tarja; Elo, Laura L; Rokka, Anne; Rantamäki, Tomi

    2016-06-15

    Anesthetics are widely used in medical practice and experimental research, yet the neurobiological basis governing their effects remains obscure. We have here used quantitative phosphoproteomics to investigate the protein phosphorylation changes produced by a 30 min isoflurane anesthesia in the adult mouse hippocampus. Altogether 318 phosphorylation alterations in total of 237 proteins between sham and isoflurane anesthesia were identified. Many of the hit proteins represent primary pharmacological targets of anesthetics. However, findings also enlighten the role of several other proteins-implicated in various biological processes including neuronal excitability, brain energy homeostasis, synaptic plasticity and transmission, and microtubule function-as putative (secondary) targets of anesthetics. In particular, isoflurane increases glycogen synthase kinase-3β (GSK3β) phosphorylation at the inhibitory Ser(9) residue and regulates the phosphorylation of multiple proteins downstream and upstream of this promiscuous kinase that regulate diverse biological functions. Along with confirmatory Western blot data for GSK3β and p44/42-MAPK (mitogen-activated protein kinase; reduced phosphorylation of the activation loop), we observed increased phosphorylation of microtubule-associated protein 2 (MAP2) on residues (Thr(1620,1623)) that have been shown to render its dissociation from microtubules and alterations in microtubule stability. We further demonstrate that diverse anesthetics (sevoflurane, urethane, ketamine) produce essentially similar phosphorylation changes on GSK3β, p44/p42-MAPK, and MAP2 as observed with isoflurane. Altogether our study demonstrates the potential of quantitative phosphoproteomics to study the mechanisms of anesthetics (and other drugs) in the mammalian brain and reveals how already a relatively brief anesthesia produces pronounced phosphorylation changes in multiple proteins in the central nervous system. PMID:27074656

  13. Data set from a comprehensive phosphoproteomic analysis of rice variety IRBB5 in response to bacterial blight

    PubMed Central

    Hou, Yuxuan; Tong, Xiaohong; Wang, Yifeng; Qiu, Jiehua; Li, Zhiyong; Zhang, Wen; Huang, Shiwen; Zhang, Jian

    2015-01-01

    Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) has become one of the most devastating diseases for rice, a major food source for over half of the world populations. To investigate the roles of protein phosphorylation in rice bacterial blight resistance, a quantitative phosphoproteomic study was conducted in rice variety IRBB5 at 0 h and 24 h after Xoo infection. 2367 and 2223 phosphosites on 1334 and 1297 representative proteins were identified in 0 h and 24 h after Xoo infection, respectively, out of which 762 proteins were found to be differentially phosphorylated. In associated with the published article “A comprehensive quantitative phosphoproteome analysis of rice in response to bacterial blight” in BMC Plant Biology (Hou et al., 2015) [1], this dataset article provided the detailed information of experimental designing, methods, features as well as the raw data of mass spectrometry (MS) identification. The MS proteomics data could be fully accessed from the ProteomeXchange Consortium with the dataset identifier PXD002222. PMID:26862573

  14. Phosphoproteomic Analyses Reveal Early Signaling Events in the Osmotic Stress Response1[W][OPEN

    PubMed Central

    E. Stecker, Kelly; Minkoff, Benjamin B.; Sussman, Michael R.

    2014-01-01

    Elucidating how plants sense and respond to water loss is important for identifying genetic and chemical interventions that may help sustain crop yields in water-limiting environments. Currently, the molecular mechanisms involved in the initial perception and response to dehydration are not well understood. Modern mass spectrometric methods for quantifying changes in the phosphoproteome provide an opportunity to identify key phosphorylation events involved in this process. Here, we have used both untargeted and targeted isotope-assisted mass spectrometric methods of phosphopeptide quantitation to characterize proteins in Arabidopsis (Arabidopsis thaliana) whose degree of phosphorylation is rapidly altered by hyperosmotic treatment. Thus, protein phosphorylation events responsive to 5 min of 0.3 m mannitol treatment were first identified using 15N metabolic labeling and untargeted mass spectrometry with a high-resolution ion-trap instrument. The results from these discovery experiments were then validated using targeted Selected Reaction Monitoring mass spectrometry with a triple quadrupole. Targeted Selected Reaction Monitoring experiments were conducted with plants treated under nine different environmental perturbations to determine whether the phosphorylation changes were specific for osmosignaling or involved cross talk with other signaling pathways. The results indicate that regulatory proteins such as members of the mitogen-activated protein kinase family are specifically phosphorylated in response to osmotic stress. Proteins involved in 5′ messenger RNA decapping and phosphatidylinositol 3,5-bisphosphate synthesis were also identified as targets of dehydration-induced phosphoregulation. The results of these experiments demonstrate the utility of targeted phosphoproteomic analysis in understanding protein regulation networks and provide new insight into cellular processes involved in the osmotic stress response. PMID:24808101

  15. Phosphoproteome Integration Reveals Patient-Specific Networks in Prostate Cancer.

    PubMed

    Drake, Justin M; Paull, Evan O; Graham, Nicholas A; Lee, John K; Smith, Bryan A; Titz, Bjoern; Stoyanova, Tanya; Faltermeier, Claire M; Uzunangelov, Vladislav; Carlin, Daniel E; Fleming, Daniel Teo; Wong, Christopher K; Newton, Yulia; Sudha, Sud; Vashisht, Ajay A; Huang, Jiaoti; Wohlschlegel, James A; Graeber, Thomas G; Witte, Owen N; Stuart, Joshua M

    2016-08-11

    We used clinical tissue from lethal metastatic castration-resistant prostate cancer (CRPC) patients obtained at rapid autopsy to evaluate diverse genomic, transcriptomic, and phosphoproteomic datasets for pathway analysis. Using Tied Diffusion through Interacting Events (TieDIE), we integrated differentially expressed master transcriptional regulators, functionally mutated genes, and differentially activated kinases in CRPC tissues to synthesize a robust signaling network consisting of druggable kinase pathways. Using MSigDB hallmark gene sets, six major signaling pathways with phosphorylation of several key residues were significantly enriched in CRPC tumors after incorporation of phosphoproteomic data. Individual autopsy profiles developed using these hallmarks revealed clinically relevant pathway information potentially suitable for patient stratification and targeted therapies in late stage prostate cancer. Here, we describe phosphorylation-based cancer hallmarks using integrated personalized signatures (pCHIPS) that shed light on the diversity of activated signaling pathways in metastatic CRPC while providing an integrative, pathway-based reference for drug prioritization in individual patients. PMID:27499020

  16. The Quantitative Criteria Based on the Fractal Dimensions, Entropy, and Lacunarity for the Spatial Distribution of Cancer Cell Nuclei Enable Identification of Low or High Aggressive Prostate Carcinomas

    PubMed Central

    Waliszewski, Przemyslaw

    2016-01-01

    relevant information. Two novel quantitative criteria based on the complexity and the diversity measures enabled the identification of low or high aggressive prostate carcinomas and should be verified in the future multicenter, randomized studies. PMID:26903883

  17. Understanding the Mechanisms Enabling an Ultra-high Efficiency Moving Wire Interface for Real-time Carbon 14 Accelerator Mass Spectrometry Quantitation of Samples Suspended in Solvent

    NASA Astrophysics Data System (ADS)

    Thomas, Avraham Thaler

    Carbon 14 (14C) quantitation by accelerator mass spectrometry (AMS) is a powerfully sensitive and uniquely quantitative tool for tracking labeled carbonaceous molecules in biological systems. This is due to 14C's low natural abundance of 1 ppt, the nominal difference in biological activity between an unlabeled and a 14C-labeled molecule, and the ability of AMS to measure isotopic ratios independently of a sample's other characteristics. To make AMS more broadly accessible, a moving wire interface for real-time coupling of high pressure liquid chromatography (HPLC) to AMS and high throughput AMS quantitation of minute single samples has been developed. Prior to this work, samples needed to be converted to solid carbon before measurement. This conversion process has many steps and requires that the sample size be large enough to allow precise handling of the resulting graphite. These factors make the process susceptible to error and time consuming, as well as requiring 0.5 ug of carbon. Samples which do not contain enough carbon, such as HPLC fractions, must be bulked up. This adds background and increases effort. The moving wire interface overcomes these limitations by automating sample processing. Samples placed on the wire are transported through a solvent removal stage followed by a combustion stage after which the combustion products are directed to a gas accepting ion source. The ion source converts the carbon from the CO2 combustion product into C ions, from which an isotopic ratio can be determined by AMS. Although moving wire interfaces have been implemented for various tasks since 1964, the efficiency of these systems at transferring fluid from an HPLC to the wire was only 3%, the efficiency of transferring combustion products from the combustion oven to ion source was only 30%, the flow and composition of the carrier gas from the combustion oven to the ion source needed to be optimized for coupling to an AMS gas accepting ion source and the drying ovens

  18. Phosphoproteomic analysis of induced resistance reveals activation of signal transduction processes by beneficial and pathogenic interaction in grapevine.

    PubMed

    Perazzolli, Michele; Palmieri, Maria Cristina; Matafora, Vittoria; Bachi, Angela; Pertot, Ilaria

    2016-05-20

    Protein phosphorylation regulates several key processes of the plant immune system. Protein kinases and phosphatases are pivotal regulators of defense mechanisms elicited by resistance inducers. However, the phosphorylation cascades that trigger the induced resistance mechanisms in plants have not yet been deeply investigated. The beneficial fungus Trichoderma harzianum T39 (T39) induces resistance against grapevine downy mildew (Plasmopara viticola), but its efficacy could be further improved by a better understanding of the cellular regulations involved. We investigated quantitative changes in the grapevine phosphoproteome during T39-induced resistance to get an overview of regulatory mechanisms of downy mildew resistance. Immunodetection experiments revealed activation of the 45 and 49kDa kinases by T39 treatment both before and after pathogen inoculation, and the phosphoproteomic analysis identified 103 phosphopeptides that were significantly affected by the phosphorylation cascades during T39-induced resistance. Peptides affected by T39 treatment showed comparable phosphorylation levels after P. viticola inoculation, indicating activation of the microbial recognition machinery before pathogen infection. Phosphorylation profiles of proteins related to photosynthetic processes and protein ubiquitination indicated a partial overlap of cellular responses in T39-treated and control plants. However, phosphorylation changes of proteins involved in response to stimuli, signal transduction, hormone signaling, gene expression regulation, and RNA metabolism were exclusively elicited by P. viticola inoculation in T39-treated plants. These results highlighted the relevance of phosphorylation changes during T39-induced resistance and identified key regulator candidates of the grapevine defense against downy mildew. PMID:27010348

  19. Preparation of Polypropylene Spin Tips Filled with Immobilized Titanium(IV) Ion Monolithic Adsorbent for Robust Phosphoproteome Analysis.

    PubMed

    Liu, Fangjie; Wan, Hao; Liu, Zhongshan; Wang, Hongwei; Mao, Jiawei; Ye, Mingliang; Zou, Hanfa

    2016-05-17

    In this study, we developed a Ti(IV) monolithic spin tip for phosphoproteome analysis of a minute amount of biological sample for the first time. The surface of polypropylene pipet tip was activated by the photoinitiator benzophenone under UV light radiation followed by polymerization of ethylene glycol methacrylate phosphate and bis-acrylamide in the tip to form a porous monolith with reactive phosphate groups. The as-prepared tips grafted with monolithic adsorbent were then chelated with titanium(IV) ion for phosphopeptide enrichment. It was found that the tips enabled fast and efficient capture of phosphopeptides from microscale complex samples. The monolithic tip was demonstrated to have a detection limit as low as 5 fmol β-casein tryptic digest, along with an exceptionally high specificity to capture phosphopeptides from complex tryptic digest mixed with an unphosphorylated protein and a phosphorylated protein at a molar ratio up to 1000:1. When the tip was applied to enrich phosphopeptides from 5 μg of tryptic digest of complex HeLa cell proteins, 1185 high confidence of phosphorylated sites were successfully identified with the specificity as high as 92.5%. So far, this is the most sensitive phosphoproteomics analysis using a standard liquid chromatography-tandem mass spectrometry (LC-MS/MS) system for proteome-wide phosphorylation analysis in mammalian cells. PMID:27101427

  20. Quantitative Analysis of Tissue Samples by Combining iTRAQ Isobaric Labeling with Selected/Multiple Reaction Monitoring (SRM/MRM).

    PubMed

    Narumi, Ryohei; Tomonaga, Takeshi

    2016-01-01

    Mass spectrometry-based phosphoproteomics is an indispensible technique used in the discovery and quantification of phosphorylation events on proteins in biological samples. The application of this technique to tissue samples is especially useful for the discovery of biomarkers as well as biological studies. We herein describe the application of a large-scale phosphoproteome analysis and SRM/MRM-based quantitation to develop a strategy for the systematic discovery and validation of biomarkers using tissue samples. PMID:26584920

  1. In vivo phosphoproteomics analysis reveals the cardiac targets of β-adrenergic receptor signaling.

    PubMed

    Lundby, Alicia; Andersen, Martin N; Steffensen, Annette B; Horn, Heiko; Kelstrup, Christian D; Francavilla, Chiara; Jensen, Lars J; Schmitt, Nicole; Thomsen, Morten B; Olsen, Jesper V

    2013-06-01

    β-Blockers are widely used to prevent cardiac arrhythmias and to treat hypertension by inhibiting β-adrenergic receptors (βARs) and thus decreasing contractility and heart rate. βARs initiate phosphorylation-dependent signaling cascades, but only a small number of the target proteins are known. We used quantitative in vivo phosphoproteomics to identify 670 site-specific phosphorylation changes in murine hearts in response to acute treatment with specific βAR agonists. The residues adjacent to the regulated phosphorylation sites exhibited a sequence-specific preference (R-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 monophosphate-dependent protein kinase) and CaMKII (calcium/calmodulin-dependent protein kinase type II). We found specific regulation of phosphorylation sites on six ion channels and transporters that mediate increased ion fluxes at higher heart rates, and we showed that phosphorylation of one of these, Ser(92) of the potassium channel KV7.1, increased current amplitude. Our data set represents a quantitative analysis of phosphorylated proteins regulated in vivo upon stimulation of seven-transmembrane receptors, and our findings reveal previously unknown phosphorylation sites that regulate myocardial contractility, suggesting new potential targets for the treatment of heart disease and hypertension. PMID:23737553

  2. Systematic Analysis of Protein Phosphorylation Networks From Phosphoproteomic Data*

    PubMed Central

    Song, Chunxia; Ye, Mingliang; Liu, Zexian; Cheng, Han; Jiang, Xinning; Han, Guanghui; Songyang, Zhou; Tan, Yexiong; Wang, Hongyang; Ren, Jian; Xue, Yu; Zou, Hanfa

    2012-01-01

    In eukaryotes, hundreds of protein kinases (PKs) specifically and precisely modify thousands of substrates at specific amino acid residues to faithfully orchestrate numerous biological processes, and reversibly determine the cellular dynamics and plasticity. Although over 100,000 phosphorylation sites (p-sites) have been experimentally identified from phosphoproteomic studies, the regulatory PKs for most of these sites still remain to be characterized. Here, we present a novel software package of iGPS for the prediction of in vivo site-specific kinase-substrate relations mainly from the phosphoproteomic data. By critical evaluations and comparisons, the performance of iGPS is satisfying and better than other existed tools. Based on the prediction results, we modeled protein phosphorylation networks and observed that the eukaryotic phospho-regulation is poorly conserved at the site and substrate levels. With an integrative procedure, we conducted a large-scale phosphorylation analysis of human liver and experimentally identified 9719 p-sites in 2998 proteins. Using iGPS, we predicted a human liver protein phosphorylation networks containing 12,819 potential site-specific kinase-substrate relations among 350 PKs and 962 substrates for 2633 p-sites. Further statistical analysis and comparison revealed that 127 PKs significantly modify more or fewer p-sites in the liver protein phosphorylation networks against the whole human protein phosphorylation network. The largest data set of the human liver phosphoproteome together with computational analyses can be useful for further experimental consideration. This work contributes to the understanding of phosphorylation mechanisms at the systemic level, and provides a powerful methodology for the general analysis of in vivo post-translational modifications regulating sub-proteomes. PMID:22798277

  3. Large-Scale Multiplexed Quantitative Discovery Proteomics Enabled by the Use of an 18O-Labeled “Universal” Reference Sample

    PubMed Central

    Qian, Wei-Jun; Liu, Tao; Petyuk, Vladislav A.; Gritsenko, Marina A.; Petritis, Brianne O.; Polpitiya, Ashoka D.; Kaushal, Amit; Xiao, Wenzhong; Finnerty, Celeste C.; Jeschke, Marc G.; Jaitly, Navdeep; Monroe, Matthew E.; Moore, Ronald J.; Moldawer, Lyle L.; Davis, Ronald W.; Tompkins, Ronald G.; Herndon, David N.; Camp, David G.; Smith, Richard D.

    2009-01-01

    The quantitative comparison of protein abundances across a large number of biological or patient samples represents an important proteomics challenge that needs to be addressed for proteomics discovery applications. Herein, we describe a strategy that incorporates a stable isotope 18O-labeled ″universal″ reference sample as a comprehensive set of internal standards for analyzing large sample sets quantitatively. As a pooled sample, the 18O-labeled ″universal″ reference sample is spiked into each individually processed unlabeled biological sample and the peptide/protein abundances are quantified based on 16O/18O isotopic peptide pair abundance ratios that compare each unlabeled sample to the identical reference sample. This approach also allows for the direct application of label-free quantitation across the sample set simultaneously along with the labeling-approach (i.e., dual-quantitation) since each biological sample is unlabeled except for the labeled reference sample that is used as internal standards. The effectiveness of this approach for large-scale quantitative proteomics is demonstrated by its application to a set of 18 plasma samples from severe burn patients. When immunoaffinity depletion and cysteinyl-peptide enrichment-based fractionation with high resolution LC-MS measurements were combined, a total of 312 plasma proteins were confidently identified and quantified with a minimum of two unique peptides per protein. The isotope labeling data was directly compared with the label-free 16O-MS intensity data extracted from the same data sets. The results showed that the 18O reference-based labeling approach had significantly better quantitative precision compared to the label-free approach. The relative abundance differences determined by the two approaches also displayed strong correlation, illustrating the complementary nature of the two quantitative methods. The simplicity of including the 18O-reference for accurate quantitation makes this

  4. A shotgun phosphoproteomics analysis of embryos in germinated maize seeds.

    PubMed

    Lu, Tian-Cong; Meng, Ling-Bo; Yang, Chuan-Ping; Liu, Gui-Feng; Liu, Guan-Jun; Ma, Wei; Wang, Bai-Chen

    2008-11-01

    To better understand the role that reversible protein phosphorylation plays in seed germination, we initiated a phosphoproteomic investigation of embryos of germinated maize seeds. A total of 776 proteins including 39 kinases, 16 phosphatases, and 33 phosphoproteins containing 36 precise in vivo phosphorylation sites were identified. All the phosphorylation sites identified, with the exception of the phosphorylation site on HSP22, have not been reported previously (Lund et al. in J Biol Chem, 276, 29924-29929, 2001). Assayed with QRT-PCR, the transcripts of ten kinase genes were found to be dramatically up-regulated during seed germination and those of four phosphatase genes were up-regulated after germination, which indicated that reversible protein phosphorylation occurred and complex regulating networks were activated during this period. At least one-third of these phosphoproteins are key components involved in biological processes which relate to seed germination, such as DNA repair, gene transcription, RNA splicing and protein translation, suggesting that protein phosphorylation plays an important role in seed germination. As far as we know, this is the first phosphoproteomic study on a monocot and it will lay a solid foundation for further study of the molecular mechanisms of seed germination and seedling development. PMID:18726113

  5. Phosphopeptide enrichment using offline titanium dioxide columns for phosphoproteomics.

    PubMed

    Yu, Li-Rong; Veenstra, Timothy

    2013-01-01

    Identification of phosphoproteins or phosphopeptides as cancer biomarkers is an emerging field in phosphoproteomics. Owing to the low stoichiometric nature of protein phosphorylation, phosphoproteins or phosphopeptides must be enriched prior to downstream mass spectrometry analysis. Titanium dioxide (TiO2) has been prevalently used to enrich phosphopeptides from complex proteome samples due to its high affinity for phosphopeptides, and the method is straightforward. In this protocol, an offline phosphopeptide enrichment procedure using TiO2 columns is described. Peptides from a proteome lysate are loaded onto a TiO2 column in an acidic environment, followed by column washing with aqueous, organic, and ammonium glutamate (NH4Glu) buffers at acidic conditions. Phosphopeptides are eluted using an ammonia solution at high pH. Use of NH4Glu significantly reduces nonspecific bindings while a high recovery rate (84 %) of phosphopeptides is retained. The method is optimized for large-scale phosphoproteomic analysis and phosphoprotein biomarker discovery starting from sub-milligram or milligrams of proteome samples. PMID:23625397

  6. Breast Cancer Proteomic and Phosphoproteomic Data Released - Office of Cancer Clinical Proteomics Research

    Cancer.gov

    National Cancer Institute (NCI) Clinical Proteomic Tumor Analysis Consortium (CPTAC) scientists have released a dataset of proteins and phophorylated phosphopeptides identified through deep proteomic and phosphoproteomic analysis of breast tumor samples, previously genomically analyzed by The Cancer Genome Atlas (TCGA).

  7. Plug-and-play analysis of the human phosphoproteome by targeted high-resolution mass spectrometry.

    PubMed

    Lawrence, Robert T; Searle, Brian C; Llovet, Ariadna; Villén, Judit

    2016-05-01

    Systematic approaches to studying cellular signaling require phosphoproteomic techniques that reproducibly measure the same phosphopeptides across multiple replicates, conditions, and time points. Here we present a method to mine information from large-scale, heterogeneous phosphoproteomics data sets to rapidly generate robust targeted mass spectrometry (MS) assays. We demonstrate the performance of our method by interrogating the IGF-1/AKT signaling pathway, showing that even rarely observed phosphorylation events can be consistently detected and precisely quantified. PMID:27018578

  8. Mechanisms of Soybean Roots' Tolerances to Salinity Revealed by Proteomic and Phosphoproteomic Comparisons Between Two Cultivars.

    PubMed

    Pi, Erxu; Qu, Liqun; Hu, Jianwen; Huang, Yingying; Qiu, Lijuan; Lu, Hongfei; Jiang, Bo; Liu, Cong; Peng, Tingting; Zhao, Ying; Wang, Huizhong; Tsai, Sau-Na; Ngai, Saiming; Du, Liqun

    2016-01-01

    Understanding molecular mechanisms underlying plant salinity tolerance provides valuable knowledgebase for effective crop improvement through genetic engineering. Current proteomic technologies, which support reliable and high-throughput analyses, have been broadly used for exploring sophisticated molecular networks in plants. In the current study, we compared phosphoproteomic and proteomic changes in roots of different soybean seedlings of a salt-tolerant cultivar (Wenfeng07) and a salt-sensitive cultivar (Union85140) induced by salt stress. The root samples of Wenfeng07 and Union85140 at three-trifoliate stage were collected at 0 h, 0.5 h, 1 h, 4 h, 12 h, 24 h, and 48 h after been treated with 150 mm NaCl. LC-MS/MS based phosphoproteomic analysis of these samples identified a total of 2692 phosphoproteins and 5509 phosphorylation sites. Of these, 2344 phosphoproteins containing 3744 phosphorylation sites were quantitatively analyzed. Our results showed that 1163 phosphorylation sites were differentially phosphorylated in the two compared cultivars. Among them, 10 MYB/MYB transcription factor like proteins were identified with fluctuating phosphorylation modifications at different time points, indicating that their crucial roles in regulating flavonol accumulation might be mediated by phosphorylated modifications. In addition, the protein expression profiles of these two cultivars were compared using LC MS/MS based shotgun proteomic analysis, and expression pattern of all the 89 differentially expressed proteins were independently confirmed by qRT-PCR. Interestingly, the enzymes involved in chalcone metabolic pathway exhibited positive correlations with salt tolerance. We confirmed the functional relevance of chalcone synthase, chalcone isomerase, and cytochrome P450 monooxygenase genes using soybean composites and Arabidopsis thaliana mutants, and found that their salt tolerance were positively regulated by chalcone synthase, but was negatively regulated by

  9. Phosphoproteomic network analysis in the sea urchin Strongylocentrotus purpuratus reveals new candidates in egg activation.

    PubMed

    Guo, Hongbo; Garcia-Vedrenne, Ana Elisa; Isserlin, Ruth; Lugowski, Andrew; Morada, Anthony; Sun, Alex; Miao, Yishen; Kuzmanov, Uros; Wan, Cuihong; Ma, Hongyue; Foltz, Kathy; Emili, Andrew

    2015-12-01

    Fertilization triggers a dynamic symphony of molecular transformations induced by a rapid rise in intracellular calcium. Most prominent are surface alterations, metabolic activation, cytoskeletal reorganization, and cell-cycle reentry. While the activation process appears to be broadly evolutionarily conserved, and protein phosphorylation is known to play a key role, the signaling networks mediating the response to fertilization are not well described. To address this gap, we performed a time course phosphoproteomic analysis of egg activation in the sea urchin Strongylocentrotus purpuratus, a system that offers biochemical tractability coupled with exquisite synchronicity. By coupling large-scale phosphopeptide enrichment with unbiased quantitative MS, we identified striking changes in global phosphoprotein patterns at 2- and 5-min postfertilization as compared to unfertilized eggs. Overall, we mapped 8796 distinct phosphosite modifications on 2833 phosphoproteins, of which 15% were differentially regulated in early egg activation. Activated kinases were identified by phosphosite mapping, while enrichment analyses revealed conserved signaling cascades not previously associated with egg activation. This work represents the most comprehensive study of signaling associated with egg activation to date, suggesting novel mechanisms that can be experimentally tested and providing a valuable resource for the broader research community. All MS data have been deposited in the ProteomeXchange with identifier PXD002239 (http://proteomecentral.proteomexchange.org/dataset/PXD002239). PMID:26227301

  10. Phosphoproteome dynamics of Saccharomyces cerevisiae under heat shock and cold stress

    PubMed Central

    Kanshin, Evgeny; Kubiniok, Peter; Thattikota, Yogitha; D'Amours, Damien; Thibault, Pierre

    2015-01-01

    The ability of cells and organisms to survive and function through changes in temperature evolved from their specific adaptations to nonoptimal growth conditions. Responses to elevated temperatures have been studied in yeast and other model organisms using transcriptome profiling and provided valuable biological insights on molecular mechanisms involved in stress tolerance and adaptation to adverse environment. In contrast, little is known about rapid signaling events associated with changes in temperature. To gain a better understanding of global changes in protein phosphorylation in response to heat and cold, we developed a high temporal resolution phosphoproteomics protocol to study cell signaling in Saccharomyces cerevisiae. The method allowed for quantitative analysis of phosphodynamics on 2,777 phosphosites from 1,228 proteins. The correlation of kinetic profiles between kinases and their substrates provided a predictive tool to identify new putative substrates for kinases such as Cdc28 and PKA. Cell cycle analyses revealed that the increased phosphorylation of Cdc28 at its inhibitory site Y19 during heat shock is an adaptive response that delays cell cycle progression under stress conditions. The cellular responses to heat and cold were associated with extensive changes in phosphorylation on proteins implicated in transcription, protein folding and degradation, cell cycle regulation and morphogenesis. PMID:26040289

  11. Phosphoproteomics reveals malaria parasite Protein Kinase G as a signalling hub regulating egress and invasion

    PubMed Central

    Alam, Mahmood M.; Solyakov, Lev; Bottrill, Andrew R.; Flueck, Christian; Siddiqui, Faiza A.; Singh, Shailja; Mistry, Sharad; Viskaduraki, Maria; Lee, Kate; Hopp, Christine S.; Chitnis, Chetan E.; Doerig, Christian; Moon, Robert W.; Green, Judith L.; Holder, Anthony A.; Baker, David A.; Tobin, Andrew B.

    2015-01-01

    Our understanding of the key phosphorylation-dependent signalling pathways in the human malaria parasite, Plasmodium falciparum, remains rudimentary. Here we address this issue for the essential cGMP-dependent protein kinase, PfPKG. By employing chemical and genetic tools in combination with quantitative global phosphoproteomics, we identify the phosphorylation sites on 69 proteins that are direct or indirect cellular targets for PfPKG. These PfPKG targets include proteins involved in cell signalling, proteolysis, gene regulation, protein export and ion and protein transport, indicating that cGMP/PfPKG acts as a signalling hub that plays a central role in a number of core parasite processes. We also show that PfPKG activity is required for parasite invasion. This correlates with the finding that the calcium-dependent protein kinase, PfCDPK1, is phosphorylated by PfPKG, as are components of the actomyosin complex, providing mechanistic insight into the essential role of PfPKG in parasite egress and invasion. PMID:26149123

  12. Phosphoproteomics reveals malaria parasite Protein Kinase G as a signalling hub regulating egress and invasion.

    PubMed

    Alam, Mahmood M; Solyakov, Lev; Bottrill, Andrew R; Flueck, Christian; Siddiqui, Faiza A; Singh, Shailja; Mistry, Sharad; Viskaduraki, Maria; Lee, Kate; Hopp, Christine S; Chitnis, Chetan E; Doerig, Christian; Moon, Robert W; Green, Judith L; Holder, Anthony A; Baker, David A; Tobin, Andrew B

    2015-01-01

    Our understanding of the key phosphorylation-dependent signalling pathways in the human malaria parasite, Plasmodium falciparum, remains rudimentary. Here we address this issue for the essential cGMP-dependent protein kinase, PfPKG. By employing chemical and genetic tools in combination with quantitative global phosphoproteomics, we identify the phosphorylation sites on 69 proteins that are direct or indirect cellular targets for PfPKG. These PfPKG targets include proteins involved in cell signalling, proteolysis, gene regulation, protein export and ion and protein transport, indicating that cGMP/PfPKG acts as a signalling hub that plays a central role in a number of core parasite processes. We also show that PfPKG activity is required for parasite invasion. This correlates with the finding that the calcium-dependent protein kinase, PfCDPK1, is phosphorylated by PfPKG, as are components of the actomyosin complex, providing mechanistic insight into the essential role of PfPKG in parasite egress and invasion. PMID:26149123

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

    PubMed

    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. PMID:26792808

  14. Integrative Phosphoproteomics Links IL-23R Signaling with Metabolic Adaptation in Lymphocytes.

    PubMed

    Lochmatter, Corinne; Fischer, Roman; Charles, Philip D; Yu, Zhanru; Powrie, Fiona; Kessler, Benedikt M

    2016-01-01

    Interleukin (IL)-23 mediated signal transduction represents a major molecular mechanism underlying the pathology of inflammatory bowel disease, Crohn's disease and ulcerative colitis. In addition, emerging evidence supports the role of IL-23-driven Th17 cells in inflammation. Components of the IL-23 signaling pathway, such as IL-23R, JAK2 and STAT3, have been characterized, but elements unique to this network as compared to other interleukins have not been readily explored. In this study, we have undertaken an integrative phosphoproteomics approach to better characterise downstream signaling events. To this end, we performed and compared phosphopeptide and phosphoprotein enrichment methodologies after activation of T lymphocytes by IL-23. We demonstrate the complementary nature of the two phosphoenrichment approaches by maximizing the capture of phosphorylation events. A total of 8202 unique phosphopeptides, and 4317 unique proteins were identified, amongst which STAT3, PKM2, CDK6 and LASP-1 showed induction of specific phosphorylation not readily observed after IL-2 stimulation. Interestingly, quantitative analysis revealed predominant phosphorylation of pre-existing STAT3 nuclear subsets in addition to translocation of phosphorylated STAT3 within 30 min after IL-23 stimulation. After IL-23R activation, a small subset of PKM2 also translocates to the nucleus and may contribute to STAT3 phosphorylation, suggesting multiple cellular responses including metabolic adaptation. PMID:27080861

  15. Integrative Phosphoproteomics Links IL-23R Signaling with Metabolic Adaptation in Lymphocytes

    PubMed Central

    Lochmatter, Corinne; Fischer, Roman; Charles, Philip D.; Yu, Zhanru; Powrie, Fiona; Kessler, Benedikt M.

    2016-01-01

    Interleukin (IL)-23 mediated signal transduction represents a major molecular mechanism underlying the pathology of inflammatory bowel disease, Crohn’s disease and ulcerative colitis. In addition, emerging evidence supports the role of IL-23-driven Th17 cells in inflammation. Components of the IL-23 signaling pathway, such as IL-23R, JAK2 and STAT3, have been characterized, but elements unique to this network as compared to other interleukins have not been readily explored. In this study, we have undertaken an integrative phosphoproteomics approach to better characterise downstream signaling events. To this end, we performed and compared phosphopeptide and phosphoprotein enrichment methodologies after activation of T lymphocytes by IL-23. We demonstrate the complementary nature of the two phosphoenrichment approaches by maximizing the capture of phosphorylation events. A total of 8202 unique phosphopeptides, and 4317 unique proteins were identified, amongst which STAT3, PKM2, CDK6 and LASP-1 showed induction of specific phosphorylation not readily observed after IL-2 stimulation. Interestingly, quantitative analysis revealed predominant phosphorylation of pre-existing STAT3 nuclear subsets in addition to translocation of phosphorylated STAT3 within 30 min after IL-23 stimulation. After IL-23R activation, a small subset of PKM2 also translocates to the nucleus and may contribute to STAT3 phosphorylation, suggesting multiple cellular responses including metabolic adaptation. PMID:27080861

  16. Human cytomegalovirus pUL97 kinase induces global changes in the infected cell phosphoproteome

    PubMed Central

    Oberstein, Adam; Perlman, David H.; Shenk, Thomas; Terry, Laura J.

    2015-01-01

    Replication of human cytomegalovirus is regulated in part by cellular kinases and the single viral Ser/Thr kinase, pUL97. The virus-coded kinase augments the replication of human cytomegalovirus (HCMV) by enabling nuclear egress and altering cell cycle progression. These roles are accomplished through direct phosphorylation of nuclear lamins and the retinoblastoma protein, respectively. In an effort to identify additional pUL97 substrates, we analyzed the phosphoproteome of SILAC-labeled human fibroblasts during infection with either wild-type HCMV or a pUL97 kinase-dead mutant virus. Phosphopeptides were enriched over a titanium dioxide matrix and analyzed by high resolution mass spectrometry. We identified 157 unambiguous phosphosites from 106 cellular and 17 viral proteins whose phosphorylation required UL97. Analysis of peptides containing these sites allowed the identification of several candidate pUL97 phosphorylation motifs, including a completely novel phosphorylation motif, LxSP. Substrates harboring the LxSP motif were enriched in nucleocytoplasmic transport functions, including a number of components of the nuclear pore complex. These results extend the known functions of pUL97 and suggest that modulation of nuclear pore function may be important during HCMV replication. PMID:25867546

  17. Semi-quantitative analysis of solid waste flows from nano-enabled consumer products in Europe, Denmark and the United Kingdom - Abundance, distribution and management.

    PubMed

    Heggelund, Laura; Hansen, Steffen Foss; Astrup, Thomas Fruergaard; Boldrin, Alessio

    2016-10-01

    Many nano-enabled consumer products are known to be in the global market. At the same, little is known about the quantity, type, location etc. of the engineered nanomaterials (ENMs) inside the products. This limits the scientific investigations of potential environmental effects of these materials, and especially the knowledge of ENM behaviour and potential effects at the end-of-life stage of the products is scarce. To gain a better understanding of the end-of-life waste treatment of nano-enabled consumer product, we provide an overview of the ENMs flowing into and throughout waste systems in Europe, Denmark and the United Kingdom. Using a nanoproduct inventory (nanodb.dk), we performed a four-step analysis to estimate the most abundant ENMs and in which waste fractions they are present. We found that in terms of number of products: (i) nano silver is the most used ENM in consumer products, and (ii) plastic from used product containers is the largest waste fraction also comprising a large variety of ENMs, though possibly in very small masses. Also, we showed that the local waste management system can influence the distribution of ENMs. It is recommended that future research focus on recycling and landfilling of nano-enabled products since these compartments represent hot spots for end-of-life nanoproducts. PMID:27311351

  18. Rapid Phosphoproteomic Effects of Abscisic Acid (ABA) on Wild-Type and ABA Receptor-Deficient A. thaliana Mutants*

    PubMed Central

    Minkoff, Benjamin B.; Stecker, Kelly E.; Sussman, Michael R.

    2015-01-01

    Abscisic acid (ABA)1 is a plant hormone that controls many aspects of plant growth, including seed germination, stomatal aperture size, and cellular drought response. ABA interacts with a unique family of 14 receptor proteins. This interaction leads to the activation of a family of protein kinases, SnRK2s, which in turn phosphorylate substrates involved in many cellular processes. The family of receptors appears functionally redundant. To observe a measurable phenotype, four of the fourteen receptors have to be mutated to create a multilocus loss-of-function quadruple receptor (QR) mutant, which is much less sensitive to ABA than wild-type (WT) plants. Given these phenotypes, we asked whether or not a difference in ABA response between the WT and QR backgrounds would manifest on a phosphorylation level as well. We tested WT and QR mutant ABA response using isotope-assisted quantitative phosphoproteomics to determine what ABA-induced phosphorylation changes occur in WT plants within 5 min of ABA treatment and how that phosphorylation pattern is altered in the QR mutant. We found multiple ABA-induced phosphorylation changes that occur within 5 min of treatment, including three SnRK2 autophosphorylation events and phosphorylation on SnRK2 substrates. The majority of robust ABA-dependent phosphorylation changes observed were partially diminished in the QR mutant, whereas many smaller ABA-dependent phosphorylation changes observed in the WT were not responsive to ABA in the mutant. A single phosphorylation event was increased in response to ABA treatment in both the WT and QR mutant. A portion of the discovery data was validated using selected reaction monitoring-based targeted measurements on a triple quadrupole mass spectrometer. These data suggest that different subsets of phosphorylation events depend upon different subsets of the ABA receptor family to occur. Altogether, these data expand our understanding of the model by which the family of ABA receptors directs

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

    PubMed Central

    Šalovská, Barbora; Fabrik, Ivo; Ďurišová, Kamila; Link, Marek; Vávrová, Jiřina; Řezáčová, Martina; Tichý, Aleš

    2014-01-01

    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-(methylsulfonyl)phenyl)-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. PMID:25003641

  20. A pipeline for 15N metabolic labeling and phosphoproteome analysis in Arabidopsis thaliana.

    PubMed

    Minkoff, Benjamin B; Burch, Heather L; Sussman, Michael R

    2014-01-01

    Within the past two decades, the biological application of mass spectrometric technology has seen great advances in terms of innovations in hardware, software, and reagents. Concurrently, the burgeoning field of proteomics has followed closely (Yates et al., Annu Rev Biomed Eng 11:49-79, 2009)-and with it, importantly, the ability to globally assay altered levels of posttranslational modifications in response to a variety of stimuli. Though many posttranslational modifications have been described, a major focus of these efforts has been protein-level phosphorylation of serine, threonine, and tyrosine residues (Schreiber et al., Proteomics 8:4416-4432, 2008). The desire to examine changes across signal transduction cascades and networks in their entirety using a single mass spectrometric analysis accounts for this push-namely, preservation and enrichment of the transient yet informative phosphoryl side group. Analyzing global changes in phosphorylation allows inferences surrounding cascades/networks as a whole to be made. Towards this same end, much work has explored ways to permit quantitation and combine experimental samples such that more than one replicate or experimental condition can be identically processed and analyzed, cutting down on experimental and instrument variability, in addition to instrument run time. One such technique that has emerged is metabolic labeling (Gouw et al., Mol Cell Proteomics 9:11-24, 2010), wherein biological samples are labeled in living cells with nonradioactive heavy isotopes such as (15)N or (13)C. Since metabolic labeling in living organisms allows one to combine the material to be processed at the earliest possible step, before the tissue is homogenized, it provides a unique and excellent method for comparing experimental samples in a high-throughput, reproducible fashion with minimal technical variability. This chapter describes a pipeline used for labeling living Arabidopsis thaliana plants with nitrogen-15 ((15)N) and how

  1. Discovery of O-GlcNAc-6-phosphate Modified Proteins in Large-scale Phosphoproteomics Data*

    PubMed Central

    Hahne, Hannes; Kuster, Bernhard

    2012-01-01

    Phosphorylated O-GlcNAc is a novel post-translational modification that has so far only been found on the neuronal protein AP180 from the rat (Graham et al., J. Proteome Res. 2011, 10, 2725–2733). Upon collision induced dissociation, the modification generates a highly mass deficient fragment ion (m/z 284.0530) that can be used as a reporter for the identification of phosphorylated O-GlcNAc. Using a publically available mouse brain phosphoproteome data set, we employed our recently developed Oscore software to re-evaluate high resolution/high accuracy tandem mass spectra and discovered the modification on 23 peptides corresponding to 11 mouse proteins. The systematic analysis of 220 candidate phosphoGlcNAc tandem mass spectra as well as a synthetic standard enabled the dissection of the major phosphoGlcNAc fragmentation pathways, suggesting that the modification is O-GlcNAc-6-phosphate. We find that the classical O-GlcNAc modification often exists on the same peptides indicating that O-GlcNAc-6-phosphate may biosynthetically arise in two steps involving the O-GlcNAc transferase and a currently unknown kinase. Many of the identified proteins are involved in synaptic transmission and for Ca2+/calmodulin kinase IV, the O-GlcNAc-6-phosphate modification was found in the vicinity of two autophosphorylation sites required for full activation of the kinase suggesting a potential regulatory role for O-GlcNAc-6-phosphate. By re-analyzing mass spectrometric data from human embryonic and induced pluripotent stem cells, our study also identified Zinc finger protein 462 (ZNF462) as the first human O-GlcNAc-6-phosphate modified protein. Collectively, the data suggests that O-GlcNAc-6-phosphate is a general post-translation modification of mammalian proteins with a variety of possible cellular functions. PMID:22826440

  2. Identification of targets of c-Src tyrosine kinase by chemical complementation and phosphoproteomics.

    PubMed

    Ferrando, Isabel Martinez; Chaerkady, Raghothama; Zhong, Jun; Molina, Henrik; Jacob, Harrys K C; Herbst-Robinson, Katie; Dancy, Beverley M; Katju, Vikram; Bose, Ron; Zhang, Jin; Pandey, Akhilesh; Cole, Philip A

    2012-08-01

    The cellular proto-oncogene c-Src is a nonreceptor tyrosine kinase involved in cell growth and cytoskeletal regulation. Despite being dysregulated in a variety of human cancers, its precise functions are not fully understood. Identification of the substrates of c-Src remains a major challenge, because there is no simple way to directly stimulate its activity. Here we combine the chemical rescue of mutant c-Src and global quantitative phosphoproteomics to obtain the first high resolution snapshot of the range of tyrosine phosphorylation events that occur in the cell immediately after specific c-Src stimulation. After enrichment by anti-phosphotyrosine antibodies, we identified 29 potential novel c-Src substrate proteins. Tyrosine phosphopeptide mapping allowed the identification of 382 nonredundant tyrosine phosphopeptides on 213 phosphoproteins. Stable isotope labeling of amino acids in cell culture-based quantitation allowed the detection of 97 nonredundant tyrosine phosphopeptides whose level of phosphorylation is increased by c-Src. A large number of previously uncharacterized c-Src putative protein targets and phosphorylation sites are presented here, a majority of which play key roles in signaling and cytoskeletal networks, particularly in cell adhesion. Integrin signaling and focal adhesion kinase signaling pathway are two of the most altered pathways upon c-Src activation through chemical rescue. In this context, our study revealed the temporal connection between c-Src activation and the GTPase Rap1, known to stimulate integrin-dependent adhesion. Chemical rescue of c-Src provided a tool to dissect the spatiotemporal mechanism of activation of the Rap1 guanine exchange factor, C3G, one of the identified potential c-Src substrates that plays a role in focal adhesion signaling. In addition to unveiling the role of c-Src in the cell and, specifically, in the Crk-C3G-Rap1 pathway, these results exemplify a strategy for obtaining a comprehensive understanding of

  3. Wrangling Phosphoproteomic Data to Elucidate Cancer Signaling Pathways

    PubMed Central

    Grimes, Mark L.; Lee, Wan-Jui; van der Maaten, Laurens; Shannon, Paul

    2013-01-01

    The interpretation of biological data sets is essential for generating hypotheses that guide research, yet modern methods of global analysis challenge our ability to discern meaningful patterns and then convey results in a way that can be easily appreciated. Proteomic data is especially challenging because mass spectrometry detectors often miss peptides in complex samples, resulting in sparsely populated data sets. Using the R programming language and techniques from the field of pattern recognition, we have devised methods to resolve and evaluate clusters of proteins related by their pattern of expression in different samples in proteomic data sets. We examined tyrosine phosphoproteomic data from lung cancer samples. We calculated dissimilarities between the proteins based on Pearson or Spearman correlations and on Euclidean distances, whilst dealing with large amounts of missing data. The dissimilarities were then used as feature vectors in clustering and visualization algorithms. The quality of the clusterings and visualizations were evaluated internally based on the primary data and externally based on gene ontology and protein interaction networks. The results show that t-distributed stochastic neighbor embedding (t-SNE) followed by minimum spanning tree methods groups sparse proteomic data into meaningful clusters more effectively than other methods such as k-means and classical multidimensional scaling. Furthermore, our results show that using a combination of Spearman correlation and Euclidean distance as a dissimilarity representation increases the resolution of clusters. Our analyses show that many clusters contain one or more tyrosine kinases and include known effectors as well as proteins with no known interactions. Visualizing these clusters as networks elucidated previously unknown tyrosine kinase signal transduction pathways that drive cancer. Our approach can be applied to other data types, and can be easily adopted because open source software

  4. Comprehensive Analysis of in Vivo Phosphoproteome of Mouse Liver Microsomes.

    PubMed

    Kwon, Oh Kwang; Sim, JuHee; Kim, Sun Ju; Sung, Eunji; Kim, Jin Young; Jeong, Tae Cheon; Lee, Sangkyu

    2015-12-01

    Protein phosphorylation at serine, threonine, and tyrosine residues are some of the most widespread reversible post-translational modifications. Microsomes are vesicle-like bodies, not ordinarily present within living cells, which form from pieces of the endoplasmic reticulum (ER), plasma membrane, mitochondria, or Golgi apparatus of broken eukaryotic cells. Here we investigated the total phosphoproteome of mouse liver microsomes (MLMs) using TiO2 enrichment of phosphopeptides coupled to on-line 2D-LC-MS/MS. In total, 699 phosphorylation sites in 527 proteins were identified in MLMs. When compared with the current phosphoSitePlus database, 155 novel phosphoproteins were identified in MLM. The distributions of phosphosites were 89.4, 8.0, and 2.6% for phosphoserine, phosphotheronine, and phosphotyrosine, respectively. By Motif-X analysis, eight Ser motifs and one Thr motif were found, and five acidic, two basophilic-, and two proline-directed motifs were assigned. The potential functions of phosphoproteins in MLM were assigned by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. In GO annotation, phosphorylated microsomal proteins were involved in mRNA processing, mRNA metabolic processes, and RNA splicing. In the KEGG pathway analysis, phosphorylated microsomal proteins were highly enriched in ribosome protein processing in ER and ribosomes and in RNA transport. Furthermore, we determined that 52 and 23 phosphoproteins were potential substrates of cAMP-dependent protein kinase A and casein kinase II, respectively, many of which are 40S/60S ribosomal proteins. Overall, our results provide an overview of features of protein phosphorylation in MLMs that should be a valuable resource for the future understanding of protein synthesis or translation involving phosphorylation. PMID:26487105

  5. Enabling comparative gene expression studies of thyroid hormone action through the development of a flexible real-time quantitative PCR assay for use across multiple anuran indicator and sentinel species.

    PubMed

    Veldhoen, Nik; Propper, Catherine R; Helbing, Caren C

    2014-03-01

    Studies performed across diverse frog species have made substantial contributions to our understanding of basic vertebrate development and the natural or anthropogenic environmental factors impacting sensitive life stages. Because, anurans are developmental models, provide ecosystems services, and act as sentinels for the identification of environmental chemical contaminants that interfere with thyroid hormone (TH) action during postembryonic development, there is demand for flexible assessment techniques that can be applied to multiple species. As part of the "thyroid assays across indicator and sentinel species" (TAXISS) initiative, we have designed and validated a series of cross-species real time quantitative PCR (qPCR) primer sets that provide information on transcriptome components in evolutionarily distant anurans. Validation for fifteen gene transcripts involved a rigorous three-tiered quality control within tissue/development-specific contexts. Assay performance was confirmed on multiple tissues (tail fin, liver, brain, and intestine) of Rana catesbeiana and Xenopus laevis tadpoles enabling comparisons between tissues and generation of response profiles to exogenous TH. This revealed notable differences in TH-responsive gene transcripts including thra, thrb, thibz, klf9, col1a2, fn1, plp1, mmp2, timm50, otc, and dio2, suggesting differential regulation and susceptibility to contaminant effects. Evidence for the applicability of the TAXISS anuran qPCR assay across seven other species is also provided with five frog families represented and its utility in defining genome structure was demonstrated. This novel validated approach will enable meaningful comparative studies between frog species and aid in extending knowledge of developmental regulatory pathways and the impact of environmental factors on TH signaling in frog species for which little or no genetic information is currently available. PMID:24503578

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

    PubMed

    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

  7. A Phosphoproteomic Comparison of B-RAFV600E and MKK1/2 Inhibitors in Melanoma Cells*

    PubMed Central

    Stuart, Scott A.; Houel, Stephane; Lee, Thomas; Wang, Nan; Old, William M.; Ahn, Natalie G.

    2015-01-01

    Inhibitors of oncogenic B-RAFV600E and MKK1/2 have yielded remarkable responses in B-RAFV600E-positive melanoma patients. However, the efficacy of these inhibitors is limited by the inevitable onset of resistance. Despite the fact that these inhibitors target the same pathway, combination treatment with B-RAFV600E and MKK1/2 inhibitors has been shown to improve both response rates and progression-free survival in B-RAFV600E melanoma patients. To provide insight into the molecular nature of the combinatorial response, we used quantitative mass spectrometry to characterize the inhibitor-dependent phosphoproteome of human melanoma cells treated with the B-RAFV600E inhibitor PLX4032 (vemurafenib) or the MKK1/2 inhibitor AZD6244 (selumetinib). In three replicate experiments, we quantified changes at a total of 23,986 phosphosites on 4784 proteins. This included 1317 phosphosites that reproducibly decreased in response to at least one inhibitor. Phosphosites that responded to both inhibitors grouped into networks that included the nuclear pore complex, growth factor signaling, and transcriptional regulators. Although the majority of phosphosites were responsive to both inhibitors, we identified 16 sites that decreased only in response to PLX4032, suggesting rare instances where oncogenic B-RAF signaling occurs in an MKK1/2-independent manner. Only two phosphosites were identified that appeared to be uniquely responsive to AZD6244. When cells were treated with the combination of AZD6244 and PLX4032 at subsaturating concentrations (30 nm), responses at nearly all phosphosites were additive. We conclude that AZD6244 does not substantially widen the range of phosphosites inhibited by PLX4032 and that the benefit of the drug combination is best explained by their additive effects on suppressing ERK1/2 signaling. Comparison of our results to another recent ERK1/2 phosphoproteomics study revealed a surprising degree of variability in the sensitivity of phosphosites to MKK1

  8. Phosphoproteomic profiling identifies focal adhesion kinase as a mediator of docetaxel resistance in castrate-resistant prostate cancer.

    PubMed

    Lee, Brian Y; Hochgräfe, Falko; Lin, Hui-Ming; Castillo, Lesley; Wu, Jianmin; Raftery, Mark J; Martin Shreeve, S; Horvath, Lisa G; Daly, Roger J

    2014-01-01

    Docetaxel remains the standard-of-care for men diagnosed with metastatic castrate-resistant prostate cancer (CRPC). However, only approximately 50% of patients benefit from treatment and all develop docetaxel-resistant disease. Here, we characterize global perturbations in tyrosine kinase signaling associated with docetaxel resistance and thereby develop a potential therapeutic strategy to reverse this phenotype. Using quantitative mass spectrometry-based phosphoproteomics, we identified that metastatic docetaxel-resistant prostate cancer cell lines (DU145-Rx and PC3-Rx) exhibit increased phosphorylation of focal adhesion kinase (FAK) on Y397 and Y576, in comparison with parental controls (DU145 and PC3, respectively). Bioinformatic analyses identified perturbations in pathways regulating focal adhesions and the actin cytoskeleton and in protein-protein interaction networks related to these pathways in docetaxel-resistant cells. Treatment with the FAK tyrosine kinase inhibitor (TKI) PF-00562271 reduced FAK phosphorylation in the resistant cells, but did not affect cell viability or Akt phosphorylation. Docetaxel administration reduced FAK and Akt phosphorylation, whereas cotreatment with PF-00562271 and docetaxel resulted in an additive attenuation of FAK and Akt phosphorylation and overcame the chemoresistant phenotype. The enhanced efficacy of cotreatment was due to increased autophagic cell death, rather than apoptosis. These data strongly support that enhanced FAK activation mediates chemoresistance in CRPC, and identify a potential clinical niche for FAK TKIs, where coadministration with docetaxel may be used in patients with CRPC to overcome chemoresistance. PMID:24194567

  9. Phosphoproteomic profiling of tumor tissues identifies HSP27 Ser82 phosphorylation as a robust marker of early ischemia

    PubMed Central

    Zahari, Muhammad Saddiq; Wu, Xinyan; Pinto, Sneha M.; Nirujogi, Raja Sekhar; Kim, Min-Sik; Fetics, Barry; Philip, Mathew; Barnes, Sheri R.; Godfrey, Beverly; Gabrielson, Edward; Nevo, Erez; Pandey, Akhilesh

    2015-01-01

    Delays between tissue collection and tissue fixation result in ischemia and ischemia-associated changes in protein phosphorylation levels, which can misguide the examination of signaling pathway status. To identify a biomarker that serves as a reliable indicator of ischemic changes that tumor tissues undergo, we subjected harvested xenograft tumors to room temperature for 0, 2, 10 and 30 minutes before freezing in liquid nitrogen. Multiplex TMT-labeling was conducted to achieve precise quantitation, followed by TiO2 phosphopeptide enrichment and high resolution mass spectrometry profiling. LC-MS/MS analyses revealed phosphorylation level changes of a number of phosphosites in the ischemic samples. The phosphorylation of one of these sites, S82 of the heat shock protein 27 kDa (HSP27), was especially abundant and consistently upregulated in tissues with delays in freezing as short as 2 minutes. In order to eliminate effects of ischemia, we employed a novel cryogenic biopsy device which begins freezing tissues in situ before they are excised. Using this device, we showed that the upregulation of phosphorylation of S82 on HSP27 was abrogated. We thus demonstrate that our cryogenic biopsy device can eliminate ischemia-induced phosphoproteome alterations, and measurements of S82 on HSP27 can be used as a robust marker of ischemia in tissues. PMID:26329039

  10. Phosphoproteomic analysis identifies the tumor suppressor PDCD4 as a RSK substrate negatively regulated by 14-3-3

    PubMed Central

    Galan, Jacob A.; Geraghty, Kathryn M.; Lavoie, Geneviève; Kanshin, Evgeny; Tcherkezian, Joseph; Calabrese, Viviane; Jeschke, Grace R.; Turk, Benjamin E.; Ballif, Bryan A.; Blenis, John; Thibault, Pierre; Roux, Philippe P.

    2014-01-01

    The Ras/MAPK signaling cascade regulates various biological functions, including cell growth and proliferation. As such, this pathway is frequently deregulated in several types of cancer, including most cases of melanoma. RSK (p90 ribosomal S6 kinase) is a MAPK-activated protein kinase required for melanoma growth and proliferation, but relatively little is known about its exact function and the nature of its substrates. Herein, we used a quantitative phosphoproteomics approach to define the signaling networks regulated by RSK in melanoma. To more accurately predict direct phosphorylation substrates, we defined the RSK consensus phosphorylation motif and found significant overlap with the binding consensus of 14-3-3 proteins. We thus characterized the phospho-dependent 14-3-3 interactome in melanoma cells and found that a large proportion of 14-3-3 binding proteins are also potential RSK substrates. Our results show that RSK phosphorylates the tumor suppressor PDCD4 (programmed cell death protein 4) on two serine residues (Ser76 and Ser457) that regulate its subcellular localization and interaction with 14-3-3 proteins. We found that 14-3-3 binding promotes PDCD4 degradation, suggesting an important role for RSK in the inactivation of PDCD4 in melanoma. In addition to this tumor suppressor, our results suggest the involvement of RSK in a vast array of unexplored biological functions with relevance in oncogenesis. PMID:25002506

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

    PubMed

    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. PMID:26841317

  12. Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1.

    PubMed

    Lai, Yu-Chiang; Kondapalli, Chandana; Lehneck, Ronny; Procter, James B; Dill, Brian D; Woodroof, Helen I; Gourlay, Robert; Peggie, Mark; Macartney, Thomas J; Corti, Olga; Corvol, Jean-Christophe; Campbell, David G; Itzen, Aymelt; Trost, Matthias; Muqit, Miratul Mk

    2015-11-12

    Mutations in the PTEN-induced kinase 1 (PINK1) are causative of autosomal recessive Parkinson's disease (PD). We have previously reported that PINK1 is activated by mitochondrial depolarisation and phosphorylates serine 65 (Ser(65)) of the ubiquitin ligase Parkin and ubiquitin to stimulate Parkin E3 ligase activity. Here, we have employed quantitative phosphoproteomics to search for novel PINK1-dependent phosphorylation targets in HEK (human embryonic kidney) 293 cells stimulated by mitochondrial depolarisation. This led to the identification of 14,213 phosphosites from 4,499 gene products. Whilst most phosphosites were unaffected, we strikingly observed three members of a sub-family of Rab GTPases namely Rab8A, 8B and 13 that are all phosphorylated at the highly conserved residue of serine 111 (Ser(111)) in response to PINK1 activation. Using phospho-specific antibodies raised against Ser(111) of each of the Rabs, we demonstrate that Rab Ser(111) phosphorylation occurs specifically in response to PINK1 activation and is abolished in HeLa PINK1 knockout cells and mutant PINK1 PD patient-derived fibroblasts stimulated by mitochondrial depolarisation. We provide evidence that Rab8A GTPase Ser(111) phosphorylation is not directly regulated by PINK1 in vitro and demonstrate in cells the time course of Ser(111) phosphorylation of Rab8A, 8B and 13 is markedly delayed compared to phosphorylation of Parkin at Ser(65). We further show mechanistically that phosphorylation at Ser(111) significantly impairs Rab8A activation by its cognate guanine nucleotide exchange factor (GEF), Rabin8 (by using the Ser111Glu phosphorylation mimic). These findings provide the first evidence that PINK1 is able to regulate the phosphorylation of Rab GTPases and indicate that monitoring phosphorylation of Rab8A/8B/13 at Ser(111) may represent novel biomarkers of PINK1 activity in vivo. Our findings also suggest that disruption of Rab GTPase-mediated signalling may represent a major mechanism

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

    PubMed

    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

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

    PubMed Central

    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. PMID:25873915

  15. Phosphoproteome of Human Glioblastoma Initiating Cells Reveals Novel Signaling Regulators Encoded by the Transcriptome

    PubMed Central

    Kozuka-Hata, Hiroko; Nasu-Nishimura, Yukiko; Koyama-Nasu, Ryo; Ao-Kondo, Hiroko; Tsumoto, Kouhei; Akiyama, Tetsu; Oyama, Masaaki

    2012-01-01

    Background Glioblastoma is one of the most aggressive tumors with poor prognosis. Although various studies have been performed so far, there are not effective treatments for patients with glioblastoma. Methodology/Principal Findings In order to systematically elucidate the aberrant signaling machinery activated in this malignant brain tumor, we investigated phosphoproteome dynamics of glioblastoma initiating cells using high-resolution nanoflow LC-MS/MS system in combination with SILAC technology. Through phosphopeptide enrichment by titanium dioxide beads, a total of 6,073 phosphopeptides from 2,282 phosphorylated proteins were identified based on the two peptide fragmentation methodologies of collision induced dissociation and higher-energy C-trap dissociation. The SILAC-based quantification described 516 up-regulated and 275 down-regulated phosphorylation sites upon epidermal growth factor stimulation, including the comprehensive status of the phosphorylation sites on stem cell markers such as nestin. Very intriguingly, our in-depth phosphoproteome analysis led to identification of novel phosphorylated molecules encoded by the undefined sequence regions of the human transcripts, one of which was regulated upon external stimulation in human glioblastoma initiating cells. Conclusions/Significance Our result unveils an expanded diversity of the regulatory phosphoproteome defined by the human transcriptome. PMID:22912867

  16. Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates.

    PubMed

    Xue, Liang; Wang, Wen-Horng; Iliuk, Anton; Hu, Lianghai; Galan, Jacob A; Yu, Shuai; Hans, Michael; Geahlen, Robert L; Tao, W Andy

    2012-04-10

    Our understanding of the molecular control of many disease pathologies requires the identification of direct substrates targeted by specific protein kinases. Here we describe an integrated proteomic strategy, termed kinase assay linked with phosphoproteomics, which combines a sensitive kinase reaction with endogenous kinase-dependent phosphoproteomics to identify direct substrates of protein kinases. The unique in vitro kinase reaction is carried out in a highly efficient manner using a pool of peptides derived directly from cellular kinase substrates and then dephosphorylated as substrate candidates. The resulting newly phosphorylated peptides are then isolated and identified by mass spectrometry. A further comparison of these in vitro phosphorylated peptides with phosphopeptides derived from endogenous proteins isolated from cells in which the kinase is either active or inhibited reveals new candidate protein substrates. The kinase assay linked with phosphoproteomics strategy was applied to identify unique substrates of spleen tyrosine kinase (Syk), a protein-tyrosine kinase with duel properties of an oncogene and a tumor suppressor in distinctive cell types. We identified 64 and 23 direct substrates of Syk specific to B cells and breast cancer cells, respectively. Both known and unique substrates, including multiple centrosomal substrates for Syk, were identified, supporting a unique mechanism that Syk negatively affects cell division through its centrosomal kinase activity. PMID:22451900

  17. Phosphoproteomic Analysis of Platelets Activated by Pro-Thrombotic Oxidized Phospholipids and Thrombin

    PubMed Central

    Zimman, Alejandro; Titz, Bjoern; Komisopoulou, Evangelia; Biswas, Sudipta; Graeber, Thomas G.; Podrez, Eugene A.

    2014-01-01

    Specific oxidized phospholipids (oxPCCD36) promote platelet hyper-reactivity and thrombosis in hyperlipidemia via the scavenger receptor CD36, however the signaling pathway(s) induced in platelets by oxPCCD36 are not well defined. We have employed mass spectrometry-based tyrosine, serine, and threonine phosphoproteomics for the unbiased analysis of platelet signaling pathways induced by oxPCCD36 as well as by the strong physiological agonist thrombin. oxPCCD36 and thrombin induced differential phosphorylation of 115 proteins (162 phosphorylation sites) and 181 proteins (334 phosphorylation sites) respectively. Most of the phosphoproteome changes induced by either agonist have never been reported in platelets; thus they provide candidates in the study of platelet signaling. Bioinformatic analyses of protein phosphorylation dependent responses were used to categorize preferential motifs for (de)phosphorylation, predict pathways and kinase activity, and construct a phosphoproteome network regulating integrin activation. A putative signaling pathway involving Src-family kinases, SYK, and PLCγ2 was identified in platelets activated by oxPCCD36. Subsequent ex vivo studies in human platelets demonstrated that this pathway is downstream of the scavenger receptor CD36 and is critical for platelet activation by oxPCCD36. Our results provide multiple insights into the mechanism of platelet activation and specifically in platelet regulation by oxPCCD36. PMID:24400094

  18. Identification of the PLK2-dependent phosphopeptidome by quantitative proteomics [corrected].

    PubMed

    Franchin, Cinzia; Cesaro, Luca; Pinna, Lorenzo A; Arrigoni, Giorgio; Salvi, Mauro

    2014-01-01

    Polo-like kinase 2 (PLK2) has been recently recognized as the major enzyme responsible for phosphorylation of α-synuclein at S129 in vitro and in vivo, suggesting that this kinase may play a key role in the pathogenesis of Parkinson's disease and other synucleinopathies. Moreover PLK2 seems to be implicated in cell division, oncogenesis, and synaptic regulation of the brain. However little is known about the phosphoproteome generated by PLK2 and, consequently the overall impact of PLK2 on cellular signaling. To fill this gap we exploited an approach based on in vitro kinase assay and quantitative phosphoproteomics. A proteome-derived peptide library obtained by digestion of undifferentiated human neuroblastoma cell line was exhaustively dephosphorylated by lambda phosphatase followed by incubation with or without PLK2 recombinant kinase. Stable isotope labeling based quantitative phosphoproteomics was applied to identify the phosphosites generated by PLK2. A total of 98 unique PLK2-dependent phosphosites from 89 proteins were identified by LC-MS/MS. Analysis of the primary structure of the identified phosphosites allowed the detailed definition of the kinase specificity and the compilation of a list of potential PLK2 targets among those retrieved in PhosphositePlus, a curated database of in cell/vivo phosphorylation sites. PMID:25338102

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

    PubMed

    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. PMID:26969016

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

    PubMed Central

    Adams, Nancy E.; Maurelli, Anthony T.

    2015-01-01

    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. PMID:25998263

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

    PubMed

    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. PMID:26573262

  2. Kicking Genomic Profiling to the Curb: How Re-wiring the Phosphoproteome Can Explain Treatment Resistance in Glioma.

    PubMed

    Lam, Fred C; Yaffe, Michael B

    2016-04-11

    In this issue of Cancer Cell, Wei et al. (2016) identify adaptive re-wiring of signaling nodes in glioma as major mechanisms of treatment resistance without genome-wide mutations. Targeting these nodes before treatment blocks resistance and underscores the importance of single-cell phosphoproteomics and network re-wiring in predicting cancer treatment responses. PMID:27070697

  3. SELPHI: correlation-based identification of kinase-associated networks from global phospho-proteomics data sets.

    PubMed

    Petsalaki, Evangelia; Helbig, Andreas O; Gopal, Anjali; Pasculescu, Adrian; Roth, Frederick P; Pawson, Tony

    2015-07-01

    While phospho-proteomics studies have shed light on the dynamics of cellular signaling, they mainly describe global effects and rarely explore mechanistic details, such as kinase/substrate relationships. Tools and databases, such as NetworKIN and PhosphoSitePlus, provide valuable regulatory details on signaling networks but rely on prior knowledge. They therefore provide limited information on less studied kinases and fewer unexpected relationships given that better studied signaling events can mask condition- or cell-specific 'network wiring'. SELPHI is a web-based tool providing in-depth analysis of phospho-proteomics data that is intuitive and accessible to non-bioinformatics experts. It uses correlation analysis of phospho-sites to extract kinase/phosphatase and phospho-peptide associations, and highlights the potential flow of signaling in the system under study. We illustrate SELPHI via analysis of phospho-proteomics data acquired in the presence of erlotinib-a tyrosine kinase inhibitor (TKI)-in cancer cells expressing TKI-resistant and -sensitive variants of the Epidermal Growth Factor Receptor. In this data set, SELPHI revealed information overlooked by the reporting study, including the known role of MET and EPHA2 kinases in conferring resistance to erlotinib in TKI sensitive strains. SELPHI can significantly enhance the analysis of phospho-proteomics data contributing to improved understanding of sample-specific signaling networks. SELPHI is freely available via http://llama.mshri.on.ca/SELPHI. PMID:25948583

  4. SELPHI: correlation-based identification of kinase-associated networks from global phospho-proteomics data sets

    PubMed Central

    Petsalaki, Evangelia; Helbig, Andreas O.; Gopal, Anjali; Pasculescu, Adrian; Roth, Frederick P.; Pawson, Tony

    2015-01-01

    While phospho-proteomics studies have shed light on the dynamics of cellular signaling, they mainly describe global effects and rarely explore mechanistic details, such as kinase/substrate relationships. Tools and databases, such as NetworKIN and PhosphoSitePlus, provide valuable regulatory details on signaling networks but rely on prior knowledge. They therefore provide limited information on less studied kinases and fewer unexpected relationships given that better studied signaling events can mask condition- or cell-specific ‘network wiring’. SELPHI is a web-based tool providing in-depth analysis of phospho-proteomics data that is intuitive and accessible to non-bioinformatics experts. It uses correlation analysis of phospho-sites to extract kinase/phosphatase and phospho-peptide associations, and highlights the potential flow of signaling in the system under study. We illustrate SELPHI via analysis of phospho-proteomics data acquired in the presence of erlotinib—a tyrosine kinase inhibitor (TKI)—in cancer cells expressing TKI-resistant and -sensitive variants of the Epidermal Growth Factor Receptor. In this data set, SELPHI revealed information overlooked by the reporting study, including the known role of MET and EPHA2 kinases in conferring resistance to erlotinib in TKI sensitive strains. SELPHI can significantly enhance the analysis of phospho-proteomics data contributing to improved understanding of sample-specific signaling networks. SELPHI is freely available via http://llama.mshri.on.ca/SELPHI. PMID:25948583

  5. Phosphorylation-Specific MS/MS Scoring for Rapid and Accurate Phosphoproteome Analysis

    PubMed Central

    Payne, Samuel H.; Yau, Margaret; Smolka, Marcus B.; Tanner, Stephen; Zhou, Huilin; Bafna, Vineet

    2008-01-01

    The promise of mass spectrometry as a tool for probing signal-transduction is predicated on reliable identification of post-translational modifications. Phosphorylations are key mediators of cellular signaling, yet are hard to detect, partly because of unusual fragmentation patterns of phosphopeptides. In addition to being accurate, MS/MS identification software must be robust and efficient to deal with increasingly large spectral data sets. Here, we present a new scoring function for the Inspect software for phosphorylated peptide tandem mass spectra for ion-trap instruments, without the need for manual validation. The scoring function was modeled by learning fragmentation patterns from 7677 validated phosphopeptide spectra. We compare our algorithm against SEQUEST and X!Tandem on testing and training data sets. At a 1% false positive rate, Inspect identified the greatest total number of phosphorylated spectra, 13% more than SEQUEST and 39% more than X!Tandem. Spectra identified by Inspect tended to score better in several spectral quality measures. Furthermore, Inspect runs much faster than either SEQUEST or X!Tandem, making desktop phosphoproteomics feasible. Finally, we used our new models to reanalyze a corpus of 423 000 LTQ spectra acquired for a phosphoproteome analysis of Saccharomyces cerevisiae DNA damage and repair pathways and discovered 43% more phosphopeptides than the previous study. PMID:18563926

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

    PubMed

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

    2014-11-01

    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. PMID:24702127

  7. The Global Phosphoproteome of Chlamydomonas reinhardtii Reveals Complex Organellar Phosphorylation in the Flagella and Thylakoid Membrane *

    PubMed Central

    Wang, Hongxia; Gau, Brian; Slade, William O.; Juergens, Matthew; Li, Ping; Hicks, Leslie M.

    2014-01-01

    Chlamydomonas reinhardtii is the most intensively-studied and well-developed model for investigation of a wide-range of microalgal processes ranging from basic development through understanding triacylglycerol production. Although proteomic technologies permit interrogation of these processes at the protein level and efforts to date indicate phosphorylation-based regulation of proteins in C. reinhardtii is essential for its underlying biology, characterization of the C. reinhardtii phosphoproteome has been limited. Herein, we report the richest exploration of the C. reinhardtii proteome to date. Complementary enrichment strategies were used to detect 4588 phosphoproteins distributed among every cellular component in C. reinhardtii. Additionally, we report 18,160 unique phosphopeptides at <1% false discovery rate, which comprise 15,862 unique phosphosites - 98% of which are novel. Given that an estimated 30% of proteins in a eukaryotic cell are subject to phosphorylation, we report the majority of the phosphoproteome (23%) of C. reinhardtii. Proteins in key biological pathways were phosphorylated, including photosynthesis, pigment production, carbon assimilation, glycolysis, and protein and carbohydrate metabolism, and it is noteworthy that hyperphosphorylation was observed in flagellar proteins. This rich data set is available via ProteomeXchange (ID: PXD000783) and will significantly enhance understanding of a range of regulatory mechanisms controlling a variety of cellular process and will serve as a critical resource for the microalgal community. PMID:24917610

  8. Phosphoproteome mapping of cardiomyocyte mitochondria in a rat model of heart failure.

    PubMed

    Giorgianni, Francesco; Usman Khan, M; Weber, Karl T; Gerling, Ivan C; Beranova-Giorgianni, Sarka

    2014-04-01

    Mitochondria are complex organelles essential to cardiomyocyte survival. Protein phosphorylation is emerging as a key regulator of mitochondrial function. In the study reported here, we analyzed subsarcolemmal (SSM) mitochondria harvested from rats who have received 4 weeks of aldosterone/salt treatment to simulate the neurohormonal profile of human congestive heart failure. Our objective was to obtain an initial qualitative inventory of the phosphoproteins in this biologic system. SSM mitochondria were harvested, and the phosphoproteome was analyzed with a gel-free bioanalytical platform. Mitochondrial proteins were digested with trypsin, and the digests were enriched for phosphopeptides with immobilized metal ion affinity chromatography. The phosphopeptides were analyzed by ion trap liquid chromatography-tandem mass spectrometry, and the phosphoproteins identified via database searches. Based on MS/MS and MS(3) data, we characterized a set of 42 phosphopeptides that encompassed 39 phosphorylation sites. These peptides mapped to 26 proteins, for example, long-chain specific acyl-CoA dehydrogenase, Complex III subunit 6, and mitochondrial import receptor TOM70. Collectively, the characterized phosphoproteins belong to diverse functional modules, including bioenergetic pathways, protein import machinery, and calcium handling. The phosphoprotein panel discovered in this study provides a foundation for future differential phosphoproteome profiling toward an integrated understanding of the role of mitochondrial phosphorylation in heart failure. PMID:24395194

  9. Phosphoproteome mapping of cardiomyocyte mitochondria in a rat model of heart failure

    PubMed Central

    Giorgianni, Francesco; Khan, M. Usman; Weber, Karl T.; Gerling, Ivan C.; Beranova-Giorgianni, Sarka

    2014-01-01

    Mitochondria are complex organelles essential to cardiomyocyte survival. Protein phosphorylation is emerging as a key regulator of mitochondrial function. In the study reported here, we analyzed subsarcolemmal mitochondria harvested from rats who have received 4 weeks of aldosterone/salt treatment to simulate the neurohormonal profile of human congestive heart failure. Our objective was to obtain an initial qualitative inventory of the phosphoproteins in this biological system. Subsarcolemmal mitochondria were harvested and the phosphoproteome was analyzed with a gel-free bioanalytical platform. Mitochondrial proteins were digested with trypsin, and the digests were enriched for phosphopeptides with Immobilized Metal Ion Affinity Chromatography (IMAC). The phosphopeptides were analyzed by ion trap LC-MS/MS, and the phosphoproteins identified via database searches. Based on MS/MS and MS3 data, we characterized a set of 42 phosphopeptides that encompassed 39 phosphorylation sites. These peptides mapped to 26 proteins, for example long-chain specific acyl-CoA dehydrogenase (LCAD), Complex III subunit 6, and mitochondrial import receptor TOM70. Collectively, the characterized phosphoproteins belong to diverse functional modules, including bioenergetic pathways, protein import machinery, and calcium handling. The phosphoprotein panel discovered in this study provides a foundation for future differential phosphoproteome profiling towards an integrated understanding of the role of mitochondrial phosphorylation in heart failure. PMID:24395194

  10. Off-line high-pH reversed-phase fractionation for in-depth phosphoproteomics.

    PubMed

    Batth, Tanveer S; Francavilla, Chiara; Olsen, Jesper V

    2014-12-01

    Protein phosphorylation is an important post-translational modification (PTM) involved in embryonic development, adult homeostasis, and disease. Over the past decade, several advances have been made in liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based technologies to identify thousands of phosphorylation sites. However, in-depth phosphoproteomics often require off-line enrichment and fractionation techniques. In this study, we provide a detailed analysis of the physicochemical characteristics of phosphopeptides, which have been fractionated by off-line high-pH chromatography (HpH) before subsequent titanium dioxide (TiO2) enrichment and LC-MS/MS analysis. Our results demonstrate that HpH is superior to standard strong-cation exchange (SCX) fractionation in the total number of phosphopeptides detected when analyzing the same number of fractions by identical LC-MS/MS gradients. From 14 HpH fractions, we routinely identified over 30,000 unique phosphopeptide variants, which is more than twice the number of that obtained from SCX fractionation. HpH chromatography displayed an exceptional ability to fractionate singly phosphorylated peptides, with minor benefits for doubly phosphorylated peptides over that with SCX. Further optimizations in the pooling and concatenation strategy increased the total number of multiphosphorylated peptides detected after HpH fractionation. In conclusion, we provide a basic framework and resource for performing in-depth phosphoproteome studies utilizing off-line basic reversed-phased fractionation. Raw data is available at ProteomeXchange (PXD001404). PMID:25338131

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

    PubMed Central

    2013-01-01

    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 < 0.05) were identified in infected compared to healthy plants, including proteins involved in photosynthesis, response to stress and the antioxidant system. Many were not differentially phosphorylated in recovered compared to healthy plants, pointing to their specific role in responding to infection, followed by a return to a steady-state phosphorylation level after remission of symptoms. Gene ontology (GO) enrichment and statistical

  12. A Phos-tag SDS-PAGE method that effectively uses phosphoproteomic data for profiling the phosphorylation dynamics of MEK1.

    PubMed

    Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Kubota, Yuji; Takekawa, Mutsuhiro; Koike, Tohru

    2016-07-01

    MEK1, an essential component of the mitogen-activated protein kinase (MAPK) pathway, is phosphorylated during activation of the pathway; 12 phosphorylation sites have been identified in human MEK1 by MS-based phosphoproteomic methods. By using Phos-tag SDS-PAGE, we found that multiple variants of MEK1 with different phosphorylation states are constitutively present in typical human cells. The Phos-tag-based strategy, which makes effective use of existing information on the location of phosphorylation sites, permits quantitative time-course profiling of MEK1 phosphospecies in their respective phosphorylation states. By subsequent immunoblotting with an anti-HaloTag antibody, we analyzed a HaloTag-fused MEK1 protein and 12 potential phosphorylation-site-directed mutants of the protein transiently expressed in HEK 293 cells. This strategy revealed that MEK1 is constitutively and mainly phosphorylated at the Thr-292, Ser-298, Thr-386, and Thr-388 residues in vivo, and that combinations of phosphorylations at these four residues produce at least six phosphorylated variants of MEK1. Like the levels of phosphorylation of the Ser-218 and Ser-222 residues by RAF1, which have been well studied, the phosphorylation statuses of Thr-292, Ser-298, Thr-386, and Thr-388 residues vary widely during activation and deactivation of the MAPK pathway. Furthermore, we demonstrated inhibitor-specific profiling of MEK1 phosphospecies by using three MEK inhibitors: TAK-733, PD98059, and U0126. PMID:27169363

  13. Unbiased phosphoproteomic method identifies the initial effects of a methacrylic acid copolymer on macrophages.

    PubMed

    Chamberlain, Michael Dean; Wells, Laura A; Lisovsky, Alexandra; Guo, Hongbo; Isserlin, Ruth; Talior-Volodarsky, Ilana; Mahou, Redouan; Emili, Andrew; Sefton, Michael V

    2015-08-25

    An unbiased phosphoproteomic method was used to identify biomaterial-associated changes in the phosphorylation patterns of macrophage-like cells. The phosphorylation differences between differentiated THP1 (dTHP1) cells treated for 10, 20, or 30 min with a vascular regenerative methacrylic acid (MAA) copolymer or a control methyl methacrylate (MM) copolymer were determined by MS. There were 1,470 peptides (corresponding to 729 proteins) that were differentially phosphorylated in dTHP1 cells treated with the two materials with a greater cellular response to MAA treatment. In addition to identifying pathways (such as integrin signaling and cytoskeletal arrangement) that are well known to change with cell-material interaction, previously unidentified pathways, such as apoptosis and mRNA splicing, were also discovered. PMID:26261332

  14. Development of the affinity materials for phosphorylated proteins/peptides enrichment in phosphoproteomics analysis.

    PubMed

    Wang, Zhi-Gang; Lv, Nan; Bi, Wen-Zhi; Zhang, Ji-Lin; Ni, Jia-Zuan

    2015-04-29

    Reversible protein phosphorylation is a key event in numerous biological processes. Mass spectrometry (MS) is the most powerful analysis tool in modern phosphoproteomics. However, the direct MS analysis of phosphorylated proteins/peptides is still a big challenge because of the low abundance and insufficient ionization of phosphorylated proteins/peptides as well as the suppression effects of nontargets. Enrichment of phosphorylated proteins/peptides by affinity materials from complex biosamples is the most widely used strategy to enhance the MS detection. The demand of efficiently enriching phosphorylated proteins/peptides has spawned diverse affinity materials based on different enrichment principles (e.g., electronic attraction, chelating). In this review, we summarize the recent development of various affinity materials for phosphorylated proteins/peptides enrichment. We will highlight the design and fabrication of these affinity materials, discuss the enrichment mechanisms involved in different affinity materials, and suggest the future challenges and research directions in this field. PMID:25845677

  15. Single-Cell Phosphoproteomics Resolves Adaptive Signaling Dynamics and Informs Targeted Combination Therapy in Glioblastoma.

    PubMed

    Wei, Wei; Shin, Young Shik; Xue, Min; Matsutani, Tomoo; Masui, Kenta; Yang, Huijun; Ikegami, Shiro; Gu, Yuchao; Herrmann, Ken; Johnson, Dazy; Ding, Xiangming; Hwang, Kiwook; Kim, Jungwoo; Zhou, Jian; Su, Yapeng; Li, Xinmin; Bonetti, Bruno; Chopra, Rajesh; James, C David; Cavenee, Webster K; Cloughesy, Timothy F; Mischel, Paul S; Heath, James R; Gini, Beatrice

    2016-04-11

    Intratumoral heterogeneity of signaling networks may contribute to targeted cancer therapy resistance, including in the highly lethal brain cancer glioblastoma (GBM). We performed single-cell phosphoproteomics on a patient-derived in vivo GBM model of mTOR kinase inhibitor resistance and coupled it to an analytical approach for detecting changes in signaling coordination. Alterations in the protein signaling coordination were resolved as early as 2.5 days after treatment, anticipating drug resistance long before it was clinically manifest. Combination therapies were identified that resulted in complete and sustained tumor suppression in vivo. This approach may identify actionable alterations in signal coordination that underlie adaptive resistance, which can be suppressed through combination drug therapy, including non-obvious drug combinations. PMID:27070703

  16. Unbiased phosphoproteomic method identifies the initial effects of a methacrylic acid copolymer on macrophages

    PubMed Central

    Chamberlain, Michael Dean; Wells, Laura A.; Lisovsky, Alexandra; Guo, Hongbo; Isserlin, Ruth; Talior-Volodarsky, Ilana; Mahou, Redouan; Emili, Andrew; Sefton, Michael V.

    2015-01-01

    An unbiased phosphoproteomic method was used to identify biomaterial-associated changes in the phosphorylation patterns of macrophage-like cells. The phosphorylation differences between differentiated THP1 (dTHP1) cells treated for 10, 20, or 30 min with a vascular regenerative methacrylic acid (MAA) copolymer or a control methyl methacrylate (MM) copolymer were determined by MS. There were 1,470 peptides (corresponding to 729 proteins) that were differentially phosphorylated in dTHP1 cells treated with the two materials with a greater cellular response to MAA treatment. In addition to identifying pathways (such as integrin signaling and cytoskeletal arrangement) that are well known to change with cell–material interaction, previously unidentified pathways, such as apoptosis and mRNA splicing, were also discovered. PMID:26261332

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

    PubMed Central

    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. PMID:26400465

  18. Characterization of the human plasma phosphoproteome using linear ion trap mass spectrometry and multiple search engines.

    PubMed

    Carrascal, Montserrat; Gay, Marina; Ovelleiro, David; Casas, Vanessa; Gelpí, Emilio; Abian, Joaquin

    2010-02-01

    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. PMID:19941383

  19. Automated Immobilized Metal Affinity Chromatography System for Enrichment of Escherichia coli Phosphoproteome

    SciTech Connect

    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.

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

    PubMed Central

    Rampitsch, Christof; Bykova, Natalia V.

    2012-01-01

    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-signaling 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 hydrogen peroxide 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. PMID:22783265

  1. Phosphatase of Regenerating Liver 3 (PRL3) Provokes a Tyrosine Phosphoproteome to Drive Prometastatic Signal Transduction*

    PubMed Central

    Walls, Chad D.; Iliuk, Anton; Bai, Yunpeng; Wang, Mu; Tao, W. Andy; Zhang, Zhong-Yin

    2013-01-01

    Phosphatase of regenerating liver 3 (PRL3) is suspected to be a causative factor toward cellular metastasis when in excess. To date, the molecular basis for PRL3 function remains an enigma, making efforts at distilling a concerted mechanism for PRL3-mediated metastatic dissemination very difficult. We previously discovered that PRL3 expressing cells exhibit a pronounced increase in protein tyrosine phosphorylation. Here we take an unbiased mass spectrometry-based approach toward identifying the phosphoproteins exhibiting enhanced levels of tyrosine phosphorylation with a goal to define the “PRL3-mediated signaling network.” Phosphoproteomic data support intracellular activation of an extensive signaling network normally governed by extracellular ligand-activated transmembrane growth factor, cytokine, and integrin receptors in the PRL3 cells. Additionally, data implicate the Src tyrosine kinase as the major intracellular kinase responsible for “hijacking” this network and provide strong evidence that aberrant Src activation is a major consequence of PRL3 overexpression. Importantly, the data support a PDGF(α/β)-, Eph (A2/B3/B4)-, and Integrin (β1/β5)-receptor array as being the predominant network coordinator in the PRL3 cells, corroborating a PRL3-induced mesenchymal-state. Within this network, we find that tyrosine phosphorylation is increased on a multitude of signaling effectors responsible for Rho-family GTPase, PI3K-Akt, STAT, and ERK activation, linking observations made by the field as a whole under Src as a primary signal transducer. Our phosphoproteomic data paint the most comprehensive picture to date of how PRL3 drives prometastatic molecular events through Src activation. PMID:24030100

  2. Multiplexed Detection of O-GlcNAcome, Phosphoproteome, and Whole Proteome within the Same Gel

    PubMed Central

    Cieniewski-Bernard, Caroline; Dupont, Erwan; Deracinois, Barbara; Lambert, Matthias; Bastide, Bruno

    2014-01-01

    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. The development of multiplexed proteomics based on fluorescent detection of proteins permits to go further in the understanding of the proteome complexity. 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. PMID:25389416

  3. Identification of kinase inhibitor targets in the lung cancer microenvironment by chemical and phosphoproteomics

    PubMed Central

    Gridling, Manuela; Ficarro, Scott B.; Breitwieser, Florian P.; Song, Lanxi; Parapatics, Katja; Colinge, Jacques; Haura, Eric B.; Marto, Jarrod A.; Superti-Furga, Giulio; Bennett, Keiryn L.; Rix, Uwe

    2014-01-01

    A growing number of gene mutations, which are recognized as cancer drivers, can be successfully targeted with drugs. The redundant and dynamic nature of oncogenic signaling networks and complex interactions between cancer cells and the microenvironment, however, can cause drug resistance. Whereas these challenges can be addressed by developing drug combinations or polypharmacology drugs, this benefits greatly from a detailed understanding of the proteome-wide target profiles. Using mass spectrometry-based chemical proteomics, we report the comprehensive characterization of the drug-protein interaction networks for the multikinase inhibitors dasatinib and sunitinib in primary lung cancer tissue specimens derived from patients. We observed in excess of 100 protein kinase targets plus various protein complexes involving, for instance, AMPK, TBK1 (sunitinib) and ILK (dasatinib). Importantly, comparison with lung cancer cell lines and mouse xenografts thereof showed that most targets were shared between cell lines and tissues. Several targets, however, were only present in tumor tissues. In xenografts, most of these proteins were of mouse origin suggesting that they originate from the tumor microenvironment. Furthermore, intersection with subsequent global phosphoproteomic analysis identified several activated signaling pathways. These included MAPK, immune and integrin signaling, which were affected by these drugs in both cancer cells and the microenvironment. Thus, the combination of chemical and phosphoproteomics can generate a systems view of proteins, complexes and signaling pathways that are simultaneously engaged by multi-targeted drugs in cancer cells and the tumor microenvironment. This may allow for the design of novel anticancer therapies that concurrently target multiple tumor compartments. PMID:25189542

  4. Structural insight into effector proteins of Gram-negative bacterial pathogens that modulate the phosphoproteome of their host

    PubMed Central

    Grishin, Andrey M; Beyrakhova, Ksenia A; Cygler, Miroslaw

    2015-01-01

    Invading pathogens manipulate cellular process of the host cell to establish a safe replicative niche. To this end they secrete a spectrum of proteins called effectors that modify cellular environment through a variety of mechanisms. One of the most important mechanisms is the manipulation of cellular signaling through modifications of the cellular phosphoproteome. Phosphorylation/dephosphorylation plays a pivotal role in eukaryotic cell signaling, with ∼500 different kinases and ∼130 phosphatases in the human genome. Pathogens affect the phosphoproteome either directly through the action of bacterial effectors, and/or indirectly through downstream effects of host proteins modified by the effectors. Here we review the current knowledge of the structure, catalytic mechanism and function of bacterial effectors that modify directly the phosphorylation state of host proteins. These effectors belong to four enzyme classes: kinases, phosphatases, phospholyases and serine/threonine acetylases. PMID:25565677

  5. The phosphoproteome in regenerating protoplasts from Physcomitrella patens protonemata shows changes paralleling postembryonic development in higher plants

    PubMed Central

    He, Yikun

    2014-01-01

    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. PMID:24700621

  6. Searching for Novel Cdk5 Substrates in Brain by Comparative Phosphoproteomics of Wild Type and Cdk5−/− Mice

    PubMed Central

    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

  7. Technology Enabled Learning. Symposium.

    ERIC Educational Resources Information Center

    2002

    This document contains three papers on technology-enabled learning and human resource development. Among results found in "Current State of Technology-enabled Learning Programs in Select Federal Government Organizations: a Case Study of Ten Organizations" (Letitia A. Combs) are the following: the dominant delivery method is traditional…

  8. Integrated approach using multistep enzyme digestion, TiO2 enrichment, and database search for in-depth phosphoproteomic profiling.

    PubMed

    Han, Dohyun; Jin, Jonghwa; Yu, Jiyoung; Kim, Kyunggon; Kim, Youngsoo

    2015-01-01

    Protein phosphorylation is a major PTM that regulates important cell signaling mechanisms. In-depth phosphoproteomic analysis provides a method of examining this complex interplay, yielding a mechanistic understanding of the cellular processes and pathogenesis of various diseases. However, the analysis of protein phosphorylation is challenging, due to the low concentration of phosphoproteins in highly complex mixtures and the high variability of phosphorylation sites. Thus, typical phosphoproteome studies that are based on MS require large amounts of starting material and extensive fractionation steps to reduce the sample complexity. To this end, we present a simple strategy (integrated multistep enzyme digestion, enrichment, database search-iMEED) to improve coverage of the phosphoproteome from lower sample amounts which is faster than other commonly used approaches. It is inexpensive and adaptable to low sample amounts and saves time and effort with regard to sample preparation and mass spectrometric analysis, allowing samples to be prepared without prefractionation or specific instruments, such as HPLC. All MS data have been deposited in the ProteomeXchange with identifier PXD001033 (http://proteomecentral.proteomexchange.org/dataset/PXD001033). PMID:25159016

  9. In-depth Analyses of Kinase-dependent Tyrosine Phosphoproteomes Based on Metal Ion-functionalized Soluble Nanopolymers*

    PubMed Central

    Iliuk, Anton B.; Martin, Victoria A.; Alicie, Bethany M.; Geahlen, Robert L.; Tao, W. Andy

    2010-01-01

    The ability to obtain in-depth understanding of signaling networks in cells is a key objective of systems biology research. Such ability depends largely on unbiased and reproducible analysis of phosphoproteomes. We present here a novel proteomics tool, polymer-based metal ion affinity capture (PolyMAC), for the highly efficient isolation of phosphopeptides to facilitate comprehensive phosphoproteome analyses. This approach uses polyamidoamine dendrimers multifunctionalized with titanium ions and aldehyde groups to allow the chelation and subsequent isolation of phosphopeptides in a homogeneous environment. Compared with current strategies based on solid phase micro- and nanoparticles, PolyMAC demonstrated outstanding reproducibility, exceptional selectivity, fast chelation times, and high phosphopeptide recovery from complex mixtures. Using the PolyMAC method combined with antibody enrichment, we identified 794 unique sites of tyrosine phosphorylation in malignant breast cancer cells, 514 of which are dependent on the expression of Syk, a protein-tyrosine kinase with unusual properties of a tumor suppressor. The superior sensitivity of PolyMAC allowed us to identify novel components in a variety of major signaling networks, including cell migration and apoptosis. PolyMAC offers a powerful and widely applicable tool for phosphoproteomics and molecular signaling. PMID:20562096

  10. Proteomic and phosphoproteomic analysis of polyethylene glycol-induced osmotic stress in root tips of common bean (Phaseolus vulgaris L.)

    PubMed Central

    Horst, Walter Johannes

    2013-01-01

    Previous studies have shown that polyethylene glycol (PEG)-induced osmotic stress (OS) reduces cell-wall (CW) porosity and limits aluminium (Al) uptake by root tips of common bean (Phaseolus vulgaris L.). A subsequent transcriptomic study suggested that genes related to CW processes are involved in adjustment to OS. In this study, a proteomic and phosphoproteomic approach was applied to identify OS-induced protein regulation to further improve our understanding of how OS affects Al accumulation. Analysis of total soluble proteins in root tips indicated that, in total, 22 proteins were differentially regulated by OS; these proteins were functionally categorized. Seventy-seven per- cent of the total expressed proteins were involved in metabolic pathways, particularly of carbohydrate and amino acid metabolism. An analysis of the apoplastic proteome revealed that OS reduced the level of five proteins and increased that of seven proteins. Investigation of the total soluble phosphoproteome suggested that dehydrin responded to OS with an enhanced phosphorylation state without a change in abundance. A cellular immunolocalization analysis indicated that dehydrin was localized mainly in the CW. This suggests that dehydrin may play a major protective role in the OS-induced physical breakdown of the CW structure and thus maintenance of the reversibility of CW extensibility during recovery from OS. The proteomic and phosphoproteomic analyses provided novel insights into the complex mechanisms of OS-induced reduction of Al accumulation in the root tips of common bean and highlight a key role for modification of CW structure. PMID:24123251

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

    PubMed Central

    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.

    2013-01-01

    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. PMID:23822953

  12. Changes in the Phosphoproteome and Metabolome Link Early Signaling Events to Rearrangement of Photosynthesis and Central Metabolism in Salinity and Oxidative Stress Response in Arabidopsis.

    PubMed

    Chen, Yanmei; Hoehenwarter, Wolfgang

    2015-12-01

    Salinity and oxidative stress are major factors affecting and limiting the productivity of agricultural crops. The molecular and biochemical processes governing the plant response to abiotic stress have often been researched in a reductionist manner. Here, we report a systemic approach combining metabolic labeling and phosphoproteomics to capture early signaling events with quantitative metabolome analysis and enzyme activity assays to determine the effects of salt and oxidative stress on plant physiology. K(+) and Na(+) transporters showed coordinated changes in their phosphorylation pattern, indicating the importance of dynamic ion homeostasis for adaptation to salt stress. Unique phosphorylation sites were found for Arabidopsis (Arabidopsis thaliana) SNF1 kinase homolog10 and 11, indicating their central roles in the stress-regulated responses. Seven Sucrose Non-fermenting1-Related Protein Kinase2 kinases showed varying levels of phosphorylation at multiple serine/threonine residues in their kinase domain upon stress, showing temporally distinct modulation of the various isoforms. Salinity and oxidative stress also lead to changes in protein phosphorylation of proteins central to photosynthesis, in particular the kinase State Transition Protein7 required for state transition and light-harvesting II complex proteins. Furthermore, stress-induced changes of the phosphorylation of enzymes of central metabolism were observed. The phosphorylation patterns of these proteins were concurrent with changes in enzyme activity. This was reflected by altered levels of metabolites, such as the sugars sucrose and fructose, glycolysis intermediates, and amino acids. Together, our study provides evidence for a link between early signaling in the salt and oxidative stress response that regulates the state transition of photosynthesis and the rearrangement of primary metabolism. PMID:26471895

  13. 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

    SciTech Connect

    Matsumura, Takayuki; Oyama, Masaaki; Kozuka-Hata, Hiroko; Ishikawa, Kosuke; Inoue, Takafumi; Muta, Tatsushi; Semba, Kentaro; Inoue, Jun-ichiro

    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.

  14. Dust control for Enabler

    NASA Technical Reports Server (NTRS)

    Hilton, Kevin; Karl, Chad; Litherland, Mark; Ritchie, David; Sun, Nancy

    1992-01-01

    The dust control group designed a system to restrict dust that is disturbed by the Enabler during its operation from interfering with astronaut or camera visibility. This design also considers the many different wheel positions made possible through the use of artinuation joints that provide the steering and wheel pitching for the Enabler. The system uses a combination of brushes and fenders to restrict the dust when the vehicle is moving in either direction and in a turn. This design also allows for each of maintenance as well as accessibility of the remainder of the vehicle.

  15. Dust control for Enabler

    NASA Technical Reports Server (NTRS)

    Hilton, Kevin; Karl, Chad; Litherland, Mark; Ritchie, David; Sun, Nancy

    1992-01-01

    The dust control group designed a system to restrict dust that is disturbed by the Enabler during its operation from interfering with astronaut or camera visibility. This design also considers the many different wheel positions made possible through the use of artinuation joints that provide the steering and wheel pitching for the Enabler. The system uses a combination of brushes and fenders to restrict the dust when the vehicle is moving in either direction and in a turn. This design also allows for ease of maintenance as well as accessibility of the remainder of the vehicle.

  16. Microsystems Enabled Photovoltaics

    ScienceCinema

    Gupta, Vipin; Nielson, Greg; Okandan, Murat, Granata, Jennifer; Nelson, Jeff; Haney, Mike; Cruz-Campa, Jose Luiz

    2014-06-23

    Sandia's microsystems enabled photovoltaic advances combine mature technology and tools currently used in microsystem production with groundbreaking advances in photovoltaics cell design, decreasing production and system costs while improving energy conversion efficiency. The technology has potential applications in buildings, houses, clothing, portable electronics, vehicles, and other contoured structures.

  17. Microsystems Enabled Photovoltaics

    SciTech Connect

    Gupta, Vipin; Nielson, Greg; Okandan, Murat, Granata, Jennifer; Nelson, Jeff; Haney, Mike; Cruz-Campa, Jose Luiz

    2012-07-02

    Sandia's microsystems enabled photovoltaic advances combine mature technology and tools currently used in microsystem production with groundbreaking advances in photovoltaics cell design, decreasing production and system costs while improving energy conversion efficiency. The technology has potential applications in buildings, houses, clothing, portable electronics, vehicles, and other contoured structures.

  18. Human embryonic stem cell phosphoproteome revealed by electron transfer dissociation tandem mass spectrometry

    PubMed Central

    Swaney, Danielle L.; Wenger, Craig D.; Thomson, James A.; Coon, Joshua J.

    2009-01-01

    Protein phosphorylation is central to the understanding of cellular signaling, and cellular signaling is suggested to play a major role in the regulation of human embryonic stem (ES) cell pluripotency. Here, we describe the use of conventional tandem mass spectrometry-based sequencing technology—collision-activated dissociation (CAD)—and the more recently developed method electron transfer dissociation (ETD) to characterize the human ES cell phosphoproteome. In total, these experiments resulted in the identification of 11,995 unique phosphopeptides, corresponding to 10,844 nonredundant phosphorylation sites, at a 1% false discovery rate (FDR). Among these phosphorylation sites are 5 localized to 2 pluripotency critical transcription factors—OCT4 and SOX2. From these experiments, we conclude that ETD identifies a larger number of unique phosphopeptides than CAD (8,087 to 3,868), more frequently localizes the phosphorylation site to a specific residue (49.8% compared with 29.6%), and sequences whole classes of phosphopeptides previously unobserved. PMID:19144917

  19. The proteome and phosphoproteome of Neurospora crassa in response to cellulose, sucrose and carbon starvation

    SciTech Connect

    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

    2014-05-29

    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 versus sucrose medium were identified, including phosphorylation sites in a major transcriptional activator for cellulase genes, CLR1, as well as a cellobionic acid transporter, CBT1. Finally, we found 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.

  20. Site-Specific Ser/Thr/Tyr Phosphoproteome of Sinorhizobium meliloti at Stationary Phase

    PubMed Central

    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. PMID:26401955

  1. The proteome and phosphoproteome of Neurospora crassa in response to cellulose, sucrose and carbon starvation

    DOE PAGESBeta

    Xiong, Yi; Coradetti, Samuel T.; Li, Xin; Gritsenko, Marina A.; Clauss, Therese; Petyuk, Vlad; Camp, David; Smith, Richard; Cate, Jamie H. D.; Yang, Feng; et al

    2014-05-29

    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 suggestsmore » 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 versus sucrose medium were identified, including phosphorylation sites in a major transcriptional activator for cellulase genes, CLR1, as well as a cellobionic acid transporter, CBT1. Finally, we found 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.« less

  2. A solid phase extraction-based platform for rapid phosphoproteomic analysis

    PubMed Central

    Dephoure, Noah; Gygi, Steven P.

    2011-01-01

    Protein phosphorylation is among the most common and intensely studied post-translational protein modification. It plays crucial roles in virtually all cellular processes and has been implicated in numerous human diseases, including cancer. Traditional biochemical and genetic methods for identifying and monitoring sites of phosphorylation are laborious and slow and in recent years have largely been replaced by mass spectrometric analysis. Improved methods for phosphopeptide enrichment coupled with faster and more sensitive mass spectrometers have led to an explosion in the size of phosphoproteomic datasets. However, wider application of these methods is limited by equipment costs and the resultant high demand for instrument time as well as by a technology gap between biologists and mass spectrometrists. Here we describe a modified two-step enrichment strategy that employs lysC digestion and step elution from self-packed strong cation exchange (SCX) solid phase extraction (SPE) columns followed by immobilized metal ion affinity chromatography (IMAC) and LC-MS/MS analysis using a hybrid LTQ Orbitrap Velos mass spectrometer. The SCX procedure does not require an HPLC system, demands little expertise, and because multiple samples can be processed in parallel, can provide a large savings of time and labor. We demonstrate this method in conjunction with stable isotope labeling to quantify peptides harboring >8,000 unique phosphorylation sites in yeast in 12 hours of instrument analysis time and examine the impact of enzyme choice and instrument platform. PMID:21440633

  3. Identification of in vivo protein phosphorylation sites in human pathogen Schistosoma japonicum by a phosphoproteomic approach.

    PubMed

    Luo, Rong; Zhou, Chunjing; Lin, Jiaojiao; Yang, Dehao; Shi, Yaojun; Cheng, Guofeng

    2012-01-01

    Schistosome is the causative agent of human schistosomiasis and related animal disease. Reversible protein phosphorylation plays a key role in signaling processing that are vital for a cell and organism. However, it remains to be undercharacterized in schistosomes. In the present study, we characterized in vivo protein phosphorylation events in different developmental stages (schistosomula and adult worms) of Schistosoma japonicum by using microvolume immobilized metal-ion affinity chromatography (IMAC) pipette tips coupled to nanoLC-ESI-MS/MS. In total, 127 distinct phosphorylation sites were identified in 92 proteins in S. japonicum. A comparison of the phosphopeptides identified between the schistosomula and the adult worms revealed 30 phosphoproteins co-detected in both of the two worms. These proteins included several signal molecules and enzymes such as 14-3-3 protein, cysteine string protein, heat shock protein 90, epidermal growth factor receptor pathway substrate 8, proliferation-associated protein 2G4, peptidyl-prolyl isomerase G, phosphofructokinase and thymidylate kinase. Additionally, the phosphorylation sites were examined for phosphorylation specific motif and evolutionarily conservation. The study represents the first attempt to determine in vivo protein phosphorylation in S. japonicum by using a phosphoproteomic approach. The results by providing an inventory of phosphorylated proteins may facilitate to further understand the mechanisms involved in schistosome development and growth, and then may result in the development of novel vaccine candidates and drug targets for schistosomiasis control. PMID:22036931

  4. The proteome and phosphoproteome of Neurospora crassa in response to cellulose, sucrose and carbon starvation

    SciTech Connect

    Xiong, Yi; Coradetti, Samuel T.; Li, Xin; Gritsenko, Marina A.; Clauss, Therese RW; Petyuk, Vladislav A.; Camp, David G.; Smith, Richard D.; Cate, Jamie H.; Yang, Feng; Glass, 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 versus 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.

  5. Phosphoproteome and Transcriptome of RA-Responsive and RA-Resistant Breast Cancer Cell Lines

    PubMed Central

    Carrier, Marilyn; Joint, Mathilde; Lutzing, Régis; Page, Adeline; Rochette-Egly, Cécile

    2016-01-01

    Retinoic acid (RA), the main active vitamin A metabolite, controls multiple biological processes such as cell proliferation and differentiation through genomic programs and kinase cascades activation. Due to these properties, RA has proven anti-cancer capacity. Several breast cancer cells respond to the antiproliferative effects of RA, while others are RA-resistant. However, the overall signaling and transcriptional pathways that are altered in such cells have not been elucidated. Here, in a large-scale analysis of the phosphoproteins and in a genome-wide analysis of the RA-regulated genes, we compared two human breast cancer cell lines, a RA-responsive one, the MCF7 cell line, and a RA-resistant one, the BT474 cell line, which depicts several alterations of the “kinome”. Using high-resolution nano-LC-LTQ-Orbitrap mass spectrometry associated to phosphopeptide enrichment, we found that several proteins involved in signaling and in transcription, are differentially phosphorylated before and after RA addition. The paradigm of these proteins is the RA receptor α (RARα), which was phosphorylated in MCF7 cells but not in BT474 cells after RA addition. The panel of the RA-regulated genes was also different. Overall our results indicate that RA resistance might correlate with the deregulation of the phosphoproteome with consequences on gene expression. PMID:27362937

  6. Phosphoproteomics reveals that Parkinson's disease kinase LRRK2 regulates a subset of Rab GTPases

    PubMed Central

    Steger, Martin; Tonelli, Francesca; Ito, Genta; Davies, Paul; Trost, Matthias; Vetter, Melanie; Wachter, Stefanie; Lorentzen, Esben; Duddy, Graham; Wilson, Stephen; Baptista, Marco AS; Fiske, Brian K; Fell, Matthew J; Morrow, John A; Reith, Alastair D; Alessi, Dario R; Mann, Matthias

    2016-01-01

    Mutations in Park8, encoding for the multidomain Leucine-rich repeat kinase 2 (LRRK2) protein, comprise the predominant genetic cause of Parkinson's disease (PD). G2019S, the most common amino acid substitution activates the kinase two- to threefold. This has motivated the development of LRRK2 kinase inhibitors; however, poor consensus on physiological LRRK2 substrates has hampered clinical development of such therapeutics. We employ a combination of phosphoproteomics, genetics, and pharmacology to unambiguously identify a subset of Rab GTPases as key LRRK2 substrates. LRRK2 directly phosphorylates these both in vivo and in vitro on an evolutionary conserved residue in the switch II domain. Pathogenic LRRK2 variants mapping to different functional domains increase phosphorylation of Rabs and this strongly decreases their affinity to regulatory proteins including Rab GDP dissociation inhibitors (GDIs). Our findings uncover a key class of bona-fide LRRK2 substrates and a novel regulatory mechanism of Rabs that connects them to PD. DOI: http://dx.doi.org/10.7554/eLife.12813.001 PMID:26824392

  7. The proteome and phosphoproteome of Neurospora crassa in response to cellulose, sucrose and carbon starvation.

    PubMed

    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. PMID:24881580

  8. Phosphoproteomics reveals that Parkinson's disease kinase LRRK2 regulates a subset of Rab GTPases.

    PubMed

    Steger, Martin; Tonelli, Francesca; Ito, Genta; Davies, Paul; Trost, Matthias; Vetter, Melanie; Wachter, Stefanie; Lorentzen, Esben; Duddy, Graham; Wilson, Stephen; Baptista, Marco As; Fiske, Brian K; Fell, Matthew J; Morrow, John A; Reith, Alastair D; Alessi, Dario R; Mann, Matthias

    2016-01-01

    Mutations in Park8, encoding for the multidomain Leucine-rich repeat kinase 2 (LRRK2) protein, comprise the predominant genetic cause of Parkinson's disease (PD). G2019S, the most common amino acid substitution activates the kinase two- to threefold. This has motivated the development of LRRK2 kinase inhibitors; however, poor consensus on physiological LRRK2 substrates has hampered clinical development of such therapeutics. We employ a combination of phosphoproteomics, genetics, and pharmacology to unambiguously identify a subset of Rab GTPases as key LRRK2 substrates. LRRK2 directly phosphorylates these both in vivo and in vitro on an evolutionary conserved residue in the switch II domain. Pathogenic LRRK2 variants mapping to different functional domains increase phosphorylation of Rabs and this strongly decreases their affinity to regulatory proteins including Rab GDP dissociation inhibitors (GDIs). Our findings uncover a key class of bona-fide LRRK2 substrates and a novel regulatory mechanism of Rabs that connects them to PD. PMID:26824392

  9. Phosphoproteomics Identified an NS5A Phosphorylation Site Involved in Hepatitis C Virus Replication.

    PubMed

    Chong, Weng Man; Hsu, Shih-Chin; Kao, Wei-Ting; Lo, Chieh-Wen; Lee, Kuan-Ying; Shao, Jheng-Syuan; Chen, Yi-Hung; Chang, Justin; Chen, Steve S-L; Yu, Ming-Jiun

    2016-02-19

    The non-structural protein 5A (NS5A) is a hepatitis C virus (HCV) protein indispensable for the viral life cycle. Many prior papers have pinpointed several serine residues in the low complexity sequence I region of NS5A responsible for NS5A phosphorylation; however, the functions of specific phosphorylation sites remained obscure. Using phosphoproteomics, we identified three phosphorylation sites (serines 222, 235, and 238) in the NS5A low complexity sequence I region. Reporter virus and replicon assays using phosphorylation-ablated alanine mutants of these sites showed that Ser-235 dominated over Ser-222 and Ser-238 in HCV replication. Immunoblotting using an Ser-235 phosphorylation-specific antibody showed a time-dependent increase in Ser-235 phosphorylation that correlated with the viral replication activity. Ser-235 phosphorylated NS5A co-localized with double-stranded RNA, consistent with its role in HCV replication. Mechanistically, Ser-235 phosphorylation probably promotes the replication complex formation via increasing NS5A interaction with the human homologue of the 33-kDa vesicle-associated membrane protein-associated protein. Casein kinase Iα (CKIα) directly phosphorylated Ser-235 in vitro. Inhibition of CKIα reduced Ser-235 phosphorylation and the HCV RNA levels in the infected cells. We concluded that NS5A Ser-235 phosphorylated by CKIα probably promotes HCV replication via increasing NS5A interaction with the 33-kDa vesicle-associated membrane protein-associated protein. PMID:26702051

  10. Phosphoproteome profiling using a fluorescent phosphosensor dye in two-dimensional polyacrylamide gel electrophoresis.

    PubMed

    Otani, Mieko; Taniguchi, Taizo; Sakai, Akiko; Seta, Jouji; Kadoyama, Keiichi; Nakamura-Hirota, Tooru; Matsuyama, Shogo; Sano, Keiji; Takano, Masaoki

    2011-07-01

    We validated the novel PhosphoQUANTI SolidBlue Complex (PQSC) dye for the sensitive fluorescent detection of phosphorylated proteins in polyacrylamide- and two-dimensional gel electrophoresis (PAGE and 2DE, respectively). PQSC can detect as little as 15.6 ng of ß-casein, a pentaphosphorylated protein, and 61.3 ng of ovalbumin, a diphosphorylated protein. Fluorescence intensity correlates with the number of phosphorylated residues on the protein. To demonstrate the specificity of PQSC for phosphoproteins, enzymatically dephosphorylated lysates of Swiss 3T3 cells were separated in 2DE gels and stained by PQSC. The fluorescence signals in these gels were markedly reduced following dephosphorylation. When the phosphorylated proteins in Swiss 3T3 cell lysates were concentrated using a phosphoprotein enrichment column, the majority of phosphoproteins showed fluorescence signals in the pI 4-5 range. Finally, we performed phosphoproteome analysis to study differences in the protein phosphorylation profiles of proliferating and quiescent Swiss 3T3 cells. Over 135 discernible protein spots were detected, from which a selection of 15 spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF-MS). The PQSC staining procedure for phosphoprotein detection is simple, reversible, and fully compatible with MALDI TOF-MS. PMID:21384102

  11. The proteome and phosphoproteome of Neurospora crassa in response to cellulose, sucrose and carbon starvation

    PubMed Central

    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

    2014-01-01

    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. PMID:24881580

  12. Plasmodiumfalciparum infection induces dynamic changes in the erythrocyte phospho-proteome.

    PubMed

    Bouyer, Guillaume; Reininger, Luc; Ramdani, Ghania; D Phillips, Lee; Sharma, Vikram; Egee, Stephane; Langsley, Gordon; Lasonder, Edwin

    2016-05-01

    The phosphorylation status of red blood cell proteins is strongly altered during the infection by the malaria parasite Plasmodium falciparum. We identify the key phosphorylation events that occur in the erythrocyte membrane and cytoskeleton during infection, by a comparative analysis of global phospho-proteome screens between infected (obtained at schizont stage) and uninfected RBCs. The meta-analysis of reported mass spectrometry studies revealed a novel compendium of 495 phosphorylation sites in 182 human proteins with regulatory roles in red cell morphology and stability, with about 25% of these sites specific to infected cells. A phosphorylation motif analysis detected 7 unique motifs that were largely mapped to kinase consensus sequences of casein kinase II and of protein kinase A/protein kinase C. This analysis highlighted prominent roles for PKA/PKC involving 78 phosphorylation sites. We then compared the phosphorylation status of PKA (PKC) specific sites in adducin, dematin, Band 3 and GLUT-1 in uninfected RBC stimulated or not by cAMP to their phosphorylation status in iRBC. We showed cAMP-induced phosphorylation of adducin S59 by immunoblotting and we were able to demonstrate parasite-induced phosphorylation for adducin S726, Band 3 and GLUT-1, corroborating the protein phosphorylation status in our erythrocyte phosphorylation site compendium. PMID:27067487

  13. Analytical bioconjugates, aptamers, enable specific quantitative detection of Listeria monocytogenes.

    PubMed

    Lee, Sang-Hee; Ahn, Ji-Young; Lee, Kyeong-Ah; Um, Hyun-Ju; Sekhon, Simranjeet Singh; Sun Park, Tae; Min, Jiho; Kim, Yang-Hoon

    2015-06-15

    As a major human pathogen in the Listeria genus, Listeria monocytogenes causes the bacterial disease listeriosis, which is a serious infection caused by eating food contaminated with the bacteria. We have developed an aptamer-based sandwich assay (ABSA) platform that demonstrates a promising potential for use in pathogen detection using aptamers as analytical bioconjugates. The whole-bacteria SELEX (WB-SELEX) strategy was adopted to generate aptamers with high affinity and specificity against live L. monocytogenes. Of the 35 aptamer candidates tested, LMCA2 and LMCA26 reacted to L. monocytogenes with high binding, and were consequently chosen as sensing probes. The ABSA platform can significantly enhance the sensitivity by employing a very specific aptamer pair for the sandwich complex. The ABSA platform exhibited a linear response over a wide concentration range of L. monocytogenes from 20 to 2×10(6) CFU per mL and was closely correlated with the following relationship: y=9533.3x+1542.3 (R(2)=0.99). Our proposed ABSA platform also provided excellent specificity for the tests to distinguish L. monocytogenes from other Listeria species and other bacterial genera (3 Listeria spp., 4 Salmonella spp., 2 Vibrio spp., 3 Escherichia coli and 3 Shigella spp.). Improvements in the sensitivity and specificity have not only facilitated the reliable detection of L. monocytogenes at extremely low concentrations, but also allowed for the development of a 96-well plate-based routine assay platform for multivalent diagnostics. PMID:25590973

  14. Liquid metal enabled pump

    PubMed Central

    Tang, Shi-Yang; Khoshmanesh, Khashayar; Sivan, Vijay; Petersen, Phred; O’Mullane, Anthony P.; Abbott, Derek; Mitchell, Arnan; Kalantar-zadeh, Kourosh

    2014-01-01

    Small-scale pumps will be the heartbeat of many future micro/nanoscale platforms. However, the integration of small-scale pumps is presently hampered by limited flow rate with respect to the input power, and their rather complicated fabrication processes. These issues arise as many conventional pumping effects require intricate moving elements. Here, we demonstrate a system that we call the liquid metal enabled pump, for driving a range of liquids without mechanical moving parts, upon the application of modest electric field. This pump incorporates a droplet of liquid metal, which induces liquid flow at high flow rates, yet with exceptionally low power consumption by electrowetting/deelectrowetting at the metal surface. We present theory explaining this pumping mechanism and show that the operation is fundamentally different from other existing pumps. The presented liquid metal enabled pump is both efficient and simple, and thus has the potential to fundamentally advance the field of microfluidics. PMID:24550485

  15. Enabling Wind Power Nationwide

    SciTech Connect

    Jose, Zayas; Michael, Derby; Patrick, Gilman; Ananthan, Shreyas; Lantz, Eric; Cotrell, Jason; Beck, Fredic; Tusing, Richard

    2015-05-01

    Leveraging this experience, the U.S. Department of Energy’s (DOE’s) Wind and Water Power Technologies Office has evaluated the potential for wind power to generate electricity in all 50 states. This report analyzes and quantifies the geographic expansion that could be enabled by accessing higher above ground heights for wind turbines and considers the means by which this new potential could be responsibly developed.

  16. Bedrails: restraints or enablers?

    PubMed

    Mullette, Betty; Zulkowski, Karen

    2004-08-01

    Bedrails presently are used as both mobility restraints and enablers in long-term care facilities. As enablers, bedrails facilitate movement and may reduce the risk of pressure ulcer development. As restraints, they impede movement and may increase risk of ulcer development. Omnibus Budget Reconciliation Act regulations on restraint use have led to confusion for state Medicare surveyors and facilities regarding the definition of appropriate bedrail use and need for supportive documentation. Consequently, some facilities receive deficiency citations for inappropriate use or documentation while others do not. The purpose of this survey was to compare responses of Directors of Nursing in long-term care facilities and Medicare state surveyors to determine how each interprets the Omnibus Budget Reconciliation Act bedrail language for use and documentation. Questionnaires on bedrail use and documentation were sent to state surveyors and Directors of Nursing. One hundred, three (103) Directors of Nursing in 45 states and 65 surveyors from 39 states participated in the survey (response rate 61%). Study results demonstrated general acceptance of bedrail use as an enabler but not as a restraint by both Directors of Nursing and state surveyors. Four percent (4%) of Directors of Nursing reported receiving a citation for bedrail use and 59% of surveyors reported issuing citations for bedrail use. Significant differences were noted between the two groups regarding appropriate bedrail use and necessary documentation. The intent of Medicare guidelines and the Centers for Medicare and Medicaid Services is to standardize care for nursing home residents in the United States; yet, current regulations are open to individual interpretation by state surveyors and confusion exists between the intent of the Omnibus Budget Reconciliation Act and the daily operations of nursing homes. Educating clinicians about the risks and benefits of bedrail use, either as restraint or enabler, and

  17. iPhos: a toolkit to streamline the alkaline phosphatase-assisted comprehensive LC-MS phosphoproteome investigation

    PubMed Central

    2014-01-01

    Background Comprehensive characterization of the phosphoproteome in living cells is critical in signal transduction research. But the low abundance of phosphopeptides among the total proteome in cells remains an obstacle in mass spectrometry-based proteomic analysis. To provide a solution, an alternative analytic strategy to confidently identify phosphorylated peptides by using the alkaline phosphatase (AP) treatment combined with high-resolution mass spectrometry was provided. While the process is applicable, the key integration along the pipeline was mostly done by tedious manual work. Results We developed a software toolkit, iPhos, to facilitate and streamline the work-flow of AP-assisted phosphoproteome characterization. The iPhos tookit includes one assister and three modules. The iPhos Peak Extraction Assister automates the batch mode peak extraction for multiple liquid chromatography mass spectrometry (LC-MS) runs. iPhos Module-1 can process the peak lists extracted from the LC-MS analyses derived from the original and dephosphorylated samples to mine out potential phosphorylated peptide signals based on mass shift caused by the loss of some multiples of phosphate groups. And iPhos Module-2 provides customized inclusion lists with peak retention time windows for subsequent targeted LC-MS/MS experiments. Finally, iPhos Module-3 facilitates to link the peptide identifications from protein search engines to the quantification results from pattern-based label-free quantification tools. We further demonstrated the utility of the iPhos toolkit on the data of human metastatic lung cancer cells (CL1-5). Conclusions In the comparison study of the control group of CL1-5 cell lysates and the treatment group of dasatinib-treated CL1-5 cell lysates, we demonstrated the applicability of the iPhos toolkit and reported the experimental results based on the iPhos-facilitated phosphoproteome investigation. And further, we also compared the strategy with pure DDA-based LC

  18. Multidimensional strategy for sensitive phosphoproteomics incorporating protein prefractionation combined with SIMAC, HILIC, and TiO(2) chromatography applied to proximal EGF signaling.

    PubMed

    Engholm-Keller, Kasper; Hansen, Thomas Aarup; Palmisano, Giuseppe; Larsen, Martin R

    2011-12-01

    Comprehensive enrichment and fractionation is essential to obtain a broad coverage of the phosphoproteome. This inevitably leads to sample loss, and thus, phosphoproteomics studies are usually only performed on highly abundant samples. Here, we present a comprehensive phosphoproteomics strategy applied to 400 μg of protein from EGF-stimulated HeLa cells. The proteins are separated into membrane and cytoplasmic fractions using sodium carbonate combined with ultracentrifugation. The phosphopeptides were separated into monophosphorylated and multiphosphorylated pools using sequential elution from IMAC (SIMAC) followed by hydrophilic interaction liquid chromatography of the mono- and nonphosphorylated peptides and subsequent titanium dioxide chromatography of the HILIC fractions. This strategy facilitated the identification of >4700 unique phosphopeptides, while 636 phosphosites were changing following short-term EGF stimulation, many of which were not previously known to be involved in EGFR signaling. We further compared three different data processing programs and found large differences in their peptide identification rates due to different implementations of recalibration and filtering. Manually validating a subset of low-scoring peptides exclusively identified using the MaxQuant software revealed a large percentage of false positive identifications. This indicates that, despite having highly accurate precursor mass determination, peptides with low fragment ion scores should not automatically be reported in phosphoproteomics studies. PMID:21955146

  19. Immobilized metal affinity chromatography on collapsed Langmuir-Blodgett iron(III) stearate films and iron(III) oxide nanoparticles for bottom-up phosphoproteomics.

    PubMed

    Gladilovich, Vladimir; Greifenhagen, Uta; Sukhodolov, Nikolai; Selyutin, Artem; Singer, David; Thieme, Domenika; Majovsky, Petra; Shirkin, Alexey; Hoehenwarter, Wolfgang; Bonitenko, Evgeny; Podolskaya, Ekaterina; Frolov, Andrej

    2016-04-22

    Phosphorylation is the enzymatic reaction of site-specific phosphate transfer from energy-rich donors to the side chains of serine, threonine, tyrosine, and histidine residues in proteins. In living cells, reversible phosphorylation underlies a universal mechanism of intracellular signal transduction. In this context, analysis of the phosphoproteome is a prerequisite to better understand the cellular regulatory networks. Conventionally, due to the low contents of signaling proteins, selective enrichment of proteolytic phosphopeptides by immobilized metal affinity chromatography (IMAC) is performed prior to their LC-MS or -MS/MS analysis. Unfortunately, this technique still suffers from low selectivity and compromised analyte recoveries. To overcome these limitations, we propose IMAC systems comprising stationary phases based on collapsed Langmuir-Blodgett films of iron(III) stearate (FF) or iron(III) oxide nanoparticles (FO) and mobile phases relying on ammonia, piperidine and heptadecafluorooctanesulfonic acid (PFOS). Experiments with model phosphopeptides and phosphoprotein tryptic digests showed superior binding capacity, selectivity and recovery for both systems in comparison to the existing commercial analogs. As evidenced by LC-MS/MS analysis of the HeLa phosphoproteome, these features of the phases resulted in increased phosphoproteome coverage in comparison to the analogous commercially available phases, indicating that our IMAC protocol is a promising chromatographic tool for in-depth phosphoproteomic research. PMID:27016113

  20. Smart Grid Enabled EVSE

    SciTech Connect

    None, None

    2014-10-15

    The combined team of GE Global Research, Federal Express, National Renewable Energy Laboratory, and Consolidated Edison has successfully achieved the established goals contained within the Department of Energy’s Smart Grid Capable Electric Vehicle Supply Equipment funding opportunity. The final program product, shown charging two vehicles in Figure 1, reduces by nearly 50% the total installed system cost of the electric vehicle supply equipment (EVSE) as well as enabling a host of new Smart Grid enabled features. These include bi-directional communications, load control, utility message exchange and transaction management information. Using the new charging system, Utilities or energy service providers will now be able to monitor transportation related electrical loads on their distribution networks, send load control commands or preferences to individual systems, and then see measured responses. Installation owners will be able to authorize usage of the stations, monitor operations, and optimally control their electricity consumption. These features and cost reductions have been developed through a total system design solution.

  1. Enable, mediate, advocate.

    PubMed

    Saan, Hans; Wise, Marilyn

    2011-12-01

    The authors of the Ottawa Charter selected the words enable, mediate and advocate to describe the core activities in what was, in 1986, the new Public Health. This article considers these concepts and the values and ideas upon which they were based. We discuss their relevance in the current context within which health promotion is being conducted, and discuss the implications of changes in the health agenda, media and globalization for practice. We consider developments within health promotion since 1986: its central role in policy rhetoric, the increasing understanding of complexities and the interlinkage with many other societal processes. So the three core activities are reviewed: they still fit well with the main health promotion challenges, but should be refreshed by new ideas and values. As the role of health promotion in the political arena has grown we have become part of the policy establishment and that is a mixed blessing. Making way for community advocates is now our challenge. Enabling requires greater sensitivity to power relations involved and an understanding of the role of health literacy. Mediating keeps its central role as it bridges vital interests of parties. We conclude that these core concepts in the Ottawa Charter need no serious revision. There are, however, lessons from the last 25 years that point to ways to address present and future challenges with greater sensitivity and effectiveness. We invite the next generation to avoid canonizing this text: as is true of every heritage, the heirs must decide on its use. PMID:22080073

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

    SciTech Connect

    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.

  3. Phosphoproteomic Approach to Characterize Protein Mono- and Poly(ADP-ribosyl)ation Sites from Cells

    PubMed Central

    2015-01-01

    Poly(ADP-ribose), or PAR, is a cellular polymer implicated in DNA/RNA metabolism, cell death, and cellular stress response via its role as a post-translational modification, signaling molecule, and scaffolding element. PAR is synthesized by a family of proteins known as poly(ADP-ribose) polymerases, or PARPs, which attach PAR polymers to various amino acids of substrate proteins. The nature of these polymers (large, charged, heterogeneous, base-labile) has made these attachment sites difficult to study by mass spectrometry. Here we propose a new pipeline that allows for the identification of mono(ADP-ribosyl)ation and poly(ADP-ribosyl)ation sites via the enzymatic product of phosphodiesterase-treated ADP-ribose, or phospho(ribose). The power of this method lies in the enrichment potential of phospho(ribose), which we show to be enriched by phosphoproteomic techniques when a neutral buffer, which allows for retention of the base-labile attachment site, is used for elution. Through the identification of PARP-1 in vitro automodification sites as well as endogenous ADP-ribosylation sites from whole cells, we have shown that ADP-ribose can exist on adjacent amino acid residues as well as both lysine and arginine in addition to known acidic modification sites. The universality of this technique has allowed us to show that enrichment of ADP-ribosylated proteins by macrodomain leads to a bias against ADP-ribose modifications conjugated to glutamic acids, suggesting that the macrodomain is either removing or selecting against these distinct protein attachments. Ultimately, the enrichment pipeline presented here offers a universal approach for characterizing the mono- and poly(ADP-ribosyl)ated proteome. PMID:24920161

  4. Use of 32P to Study Dynamics of the Mitochondrial Phosphoproteome

    PubMed Central

    Aponte, Angel M.; Phillips, Darci; Hopper, Rachel K.; Johnson, D. Thor; Harris, Robert A.; Blinova, Ksenia; Boja, Emily S.; French, Stephanie; Balaban, Robert S.

    2009-01-01

    Protein phosphorylation is a well characterized regulatory mechanism in the cytosol, but remains poorly defined in the mitochondrion. In this study, we characterized the use of 32P-labeling to monitor the turnover of protein phosphorylation in the heart and liver mitochondria matrix. The 32P labeling technique was compared and contrasted to Phos-tag protein phosphorylation fluorescent stain and 2D isoelectric focusing. Of the 64 proteins identified by MS spectroscopy in the Phos-Tag gels, over 20 proteins were correlated with 32P labeling. The high sensitivity of 32P incorporation detected proteins well below the mass spectrometry and even 2D gel protein detection limits. Phosphate-chase experiments revealed both turnover and phosphate associated protein pool size alterations dependent on initial incubation conditions. Extensive weak phosphate/phosphate metabolite interactions were observed using non-disruptive native gels, providing a novel approach to screen for potential allosteric interactions of phosphate metabolites with matrix proteins. We confirmed the phosphate associations in Complexes V and I due to their critical role in oxidative phosphorylation and to validate the 2D methods. These complexes were isolated by immunocapture, after 32P labeling in the intact mitochondria, and revealed 32P-incorporation for the α, β, γ, OSCP, and d subunits in Complex V and the 75kDa, 51kDa, 42kDa, 23kDa, and 13a kDa subunits in Complex I. These results demonstrate that a dynamic and extensive mitochondrial matrix phosphoproteome exists in heart and liver. PMID:19351177

  5. Salt-Induced Changes in Cardiac Phosphoproteome in a Rat Model of Chronic Renal Failure

    PubMed Central

    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. PMID:24945867

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

    PubMed Central

    Højlund, Kurt; Bowen, Benjamin P.; Hwang, Hyonson; Flynn, Charles R.; Madireddy, Lohith; Thangiah, Geetha; Langlais, Paul; Meyer, Christian; Mandarino, Lawrence J.; Yi, Zhengping

    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 volunteers. Trypsin digestion of 3-5 mg human skeletal muscle protein was followed by phosphopeptide enrichment using SCX and TiO2. The resulting phosphopeptides were analyzed by HPLC-ESI-MS/MS. Using this unbiased approach, we identified 306 distinct in vivo phosphorylation sites in 127 proteins, including 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-contraction coupling (the Ca2+-cycle), glycolysis and glycogen metabolism. Of particular interest, we identified multiple novel phosphorylation sites on several sarcomeric Z-disc proteins known to be involved in signaling and muscle disorders. These results provide numerous new targets for the investigation of human skeletal muscle phosphoproteins in health and disease and demonstrate feasibility of phosphoproteomics research of human skeletal muscle in vivo. PMID:19764811

  7. Alterations in the Cerebellar (Phospho)Proteome of a Cyclic Guanosine Monophosphate (cGMP)-dependent Protein Kinase Knockout Mouse*

    PubMed Central

    Corradini, Eleonora; Vallur, Raghavan; Raaijmakers, Linsey M.; Feil, Susanne; Feil, Robert; Heck, Albert J. R.; Scholten, Arjen

    2014-01-01

    The cyclic nucleotide cyclic guanosine monophosphate (cGMP) plays an important role in learning and memory, but its signaling mechanisms in the mammalian brain are not fully understood. Using mass-spectrometry-based proteomics, we evaluated how the cerebellum adapts its (phospho)proteome in a knockout mouse model of cGMP-dependent protein kinase type I (cGKI). Our data reveal that a small subset of proteins in the cerebellum (∼3% of the quantified proteins) became substantially differentially expressed in the absence of cGKI. More changes were observed at the phosphoproteome level, with hundreds of sites being differentially phosphorylated between wild-type and knockout cerebellum. Most of these phosphorylated sites do not represent known cGKI substrates. An integrative computational network analysis of the data indicated that the differentially expressed proteins and proteins harboring differentially phosphorylated sites largely belong to a tight network in the Purkinje cells of the cerebellum involving important cGMP/cAMP signaling nodes (e.g. PDE5 and PKARIIβ) and Ca2+ signaling (e.g. SERCA3). In this way, removal of cGKI could be linked to impaired cerebellar long-term depression at Purkinje cell synapses. In addition, we were able to identify a set of novel putative (phospho)proteins to be considered in this network. Overall, our data improve our understanding of cerebellar cGKI signaling and suggest novel players in cGKI-regulated synaptic plasticity. PMID:24925903

  8. Biosynthesis and Regulation of Wheat Amylose and Amylopectin from Proteomic and Phosphoproteomic Characterization of Granule-binding Proteins

    PubMed Central

    Chen, Guan-Xing; Zhou, Jian-Wen; Liu, Yan-Lin; Lu, Xiao-Bing; Han, Cai-Xia; Zhang, Wen-Ying; Xu, Yan-Hao; Yan, Yue-Ming

    2016-01-01

    Waxy starch has an important influence on the qualities of breads. Generally, grain weight and yield in waxy wheat (Triticum aestivum L.) are significantly lower than in bread wheat. In this study, we performed the first proteomic and phosphoproteomic analyses of starch granule-binding proteins by comparing the waxy wheat cultivar Shannong 119 and the bread wheat cultivar Nongda 5181. These results indicate that reduced amylose content does not affect amylopectin synthesis, but it causes significant reduction of total starch biosynthesis, grain size, weight and grain yield. Two-dimensional differential in-gel electrophoresis identified 40 differentially expressed protein (DEP) spots in waxy and non-waxy wheats, which belonged mainly to starch synthase (SS) I, SS IIa and granule-bound SS I. Most DEPs involved in amylopectin synthesis showed a similar expression pattern during grain development, suggesting relatively independent amylose and amylopectin synthesis pathways. Phosphoproteome analysis of starch granule-binding proteins, using TiO2 microcolumns and LC-MS/MS, showed that the total number of phosphoproteins and their phosphorylation levels in ND5181 were significantly higher than in SN119, but proteins controlling amylopectin synthesis had similar phosphorylation levels. Our results revealed the lack of amylose did not affect the expression and phosphorylation of the starch granule-binding proteins involved in amylopectin biosynthesis. PMID:27604546

  9. Biosynthesis and Regulation of Wheat Amylose and Amylopectin from Proteomic and Phosphoproteomic Characterization of Granule-binding Proteins.

    PubMed

    Chen, Guan-Xing; Zhou, Jian-Wen; Liu, Yan-Lin; Lu, Xiao-Bing; Han, Cai-Xia; Zhang, Wen-Ying; Xu, Yan-Hao; Yan, Yue-Ming

    2016-01-01

    Waxy starch has an important influence on the qualities of breads. Generally, grain weight and yield in waxy wheat (Triticum aestivum L.) are significantly lower than in bread wheat. In this study, we performed the first proteomic and phosphoproteomic analyses of starch granule-binding proteins by comparing the waxy wheat cultivar Shannong 119 and the bread wheat cultivar Nongda 5181. These results indicate that reduced amylose content does not affect amylopectin synthesis, but it causes significant reduction of total starch biosynthesis, grain size, weight and grain yield. Two-dimensional differential in-gel electrophoresis identified 40 differentially expressed protein (DEP) spots in waxy and non-waxy wheats, which belonged mainly to starch synthase (SS) I, SS IIa and granule-bound SS I. Most DEPs involved in amylopectin synthesis showed a similar expression pattern during grain development, suggesting relatively independent amylose and amylopectin synthesis pathways. Phosphoproteome analysis of starch granule-binding proteins, using TiO2 microcolumns and LC-MS/MS, showed that the total number of phosphoproteins and their phosphorylation levels in ND5181 were significantly higher than in SN119, but proteins controlling amylopectin synthesis had similar phosphorylation levels. Our results revealed the lack of amylose did not affect the expression and phosphorylation of the starch granule-binding proteins involved in amylopectin biosynthesis. PMID:27604546

  10. Meta-Analysis of Arabidopsis thaliana Phospho-Proteomics Data Reveals Compartmentalization of Phosphorylation Motifs[C][W

    PubMed Central

    van Wijk, Klaas J.; Friso, Giulia; Walther, Dirk; Schulze, Waltraud X.

    2014-01-01

    Protein (de)phosphorylation plays an important role in plants. To provide a robust foundation for subcellular phosphorylation signaling network analysis and kinase-substrate relationships, we performed a meta-analysis of 27 published and unpublished in-house mass spectrometry–based phospho-proteome data sets for Arabidopsis thaliana covering a range of processes, (non)photosynthetic tissue types, and cell cultures. This resulted in an assembly of 60,366 phospho-peptides matching to 8141 nonredundant proteins. Filtering the data for quality and consistency generated a set of medium and a set of high confidence phospho-proteins and their assigned phospho-sites. The relation between single and multiphosphorylated peptides is discussed. The distribution of p-proteins across cellular functions and subcellular compartments was determined and showed overrepresentation of protein kinases. Extensive differences in frequency of pY were found between individual studies due to proteomics and mass spectrometry workflows. Interestingly, pY was underrepresented in peroxisomes but overrepresented in mitochondria. Using motif-finding algorithms motif-x and MMFPh at high stringency, we identified compartmentalization of phosphorylation motifs likely reflecting localized kinase activity. The filtering of the data assembly improved signal/noise ratio for such motifs. Identified motifs were linked to kinases through (bioinformatic) enrichment analysis. This study also provides insight into the challenges/pitfalls of using large-scale phospho-proteomic data sets to nonexperts. PMID:24894044

  11. Enabling graphene nanoelectronics.

    SciTech Connect

    Pan, Wei; Ohta, Taisuke; Biedermann, Laura Butler; Gutierrez, Carlos; Nolen, C. M.; Howell, Stephen Wayne; Beechem Iii, Thomas Edwin; McCarty, Kevin F.; Ross, Anthony Joseph, III

    2011-09-01

    Recent work has shown that graphene, a 2D electronic material amenable to the planar semiconductor fabrication processing, possesses tunable electronic material properties potentially far superior to metals and other standard semiconductors. Despite its phenomenal electronic properties, focused research is still required to develop techniques for depositing and synthesizing graphene over large areas, thereby enabling the reproducible mass-fabrication of graphene-based devices. To address these issues, we combined an array of growth approaches and characterization resources to investigate several innovative and synergistic approaches for the synthesis of high quality graphene films on technologically relevant substrate (SiC and metals). Our work focused on developing the fundamental scientific understanding necessary to generate large-area graphene films that exhibit highly uniform electronic properties and record carrier mobility, as well as developing techniques to transfer graphene onto other substrates.

  12. Enabling distributed petascale science.

    SciTech Connect

    Baranovski, A.; Bharathi, S.; Bresnahan, J.; chervenak, A.; Foster, I.; Fraser, D.; Freeman, T.; Gunter, D.; Jackson, K.; Keahey, K.; Kesselman, C.; Konerding, D. E.; Leroy, N.; Link, M.; Livny, M.; Miller, N.; Miller, R.; Oleynik, G.; Pearlman, L.; Schopf, J. M.; Schuler, R.; Tierney, B.; Mathematics and Computer Science; FNL; Univ. of Southern California; Univ. of Chicago; LBNL; Univ. of Wisconsin

    2007-01-01

    Petascale science is an end-to-end endeavour, involving not only the creation of massive datasets at supercomputers or experimental facilities, but the subsequent analysis of that data by a user community that may be distributed across many laboratories and universities. The new SciDAC Center for Enabling Distributed Petascale Science (CEDPS) is developing tools to support this end-to-end process. These tools include data placement services for the reliable, high-performance, secure, and policy-driven placement of data within a distributed science environment; tools and techniques for the construction, operation, and provisioning of scalable science services; and tools for the detection and diagnosis of failures in end-to-end data placement and distributed application hosting configurations. In each area, we build on a strong base of existing technology and have made useful progress in the first year of the project. For example, we have recently achieved order-of-magnitude improvements in transfer times (for lots of small files) and implemented asynchronous data staging capabilities; demonstrated dynamic deployment of complex application stacks for the STAR experiment; and designed and deployed end-to-end troubleshooting services. We look forward to working with SciDAC application and technology projects to realize the promise of petascale science.

  13. Enabling immersive simulation.

    SciTech Connect

    McCoy, Josh; Mateas, Michael; Hart, Derek H.; Whetzel, Jonathan; Basilico, Justin Derrick; Glickman, Matthew R.; Abbott, Robert G.

    2009-02-01

    The object of the 'Enabling Immersive Simulation for Complex Systems Analysis and Training' LDRD has been to research, design, and engineer a capability to develop simulations which (1) provide a rich, immersive interface for participation by real humans (exploiting existing high-performance game-engine technology wherever possible), and (2) can leverage Sandia's substantial investment in high-fidelity physical and cognitive models implemented in the Umbra simulation framework. We report here on these efforts. First, we describe the integration of Sandia's Umbra modular simulation framework with the open-source Delta3D game engine. Next, we report on Umbra's integration with Sandia's Cognitive Foundry, specifically to provide for learning behaviors for 'virtual teammates' directly from observed human behavior. Finally, we describe the integration of Delta3D with the ABL behavior engine, and report on research into establishing the theoretical framework that will be required to make use of tools like ABL to scale up to increasingly rich and realistic virtual characters.

  14. Grid-Enabled Measures

    PubMed Central

    Moser, Richard P.; Hesse, Bradford W.; Shaikh, Abdul R.; Courtney, Paul; Morgan, Glen; Augustson, Erik; Kobrin, Sarah; Levin, Kerry; Helba, Cynthia; Garner, David; Dunn, Marsha; Coa, Kisha

    2011-01-01

    Scientists are taking advantage of the Internet and collaborative web technology to accelerate discovery in a massively connected, participative environment —a phenomenon referred to by some as Science 2.0. As a new way of doing science, this phenomenon has the potential to push science forward in a more efficient manner than was previously possible. The Grid-Enabled Measures (GEM) database has been conceptualized as an instantiation of Science 2.0 principles by the National Cancer Institute with two overarching goals: (1) Promote the use of standardized measures, which are tied to theoretically based constructs; and (2) Facilitate the ability to share harmonized data resulting from the use of standardized measures. This is done by creating an online venue connected to the Cancer Biomedical Informatics Grid (caBIG®) where a virtual community of researchers can collaborate together and come to consensus on measures by rating, commenting and viewing meta-data about the measures and associated constructs. This paper will describe the web 2.0 principles on which the GEM database is based, describe its functionality, and discuss some of the important issues involved with creating the GEM database, such as the role of mutually agreed-on ontologies (i.e., knowledge categories and the relationships among these categories— for data sharing). PMID:21521586

  15. Enabling cleanup technology transfer.

    SciTech Connect

    Ditmars, J. D.

    2002-08-12

    Technology transfer in the environmental restoration, or cleanup, area has been challenging. While there is little doubt that innovative technologies are needed to reduce the times, risks, and costs associated with the cleanup of federal sites, particularly those of the Departments of Energy (DOE) and Defense, the use of such technologies in actual cleanups has been relatively limited. There are, of course, many reasons why technologies do not reach the implementation phase or do not get transferred from developing entities to the user community. For example, many past cleanup contracts provided few incentives for performance that would compel a contractor to seek improvement via technology applications. While performance-based contracts are becoming more common, they alone will not drive increased technology applications. This paper focuses on some applications of cleanup methodologies and technologies that have been successful and are illustrative of a more general principle. The principle is at once obvious and not widely practiced. It is that, with few exceptions, innovative cleanup technologies are rarely implemented successfully alone but rather are implemented in the context of enabling processes and methodologies. And, since cleanup is conducted in a regulatory environment, the stage is better set for technology transfer when the context includes substantive interactions with the relevant stakeholders. Examples of this principle are drawn from Argonne National Laboratory's experiences in Adaptive Sampling and Analysis Programs (ASAPs), Precise Excavation, and the DOE Technology Connection (TechCon) Program. The lessons learned may be applicable to the continuing challenges posed by the cleanup and long-term stewardship of radioactive contaminants and unexploded ordnance (UXO) at federal sites.

  16. Unraveling Kinase Activation Dynamics Using Kinase-Substrate Relationships from Temporal Large-Scale Phosphoproteomics Studies

    PubMed Central

    Chaudhuri, Rima; Yang, Pengyi; Vafaee, Fatemeh; Fazakerley, Daniel; Humphrey, Sean; James, David; Kuncic, Zdenka

    2016-01-01

    In response to stimuli, biological processes are tightly controlled by dynamic cellular signaling mechanisms. Reversible protein phosphorylation occurs on rapid time-scales (milliseconds to seconds), making it an ideal carrier of these signals. Advances in mass spectrometry-based proteomics have led to the identification of many tens of thousands of phosphorylation sites, yet for the majority of these the kinase is unknown and the underlying network topology of signaling networks therefore remains obscured. Identifying kinase substrate relationships (KSRs) is therefore an important goal in cell signaling research. Existing consensus sequence motif based prediction algorithms do not consider the biological context of KSRs, and are therefore insensitive to many other mechanisms guiding kinase-substrate recognition in cellular contexts. Here, we use temporal information to identify biologically relevant KSRs from Large-scale In Vivo Experiments (KSR-LIVE) in a data-dependent and automated fashion. First, we used available phosphorylation databases to construct a repository of existing experimentally-predicted KSRs. For each kinase in this database, we used time-resolved phosphoproteomics data to examine how its substrates changed in phosphorylation over time. Although substrates for a particular kinase clustered together, they often exhibited a different temporal pattern to the phosphorylation of the kinase. Therefore, although phosphorylation regulates kinase activity, our findings imply that substrate phosphorylation likely serve as a better proxy for kinase activity than kinase phosphorylation. KSR-LIVE can thereby infer which kinases are regulated within a biological context. Moreover, KSR-LIVE can also be used to automatically generate positive training sets for the subsequent prediction of novel KSRs using machine learning approaches. We demonstrate that this approach can distinguish between Akt and Rps6kb1, two kinases that share the same linear consensus motif

  17. Unraveling Kinase Activation Dynamics Using Kinase-Substrate Relationships from Temporal Large-Scale Phosphoproteomics Studies.

    PubMed

    Domanova, Westa; Krycer, James; Chaudhuri, Rima; Yang, Pengyi; Vafaee, Fatemeh; Fazakerley, Daniel; Humphrey, Sean; James, David; Kuncic, Zdenka

    2016-01-01

    In response to stimuli, biological processes are tightly controlled by dynamic cellular signaling mechanisms. Reversible protein phosphorylation occurs on rapid time-scales (milliseconds to seconds), making it an ideal carrier of these signals. Advances in mass spectrometry-based proteomics have led to the identification of many tens of thousands of phosphorylation sites, yet for the majority of these the kinase is unknown and the underlying network topology of signaling networks therefore remains obscured. Identifying kinase substrate relationships (KSRs) is therefore an important goal in cell signaling research. Existing consensus sequence motif based prediction algorithms do not consider the biological context of KSRs, and are therefore insensitive to many other mechanisms guiding kinase-substrate recognition in cellular contexts. Here, we use temporal information to identify biologically relevant KSRs from Large-scale In Vivo Experiments (KSR-LIVE) in a data-dependent and automated fashion. First, we used available phosphorylation databases to construct a repository of existing experimentally-predicted KSRs. For each kinase in this database, we used time-resolved phosphoproteomics data to examine how its substrates changed in phosphorylation over time. Although substrates for a particular kinase clustered together, they often exhibited a different temporal pattern to the phosphorylation of the kinase. Therefore, although phosphorylation regulates kinase activity, our findings imply that substrate phosphorylation likely serve as a better proxy for kinase activity than kinase phosphorylation. KSR-LIVE can thereby infer which kinases are regulated within a biological context. Moreover, KSR-LIVE can also be used to automatically generate positive training sets for the subsequent prediction of novel KSRs using machine learning approaches. We demonstrate that this approach can distinguish between Akt and Rps6kb1, two kinases that share the same linear consensus motif

  18. FOILFEST :community enabled security.

    SciTech Connect

    Moore, Judy Hennessey; Johnson, Curtis Martin; Whitley, John B.; Drayer, Darryl Donald; Cummings, John C., Jr.

    2005-09-01

    The Advanced Concepts Group of Sandia National Laboratories hosted a workshop, ''FOILFest: Community Enabled Security'', on July 18-21, 2005, in Albuquerque, NM. This was a far-reaching look into the future of physical protection consisting of a series of structured brainstorming sessions focused on preventing and foiling attacks on public places and soft targets such as airports, shopping malls, hotels, and public events. These facilities are difficult to protect using traditional security devices since they could easily be pushed out of business through the addition of arduous and expensive security measures. The idea behind this Fest was to explore how the public, which is vital to the function of these institutions, can be leveraged as part of a physical protection system. The workshop considered procedures, space design, and approaches for building community through technology. The workshop explored ways to make the ''good guys'' in public places feel safe and be vigilant while making potential perpetrators of harm feel exposed and convinced that they will not succeed. Participants in the Fest included operators of public places, social scientists, technology experts, representatives of government agencies including DHS and the intelligence community, writers and media experts. Many innovative ideas were explored during the fest with most of the time spent on airports, including consideration of the local airport, the Albuquerque Sunport. Some provocative ideas included: (1) sniffers installed in passage areas like revolving door, escalators, (2) a ''jumbotron'' showing current camera shots in the public space, (3) transparent portal screeners allowing viewing of the screening, (4) a layered open/funnel/open/funnel design where open spaces are used to encourage a sense of ''communitas'' and take advantage of citizen ''sensing'' and funnels are technological tunnels of sensors (the tunnels of truth), (5) curved benches with blast proof walls or backs, (6

  19. Evaluation of Quantitative Performance of Sequential Immobilized Metal Affinity Chromatographic Enrichment for Phosphopeptides

    PubMed Central

    Sun, Zeyu; Hamilton, Karyn L.; Reardon, Kenneth F.

    2014-01-01

    We evaluated a sequential elution protocol from immobilized metal affinity chromatography (SIMAC) employing gallium-based immobilized metal affinity chromatography (IMAC) in conjunction with titanium-dioxide-based metal oxide affinity chromatography (MOAC). The quantitative performance of this SIMAC enrichment approach, assessed in terms of repeatability, dynamic range, and linearity, was evaluated using a mixture composed of tryptic peptides from caseins, bovine serum albumin, and phosphopeptide standards. While our data demonstrate the overall consistent performance of the SIMAC approach under various loading conditions, the results also revealed that the method had limited repeatability and linearity for most phosphopeptides tested, and different phosphopeptides were found to have different linear ranges. These data suggest that, unless additional strategies are used, SIMAC should be regarded as a semi-quantitative method when used in large-scale phosphoproteomics studies in complex backgrounds. PMID:24096195

  20. Data set from the phosphoproteomic analysis of Magnaporthe oryzae-responsive proteins in susceptible and resistant rice cultivars

    PubMed Central

    Li, Yunfeng; Ye, Zhijian; Nie, Yanfang; Zhang, Jian; Wang, Guo-Liang; Wang, Zhenzhong

    2015-01-01

    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. PMID:26217708

  1. Phosphoproteomic Profiling of In Vivo Signaling in Liver by the Mammalian Target of Rapamycin Complex 1 (mTORC1)

    PubMed Central

    Demirkan, Gokhan; Yu, Kebing; Boylan, Joan M.; Salomon, Arthur R.; Gruppuso, Philip A.

    2011-01-01

    Background 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. Methodology/Principal Findings 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, TiO2 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. Conclusions/Significance 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. PMID:21738781

  2. Phosphoproteomes of Strongylocentrotus purpuratus shell and tooth matrix: identification of a major acidic sea urchin tooth phosphoprotein, phosphodontin

    PubMed Central

    2010-01-01

    Background Sea urchin is a major model organism for developmental biology and biomineralization research. However, identification of proteins involved in larval skeleton formation and mineralization processes in the embryo and adult, and the molecular characterization of such proteins, has just gained momentum with the sequencing of the Strongylocentrotus purpuratus genome and the introduction of high-throughput proteomics into the field. Results The present report contains the determination of test (shell) and tooth organic matrix phosphoproteomes. Altogether 34 phosphoproteins were identified in the biomineral organic matrices. Most phosphoproteins were specific for one compartment, only two were identified in both matrices. The sea urchin phosphoproteomes contained several obvious orthologs of mammalian proteins, such as a Src family tyrosine kinase, protein kinase C-delta 1, Dickkopf-1 and other signal transduction components, or nucleobindin. In most cases phosphorylation sites were conserved between sea urchin and mammalian proteins. However, the majority of phosphoproteins had no mammalian counterpart. The most interesting of the sea urchin-specific phosphoproteins, from the perspective of biomineralization research, was an abundant highly phosphorylated and very acidic tooth matrix protein composed of 35 very similar short sequence repeats, a predicted N-terminal secretion signal sequence, and an Asp-rich C-terminal motif, contained in [Glean3:18919]. Conclusions The 64 phosphorylation sites determined represent the most comprehensive list of experimentally identified sea urchin protein phosphorylation sites at present and are an important addition to the recently analyzed Strongylocentrotus purpuratus shell and tooth proteomes. The identified phosphoproteins included a major, highly phosphorylated protein, [Glean3:18919], for which we suggest the name phosphodontin. Although not sequence-related to such highly phosphorylated acidic mammalian dental

  3. Proteome and Phosphoproteome Characterization Reveals New Response and Defense Mechanisms of Brachypodium distachyon Leaves under Salt Stress*

    PubMed Central

    Lv, Dong-Wen; Subburaj, Saminathan; Cao, Min; Yan, Xing; Li, Xiaohui; Appels, Rudi; Sun, Dong-Fa; Ma, Wujun; Yan, Yue-Ming

    2014-01-01

    Salinity is a major abiotic stress affecting plant growth and development. Understanding the molecular mechanisms of salt response and defense in plants will help in efforts to improve the salt tolerance of crops. Brachypodium distachyon is a new model plant for wheat, barley, and several potential biofuel grasses. In the current study, proteome and phosphoproteome changes induced by salt stress were the focus. The Bd21 leaves were initially treated with salt in concentrations ranging from 80 to 320 mm and then underwent a recovery process prior to proteome analysis. A total of 80 differentially expressed protein spots corresponding to 60 unique proteins were identified. The sample treated with a median salt level of 240 mm and the control were selected for phosphopeptide purification using TiO2 microcolumns and LC-MS/MS for phosphoproteome analysis to identify the phosphorylation sites and phosphoproteins. A total of 1509 phosphoproteins and 2839 phosphorylation sites were identified. Among them, 468 phosphoproteins containing 496 phosphorylation sites demonstrated significant changes at the phosphorylation level. Nine phosphorylation motifs were extracted from the 496 phosphorylation sites. Of the 60 unique differentially expressed proteins, 14 were also identified as phosphoproteins. Many proteins and phosphoproteins, as well as potential signal pathways associated with salt response and defense, were found, including three 14-3-3s (GF14A, GF14B, and 14-3-3A) for signal transduction and several ABA signal-associated proteins such as ABF2, TRAB1, and SAPK8. Finally, a schematic salt response and defense mechanism in B. distachyon was proposed. PMID:24335353

  4. Comparative Ser/Thr/Tyr phosphoproteomics between two mycobacterial species: the fast growing Mycobacterium smegmatis and the slow growing Mycobacterium bovis BCG

    PubMed Central

    Nakedi, Kehilwe C.; Nel, Andrew J. M.; Garnett, Shaun; Blackburn, Jonathan M.; Soares, Nelson C.

    2015-01-01

    Ser/Thr/Tyr protein phosphorylation plays a critical role in regulating mycobacterial growth and development. Understanding the mechanistic link between protein phosphorylation signaling network and mycobacterial growth rate requires a global view of the phosphorylation events taking place at a given time under defined conditions. In the present study we employed a phosphopeptide enrichment and high throughput mass spectrometry-based strategy to investigate and qualitatively compare the phosphoproteome of two mycobacterial model organisms: the fast growing Mycobacterium smegmatis and the slow growing Mycobacterium bovis BCG. Cells were harvested during exponential phase and our analysis detected a total of 185 phospho-sites in M. smegmatis, of which 106 were confidently localized [localization probability (LP) = 0.75; PEP = 0.01]. By contrast, in M. bovis BCG the phosphoproteome comprised 442 phospho-sites, of which 289 were confidently localized. The percentage distribution of Ser/Thr/Tyr phosphorylation was 39.47, 57.02, and 3.51% for M. smegmatis and 35, 61.6, and 3.1% for M. bovis BCG. Moreover, our study identified a number of conserved Ser/Thr phosphorylated sites and conserved Tyr phosphorylated sites across different mycobacterial species. Overall a qualitative comparison of the fast and slow growing mycobacteria suggests that the phosphoproteome of M. smegmatis is a simpler version of that of M. bovis BCG. In particular, M. bovis BCG exponential cells exhibited a much more complex and sophisticated protein phosphorylation network regulating important cellular cycle events such as cell wall biosynthesis, elongation, cell division including immediately response to stress. The differences in the two phosphoproteomes are discussed in light of different mycobacterial growth rates. PMID:25904896

  5. Comparative Ser/Thr/Tyr phosphoproteomics between two mycobacterial species: the fast growing Mycobacterium smegmatis and the slow growing Mycobacterium bovis BCG.

    PubMed

    Nakedi, Kehilwe C; Nel, Andrew J M; Garnett, Shaun; Blackburn, Jonathan M; Soares, Nelson C

    2015-01-01

    Ser/Thr/Tyr protein phosphorylation plays a critical role in regulating mycobacterial growth and development. Understanding the mechanistic link between protein phosphorylation signaling network and mycobacterial growth rate requires a global view of the phosphorylation events taking place at a given time under defined conditions. In the present study we employed a phosphopeptide enrichment and high throughput mass spectrometry-based strategy to investigate and qualitatively compare the phosphoproteome of two mycobacterial model organisms: the fast growing Mycobacterium smegmatis and the slow growing Mycobacterium bovis BCG. Cells were harvested during exponential phase and our analysis detected a total of 185 phospho-sites in M. smegmatis, of which 106 were confidently localized [localization probability (LP) = 0.75; PEP = 0.01]. By contrast, in M. bovis BCG the phosphoproteome comprised 442 phospho-sites, of which 289 were confidently localized. The percentage distribution of Ser/Thr/Tyr phosphorylation was 39.47, 57.02, and 3.51% for M. smegmatis and 35, 61.6, and 3.1% for M. bovis BCG. Moreover, our study identified a number of conserved Ser/Thr phosphorylated sites and conserved Tyr phosphorylated sites across different mycobacterial species. Overall a qualitative comparison of the fast and slow growing mycobacteria suggests that the phosphoproteome of M. smegmatis is a simpler version of that of M. bovis BCG. In particular, M. bovis BCG exponential cells exhibited a much more complex and sophisticated protein phosphorylation network regulating important cellular cycle events such as cell wall biosynthesis, elongation, cell division including immediately response to stress. The differences in the two phosphoproteomes are discussed in light of different mycobacterial growth rates. PMID:25904896

  6. Evaluating Multiplexed Quantitative Phosphopeptide Analysis on a Hybrid Quadrupole Mass Filter/Linear Ion Trap/Orbitrap Mass Spectrometer

    PubMed Central

    2015-01-01

    As a driver for many biological processes, phosphorylation remains an area of intense research interest. Advances in multiplexed quantitation utilizing isobaric tags (e.g., TMT and iTRAQ) have the potential to create a new paradigm in quantitative proteomics. New instrumentation and software are propelling these multiplexed workflows forward, which results in more accurate, sensitive, and reproducible quantitation across tens of thousands of phosphopeptides. This study assesses the performance of multiplexed quantitative phosphoproteomics on the Orbitrap Fusion mass spectrometer. Utilizing a two-phosphoproteome model of precursor ion interference, we assessed the accuracy of phosphopeptide quantitation across a variety of experimental approaches. These methods included the use of synchronous precursor selection (SPS) to enhance TMT reporter ion intensity and accuracy. We found that (i) ratio distortion remained a problem for phosphopeptide analysis in multiplexed quantitative workflows, (ii) ratio distortion can be overcome by the use of an SPS-MS3 scan, (iii) interfering ions generally possessed a different charge state than the target precursor, and (iv) selecting only the phosphate neutral loss peak (single notch) for the MS3 scan still provided accurate ratio measurements. Remarkably, these data suggest that the underlying cause of interference may not be due to coeluting and cofragmented peptides but instead from consistent, low level background fragmentation. Finally, as a proof-of-concept 10-plex experiment, we compared phosphopeptide levels from five murine brains to five livers. In total, the SPS-MS3 method quantified 38 247 phosphopeptides, corresponding to 11 000 phosphorylation sites. With 10 measurements recorded for each phosphopeptide, this equates to more than 628 000 binary comparisons collected in less than 48 h. PMID:25521595

  7. Comparative phosphoproteome analysis of the developing grains in bread wheat (Triticum aestivum L.) under well-watered and water-deficit conditions.

    PubMed

    Zhang, Ming; Ma, Cao-Ying; Lv, Dong-Wen; Zhen, Shou-Min; Li, Xiao-Hui; Yan, Yue-Ming

    2014-10-01

    Wheat (Triticum aestivum), one of the most important cereal crops, is often threatened by drought. In this study, water deficit significantly reduced the height of plants and yield of grains. To explore further the effect of drought stress on the development and yield of grains, we first performed a large scale phosphoproteome analysis of developing grains in wheat. A total of 590 unique phosphopeptides, representing 471 phosphoproteins, were identified under well-watered conditions. Motif-X analysis showed that four motifs were enriched, including [sP], [Rxxs], [sDxE], and [sxD]. Through comparative phosphoproteome analysis between well-watered and water-deficit conditions, we found that 63 unique phosphopeptides, corresponding to 61 phosphoproteins, showed significant changes in phosphorylation level (≥2-fold intensities). Functional analysis suggested that some of these proteins may be involved in signal transduction, embryo and endosperm development of grains, and drought response and defense under water-deficit conditions. Moreover, we also found that some chaperones may play important roles in protein refolding or degradation when the plant is subjected to water stress. These results provide a detailed insight into the stress response and defense mechanisms of developmental grains at the phosphoproteome level. They also suggested some potential candidates for further study of transgenosis and drought stress as well as incorporation into molecular breeding for drought resistance. PMID:25145454

  8. Quantitative Thinking.

    ERIC Educational Resources Information Center

    DuBridge, Lee A.

    An appeal for more research to determine how to educate children as effectively as possible is made. Mathematics teachers can readily examine the educational problems of today in their classrooms since learning progress in mathematics can easily be measured and evaluated. Since mathematics teachers have learned to think in quantitative terms and…

  9. On Quantitizing

    ERIC Educational Resources Information Center

    Sandelowski, Margarete; Voils, Corrine I.; Knafl, George

    2009-01-01

    "Quantitizing", commonly understood to refer to the numerical translation, transformation, or conversion of qualitative data, has become a staple of mixed methods research. Typically glossed are the foundational assumptions, judgments, and compromises involved in converting disparate data sets into each other and whether such conversions advance…

  10. QUANTITATIVE MORPHOLOGY

    EPA Science Inventory

    Abstract: In toxicology, the role of quantitative assessment of brain morphology can be understood in the context of two types of treatment-related alterations. One type of alteration is specifically associated with treatment and is not observed in control animals. Measurement ...

  11. Berkeley Quantitative Genome Browser

    SciTech Connect

    Hechmer, Aaron

    2008-02-29

    The Berkeley Quantitative Genome Browser provides graphical browsing functionality for genomic data organized, at a minimum, by sequence and position. While supporting the annotation browsing features typical of many other genomic browsers, additional emphasis is placed on viewing and utilizing quantitative data. Data may be read from GFF, SGR, FASTA or any column delimited format. Once the data has been read into the browser's buffer, it may be searched. filtered or subjected to mathematical transformation. The browser also supplies some graphical design manipulation functionality geared towards preparing figures for presentations or publication. A plug-in mechanism enables development outside the core functionality that adds more advanced or esoteric analysis capabilities. BBrowse's development and distribution is open-source and has been built to run on Linux, OSX and MS Windows operating systems.

  12. Berkeley Quantitative Genome Browser

    Energy Science and Technology Software Center (ESTSC)

    2008-02-29

    The Berkeley Quantitative Genome Browser provides graphical browsing functionality for genomic data organized, at a minimum, by sequence and position. While supporting the annotation browsing features typical of many other genomic browsers, additional emphasis is placed on viewing and utilizing quantitative data. Data may be read from GFF, SGR, FASTA or any column delimited format. Once the data has been read into the browser's buffer, it may be searched. filtered or subjected to mathematical transformation.more » The browser also supplies some graphical design manipulation functionality geared towards preparing figures for presentations or publication. A plug-in mechanism enables development outside the core functionality that adds more advanced or esoteric analysis capabilities. BBrowse's development and distribution is open-source and has been built to run on Linux, OSX and MS Windows operating systems.« less

  13. Toward genome-enabled mycology.

    PubMed

    Hibbett, David S; Stajich, Jason E; Spatafora, Joseph W

    2013-01-01

    Genome-enabled mycology is a rapidly expanding field that is characterized by the pervasive use of genome-scale data and associated computational tools in all aspects of fungal biology. Genome-enabled mycology is integrative and often requires teams of researchers with diverse skills in organismal mycology, bioinformatics and molecular biology. This issue of Mycologia presents the first complete fungal genomes in the history of the journal, reflecting the ongoing transformation of mycology into a genome-enabled science. Here, we consider the prospects for genome-enabled mycology and the technical and social challenges that will need to be overcome to grow the database of complete fungal genomes and enable all fungal biologists to make use of the new data. PMID:23928422

  14. Delivering compassionate care: the enablers and barriers.

    PubMed

    Christiansen, Angela; O'Brien, Mary R; Kirton, Jennifer A; Zubairu, Kate; Bray, Lucy

    The importance of providing compassionate care to patients is well established. While compassionate care can be understood as an individual response to others' vulnerability, it is acknowledged that healthcare environments can impact significantly on this aspect of practice. This study sought to explore how health professionals and pre-qualifying healthcare students (HCS) understand compassionate care and factors that hinder or enable them to practice compassionately. The perceptions of health professionals (n=146) and HCS (n=166) registered at a university in Northwest England were explored using mixed methods. This article reports on the data gained from the qualitative interviews and responses to open-text questions from the mainly quantitative questionnaire. The findings are discussed under the following themes: individual and relationship factors that impact on compassionate care practice; organisational factors that impact on the clinical environment and team; and leadership factors that hinder or enable a compassionate care culture. This article argues that there are a number of enabling factors that enhance a culture conducive to providing compassionate care. These include leaders who act as positive role models, good relationships between team members and a focus on staff wellbeing. PMID:26355360

  15. An Analysis of Enabling School Structure: Theoretical, Empirical, and Research Considerations

    ERIC Educational Resources Information Center

    Sinden, James E.; Hoy, Wayne K.; Sweetland, Scott R.

    2004-01-01

    The construct of enabling school structure is empirically analyzed in this qualitative study of high schools. First, the theoretical underpinning of enabling school structure is developed. Then, six high schools, which were determined to have enabling structures in a large quantitative study of Ohio schools, were analyzed in depth using…

  16. Computer Security Systems Enable Access.

    ERIC Educational Resources Information Center

    Riggen, Gary

    1989-01-01

    A good security system enables access and protects information from damage or tampering, but the most important aspects of a security system aren't technical. A security procedures manual addresses the human element of computer security. (MLW)

  17. Enabling Space Science and Exploration

    NASA Technical Reports Server (NTRS)

    Weber, William J.

    2006-01-01

    This viewgraph presentation on enabling space science and exploration covers the following topics: 1) Today s Deep Space Network; 2) Next Generation Deep Space Network; 3) Needed technologies; 4) Mission IT and networking; and 5) Multi-mission operations.

  18. Multiplex staining of 2-DE gels for an initial phosphoproteome analysis of germinating seeds and early grown seedlings from a non-orthodox specie: Quercus ilex L. subsp. ballota [Desf.] Samp.

    PubMed Central

    Romero-Rodríguez, M. Cristina; Abril, Nieves; Sánchez-Lucas, Rosa; Jorrín-Novo, Jesús V.

    2015-01-01

    As a preliminary step in the phosphoproteome analysis of germinating seeds (0 and 24 h after seed imbibition) and early grown seedlings (216 h after seed imbibition) from a non-orthodox sp. Quercus ilex, a multiplex (SYPRO-Ruby and Pro-Q DPS) staining of high-resolution 2-DE gels was used. By using this protocol it was possible to detect changes in protein-abundance and/or phosphorylation status. This simple approach could be a good complementary alternative to the enrichment protocols used in the search for phosphoprotein candidates. While 482 spots were visualized with SYPRO-Ruby, 222 were with Pro-Q DPS. Statistically significant differences in spot intensity were observed among samples, these corresponding to 85 SYPRO-Ruby-, 20 Pro-Q-DPS-, and 35 SYPRO-Ruby and Pro-Q-DPS-stained spots. Fifty-five phosphoprotein candidates showing qualitative or quantitative differences between samples were subjected to MALDI-TOF-TOF MS analysis, with 20 of them being identified. Identified proteins belonged to five different functional categories, namely: carbohydrate and amino acid metabolism, defense, protein folding, and oxidation-reduction processes. With the exception of a putative cyclase, the other 19 proteins had at least one orthologous phosphoprotein in Arabidopsis thaliana, Medicago truncatula, N. tabacum, and Glycine max. Out of the 20 identified, seven showed differences in intensity in Pro-Q-DPS but not in SYPRO-Ruby-stained gels, including enzymes of the glycolysis and amino acid metabolism. This bears out that theory the regulation of these enzymes occurs at the post-translational level by phosphorylation with no changes at the transcriptional or translational level. This is different from the mechanism reported in orthodox seeds, in which concomitant changes in abundance and phosphorylation status have been observed for these enzymes. PMID:26322061

  19. The Tec Kinase-Regulated Phosphoproteome Reveals a Mechanism for the Regulation of Inhibitory Signals in Murine Macrophages.

    PubMed

    Tampella, Giacomo; Kerns, Hannah M; Niu, Deqiang; Singh, Swati; Khim, Socheath; Bosch, Katherine A; Garrett, Meghan E; Moguche, Albanus; Evans, Erica; Browning, Beth; Jahan, Tahmina A; Nacht, Mariana; Wolf-Yadlin, Alejandro; Plebani, Alessandro; Hamerman, Jessica A; Rawlings, David J; James, Richard G

    2015-07-01

    Previous work has shown conflicting roles for Tec family kinases in regulation of TLR-dependent signaling in myeloid cells. In the present study, we performed a detailed investigation of the role of the Tec kinases Btk and Tec kinases in regulating TLR signaling in several types of primary murine macrophages. We demonstrate that primary resident peritoneal macrophages deficient for Btk and Tec secrete less proinflammatory cytokines in response to TLR stimulation than do wild-type cells. In contrast, we found that bone marrow-derived and thioglycollate-elicited peritoneal macrophages deficient for Btk and Tec secrete more proinflammatory cytokines than do wild-type cells. We then compared the phosphoproteome regulated by Tec kinases and LPS in primary peritoneal and bone marrow-derived macrophages. From this analysis we determined that Tec kinases regulate different signaling programs in these cell types. In additional studies using bone marrow-derived macrophages, we found that Tec and Btk promote phosphorylation events necessary for immunoreceptor-mediated inhibition of TLR signaling. Taken together, our results are consistent with a model where Tec kinases (Btk, Tec, Bmx) are required for TLR-dependent signaling in many types of myeloid cells. However, our data also support a cell type-specific TLR inhibitory role for Btk and Tec that is mediated by immunoreceptor activation and signaling via PI3K. PMID:26026062

  20. The Tec kinase-regulated phosphoproteome reveals a mechanism for the regulation of inhibitory signals in murine macrophages

    PubMed Central

    Tampella, Giacomo; Kerns, Hannah M.; Niu, Deqiang; Singh, Swati; Khim, Socheath; Bosch, Katherine A.; Garrett, Meghan E.; Moguche, Albanus; Evans, Erica; Browning, Beth; Jahan, Tahmina A.; Nacht, Mariana; Wolf-Yadlin, Alejandro; Plebani, Alessandro; Hamerman, Jessica A.; Rawlings, David J.; James, Richard G.

    2015-01-01

    Previous work has shown conflicting roles for Tec family kinases in regulation of Toll-like receptor (TLR)-dependent signalling in myeloid cells. In the present study, we performed a detailed investigation of the role of Btk and Tec kinases in regulating TLR signalling in several types of primary murine macrophages. We demonstrate that primary resident peritoneal macrophages deficient for Btk and Tec secrete less pro-inflammatory cytokines in response to TLR stimulation than wild type cells. In contrast, we found that bone marrow-derived and thioglycollate-elicited peritoneal macrophages deficient for Btk and Tec secrete more pro-inflammatory cytokines than wild type cells. We then compared the phosphoproteome regulated by Tec kinases and lipopolysaccharide in primary peritoneal and bone marrow derived macrophages. From this analysis we determined that Tec kinases regulate different signalling programs in these cell types. In additional studies using bone marrow-derived macrophages, we find that Tec and Btk promote phosphorylation events necessary for immunoreceptor-mediated inhibition of TLR signalling. Taken together, our results are consistent with a model where Tec kinases (Btk, Tec, Bmx) are required for TLR-dependent signalling in many types of myeloid cells. However, our data also support a cell type-specific TLR-inhibitory role for Btk and Tec that is mediated by immunoreceptor activation and signalling via PI3K. PMID:26026062

  1. Comparative phosphoproteome profiling reveals a function of the STN8 kinase in fine-tuning of cyclic electron flow (CEF)

    PubMed Central

    Reiland, Sonja; Finazzi, Giovanni; Endler, Anne; Willig, Adrian; Baerenfaller, Katja; Grossmann, Jonas; Gerrits, Bertran; Rutishauser, Dorothea; Gruissem, Wilhelm; Rochaix, Jean-David; Baginsky, Sacha

    2011-01-01

    Important aspects of photosynthetic electron transport efficiency in chloroplasts are controlled by protein phosphorylation. Two thylakoid-associated kinases, STN7 and STN8, have distinct roles in short- and long-term photosynthetic acclimation to changes in light quality and quantity. Although some substrates of STN7 and STN8 are known, the complexity of this regulatory kinase system implies that currently unknown substrates connect photosynthetic performance with the regulation of metabolic and regulatory functions. We performed an unbiased phosphoproteome-wide screen with Arabidopsis WT and stn8 mutant plants to identify unique STN8 targets. The phosphorylation status of STN7 was not affected in stn8, indicating that kinases other than STN8 phosphorylate STN7 under standard growth conditions. Among several putative STN8 substrates, PGRL1-A is of particular importance because of its possible role in the modulation of cyclic electron transfer. The STN8 phosphorylation site on PGRL1-A is absent in both monocotyledonous plants and algae. In dicots, spectroscopic measurements with Arabidopsis WT, stn7, stn8, and stn7/stn8 double-mutant plants indicate a STN8-mediated slowing down of the transition from cyclic to linear electron flow at the onset of illumination. This finding suggests a possible link between protein phosphorylation by STN8 and fine-tuning of cyclic electron flow during this critical step of photosynthesis, when the carbon assimilation is not commensurate to the electron flow capacity of the chloroplast. PMID:21768351

  2. Phosphoproteomics reveals resveratrol-dependent inhibition of Akt/mTORC1/S6K1 signaling.

    PubMed

    Alayev, Anya; Doubleday, Peter F; Berger, Sara Malka; Ballif, Bryan A; Holz, Marina K

    2014-12-01

    Resveratrol, a plant-derived polyphenol, regulates many cellular processes, including cell proliferation, aging and autophagy. However, the molecular mechanisms of resveratrol action in cells are not completely understood. Intriguingly, resveratrol treatment of cells growing in nutrient-rich conditions induces autophagy, while acute resveratrol treatment of cells in a serum-deprived state inhibits autophagy. In this study, we performed a phosphoproteomic analysis after applying resveratrol to serum-starved cells with the goal of identifying the acute signaling events initiated by resveratrol in a serum-deprived state. We determined that resveratrol in serum-starved conditions reduces the phosphorylation of several proteins belonging to the mTORC1 signaling pathway, most significantly, PRAS40 at T246 and S183. Under these same conditions, we also found that resveratrol altered the phosphorylation of several proteins involved in various biological processes, most notably transcriptional modulators, represented by p53, FOXA1, and AATF. Together these data provide a more comprehensive view of both the spectrum of phosphoproteins upon which resveratrol acts as well as the potential mechanisms by which it inhibits autophagy in serum-deprived cells. PMID:25311616

  3. Large-Scale Phosphoproteome of Human Whole Saliva Using Disulphide-Thiol-Interchange Covalent Chromatography and Mass Spectrometry

    PubMed Central

    Salih, Erdjan; Siqueira, Walter L.; Helmerhorst, Eva J.; Oppenheim, Frank G.

    2010-01-01

    Thus far only a handful of phosphoproteins with important biological functions have been identified and characterized in oral fluids and these include some of the abundant protein constituents of saliva. Whole saliva (WS) samples were trypsin digested followed by chemical derivatization using dithiothreitol (DTT) of the phospho-serine/threonine containing peptides. The DTT-phosphopeptides were enriched by covalent disulphide-thiol-interchange chromatography and analysis by nano-flow LC-ESI-MS/MS. The specificity of DTT chemical derivatization was evaluated separately under different base-catalyzed conditions with NaOH and Ba(OH)2, blocking cysteine residues by iodoacetamide and enzymatic O-deglycosylation prior to DTT reaction. Further analysis of WS samples which were subjected to either of these conditions provided supporting evidence for phosphoprotein identifications. The combined chemical strategies and mass spectrometric analyses identified 65 phosphoproteins in WS of which 28 were based on two or more peptide identification criteria with high confidence, and 37 were based on a single phosphopeptide identification. Most of the identified proteins, ~80%, were hitherto unknown phosphoprotein components. This study represents the first large-scale documentation of phosphoproteins of WS. The origins and identity of WS phosphoproteome suggest significant implications for both basic science and the development of novel biomarkers/diagnostic tools for both systemic and oral disease states. PMID:20659418

  4. Phosphoproteomic analysis of basal and therapy-induced adaptive signaling networks in BRAF and NRAS mutant melanoma

    PubMed Central

    Fedorenko, Inna V.; Fang, Bin; Munko, A. Cecelia; Gibney, Geoffrey T.; Koomen, John M.; Smalley, Keiran S.M.

    2015-01-01

    Basal and kinase inhibitor-driven adaptive signaling has been examined in a panel of melanoma cell lines using phosphoproteomics in conjunction with pathway analysis. A considerable divergence in the spectrum of tyrosine-phosphorylated peptides was noted at the cell line level. The unification of genotype-specific cell line data revealed the enrichment for the tyrosine-phosphorylated cytoskeletal proteins to be associated with the presence of a BRAF mutation and oncogenic NRAS to be associated with increased receptor tyrosine kinase phosphorylation. A number of proteins including cell cycle regulators (CDK1, CDK2 and CDK3), MAPK pathway components (ERK1 and ERK2), interferon regulators (TYK2), GTPase regulators (RIN1) and controllers of protein tyrosine phosphorylation (DYR1A and PTPRA) were common to all genotypes. Treatment of a BRAF-mutant/PTEN-null melanoma cell line with vemurafenib led to decreased phosphorylation of ERK, phospholipase C1 and β-catenin with increases in RTK phosphorylation, STAT3 and GSK3α noted. In NRAS-mutant melanoma, MEK inhibition led to increased phosphorylation of EGFR signaling pathway components, Src family kinases and PKCδ with decreased phosphorylation seen in STAT3 and ERK1/2. Together these data present the first systems level view of adaptive and basal phosphotyrosine signaling in BRAF- and NRAS-mutant melanoma. PMID:25339196

  5. A novel double-component MOAC honeycomb composite with pollen grains as a template for phosphoproteomics research.

    PubMed

    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. PMID:27154659

  6. Comparative Phosphoproteomics Analysis of VEGF and Angiopoietin-1 Signaling Reveals ZO-1 as a Critical Regulator of Endothelial Cell Proliferation.

    PubMed

    Chidiac, Rony; Zhang, Ying; Tessier, Sylvain; Faubert, Denis; Delisle, Chantal; Gratton, Jean-Philippe

    2016-05-01

    VEGF and angiopoietin-1 (Ang-1) are essential factors to promote angiogenesis through regulation of a plethora of signaling events in endothelial cells (ECs). Although pathways activated by VEGF and Ang-1 are being established, the unique signaling nodes conferring specific responses to each factor remain poorly defined. Thus, we conducted a large-scale comparative phosphoproteomic analysis of signaling pathways activated by VEGF and Ang-1 in ECs using mass spectrometry. Analysis of VEGF and Ang-1 networks of regulated phosphoproteins revealed that the junctional proteins ZO-1, ZO-2, JUP and p120-catenin are part of a cluster of proteins phosphorylated following VEGF stimulation that are linked to MAPK1 activation. Down-regulation of these junctional proteins led to MAPK1 activation and accordingly, increased proliferation of ECs stimulated specifically by VEGF, but not by Ang-1. We identified ZO-1 as the central regulator of this effect and showed that modulation of cellular ZO-1 levels is necessary for EC proliferation during vascular development of the mouse postnatal retina. In conclusion, we uncovered ZO-1 as part of a signaling node activated by VEGF, but not Ang-1, that specifically modulates EC proliferation during angiogenesis. PMID:26846344

  7. MALDI-TOF and nESI Orbitrap MS/MS identify orthogonal parts of the phosphoproteome.

    PubMed

    Ruprecht, Benjamin; Roesli, Christoph; Lemeer, Simone; Kuster, Bernhard

    2016-05-01

    Phosphorylation is a reversible posttranslational protein modification which plays a pivotal role in intracellular signaling. Despite extensive efforts, phosphorylation site mapping of proteomes is still incomplete motivating the exploration of alternative methods that complement existing workflows. In this study, we compared tandem mass spectrometry (MS/MS) on matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) and nano-electrospray ionization (nESI) Orbitrap instruments with respect to their ability to identify phosphopeptides from complex proteome digests. Phosphopeptides were enriched from tryptic digests of cell lines using Fe-IMAC column chromatography and subjected to LC-MS/MS analysis. We found that the two analytical workflows exhibited considerable orthogonality. For instance, MALDI-TOF MS/MS favored the identification of phosphopeptides encompassing clear motif signatures for acidic residue directed kinases. The extent of orthogonality of the two LC-MS/MS systems was comparable to that of using alternative proteases such as Asp-N, Arg-C, chymotrypsin, Glu-C and Lys-C on just one LC-MS/MS instrument. Notably, MALDI-TOF MS/MS identified an unexpectedly high number and percentage of phosphotyrosine sites (∼20% of all sites), possibly as a direct consequence of more efficient ionization. The data clearly show that LC-MALDI MS/MS can be a useful complement to LC-nESI MS/MS for phosphoproteome mapping and particularly so for acidic and phosphotyrosine containing peptides. PMID:26990019

  8. Global Phosphoproteomic Analysis of Human Skeletal Muscle Reveals a Network of Exercise-Regulated Kinases and AMPK Substrates.

    PubMed

    Hoffman, Nolan J; Parker, Benjamin L; Chaudhuri, Rima; Fisher-Wellman, Kelsey H; Kleinert, Maximilian; Humphrey, Sean J; Yang, Pengyi; Holliday, Mira; Trefely, Sophie; Fazakerley, Daniel J; Stöckli, Jacqueline; Burchfield, James G; Jensen, Thomas E; Jothi, Raja; Kiens, Bente; Wojtaszewski, Jørgen F P; Richter, Erik A; James, David E

    2015-11-01

    Exercise is essential in regulating energy metabolism and whole-body insulin sensitivity. To explore the exercise signaling network, we undertook a global analysis of protein phosphorylation in human skeletal muscle biopsies from untrained healthy males before and after a single high-intensity exercise bout, revealing 1,004 unique exercise-regulated phosphosites on 562 proteins. These included substrates of known exercise-regulated kinases (AMPK, PKA, CaMK, MAPK, mTOR), yet the majority of kinases and substrate phosphosites have not previously been implicated in exercise signaling. Given 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 an undescribed role for AMPK-dependent phosphorylation of AKAP1 in mitochondrial respiration. These data expose the unexplored complexity of acute exercise signaling and provide insights into the role of AMPK in mitochondrial biochemistry. PMID:26437602

  9. Nuclear Phosphoproteomic Screen Uncovers ACLY as Mediator of IL-2-induced Proliferation of CD4+ T lymphocytes.

    PubMed

    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. PMID:27067055

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

    PubMed

    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). PMID:24825570

  11. Phosphoproteomic analysis of Methanohalophilus portucalensis FDF1(T) identified the role of protein phosphorylation in methanogenesis and osmoregulation.

    PubMed

    Wu, Wan-Ling; Lai, Shu-Jung; Yang, Jhih-Tian; Chern, Jeffy; Liang, Suh-Yuen; Chou, Chi-Chi; Kuo, Chih-Horng; Lai, Mei-Chin; Wu, Shih-Hsiung

    2016-01-01

    Methanogens have gained much attention for their metabolic product, methane, which could be an energy substitute but also contributes to the greenhouse effect. One factor that controls methane emission, reversible protein phosphorylation, is a crucial signaling switch, and phosphoproteomics has become a powerful tool for large-scale surveying. Here, we conducted the first phosphorylation-mediated regulation study in halophilic Methanohalophilus portucalensis FDF1(T), a model strain for studying stress response mechanisms in osmoadaptation. A shotgun approach and MS-based analysis identified 149 unique phosphoproteins. Among them, 26% participated in methanogenesis and osmolytes biosynthesis pathways. Of note, we uncovered that protein phosphorylation might be a crucial factor to modulate the pyrrolysine (Pyl) incorporation and Pyl-mediated methylotrophic methanogenesis. Furthermore, heterologous expression of glycine sarcosine N-methyltransferase (GSMT) mutant derivatives in the osmosensitive Escherichia coli MKH13 revealed that the nonphosphorylated T68A mutant resulted in increased salt tolerance. In contrast, mimic phosphorylated mutant T68D proved defective in both enzymatic activity and salinity tolerance for growth. Our study provides new insights into phosphorylation modification as a crucial role of both methanogenesis and osmoadaptation in methanoarchaea, promoting biogas production or reducing future methane emission in response to global warming and climate change. PMID:27357474

  12. Phosphoproteomic analysis of Methanohalophilus portucalensis FDF1T identified the role of protein phosphorylation in methanogenesis and osmoregulation

    PubMed Central

    Wu, Wan-Ling; Lai, Shu-Jung; Yang, Jhih-Tian; Chern, Jeffy; Liang, Suh-Yuen; Chou, Chi-Chi; Kuo, Chih-Horng; Lai, Mei-Chin; Wu, Shih-Hsiung

    2016-01-01

    Methanogens have gained much attention for their metabolic product, methane, which could be an energy substitute but also contributes to the greenhouse effect. One factor that controls methane emission, reversible protein phosphorylation, is a crucial signaling switch, and phosphoproteomics has become a powerful tool for large-scale surveying. Here, we conducted the first phosphorylation-mediated regulation study in halophilic Methanohalophilus portucalensis FDF1T, a model strain for studying stress response mechanisms in osmoadaptation. A shotgun approach and MS-based analysis identified 149 unique phosphoproteins. Among them, 26% participated in methanogenesis and osmolytes biosynthesis pathways. Of note, we uncovered that protein phosphorylation might be a crucial factor to modulate the pyrrolysine (Pyl) incorporation and Pyl-mediated methylotrophic methanogenesis. Furthermore, heterologous expression of glycine sarcosine N-methyltransferase (GSMT) mutant derivatives in the osmosensitive Escherichia coli MKH13 revealed that the nonphosphorylated T68A mutant resulted in increased salt tolerance. In contrast, mimic phosphorylated mutant T68D proved defective in both enzymatic activity and salinity tolerance for growth. Our study provides new insights into phosphorylation modification as a crucial role of both methanogenesis and osmoadaptation in methanoarchaea, promoting biogas production or reducing future methane emission in response to global warming and climate change. PMID:27357474

  13. Fast and easy phosphopeptide fractionation by combinatorial ERLIC-SCX solid-phase extraction for in-depth phosphoproteome analysis.

    PubMed

    Zarei, Mostafa; Sprenger, Adrian; Rackiewicz, Michal; Dengjel, Joern

    2016-01-01

    Mass spectrometry-based phosphoproteomic analysis is a powerful method for gaining a global, unbiased understanding of cellular signaling. Its accuracy and comprehensiveness stands or falls with the quality and choice of the applied phosphopeptide prefractionation strategy. This protocol covers a powerful but simple and rapid strategy for phosphopeptide prefractionation. The combinatorial use of two distinct chromatographic techniques that address the inverse physicochemical properties of peptides allows for superior fractionation efficiency of multiple phosphorylated peptides. In the first step, multiphosphorylated peptides are separated according to the number of negatively charged phosphosites by electrostatic repulsion-hydrophilic interaction chromatography (ERLIC). A subsequent strong cation exchange (SCX) step separates mostly singly phosphorylated peptides in the ERLIC flow-through according to their positive charge. The presented strategy is inexpensive and adaptable to large and small amounts of starting material, and it allows highly multiplexed sample preparation. Because of its implementation as solid-phase extraction, the entire workflow takes only 2 h to complete. PMID:26633130

  14. Proteomic and Phospho-Proteomic Profile of Human Platelets in Basal, Resting State: Insights into Integrin Signaling

    PubMed Central

    Maiguel, Dony; Faridi, Mohd Hafeez; Barth, Constantinos J.; Salem, Saeed M.; Singhal, Mudita; Stoub, Darren; Krastins, Bryan; Ogihara, Mitsunori; Zaki, Mohammed J.; Gupta, Vineet

    2009-01-01

    During atherogenesis and vascular inflammation quiescent platelets are activated to increase the surface expression and ligand affinity of the integrin αIIbβ3 via inside-out signaling. Diverse signals such as thrombin, ADP and epinephrine transduce signals through their respective GPCRs to activate protein kinases that ultimately lead to the phosphorylation of the cytoplasmic tail of the integrin αIIbβ3 and augment its function. The signaling pathways that transmit signals from the GPCR to the cytosolic domain of the integrin are not well defined. In an effort to better understand these pathways, we employed a combination of proteomic profiling and computational analyses of isolated human platelets. We analyzed ten independent human samples and identified a total of 1507 unique proteins in platelets. This is the most comprehensive platelet proteome assembled to date and includes 190 membrane-associated and 262 phosphorylated proteins, which were identified via independent proteomic and phospho-proteomic profiling. We used this proteomic dataset to create a platelet protein-protein interaction (PPI) network and applied novel contextual information about the phosphorylation step to introduce limited directionality in the PPI graph. This newly developed contextual PPI network computationally recapitulated an integrin signaling pathway. Most importantly, our approach not only provided insights into the mechanism of integrin αIIbβ3 activation in resting platelets but also provides an improved model for analysis and discovery of PPI dynamics and signaling pathways in the future. PMID:19859549

  15. Using multiplex-staining to study changes in the maize leaf phosphoproteome in response to mechanical wounding.

    PubMed

    Lewandowska-Gnatowska, Elżbieta; Johnston, Mark L; Antoine, Wesner; Szczegielniak, Jadwiga; Muszyńska, Grażyna; Miernyk, Ján A

    2011-07-01

    Mechanical wounding of 2-week-old maize (Zea mays L.) leaves, one of the first steps in both pathogen infection and herbivore attack, stimulates metabolism and activates signal transduction pathways dedicated to defense and recovery. The signaling pathways include reversible protein phosphorylation which can modulate protein activities, and transmit signals within cellular pathways and networks. We have used multiplex-staining of high-resolution 2D gels for protein (Sypro Ruby) and phosphorylation (Pro-Q Diamond) as a strategy for quantifying changes in the stoichiometry of phosphorylation after wounding for 270 protein spots. Rigorous statistical analysis of the time-index data allowed us to accept patterns of change in 125 of the spots as non-random, and these patterns were assigned to five clusters. A reliable identity was assigned to 21 selected proteins, most of which have been previously described as phospho-proteins. The results suggest that analysis of protein spots from high-resolution 2D gels by multiplex-staining for protein plus phosphorylation is a strategy that can be broadly useful for study of how the phospho-proteome responds to abiotic stress. PMID:21334701

  16. Nanoplasmon-enabled macroscopic thermal management

    NASA Astrophysics Data System (ADS)

    Jonsson, Gustav Edman; Miljkovic, Vladimir; Dmitriev, Alexandre

    2014-05-01

    In numerous applications of energy harvesting via transformation of light into heat the focus recently shifted towards highly absorptive nanoplasmonic materials. It is currently established that noble metals-based absorptive plasmonic platforms deliver significant light-capturing capability and can be viewed as super-absorbers of optical radiation. Naturally, approaches to the direct experimental probing of macroscopic temperature increase resulting from these absorbers are welcomed. Here we derive a general quantitative method of characterizing heat-generating properties of optically absorptive layers via macroscopic thermal imaging. We further monitor macroscopic areas that are homogeneously heated by several degrees with nanostructures that occupy a mere 8% of the surface, leaving it essentially transparent and evidencing significant heat generation capability of nanoplasmon-enabled light capture. This has a direct bearing to a large number of applications where thermal management is crucial.

  17. Nanoplasmon-enabled macroscopic thermal management

    PubMed Central

    Jonsson, Gustav Edman; Miljkovic, Vladimir; Dmitriev, Alexandre

    2014-01-01

    In numerous applications of energy harvesting via transformation of light into heat the focus recently shifted towards highly absorptive nanoplasmonic materials. It is currently established that noble metals-based absorptive plasmonic platforms deliver significant light-capturing capability and can be viewed as super-absorbers of optical radiation. Naturally, approaches to the direct experimental probing of macroscopic temperature increase resulting from these absorbers are welcomed. Here we derive a general quantitative method of characterizing heat-generating properties of optically absorptive layers via macroscopic thermal imaging. We further monitor macroscopic areas that are homogeneously heated by several degrees with nanostructures that occupy a mere 8% of the surface, leaving it essentially transparent and evidencing significant heat generation capability of nanoplasmon-enabled light capture. This has a direct bearing to a large number of applications where thermal management is crucial. PMID:24870613

  18. [Quantitative ultrasound].

    PubMed

    Barkmann, R; Glüer, C-C

    2006-10-01

    Methods of quantitative ultrasound (QUS) can be used to obtain knowledge about bone fragility. Comprehensive study results exist showing the power of QUS for the estimation of osteoporotic fracture risk. Nevertheless, the variety of technologies, devices, and variables as well as different degrees of validation of the single devices have to be taken into account. Using methods to simulate ultrasound propagation, the complex interaction between ultrasound and bone could be understood and the propagation could be visualized. Preceding widespread clinical use, it has to be clarified if patients with low QUS values will profit from therapy, as it has been shown for DXA. Moreover, the introduction of quality assurance measures is essential. The user should know the limitations of the methods and be able to interpret the results correctly. Applied in an adequate manner QUS methods could then, due to lower costs and absence of ionizing radiation, become important players in osteoporosis management. PMID:16896637

  19. Enable: Developing Instructional Language Skills.

    ERIC Educational Resources Information Center

    Witt, Beth

    The program presented in this manual provides a structure and activities for systematic development of effective listening comprehension in typical and atypical children. The complete ENABLE kit comes with pictures, cut-outs, and puppets to illustrate the directives, questions, and narrative activities. The manual includes an organizational and…

  20. Exogenous Attention Enables Perceptual Learning

    PubMed Central

    Szpiro, Sarit F. A.; Carrasco, Marisa

    2015-01-01

    Practice can improve visual perception, and these improvements are considered to be a form of brain plasticity. Training-induced learning is time-consuming and requires hundreds of trials across multiple days. The process of learning acquisition is understudied. Can learning acquisition be potentiated by manipulating visual attentional cues? We developed a protocol in which we used task-irrelevant cues for between-groups manipulation of attention during training. We found that training with exogenous attention can enable the acquisition of learning. Remarkably, this learning was maintained even when observers were subsequently tested under neutral conditions, which indicates that a change in perception was involved. Our study is the first to isolate the effects of exogenous attention and to demonstrate its efficacy to enable learning. We propose that exogenous attention boosts perceptual learning by enhancing stimulus encoding. PMID:26502745

  1. Nanofluidics: enabling processes for biotech

    NASA Astrophysics Data System (ADS)

    Ulmanella, Umberto; Ho, Chih-Ming

    2001-10-01

    The advance of micro and nanodevice manufacturing technology enables us to carry out biological and chemical processes in a more efficient manner. In fact, fluidic processes connect the macro and the micro/nano worlds. For devices approaching the size of the fluid molecules, many physical phenomena occur that are not observed in macro flows. In this brief review, we discuss a few selected topics which of are interest for basic research and are important for applications in biotechnology.

  2. Echo-Enabled Harmonic Generation

    SciTech Connect

    Stupakov, Gennady; /SLAC

    2012-06-28

    A recently proposed concept of the Echo-Enabled Harmonic Generation (EEHG) FEL uses two laser modulators in combination with two dispersion sections to generate a high-harmonic density modulation in a relativistic beam. This seeding technique holds promise of a one-stage soft x-ray FEL that radiates not only transversely but also longitudinally coherent pulses. Currently, an experimental verification of the concept is being conducted at the SLAC National Accelerator Laboratory aimed at the demonstration of the EEHG.

  3. Technologies for Networked Enabled Operations

    NASA Technical Reports Server (NTRS)

    Glass, B.; Levine, J.

    2005-01-01

    Current point-to-point data links will not scale to support future integration of surveillance, security, and globally-distributed air traffic data, and already hinders efficiency and capacity. While the FAA and industry focus on a transition to initial system-wide information management (SWIM) capabilities, this paper describes a set of initial studies of NAS network-enabled operations technology gaps targeted for maturity in later SWIM spirals (201 5-2020 timeframe).

  4. MEMS: Enabled Drug Delivery Systems.

    PubMed

    Cobo, Angelica; Sheybani, Roya; Meng, Ellis

    2015-05-01

    Drug delivery systems play a crucial role in the treatment and management of medical conditions. Microelectromechanical systems (MEMS) technologies have allowed the development of advanced miniaturized devices for medical and biological applications. This Review presents the use of MEMS technologies to produce drug delivery devices detailing the delivery mechanisms, device formats employed, and various biomedical applications. The integration of dosing control systems, examples of commercially available microtechnology-enabled drug delivery devices, remaining challenges, and future outlook are also discussed. PMID:25703045

  5. New Generation Sensor Web Enablement

    PubMed Central

    Bröring, Arne; Echterhoff, Johannes; Jirka, Simon; Simonis, Ingo; Everding, Thomas; Stasch, Christoph; Liang, Steve; Lemmens, Rob

    2011-01-01

    Many sensor networks have been deployed to monitor Earth’s environment, and more will follow in the future. Environmental sensors have improved continuously by becoming smaller, cheaper, and more intelligent. Due to the large number of sensor manufacturers and differing accompanying protocols, integrating diverse sensors into observation systems is not straightforward. A coherent infrastructure is needed to treat sensors in an interoperable, platform-independent and uniform way. The concept of the Sensor Web reflects such a kind of infrastructure for sharing, finding, and accessing sensors and their data across different applications. It hides the heterogeneous sensor hardware and communication protocols from the applications built on top of it. The Sensor Web Enablement initiative of the Open Geospatial Consortium standardizes web service interfaces and data encodings which can be used as building blocks for a Sensor Web. This article illustrates and analyzes the recent developments of the new generation of the Sensor Web Enablement specification framework. Further, we relate the Sensor Web to other emerging concepts such as the Web of Things and point out challenges and resulting future work topics for research on Sensor Web Enablement. PMID:22163760

  6. 'Ethos' Enabling Organisational Knowledge Creation

    NASA Astrophysics Data System (ADS)

    Matsudaira, Yoshito

    This paper examines knowledge creation in relation to improvements on the production line in the manufacturing department of Nissan Motor Company and aims to clarify embodied knowledge observed in the actions of organisational members who enable knowledge creation will be clarified. For that purpose, this study adopts an approach that adds a first, second, and third-person's viewpoint to the theory of knowledge creation. Embodied knowledge, observed in the actions of organisational members who enable knowledge creation, is the continued practice of 'ethos' (in Greek) founded in Nissan Production Way as an ethical basis. Ethos is knowledge (intangible) assets for knowledge creating companies. Substantiated analysis classifies ethos into three categories: the individual, team and organisation. This indicates the precise actions of the organisational members in each category during the knowledge creation process. This research will be successful in its role of showing the indispensability of ethos - the new concept of knowledge assets, which enables knowledge creation -for future knowledge-based management in the knowledge society.

  7. Studying mechanisms of cAMP and cyclic nucleotide phosphodiesterase signaling in Leydig cell function with phosphoproteomics.

    PubMed

    Golkowski, Martin; Shimizu-Albergine, Masami; Suh, Hyong Won; Beavo, Joseph A; Ong, Shao-En

    2016-07-01

    Many cellular processes are modulated by cyclic AMP and nucleotide phosphodiesterases (PDEs) regulate this second messenger by catalyzing its breakdown. The major unique function of testicular Leydig cells is to produce testosterone in response to luteinizing hormone (LH). Treatment of Leydig cells with PDE inhibitors increases cAMP levels and the activity of its downstream effector, cAMP-dependent protein kinase (PKA), leading to a series of kinase-dependent signaling and transcription events that ultimately increase testosterone release. We have recently shown that PDE4B and PDE4C as well as PDE8A and PDE8B are expressed in rodent Leydig cells and that combined inhibition of PDE4 and PDE8 leads to dramatically increased steroid biosynthesis. Here we investigated the effect of PDE4 and PDE8 inhibition on the molecular mechanisms of cAMP actions in a mouse MA10 Leydig cell line model with SILAC mass spectrometry-based phosphoproteomics. We treated MA10 cells either with PDE4 family specific inhibitor (Rolipram) and PDE8 family specific inhibitor (PF-04957325) alone or in combination and quantified the resulting phosphorylation changes at five different time points between 0 and 180min. We identified 28,336 phosphosites from 4837 proteins and observed significant regulation of 749 sites in response to PDE4 and PDE8 inhibitor treatment. Of these, 132 phosphosites were consensus PKA sites. Our data strongly suggest that PDE4 and PDE8 inhibitors synergistically regulate phosphorylation of proteins required for many different cellular processes, including cell cycle progression, lipid and glucose metabolism, transcription, endocytosis and vesicle transport. Our data suggests that cAMP, PDE4 and PDE8 coordinate steroidogenesis by acting on not one rate-limiting step but rather multiple pathways. Moreover, the pools of cAMP controlled by these PDEs also coordinate many other metabolic processes that may be regulated to assure timely and sufficient testosterone secretion

  8. Exploratory investigation on nitro- and phospho-proteome cerebellum changes in hyperammonemia and hepatic encephalopathy rat models.

    PubMed

    Brunelli, Laura; Campagna, Roberta; Airoldi, Luisa; Cauli, Omar; Llansola, Marta; Boix, Jordi; Felipo, Vicente; Pastorelli, Roberta

    2012-03-01

    Hepatic encephalopathy (HE) is a neurological disease associated with hepatic dysfunction. Current knowledge suggests that hyperammonemia, related to liver failure, is a main factor contributing to the cerebral alterations in HE and that hyperammonemia might impair signal transduction associated with post-translational modification of proteins such as tyrosine-nitration and phosphorylation. However, the molecular bases of the HE remain unclear and very little is known about the occurrence of post-translational modification on in vivo proteins. In this exploratory study we look for evidence of post-translation modifications of proteins in the cerebellum of experimental HE rat models using a proteomic approach. For the first time we showed that hyperammonemia without liver failure (HA rats) and experimental HE with liver failure due to portacaval shunt (PCS rats) lead to a reduced protein nitration in rat cerebellum, where the undernitrated proteins were involved in energy metabolism and cytoskeleton remodelling. Moreover we showed that tyrosine nitration loss of these proteins was not necessarily associated to a change in their phosphorylation state as result of the disease. Interestingly the rat cerebellum phosphoproteome was mainly perturbed in PCS rats, whereas HA rats did not shown appreciable changes in their phosphoprotein profile. Since the protein nitration level decreased similarly in the cerebellum of both HA and PCS rats, this implies that the two disease models share common effects but also present some differential signalling effects in the cerebellum of the same animals. This study highlights the interest for studying the concerted action of multiple signalling pathways in HE development. PMID:22083566

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

    SciTech Connect

    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

  10. The G Protein-Coupled Estrogen Receptor Agonist G-1 Inhibits Nuclear Estrogen Receptor Activity and Stimulates Novel Phosphoproteomic Signatures.

    PubMed

    Smith, L Cody; Ralston-Hooper, Kimberly J; Ferguson, P Lee; Sabo-Attwood, Tara

    2016-06-01

    Estrogen exerts cellular effects through both nuclear (ESR1 and ESR2) and membrane-bound estrogen receptors (G-protein coupled estrogen receptor, GPER); however, it is unclear if they act independently or engage in crosstalk to influence hormonal responses. To investigate each receptor's role in proliferation, transcriptional activation, and protein phosphorylation in breast cancer cells (MCF-7), we employed selective agonists for ESR1 propyl-pyrazole-triol (PPT), ESR2 diarylpropionitrile (DPN), and GPER (G-1) and also determined the impact of xenoestrogens bisphenol-A (BPA) and genistein on these effects. As anticipated, 17β-estradiol (E2), PPT, DPN, BPA, and genistein each enhanced proliferation and activation of an ERE-driven reporter gene whereas G-1 had no significant impact. However, G-1 significantly reduced E2-, PPT-, DPN-, BPA-, and genistein-induced proliferation and ERE activation at doses greater than 500 nM indicating that G-1 mediated inhibition is not ESR isotype specific. As membrane receptors initiate cascades of phosphorylation events, we performed a global phosphoproteomic analysis on cells exposed to E2 or G-1 to identify potential targets of receptor crosstalk via downstream protein phosphorylation targets. Of the 211 phosphorylated proteins identified, 40 and 13 phosphoproteins were specifically modified by E2 and G-1, respectively. Subnetwork enrichment analysis revealed several processes related to cell cycle were specifically enriched by G-1 compared with E2. Further there existed a number of newly identified proteins that were specifically phosphorylated by G-1. These phosphorylation networks highlight specific proteins that may modulate the inhibitory effects of G-1 and suggest a novel role for interference with nuclear receptor activity driven by E2 and xenoestrogens. PMID:27026707

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

    PubMed Central

    Avey, Denis; Tepper, Sarah; Li, Wenwei; Turpin, Zachary; Zhu, Fanxiu

    2015-01-01

    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 manipulates

  12. Proteome and phosphoproteome analysis of honeybee (Apis mellifera) venom collected from electrical stimulation and manual extraction of the venom gland

    PubMed Central

    2013-01-01

    Background Honeybee venom is a complicated defensive toxin that has a wide range of pharmacologically active compounds. Some of these compounds are useful for human therapeutics. There are two major forms of honeybee venom used in pharmacological applications: manually (or reservoir disrupting) extracted glandular venom (GV), and venom extracted through the use of electrical stimulation (ESV). A proteome comparison of these two venom forms and an understanding of the phosphorylation status of ESV, are still very limited. Here, the proteomes of GV and ESV were compared using both gel-based and gel-free proteomics approaches and the phosphoproteome of ESV was determined through the use of TiO2 enrichment. Results Of the 43 proteins identified in GV, < 40% were venom toxins, and > 60% of the proteins were non-toxic proteins resulting from contamination by gland tissue damage during extraction and bee death. Of the 17 proteins identified in ESV, 14 proteins (>80%) were venom toxic proteins and most of them were found in higher abundance than in GV. Moreover, two novel proteins (dehydrogenase/reductase SDR family member 11-like and histone H2B.3-like) and three novel phosphorylation sites (icarapin (S43), phospholipase A-2 (T145), and apamin (T23)) were identified. Conclusions Our data demonstrate that venom extracted manually is different from venom extracted using ESV, and these differences may be important in their use as pharmacological agents. ESV may be more efficient than GV as a potential pharmacological source because of its higher venom protein content, production efficiency, and without the need to kill honeybee. The three newly identified phosphorylated venom proteins in ESV may elicit a different immune response through the specific recognition of antigenic determinants. The two novel venom proteins extend our proteome coverage of honeybee venom. PMID:24199871

  13. Improved Proteome and Phosphoproteome Analysis on a Cation Exchanger by a Combined Acid and Salt Gradient.

    PubMed

    Adachi, Jun; Hashiguchi, Kazunari; Nagano, Maiko; Sato, Misako; Sato, Ayako; Fukamizu, Kazuna; Ishihama, Yasushi; Tomonaga, Takeshi

    2016-08-16

    Currently used elution methods for strong cation exchange (SCX) chromatography are based on two principles: salt and pH gradient. In this paper, we report the first observation of peptide elution by acid gradient. The degree of peptide separation using C18-SCX StageTip was greatly improved by our acid and salt-based elution method compared with a salt-based elution method. This development enabled us to identify over 22 000 phosphopeptides from 2 mg of protein without labor-intensive sample preparation. Our method is simple, robust, scalable, and low-cost and can be easily implemented without any special equipment or techniques. PMID:27436111

  14. A Comparison of the Chicken and Turkey Proteomes and Phosphoproteomes in the Development of Poultry-Specific Immuno-Metabolism Kinome Peptide Arrays

    PubMed Central

    Arsenault, Ryan J.; Trost, Brett; Kogut, Michael H.

    2014-01-01

    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. PMID:26664921

  15. Optimized microsystems-enabled photovoltaics

    SciTech Connect

    Cruz-Campa, Jose Luis; Nielson, Gregory N.; Young, Ralph W.; Resnick, Paul J.; Okandan, Murat; Gupta, Vipin P.

    2015-09-22

    Technologies pertaining to designing microsystems-enabled photovoltaic (MEPV) cells are described herein. A first restriction for a first parameter of an MEPV cell is received. Subsequently, a selection of a second parameter of the MEPV cell is received. Values for a plurality of parameters of the MEPV cell are computed such that the MEPV cell is optimized with respect to the second parameter, wherein the values for the plurality of parameters are computed based at least in part upon the restriction for the first parameter.

  16. Directory Enabled Policy Based Networking

    SciTech Connect

    KELIIAA, CURTIS M.

    2001-10-01

    This report presents a discussion of directory-enabled policy-based networking with an emphasis on its role as the foundation for securely scalable enterprise networks. A directory service provides the object-oriented logical environment for interactive cyber-policy implementation. Cyber-policy implementation includes security, network management, operational process and quality of service policies. The leading network-technology vendors have invested in these technologies for secure universal connectivity that transverses Internet, extranet and intranet boundaries. Industry standards are established that provide the fundamental guidelines for directory deployment scalable to global networks. The integration of policy-based networking with directory-service technologies provides for intelligent management of the enterprise network environment as an end-to-end system of related clients, services and resources. This architecture allows logical policies to protect data, manage security and provision critical network services permitting a proactive defense-in-depth cyber-security posture. Enterprise networking imposes the consideration of supporting multiple computing platforms, sites and business-operation models. An industry-standards based approach combined with principled systems engineering in the deployment of these technologies allows these issues to be successfully addressed. This discussion is focused on a directory-based policy architecture for the heterogeneous enterprise network-computing environment and does not propose specific vendor solutions. This document is written to present practical design methodology and provide an understanding of the risks, complexities and most important, the benefits of directory-enabled policy-based networking.

  17. Nanomaterial-Enabled Neural Stimulation.

    PubMed

    Wang, Yongchen; Guo, Liang

    2016-01-01

    Neural stimulation is a critical technique in treating neurological diseases and investigating brain functions. Traditional electrical stimulation uses electrodes to directly create intervening electric fields in the immediate vicinity of neural tissues. Second-generation stimulation techniques directly use light, magnetic fields or ultrasound in a non-contact manner. An emerging generation of non- or minimally invasive neural stimulation techniques is enabled by nanotechnology to achieve a high spatial resolution and cell-type specificity. In these techniques, a nanomaterial converts a remotely transmitted primary stimulus such as a light, magnetic or ultrasonic signal to a localized secondary stimulus such as an electric field or heat to stimulate neurons. The ease of surface modification and bio-conjugation of nanomaterials facilitates cell-type-specific targeting, designated placement and highly localized membrane activation. This review focuses on nanomaterial-enabled neural stimulation techniques primarily involving opto-electric, opto-thermal, magneto-electric, magneto-thermal and acousto-electric transduction mechanisms. Stimulation techniques based on other possible transduction schemes and general consideration for these emerging neurotechnologies are also discussed. PMID:27013938

  18. Enabling Exploration Through Docking Standards

    NASA Technical Reports Server (NTRS)

    Hatfield, Caris A.

    2012-01-01

    Human exploration missions beyond low earth orbit will likely require international cooperation in order to leverage limited resources. International standards can help enable cooperative missions by providing well understood, predefined interfaces allowing compatibility between unique spacecraft and systems. The International Space Station (ISS) partnership has developed a publicly available International Docking System Standard (IDSS) that provides a solution to one of these key interfaces by defining a common docking interface. The docking interface provides a way for even dissimilar spacecraft to dock for exchange of crew and cargo, as well as enabling the assembly of large space systems. This paper provides an overview of the key attributes of the IDSS, an overview of the NASA Docking System (NDS), and the plans for updating the ISS with IDSS compatible interfaces. The NDS provides a state of the art, low impact docking system that will initially be made available to commercial crew and cargo providers. The ISS will be used to demonstrate the operational utility of the IDSS interface as a foundational technology for cooperative exploration.

  19. Nanomaterial-Enabled Neural Stimulation

    PubMed Central

    Wang, Yongchen; Guo, Liang

    2016-01-01

    Neural stimulation is a critical technique in treating neurological diseases and investigating brain functions. Traditional electrical stimulation uses electrodes to directly create intervening electric fields in the immediate vicinity of neural tissues. Second-generation stimulation techniques directly use light, magnetic fields or ultrasound in a non-contact manner. An emerging generation of non- or minimally invasive neural stimulation techniques is enabled by nanotechnology to achieve a high spatial resolution and cell-type specificity. In these techniques, a nanomaterial converts a remotely transmitted primary stimulus such as a light, magnetic or ultrasonic signal to a localized secondary stimulus such as an electric field or heat to stimulate neurons. The ease of surface modification and bio-conjugation of nanomaterials facilitates cell-type-specific targeting, designated placement and highly localized membrane activation. This review focuses on nanomaterial-enabled neural stimulation techniques primarily involving opto-electric, opto-thermal, magneto-electric, magneto-thermal and acousto-electric transduction mechanisms. Stimulation techniques based on other possible transduction schemes and general consideration for these emerging neurotechnologies are also discussed. PMID:27013938

  20. Rotational propulsion enabled by inertia.

    PubMed

    Nadal, François; Pak, On Shun; Zhu, LaiLai; Brandt, Luca; Lauga, Eric

    2014-07-01

    The fluid mechanics of small-scale locomotion has recently attracted considerable attention, due to its importance in cell motility and the design of artificial micro-swimmers for biomedical applications. Most studies on the topic consider the ideal limit of zero Reynolds number. In this paper, we investigate a simple propulsion mechanism --an up-down asymmetric dumbbell rotating about its axis of symmetry-- unable to propel in the absence of inertia in a Newtonian fluid. Inertial forces lead to continuous propulsion for all finite values of the Reynolds number. We study computationally its propulsive characteristics as well as analytically in the small-Reynolds-number limit. We also derive the optimal dumbbell geometry. The direction of propulsion enabled by inertia is opposite to that induced by viscoelasticity. PMID:25034393

  1. Simulation Enabled Safeguards Assessment Methodology

    SciTech Connect

    Robert Bean; Trond Bjornard; Thomas Larson

    2007-09-01

    It is expected that nuclear energy will be a significant component of future supplies. New facilities, operating under a strengthened international nonproliferation regime will be needed. There is good reason to believe virtual engineering applied to the facility design, as well as to the safeguards system design will reduce total project cost and improve efficiency in the design cycle. Simulation Enabled Safeguards Assessment MEthodology (SESAME) has been developed as a software package to provide this capability for nuclear reprocessing facilities. The software architecture is specifically designed for distributed computing, collaborative design efforts, and modular construction to allow step improvements in functionality. Drag and drop wireframe construction allows the user to select the desired components from a component warehouse, render the system for 3D visualization, and, linked to a set of physics libraries and/or computational codes, conduct process evaluations of the system they have designed.

  2. Context-Enabled Business Intelligence

    SciTech Connect

    Troy Hiltbrand

    2012-04-01

    To truly understand context and apply it in business intelligence, it is vital to understand what context is and how it can be applied in addressing organizational needs. Context describes the facets of the environment that impact the way that end users interact with the system. Context includes aspects of location, chronology, access method, demographics, social influence/ relationships, end-user attitude/ emotional state, behavior/ past behavior, and presence. To be successful in making Business Intelligence content enabled, it is important to be able to capture the context of use user. With advances in technology, there are a number of ways in which this user based information can be gathered and exposed to enhance the overall end user experience.

  3. Imaging enabled platforms for development of therapeutics

    NASA Astrophysics Data System (ADS)

    Celli, Jonathan; Rizvi, Imran; Blanden, Adam R.; Evans, Conor L.; Abu-Yousif, Adnan O.; Spring, Bryan Q.; Muzikansky, Alona; Pogue, Brian W.; Finkelstein, Dianne M.; Hasan, Tayyaba

    2011-03-01

    Advances in imaging and spectroscopic technologies have enabled the optimization of many therapeutic modalities in cancer and noncancer pathologies either by earlier disease detection or by allowing therapy monitoring. Amongst the therapeutic options benefiting from developments in imaging technologies, photodynamic therapy (PDT) is exceptional. PDT is a photochemistry-based therapeutic approach where a light-sensitive molecule (photosensitizer) is activated with light of appropriate energy (wavelength) to produce reactive molecular species such as free radicals and singlet oxygen. These molecular entities then react with biological targets such as DNA, membranes and other cellular components to impair their function and lead to eventual cell and tissue death. Development of PDT-based imaging also provides a platform for rapid screening of new therapeutics in novel in vitro models prior to expensive and labor-intensive animal studies. In this study we demonstrate how an imaging platform can be used for strategizing a novel combination treatment strategy for multifocal ovarian cancer. Using an in vitro 3D model for micrometastatic ovarian cancer in conjunction with quantitative imaging we examine dose and scheduling strategies for PDT in combination with carboplatin, a chemotherapeutic agent presently in clinical use for management of this deadly form of cancer.

  4. Activating Mutations in PIK3CA Lead to Widespread Modulation of the Tyrosine Phosphoproteome

    PubMed Central

    Blair, Brian G.; Pinto, Sneha M.; Nirujogi, Raja S.; Jelinek, Christine A.; Malhotra, Radhika; Kim, Min-Sik; Park, Ben Ho; Pandey, Akhilesh

    2015-01-01

    The human oncogene PIK3CA is frequently mutated in human cancers. Two hotspot mutations in PIK3CA, E545K and H1047R, have been shown to regulate widespread signaling events downstream of AKT, leading to increased cell proliferation, growth, survival, and motility. We used quantitative mass spectrometry to profile the global phosphotyrosine proteome of isogenic knock-in cell lines containing these activating mutations, where we identified 824 unique phosphopeptides. Although it is well understood that these mutations result in hyperactivation of the serine/threonine kinase AKT, we found a surprisingly widespread modulation of tyrosine phosphorylation levels of proteins in the mutant cells. In the tyrosine kinome alone, 29 tyrosine kinases were altered in their phosphorylation status. Many of the regulated phosphosites that we identified were located in the kinase domain or the canonical activation sites, indicating that these kinases and their downstream signaling pathways were activated. Our study demonstrates that there is frequent and unexpected cross-talk that occurs between tyrosine signaling pathways and serine/threonine signaling pathways activated by the canonical PI3K-AKT axis. PMID:26267517

  5. Relative quantification of phosphoproteomic changes in grapevine (Vitis vinifera L.) leaves in response to abscisic acid.

    PubMed

    Rattanakan, Supakan; George, Iniga; Haynes, Paul A; Cramer, Grant R

    2016-01-01

    In a previous transcriptomic analysis, abscisic acid (ABA) was found to affect the abundance of a number of transcripts in leaves of Cabernet Sauvignon grapevines with roots that had been exposed to 10 μm ABA for 2 h. Other work has indicated that ABA affects protein abundance and protein phosphorylation as well. In this study we investigated changes in protein abundance and phosphorylation of Cabernet Sauvignon grapevine leaves. Protein abundance was assessed by both label-free and isobaric-label quantitive proteomic methods. Each identified common proteins, but also additional proteins not found with the other method. Overall, several thousand proteins were identified and several hundred were quantified. In addition, hundreds of phosphoproteins were identified. Tens of proteins were found to be affected in the leaf after the roots had been exposed to ABA for 2 h, more than half of them were phosphorylated proteins. Many phosphosites were confirmed and several new ones were identified. ABA increased the abundance of some proteins, but the majority of the proteins had their protein abundance decreased. Many of these proteins were involved in growth and plant organ development, including proteins involved in protein synthesis, photosynthesis, sugar and amino-acid metabolism. This study provides new insights into how ABA regulates plant responses and acclimation to water deficits. PMID:27366326

  6. Relative quantification of phosphoproteomic changes in grapevine (Vitis vinifera L.) leaves in response to abscisic acid

    PubMed Central

    Rattanakan, Supakan; George, Iniga; Haynes, Paul A; Cramer, Grant R

    2016-01-01

    In a previous transcriptomic analysis, abscisic acid (ABA) was found to affect the abundance of a number of transcripts in leaves of Cabernet Sauvignon grapevines with roots that had been exposed to 10 μm ABA for 2 h. Other work has indicated that ABA affects protein abundance and protein phosphorylation as well. In this study we investigated changes in protein abundance and phosphorylation of Cabernet Sauvignon grapevine leaves. Protein abundance was assessed by both label-free and isobaric-label quantitive proteomic methods. Each identified common proteins, but also additional proteins not found with the other method. Overall, several thousand proteins were identified and several hundred were quantified. In addition, hundreds of phosphoproteins were identified. Tens of proteins were found to be affected in the leaf after the roots had been exposed to ABA for 2 h, more than half of them were phosphorylated proteins. Many phosphosites were confirmed and several new ones were identified. ABA increased the abundance of some proteins, but the majority of the proteins had their protein abundance decreased. Many of these proteins were involved in growth and plant organ development, including proteins involved in protein synthesis, photosynthesis, sugar and amino-acid metabolism. This study provides new insights into how ABA regulates plant responses and acclimation to water deficits. PMID:27366326

  7. Global analysis of phosphoproteome dynamics in embryonic development of zebrafish (Danio rerio).

    PubMed

    Kwon, Oh Kwang; Kim, Sun Ju; Lee, You-Mie; Lee, Young-Hoon; Bae, Young-Seuk; Kim, Jin Young; Peng, Xiaojun; Cheng, Zhongyi; Zhao, Yingming; Lee, Sangkyu

    2016-01-01

    The zebrafish (Danio rerio) is a popular animal model used for studies on vertebrate development and organogenesis. Recent research has shown a similarity of approximately 70% between the human and zebrafish genomes and about 84% of human disease-causing genes have common ancestry with that of the zebrafish genes. Zebrafish embryos have a number of desirable features, including transparency, a large size, and rapid embryogenesis. Protein phosphorylation is a well-known PTM, which can carry out various biological functions. Recent MS developments have enabled the study of global phosphorylation patterns by using MS-based proteomics coupled with titanium dioxide phosphopeptide enrichment. In the present study, we identified 3500 nonredundant phosphorylation sites on 2166 phosphoproteins and quantified 1564 phosphoproteins in developing embryos of zebrafish. The distribution of Ser/Thr/Tyr phosphorylation sites in zebrafish embryos was found to be 88.9, 10.2, and 0.9%, respectively. A potential kinase motif was predicted using Motif-X analysis, for 80% of the identified phosphorylation sites, with the proline-directed motif appearing most frequently, and 35 phosphorylation sites having the pSF motif. In addition, we created six phosphoprotein clusters based on their dynamic pattern during the development of zebrafish embryos. Here, we report the largest dataset of phosphoproteins in zebrafish embryos and our results can be used for further studies on phosphorylation sites or phosphoprotein dynamics in zebrafish embryos. PMID:26449285

  8. Functional phosphoproteomic profiling of phosphorylation sites in membrane fractions of salt-stressed Arabidopsis thaliana

    PubMed Central

    2009-01-01

    Background Under conditions of salt stress, plants respond by initiating phosphorylation cascades. Many key phosphorylation events occur at the membrane. However, to date only limited sites have been identified that are phosphorylated in response to salt stress in plants. Results Membrane fractions from three-day and 200 mM salt-treated Arabidopsis suspension plants were isolated, followed by protease shaving and enrichment using Zirconium ion-charged magnetic beads, and tandem mass spectrometry analyses. From this isolation, 18 phosphorylation sites from 15 Arabidopsis proteins were identified. A unique phosphorylation site in 14-3-3-interacting protein AHA1 was predominately identified in 200 mM salt-treated plants. We also identified some phosphorylation sites in aquaporins. A doubly phosphorylated peptide of PIP2;1 as well as a phosphopeptide containing a single phosphorylation site (Ser-283) and a phosphopeptide containing another site (Ser-286) of aquaporin PIP2;4 were identified respectively. These two sites appeared to be novel of which were not reported before. In addition, quantitative analyses of protein phosphorylation with either label-free or stable-isotope labeling were also employed in this study. The results indicated that level of phosphopeptides on five membrane proteins such as AHA1, STP1, Patellin-2, probable inactive receptor kinase (At3g02880), and probable purine permease 18 showed at least two-fold increase in comparison to control in response to 200 mM salt-stress. Conclusion In this study, we successfully identified novel salt stress-responsive protein phosphorylation sites from membrane isolates of abiotic-stressed plants by membrane shaving followed by Zr4+-IMAC enrichment. The identified phosphorylation sites can be important in the salt stress response in plants. PMID:19900291

  9. Identifying novel targets of oncogenic EGF receptor signaling in lung cancer through global phosphoproteomics.

    PubMed

    Zhang, Xu; Belkina, Natalya; Jacob, Harrys Kishore Charles; Maity, Tapan; Biswas, Romi; Venugopalan, Abhilash; Shaw, Patrick G; Kim, Min-Sik; Chaerkady, Raghothama; Pandey, Akhilesh; Guha, Udayan

    2015-01-01

    Mutations in the epidermal growth factor receptor (EGFR) kinase domain occur in 10-30% of lung adenocarcinoma and are associated with tyrosine kinase inhibitor (TKI) sensitivity. We sought to identify the immediate direct and indirect phosphorylation targets of mutant EGFRs in lung adenocarcinoma. We undertook SILAC strategy, phosphopeptide enrichment, and quantitative MS to identify dynamic changes of phosphorylation downstream of mutant EGFRs in lung adenocarcinoma cells harboring EGFR(L858R) and EGFR(L858R/T790M) , the TKI-sensitive, and TKI-resistant mutations, respectively. Top canonical pathways that were inhibited upon erlotinib treatment in sensitive cells, but not in the resistant cells include EGFR, insulin receptor, hepatocyte growth factor, mitogen-activated protein kinase, mechanistic target of rapamycin, ribosomal protein S6 kinase beta 1, and Janus kinase/signal transducer and activator of transcription signaling. We identified phosphosites in proteins of the autophagy network, such as ULK1 (S623) that is constitutively phosphorylated in these lung adenocarcinoma cells; phosphorylation is inhibited upon erlotinib treatment in sensitive cells, but not in resistant cells. Finally, kinase-substrate prediction analysis from our data indicated that substrates of basophilic kinases from, AGC and Calcium and calmodulin-dependent kinase groups, as well as STE group kinases were significantly enriched and those of proline-directed kinases from, CMGC and Casein kinase groups were significantly depleted among substrates that exhibited increased phosphorylation upon EGF stimulation and reduced phosphorylation upon TKI inhibition. This is the first study to date to examine global phosphorylation changes upon erlotinib treatment of lung adenocarcinoma cells and results from this study provide new insights into signaling downstream of mutant EGFRs in lung adenocarcinoma. All MS data have been deposited in the ProteomeXchange with identifier PXD001101 (http

  10. Global Phosphoproteome Profiling Reveals Unanticipated Networks Responsive to Cisplatin Treatment of Embryonic Stem Cells ▿ †

    PubMed Central

    Pines, Alex; Kelstrup, Christian D.; Vrouwe, Mischa G.; Puigvert, Jordi C.; Typas, Dimitris; Misovic, Branislav; de Groot, Anton; von Stechow, Louise; van de Water, Bob; Danen, Erik H. J.; Vrieling, Harry; Mullenders, Leon H. F.; Olsen, Jesper V.

    2011-01-01

    Cellular responses to DNA-damaging agents involve the activation of various DNA damage signaling and transduction pathways. Using quantitative and high-resolution tandem mass spectrometry, we determined global changes in protein level and phosphorylation site profiles following treatment of SILAC (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 telangiectasia mutated) and ATR (ATM and Rad3-related) consensus sequence (S/T-Q motif) was significantly overrepresented among hyperphosphorylated peptides, about half of the >2-fold-upregulated phosphorylation sites based on the consensus sequence were not direct substrates of ATM and ATR. Eleven protein kinases mainly belonging to the mitogen-activated protein kinase (MAPK) family were identified as being regulated in their kinase domain activation loop. The biological importance of three of these kinases (cyclin-dependent kinase 7 [CDK7], Plk1, and KPCD1) in the protection against cisplatin-induced cytotoxicity was demonstrated by small interfering RNA (siRNA)-mediated knockdown. Our results indicate that the cellular response to cisplatin involves a variety of kinases and phosphatases not only acting in the nucleus but also regulating cytoplasmic targets, resulting in extensive cytoskeletal rearrangements. Integration of transcriptomic and proteomic data revealed a poor correlation between changes in the relative levels of transcripts and their corresponding proteins, but a large overlap in affected pathways at the levels of mRNA, protein, and phosphoprotein. This study provides an integrated view of pathways activated by genotoxic stress and deciphers kinases that play a pivotal role in regulating cellular processes other than the DNA damage response. PMID:22006019

  11. Enabling technology for human collaboration.

    SciTech Connect

    Murphy, Tim Andrew; Jones, Wendell Bruce; Warner, David Jay; Doser, Adele Beatrice; Johnson, Curtis Martin; Merkle, Peter Benedict

    2003-11-01

    This report summarizes the results of a five-month LDRD late start project which explored the potential of enabling technology to improve the performance of small groups. The purpose was to investigate and develop new methods to assist groups working in high consequence, high stress, ambiguous and time critical situations, especially those for which it is impractical to adequately train or prepare. A testbed was constructed for exploratory analysis of a small group engaged in tasks with high cognitive and communication performance requirements. The system consisted of five computer stations, four with special devices equipped to collect physiologic, somatic, audio and video data. Test subjects were recruited and engaged in a cooperative video game. Each team member was provided with a sensor array for physiologic and somatic data collection while playing the video game. We explored the potential for real-time signal analysis to provide information that enables emergent and desirable group behavior and improved task performance. The data collected in this study included audio, video, game scores, physiological, somatic, keystroke, and mouse movement data. The use of self-organizing maps (SOMs) was explored to search for emergent trends in the physiological data as it correlated with the video, audio and game scores. This exploration resulted in the development of two approaches for analysis, to be used concurrently, an individual SOM and a group SOM. The individual SOM was trained using the unique data of each person, and was used to monitor the effectiveness and stress level of each member of the group. The group SOM was trained using the data of the entire group, and was used to monitor the group effectiveness and dynamics. Results suggested that both types of SOMs were required to adequately track evolutions and shifts in group effectiveness. Four subjects were used in the data collection and development of these tools. This report documents a proof of concept

  12. Enabling Participation In Exoplanet Science

    NASA Astrophysics Data System (ADS)

    Taylor, Stuart F.

    2015-08-01

    Determining the distribution of exoplanets has required the contributions of a community of astronomers, who all require the support of colleagues to finish their projects in a manner to enable them to enter new collaborations to continue to contribute to understanding exoplanet science.The contributions of each member of the astronomy community are to be encouraged and must never be intentionally obstructed.We present a member’s long pursuit to be a contributing part of the exoplanet community through doing transit photometry as a means of commissioning the telescopes for a new observatory, followed by pursuit of interpreting the distributions in exoplanet parameter data.We present how the photometry projects have been presented as successful by the others who have claimed to have completed them, but how by requiring its employees to present results while omitting one member has been obstructive against members working together and has prevented the results from being published in what can genuinely be called a peer-reviewed fashion.We present how by tolerating one group to obstruct one member from finishing participation and then falsely denying credit is counterproductive to doing science.We show how expecting one member to attempt to go around an ostracizing group by starting something different is destructive to the entire profession. We repeat previously published appeals to help ostracized members to “go around the observatory” by calling for discussion on how the community must act to reverse cases of shunning, bullying, and other abuses. Without better recourse and support from the community, actions that do not meet standard good collegial behavior end up forcing good members from the community. The most important actions are to enable an ostracized member to have recourse to participating in group papers by either working through other authors or through the journal. All journals and authors must expect that no co-author is keeping out a major

  13. DMD-enabled confocal microendoscopy

    NASA Astrophysics Data System (ADS)

    Lane, Pierre M.; Dlugan, Andrew L. P.; MacAulay, Calum E.

    2001-05-01

    Conventional endoscopy is limited to imaging macroscopic views of tissue. The British Columbia Cancer Research Center, in collaboration with Digital Optical Imaging Corp., is developing a fiber-bundle based microendoscopy system to enable in vivo confocal imaging of cells and tissue structure through the biopsy channel of an endoscope, hypodermic needle, or catheter. The feasibility of imaging individual cells and tissue architecture will be presented using both reflectance and tissue auto-fluorescence modes of imaging. The system consists of a coherent fiber bundle, low-magnification high-NA objective lens, Digital Micromirror DeviceTM(DMD), light source, and CCD camera. The novel approach is the precise control and manipulation of light flow into and out of individual optical fibers. This control is achieved by employing a DMD to illuminate and detect light from selected fibers such that only the core of each fiber is illuminated or detected. The objective of the research is to develop a low-cost, clinically viable microendoscopy system for a range of detection, diagnostic, localization and differentiation uses associated with cancer and pre-cancerous conditions. Currently, multi-wavelength reflectance confocal images with 1 micrometers lateral resolution and 1.6 micrometers axial resolution have been achieved using a 0.95 mm bundle with 30,000 fibers.

  14. Hydrophilic polydopamine-coated graphene for metal ion immobilization as a novel immobilized metal ion affinity chromatography platform for phosphoproteome analysis.

    PubMed

    Yan, Yinghua; Zheng, Zhifang; Deng, Chunhui; Li, Yan; Zhang, Xiangmin; Yang, Pengyuan

    2013-09-17

    To discover trace phosphorylated proteins or peptides with great biological significance for in-depth phosphoproteome analysis, it is urgent to develop a novel technique for highly selective and effective enrichment of phosphopeptides. In this work, an IMAC (immobilized metal ion affinity chromatography) material with polydopamine coated on the surface of graphene and functionalized with titanium ions (denoted as Ti(4+)-G@PD) was initially designed and synthesized. The newly prepared Ti(4+)-G@PD with enhanced hydrophilicity and biological compatibility was characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and infrared (IR), and its performance for selective and effective enrichment of phosphopeptide was evaluated with both standard peptide mixtures and human serum. PMID:23941301

  15. Enabling individualized therapy through nanotechnology

    PubMed Central

    Sakamoto, Jason H.; van de Ven, Anne L.; Godin, Biana; Blanco, Elvin; Serda, Rita E.; Grattoni, Alessandro; Ziemys, Arturas; Bouamrani, Ali; Hu, Tony; Ranganathan, Shivakumar I.; De Rosa, Enrica; Martinez, Jonathan O.; Smid, Christine A.; Buchanan, Rachel M.; Lee, Sei-Young; Srinivasan, Srimeenakshi; Landry, Matthew; Meyn, Anne; Tasciotti, Ennio; Liu, Xuewu; Decuzzi, Paolo; Ferrari, Mauro

    2010-01-01

    Individualized medicine is the healthcare strategy that rebukes the idiomatic dogma of ‘losing sight of the forest for the trees’. We are entering a new era of healthcare where it is no longer acceptable to develop and market a drug that is effective for only 80% of the patient population. The emergence of “-omic” technologies (e.g. genomics, transcriptomics, proteomics, metabolomics) and advances in systems biology are magnifying the deficiencies of standardized therapy, which often provide little treatment latitude for accommodating patient physiologic idiosyncrasies. A personalized approach to medicine is not a novel concept. Ever since the scientific community began unraveling the mysteries of the genome, the promise of discarding generic treatment regimens in favor of patient-specific therapies became more feasible and realistic. One of the major scientific impediments of this movement towards personalized medicine has been the need for technological enablement. Nanotechnology is projected to play a critical role in patient-specific therapy; however, this transition will depend heavily upon the evolutionary development of a systems biology approach to clinical medicine based upon “-omic” technology analysis and integration. This manuscript provides a forward looking assessment of the promise of nanomedicine as it pertains to individualized medicine and establishes a technology “snapshot” of the current state of nano-based products over a vast array of clinical indications and range of patient specificity. Other issues such as market driven hurdles and regulatory compliance reform are anticipated to “self-correct” in accordance to scientific advancement and healthcare demand. These peripheral, non-scientific concerns are not addressed at length in this manuscript; however they do exist, and their impact to the paradigm shifting healthcare transformation towards individualized medicine will be critical for its success. PMID:20045055

  16. Solar Glitter -- Microsystems Enabled Photovoltaics

    NASA Astrophysics Data System (ADS)

    Nielson, Gregory N.

    2012-02-01

    Many products have significantly benefitted from, or been enabled by, the ability to manufacture structures at an ever decreasing length scale. Obvious examples of this include integrated circuits, flat panel displays, micro-scale sensors, and LED lighting. These industries have benefited from length scale effects in terms of improved performance, reduced cost, or new functionality (or a combination of these). In a similar manner, we are working to take advantage of length scale effects that exist within solar photovoltaic (PV) systems. While this is a significant step away from traditional approaches to solar power systems, the benefits in terms of new functionality, improved performance, and reduced cost for solar power are compelling. We are exploring scale effects that result from the size of the solar cells within the system. We have developed unique cells of both crystalline silicon and III-V materials that are very thin (5-20 microns thick) and have very small lateral dimensions (on the order of hundreds of microns across). These cells minimize the amount of expensive semiconductor material required for the system, allow improved cell performance, and provide an expanded design space for both module and system concepts allowing optimized power output and reduced module and balance of system costs. Furthermore, the small size of the cells allows for unique high-efficiency, high-flexibility PV panels and new building-integrated PV options that are currently unavailable. These benefits provide a pathway for PV power to become cost competitive with grid power and allow unique power solutions independent of grid power.

  17. Quantitative multiplexed quantum dot immunohistochemistry

    SciTech Connect

    Sweeney, E.; Ward, T.H.; Gray, N.; Womack, C.; Jayson, G.; Hughes, A.; Dive, C.; Byers, R.

    2008-09-19

    Quantum dots are photostable fluorescent semiconductor nanocrystals possessing wide excitation and bright narrow, symmetrical, emission spectra. These characteristics have engendered considerable interest in their application in multiplex immunohistochemistry for biomarker quantification and co-localisation in clinical samples. Robust quantitation allows biomarker validation, and there is growing need for multiplex staining due to limited quantity of clinical samples. Most reported multiplexed quantum dot staining used sequential methods that are laborious and impractical in a high-throughput setting. Problems associated with sequential multiplex staining have been investigated and a method developed using QDs conjugated to biotinylated primary antibodies, enabling simultaneous multiplex staining with three antibodies. CD34, Cytokeratin 18 and cleaved Caspase 3 were triplexed in tonsillar tissue using an 8 h protocol, each localised to separate cellular compartments. This demonstrates utility of the method for biomarker measurement enabling rapid measurement of multiple co-localised biomarkers on single paraffin tissue sections, of importance for clinical trial studies.

  18. Two Types of Bureaucracy: Enabling and Coercive.

    ERIC Educational Resources Information Center

    Adler, Paul S.; Borys, Bryan

    1996-01-01

    Proposes a conceptualization of workflow formalization that helps reconcile contrasting assessments of bureaucracy as alienating or enabling to employees. Uses research on equipment technology design to identify enabling and coercive types of formalization. Identifies some forces tending to discourage an enabling orientation and some persistent…

  19. Global quantitative analysis of phosphorylation underlying phencyclidine signaling and sensorimotor gating in the prefrontal cortex

    PubMed Central

    McClatchy, Daniel B.; Savas, Jeffrey N.; Martínez-Bartolomé, Salvador; Park, Sung Kyu; Maher, Pamela; Powell, Susan B.; Yates, John R.

    2015-01-01

    Prepulse inhibition (PPI) is an example of sensorimotor gating and deficits in PPI have been demonstrated in schizophrenia patients. Phencyclidine (PCP) suppression of PPI in animals has been studied to elucidate the pathological elements of schizophrenia. However, the molecular mechanisms underlying PCP treatment or PPI in the brain are still poorly understood. In this study, quantitative phosphoproteomic analysis was performed on the prefrontal cortex from rats that were subjected to PPI after being systemically injected with PCP or saline. PCP down-regulated phosphorylation events were significantly enriched in proteins associated with long-term potentiation (LTP). Importantly, this dataset identifies functionally novel phosphorylation sites on known LTP-associated signaling molecules. In addition, mutagenesis of a significantly altered phosphorylation site on xCT (SLC7A11), the light chain of system xc-, the cystine/glutamate antiporter, suggests that PCP also regulates the activity of this protein. Finally, new insights were also derived on PPI signaling independent of PCP treatment. This is the first quantitative phosphorylation proteomic analysis providing new molecular insights into sensorimotor gating. PMID:25869802

  20. A Semantic Image Annotation Model to Enable Integrative Translational Research

    PubMed Central

    Rubin, Daniel L.; Mongkolwat, Pattanasak; Channin, David S.

    2009-01-01

    Integrating and relating images with clinical and molecular data is a crucial activity in translational research, but challenging because the information in images is not explicit in standard computer-accessible formats. We have developed an ontology-based representation of the semantic contents of radiology images called AIM (Annotation and Image Markup). AIM specifies the quantitative and qualitative content that researchers extract from images. The AIM ontology enables semantic image annotation and markup, specifying the entities and relations necessary to describe images. AIM annotations, represented as instances in the ontology, enable key use cases for images in translational research such as disease status assessment, query, and inter-observer variation analysis. AIM will enable ontology-based query and mining of images, and integration of images with data in other ontology-annotated bioinformatics databases. Our ultimate goal is to enable researchers to link images with related scientific data so they can learn the biological and physiological significance of the image content. PMID:21347180

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

    PubMed

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

    2015-08-01

    Labeling-based proteomics is a powerful method for detection of differentially expressed proteins (DEPs). The current data analysis platform typically relies on protein-level ratios, which is obtained by summarizing peptide-level ratios for each protein. In shotgun proteomics, however, some proteins are quantified with more peptides than others, and this reproducibility information is not incorporated into the differential expression (DE) analysis. Here, we propose a novel probabilistic framework EBprot that directly models the peptide-protein hierarchy and rewards the proteins with reproducible evidence of DE over multiple peptides. To evaluate its performance with known DE states, we conducted a simulation study to show that the peptide-level analysis of EBprot provides better receiver-operating characteristic and more accurate estimation of the false discovery rates than the methods based on protein-level ratios. We also demonstrate superior classification performance of peptide-level EBprot analysis in a spike-in dataset. To illustrate the wide applicability of EBprot in different experimental designs, we applied EBprot to a dataset for lung cancer subtype analysis with biological replicates and another dataset for time course phosphoproteome analysis of EGF-stimulated HeLa cells with multiplexed labeling. Through these examples, we show that the peptide-level analysis of EBprot is a robust alternative to the existing statistical methods for the DE analysis of labeling-based quantitative datasets. The software suite is freely available on the Sourceforge website http://ebprot.sourceforge.net/. All MS data have been deposited in the ProteomeXchange with identifier PXD001426 (http://proteomecentral.proteomexchange.org/dataset/PXD001426/). PMID:25913743

  2. An Internet enabled impact limiter material database

    SciTech Connect

    Wix, S.; Kanipe, F.; McMurtry, W.

    1998-09-01

    This paper presents a detailed explanation of the construction of an interest enabled database, also known as a database driven web site. The data contained in the internet enabled database are impact limiter material and seal properties. The technique used in constructing the internet enabled database presented in this paper are applicable when information that is changing in content needs to be disseminated to a wide audience.

  3. Electronic Health Record Application Support Service Enablers.

    PubMed

    Neofytou, M S; Neokleous, K; Aristodemou, A; Constantinou, I; Antoniou, Z; Schiza, E C; Pattichis, C S; Schizas, C N

    2015-08-01

    There is a huge need for open source software solutions in the healthcare domain, given the flexibility, interoperability and resource savings characteristics they offer. In this context, this paper presents the development of three open source libraries - Specific Enablers (SEs) for eHealth applications that were developed under the European project titled "Future Internet Social and Technological Alignment Research" (FI-STAR) funded under the "Future Internet Public Private Partnership" (FI-PPP) program. The three SEs developed under the Electronic Health Record Application Support Service Enablers (EHR-EN) correspond to: a) an Electronic Health Record enabler (EHR SE), b) a patient summary enabler based on the EU project "European patient Summary Open Source services" (epSOS SE) supporting patient mobility and the offering of interoperable services, and c) a Picture Archiving and Communications System (PACS) enabler (PACS SE) based on the dcm4che open source system for the support of medical imaging functionality. The EHR SE follows the HL7 Clinical Document Architecture (CDA) V2.0 and supports the Integrating the Healthcare Enterprise (IHE) profiles (recently awarded in Connectathon 2015). These three FI-STAR platform enablers are designed to facilitate the deployment of innovative applications and value added services in the health care sector. They can be downloaded from the FI-STAR cataloque website. Work in progress focuses in the validation and evaluation scenarios for the proving and demonstration of the usability, applicability and adaptability of the proposed enablers. PMID:26736531

  4. Quantitative autoradiography of neurochemicals

    SciTech Connect

    Rainbow, T.C.; Biegon, A.; Bleisch, W.V.

    1982-05-24

    Several new methods have been developed that apply quantitative autoradiography to neurochemistry. These methods are derived from the 2-deoxyglucose (2DG) technique of Sokoloff (1), which uses quantitative autoradiography to measure the rate of glucose utilization in brain structures. The new methods allow the measurement of the rate of cerbral protein synthesis and the levels of particular neurotransmitter receptors by quantitative autoradiography. As with the 2DG method, the new techniques can measure molecular levels in micron-sized brain structures; and can be used in conjunction with computerized systems of image processing. It is possible that many neurochemical measurements could be made by computerized analysis of quantitative autoradiograms.

  5. Supramolecular Probes for Assessing Glutamine Uptake Enable Semi-Quantitative Metabolic Models in Single Cells

    PubMed Central

    Xue, Min; Wei, Wei; Su, Yapeng; Johnson, Dazy; Heath, James R.

    2016-01-01

    We describe a supramolecular surface competition assay for quantifying glutamine uptake from single cells. Cy3-labeled cyclodextrins were immobilized on a glass surface as a supramolecular host/FRET donor, and adamantane-BHQ2 conjugates were employed as the guest/quencher. An adamantane-labeled glutamine analog was selected through screening a library of compounds and validated by cell uptake experiments. When integrated onto a single cell barcode chip (SCBC) with a multiplex panel of 15 other metabolites, associated metabolic enzymes, and phosphoproteins, the resultant data provided input for a steady state model that describes energy potential in single cells, and correlates that potential with receptor tyrosine kinase signaling. We utilize this integrated assay to interrogate a dose-dependent response of model brain cancer cells to EGFR inhibition. We find that low dose (1 μM erlotinib) drugging actually increases cellular energy potential even as glucose uptake and phosphoprotein signaling is repressed. We also identify new interactions between phosphoprotein signaling and cellular energy processes that may help explain the facile resistance exhibited by certain cancer patients to EGFR inhibitors. PMID:26916347

  6. Tagging CO2 to Enable Quantitative Inventories of Geological Carbon Storage

    SciTech Connect

    Lackner, Klaus; Matter, Juerg; Park, Ah-Hyung; Stute, Martin; Carson, Cantwell; Ji, Yinghuang

    2014-06-30

    In the wake of concerns about the long term integrity and containment of sub-surface CO2 sequestration reservoirs, many efforts have been made to improve the monitoring, verification, and accounting methods for geo-sequestered CO2. Our project aimed to demonstrate the feasibility of a system designed to tag CO2 with carbon isotope 14C immediately prior to sequestration to a level that is normal on the surface (one part per trillion). Because carbon found at depth is naturally free of 14C, this tag would easily differentiate pre-existing carbon from anthropogenic injected carbon and provide an excellent handle for monitoring its whereabouts in the subsurface. It also creates an excellent handle for adding up anthropogenic carbon inventories. Future inventories in effect count 14C atoms. Accordingly, we have developed a 14C tagging system suitable for use at the part-per-trillion level. This system consists of a gas-exchange apparatus to make disposable cartridges ready for controlled injection into a fast flowing stream of pressurized CO2. We built a high-pressure injection and tagging system, and a 14C detection system. The disposable cartridge and injection system have been successfully demonstrated in the lab with a high-pressure flow reactor, as well as in the field at the CarbFix CO2 sequestration site in Iceland. The laser-based 14C detection system originally conceived has been shown to possess inadequate sensitivity for ambient levels. Alternative methods for detecting 14C, such as saturated cavity absorption ringdown spectroscopy and scintillation counting, may still be suitable. KEYWORDS

  7. Supramolecular Probes for Assessing Glutamine Uptake Enable Semi-Quantitative Metabolic Models in Single Cells.

    PubMed

    Xue, Min; Wei, Wei; Su, Yapeng; Johnson, Dazy; Heath, James R

    2016-03-01

    We describe a supramolecular surface competition assay for quantifying glutamine uptake from single cells. Cy3-labeled cyclodextrins were immobilized on a glass surface as a supramolecular host/FRET donor, and adamantane-BHQ2 conjugates were employed as the guest/quencher. An adamantane-labeled glutamine analog was selected through screening a library of compounds and validated by cell uptake experiments. When integrated onto a single cell barcode chip with a multiplex panel of 15 other metabolites, associated metabolic enzymes, and phosphoproteins, the resultant data provided input for a steady-state model that describes energy potential in single cells and correlates that potential with receptor tyrosine kinase signaling. We utilize this integrated assay to interrogate a dose-dependent response of model brain cancer cells to EGFR inhibition. We find that low-dose (1 μM erlotinib) drugging actually increases cellular energy potential even as glucose uptake and phosphoprotein signaling is repressed. We also identify new interactions between phosphoprotein signaling and cellular energy processes that may help explain the facile resistance exhibited by certain cancer patients to EGFR inhibitors. PMID:26916347

  8. Confocal Fluorescence Imaging Enables Noninvasive Quantitative Assessment of Host Cell Populations In Vivo Following Photodynamic Therapy

    PubMed Central

    Mitra, Soumya; Mironov, Oleg; Foster, Thomas H.

    2012-01-01

    We report the use of optical imaging strategies to noninvasively examine photosensitizer distribution and physiological and host responses to 2-[1-hexyloxyethyl]-2 devinyl pyropheophorbide-a (HPPH)-mediated photodynamic therapy (PDT) of EMT6 tumors established in the ears of BALB/c mice. 24 h following intravenous (IV) administration of 1 μmol kg-1 HPPH, wide-field fluorescence imaging reveals tumor selectivity with an approximately 2-3-fold differential between tumor and adjacent normal tissue. Confocal microscopy demonstrates a relatively homogeneous intratumor HPPH distribution. Labeling of host cells using fluorophore-conjugated antibodies allowed the visualization of Gr1+/CD11b+ leukocytes and major histocompatibility complex class II (MHC-II)+ cells in vivo. Imaging of the treated site at different time-points following irradiation shows significant and rapid increases in Gr1+ cells in response to therapy. The maximum accumulation of Gr1+ cells is found at 24 h post-irradiation, followed by a decrease at the 48 h time-point. Using IV-injected FITC-conjugated dextran as a fluorescent perfusion marker, we imaged tissue perfusion at different times post-irradiation and found that the reduced Gr1+ cell density at 48 h correlated strongly with functional damage to the vasculature as reported via decreased perfusion status. Dual color confocal imaging experiments demonstrates that about 90% of the anti-Gr1 cell population co-localized with anti-CD11b labeling, thus indicating that majority of the Gr1-labeled cells were neutrophils. At 24 h post-PDT, an approximately 2-fold increase in MHC-II+ cells relative to untreated control is also observed. Co-localization analysis reveals an increase in the fraction of Gr1+ cells expressing MHC-II, suggesting that HPPH-PDT is stimulating neutrophils to express an antigen-presenting phenotype. PMID:23082097

  9. Sequential Enrichment with Titania-coated Magnetic Mesoporous Hollow Silica Microspheres and Zirconium Arsenate-modified Magnetic Nanoparticles for the Study of Phosphoproteome of HL60 Cells

    PubMed Central

    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

  10. A High-Resolution Tissue-Specific Proteome and Phosphoproteome Atlas of Maize Primary Roots Reveals Functional Gradients along the Root Axes1[OPEN

    PubMed Central

    Malik, Waqas Ahmed; Shen, Zhouxin; Paschold, Anja

    2015-01-01

    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. PMID:25780097

  11. Phosphoproteomic Analysis Reveals the Effects of PilF Phosphorylation on Type IV Pilus and Biofilm Formation in Thermus thermophilus HB27*

    PubMed Central

    Wu, Wan-Ling; Liao, Jiahn-Haur; Lin, Guang-Huey; Lin, Miao-Hsia; Chang, Ying-Che; Liang, Suh-Yuen; Yang, Feng-Ling; Khoo, Kay-Hooi; Wu, Shih-Hsiung

    2013-01-01

    Thermus thermophilus HB27 is an extremely thermophilic eubacteria with a high frequency of natural competence. This organism is therefore often used as a thermophilic model to investigate the molecular basis of type IV pili–mediated functions, such as the uptake of free DNA, adhesion, twitching motility, and biofilm formation, in hot environments. In this study, the phosphoproteome of T. thermophilus HB27 was analyzed via a shotgun approach and high-accuracy mass spectrometry. Ninety-three unique phosphopeptides, including 67 in vivo phosphorylated sites on 53 phosphoproteins, were identified. The distribution of Ser/Thr/Tyr phosphorylation sites was 57%/36%/7%. The phosphoproteins were mostly involved in central metabolic pathways and protein/cell envelope biosynthesis. According to this analysis, the ATPase motor PilF, a type IV pili–related component, was first found to be phosphorylated on Thr-368 and Ser-372. Through the point mutation of PilF, mimic phosphorylated mutants T368D and S372E resulted in nonpiliated and nontwitching phenotypes, whereas nonphosphorylated mutants T368V and S372A displayed piliation and twitching motility. In addition, mimic phosphorylated mutants showed elevated biofilm-forming abilities with a higher initial attachment rate, caused by increasing exopolysaccharide production. In summary, the phosphorylation of PilF might regulate the pili and biofilm formation associated with exopolysaccharide production. PMID:23828892

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

    PubMed

    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. PMID:25780097

  13. Identification of Extracellular Signal-regulated Kinase 1 (ERK1) Direct Substrates using Stable Isotope Labeled Kinase Assay-Linked Phosphoproteomics*

    PubMed Central

    Xue, Liang; Wang, Pengcheng; Cao, Pianpian; Zhu, Jian-kang; Tao, W. Andy

    2014-01-01

    Kinase mediated phosphorylation signaling is extensively involved in cellular functions and human diseases, and unraveling phosphorylation networks requires the identification of substrates targeted by kinases, which has remained challenging. We report here a novel proteomic strategy to identify the specificity and direct substrates of kinases by coupling phosphoproteomics with a sensitive stable isotope labeled kinase reaction. A whole cell extract was moderately dephosphorylated and subjected to in vitro kinase reaction under the condition in which 18O-ATP is the phosphate donor. The phosphorylated proteins are then isolated and identified by mass spectrometry, in which the heavy phosphate (+85.979 Da) labeled phosphopeptides reveal the kinase specificity. The in vitro phosphorylated proteins with heavy phosphates are further overlapped with in vivo kinase-dependent phosphoproteins for the identification of direct substrates with high confidence. The strategy allowed us to identify 46 phosphorylation sites on 38 direct substrates of extracellular signal-regulated kinase 1, including multiple known substrates and novel substrates, highlighting the ability of this high throughput method for direct kinase substrate screening. PMID:25022875

  14. Phosphoproteome Profiling of SH-SY5y Neuroblastoma Cells Treated with Anesthetics: Sevoflurane and Isoflurane Affect the Phosphorylation of Proteins Involved in Cytoskeletal Regulation.

    PubMed

    Lee, Joomin; Ahn, Eunsook; Park, Wyun Kon; Park, Seyeon

    2016-01-01

    Inhalation anesthetics are used to decrease the spinal cord transmission of painful stimuli. However, the molecular or biochemical processes within cells that regulate anesthetic-induced responses at the cellular level are largely unknown. Here, we report the phosphoproteome profile of SH-SY5y human neuroblastoma cells treated with sevoflurane, a clinically used anesthetic. Phosphoproteins were isolated from cell lysates and analyzed using two-dimensional gel electrophoresis. The phosphorylation of putative anesthetic-responsive marker proteins was validated using western blot analysis in cells treated with both sevoflurane and isoflurane. A total of 25 phosphoproteins were identified as differentially phosphorylated proteins. These included key regulators that signal cytoskeletal remodeling steps in pathways related to vesicle trafficking, axonal growth, and cell migration. These proteins included the Rho GTPase, Ras-GAP SH3 binding protein, Rho GTPase activating protein, actin-related protein, and actin. Sevoflurane and isoflurane also resulted in the dissolution of F-actin fibers in SH-SY5y cells. Our results show that anesthetics affect the phosphorylation of proteins involved in cytoskeletal remodeling pathways. PMID:27611435

  15. Quantitative assessment of fluorescent proteins.

    PubMed

    Cranfill, Paula J; Sell, Brittney R; Baird, Michelle A; Allen, John R; Lavagnino, Zeno; de Gruiter, H Martijn; Kremers, Gert-Jan; Davidson, Michael W; Ustione, Alessandro; Piston, David W

    2016-07-01

    The advent of fluorescent proteins (FPs) for genetic labeling of molecules and cells has revolutionized fluorescence microscopy. Genetic manipulations have created a vast array of bright and stable FPs spanning blue to red spectral regions. Common to autofluorescent FPs is their tight β-barrel structure, which provides the rigidity and chemical environment needed for effectual fluorescence. Despite the common structure, each FP has unique properties. Thus, there is no single 'best' FP for every circumstance, and each FP has advantages and disadvantages. To guide decisions about which FP is right for a given application, we have quantitatively characterized the brightness, photostability, pH stability and monomeric properties of more than 40 FPs to enable straightforward and direct comparison between them. We focus on popular and/or top-performing FPs in each spectral region. PMID:27240257

  16. Utility Energy Services Contracts: Enabling Documents

    SciTech Connect

    2009-05-01

    Utility Energy Services Contracts: Enabling Documents provides materials that clarify the authority for Federal agencies to enter into utility energy services contracts (UESCs), as well as sample documents and resources to ease utility partnership contracting.

  17. Quantitative roadmap of holographic media performance

    NASA Astrophysics Data System (ADS)

    Kowalski, Benjamin A.; McLeod, Robert R.

    2015-09-01

    For holographic photopolymer media, the "formula limit" concept enables facile calculation of the fraction of writing chemistry that is usefully patterned, and the fraction that is wasted. This provides a quantitative context to compare the performance of a diverse range of media formulations from the literature, using only information already reported in the original works. Finally, this analysis is extended to estimate the scope of achievable future performance improvements.

  18. An Architecture to Enable Future Sensor Webs

    NASA Technical Reports Server (NTRS)

    Mandl, Dan; Caffrey, Robert; Frye, Stu; Grosvenor, Sandra; Hess, Melissa; Chien, Steve; Sherwood, Rob; Davies, Ashley; Hayden, Sandra; Sweet, Adam

    2004-01-01

    A sensor web is a coherent set of distributed 'nodes', interconnected by a communications fabric, that collectively behave as a single dynamic observing system. A 'plug and play' mission architecture enables progressive mission autonomy and rapid assembly and thereby enables sensor webs. This viewgraph presentation addresses: Target mission messaging architecture; Strategy to establish architecture; Progressive autonomy with onboard sensor web; EO-1; Adaptive array antennas (smart antennas) for satellite ground stations.

  19. ISS - Enabling Exploration Through Docking Standards

    NASA Technical Reports Server (NTRS)

    Hatfield, Caris A.

    2011-01-01

    NASA and the ISS partnership are jointly developing a key standard to enable future collaborative exploration. The IDSS is based on flight proven design while incorporating new low impact technology. Low impact technology accommodates a wide range of vehicle contact and capture conditions. This standard will get early demonstration on the ISS. Experience gained here will enable operational experience and the opportunity to refine the standard.

  20. Integrative Quantitative Proteomics Unveils Proteostasis Imbalance in Human Hepatocellular Carcinoma Developed on Nonfibrotic Livers*

    PubMed Central

    Negroni, Luc; Taouji, Said; Arma, Daniela; Pallares-Lupon, Nestor; Leong, Kristen; Beausang, Lee Anne; Latterich, Martin; Bossé, Roger; Balabaud, Charles; Schmitter, Jean-Marie; Bioulac-Sage, Paulette; Zucman-Rossi, Jessica; Rosenbaum, Jean; Chevet, Eric

    2014-01-01

    Proteomics-based clinical studies represent promising resources for the discovery of novel biomarkers or for unraveling molecular mechanisms underlying particular diseases. Here, we present a discovery study of hepatocellular carcinoma developed on nonfibrotic liver (nfHCC) that combines complementary quantitative iTRAQ-based proteomics and phosphoproteomics approaches. Using both approaches, we compared a set of 24 samples (18 nfHCC versus six nontumor liver tissue). We identified 43 proteins (67 peptides) differentially expressed and 32 peptides differentially phosphorylated between the experimental groups. The functional analysis of the two data sets pointed toward the deregulation of a protein homeostasis (proteostasis) network including the up-regulation of the Endoplasmic Reticulum (ER) resident HSPA5, HSP90B1, PDIA6, and P4HB and of the cytosolic HSPA1B, HSP90AA1, HSPA9, UBC, CNDP2, TXN, and VCP as well as the increased phosphorylation of the ER resident calnexin at Ser583. Antibody-based validation approaches (immunohistochemistry, immunoblot, Alphascreen®, and AMMP®) on independent nfHCC tumor sets (up to 77 samples) confirmed these observations, thereby indicating a common mechanism occurring in nfHCC tumors. Based on these results we propose that adaptation to proteostasis imbalance in nfHCC tumors might confer selective advantages to those tumors. As such, this model could provide an additional therapeutic opportunity for those tumors arising on normal liver by targeting the tumor proteostasis network. Data are available via ProteomeXchange with identifier PXD001253. PMID:25225353

  1. Non Linear Programming (NLP) formulation for quantitative modeling of protein signal transduction pathways.

    PubMed

    Mitsos, Alexander; Melas, Ioannis N; Morris, Melody K; Saez-Rodriguez, Julio; Lauffenburger, Douglas A; Alexopoulos, Leonidas G

    2012-01-01

    Modeling of signal transduction pathways plays a major role in understanding cells' function and predicting cellular response. Mathematical formalisms based on a logic formalism are relatively simple but can describe how signals propagate from one protein to the next and have led to the construction of models that simulate the cells response to environmental or other perturbations. Constrained fuzzy logic was recently introduced to train models to cell specific data to result in quantitative pathway models of the specific cellular behavior. There are two major issues in this pathway optimization: i) excessive CPU time requirements and ii) loosely constrained optimization problem due to lack of data with respect to large signaling pathways. Herein, we address both issues: the former by reformulating the pathway optimization as a regular nonlinear optimization problem; and the latter by enhanced algorithms to pre/post-process the signaling network to remove parts that cannot be identified given the experimental conditions. As a case study, we tackle the construction of cell type specific pathways in normal and transformed hepatocytes using medium and large-scale functional phosphoproteomic datasets. The proposed Non Linear Programming (NLP) formulation allows for fast optimization of signaling topologies by combining the versatile nature of logic modeling with state of the art optimization algorithms. PMID:23226239

  2. Quantitative analysis of blood vessel geometry

    NASA Astrophysics Data System (ADS)

    Fuhrman, Michael G.; Abdul-Karim, Othman; Shah, Sujal; Gilbert, Steven G.; Van Bibber, Richard

    2001-07-01

    Re-narrowing or restenosis of a human coronary artery occurs within six months in one third of balloon angioplasty procedures. Accurate and repeatable quantitative analysis of vessel shape is important to characterize the progression and type of restenosis, and to evaluate effects new therapies might have. A combination of complicated geometry and image variability, and the need for high resolution and large image size makes visual/manual analysis slow, difficult, and prone to error. The image processing and analysis described here was developed to automate feature extraction of the lumen, internal elastic lamina, neointima, external elastic lamina, and tunica adventitia and to enable an objective, quantitative definition of blood vessel geometry. The quantitative geometrical analysis enables the measurement of several features including perimeter, area, and other metrics of vessel damage. Automation of feature extraction creates a high throughput capability that enables analysis of serial sections for more accurate measurement of restenosis dimensions. Measurement results are input into a relational database where they can be statistically analyzed compared across studies. As part of the integrated process, results are also imprinted on the images themselves to facilitate auditing of the results. The analysis is fast, repeatable and accurate while allowing the pathologist to control the measurement process.

  3. Quantitative Analysis of Face Symmetry.

    PubMed

    Tamir, Abraham

    2015-06-01

    The major objective of this article was to report quantitatively the degree of human face symmetry for reported images taken from the Internet. From the original image of a certain person that appears in the center of each triplet, 2 symmetric combinations were constructed that are based on the left part of the image and its mirror image (left-left) and on the right part of the image and its mirror image (right-right). By applying a computer software that enables to determine length, surface area, and perimeter of any geometric shape, the following measurements were obtained for each triplet: face perimeter and area; distance between the pupils; mouth length; its perimeter and area; nose length and face length, usually below the ears; as well as the area and perimeter of the pupils. Then, for each of the above measurements, the value C, which characterizes the degree of symmetry of the real image with respect to the combinations right-right and left-left, was calculated. C appears on the right-hand side below each image. A high value of C indicates a low symmetry, and as the value is decreasing, the symmetry is increasing. The magnitude on the left relates to the pupils and compares the difference between the area and perimeter of the 2 pupils. The major conclusion arrived at here is that the human face is asymmetric to some degree; the degree of asymmetry is reported quantitatively under each portrait. PMID:26080172

  4. A microelectromechanical systems-enabled, miniature triple quadrupole mass spectrometer.

    PubMed

    Wright, Steven; Malcolm, Andrew; Wright, Christopher; O'Prey, Shane; Crichton, Edward; Dash, Neil; Moseley, Richard W; Zaczek, Wojciech; Edwards, Peter; Fussell, Richard J; Syms, Richard R A

    2015-03-17

    Miniaturized mass spectrometers are becoming increasingly capable, enabling the development of many novel field and laboratory applications. However, to date, triple quadrupole tandem mass spectrometers, the workhorses of quantitative analysis, have not been significantly reduced in size. Here, the basis of a field-deployable triple quadrupole is described. The key development is a highly miniaturized ion optical assembly in which a sequence of six microengineered components is employed to generate ions at atmospheric pressure, provide a vacuum interface, effect ion guiding, and perform fragmentation and mass analysis. Despite its small dimensions, the collision cell efficiently fragments precursor ions and yields product ion spectra that are very similar to those recorded using conventional instruments. The miniature triple quadrupole has been used to detect thiabendazole, a common pesticide, in apples at a level of 10 ng/g. PMID:25708099

  5. Structures and practices enabling staff nurses to control their practice.

    PubMed

    Kramer, Marlene; Schmalenberg, Claudia; Maguire, Patricia; Brewer, Barbara B; Burke, Rebecca; Chmielewski, Linda; Cox, Karen; Kishner, Janice; Krugman, Mary; Meeks-Sjostrom, Diana; Waldo, Mary

    2008-08-01

    This mixed-methods study uses interviews, participant observations, and the CWEQII empowerment tool to identify structures and attributes of structures that promote control over nursing practice (CNP). Nearly 3,000 staff nurses completed the Essentials of Magnetism (EOM), an instrument that measures CNP, one of the eight staff nurse-identified essential attributes of a productive work environment. Strategic sampling is used to identify 101 high CNP-scoring clinical units in 8 high-EOM scoring magnet hospitals. In addition to 446 staff nurses, managers, and physicians on these high-scoring units, chief nursing officers, chief operating officers, and representatives from other professional departments are interviewed; participant observations are made of all unit/departmental/hospital council and interdisciplinary meetings held during a 4 to 6 day site visit. Structures and components of viable shared governance structures that enabled CNP are identified through constant comparative analysis of interviews and observations, and through analysis of quantitative measures. PMID:18195080

  6. New Labour and the enabling state.

    PubMed

    Taylor, Ian

    2000-11-01

    The notion of the 'enabling state' gained currency in the UK during the 1990s as an alternative to the 'providing' or the welfare state. It reflected the process of contracting out in the NHS and compulsory competitive tendering (CCT) in local government during the 1980s, but was also associated with developments during the 1990s in health, social care and education in particular. The creation of an internal market in the NHS and the associated purchaser-provider split appeared to transfer 'ownership' of services increasingly to the providers - hospitals, General Practitioners (GPs) and schools. The mixed economy of care that was stimulated by the 1990 NHS and Community Care Act appeared to offer local authorities the opportunity to enable non state providers to offer care services in the community. The new service charters were part of the enablement process because they offered users more opportunity to influence provision. This article examines how far service providers were enabled and assesses the extent to which new Labour's policies enhance or reject the 'enabling state' in favour of more direct provision. PMID:11560707

  7. Energy Savings Performance Contract (ESPC) ENABLE Program

    SciTech Connect

    2012-06-01

    The Energy Savings Performance Contract (ESPC) ENABLE program, a new project funding approach, allows small Federal facilities to realize energy and water savings in six months or less. ESPC ENABLE provides a standardized and streamlined process to install targeted energy conservation measures (ECMs) such as lighting, water, and controls with measurement and verification (M&V) appropriate for the size and scope of the project. This allows Federal facilities smaller than 200,000 square feet to make progress towards important energy efficiency and water conservation requirements.

  8. Enabling human HUMS with data modeling

    NASA Astrophysics Data System (ADS)

    Jaenisch, Holger M.; Handley, James W.; Jaenisch, Kristina K.; Hicklen, Michael L.

    2006-05-01

    We simulate a notional Navy SEAL rebreather diver on an extended mission using Model Predictive Control (MPC) theory. A mathematical framework for enabling physiological HUMS (Health Usage Management Systems) is shown. A rebreather simulation is used to derive MPC baseline Data Models of diver status by converting the simulation first into differential equations and then into lookup tables (LUT). When abnormal readings are indicated, sensor data from the diver is published to the ad hoc network, enabling intermittent upload. Mission success confidence is updated and determined during the mission. A novel method of converting MPC Data Models into lookup tables worn by the diver is given.

  9. Origami-enabled deformable silicon solar cells

    SciTech Connect

    Tang, Rui; Huang, Hai; Liang, Hanshuang; Liang, Mengbing; Tu, Hongen; Xu, Yong; Song, Zeming; Jiang, Hanqing; Yu, Hongyu

    2014-02-24

    Deformable electronics have found various applications and elastomeric materials have been widely used to reach flexibility and stretchability. In this Letter, we report an alternative approach to enable deformability through origami. In this approach, the deformability is achieved through folding and unfolding at the creases while the functional devices do not experience strain. We have demonstrated an example of origami-enabled silicon solar cells and showed that this solar cell can reach up to 644% areal compactness while maintaining reasonable good performance upon cyclic folding/unfolding. This approach opens an alternative direction of producing flexible, stretchable, and deformable electronics.

  10. Networking Technologies Enable Advances in Earth Science

    NASA Technical Reports Server (NTRS)

    Johnson, Marjory; Freeman, Kenneth; Gilstrap, Raymond; Beck, Richard

    2004-01-01

    This paper describes an experiment to prototype a new way of conducting science by applying networking and distributed computing technologies to an Earth Science application. A combination of satellite, wireless, and terrestrial networking provided geologists at a remote field site with interactive access to supercomputer facilities at two NASA centers, thus enabling them to validate and calibrate remotely sensed geological data in near-real time. This represents a fundamental shift in the way that Earth scientists analyze remotely sensed data. In this paper we describe the experiment and the network infrastructure that enabled it, analyze the data flow during the experiment, and discuss the scientific impact of the results.

  11. Upgraded NERVA systems: Enabler nuclear system

    NASA Technical Reports Server (NTRS)

    Farbman, Gerry

    1991-01-01

    The NERVA/Rover Enabler technology enables to go on a low risk, short-term program to meet the requirements of the Mars mission and maybe some lunar missions. The following subject areas are covered: NERVA technology - the foundation for tomorrow's space missions; NERVA/Rover reactor system test sequence; NERVA engine development program; nuclear thermal reactor capability based on many related Westinghouse technology programs; investment in Rover/Nerva technology; synergistic applications of NERVA technology; flow schematic of the NDR engine; the NERVA nuclear subsystem; and technology evolution.

  12. Recapturing Quantitative Biology.

    ERIC Educational Resources Information Center

    Pernezny, Ken; And Others

    1996-01-01

    Presents a classroom activity on estimating animal populations. Uses shoe boxes and candies to emphasize the importance of mathematics in biology while introducing the methods of quantitative ecology. (JRH)

  13. On Quantitative Rorschach Scales.

    ERIC Educational Resources Information Center

    Haggard, Ernest A.

    1978-01-01

    Two types of quantitative Rorschach scales are discussed: first, those based on the response categories of content, location, and the determinants, and second, global scales based on the subject's responses to all ten stimulus cards. (Author/JKS)

  14. Quantitative film radiography

    SciTech Connect

    Devine, G.; Dobie, D.; Fugina, J.; Hernandez, J.; Logan, C.; Mohr, P.; Moss, R.; Schumacher, B.; Updike, E.; Weirup, D.

    1991-02-26

    We have developed a system of quantitative radiography in order to produce quantitative images displaying homogeneity of parts. The materials that we characterize are synthetic composites and may contain important subtle density variations not discernible by examining a raw film x-radiograph. In order to quantitatively interpret film radiographs, it is necessary to digitize, interpret, and display the images. Our integrated system of quantitative radiography displays accurate, high-resolution pseudo-color images in units of density. We characterize approximately 10,000 parts per year in hundreds of different configurations and compositions with this system. This report discusses: the method; film processor monitoring and control; verifying film and processor performance; and correction of scatter effects.

  15. Quantitative receptor autoradiography

    SciTech Connect

    Boast, C.A.; Snowhill, E.W.; Altar, C.A.

    1986-01-01

    Quantitative receptor autoradiography addresses the topic of technical and scientific advances in the sphere of quantitative autoradiography. The volume opens with a overview of the field from a historical and critical perspective. Following is a detailed discussion of in vitro data obtained from a variety of neurotransmitter systems. The next section explores applications of autoradiography, and the final two chapters consider experimental models. Methodological considerations are emphasized, including the use of computers for image analysis.

  16. Apparatus enables automatic microanalysis of body fluids

    NASA Technical Reports Server (NTRS)

    Soffen, G. A.; Stuart, J. L.

    1966-01-01

    Apparatus will automatically and quantitatively determine body fluid constituents which are amenable to analysis by fluorometry or colorimetry. The results of the tests are displayed as percentages of full scale deflection on a strip-chart recorder. The apparatus can also be adapted for microanalysis of various other fluids.

  17. Phosphoproteomic analysis of anaplastic lymphoma kinase (ALK) downstream signaling pathways identifies signal transducer and activator of transcription 3 as a functional target of activated ALK in neuroblastoma cells

    PubMed Central

    Sattu, Kamaraj; Hochgräfe, Falko; Wu, Jianmin; Umapathy, Ganesh; Schönherr, Christina; Ruuth, Kristina; Chand, Damini; Witek, Barbara; Fuchs, James; Li, Pui-Kai; Hugosson, Fredrik; Daly, Roger J; Palmer, Ruth H; Hallberg, Bengt

    2013-01-01

    Activation of the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase is a key oncogenic mechanism in a growing number of tumor types. In the majority of cases, ALK is activated by fusion with a dimerizing partner protein as a result of chromosomal translocation events, most studied in the case of the nucleophosmin–ALK and echinoderm microtubule-associated protein-like 4–ALK oncoproteins. It is now also appreciated that the full-length ALK receptor can be activated by point mutations and by deletions within the extracellular domain, such as those observed in neuroblastoma. Several studies have employed phosphoproteomics approaches to find substrates of ALK fusion proteins. In this study, we used MS-based phosphotyrosine profiling to characterize phosphotyrosine signaling events associated with the full-length ALK receptor. A number of previously identified and novel targets were identified. One of these, signal transducer and activator of transcription 3 (STAT3), has previously been observed to be activated in response to oncogenic ALK signaling, but the significance of this in signaling from the full-length ALK receptor has not been explored further. We show here that activated ALK robustly activates STAT3 on Tyr705 in a number of independent neuroblastoma cell lines. Furthermore, knockdown of STAT3 by RNA interference resulted in a reduction in myelocytomatosis neuroblastom (MYCN) protein levels downstream of ALK signaling. These observations, together with a decreased level of MYCN and inhibition of neuroblastoma cell growth in the presence of STAT3 inhibitors, suggest that activation of STAT3 is important for ALK signaling activity in neuroblastoma. PMID:23889739

  18. Proteomic and Phosphoproteomic Insights into a Signaling Hub Role for Cdc14 in Asexual Development and Multiple Stress Responses in Beauveria bassiana

    PubMed Central

    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. PMID:27055109

  19. Phosphoproteomic identification of targets of the Arabidopsis sucrose nonfermenting-like kinase SnRK2.8 reveals a connection to metabolic processes

    PubMed Central

    Shin, Ryoung; Alvarez, Sophie; Burch, Adrien Y.; Jez, Joseph M.; Schachtman, Daniel P.

    2007-01-01

    SnRK2.8 is a member of the sucrose nonfermenting-related kinase family that is down-regulated when plants are deprived of nutrients and growth is reduced. When this kinase is over expressed in Arabidopsis, the plants grow larger. To understand how this kinase modulates growth, we identified some of the proteins that are phosphorylated by this kinase. A new phosphoproteomic method was used in which total protein from plants overexpressing the kinase was compared with total protein from plants in which the kinase was inactivated. Protein profiles were compared on two-dimensional gels following staining by a dye that recognizes phosphorylated amino acids. Candidate target proteins were confirmed with in vitro phosphorylation assays, using the kinase and target proteins that were purified from Escherichia coli. Seven target proteins were confirmed as being phosphorylated by SnRK2.8. Certain targets, such as 14-3-3 proteins, regulate as yet unidentified proteins, whereas other targets, such as glyoxalase I and ribose 5-phosphate isomerase, detoxify byproducts from glycolysis and catalyze one of the final steps in carbon fixation, respectively. Also, adenosine kinase and 60S ribosomal protein were confirmed as targets of SnRK2.8. Using mass spectrometry, we identified phosphorylated residues in the SnRK2.8, the 14-3-3κ, and the 14-3-3χ. These data show that the expression of SnRK2.8 is correlated with plant growth, which may in part be due to the phosphorylation of enzymes involved in metabolic processes. PMID:17404219

  20. Proteomic and Phosphoproteomic Insights into a Signaling Hub Role for Cdc14 in Asexual Development and Multiple Stress Responses in Beauveria bassiana.

    PubMed

    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. PMID:27055109

  1. The Role of Phospholipase D and MAPK Signaling Cascades in the Adaption of Lichen Microalgae to Desiccation: Changes in Membrane Lipids and Phosphoproteome.

    PubMed

    Gasulla, Francisco; Barreno, Eva; Parages, María L; Cámara, Joaquín; Jiménez, Carlos; Dörmann, Peter; Bartels, Dorothea

    2016-09-01

    Classically, lichen phycobionts are described as poikilohydric organisms able to undergo desiccation due to the constitutive presence of molecular protection mechanisms. However, little is known about the induction of cellular responses in lichen phycobionts during drying. The analysis of the lipid composition of the desiccated lichen microalga Asterochloris erici revealed the unusual accumulation of highly polar lipids (oligogalactolipids and phosphatidylinositol), which prevents the fusion of membranes during stress, but also the active degradation of cone-shaped lipids (monogalactosyldiacylglycerol and phosphatidylethanolamine) to stabilize membranes in desiccated cells. The level of phosphatidic acid increased 7-fold during desiccation, implicating a possible role for phospholipase D (PLD) in the response to osmotic stress. Inhibition of PLD with 1-butanol markedly impaired the recovery of photosynthesis activity in A. erici upon desiccation and salt stress (2 M NaCl). These two hyperosmotic stresses caused the phosphorylation of c-Jun N-terminal kinase (JNK) and p38-like mitogen-activated protein kinase (MAPK) and the dephosphorylation of extracellular signal-regulated kinase (ERK). The incubation with 1-butanol reduced the phosphorylation of JNK-like proteins and increased the dephosphorylation of ERK-like proteins, which indicates an upstream control of MAPK cascades by PLD. The phosphoproteome showed that desiccation caused the phosphorylation of several proteins in A. erici, most of them involved in protein turnover. The results demonstrate that lichen phycobionts possess both constitutive and inducible protective mechanisms to acquire desiccation tolerance. Among others, these responses are controlled by the PLD pathway through the activation of MAPK cascades. PMID:27335354

  2. Nanotechnologv Enabled Biological and Chemical Sensors

    NASA Technical Reports Server (NTRS)

    Koehne, Jessica; Meyyappan, M.

    2011-01-01

    Nanotechnology is an enabling technology that will impact almost all economic sectors: one of the most important and with great potential is the health/medical sector. - Nanomaterials for drug delivery - Early warning sensors - Implantable devices - Artificial parts with improved characteristics Carbon nanotubes and nanofibers show promise for use in sensor development, electrodes and other biomedical applications.

  3. Technology-Enabled Crime, Policing and Security

    ERIC Educational Resources Information Center

    McQuade, Sam

    2006-01-01

    Crime, policing and security are enabled by and co-evolve with technologies that make them possible. As criminals compete with security and policing officials for technological advantage perpetually complex crime, policing and security results in relatively confusing and therefore unmanageable threats to society. New, adaptive and ordinary crimes…

  4. Action Learning: Avoiding Conflict or Enabling Action

    ERIC Educational Resources Information Center

    Corley, Aileen; Thorne, Ann

    2006-01-01

    Action learning is based on the premise that action and learning are inextricably entwined and it is this potential, to enable action, which has contributed to the growth of action learning within education and management development programmes. However has this growth in action learning lead to an evolution or a dilution of Revan's classical…

  5. Enabling Family-Friendly Cultural Change

    ERIC Educational Resources Information Center

    Quinn, Kate; Yen, Joyce W.; Riskin, Eve A.; Lange, Sheila Edwards

    2007-01-01

    Strategies to address the problem of work and family balance have begun emerging in recent years. Many American college and universities have begun to adopt this "family-friendly policies," such as tenure-clock extensions. Each of the policies to enable work and family balance, however, is situated within the broader academic culture. Departmental…

  6. Safely Enabling Low-Altitude Airspace Operations

    NASA Technical Reports Server (NTRS)

    Kopardekar, Parimal

    2015-01-01

    Near-term Goal: Enable initial low-altitude airspace and UAS operations with demonstrated safety as early as possible, within 5 years. Long-term Goal: Accommodate increased UAS operations with highest safety, efficiency, and capacity as much autonomously as possible (10-15 years).

  7. Safely Enabling Low-Altitude Airspace Operations

    NASA Technical Reports Server (NTRS)

    Kopardekar, Parimal

    2015-01-01

    Near-term Goal - Enable initial low-altitude airspace and UAS operations with demonstrated safety as early as possible, within 5 years. Long-term Goal - Accommodate increased UAS operations with highest safety, efficiency, and capacity as much autonomously as possible (10-15 years).

  8. Fluorescent particles enable visualization of gas flow

    NASA Technical Reports Server (NTRS)

    Wilson, A. J.

    1968-01-01

    Fluorescent particles enable visualization of the flow patterns of gases at slow velocities. Through a transparent section in the gas line, a camera views the visible light emitted by the particles carried by the gas stream. Fine definition of the particle tracks are obtained at slow camera shutter speeds.

  9. 75 FR 13235 - IP-Enabled Services

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-19

    ... 47 CFR 63.60(a) and (f), published on August 7, 2009 (74 FR 39551), were approved by the Office of... published a document in the Federal Register, 74 FR 39551, August 7, 2009, that sets forth an effective date... COMMISSION 47 CFR Part 63 IP-Enabled Services AGENCY: Federal Communications Commission ACTION: Final...

  10. Enabling a Comprehensive Teaching Strategy: Video Lectures

    ERIC Educational Resources Information Center

    Brecht, H. David; Ogilby, Suzanne M.

    2008-01-01

    This study empirically tests the feasibility and effectiveness of video lectures as a form of video instruction that enables a comprehensive teaching strategy used throughout a traditional classroom course. It examines student use patterns and the videos' effects on student learning, using qualitative and nonparametric statistical analyses of…

  11. Implementation of Quantitative and Systems Pharmacology in Large Pharma

    PubMed Central

    Visser, S A G; de Alwis, D P; Kerbusch, T; Stone, J A; Allerheiligen, S R B

    2014-01-01

    Quantitative and systems pharmacology concepts and tools are the foundation of the model-informed drug development paradigm at Merck for integrating knowledge, enabling decisions, and enhancing submissions. Rigorous prioritization of modeling and simulation activities has enabled key drug development decisions and led to a high return on investment through significant cost avoidance. Critical factors for the successful implementation, examples on impact on decision making with associated return of investment, and drivers for continued success are discussed. PMID:25338195

  12. NASP - Enabling new space launch options

    NASA Technical Reports Server (NTRS)

    Froning, David; Gaubatz, William; Mathews, George

    1990-01-01

    Successful NASP developments in the United States are bringing about the possibility of effective, fully reusable vehicles for transport of people and cargo between earth and space. These developments include: extension of airbreathing propulsion to a much higher speed; densification of propellants for greater energy per unit volume of mass; structures with much greater strength-to-weight at high temperatures; computational advancements that enable more optimal design and integration of airframes, engines and controls; and advances in avionics, robotics, artificial intelligence and automation that enable accomplishment of earth-to-orbit (ETO) operations with much less manpower support and cost. This paper describes the relative magnitude of improvement that these developments may provide.

  13. Enabling Semantic Interoperability for Earth System Science

    NASA Astrophysics Data System (ADS)

    Raskin, R.

    2004-12-01

    Data interoperability across heterogeneous systems can be hampered by differences in terminology, particularly when multiple scientific communities are involved. To reconcile differences in semantics, a common semantic framework was created as a collection of ontologies. Such a shared understanding of concepts enables ontology-aware software tools to understand the meaning of terms in documents and web pages. The ontologies were created as part of the Semantic Web for Earth and Environmental Terminology (SWEET) prototype. The ontologies provide a representation of Earth system science knowledge and associated data, organized in a scalable structure, bulding on the keywords developed by the NASA Global Change Master Directory (GCMD). An integrated search tool consults the ontologies to enable searches without an exact term match. The ontologies can be used within other applications (such as Earth Science Markup Language descriptors) and future semantic web services in Earth system science.

  14. Femtosecond laser enabled keratoplasty for advanced keratoconus

    PubMed Central

    Shivanna, Yathish; Nagaraja, Harsha; Kugar, Thungappa; Shetty, Rohit

    2013-01-01

    Purpose: To assess the efficacy and advantages of femtosecond laser enabled keratoplasty (FLEK) over conventional penetrating keratoplasty (PKP) in advanced keratoconus. Materials and Methods: Detailed review of literature of published randomized controlled trials of operative techniques in PKP and FLEK. Results: Fifteen studies were identified, analyzed, and compared with our outcome. FLEK was found to have better outcome in view of better and earlier stabilization uncorrected visual acuity (UCVA), best corrected visual acuity (BCVA), and better refractive outcomes with low astigmatism as compared with conventional PKP. Wound healing also was noticed to be earlier, enabling early suture removal in FLEK. Conclusions: Studies relating to FLEK have shown better results than conventional PKP, however further studies are needed to assess the safety and intraoperative complications of the procedure. PMID:23925340

  15. A novel guide catheter enabling intracranial placement.

    PubMed

    Hurley, Michael C; Sherma, Arun K; Surdell, Daniel; Shaibani, Ali; Bendok, Bernard R

    2009-11-15

    We describe use of a novel guide, catheter with a soft and pliable, 6-cm or 12-cm distal segment that enables distal, including intracranial, placement--the Neuron guide catheter (Penumbra, San Leandro, CA)--in the treatment of 11 cases with a range of neuroendovascular lesions. We were able to advance the Neuron guide catheter to the intended level in each case and suffered no complications related to catheter spasm, dissection, thrombosis or thromboembolism. PMID:19670314

  16. Enabling Technologies for Petascale Electromagnetic Accelerator Simulation

    SciTech Connect

    Lee, Lie-Quan; Akcelik, Volkan; Chen, Sheng; Ge, Li-Xin; Prudencio, Ernesto; Schussman, Greg; Uplenchwar, Ravi; Ng, Cho; Ko, Kwok; Luo, Xiaojun; Shephard, Mark; /Rensselaer Poly.

    2007-11-09

    The SciDAC2 accelerator project at SLAC aims to simulate an entire three-cryomodule radio frequency (RF) unit of the International Linear Collider (ILC) main Linac. Petascale computing resources supported by advances in Applied Mathematics (AM) and Computer Science (CS) and INCITE Program are essential to enable such very large-scale electromagnetic accelerator simulations required by the ILC Global Design Effort. This poster presents the recent advances and achievements in the areas of CS/AM through collaborations.

  17. Enabling international adoption of LOINC through translation

    PubMed Central

    Vreeman, Daniel J.; Chiaravalloti, Maria Teresa; Hook, John; McDonald, Clement J.

    2012-01-01

    Interoperable health information exchange depends on adoption of terminology standards, but international use of such standards can be challenging because of language differences between local concept names and the standard terminology. To address this important barrier, we describe the evolution of an efficient process for constructing translations of LOINC terms names, the foreign language functions in RELMA, and the current state of translations in LOINC. We also present the development of the Italian translation to illustrate how translation is enabling adoption in international contexts. We built a tool that finds the unique list of LOINC Parts that make up a given set of LOINC terms. This list enables translation of smaller pieces like the core component “hepatitis c virus” separately from all the suffixes that could appear with it, such “Ab.IgG”, “DNA”, and “RNA”. We built another tool that generates a translation of a full LOINC name from all of these atomic pieces. As of version 2.36 (June 2011), LOINC terms have been translated into 9 languages from 15 linguistic variants other than its native English. The five largest linguistic variants have all used the Part-based translation mechanism. However, even with efficient tools and processes, translation of standard terminology is a complex undertaking. Two of the prominent linguistic challenges that translators have faced include: the approach to handling acronyms and abbreviations, and the differences in linguistic syntax (e.g. word order) between languages. LOINC’s open and customizable approach has enabled many different groups to create translations that met their needs and matched their resources. Distributing the standard and its many language translations at no cost worldwide accelerates LOINC adoption globally, and is an important enabler of interoperable health information exchange PMID:22285984

  18. NASA Missions Enabled by Space Nuclear Systems

    NASA Technical Reports Server (NTRS)

    Scott, John H.; Schmidt, George R.

    2009-01-01

    This viewgraph presentation reviews NASA Space Missions that are enabled by Space Nuclear Systems. The topics include: 1) Space Nuclear System Applications; 2) Trade Space for Electric Power Systems; 3) Power Generation Specific Energy Trade Space; 4) Radioisotope Power Generation; 5) Radioisotope Missions; 6) Fission Power Generation; 7) Solar Powered Lunar Outpost; 8) Fission Powered Lunar Outpost; 9) Fission Electric Power Generation; and 10) Fission Nuclear Thermal Propulsion.

  19. Design and Simulation of MEMS Enabled Systems

    NASA Astrophysics Data System (ADS)

    da Silva, Mark

    2001-03-01

    Over the past two decades considerable progress in microsystems (MEMS) fabrication technologies has been made resulting in a variety of commercially successful devices. Most of these devices have required application specific fabrication steps, which must be developed, and the lack of proper design tools often resulted in repeated prototyping that was expensive and time consuming. Further development of MEMS enabled commercial products and reduction of the time to market requires implementation of a concurrent design methodology through better design tools and standardization of the fabrication processes. The cross-disciplinary nature of MEMS-Enabled Systems necessitates designers with different backgrounds to work together in understanding the effects of one sub-system on another and this requires a top-down approach to integrated system design. Design tools that can facilitate this communication and reduce the need for excessive prototype fabrication and test iterations and significantly reduce cost and time-to-market are vitally important. The main focus of this article is to describe the top-down design methodology and and ongoing research on tools that facilitate concurrent design of MEMS enabled systems.

  20. Technology Enabling the First 100 Exoplanets

    NASA Astrophysics Data System (ADS)

    Marcy, Geoffrey W.

    2014-01-01

    The discoveries of the first 100 exoplanets by precise radial velocities in the late 1990's at Lick Observatory and Observatoire de Haute-Provence were enabled by several technological advances and a cultural one. A key ingredient was a cross-dispersed echelle spectrometer at a stable, coude focus, with a CCD detector, offering high spectral resolution, large wavelength coverage, and a linear response to photons. A second ingredient was a computer capable of storing the megabyte images from such spectrometers and analyzing them for Doppler shifts. Both Lick and OHP depended on these advents. A third ingredient was a stable wavelength calibration. Here, two technologies emerged independently, with iodine gas employed by Marcy's group (used first by solar physicists doing helioseismology) and simultaneous thorium-argon spectra (enabled by fiber optics) used by Mayor's group. A final ingredient was a new culture emerging in the 1990's of forward-modeling of spectra on computers, enabled by the well-behaved photon noise of CCDs, giving Poisson errors amenable to rigorous statistical algorithms for measuring millipixel Doppler shifts. The prospect of detecting the 12 meter/sec reflex velocity (1/100 pixel) of a Jupiter-like planet was considered impossible, except to a few who asked, "What actually limits Doppler precision?". Inspired insights were provided by Robert Howard, Paul Schechter, Bruce Campbell, and Gordon Walker, leading to the first 100 exoplanets.

  1. Quantitative Hydrocarbon Surface Analysis

    NASA Technical Reports Server (NTRS)

    Douglas, Vonnie M.

    2000-01-01

    The elimination of ozone depleting substances, such as carbon tetrachloride, has resulted in the use of new analytical techniques for cleanliness verification and contamination sampling. The last remaining application at Rocketdyne which required a replacement technique was the quantitative analysis of hydrocarbons by infrared spectrometry. This application, which previously utilized carbon tetrachloride, was successfully modified using the SOC-400, a compact portable FTIR manufactured by Surface Optics Corporation. This instrument can quantitatively measure and identify hydrocarbons from solvent flush of hardware as well as directly analyze the surface of metallic components without the use of ozone depleting chemicals. Several sampling accessories are utilized to perform analysis for various applications.

  2. Quantitative photoacoustic tomography

    PubMed Central

    Yuan, Zhen; Jiang, Huabei

    2009-01-01

    In this paper, several algorithms that allow for quantitative photoacoustic reconstruction of tissue optical, acoustic and physiological properties are described in a finite-element method based framework. These quantitative reconstruction algorithms are compared, and the merits and limitations associated with these methods are discussed. In addition, a multispectral approach is presented for concurrent reconstructions of multiple parameters including deoxyhaemoglobin, oxyhaemoglobin and water concentrations as well as acoustic speed. Simulation and in vivo experiments are used to demonstrate the effectiveness of the reconstruction algorithms presented. PMID:19581254

  3. Quantitative Decision Support Requires Quantitative User Guidance

    NASA Astrophysics Data System (ADS)

    Smith, L. A.

    2009-12-01

    Is it conceivable that models run on 2007 computer hardware could provide robust and credible probabilistic information for decision support and user guidance at the ZIP code level for sub-daily meteorological events in 2060? In 2090? Retrospectively, how informative would output from today’s models have proven in 2003? or the 1930’s? Consultancies in the United Kingdom, including the Met Office, are offering services to “future-proof” their customers from climate change. How is a US or European based user or policy maker to determine the extent to which exciting new Bayesian methods are relevant here? or when a commercial supplier is vastly overselling the insights of today’s climate science? How are policy makers and academic economists to make the closely related decisions facing them? How can we communicate deep uncertainty in the future at small length-scales without undermining the firm foundation established by climate science regarding global trends? Three distinct aspects of the communication of the uses of climate model output targeting users and policy makers, as well as other specialist adaptation scientists, are discussed. First, a brief scientific evaluation of the length and time scales at which climate model output is likely to become uninformative is provided, including a note on the applicability the latest Bayesian methodology to current state-of-the-art general circulation models output. Second, a critical evaluation of the language often employed in communication of climate model output, a language which accurately states that models are “better”, have “improved” and now “include” and “simulate” relevant meteorological processed, without clearly identifying where the current information is thought to be uninformative and misleads, both for the current climate and as a function of the state of the (each) climate simulation. And thirdly, a general approach for evaluating the relevance of quantitative climate model output

  4. Web-enabling technologies for the factory floor: a web-enabling strategy for emanufacturing

    NASA Astrophysics Data System (ADS)

    Velez, Ricardo; Lastra, Jose L. M.; Tuokko, Reijo O.

    2001-10-01

    This paper is intended to address the different technologies available for Web-enabling of the factory floor. It will give an overview of the importance of Web-enabling of the factory floor, in the application of the concepts of flexible and intelligent manufacturing, in conjunction with e-commerce. As a last section, it will try to define a Web-enabling strategy for the application in eManufacturing. This is made under the scope of the electronics manufacturing industry, so every application, technology or related matter is presented under such scope.

  5. Quantitative Decision Making.

    ERIC Educational Resources Information Center

    Baldwin, Grover H.

    The use of quantitative decision making tools provides the decision maker with a range of alternatives among which to decide, permits acceptance and use of the optimal solution, and decreases risk. Training line administrators in the use of these tools can help school business officials obtain reliable information upon which to base district…

  6. Quantitative Graphics in Newspapers.

    ERIC Educational Resources Information Center

    Tankard, James W., Jr.

    The use of quantitative graphics in newspapers requires achieving a balance between being accurate and getting the attention of the reader. The statistical representations in newspapers are drawn by graphic designers whose key technique is fusion--the striking combination of two visual images. This technique often results in visual puns,…

  7. Quantitative Simulation Games

    NASA Astrophysics Data System (ADS)

    Černý, Pavol; Henzinger, Thomas A.; Radhakrishna, Arjun

    While a boolean notion of correctness is given by a preorder on systems and properties, a quantitative notion of correctness is defined by a distance function on systems and properties, where the distance between a system and a property provides a measure of "fit" or "desirability." In this article, we explore several ways how the simulation preorder can be generalized to a distance function. This is done by equipping the classical simulation game between a system and a property with quantitative objectives. In particular, for systems that satisfy a property, a quantitative simulation game can measure the "robustness" of the satisfaction, that is, how much the system can deviate from its nominal behavior while still satisfying the property. For systems that violate a property, a quantitative simulation game can measure the "seriousness" of the violation, that is, how much the property has to be modified so that it is satisfied by the system. These distances can be computed in polynomial time, since the computation reduces to the value problem in limit average games with constant weights. Finally, we demonstrate how the robustness distance can be used to measure how many transmission errors are tolerated by error correcting codes.

  8. Critical Quantitative Inquiry in Context

    ERIC Educational Resources Information Center

    Stage, Frances K.; Wells, Ryan S.

    2014-01-01

    This chapter briefly traces the development of the concept of critical quantitative inquiry, provides an expanded conceptualization of the tasks of critical quantitative research, offers theoretical explanation and justification for critical research using quantitative methods, and previews the work of quantitative criticalists presented in this…

  9. A wireless sensor enabled by wireless power.

    PubMed

    Lee, Da-Sheng; Liu, Yu-Hong; Lin, Chii-Ruey

    2012-01-01

    Through harvesting energy by wireless charging and delivering data by wireless communication, this study proposes the concept of a wireless sensor enabled by wireless power (WPWS) and reports the fabrication of a prototype for functional tests. One WPWS node consists of wireless power module and sensor module with different chip-type sensors. Its main feature is the dual antenna structure. Following RFID system architecture, a power harvesting antenna was designed to gather power from a standard reader working in the 915 MHz band. Referring to the Modbus protocol, the other wireless communication antenna was integrated on a node to send sensor data in parallel. The dual antenna structure integrates both the advantages of an RFID system and a wireless sensor. Using a standard UHF RFID reader, WPWS can be enabled in a distributed area with a diameter up to 4 m. Working status is similar to that of a passive tag, except that a tag can only be queried statically, while the WPWS can send dynamic data from the sensors. The function is the same as a wireless sensor node. Different WPWSs equipped with temperature and humidity, optical and airflow velocity sensors are tested in this study. All sensors can send back detection data within 8 s. The accuracy is within 8% deviation compared with laboratory equipment. A wireless sensor network enabled by wireless power should be a totally wireless sensor network using WPWS. However, distributed WPWSs only can form a star topology, the simplest topology for constructing a sensor network. Because of shielding effects, it is difficult to apply other complex topologies. Despite this limitation, WPWS still can be used to extend sensor network applications in hazardous environments. Further research is needed to improve WPWS to realize a totally wireless sensor network. PMID:23443370

  10. Sensor Web Enablement for Coastal Buoy Systems

    NASA Astrophysics Data System (ADS)

    Ling, Y.; Durbha, S. S.; King, R. L.

    2006-12-01

    Coastal buoys and stations provide frequent, high-quality marine observations for weather service, public safety, atmospheric, and oceanographic study. However, sharing of the generated datasets, information, and results, between geographically distributed organizations often proves to be challenging. This is due to the complicated steps involved in data discovery and conversion of the data into usable information due to problems of syntactic, structural, and semantic heterogeneity in the datasets. Therefore, a standardized modeling framework is desired for the coastal buoys to provide enhanced capabilities for interoperability and to better disseminate the information. This study is developing an interoperable framework for coastal buoys using Sensor Model Language (SensorML) and other components (e.g., Observations & Measurements Schema (O&M), Transducer Markup Language (TransducerML), Sensor Observation Service (SOS), etc) of the OpenGeospatial Consortium (OGC) Sensor Web Enablement (SWE). SensorML is a standard for the description of measurement devices and more complex measurement systems, in order to enable automatic processing of sensor data by generic software. In this study, buoys operated by the National Data Buoy Center (NDBC) with different payloads (e.g., ARES, DACT, DART, GSBP, MARS, and VEEP) were described using SensorML. Each of these payloads has a variety of sensors used to measure the marine parameters (e.g., sea surface temperature, wind direction, wind speed, water level). Our framework of the proposed Coastal Sensor Web Enablement (CSWE) middleware for buoy systems is built upon the existing OGC web services. The Web service specifications such as Sensor Planning Service (SPS), Sensor Observation Service (SOS), and Sensor Alert Service (SAS) define how data collection requests are expressed, observations retrieved, and alert or alarm conditions defined. The integration of these components in the proposed architecture provides access to

  11. Astronomy Enabled by Ares V -- A Workshop

    NASA Astrophysics Data System (ADS)

    Lester, Daniel F.; Langhoff, S.; Worden, S. P.; Thronson, H.; Correll, R.

    2009-01-01

    On April 26th and 27th, 2008, NASA Ames Research Center hosted a two-day weekend workshop entitled "Astronomy Enabled by Ares V.” The primary goal of the workshop was to begin the process of bringing the Ares V designers together with senior representatives of the astronomical community to discuss the feasibility of using the Ares V heavy-lift launch vehicle to enable both new astronomical telescope architectures and new science. When developed in the latter part of the upcoming decade Ares V will be by far the most capable launch vehicle, with mass and volume launch capability many times that now available. The vehicle is understood to be the main workhorse in carrying humans and cargo to the Moon and beyond and, as such, is a key lynchpin for NASA's new space transportation architecture. Participants included experts from academia, industry, and NASA, including representatives of the Constellation architecture. Participants considered, in the context of identified astronomy needs: (1) Are there telescope concepts or missions capable of breakthrough science that are either enabled or significantly enhanced by the capabilities of an Ares V? (2) What demands do large telescopes place on the payload environment of the Ares V, such as mass, volume, fairing shape, cleanliness, acoustics, etc.? (3) What technology and environmental issues need to be addressed to facilitate launching observatories on an Ares V? (4) Is there a trade-off between mass and complexity that could reduce launch risk and, thereby, the cost of building large telescopes? We report on the results of this workshop, which included discussion on the operations model for such large-investment astronomical facilities. Such an operations model might well involve human and or robotic maintenance and servicing, in order to fully capitalize on the science potential of such facilities.

  12. A Wireless Sensor Enabled by Wireless Power

    PubMed Central

    Lee, Da-Sheng; Liu, Yu-Hong; Lin, Chii-Ruey

    2012-01-01

    Through harvesting energy by wireless charging and delivering data by wireless communication, this study proposes the concept of a wireless sensor enabled by wireless power (WPWS) and reports the fabrication of a prototype for functional tests. One WPWS node consists of wireless power module and sensor module with different chip-type sensors. Its main feature is the dual antenna structure. Following RFID system architecture, a power harvesting antenna was designed to gather power from a standard reader working in the 915 MHz band. Referring to the Modbus protocol, the other wireless communication antenna was integrated on a node to send sensor data in parallel. The dual antenna structure integrates both the advantages of an RFID system and a wireless sensor. Using a standard UHF RFID reader, WPWS can be enabled in a distributed area with a diameter up to 4 m. Working status is similar to that of a passive tag, except that a tag can only be queried statically, while the WPWS can send dynamic data from the sensors. The function is the same as a wireless sensor node. Different WPWSs equipped with temperature and humidity, optical and airflow velocity sensors are tested in this study. All sensors can send back detection data within 8 s. The accuracy is within 8% deviation compared with laboratory equipment. A wireless sensor network enabled by wireless power should be a totally wireless sensor network using WPWS. However, distributed WPWSs only can form a star topology, the simplest topology for constructing a sensor network. Because of shielding effects, it is difficult to apply other complex topologies. Despite this limitation, WPWS still can be used to extend sensor network applications in hazardous environments. Further research is needed to improve WPWS to realize a totally wireless sensor network. PMID:23443370

  13. Small-RPS Enabled Mars Rover Concept

    NASA Technical Reports Server (NTRS)

    Balint, Tibor S.

    2004-01-01

    Both the MER and the Mars Pathfinder rovers operated on Mars in an energy-limited mode, since the solar panels generated power during daylight hours only. At other times the rovers relied on power stored in batteries. In comparison, Radioisotope Power Systems (RPS) offer a power-enabled paradigm, where power can be generated for long mission durations (measured in years), independently from the Sun, and on a continuous basis. A study was performed at PL to assess the feasibility of a small-RPS enabled MER-class rover concept and any associated advantages of its mission on Mars. The rover concept relied on design heritage from MER with two significant changes. First, the solar panels were replaced with two single GPHS module based small-RPSs. Second, the Mossbauer spectroscope was substituted with a laser Raman spectroscope, in order to move towards MEPAG defined astrobiology driven science goals. The highest power requirements were contributed to mobility and telecommunication type operating modes, hence influencing power system sizing. The resulting hybrid power system included two small-RPSs and two batteries. Each small-RPS was assumed to generate 50We of power or 62OWh/sol of energy (BOL), comparable to that of MER. The two 8Ah batteries were considered available during peak power usage. Mission architecture, power trades, science instruments, data, communication, thermal and radiation environments, mobility, mass issues were also addressed. The study demonstrated that a new set of RPS-enabled rover missions could be envisioned for Mars exploration within the next decade, targeting astrobiology oriented science objectives, while powered by 2 to 4 GPHS modules.

  14. Camera-enabled techniques for organic synthesis

    PubMed Central

    Ingham, Richard J; O’Brien, Matthew; Browne, Duncan L

    2013-01-01

    Summary A great deal of time is spent within synthetic chemistry laboratories on non-value-adding activities such as sample preparation and work-up operations, and labour intensive activities such as extended periods of continued data collection. Using digital cameras connected to computer vision algorithms, camera-enabled apparatus can perform some of these processes in an automated fashion, allowing skilled chemists to spend their time more productively. In this review we describe recent advances in this field of chemical synthesis and discuss how they will lead to advanced synthesis laboratories of the future. PMID:23766820

  15. The role of CORBA in enabling telemedicine

    SciTech Connect

    Forslund, D.W.

    1997-07-01

    One of the most powerful tools available for telemedicine is a multimedia medical record accessible over a wide area and simultaneously editable by multiple physicians. The ability to do this through an intuitive interface linking multiple distributed data repositories while maintaining full data integrity is a fundamental enabling technology in healthcare. The author discusses the role of distributed object technology using CORBA in providing this capability including an example of such a system (TeleMed) which can be accessed through the World Wide Web. Issues of security, scalability, data integrity, and useability are emphasized.

  16. Transparent displays enabled by resonant nanoparticle scattering

    NASA Astrophysics Data System (ADS)

    Hsu, Chia Wei; Zhen, Bo; Qiu, Wenjun; Shapira, Ofer; Delacy, Brendan G.; Joannopoulos, John D.; Soljačić, Marin

    2014-01-01

    The ability to display graphics and texts on a transparent screen can enable many useful applications. Here we create a transparent display by projecting monochromatic images onto a transparent medium embedded with nanoparticles that selectively scatter light at the projected wavelength. We describe the optimal design of such nanoparticles, and experimentally demonstrate this concept with a blue-color transparent display made of silver nanoparticles in a polymer matrix. This approach has attractive features including simplicity, wide viewing angle, scalability to large sizes and low cost.

  17. WSKE: Web Server Key Enabled Cookies

    NASA Astrophysics Data System (ADS)

    Masone, Chris; Baek, Kwang-Hyun; Smith, Sean

    In this paper, we present the design and prototype of a new approach to cookie management: if a server deposits a cookie only after authenticating itself via the SSL handshake, the browser will return the cookie only to a server that can authenticate itself, via SSL, to the same keypair. This approach can enable usable but secure client authentication. This approach can improve the usability of server authentication by clients. This approach is superior to the prior work on Active Cookies in that it defends against both DNS spoofing and IP spoofing—and does not require binding a user's interaction with a server to individual IP addresses.

  18. Solar vapor generation enabled by nanoparticles.

    PubMed

    Neumann, Oara; Urban, Alexander S; Day, Jared; Lal, Surbhi; Nordlander, Peter; Halas, Naomi J

    2013-01-22

    Solar illumination of broadly absorbing metal or carbon nanoparticles dispersed in a liquid produces vapor without the requirement of heating the fluid volume. When particles are dispersed in water at ambient temperature, energy is directed primarily to vaporization of water into steam, with a much smaller fraction resulting in heating of the fluid. Sunlight-illuminated particles can also drive H(2)O-ethanol distillation, yielding fractions significantly richer in ethanol content than simple thermal distillation. These phenomena can also enable important compact solar applications such as sterilization of waste and surgical instruments in resource-poor locations. PMID:23157159

  19. Agility enabled by the SEMATECH CIM framework

    NASA Astrophysics Data System (ADS)

    Hawker, Scott; Waskiewicz, Fred

    1997-01-01

    The survivor in today's market environment is agile: able to survive and thrive in a market place marked by rapid, continuous change. For manufacturers, this includes an ability to rapidly develop, deploy and reconfigure manufacturing information and control systems. The SEMATECH CIM framework defines an application integration architecture and standard application components that enable agile manufacturing information and control systems. Further, the CIM framework and its evolution process foster virtual organizations of suppliers and manufacturers, combining their products and capabilities into an agile manufacturing information and control system.

  20. ENABLER Nuclear Propulsion System Conceptual Design

    NASA Astrophysics Data System (ADS)

    Pauley, Keith A.; Woodham, Kurt; Ohi, Don; Haga, Heath; Henderson, Bo

    2004-02-01

    The Titan Corporation conducted a systems engineering study to develop an overall architecture that meets both the articulated and unarticulated requirements on the Prometheus Program with the least development effort. Key elements of the Titan-designed ENABLER system include a thermal fission reactor, thermionic power converters, sodium heat pipes, ion thruster engines, and a radiation shield and deployable truss to protect the payload. The overall design is scaleable over a wide range of power requirements from 10s of kilowatts to 10s of megawatts.

  1. Enabling Rapid Naval Architecture Design Space Exploration

    NASA Technical Reports Server (NTRS)

    Mueller, Michael A.; Dufresne, Stephane; Balestrini-Robinson, Santiago; Mavris, Dimitri

    2011-01-01

    Well accepted conceptual ship design tools can be used to explore a design space, but more precise results can be found using detailed models in full-feature computer aided design programs. However, defining a detailed model can be a time intensive task and hence there is an incentive for time sensitive projects to use conceptual design tools to explore the design space. In this project, the combination of advanced aerospace systems design methods and an accepted conceptual design tool facilitates the creation of a tool that enables the user to not only visualize ship geometry but also determine design feasibility and estimate the performance of a design.

  2. Biomolecular Plasmonics for Quantitative Biology and Nanomedicine

    PubMed Central

    Lee, Somin Eunice; Lee, Luke P.

    2012-01-01

    Free electrons in a noble metal nanoparticle can be resonantly excited, leading to their collective oscillation termed as a surface plasmon. These surface plasmons enable nanoparticles to absorb light, generate heat, transfer energy, and re-radiate incident photons. Creative designs of nanoplasmonic optical antennae (i.e. plasmon resonant nanoparticles) have become a new foundation of quantitative biology and nanomedicine. This review focuses on the recent developments in dual-functional nanoplasmonic optical antennae for label-free biosensors and nanoplasmonic gene switches. Nanoplasmonic optical antennae, functioning as biosensors to significantly enhance biochemical-specific spectral information via plasmon resonance energy transfer (PRET) and surface-enhanced Raman spectroscopy (SERS), are discussed. Nanoplasmonic optical antennae, functioning as nanoplasmonic gene switches to enable spatiotemporal regulation of genetic activity, are also reviewed. Nanoplasmonic molecular rulers and integrated photoacoustic-photothermal contrast agents are also described. PMID:20801636

  3. Quantitative Radiological Diagnosis Of The Temporomandibular Joint

    NASA Astrophysics Data System (ADS)

    Jordan, Steven L.; Heffez, Leslie B.

    1989-05-01

    Recent impressive technological advances in imaging techniques for the human temporomandibular (tm) joint, and in enabling geometric algorithms have outpaced diagnostic analyses. The authors present a basis for systematic quantitative diagnoses that exploit the imaging advancements. A reference line, coordinate system, and transformations are described that are appropriate for tomography of the tm joint. These yield radiographic measurements (disk displacement) and observations (beaking of radiopaque dye and disk shape) that refine diagnostic classifications of anterior displacement of the condylar disk. The relevance of these techniques has been clinically confirmed. Additional geometric invariants and procedures are proposed for future clinical verification.

  4. On the Distinction Between Quantitative and Qualitative Research.

    ERIC Educational Resources Information Center

    Smith, P. L.

    Quantitative and qualitative research are differing modes of measurement, one using numbers and the other not. The assignment of numerals to represent properties enables a researcher to distinguish minutely between different properties. The major issue dividing these approaches to empirical research represents a philosophical dispute which has…

  5. Identification of Phosphorylated Cyclin-Dependent Kinase 1 Associated with Colorectal Cancer Survival Using Label-Free Quantitative Analyses

    PubMed Central

    Tyan, Yu-Chang; Hsiao, Eric S. L.; Chu, Po-Chen; Lee, Chung-Ta; Lee, Jenq-Chang; Chen, Yi-Ming Arthur; Liao, Pao-Chi

    2016-01-01

    Colorectal cancer is the most common form of cancer in the world, and the five-year survival rate is estimated to be almost 90% in the early stages. Therefore, the identification of potential biomarkers to assess the prognosis of early stage colorectal cancer patients is critical for further clinical treatment. Dysregulated tyrosine phosphorylation has been found in several diseases that play a significant regulator of signaling in cellular pathways. In this study, this strategy was used to characterize the tyrosine phosphoproteome of colorectal cell lines with different progression abilities (SW480 and SW620). We identified a total of 280 phosphotyrosine (pTyr) peptides comprising 287 pTyr sites from 261 proteins. Label-free quantitative analysis revealed the differential level of a total of 103 pTyr peptides between SW480 and SW620 cells. We showed that cyclin-dependent kinase I (CDK1) pTyr15 level in SW480 cells was 3.3-fold greater than in SW620 cells, and these data corresponded with the label-free mass spectrometry-based proteomic quantification analysis. High level CDK1 pTyr15 was associated with prolonged disease-free survival for stage II colorectal cancer patients (n = 79). Taken together, our results suggest that the CDK1 pTyr15 protein is a potential indicator of the progression of colorectal cancer. PMID:27383761

  6. High-performance microlasers enable display applications

    NASA Astrophysics Data System (ADS)

    Takeuchi, Eric B.; Hargis, David E.; Bergstedt, Robert; Dion, Al; Hurtado, Randy; Solone, Paul J.

    1999-08-01

    Recent advances in compact, air-cooled, diode-pumped solid- state visible microlasers have enabled the development of portable laser display systems. In addition to the added benefits of large color gamut, invariant color accuracy, image uniformity, high contrast, and large depth of focus inherent in the microlaser design, the reliability of these all-solid state red-green-blue (RGB) sources make them attractive for display applications. Compact, multi-watt laser modules have been demonstrated for use as a high brightness 'laser light engine' for replacing arc lamps in LCD/DMD type display configurations. Using this 'backlit' approach, a microlaser- based projector has been demonstrated, providing greater than 500 lumens at 1280 X 1024 resolution using reflective AMLCD light valves. Also being developed is an airborne tactical HMD system wherein the laser module is remotely coupled to a subtractive color LCD assembly through an optical fiber to provide a more than 24,000,000 (twenty-four million) cd/m2 luminance for illuminating the LCD assembly. This technology could be applied to a variety of cockpit displays providing sunlight readable illumination for both head-down and head-up backlit display configurations. The advantages of the microlaser technology will enable further applications in other military platforms such as command and control centers, simulators and HMDs. Longer term potential includes high end CAD workstations, entertainment systems, and electronic cinema.

  7. Blue space geographies: Enabling health in place.

    PubMed

    Foley, Ronan; Kistemann, Thomas

    2015-09-01

    Drawing from research on therapeutic landscapes and relationships between environment, health and wellbeing, we propose the idea of 'healthy blue space' as an important new development Complementing research on healthy green space, blue space is defined as; 'health-enabling places and spaces, where water is at the centre of a range of environments with identifiable potential for the promotion of human wellbeing'. Using theoretical ideas from emotional and relational geographies and critical understandings of salutogenesis, the value of blue space to health and wellbeing is recognised and evaluated. Six individual papers from five different countries consider how health can be enabled in mixed blue space settings. Four sub-themes; embodiment, inter-subjectivity, activity and meaning, document multiple experiences within a range of healthy blue spaces. Finally, we suggest a considerable research agenda - theoretical, methodological and applied - for future work within different forms of blue space. All are suggested as having public health policy relevance in social and public space. PMID:26238330

  8. The Master Enabler: In Orbit Servicing

    NASA Technical Reports Server (NTRS)

    Reed, Benjamin B.; Kienlen, Michael; Naasz, Bo; Roberts, Brian; Deweese, Keith; Cassidy, Justin

    2015-01-01

    Some of the most noteworthy missions in space exploration have occurred in the last two decades and owe their success to on-orbit servicing. The tremendously successful Hubble Space Telescope repair and upgrade missions, as well as the completed assembly of the International Space Station (ISS) and its full utilization, lead us to the next chapter and set of challenges. These include fully exploiting the many space systems already launched, assembling large structures in situ thereby enabling new scientific discoveries, and providing systems that reliably and cost-effectively support the next steps in space exploration. In-orbit servicing is a tool--a tool that can serve as the master enabler to create space architectures that would otherwise be unattainable. This paper will survey how NASA's satellite-servicing technology development efforts are being applied to the planning and execution of two such ambitious missions, specifically asteroid capture and the in-space assembly of a very large life-finding telescope.

  9. In-Orbit Servicing: The Master Enabler

    NASA Technical Reports Server (NTRS)

    Reed, Benjamin B.; Kienlen, Michael; Naasz, Bo; Roberts, Brian; Deweese, Keith

    2015-01-01

    Some of the most noteworthy missions in space exploration have occurred in the last two decades and owe their success to on-orbit servicing. The tremendously successful Hubble Space Telescope repair and upgrade missions, as well as the completed assembly of the International Space Station (ISS) and its full utilization, lead us to the next chapter and set of challenges. These include fully exploiting the many space systems already launched, assembling large structures in situ thereby enabling new scientific discoveries, and providing systems that reliably and cost-effectively support the next steps in space exploration. In-orbit servicing is a tool--a tool that can serve as the master enabler to create space architectures that would otherwise be unattainable. This paper will survey how NASA's satellite-servicing technology development efforts are being applied to the planning and execution of two such ambitious missions, specifically asteroid capture and the in-space assembly of a very large life-finding telescope.

  10. The "Master Enabler" - In-Orbit Servicing

    NASA Technical Reports Server (NTRS)

    Reed, Benjamin; Kienlen, Michael; Naasz, Bo; Roberts, Brian; Deweese, Keith; Cassidy, Justin

    2015-01-01

    Some of the most noteworthy missions in space exploration have occurred in the last two decades and owe their success to on-orbit servicing. The tremendously successful Hubble Space Telescope repair and upgrade missions, as well as the completed assembly of the International Space Station (ISS) and its full utilization, lead us to the next chapter and set of challenges. These include fully exploiting the many space systems already launched, assembling large structures in situ thereby enabling new scientific discoveries, and providing systems that reliably and cost-effectively support the next steps in space exploration. In-orbit servicing is a tool-a tool that can serve as the master enabler to create space architectures that would otherwise be unattainable. This paper will survey how NASA's satellite-servicing technology development efforts are being applied to the planning and execution of two such ambitious missions, specifically asteroid capture and the in-space assembly of a very large life-finding telescope.

  11. Interaction-enabled topological crystalline phases

    NASA Astrophysics Data System (ADS)

    Lapa, Matthew F.; Teo, Jeffrey C. Y.; Hughes, Taylor L.

    2016-03-01

    In this article we provide a general mechanism for generating interaction-enabled fermionic topological phases. We illustrate the mechanism with crystalline symmetry-protected topological phases in one, two, and three spatial dimensions. These nontrivial phases require interactions for their existence, and in the cases we consider, the free-fermion classification yields only a trivial phase. For the one- and two-dimensional phases we consider, we provide explicit exactly solvable models which realize the interaction-enabled phases. Similar to the interpretation of the Kitaev Majorana wire as a mean-field p -wave superconductor Hamiltonian arising from an interacting model with quartic interactions, we show that our systems can be interpreted as "mean-field" charge-4 e superconductors arising, e.g., from an interacting model with eight-body interactions or through another physical mechanism. The quartet superconducting nature allows for the teleportation of full Cooper pairs and, in two dimensions, for interesting semiclassical crystalline defects with non-Abelian anyon bound states.

  12. MENTOR: an enabler for interoperable intelligent systems

    NASA Astrophysics Data System (ADS)

    Sarraipa, João; Jardim-Goncalves, Ricardo; Steiger-Garcao, Adolfo

    2010-07-01

    A community with knowledge organisation based on ontologies will enable an increase in the computational intelligence of its information systems. However, due to the worldwide diversity of communities, a high number of knowledge representation elements, which are not semantically coincident, have appeared representing the same segment of reality, becoming a barrier to business communications. Even if a domain community uses the same kind of technologies in its information systems, such as ontologies, it doesn't solve its semantics differences. In order to solve this interoperability problem, a solution is to use a reference ontology as an intermediary in the communications between the community enterprises and the outside, while allowing the enterprises to keep their own ontology and semantics unchanged internally. This work proposes MENTOR, a methodology to support the development of a common reference ontology for a group of organisations sharing the same business domain. This methodology is based on the mediator ontology (MO) concept, which assists the semantic transformations among each enterprise's ontology and the referential one. The MO enables each organisation to keep its own terminology, glossary and ontological structures, while providing seamless communication and interaction with the others.

  13. Nano-Enabled SERS Reporting Photosensitizers

    PubMed Central

    Farhadi, Arash; Roxin, Áron; Wilson, Brian C.; Zheng, Gang

    2015-01-01

    To impart effective cellular damage via photodynamic therapy (PDT), it is vital to deliver the appropriate light dose and photosensitizer concentration, and to monitor the PDT dose delivered at the site of interest. In vivo monitoring of photosensitizers has in large part relied on their fluorescence emission. Palladium-containing photosensitizers have shown promising clinical results by demonstrating near full conversion of light to PDT activity at the cost of having undetectable fluorescence. We demonstrate that, through the coupling of plasmonic nanoparticles with palladium-photosensitizers, surface-enhanced Raman scattering (SERS) provides both reporting and monitoring capability to otherwise quiescent molecules. Nano-enabled SERS reporting of photosensitizers allows for the decoupling of the therapeutic and imaging mechanisms so that both phenomena can be optimized independently. Most importantly, the design enables the use of the same laser wavelength to stimulate both the PDT and imaging features, opening the potential for real-time dosimetry of photosensitizer concentration and PDT dose delivery by SERS monitoring. PMID:25767614

  14. Ultra-precision: enabling our future.

    PubMed

    Shore, Paul; Morantz, Paul

    2012-08-28

    This paper provides a perspective on the development of ultra-precision technologies: What drove their evolution and what do they now promise for the future as we face the consequences of consumption of the Earth's finite resources? Improved application of measurement is introduced as a major enabler of mass production, and its resultant impact on wealth generation is considered. This paper identifies the ambitions of the defence, automotive and microelectronics sectors as important drivers of improved manufacturing accuracy capability and ever smaller feature creation. It then describes how science fields such as astronomy have presented significant precision engineering challenges, illustrating how these fields of science have achieved unprecedented levels of accuracy, sensitivity and sheer scale. Notwithstanding their importance to science understanding, many science-driven ultra-precision technologies became key enablers for wealth generation and other well-being issues. Specific ultra-precision machine tools important to major astronomy programmes are discussed, as well as the way in which subsequently evolved machine tools made at the beginning of the twenty-first century, now provide much wider benefits. PMID:22802499

  15. Enabling techniques for asynchronous coherent OCDMA

    NASA Astrophysics Data System (ADS)

    Wang, Xu; Wada, Naoya; Kitayama, Ken-ichi

    2005-11-01

    The coherent OCDMA system could suffer from severe multiple access interference (MAI) and beat noise, which limit the maximum number of active users that can be supported in a network. One effective method to reduce the beat noise as well as the MAI noise is to lower the interference level by adopting ultra-long optical code. Applying optical thresholding technique is also crucial to enable data-rate detection for achieving a practical OCDMA system. In this paper, we review the recent progress in the key enabling techniques for asynchronous coherent OCDMA: the novel encoder/decoders including spatial lightwave phase modulator, micro-ring resonator for spectral phase coding and superstructured FBG (SSFBG) and AWG type encode/decoder for time-spreading coding; optical thresholding techniques with PPLN and nonlinearity in fiber. The FEC has also been applied in OCDMA system recently. With 511-chip SSFBG and SC-based optical thresholder, 10-user, truly-asynchronous gigabit OCDMA transmission has been successfully achieved. Most recently, a record throughput 12×10.71 Gbps truly-asynchronous OCDMA has been demonstrated by using the 16×16 ports AWG-type encoder/decoder and FEC transmit ITU-T G.709 OTN frames.

  16. Phosphoproteomic Profiling Reveals Epstein-Barr Virus Protein Kinase Integration of DNA Damage Response and Mitotic Signaling

    PubMed Central

    Li, Renfeng; Pinto, Sneha M.; Shaw, Patrick G.; Huang, Tai-Chung; Wan, Jun; Qian, Jiang; Gowda, Harsha; Wu, Xinyan; Lv, Dong-Wen; Zhang, Kun; Manda, Srikanth S.; Pandey, Akhilesh; Hayward, S. Diane

    2015-01-01

    Epstein-Barr virus (EBV) is etiologically linked to infectious mononucleosis and several human cancers. EBV encodes a conserved protein kinase BGLF4 that plays a key role in the viral life cycle. To provide new insight into the host proteins regulated by BGLF4, we utilized stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics to compare site-specific phosphorylation in BGLF4-expressing Akata B cells. Our analysis revealed BGLF4-mediated hyperphosphorylation of 3,046 unique sites corresponding to 1,328 proteins. Frequency analysis of these phosphosites revealed a proline-rich motif signature downstream of BGLF4, indicating a broader substrate recognition for BGLF4 than its cellular ortholog cyclin-dependent kinase 1 (CDK1). Further, motif analysis of the hyperphosphorylated sites revealed enrichment in ATM, ATR and Aurora kinase substrates while functional analyses revealed significant enrichment of pathways related to the DNA damage response (DDR), mitosis and cell cycle. Phosphorylation of proteins associated with the mitotic spindle assembly checkpoint (SAC) indicated checkpoint activation, an event that inactivates the anaphase promoting complex/cyclosome, APC/C. Furthermore, we demonstrated that BGLF4 binds to and directly phosphorylates the key cellular proteins PP1, MPS1 and CDC20 that lie upstream of SAC activation and APC/C inhibition. Consistent with APC/C inactivation, we found that BGLF4 stabilizes the expression of many known APC/C substrates. We also noted hyperphosphorylation of 22 proteins associated the nuclear pore complex, which may contribute to nuclear pore disassembly and SAC activation. A drug that inhibits mitotic checkpoint activation also suppressed the accumulation of extracellular EBV virus. Taken together, our data reveal that, in addition to the DDR, manipulation of mitotic kinase signaling and SAC activation are mechanisms associated with lytic EBV replication. All MS data have been deposited in

  17. Phosphoproteomic Profiling Reveals Epstein-Barr Virus Protein Kinase Integration of DNA Damage Response and Mitotic Signaling.

    PubMed

    Li, Renfeng; Liao, Gangling; Nirujogi, Raja Sekhar; Pinto, Sneha M; Shaw, Patrick G; Huang, Tai-Chung; Wan, Jun; Qian, Jiang; Gowda, Harsha; Wu, Xinyan; Lv, Dong-Wen; Zhang, Kun; Manda, Srikanth S; Pandey, Akhilesh; Hayward, S Diane

    2015-12-01

    Epstein-Barr virus (EBV) is etiologically linked to infectious mononucleosis and several human cancers. EBV encodes a conserved protein kinase BGLF4 that plays a key role in the viral life cycle. To provide new insight into the host proteins regulated by BGLF4, we utilized stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics to compare site-specific phosphorylation in BGLF4-expressing Akata B cells. Our analysis revealed BGLF4-mediated hyperphosphorylation of 3,046 unique sites corresponding to 1,328 proteins. Frequency analysis of these phosphosites revealed a proline-rich motif signature downstream of BGLF4, indicating a broader substrate recognition for BGLF4 than its cellular ortholog cyclin-dependent kinase 1 (CDK1). Further, motif analysis of the hyperphosphorylated sites revealed enrichment in ATM, ATR and Aurora kinase substrates while functional analyses revealed significant enrichment of pathways related to the DNA damage response (DDR), mitosis and cell cycle. Phosphorylation of proteins associated with the mitotic spindle assembly checkpoint (SAC) indicated checkpoint activation, an event that inactivates the anaphase promoting complex/cyclosome, APC/C. Furthermore, we demonstrated that BGLF4 binds to and directly phosphorylates the key cellular proteins PP1, MPS1 and CDC20 that lie upstream of SAC activation and APC/C inhibition. Consistent with APC/C inactivation, we found that BGLF4 stabilizes the expression of many known APC/C substrates. We also noted hyperphosphorylation of 22 proteins associated the nuclear pore complex, which may contribute to nuclear pore disassembly and SAC activation. A drug that inhibits mitotic checkpoint activation also suppressed the accumulation of extracellular EBV virus. Taken together, our data reveal that, in addition to the DDR, manipulation of mitotic kinase signaling and SAC activation are mechanisms associated with lytic EBV replication. All MS data have been deposited in

  18. Reference-facilitated phosphoproteomics: fast and reliable phosphopeptide validation by microLC-ESI-Q-TOF MS/MS.

    PubMed

    Imanishi, Susumu Y; Kochin, Vitaly; Ferraris, Saima E; de Thonel, Aurélie; Pallari, Hanna-Mari; Corthals, Garry L; Eriksson, John E

    2007-08-01

    Recent advances in instrument control and enrichment procedures have enabled us to quantify large numbers of phosphoproteins and record site-specific phosphorylation events. An intriguing problem that has arisen with these advances is to accurately validate where phosphorylation events occur, if possible, in an automated manner. The problem is difficult because MS/MS spectra of phosphopeptides are generally more complicated than those of unmodified peptides. For large scale studies, the problem is even more evident because phosphorylation sites are based on single peptide identifications in contrast to protein identifications where at least two peptides from the same protein are required for identification. To address this problem we have developed an integrated strategy that increases the reliability and ease for phosphopeptide validation. We have developed an off-line titanium dioxide (TiO(2)) selective phosphopeptide enrichment procedure for crude cell lysates. Following enrichment, half of the phosphopeptide fractionated sample is enzymatically dephosphorylated, after which both samples are subjected to LC-MS/MS. From the resulting MS/MS analyses, the dephosphorylated peptide is used as a reference spectrum against the original phosphopeptide spectrum, in effect generating two peptide spectra for the same amino acid sequence, thereby enhancing the probability of a correct identification. The integrated procedure is summarized as follows: 1) enrichment for phosphopeptides by TiO(2) chromatography, 2) dephosphorylation of half the sample, 3) LC-MS/MS-based analysis of phosphopeptides and corresponding dephosphorylated peptides, 4) comparison of peptide elution profiles before and after dephosphorylation to confirm phosphorylation, and 5) comparison of MS/MS spectra before and after dephosphorylation to validate the phosphopeptide and its phosphorylation site. This phosphopeptide identification represents a major improvement as compared with identifications based

  19. Energy & Climate: Getting Quantitative

    NASA Astrophysics Data System (ADS)

    Wolfson, Richard

    2011-11-01

    A noted environmentalist claims that buying an SUV instead of a regular car is energetically equivalent to leaving your refrigerator door open for seven years. A fossil-fuel apologist argues that solar energy is a pie-in-the-sky dream promulgated by na"ive environmentalists, because there's nowhere near enough solar energy to meet humankind's energy demand. A group advocating shutdown of the Vermont Yankee nuclear plant claims that 70% of its electrical energy is lost in transmission lines. Around the world, thousands agitate for climate action, under the numerical banner ``350.'' Neither the environmentalist, the fossil-fuel apologist, the antinuclear activists, nor most of those marching under the ``350'' banner can back up their assertions with quantitative arguments. Yet questions about energy and its environmental impacts almost always require quantitative answers. Physics can help! This poster gives some cogent examples, based on the newly published 2^nd edition of the author's textbook Energy, Environment, and Climate.

  20. Primary enzyme quantitation

    DOEpatents

    Saunders, G.C.

    1982-03-04

    The disclosure relates to the quantitation of a primary enzyme concentration by utilizing a substrate for the primary enzyme labeled with a second enzyme which is an indicator enzyme. Enzyme catalysis of the substrate occurs and results in release of the indicator enzyme in an amount directly proportional to the amount of primary enzyme present. By quantifying the free indicator enzyme one determines the amount of primary enzyme present.

  1. Computational vaccinology: quantitative approaches.

    PubMed

    Flower, Darren R; McSparron, Helen; Blythe, Martin J; Zygouri, Christianna; Taylor, Debra; Guan, Pingping; Wan, Shouzhan; Coveney, Peter V; Walshe, Valerie; Borrow, Persephone; Doytchinova, Irini A

    2003-01-01

    The immune system is hierarchical and has many levels, exhibiting much emergent behaviour. However, at its heart are molecular recognition events that are indistinguishable from other types of biomacromolecular interaction. These can be addressed well by quantitative experimental and theoretical biophysical techniques, and particularly by methods from drug design. We review here our approach to computational immunovaccinology. In particular, we describe the JenPep database and two new techniques for T cell epitope prediction. One is based on quantitative structure-activity relationships (a 3D-QSAR method based on CoMSIA and another 2D method based on the Free-Wilson approach) and the other on atomistic molecular dynamic simulations using high performance computing. JenPep (http://www.jenner.ar.uk/ JenPep) is a relational database system supporting quantitative data on peptide binding to major histocompatibility complexes, TAP transporters, TCR-pMHC complexes, and an annotated list of B cell and T cell epitopes. Our 2D-QSAR method factors the contribution to peptide binding from individual amino acids as well as 1-2 and 1-3 residue interactions. In the 3D-QSAR approach, the influence of five physicochemical properties (volume, electrostatic potential, hydrophobicity, hydrogen-bond donor and acceptor abilities) on peptide affinity were considered. Both methods are exemplified through their application to the well-studied problem of peptide binding to the human class I MHC molecule HLA-A*0201. PMID:14712934

  2. Enabling a New Planning and Scheduling Paradigm

    NASA Technical Reports Server (NTRS)

    Jaap, John; Davis, Elizabeth

    2004-01-01

    The Flight Projects Directorate at NASA's Marshall Space Flight Center is developing a new planning and scheduling environment and a new scheduling algorithm to enable a paradigm shift in planning and scheduling concepts. Over the past 33 years Marshall has developed and evolved a paradigm for generating payload timelines for Skylab, Spacelab, various other Shuttle payloads, and the International Space Station. The current paradigm starts by collecting the requirements, called "tasks models," from the scientists and technologists for the tasks that they want to be done. Because of shortcomings in the current modeling schema, some requirements are entered as notes. Next a cadre with knowledge of vehicle and hardware modifies these models to encompass and be compatible with the hardware model; again, notes are added when the modeling schema does not provide a better way to represent the requirements. Finally, another cadre further modifies the models to be compatible with the scheduling engine. This last cadre also submits the models to the scheduling engine or builds the timeline manually to accommodate requirements that are expressed in notes. A future paradigm would provide a scheduling engine that accepts separate science models and hardware models. The modeling schema would have the capability to represent all the requirements without resorting to notes. Furthermore, the scheduling engine would not require that the models be modified to account for the capabilities (limitations) of the scheduling engine. The enabling technology under development at Marshall has three major components. (1) A new modeling schema allows expressing all the requirements of the tasks without resorting to notes or awkward contrivances. The chosen modeling schema is both maximally expressive and easy to use. It utilizes graphics methods to show hierarchies of task constraints and networks of temporal relationships. (2) A new scheduling algorithm automatically schedules the models

  3. Enabling compassionate healthcare: perils, prospects and perspectives

    PubMed Central

    Mannion, Russell

    2014-01-01

    There is an emerging consensus that caring and compassion are under threat in the frenetic environment of modern healthcare. Enabling and sustaining compassionate care requires not only a focus on the needs of the patient, but also on those of the care giver. As such, threats and exhortations to health professionals are likely to have limited and perverse effects and it is to the organisational and system arrangements which support staff that attention should shift. Any approach to supporting compassionate care may work for some services, for some patients and staff, some of the time. No single approach is likely to be a panacea. Unravelling the contexts within which different approaches are effectual will allow for more selective development of support systems and interventions. PMID:24757687

  4. Health-Enabled Smart Sensor Fusion Technology

    NASA Technical Reports Server (NTRS)

    Wang, Ray

    2012-01-01

    A process was designed to fuse data from multiple sensors in order to make a more accurate estimation of the environment and overall health in an intelligent rocket test facility (IRTF), to provide reliable, high-confidence measurements for a variety of propulsion test articles. The object of the technology is to provide sensor fusion based on a distributed architecture. Specifically, the fusion technology is intended to succeed in providing health condition monitoring capability at the intelligent transceiver, such as RF signal strength, battery reading, computing resource monitoring, and sensor data reading. The technology also provides analytic and diagnostic intelligence at the intelligent transceiver, enhancing the IEEE 1451.x-based standard for sensor data management and distributions, as well as providing appropriate communications protocols to enable complex interactions to support timely and high-quality flow of information among the system elements.

  5. Enabling communication concurrency through flexible MPI endpoints

    DOE PAGESBeta

    Dinan, James; Grant, Ryan E.; Balaji, Pavan; Goodell, David; Miller, Douglas; Snir, Marc; Thakur, Rajeev

    2014-09-23

    MPI defines a one-to-one relationship between MPI processes and ranks. This model captures many use cases effectively; however, it also limits communication concurrency and interoperability between MPI and programming models that utilize threads. Our paper describes the MPI endpoints extension, which relaxes the longstanding one-to-one relationship between MPI processes and ranks. Using endpoints, an MPI implementation can map separate communication contexts to threads, allowing them to drive communication independently. Also, endpoints enable threads to be addressable in MPI operations, enhancing interoperability between MPI and other programming models. Furthermore, these characteristics are illustrated through several examples and an empirical study thatmore » contrasts current multithreaded communication performance with the need for high degrees of communication concurrency to achieve peak communication performance.« less

  6. Laboratory Astrophysics: Enabling Scientific Discovery and Understanding

    NASA Technical Reports Server (NTRS)

    Kirby, K.

    2006-01-01

    NASA's Science Strategic Roadmap for Universe Exploration lays out a series of science objectives on a grand scale and discusses the various missions, over a wide range of wavelengths, which will enable discovery. Astronomical spectroscopy is arguably the most powerful tool we have for exploring the Universe. Experimental and theoretical studies in Laboratory Astrophysics convert "hard-won data into scientific understanding". However, the development of instruments with increasingly high spectroscopic resolution demands atomic and molecular data of unprecedented accuracy and completeness. How to meet these needs, in a time of severe budgetary constraints, poses a significant challenge both to NASA, the astronomical observers and model-builders, and the laboratory astrophysics community. I will discuss these issues, together with some recent examples of productive astronomy/lab astro collaborations.

  7. Enabling technologies to advance microbial isoprenoid production.

    PubMed

    Chen, Yun; Zhou, Yongjin J; Siewers, Verena; Nielsen, Jens

    2015-01-01

    Microbial production of isoprenoids provides an attractive alternative to biomass extraction and chemical synthesis. Although widespread research aims for isoprenoid biosynthesis, it is still in its infancy in terms of delivering commercial products. Large barriers remain in realizing a cost-competitive process, for example, developing an optimal microbial cell factory. Here, we summarize the many tools and methods that have been developed in the metabolic engineering of isoprenoid production, with the advent of systems biology and synthetic biology, and discuss how these technologies advance to accelerate the design-build-test engineering cycle to obtain optimum microbial systems. It is anticipated that innovative combinations of new and existing technologies will continue to emerge, which will enable further development of microbial cell factories for commercial isoprenoid production. PMID:25549781

  8. Microsystem enabled photovoltaic modules and systems

    DOEpatents

    Nielson, Gregory N; Sweatt, William C; Okandan, Murat

    2015-05-12

    A microsystem enabled photovoltaic (MEPV) module including: an absorber layer; a fixed optic layer coupled to the absorber layer; a translatable optic layer; a translation stage coupled between the fixed and translatable optic layers; and a motion processor electrically coupled to the translation stage to controls motion of the translatable optic layer relative to the fixed optic layer. The absorber layer includes an array of photovoltaic (PV) elements. The fixed optic layer includes an array of quasi-collimating (QC) micro-optical elements designed and arranged to couple incident radiation from an intermediate image formed by the translatable optic layer into one of the PV elements such that it is quasi-collimated. The translatable optic layer includes an array of focusing micro-optical elements corresponding to the QC micro-optical element array. Each focusing micro-optical element is designed to produce a quasi-telecentric intermediate image from substantially collimated radiation incident within a predetermined field of view.

  9. Corrigendum: Exogenous Attention Enables Perceptual Learning.

    PubMed

    2016-04-01

    Szpiro, S. F. A., & Carrasco, M. (2015). Exogenous attention enables perceptual learning.Psychological Science, 26, 1854-1862. (Original DOI:10.1177/0956797615598976)In the second paragraph of the Testing Sessions section of this article, thetvalue for the between-group difference in spatial-frequency differences was incorrectly reported as 9.49,p> .1, rather than 0.95,p> .1. The sentence should read as follows:There was no significant difference between groups for the orientation differences,t(12) = 1.51,p> .1, or for the spatial-frequency differences,t(12) = 0.95,p> .1.Thus, the conclusion regarding the lack of significance remains the same. PMID:26935483

  10. Telexistence: Enabling Humans to Be Virtually Ubiquitous.

    PubMed

    Tachi, Susumu

    2016-01-01

    Telecommunication and remote-controlled operations are becoming increasingly common in our daily lives. While performing these operations, ideally users would feel as if they were actually present at the remote sites. However, the present commercially available telecommunication and telepresence systems do not provide the sensation of self-presence or self-existence, and hence, users do not get the feeling of being spatially present or that they are directly performing spatial tasks, rather than simply controlling them remotely. This article describes the TELESAR V telexistence master-slave system that enables a human user to feel present in a remote environment. TELESAR V can transmit not only visual and auditory sensations, but also haptic sensations, which are conveyed using the principle of haptic primary colors. PMID:26780759

  11. Enabling Computational Technologies for Terascale Scientific Simulations

    SciTech Connect

    Ashby, S.F.

    2000-08-24

    We develop scalable algorithms and object-oriented code frameworks for terascale scientific simulations on massively parallel processors (MPPs). Our research in multigrid-based linear solvers and adaptive mesh refinement enables Laboratory programs to use MPPs to explore important physical phenomena. For example, our research aids stockpile stewardship by making practical detailed 3D simulations of radiation transport. The need to solve large linear systems arises in many applications, including radiation transport, structural dynamics, combustion, and flow in porous media. These systems result from discretizations of partial differential equations on computational meshes. Our first research objective is to develop multigrid preconditioned iterative methods for such problems and to demonstrate their scalability on MPPs. Scalability describes how total computational work grows with problem size; it measures how effectively additional resources can help solve increasingly larger problems. Many factors contribute to scalability: computer architecture, parallel implementation, and choice of algorithm. Scalable algorithms have been shown to decrease simulation times by several orders of magnitude.

  12. Enabling Communication in Emergency Response Environments

    PubMed Central

    Aldunate, Roberto G.; Schmidt, Klaus Nicholas; Herrera, Oriel

    2012-01-01

    Effective communication among first responders during response to natural and human-made large-scale catastrophes has increased tremendously during the last decade. However, most efforts to achieve a higher degree of effectiveness in communication lack synergy between the environment and the technology involved to support first responders operations. This article presents a natural and intuitive interface to support Stigmergy; or communication through the environment, based on intuitively marking and retrieving information from the environment with a pointer. A prototype of the system was built and tested in the field, however the pointing activity revealed challenges regarding accuracy due to limitations of the sensors used. The results obtained from these field tests were the basis for this research effort and will have the potential to enable communication through the environment for first responders operating in highly dynamical and inhospitable disaster relief environments. PMID:22778647

  13. Photonically-enabled RF spectrum analyzer demonstration

    NASA Astrophysics Data System (ADS)

    Kunkee, Elizabeth T.; Tsai, Ken; Smith, Andrew D.; Jung, T.; Lembo, Larry; Davis, Richard; Babbitt, W. Randall; Krishna-Mohan, R.; Cole, Zachary; Merkel, Kristian D.; Wagner, Kelvin H.

    2008-04-01

    A RF spectrum analyzer with high performance and unique capabilities that traditional all-electronic spectrum analyzers do not exhibit is demonstrated. The system is based on photonic signal processing techniques that have enabled us to demonstrate the spectral analysis of a 1.5 GHz bandwidth with a 1.4 ms update time and a resolution bandwidth of 31 kHz. We observed a 100% probability of intercept for all signals, including short pulses, during the measurement window. The spectrum analyzer operated over the 0.5 to 2.0 GHz range and exhibited a spur-free dynamic range of 42 dB. The potential applications of such a system are extensive and include: detection and location of transient electromagnetic signals, spectrum monitoring for adaptive communications such as spectrum-sensing cognitive radio, and battlefield spectrum management.

  14. Metasurface-Enabled Remote Quantum Interference.

    PubMed

    Jha, Pankaj K; Ni, Xingjie; Wu, Chihhui; Wang, Yuan; Zhang, Xiang

    2015-07-10

    An anisotropic quantum vacuum (AQV) opens novel pathways for controlling light-matter interaction in quantum optics, condensed matter physics, etc. Here, we theoretically demonstrate a strong AQV over macroscopic distances enabled by a judiciously designed array of subwavelength-scale nanoantennas-a metasurface. We harness the phase-control ability and the polarization-dependent response of the metasurface to achieve strong anisotropy in the decay rate of a quantum emitter located over distances of hundreds of wavelengths. Such an AQV induces quantum interference among radiative decay channels in an atom with orthogonal transitions. Quantum vacuum engineering with metasurfaces holds promise for exploring new paradigms of long-range light-matter interaction for atom optics, solid-state quantum optics, quantum information processing, etc. PMID:26207477

  15. The network-enabled optimization system server

    SciTech Connect

    Mesnier, M.P.

    1995-08-01

    Mathematical optimization is a technology under constant change and advancement, drawing upon the most efficient and accurate numerical methods to date. Further, these methods can be tailored for a specific application or generalized to accommodate a wider range of problems. This perpetual change creates an ever growing field, one that is often difficult to stay abreast of. Hence, the impetus behind the Network-Enabled Optimization System (NEOS) server, which aims to provide users, both novice and expert, with a guided tour through the expanding world of optimization. The NEOS server is responsible for bridging the gap between users and the optimization software they seek. More specifically, the NEOS server will accept optimization problems over the Internet and return a solution to the user either interactively or by e-mail. This paper discusses the current implementation of the server.

  16. Science Missions Enabled by the Ares V

    NASA Technical Reports Server (NTRS)

    Worden, Simon Peter; Weiler, Edward J.

    2008-01-01

    NASA's planned heavy-lift Ares V rocket is a centerpiece of U.S. Space Exploration Policy. With approximately 30% more capacity to Trans-Lunar Injection (TLI) than the Saturn V, Ares V could also enable additional science and exploration missions currently unachievable or extremely unworkable under current launch vehicle architectures. During the spring and summer of 2008, NASA held two workshops dedicated to the discussion of these new mission concepts for the Ares V rocket. The first workshop dealt with astronomy and astrophysics, and the second dealt primarily with planetary science and exploration, but did touch on Earth science and heliophysics. We present here the summary results and outcomes of these meetings, including a discussion of specific mission concepts and ideas, as well as suggestions on design for the Ares V fairing and flight configurations that improve science return.

  17. Enabling opportunistic resources for CMS Computing Operations

    NASA Astrophysics Data System (ADS)

    Hufnagel, D.; CMS Collaboration

    2015-12-01

    With the increased pressure on computing brought by the higher energy and luminosity from the LHC in Run 2, CMS Computing Operations expects to require the ability to utilize opportunistic resources resources not owned by, or a priori configured for CMS to meet peak demands. In addition to our dedicated resources we look to add computing resources from non CMS grids, cloud resources, and national supercomputing centers. CMS uses the HTCondor/glideinWMS job submission infrastructure for all its batch processing, so such resources will need to be transparently integrated into its glideinWMS pool. Bosco and parrot wrappers are used to enable access and bring the CMS environment into these non CMS resources. Here we describe our strategy to supplement our native capabilities with opportunistic resources and our experience so far using them.

  18. Enabling communication concurrency through flexible MPI endpoints

    SciTech Connect

    Dinan, James; Grant, Ryan E.; Balaji, Pavan; Goodell, David; Miller, Douglas; Snir, Marc; Thakur, Rajeev

    2014-09-23

    MPI defines a one-to-one relationship between MPI processes and ranks. This model captures many use cases effectively; however, it also limits communication concurrency and interoperability between MPI and programming models that utilize threads. Our paper describes the MPI endpoints extension, which relaxes the longstanding one-to-one relationship between MPI processes and ranks. Using endpoints, an MPI implementation can map separate communication contexts to threads, allowing them to drive communication independently. Also, endpoints enable threads to be addressable in MPI operations, enhancing interoperability between MPI and other programming models. Furthermore, these characteristics are illustrated through several examples and an empirical study that contrasts current multithreaded communication performance with the need for high degrees of communication concurrency to achieve peak communication performance.

  19. Grid Enabled Geospatial Catalogue Web Service

    NASA Technical Reports Server (NTRS)

    Chen, Ai-Jun; Di, Li-Ping; Wei, Ya-Xing; Liu, Yang; Bui, Yu-Qi; Hu, Chau-Min; Mehrotra, Piyush

    2004-01-01

    Geospatial Catalogue Web Service is a vital service for sharing and interoperating volumes of distributed heterogeneous geospatial resources, such as data, services, applications, and their replicas over the web. Based on the Grid technology and the Open Geospatial Consortium (0GC) s Catalogue Service - Web Information Model, this paper proposes a new information model for Geospatial Catalogue Web Service, named as GCWS which can securely provides Grid-based publishing, managing and querying geospatial data and services, and the transparent access to the replica data and related services under the Grid environment. This information model integrates the information model of the Grid Replica Location Service (RLS)/Monitoring & Discovery Service (MDS) with the information model of OGC Catalogue Service (CSW), and refers to the geospatial data metadata standards from IS0 19115, FGDC and NASA EOS Core System and service metadata standards from IS0 191 19 to extend itself for expressing geospatial resources. Using GCWS, any valid geospatial user, who belongs to an authorized Virtual Organization (VO), can securely publish and manage geospatial resources, especially query on-demand data in the virtual community and get back it through the data-related services which provide functions such as subsetting, reformatting, reprojection etc. This work facilitates the geospatial resources sharing and interoperating under the Grid environment, and implements geospatial resources Grid enabled and Grid technologies geospatial enabled. It 2!so makes researcher to focus on science, 2nd not cn issues with computing ability, data locztic, processir,g and management. GCWS also is a key component for workflow-based virtual geospatial data producing.

  20. Stratified charge rotary engine critical technology enablement. Volume 2: Appendixes

    NASA Technical Reports Server (NTRS)

    Irion, C. E.; Mount, R. E.

    1992-01-01

    This second volume of appendixes is a companion to Volume 1 of this report which summarizes results of a critical technology enablement effort with the stratified charge rotary engine (SCRE) focusing on a power section of 0.67 liters (40 cu. in.) per rotor in single and two rotor versions. The work is a continuation of prior NASA Contracts NAS3-23056 and NAS3-24628. Technical objectives are multi-fuel capability, including civil and military jet fuel and DF-2, fuel efficiency of 0.355 Lbs/BHP-Hr. at best cruise condition above 50 percent power, altitude capability of up to 10Km (33,000 ft.) cruise, 2000 hour TBO and reduced coolant heat rejection. Critical technologies for SCRE's that have the potential for competitive performance and cost in a representative light-aircraft environment were examined. Objectives were: the development and utilization of advanced analytical tools, i.e. higher speed and enhanced three dimensional combustion modeling; identification of critical technologies; development of improved instrumentation; and to isolate and quantitatively identify the contribution to performance and efficiency of critical components or subsystems. A family of four-stage third-order explicit Runge-Kutta schemes is derived that required only two locations and has desirable stability characteristics. Error control is achieved by embedding a second-order scheme within the four-stage procedure. Certain schemes are identified that are as efficient and accurate as conventional embedded schemes of comparable order and require fewer storage locations.

  1. Stratified Charge Rotary Engine Critical Technology Enablement, Volume 1

    NASA Technical Reports Server (NTRS)

    Irion, C. E.; Mount, R. E.

    1992-01-01

    This report summarizes results of a critical technology enablement effort with the stratified charge rotary engine (SCRE) focusing on a power section of 0.67 liters (40 cu. in.) per rotor in single and two rotor versions. The work is a continuation of prior NASA Contracts NAS3-23056 and NAS3-24628. Technical objectives are multi-fuel capability, including civil and military jet fuel and DF-2, fuel efficiency of 0.355 Lbs/BHP-Hr. at best cruise condition above 50 percent power, altitude capability of up to 10Km (33,000 ft.) cruise, 2000 hour TBO and reduced coolant heat rejection. Critical technologies for SCRE's that have the potential for competitive performance and cost in a representative light-aircraft environment were examined. Objectives were: the development and utilization of advanced analytical tools, i.e. higher speed and enhanced three dimensional combustion modeling; identification of critical technologies; development of improved instrumentation, and to isolate and quantitatively identify the contribution to performance and efficiency of critical components or subsystems.

  2. Realising the Uncertainty Enabled Model Web

    NASA Astrophysics Data System (ADS)

    Cornford, D.; Bastin, L.; Pebesma, E. J.; Williams, M.; Stasch, C.; Jones, R.; Gerharz, L.

    2012-12-01

    The FP7 funded UncertWeb project aims to create the "uncertainty enabled model web". The central concept here is that geospatial models and data resources are exposed via standard web service interfaces, such as the Open Geospatial Consortium (OGC) suite of encodings and interface standards, allowing the creation of complex workflows combining both data and models. The focus of UncertWeb is on the issue of managing uncertainty in such workflows, and providing the standards, architecture, tools and software support necessary to realise the "uncertainty enabled model web". In this paper we summarise the developments in the first two years of UncertWeb, illustrating several key points with examples taken from the use case requirements that motivate the project. Firstly we address the issue of encoding specifications. We explain the usage of UncertML 2.0, a flexible encoding for representing uncertainty based on a probabilistic approach. This is designed to be used within existing standards such as Observations and Measurements (O&M) and data quality elements of ISO19115 / 19139 (geographic information metadata and encoding specifications) as well as more broadly outside the OGC domain. We show profiles of O&M that have been developed within UncertWeb and how UncertML 2.0 is used within these. We also show encodings based on NetCDF and discuss possible future directions for encodings in JSON. We then discuss the issues of workflow construction, considering discovery of resources (both data and models). We discuss why a brokering approach to service composition is necessary in a world where the web service interfaces remain relatively heterogeneous, including many non-OGC approaches, in particular the more mainstream SOAP and WSDL approaches. We discuss the trade-offs between delegating uncertainty management functions to the service interfaces themselves and integrating the functions in the workflow management system. We describe two utility services to address

  3. Enabling Technologies for Ceramic Hot Section Components

    SciTech Connect

    Venkat Vedula; Tania Bhatia

    2009-04-30

    Silicon-based ceramics are attractive materials for use in gas turbine engine hot sections due to their high temperature mechanical and physical properties as well as lower density than metals. The advantages of utilizing ceramic hot section components include weight reduction, and improved efficiency as well as enhanced power output and lower emissions as a result of reducing or eliminating cooling. Potential gas turbine ceramic components for industrial, commercial and/or military high temperature turbine applications include combustor liners, vanes, rotors, and shrouds. These components require materials that can withstand high temperatures and pressures for long duration under steam-rich environments. For Navy applications, ceramic hot section components have the potential to increase the operation range. The amount of weight reduced by utilizing a lighter gas turbine can be used to increase fuel storage capacity while a more efficient gas turbine consumes less fuel. Both improvements enable a longer operation range for Navy ships and aircraft. Ceramic hot section components will also be beneficial to the Navy's Growth Joint Strike Fighter (JSF) and VAATE (Versatile Affordable Advanced Turbine Engines) initiatives in terms of reduced weight, cooling air savings, and capability/cost index (CCI). For DOE applications, ceramic hot section components provide an avenue to achieve low emissions while improving efficiency. Combustors made of ceramic material can withstand higher wall temperatures and require less cooling air. Ability of the ceramics to withstand high temperatures enables novel combustor designs that have reduced NO{sub x}, smoke and CO levels. In the turbine section, ceramic vanes and blades do not require sophisticated cooling schemes currently used for metal components. The saved cooling air could be used to further improve efficiency and power output. The objectives of this contract were to develop technologies critical for ceramic hot section

  4. Quantitative biomedical mass spectrometry

    NASA Astrophysics Data System (ADS)

    de Leenheer, Andrép; Thienpont, Linda M.

    1992-09-01

    The scope of this contribution is an illustration of the capabilities of isotope dilution mass spectrometry (IDMS) for quantification of target substances in the biomedical field. After a brief discussion of the general principles of quantitative MS in biological samples, special attention will be paid to new technological developments or trends in IDMS from selected examples from the literature. The final section will deal with the use of IDMS for accuracy assessment in clinical chemistry. Methodological aspects considered crucial for avoiding sources of error will be discussed.

  5. Label-free quantitative cell division monitoring of endothelial cells by digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Kemper, Björn; Bauwens, Andreas; Vollmer, Angelika; Ketelhut, Steffi; Langehanenberg, Patrik; Müthing, Johannes; Karch, Helge; von Bally, Gert

    2010-05-01

    Digital holographic microscopy (DHM) enables quantitative multifocus phase contrast imaging for nondestructive technical inspection and live cell analysis. Time-lapse investigations on human brain microvascular endothelial cells demonstrate the use of DHM for label-free dynamic quantitative monitoring of cell division of mother cells into daughter cells. Cytokinetic DHM analysis provides future applications in toxicology and cancer research.

  6. Heterogeneity of pancreatic cancer metastases in a single patient revealed by quantitative proteomics.

    PubMed

    Kim, Min-Sik; Zhong, Yi; Yachida, Shinichi; Rajeshkumar, N V; Abel, Melissa L; Marimuthu, Arivusudar; Mudgal, Keshav; Hruban, Ralph H; Poling, Justin S; Tyner, Jeffrey W; Maitra, Anirban; Iacobuzio-Donahue, Christine A; Pandey, Akhilesh

    2014-11-01

    Many patients with pancreatic cancer have metastases to distant organs at the time of initial presentation. Recent studies examining the evolution of pancreatic cancer at the genetic level have shown that clonal complexity of metastatic pancreatic cancer is already initiated within primary tumors, and organ-specific metastases are derived from different subclones. However, we do not yet understand to what extent the evolution of pancreatic cancer contributes to proteomic and signaling alterations. We hypothesized that genetic heterogeneity of metastatic pancreatic cancer results in heterogeneity at the proteome level. To address this, we employed a model system in which cells isolated from three sites of metastasis (liver, lung, and peritoneum) from a single patient were compared. We used a SILAC-based accurate quantitative proteomic strategy combined with high-resolution mass spectrometry to analyze the total proteome and tyrosine phosphoproteome of each of the distal metastases. Our data revealed distinct patterns of both overall proteome expression and tyrosine kinase activities across the three different metastatic lesions. This heterogeneity was significant because it led to differential sensitivity of the neoplastic cells to small molecule inhibitors targeting various kinases and other pathways. For example, R428, a tyrosine kinase inhibitor that targets Axl receptor tyrosine kinase, was able to inhibit cells derived from lung and liver metastases much more effectively than cells from the peritoneal metastasis. Finally, we confirmed that administration of R428 in mice bearing xenografts of cells derived from the three different metastatic sites significantly diminished tumors formed from liver- and lung-metastasis-derived cell lines as compared with tumors derived from the peritoneal metastasis cell line. Overall, our data provide proof-of-principle support that personalized therapy of multiple organ metastases in a single patient should involve the

  7. Technology-enabled Airborne Spacing and Merging

    NASA Technical Reports Server (NTRS)

    Hull, James; Barmore, Bryan; Abbott, Tetence

    2005-01-01

    Over the last several decades, advances in airborne and groundside technologies have allowed the Air Traffic Service Provider (ATSP) to give safer and more efficient service, reduce workload and frequency congestion, and help accommodate a critically escalating traffic volume. These new technologies have included advanced radar displays, and data and communication automation to name a few. In step with such advances, NASA Langley is developing a precision spacing concept designed to increase runway throughput by enabling the flight crews to manage their inter-arrival spacing from TRACON entry to the runway threshold. This concept is being developed as part of NASA s Distributed Air/Ground Traffic Management (DAG-TM) project under the Advanced Air Transportation Technologies Program. Precision spacing is enabled by Automatic Dependent Surveillance-Broadcast (ADS-B), which provides air-to-air data exchange including position and velocity reports; real-time wind information and other necessary data. On the flight deck, a research prototype system called Airborne Merging and Spacing for Terminal Arrivals (AMSTAR) processes this information and provides speed guidance to the flight crew to achieve the desired inter-arrival spacing. AMSTAR is designed to support current ATC operations, provide operationally acceptable system-wide increases in approach spacing performance and increase runway throughput through system stability, predictability and precision spacing. This paper describes problems and costs associated with an imprecise arrival flow. It also discusses methods by which Air Traffic Controllers achieve and maintain an optimum interarrival interval, and explores means by which AMSTAR can assist in this pursuit. AMSTAR is an extension of NASA s previous work on in-trail spacing that was successfully demonstrated in a flight evaluation at Chicago O Hare International Airport in September 2002. In addition to providing for precision inter-arrival spacing, AMSTAR

  8. Willing and Enabled: The Academic Outcomes of a Tertiary Enabling Program in Regional Australia

    ERIC Educational Resources Information Center

    Andrewartha, Lisa; Harvey, Andrew

    2014-01-01

    This paper examines the achievement levels of students undertaking the Tertiary Enabling Program (TEP) at La Trobe University. The TEP is an alternative pathway program that traverses multiple institutions, campuses, and disciplinary areas, and is designed to prepare a diverse student cohort for tertiary study. The Program integrates several…

  9. Smart Sensors Enable Smart Air Conditioning Control

    PubMed Central

    Cheng, Chin-Chi; Lee, Dasheng

    2014-01-01

    In this study, mobile phones, wearable devices, temperature and human motion detectors are integrated as smart sensors for enabling smart air conditioning control. Smart sensors obtain feedback, especially occupants' information, from mobile phones and wearable devices placed on human body. The information can be used to adjust air conditioners in advance according to humans' intentions, in so-called intention causing control. Experimental results show that the indoor temperature can be controlled accurately with errors of less than ±0.1 °C. Rapid cool down can be achieved within 2 min to the optimized indoor capacity after occupants enter a room. It's also noted that within two-hour operation the total compressor output of the smart air conditioner is 48.4% less than that of the one using On-Off control. The smart air conditioner with wearable devices could detect the human temperature and activity during sleep to determine the sleeping state and adjusting the sleeping function flexibly. The sleeping function optimized by the smart air conditioner with wearable devices could reduce the energy consumption up to 46.9% and keep the human health. The presented smart air conditioner could provide a comfortable environment and achieve the goals of energy conservation and environmental protection. PMID:24961213

  10. Smart sensors enable smart air conditioning control.

    PubMed

    Cheng, Chin-Chi; Lee, Dasheng

    2014-01-01

    In this study, mobile phones, wearable devices, temperature and human motion detectors are integrated as smart sensors for enabling smart air conditioning control. Smart sensors obtain feedback, especially occupants' information, from mobile phones and wearable devices placed on human body. The information can be used to adjust air conditioners in advance according to humans' intentions, in so-called intention causing control. Experimental results show that the indoor temperature can be controlled accurately with errors of less than ±0.1 °C. Rapid cool down can be achieved within 2 min to the optimized indoor capacity after occupants enter a room. It's also noted that within two-hour operation the total compressor output of the smart air conditioner is 48.4% less than that of the one using On-Off control. The smart air conditioner with wearable devices could detect the human temperature and activity during sleep to determine the sleeping state and adjusting the sleeping function flexibly. The sleeping function optimized by the smart air conditioner with wearable devices could reduce the energy consumption up to 46.9% and keep the human health. The presented smart air conditioner could provide a comfortable environment and achieve the goals of energy conservation and environmental protection. PMID:24961213

  11. Nanomaterial-enabled membranes for water treatment

    NASA Astrophysics Data System (ADS)

    Rogensues, Adam Roy

    Incorporating engineered nanomaterials as components of synthetic membranes can improve their separation performance and endow membranes with additional functions. This work explores two approaches to the design of membranes modified with nanomaterials. In the first chapter, exfoliated graphite nanoplatelets (xGnP) decorated with gold nanoparticles were embedded in a polysulfone matrix to fabricate phase inversion nanocomposite membranes. The cast membranes were evaluated as flow-through membrane reactors in experiments on the catalytic reduction of 4-nitrophenol. The nanocomposite membranes were not as catalytically efficient as those fabricated by modifying anodized alumina membranes polyelectrolyte multilayers (PEMs) containing gold nanoparticles. However, because of the facility of membrane casting by phase inversion and new opportunities enabled by the demonstrated hierarchy-based approach to nanocomposite membrane design, such membrane may hold commercial promise. In the second part of the study, the practicability of PEM-based nanofiltration was evaluated under conditions of precipitative fouling (i.e. scaling) by calcium sulfate. Polyelectrolytes were deposited onto 50 kDa polyethersulfone membranes to create PEM-based nanofiltration membranes. The prepared membranes were compared with the commercial NF270 membrane in terms of flux and rejection performance, as well as the morphology of gypsum crystals formed on the membrane surface. None of the PEM coatings tested inhibited scale formation.

  12. Enabling scientific workflows in virtual reality

    USGS Publications Warehouse

    Kreylos, O.; Bawden, G.; Bernardin, T.; Billen, M.I.; Cowgill, E.S.; Gold, R.D.; Hamann, B.; Jadamec, M.; Kellogg, L.H.; Staadt, O.G.; Sumner, D.Y.

    2006-01-01

    To advance research and improve the scientific return on data collection and interpretation efforts in the geosciences, we have developed methods of interactive visualization, with a special focus on immersive virtual reality (VR) environments. Earth sciences employ a strongly visual approach to the measurement and analysis of geologic data due to the spatial and temporal scales over which such data ranges, As observations and simulations increase in size and complexity, the Earth sciences are challenged to manage and interpret increasing amounts of data. Reaping the full intellectual benefits of immersive VR requires us to tailor exploratory approaches to scientific problems. These applications build on the visualization method's strengths, using both 3D perception and interaction with data and models, to take advantage of the skills and training of the geological scientists exploring their data in the VR environment. This interactive approach has enabled us to develop a suite of tools that are adaptable to a range of problems in the geosciences and beyond. Copyright ?? 2008 by the Association for Computing Machinery, Inc.

  13. Survey of Enabling Technologies for CAPS

    NASA Technical Reports Server (NTRS)

    Antol, Jeffrey; Mazanek, Daniel D.; Koons, Robert H.

    2005-01-01

    The enabling technologies required for the development of a viable Comet/Asteroid Protection System (CAPS) can be divided into two principal areas: detection and deflection/orbit modification. With the proper funding levels, many of the technologies needed to support a CAPS architecture could be achievable within the next 15 to 20 years. In fact, many advanced detection technologies are currently in development for future in-space telescope systems such as the James Webb Space Telescope (JWST), formerly known as the Next Generation Space Telescope. It is anticipated that many of the JWST technologies would be available for application for CAPS detection concepts. Deflection/orbit modification technologies are also currently being studied as part of advanced power and propulsion research. However, many of these technologies, such as extremely high-output power systems, advanced propulsion, heat rejection, and directed energy systems, would likely be farther term in availability than many of the detection technologies. Discussed subsequently is a preliminary examination of the main technologies that have been identified as being essential to providing the element functionality defined during the CAPS conceptual study. The detailed requirements for many of the technology areas are still unknown, and many additional technologies will be identified as future in-depth studies are conducted in this area.

  14. Powered wheelchairs: are we enabling or disabling?

    PubMed

    Beaumont-White, S; Ham, R O

    1997-04-01

    Following the unsuccessful issue of three powered indoor National Health Service (NHS) wheelchairs, a survey was carried out of 40 users in a London wheelchair service to identify the problems with issue and possible areas for improvement to practice. The survey identified improvements that were necessary both from the service and the manufacturers' booklets. The improvements include the issue of written instructions and information to complement verbal instruction given at handover. Such information should be as interesting to read as possible, make use of appropriate language and diagrams (especially in area where English is often not the first language), colour, text and print size to maximise comprehension to these severely disabled users and often their elderly relatives or carers. The importance of the role of the rehabilitation engineer in training the user, giving instruction at handover and annual review are highlighted to ensure that the equipment remains working, suitable and up to date for the individual's needs. Training in interpersonal and communication skills and the importance of recall should also be emphasised. The implementation of the findings should lead to increasing contact with the service by the user, reduction in repair and replacement costs, regular review, correct supply and will therefore enable users to increase their independence with appropriate equipment. PMID:9141127

  15. Nanocrystal-enabled solid state bonding.

    SciTech Connect

    San Diego State University, San Diego, CA; Puskar, Joseph David; Tikare, Veena; Garcia Cardona, Cristina; Reece, Mark; Brewer, Luke N.; Holm, Elizabeth Ann

    2010-10-01

    In this project, we performed a preliminary set of sintering experiments to examine nanocrystal-enabled diffusion bonding (NEDB) in Ag-on-Ag and Cu-on-Cu using Ag nanoparticles. The experimental test matrix included the effects of material system, temperature, pressure, and particle size. The nanoparticle compacts were bonded between plates using a customized hot press, tested in shear, and examined post mortem using microscopy techniques. NEDB was found to be a feasible mechanism for low-temperature, low-pressure, solid-state bonding of like materials, creating bonded interfaces that were able to support substantial loads. The maximum supported shear strength varied substantially within sample cohorts due to variation in bonded area; however, systematic variation with fabrication conditions was also observed. Mesoscale sintering simulations were performed in order to understand whether sintering models can aid in understanding the NEDB process. A pressure-assisted sintering model was incorporated into the SPPARKS kinetic Monte Carlo sintering code. Results reproduce most of the qualitative behavior observed in experiments, indicating that simulation can augment experiments during the development of the NEDB process. Because NEDB offers a promising route to low-temperature, low-pressure, solid-state bonding, we recommend further research and development with a goal of devising new NEDB bonding processes to support Sandia's customers.

  16. Glass ceramic ZERODUR enabling nanometer precision

    NASA Astrophysics Data System (ADS)

    Jedamzik, Ralf; Kunisch, Clemens; Nieder, Johannes; Westerhoff, Thomas

    2014-03-01

    The IC Lithography roadmap foresees manufacturing of devices with critical dimension of < 20 nm. Overlay specification of single digit nanometer asking for nanometer positioning accuracy requiring sub nanometer position measurement accuracy. The glass ceramic ZERODUR® is a well-established material in critical components of microlithography wafer stepper and offered with an extremely low coefficient of thermal expansion (CTE), the tightest tolerance available on market. SCHOTT is continuously improving manufacturing processes and it's method to measure and characterize the CTE behavior of ZERODUR® to full fill the ever tighter CTE specification for wafer stepper components. In this paper we present the ZERODUR® Lithography Roadmap on the CTE metrology and tolerance. Additionally, simulation calculations based on a physical model are presented predicting the long term CTE behavior of ZERODUR® components to optimize dimensional stability of precision positioning devices. CTE data of several low thermal expansion materials are compared regarding their temperature dependence between - 50°C and + 100°C. ZERODUR® TAILORED 22°C is full filling the tight CTE tolerance of +/- 10 ppb / K within the broadest temperature interval compared to all other materials of this investigation. The data presented in this paper explicitly demonstrates the capability of ZERODUR® to enable the nanometer precision required for future generation of lithography equipment and processes.

  17. Water: A Critical Material Enabling Space Exploration

    NASA Technical Reports Server (NTRS)

    Pickering, Karen D.

    2014-01-01

    Water is one of the most critical materials in human spaceflight. The availability of water defines the duration of a space mission; the volume of water required for a long-duration space mission becomes too large, heavy, and expensive for launch vehicles to carry. Since the mission duration is limited by the amount of water a space vehicle can carry, the capability to recycle water enables space exploration. In addition, water management in microgravity impacts spaceflight in other respects, such as the recent emergency termination of a spacewalk caused by free water in an astronaut's spacesuit helmet. A variety of separation technologies are used onboard spacecraft to ensure that water is always available for use, and meets the stringent water quality required for human space exploration. These separation technologies are often adapted for use in a microgravity environment, where water behaves in unique ways. The use of distillation, membrane processes, ion exchange and granular activated carbon will be reviewed. Examples of microgravity effects on operations will also be presented. A roadmap for future technologies, needed to supply water resources for the exploration of Mars, will also be reviewed.

  18. Enabling electroweak baryogenesis through dark matter

    NASA Astrophysics Data System (ADS)

    Lewicki, Marek; Rindler-Daller, Tanja; Wells, James D.

    2016-06-01

    We study the impact on electroweak baryogenesis from a swifter cosmological expansion induced by dark matter. We detail the experimental bounds that one can place on models that realize it, and we investigate the modifications of these bounds that result from a non-standard cosmological history. The modifications can be sizeable if the expansion rate of the Universe increases by several orders of magnitude. We illustrate the impact through the example of scalar field dark matter, which can alter the cosmological history enough to enable a strong-enough first-order phase transition in the Standard Model when it is supplemented by a dimension six operator directly modifying the Higgs boson potential. We show that due to the modified cosmological history, electroweak baryogenesis can be realized, while keeping deviations of the triple Higgs coupling below HL-LHC sensitivies. The required scale of new physics to effectuate a strong-enough first order phase transition can change by as much as twenty percent as the expansion rate increases by six orders of magnitude.

  19. BEST: Barcode Enabled Sequencing of Tetrads

    PubMed Central

    Scott, Adrian C.; Ludlow, Catherine L.; Cromie, Gareth A.; Dudley, Aimée M.

    2014-01-01

    Tetrad analysis is a valuable tool for yeast genetics, but the laborious manual nature of the process has hindered its application on large scales. Barcode Enabled Sequencing of Tetrads (BEST)1 replaces the manual processes of isolating, disrupting and spacing tetrads. BEST isolates tetrads by virtue of a sporulation-specific GFP fusion protein that permits fluorescence-activated cell sorting of tetrads directly onto agar plates, where the ascus is enzymatically digested and the spores are disrupted and randomly arrayed by glass bead plating. The haploid colonies are then assigned sister spore relationships, i.e. information about which spores originated from the same tetrad, using molecular barcodes read during genotyping. By removing the bottleneck of manual dissection, hundreds or even thousands of tetrads can be isolated in minutes. Here we present a detailed description of the experimental procedures required to perform BEST in the yeast Saccharomyces cerevisiae, starting with a heterozygous diploid strain through the isolation of colonies derived from the haploid meiotic progeny. PMID:24836713

  20. Enabler for the agile virtual enterprise

    NASA Astrophysics Data System (ADS)

    Fuerst, Karl; Schmidt, Thomas; Wippel, Gerald

    2001-10-01

    In this presentation, a new approach for a flexible low-cost Internet extended enterprise (project FLoCI-EE) will be presented. FLoCI-EE is a project in the fifth framework program of the European commission with 8 partners from 4 countries, which started in January 2001 and will be finished in December 2003. The main objective of FLoCI-EE is the development of a software prototype, which enables flexible enterprise cooperation with the aim to design, manufacture and sell products commonly, independent of enterprise borderlines. The needed IT-support includes functions of product data management (PDM), enterprise resource planning (ERP), supply chain management (SCM) and customer relationship management (CRM). Especially for small and medium sized enterprises, existing solutions are too expensive and inflexible to be of use under current turbulent market conditions. The second part of this paper covers the item Web Services, because in the role-specific support approach of FLoCI-EE, there are user- interface-components, which are tailored for specific roles in an enterprise. These components integrate automatically the services of the so-called basic-components, and the externally offered Web Services like UDDI.

  1. Bandwidth Enabled Flight Operations: Examining the Possibilities

    NASA Technical Reports Server (NTRS)

    Pisanich, Greg; Renema, Fritz; Clancy, Dan (Technical Monitor)

    2002-01-01

    The Bandwidth Enabled Flight Operations project is a research effort at the NASA Ames Research Center to investigate the use of satellite communications to improve aviation safety and capacity. This project is a follow on to the AeroSAPIENT Project, which demonstrated methods for transmitting high bandwidth data in various configurations. For this research, we set a goal to nominally use only 10 percent of the available bandwidth demonstrated by AeroSAPIENT or projected by near-term technology advances. This paper describes the results of our research, including available satellite bandwidth, commercial and research efforts to provide these services, and some of the limiting factors inherent with this communications medium. It also describes our investigation into the needs of the stakeholders (Airlines, Pilots, Cabin Crews, ATC, Maintenance, etc). The paper also describes our development of low-cost networked flight deck and airline operations center simulations that were used to demonstrate two application areas: Providing real time weather information to the commercial flight deck, and enhanced crew monitoring and control for airline operations centers.

  2. Individualized grid-enabled mammographic training system

    NASA Astrophysics Data System (ADS)

    Yap, M. H.; Gale, A. G.

    2009-02-01

    The PERFORMS self-assessment scheme measures individuals skills in identifying key mammographic features on sets of known cases. One aspect of this is that it allows radiologists' skills to be trained, based on their data from this scheme. Consequently, a new strategy is introduced to provide revision training based on mammographic features that the radiologist has had difficulty with in these sets. To do this requires a lot of random cases to provide dynamic, unique, and up-to-date training modules for each individual. We propose GIMI (Generic Infrastructure in Medical Informatics) middleware as the solution to harvest cases from distributed grid servers. The GIMI middleware enables existing and legacy data to support healthcare delivery, research, and training. It is technology-agnostic, data-agnostic, and has a security policy. The trainee examines each case, indicating the location of regions of interest, and completes an evaluation form, to determine mammographic feature labelling, diagnosis, and decisions. For feedback, the trainee can choose to have immediate feedback after examining each case or batch feedback after examining a number of cases. All the trainees' result are recorded in a database which also contains their trainee profile. A full report can be prepared for the trainee after they have completed their training. This project demonstrates the practicality of a grid-based individualised training strategy and the efficacy in generating dynamic training modules within the coverage/outreach of the GIMI middleware. The advantages and limitations of the approach are discussed together with future plans.

  3. Quantitative Hyperspectral Reflectance Imaging

    PubMed Central

    Klein, Marvin E.; Aalderink, Bernard J.; Padoan, Roberto; de Bruin, Gerrit; Steemers, Ted A.G.

    2008-01-01

    Hyperspectral imaging is a non-destructive optical analysis technique that can for instance be used to obtain information from cultural heritage objects unavailable with conventional colour or multi-spectral photography. This technique can be used to distinguish and recognize materials, to enhance the visibility of faint or obscured features, to detect signs of degradation and study the effect of environmental conditions on the object. We describe the basic concept, working principles, construction and performance of a laboratory instrument specifically developed for the analysis of historical documents. The instrument measures calibrated spectral reflectance images at 70 wavelengths ranging from 365 to 1100 nm (near-ultraviolet, visible and near-infrared). By using a wavelength tunable narrow-bandwidth light-source, the light energy used to illuminate the measured object is minimal, so that any light-induced degradation can be excluded. Basic analysis of the hyperspectral data includes a qualitative comparison of the spectral images and the extraction of quantitative data such as mean spectral reflectance curves and statistical information from user-defined regions-of-interest. More sophisticated mathematical feature extraction and classification techniques can be used to map areas on the document, where different types of ink had been applied or where one ink shows various degrees of degradation. The developed quantitative hyperspectral imager is currently in use by the Nationaal Archief (National Archives of The Netherlands) to study degradation effects of artificial samples and original documents, exposed in their permanent exhibition area or stored in their deposit rooms.

  4. Quantitative Imaging in Cancer Evolution and Ecology

    PubMed Central

    Grove, Olya; Gillies, Robert J.

    2013-01-01

    Cancer therapy, even when highly targeted, typically fails because of the remarkable capacity of malignant cells to evolve effective adaptations. These evolutionary dynamics are both a cause and a consequence of cancer system heterogeneity at many scales, ranging from genetic properties of individual cells to large-scale imaging features. Tumors of the same organ and cell type can have remarkably diverse appearances in different patients. Furthermore, even within a single tumor, marked variations in imaging features, such as necrosis or contrast enhancement, are common. Similar spatial variations recently have been reported in genetic profiles. Radiologic heterogeneity within tumors is usually governed by variations in blood flow, whereas genetic heterogeneity is typically ascribed to random mutations. However, evolution within tumors, as in all living systems, is subject to Darwinian principles; thus, it is governed by predictable and reproducible interactions between environmental selection forces and cell phenotype (not genotype). This link between regional variations in environmental properties and cellular adaptive strategies may permit clinical imaging to be used to assess and monitor intratumoral evolution in individual patients. This approach is enabled by new methods that extract, report, and analyze quantitative, reproducible, and mineable clinical imaging data. However, most current quantitative metrics lack spatialness, expressing quantitative radiologic features as a single value for a region of interest encompassing the whole tumor. In contrast, spatially explicit image analysis recognizes that tumors are heterogeneous but not well mixed and defines regionally distinct habitats, some of which appear to harbor tumor populations that are more aggressive and less treatable than others. By identifying regional variations in key environmental selection forces and evidence of cellular adaptation, clinical imaging can enable us to define intratumoral

  5. Quantitative Ultrasound for Nondestructive Characterization of Engineered Tissues and Biomaterials.

    PubMed

    Dalecki, Diane; Mercado, Karla P; Hocking, Denise C

    2016-03-01

    Non-invasive, non-destructive technologies for imaging and quantitatively monitoring the development of artificial tissues are critical for the advancement of tissue engineering. Current standard techniques for evaluating engineered tissues, including histology, biochemical assays and mechanical testing, are destructive approaches. Ultrasound is emerging as a valuable tool for imaging and quantitatively monitoring the properties of engineered tissues and biomaterials longitudinally during fabrication and post-implantation. Ultrasound techniques are rapid, non-invasive, non-destructive and can be easily integrated into sterile environments necessary for tissue engineering. Furthermore, high-frequency quantitative ultrasound techniques can enable volumetric characterization of the structural, biological, and mechanical properties of engineered tissues during fabrication and post-implantation. This review provides an overview of ultrasound imaging, quantitative ultrasound techniques, and elastography, with representative examples of applications of these ultrasound-based techniques to the field of tissue engineering. PMID:26581347

  6. Quantitative imaging of gene expression in Drosophila embryos.

    PubMed

    Surkova, Svetlana; Myasnikova, Ekaterina; Kozlov, Konstantin N; Pisarev, Andrei; Reinitz, John; Samsonova, Maria

    2013-06-01

    Quantitative measurements derived using sophisticated microscopy techniques are essential for understanding the basic principles that control the behavior of biological systems. Here we describe a data pipeline developed to extract quantitative data on segmentation gene expression from confocal images of gene expression patterns in Drosophila. The pipeline consists of image segmentation, background removal, temporal characterization of an embryo, data registration, and data averaging. This pipeline has been successfully applied to obtain quantitative gene expression data at cellular resolution in space and at 6.5-min resolution in time. It has also enabled the construction of a spatiotemporal atlas of segmentation gene expression. We describe the software used to construct a workflow for extracting quantitative data on segmentation gene expression and the BREReA package, which implements the methods for background removal and registration of segmentation gene expression patterns. PMID:23734022

  7. Enabling performance skills: Assessment in engineering education

    NASA Astrophysics Data System (ADS)

    Ferrone, Jenny Kristina

    Current reform in engineering education is part of a national trend emphasizing student learning as well as accountability in instruction. Assessing student performance to demonstrate accountability has become a necessity in academia. In newly adopted criterion proposed by the Accreditation Board for Engineering and Technology (ABET), undergraduates are expected to demonstrate proficiency in outcomes considered essential for graduating engineers. The case study was designed as a formative evaluation of freshman engineering students to assess the perceived effectiveness of performance skills in a design laboratory environment. The mixed methodology used both quantitative and qualitative approaches to assess students' performance skills and congruency among the respondents, based on individual, team, and faculty perceptions of team effectiveness in three ABET areas: Communications Skills. Design Skills, and Teamwork. The findings of the research were used to address future use of the assessment tool and process. The results of the study found statistically significant differences in perceptions of Teamwork Skills (p < .05). When groups composed of students and professors were compared, professors were less likely to perceive student's teaming skills as effective. The study indicated the need to: (1) improve non-technical performance skills, such as teamwork, among freshman engineering students; (2) incorporate feedback into the learning process; (3) strengthen the assessment process with a follow-up plan that specifically targets performance skill deficiencies, and (4) integrate the assessment instrument and practice with ongoing curriculum development. The findings generated by this study provides engineering departments engaged in assessment activity, opportunity to reflect, refine, and develop their programs as it continues. It also extends research on ABET competencies of engineering students in an under-investigated topic of factors correlated with team

  8. Web enabled data management with DPM & LFC

    NASA Astrophysics Data System (ADS)

    Alvarez Ayllon, Alejandro; Beche, Alexandre; Furano, Fabrizio; Hellmich, Martin; Keeble and, Oliver; Brito Da Rocha, Ricardo

    2012-12-01

    The Disk Pool Manager (DPM) and LCG File Catalog (LFC) are two grid data management components currently used in production with more than 240 endpoints. Together with a set of grid client tools they give the users a unified view of their data, hiding most details concerning data location and access. Recently we've put a lot of effort in developing a reliable and high performance HTTP/WebDAV frontend to both our grid catalog and storage components, exposing the existing functionality to users accessing the services via standard clients - e.g. web browsers, curl - present in all operating systems, giving users a simple and straight-forward way of interaction. In addition, as other relevant grid storage components (like dCache) expose their data using the same protocol, for the first time we had the opportunity of attempting a unified view of all grid storage using HTTP. We describe the HTTP redirection mechanism used to integrate the grid catalog(s) with the multiple storage components, including details on some assumptions made to allow integration with other implementations. We describe the way we hide the details regarding site availability or catalog inconsistencies by switching the standard HTTP client automatically between multiple replicas. We also present measurements of access performance, and the relevant factors regarding replica selection - current throughput and load, geographic proximity, etc. Finally, we report on some additional work done to have this system as a viable alternative to GridFTP, providing multi-stream transfers and exploiting some additional features of WebDAV to enable third party copies - essential for managing data movements between storage systems - with equivalent performance.

  9. Enabling a Scientific Cloud Marketplace: VGL (Invited)

    NASA Astrophysics Data System (ADS)

    Fraser, R.; Woodcock, R.; Wyborn, L. A.; Vote, J.; Rankine, T.; Cox, S. J.

    2013-12-01

    The Virtual Geophysics Laboratory (VGL) provides a flexible, web based environment where researchers can browse data and use a variety of scientific software packaged into tool kits that run in the Cloud. Both data and tool kits are published by multiple researchers and registered with the VGL infrastructure forming a data and application marketplace. The VGL provides the basic work flow of Discovery and Access to the disparate data sources and a Library for tool kits and scripting to drive the scientific codes. Computation is then performed on the Research or Commercial Clouds. Provenance information is collected throughout the work flow and can be published alongside the results allowing for experiment comparison and sharing with other researchers. VGL's "mix and match" approach to data, computational resources and scientific codes, enables a dynamic approach to scientific collaboration. VGL allows scientists to publish their specific contribution, be it data, code, compute or work flow, knowing the VGL framework will provide other components needed for a complete application. Other scientists can choose the pieces that suit them best to assemble an experiment. The coarse grain workflow of the VGL framework combined with the flexibility of the scripting library and computational toolkits allows for significant customisation and sharing amongst the community. The VGL utilises the cloud computational and storage resources from the Australian academic research cloud provided by the NeCTAR initiative and a large variety of data accessible from national and state agencies via the Spatial Information Services Stack (SISS - http://siss.auscope.org). VGL v1.2 screenshot - http://vgl.auscope.org

  10. New Catalog of Resources Enables Paleogeosciences Research

    NASA Astrophysics Data System (ADS)

    Lingo, R. C.; Horlick, K. A.; Anderson, D. M.

    2014-12-01

    The 21st century promises a new era for scientists of all disciplines, the age where cyber infrastructure enables research and education and fuels discovery. EarthCube is a working community of over 2,500 scientists and students of many Earth Science disciplines who are looking to build bridges between disciplines. The EarthCube initiative will create a digital infrastructure that connects databases, software, and repositories. A catalog of resources (databases, software, repositories) has been produced by the Research Coordination Network for Paleogeosciences to improve the discoverability of resources. The Catalog is currently made available within the larger-scope CINERGI geosciences portal (http://hydro10.sdsc.edu/geoportal/catalog/main/home.page). Other distribution points and web services are planned, using linked data, content services for the web, and XML descriptions that can be harvested using metadata protocols. The databases provide searchable interfaces to find data sets that would otherwise remain dark data, hidden in drawers and on personal computers. The software will be described in catalog entries so just one click will lead users to methods and analytical tools that many geoscientists were unaware of. The repositories listed in the Paleogeosciences Catalog contain physical samples found all across the globe, from natural history museums to the basements of university buildings. EarthCube has over 250 databases, 300 software systems, and 200 repositories which will grow in the coming year. When completed, geoscientists across the world will be connected into a productive workflow for managing, sharing, and exploring geoscience data and information that expedites collaboration and innovation within the paleogeosciences, potentially bringing about new interdisciplinary discoveries.

  11. "Nanotechnology Enabled Advanced Industrial Heat Transfer Fluids"

    SciTech Connect

    Dr. Ganesh Skandan; Dr. Amit Singhal; Mr. Kenneth Eberts; Mr. Damian Sobrevilla; Prof. Jerry Shan; Stephen Tse; Toby Rossmann

    2008-06-12

    ABSTRACT Nanotechnology Enabled Advanced industrial Heat Transfer Fluids” Improving the efficiency of Industrial Heat Exchangers offers a great opportunity to improve overall process efficiencies in diverse industries such as pharmaceutical, materials manufacturing and food processing. The higher efficiencies can come in part from improved heat transfer during both cooling and heating of the material being processed. Additionally, there is great interest in enhancing the performance and reducing the weight of heat exchangers used in automotives in order to increase fuel efficiency. The goal of the Phase I program was to develop nanoparticle containing heat transfer fluids (e.g., antifreeze, water, silicone and hydrocarbon-based oils) that are used in transportation and in the chemical industry for heating, cooling and recovering waste heat. Much work has been done to date at investigating the potential use of nanoparticle-enhanced thermal fluids to improve heat transfer in heat exchangers. In most cases the effect in a commercial heat transfer fluid has been marginal at best. In the Phase I work, we demonstrated that the thermal conductivity, and hence heat transfer, of a fluid containing nanoparticles can be dramatically increased when subjected to an external influence. The increase in thermal conductivity was significantly larger than what is predicted by commonly used thermal models for two-phase materials. Additionally, the surface of the nanoparticles was engineered so as to have a minimal influence on the viscosity of the fluid. As a result, a nanoparticle-laden fluid was successfully developed that can lead to enhanced heat transfer in both industrial and automotive heat exchangers

  12. Quantitative velocity modulation spectroscopy.

    PubMed

    Hodges, James N; McCall, Benjamin J

    2016-05-14

    Velocity Modulation Spectroscopy (VMS) is arguably the most important development in the 20th century for spectroscopic study of molecular ions. For decades, interpretation of VMS lineshapes has presented challenges due to the intrinsic covariance of fit parameters including velocity modulation amplitude, linewidth, and intensity. This limitation has stifled the growth of this technique into the quantitative realm. In this work, we show that subtle changes in the lineshape can be used to help address this complexity. This allows for determination of the linewidth, intensity relative to other transitions, velocity modulation amplitude, and electric field strength in the positive column of a glow discharge. Additionally, we explain the large homogeneous component of the linewidth that has been previously described. Using this component, the ion mobility can be determined. PMID:27179476

  13. Quantitative velocity modulation spectroscopy

    NASA Astrophysics Data System (ADS)

    Hodges, James N.; McCall, Benjamin J.

    2016-05-01

    Velocity Modulation Spectroscopy (VMS) is arguably the most important development in the 20th century for spectroscopic study of molecular ions. For decades, interpretation of VMS lineshapes has presented challenges due to the intrinsic covariance of fit parameters including velocity modulation amplitude, linewidth, and intensity. This limitation has stifled the growth of this technique into the quantitative realm. In this work, we show that subtle changes in the lineshape can be used to help address this complexity. This allows for determination of the linewidth, intensity relative to other transitions, velocity modulation amplitude, and electric field strength in the positive column of a glow discharge. Additionally, we explain the large homogeneous component of the linewidth that has been previously described. Using this component, the ion mobility can be determined.

  14. Quantitative evolutionary design

    PubMed Central

    Diamond, Jared

    2002-01-01

    The field of quantitative evolutionary design uses evolutionary reasoning (in terms of natural selection and ultimate causation) to understand the magnitudes of biological reserve capacities, i.e. excesses of capacities over natural loads. Ratios of capacities to loads, defined as safety factors, fall in the range 1.2-10 for most engineered and biological components, even though engineered safety factors are specified intentionally by humans while biological safety factors arise through natural selection. Familiar examples of engineered safety factors include those of buildings, bridges and elevators (lifts), while biological examples include factors of bones and other structural elements, of enzymes and transporters, and of organ metabolic performances. Safety factors serve to minimize the overlap zone (resulting in performance failure) between the low tail of capacity distributions and the high tail of load distributions. Safety factors increase with coefficients of variation of load and capacity, with capacity deterioration with time, and with cost of failure, and decrease with costs of initial construction, maintenance, operation, and opportunity. Adaptive regulation of many biological systems involves capacity increases with increasing load; several quantitative examples suggest sublinear increases, such that safety factors decrease towards 1.0. Unsolved questions include safety factors of series systems, parallel or branched pathways, elements with multiple functions, enzyme reaction chains, and equilibrium enzymes. The modest sizes of safety factors imply the existence of costs that penalize excess capacities. Those costs are likely to involve wasted energy or space for large or expensive components, but opportunity costs of wasted space at the molecular level for minor components. PMID:12122135

  15. Hydrologic Prediction Through Earthcube Enabled Hydrogeophysical Cyberinfrastructure

    NASA Astrophysics Data System (ADS)

    Versteeg, R. J.; Johnson, D.

    2012-12-01

    to "develop a framework to understand and predict responses of the Earth as a system— from the space-atmosphere boundary to the core, including the influences of humans and ecosystems." Effective development of hydrologic prediction tools will require the hydrogeophysical community to engage in and become conversant with the cyberinfrastructure community. In my presentation I will provide several examples of how such tools could look like, and what some of the opportunities are for getting this engagement going and develop cyberinfrastructure enabled hydrologic prediction tools.

  16. The OGC Sensor Web Enablement framework

    NASA Astrophysics Data System (ADS)

    Cox, S. J.; Botts, M.

    2006-12-01

    Sensor observations are at the core of natural sciences. Improvements in data-sharing technologies offer the promise of much greater utilisation of observational data. A key to this is interoperable data standards. The Open Geospatial Consortium's (OGC) Sensor Web Enablement initiative (SWE) is developing open standards for web interfaces for the discovery, exchange and processing of sensor observations, and tasking of sensor systems. The goal is to support the construction of complex sensor applications through real-time composition of service chains from standard components. The framework is based around a suite of standard interfaces, and standard encodings for the message transferred between services. The SWE interfaces include: Sensor Observation Service (SOS)-parameterized observation requests (by observation time, feature of interest, property, sensor); Sensor Planning Service (SPS)-tasking a sensor- system to undertake future observations; Sensor Alert Service (SAS)-subscription to an alert, usually triggered by a sensor result exceeding some value. The interface design generally follows the pattern established in the OGC Web Map Service (WMS) and Web Feature Service (WFS) interfaces, where the interaction between a client and service follows a standard sequence of requests and responses. The first obtains a general description of the service capabilities, followed by obtaining detail required to formulate a data request, and finally a request for a data instance or stream. These may be implemented in a stateless "REST" idiom, or using conventional "web-services" (SOAP) messaging. In a deployed system, the SWE interfaces are supplemented by Catalogue, data (WFS) and portrayal (WMS) services, as well as authentication and rights management. The standard SWE data formats are Observations and Measurements (O&M) which encodes observation metadata and results, Sensor Model Language (SensorML) which describes sensor-systems, Transducer Model Language (TML) which

  17. Quantitative microbial ecology through stable isotope probing.

    PubMed

    Hungate, Bruce A; Mau, Rebecca L; Schwartz, Egbert; Caporaso, J Gregory; Dijkstra, Paul; van Gestel, Natasja; Koch, Benjamin J; Liu, Cindy M; McHugh, Theresa A; Marks, Jane C; Morrissey, Ember M; Price, Lance B

    2015-11-01

    Bacteria grow and transform eleme