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Sample records for based phosphoproteomic analysis

  1. Phosphoproteomics-based systems analysis of signal transduction networks

    Directory of Open Access Journals (Sweden)

    Hiroko eKozuka-Hata

    2012-01-01

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

  2. Recent developments in nanoparticle-based MALDI mass spectrometric analysis of phosphoproteomes

    International Nuclear Information System (INIS)

    Mass spectrometry-based strategies are widely used for mapping of post-translational modifications of phosphoproteins. However, the presence of large amounts of non-phosphopeptides seriously interferes by suppressing the intensities of signals for phosphopeptides in direct MALDI-MS techniques due to the low stoichiometry of protein phosphorylation. Several MALDI-MS approaches are known which use either nanoparticles (NPs) as affinity probes, or NPs as microwave heat absorbers. They assist in the enrichment of trace levels of phosphopeptides from complex protein digests and require minimal sample pretreatment, digestion times, and sample volume. This leads to enhance sensitivity and selectivity in the analysis of the phosphoproteomes. This review (with 89 refs.) summarizes and discusses recent developments in the field, with a particular focus on the potential use of nanomaterials such as metal oxides, metal NPs, NPs-coated target plates, and as core-shell nanocomposites acting as affinity probes and as heat absorbers in MALDI-MS analysis of phosphoproteomes. (author)

  3. SILAC-Based Temporal Phosphoproteomics

    DEFF Research Database (Denmark)

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

    2014-01-01

    signaling events. Here we provide an optimized SILAC-based proteomic workflow to analyze temporal changes in phosphoproteomes, which involve a generic three step enrichment protocol for phosphopeptides. SILAC-labeled peptides from digested whole cell lysates are as a first step enriched for phosphorylated...

  4. Knowledge-Based Analysis for Detecting Key Signaling Events from Time-Series Phosphoproteomics Data.

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    Pengyi Yang

    2015-08-01

    Full Text Available Cell signaling underlies transcription/epigenetic control of a vast majority of cell-fate decisions. A key goal in cell signaling studies is to identify the set of kinases that underlie key signaling events. In a typical phosphoproteomics study, phosphorylation sites (substrates of active kinases are quantified proteome-wide. By analyzing the activities of phosphorylation sites over a time-course, the temporal dynamics of signaling cascades can be elucidated. Since many substrates of a given kinase have similar temporal kinetics, clustering phosphorylation sites into distinctive clusters can facilitate identification of their respective kinases. Here we present a knowledge-based CLUster Evaluation (CLUE approach for identifying the most informative partitioning of a given temporal phosphoproteomics data. Our approach utilizes prior knowledge, annotated kinase-substrate relationships mined from literature and curated databases, to first generate biologically meaningful partitioning of the phosphorylation sites and then determine key kinases associated with each cluster. We demonstrate the utility of the proposed approach on two time-series phosphoproteomics datasets and identify key kinases associated with human embryonic stem cell differentiation and insulin signaling pathway. The proposed approach will be a valuable resource in the identification and characterizing of signaling networks from phosphoproteomics data.

  5. Selecting Sample Preparation Workflows for Mass Spectrometry-Based Proteomic and Phosphoproteomic Analysis of Patient Samples with Acute Myeloid Leukemia

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    Maria Hernandez-Valladares

    2016-08-01

    Full Text Available Global mass spectrometry (MS-based proteomic and phosphoproteomic studies of acute myeloid leukemia (AML biomarkers represent a powerful strategy to identify and confirm proteins and their phosphorylated modifications that could be applied in diagnosis and prognosis, as a support for individual treatment regimens and selection of patients for bone marrow transplant. MS-based studies require optimal and reproducible workflows that allow a satisfactory coverage of the proteome and its modifications. Preparation of samples for global MS analysis is a crucial step and it usually requires method testing, tuning and optimization. Different proteomic workflows that have been used to prepare AML patient samples for global MS analysis usually include a standard protein in-solution digestion procedure with a urea-based lysis buffer. The enrichment of phosphopeptides from AML patient samples has previously been carried out either with immobilized metal affinity chromatography (IMAC or metal oxide affinity chromatography (MOAC. We have recently tested several methods of sample preparation for MS analysis of the AML proteome and phosphoproteome and introduced filter-aided sample preparation (FASP as a superior methodology for the sensitive and reproducible generation of peptides from patient samples. FASP-prepared peptides can be further fractionated or IMAC-enriched for proteome or phosphoproteome analyses. Herein, we will review both in-solution and FASP-based sample preparation workflows and encourage the use of the latter for the highest protein and phosphorylation coverage and reproducibility.

  6. Analytical strategies in mass spectrometry-based phosphoproteomics

    DEFF Research Database (Denmark)

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

    2011-01-01

    discuss various tandem mass spectrometry approaches for phosphopeptide sequencing and quantification, and we consider aspects of phosphoproteome data analysis and interpretation. Efficient integration of these stages of phosphoproteome analysis is highly important to ensure a successful outcome of large...

  7. Systems Analysis for Interpretation of Phosphoproteomics Data

    DEFF Research Database (Denmark)

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

    2016-01-01

    Global phosphoproteomics investigations yield overwhelming datasets with up to tens of thousands of quantified phosphosites. The main challenge after acquiring such large-scale data is to extract the biological meaning and relate this to the experimental question at hand. Systems level analysis p...... appropriate tools for systems analysis of phosphoproteomics datasets, and the considerations that are critical for acquiring meaningful output.......Global phosphoproteomics investigations yield overwhelming datasets with up to tens of thousands of quantified phosphosites. The main challenge after acquiring such large-scale data is to extract the biological meaning and relate this to the experimental question at hand. Systems level analysis...... provides the best means for extracting functional insights from such types of datasets, and this has primed a rapid development of bioinformatics tools and resources over the last decade. Many of these tools are specialized databases that can be mined for annotation and pathway enrichment, whereas others...

  8. Phosphoproteomic analysis of cell-based resistance to BRAF inhibitor therapy in melanoma

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    Robert eParker

    2015-05-01

    Full Text Available 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.

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

    DEFF Research Database (Denmark)

    Thingholm, Tine; Larsen, Martin Røssel; Ingrell, Christian; Kassem, Moustapha; Jensen, Ole Nørregaard

    2008-01-01

    Phosphorylation of plasma membrane proteins frequently initiates signal transduction pathways or attenuate plasma membrane transport processes. Because of the low abundance and hydrophobic features of many plasma membrane proteins and the low stoichiometry of protein phosphorylation, studies of the...... plasma membrane phosphoproteome are challenging. We present an optimized analytical strategy for plasma membrane phosphoproteomics that combines efficient plasma membrane protein preparation with TiO 2-based phosphopeptide enrichment and high-performance mass spectrometry for phosphopeptide sequencing....... We used sucrose centrifugation in combination with sodium carbonate extraction to achieve efficient and reproducible purification of low microgram levels of plasma membrane proteins from human mesenchymal stem cells (hMSCs, 10 (7) cells), achieving more than 70% yield of membrane proteins...

  10. Variable Digestion Strategies for Phosphoproteomics Analysis.

    Science.gov (United States)

    Gonczarowska-Jorge, Humberto; Dell'Aica, Margherita; Dickhut, Clarissa; Zahedi, René P

    2016-01-01

    In recent years, mass spectrometry-based phosphoproteomics has propelled our knowledge about the regulation of cellular pathways. Nevertheless, typically applied bottom-up strategies have several limitations. Trypsin, the preferentially used proteolytic enzyme shows impaired cleavage efficiency in the vicinity of phosphorylation sites. Moreover, depending on the frequency and distribution of tryptic cleavage sites (Arg/Lys), generated peptides can be either too short or too long for confident identification using standard LC-MS approaches. To overcome these limitations, we introduce an alternative and simple approach based on the usage of the nonspecific serine protease subtilisin, which enables a fast and reproducible digestion and provides access to "hidden" areas of the proteome. Thus, in a single LC-MS experiment >1800 phosphopeptides were confidently identified and localized from 125 μg of HeLa digest, compared to >2100 sites after tryptic digestion. While the overlap was less than 20 %, subtilisin allowed the identification of many phosphorylation sites that are theoretically not accessible via tryptic digestion, thus considerably increasing the coverage of the phosphoproteome. PMID:26584929

  11. Assessment of phosphopeptide enrichment/precipitation method for LC-MS/MS based phosphoproteomic analysis of plant tissue

    DEFF Research Database (Denmark)

    Ye, Juanying; Rudashevskaya, Elena; Hansen, Thomas Aarup;

    stardand sample preparation protocols. Here, we combine 3 phosphpeptide enrichment methods (IMAC, TiO2 and Calcium Phosphate Precipitation (CPP)), and apply them to phosphoproteomic analysis of Arabidopsis thaliana plasma membrane preparation. Method Plant plasma membranes were isolated from Arabidopsis...... thaliana (Col-0) leaves using a two-phase partitioning system. The concentration of plasma membrane protein was determined by Bradford assay. Protein was digested with Lys-C for 4 hours and then by trypsin overnight. The peptide mixture was purified with IMAC, TiO2, CPP, SIMAC (IMAC+TiO2), the combination...... of CPP and IMAC, and the combination of CPP and TiO2, respectively. Nano-LC-MS was performed using LTQ-Orbitrap XL and LTQ-Orbitrap-XL/ETD mass spectrometer (Thermo Electron, Bremen, Germany) connected to an EASY nano-LC system (Proxeon Biosystems, Odense, Denmark). In CID mode, multi...

  12. Evaluating experimental bias and completeness in comparative phosphoproteomics analysis.

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    Jos Boekhorst

    Full Text Available Unraveling the functional dynamics of phosphorylation networks is a crucial step in understanding the way in which biological networks form a living cell. Recently there has been an enormous increase in the number of measured phosphorylation events. Nevertheless, comparative and integrative analysis of phosphoproteomes is confounded by incomplete coverage and biases introduced by different experimental workflows. As a result, we cannot differentiate whether phosphosites indentified in only one or two samples are the result of condition or species specific phosphorylation, or reflect missing data. Here, we evaluate the impact of incomplete phosphoproteomics datasets on comparative analysis, and we present bioinformatics strategies to quantify the impact of different experimental workflows on measured phosphoproteomes. We show that plotting the saturation in observed phosphosites in replicates provides a reproducible picture of the extent of a particular phosphoproteome. Still, we are still far away from a complete picture of the total human phosphoproteome. The impact of different experimental techniques on the similarity between phosphoproteomes can be estimated by comparing datasets from different experimental pipelines to a common reference. Our results show that comparative analysis is most powerful when datasets have been generated using the same experimental workflow. We show this experimentally by measuring the tyrosine phosphoproteome from Caenorhabditis elegans and comparing it to the tyrosine phosphoproteome of HeLa cells, resulting in an overlap of about 4%. This overlap between very different organisms represents a three-fold increase when compared to dataset of older studies, wherein different workflows were used. The strategies we suggest enable an estimation of the impact of differences in experimental workflows on the overlap between datasets. This will allow us to perform comparative analyses not only on datasets specifically

  13. Interleukin-2 signaling pathway analysis by quantitative phosphoproteomics

    DEFF Research Database (Denmark)

    Osinalde, Nerea; Moss, Helle; Arrizabalaga, Onetsine;

    2011-01-01

    by IL-2, we aimed to define the global tyrosine-phosphoproteome of IL-2 pathway in human T cell line Kit225 using high resolution mass spectrometry combined with phosphotyrosine immunoprecipitation and SILAC. The molecular snapshot at 5min of IL-2 stimulation resulted in identification of 172...... proteins among which 79 were found with increased abundance in the tyrosine-phosphorylated complexes, including several previously not reported IL-2 downstream effectors. Combinatorial site-specific phosphoproteomic analysis resulted in identification of 99 phosphorylated sites mapping to the identified...

  14. Quantitative Proteomic and Phosphoproteomic Analysis of Trypanosoma cruzi Amastigogenesis

    DEFF Research Database (Denmark)

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

    2014-01-01

    well-established differentiation protocol to perform a comprehensive quantitative proteomic and phosphoproteomic analysis of the T. cruzi amastigogenesis. Samples from fully differentiated forms and two biologically relevant intermediate time points were Lys-C/trypsin digested, iTRAQ-labeled and...

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

    Institute of Scientific and Technical Information of China (English)

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

    2015-01-01

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

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

    DEFF Research Database (Denmark)

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

    2014-01-01

    -arrestin dependent signalling. Two complimentary global phosphoproteomics studies have analyzed the complex signalling induced by the AT1aR. Here we integrate the data sets from these studies and perform a joint analysis using a novel method for prediction of differential kinase activity from phosphoproteomics data...... developed a new method for kinase-centric analysis of phosphoproteomes to pinpoint differential kinase activity in large-scale data sets....

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

    Science.gov (United States)

    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

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

    OpenAIRE

    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, ...

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

    DEFF Research Database (Denmark)

    Zanivan, Sara; Meves, Alexander; Behrendt, Kristina;

    2013-01-01

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

  20. Quantitative Phosphoproteomics Analysis of ERBB3/ERBB4 Signaling.

    Science.gov (United States)

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

    2016-01-01

    The four members of the epidermal growth factor receptor (EGFR/ERBB) family form homo- and heterodimers which mediate ligand-specific regulation of many key cellular processes in normal and cancer tissues. While signaling through the EGFR has been extensively studied on the molecular level, signal transduction through ERBB3/ERBB4 heterodimers is less well understood. Here, we generated isogenic mouse Ba/F3 cells that express full-length and functional membrane-integrated ERBB3 and ERBB4 or ERBB4 alone, to serve as a defined cellular model for biological and phosphoproteomics analysis of ERBB3/ERBB4 signaling. ERBB3 co-expression significantly enhanced Ba/F3 cell proliferation upon neuregulin-1 (NRG1) treatment. For comprehensive signaling studies we performed quantitative mass spectrometry (MS) experiments to compare the basal ERBB3/ERBB4 cell phosphoproteome to NRG1 treatment of ERBB3/ERBB4 and ERBB4 cells. We employed a workflow comprising differential isotope labeling with mTRAQ reagents followed by chromatographic peptide separation and final phosphopeptide enrichment prior to MS analysis. Overall, we identified 9686 phosphorylation sites which could be confidently localized to specific residues. Statistical analysis of three replicate experiments revealed 492 phosphorylation sites which were significantly changed in NRG1-treated ERBB3/ERBB4 cells. Bioinformatics data analysis recapitulated regulation of mitogen-activated protein kinase and Akt pathways, but also indicated signaling links to cytoskeletal functions and nuclear biology. Comparative assessment of NRG1-stimulated ERBB4 Ba/F3 cells revealed that ERBB3 did not trigger defined signaling pathways but more broadly enhanced phosphoproteome regulation in cells expressing both receptors. In conclusion, our data provide the first global picture of ERBB3/ERBB4 signaling and provide numerous potential starting points for further mechanistic studies. PMID:26745281

  1. Quantitative Phosphoproteomics Analysis of ERBB3/ERBB4 Signaling.

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    Sebastian K Wandinger

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

  2. Phosphoproteomics-based modeling defines the regulatory mechanism underlying aberrant EGFR signaling.

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    Shinya Tasaki

    Full Text Available BACKGROUND: Mutation of the epidermal growth factor receptor (EGFR results in a discordant cell signaling, leading to the development of various diseases. However, the mechanism underlying the alteration of downstream signaling due to such mutation has not yet been completely understood at the system level. Here, we report a phosphoproteomics-based methodology for characterizing the regulatory mechanism underlying aberrant EGFR signaling using computational network modeling. METHODOLOGY/PRINCIPAL FINDINGS: Our phosphoproteomic analysis of the mutation at tyrosine 992 (Y992, one of the multifunctional docking sites of EGFR, revealed network-wide effects of the mutation on EGF signaling in a time-resolved manner. Computational modeling based on the temporal activation profiles enabled us to not only rediscover already-known protein interactions with Y992 and internalization property of mutated EGFR but also further gain model-driven insights into the effect of cellular content and the regulation of EGFR degradation. Our kinetic model also suggested critical reactions facilitating the reconstruction of the diverse effects of the mutation on phosphoproteome dynamics. CONCLUSIONS/SIGNIFICANCE: Our integrative approach provided a mechanistic description of the disorders of mutated EGFR signaling networks, which could facilitate the development of a systematic strategy toward controlling disease-related cell signaling.

  3. Phosphoproteomic analysis of platelets activated by pro-thrombotic oxidized phospholipids and thrombin.

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    Alejandro Zimman

    Full Text Available 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 (dephosphorylation, 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.

  4. Gel-based phosphoproteomics analysis of sarcoplasmic proteins in postmortem porcine muscle with pH decline rate and time differences

    DEFF Research Database (Denmark)

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

    2011-01-01

    Meat quality development is highly influenced by the pH decline caused by the postmortem (PM) glycolysis. Protein phosphorylation is an important mechanism in regulating the activity of glycometabolic enzymes. Here, a gel-based phosphoproteomic study was performed to analyze the protein...... phosphorylation in sarcoplasmic proteins from three groups of pigs with different pH decline rates from PM 1 to 24¿h. Globally, the fast pH decline group had the highest phosphorylation level at PM 1¿h, but lowest at 24¿h, whereas the slow pH decline group showed the reverse case. The same pattern was also...... observed in most individual bands in 1-DE. The protein phosphorylation levels of 12 bands were significantly affected by the synergy effects of pH and time (p...

  5. Phosphoproteomics analysis of a clinical Mycobacterium tuberculosis Beijing isolate: Expanding the mycobacterial phosphoproteome catalogue

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    Suereta eFortuin

    2015-02-01

    Full Text Available 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 forty five 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.

  6. Assessment of phosphopeptide enrichment/precipitation method for LC-MS/MS based phosphoproteomic analysis of plant tissue

    OpenAIRE

    Ye, Juanying; Rudashevskaya, Elena; Hansen, Thomas Aarup; Fuglsang, Anja T.; Palmgren, Michael G.; Jensen, Ole Nørregaard

    2009-01-01

      IntroductionMass spectrometry (MS) is a powerful technology for study of PTMs, including protein phosphorylation. Due to the low abundance of many phosphoproteins and the relatively poor ionization efficiency of phosphopeptides, specific enrichment of phosphopeptides prior to MS analysis is necessary. At present, numerous phosphopeptide enrichment approaches have been established and applied to complex biological samples. We and others have reported that multi-step phosphopeptide purificati...

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

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    Sara Zanivan

    2013-02-01

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

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

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    Hong-Mei Xu

    2015-01-01

    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.

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

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    Xiuli eHu

    2015-05-01

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

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

    DEFF Research Database (Denmark)

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

    2012-01-01

    The aim of this study was to characterize the protein phosphorylation in postmortem (PM) muscle and reveal the change during meat quality development. The gel-based phosphoproteomic analysis of PM porcine muscle was performed in three pig groups with different pH decline rates from PM 1h to 24 h....... isomerase-1, tropomyosin and myosin regulatory light chain 2 showed to be related to PM muscle pH decline rate and time. Our work sheds light on the potential role of protein phosphorylation on regulation of meat quality development.......The aim of this study was to characterize the protein phosphorylation in postmortem (PM) muscle and reveal the change during meat quality development. The gel-based phosphoproteomic analysis of PM porcine muscle was performed in three pig groups with different pH decline rates from PM 1h to 24 h....... The sarcoplasmic and myofibrillar fractions were analyzed using gel electrophoresis in combination with a phosphoprotein specific staining. Globally, the group with fast pH decline rate had the highest phosphorylation level at PM 1 h, but lowest at PM 24 h, whereas the group with slow pH decline rate...

  11. Motif-specific sampling of phosphoproteomes

    OpenAIRE

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

    2008-01-01

    Phosphoproteomics, the targeted study of a subfraction of the proteome which is modified by phosphorylation, has become an indispensable tool to study cell signaling dynamics. We described a methodology that linked phosphoproteome and proteome analysis based on Ba2+ binding properties of amino acids. This technology selected motif-specific phosphopeptides independent of the system under analysis. MudPIT (Multidimensional Identification Technology) identified 1037 precipitated phosphopeptides ...

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  14. Enhanced Phosphoproteomic Profiling Workflow For Growth Factor Signaling Analysis

    DEFF Research Database (Denmark)

    Sylvester, Marc; Burbridge, Mike; Leclerc, Gregory;

    2010-01-01

    understanding of pathological processes. In our study we create and integrate data on phosphorylations that are initiated by several growth factor receptors. We present an approach for quantitative, time-resolved phosphoproteomic profiling that integrates the important contributions by phosphotyrosines. Methods...

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

    DEFF Research Database (Denmark)

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

    2008-01-01

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

  16. Kinomic and phospho-proteomic analysis of breast cancer stem-like cells

    DEFF Research Database (Denmark)

    Leth-Larsen, Rikke; Christensen, Anne Geske Lindhard; Ehmsen, Sidse;

    Kinomic and phospho-proteomic analysis of breast cancer stem-like cells Rikke Leth-Larsen1, Anne G Christensen1, Sidse Ehmsen1, Mark Møller1, Giuseppe Palmisano2, Martin R Larsen2, Henrik J Ditzel1,3 1Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark 2Institute of...

  17. Global analysis of neuronal phosphoproteome regulation by chondroitin sulfate proteoglycans.

    Directory of Open Access Journals (Sweden)

    Panpan Yu

    Full Text Available Chondroitin sulfate proteoglycans (CSPGs are major components of the extracellular matrix which mediate inhibition of axonal regeneration after injury to the central nervous system (CNS. Several neuronal receptors for CSPGs have recently been identified; however, the signaling pathways by which CSPGs restrict axonal growth are still largely unknown. In this study, we applied quantitative phosphoproteomics to investigate the global changes in protein phosphorylation induced by CSPGs in primary neurons. In combination with isobaric Tags for Relative and Absolute Quantitation (iTRAQ labeling, strong cation exchange chromatography (SCX fractionation, immobilized metal affinity chromatography (IMAC and LC-MS/MS, we identified and quantified 2214 unique phosphopeptides corresponding to 1118 phosphoproteins, with 118 changing significantly in abundance with CSPG treatment. The proteins that were regulated by CSPGs included key components of synaptic vesicle trafficking, axon guidance mediated by semaphorins, integrin signaling, cadherin signaling and EGF receptor signaling pathways. A significant number of the regulated proteins are cytoskeletal and related proteins that have been implicated in regulating neurite growth. Another highly represented protein category regulated by CSPGs is nucleic acid binding proteins involved in RNA post-transcriptional regulation. Together, by screening the overall phosphoproteome changes induced by CSPGs, this data expand our understanding of CSPG signaling, which provides new insights into development of strategies for overcoming CSPG inhibition and promoting axonal regeneration after CNS injury.

  18. Predicting kinase activity in angiotensin receptor phosphoproteomes based on sequence-motifs and interactions.

    Directory of Open Access Journals (Sweden)

    Rikke Bøgebo

    Full Text Available Recent progress in the understanding of seven-transmembrane receptor (7TMR signalling has promoted the development of a new generation of pathway selective ligands. The angiotensin II type I receptor (AT1aR is one of the most studied 7TMRs with respect to selective activation of the β-arrestin dependent signalling. Two complimentary global phosphoproteomics studies have analyzed the complex signalling induced by the AT1aR. Here we integrate the data sets from these studies and perform a joint analysis using a novel method for prediction of differential kinase activity from phosphoproteomics data. The method builds upon NetworKIN, which applies sophisticated linear motif analysis in combination with contextual network modelling to predict kinase-substrate associations with high accuracy and sensitivity. These predictions form the basis for subsequently nonparametric statistical analysis to identify likely activated kinases. This suggested that AT1aR-dependent signalling activates 48 of the 285 kinases detected in HEK293 cells. Of these, Aurora B, CLK3 and PKG1 have not previously been described in the pathway whereas others, such as PKA, PKB and PKC, are well known. In summary, we have developed a new method for kinase-centric analysis of phosphoproteomes to pinpoint differential kinase activity in large-scale data sets.

  19. An Automated Platform for Analysis of Phosphoproteomic Datasets: Application to Kidney Collecting Duct Phosphoproteins

    OpenAIRE

    Jason D. Hoffert; Wang, Guanghui; Pisitkun, Trairak; Shen, Rong-Fong; Knepper, Mark A.

    2007-01-01

    Large-scale phosphoproteomic analysis employing liquid chromatography-tandem mass spectrometry (LC–MS/MS) often requires a significant amount of manual manipulation of phosphopeptide datasets in the postacquisition phase. To assist in this process, we have created software, PhosphoPIC (PhosphoPeptide Identification and Compilation), which can perform a variety of useful functions including automated selection and compilation of phosphopeptide identifications from multiple MS levels, estimatio...

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

    OpenAIRE

    Fan, Shuli; Meng, Yanyan; 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 phospho...

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

    OpenAIRE

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

    2013-01-01

    Background Progress in the fields of protein separation and identification technologies has accelerated research into biofluids proteomics for protein biomarker discovery. Urine has become an ideal and rich source of biomarkers in clinical proteomics. Here we performed a proteomic analysis of urine samples from pregnant and non-pregnant patients using gel electrophoresis and high-resolution mass spectrometry. Furthermore, we also apply a non-prefractionation quantitative phosphoproteomic appr...

  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.

    Science.gov (United States)

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-11-11

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

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

    Directory of Open Access Journals (Sweden)

    Roytrakul Sittiruk

    2011-06-01

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

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

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    Barbora Šalovská

    2014-07-01

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

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

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    He Qiu-Yan

    2011-06-01

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

  7. Comparative gel-based phosphoproteomics in response to signaling molecules

    KAUST Repository

    Marondedze, Claudius

    2013-09-03

    The gel-based proteomics approach is a valuable technique for studying the characteristics of proteins. This technique has diverse applications ranging from analysis of a single protein to the study of the total cellular proteins. Further, protein quality and to some extent distribution can be first assessed by means of one-dimensional gel electrophoresis and then more informatively, for comparative analysis, using the two-dimensional gel electrophoresis technique. Here, we describe how to take advantage of the availability of fluorescent dyes to stain for a selective class of proteins on the same gel for the detection of both phospho- and total proteomes. This enables the co-detection of phosphoproteins as well as total proteins from the same gel and is accomplished by utilizing two different fluorescent stains, the ProQ-Diamond, which stains only phosphorylated proteins, and Sypro Ruby, which stains the entire subset of proteins. This workflow can be applied to gain insights into the regulatory mechanisms induced by signaling molecules such as cyclic nucleotides through the quantification and subsequent identification of responsive phospho- and total proteins. © Springer Science+Business Media New York 2013.

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

    Science.gov (United States)

    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

  9. Quantitative phosphoproteomics analysis of nitric oxide-responsive phosphoproteins in cotton leaf.

    Directory of Open Access Journals (Sweden)

    Shuli Fan

    Full Text Available 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.

  10. Functional phosphoproteomics for current immunology research

    Directory of Open Access Journals (Sweden)

    Paulino Gómez-Puertas

    2011-01-01

    Full Text Available Signaling networks are key elements in all major aspects of cellular life, playing a major role in inter- and intracellular communications. They are involved in diverse processes such as cell-cycle progression, cellular metabolism, cell-cell communication and appropriate response to the cellular environment. The latter comprises a whole range of networks that are involved in regulation of cell development, differentiation, proliferation, apoptosis, and immunologic responses. The key mechanism involves the transduction of extracellular signals across the cell sur-face to different effectors in the cytosol and the nucleus. Dysregulation of these pathways is often associated with immunology disorders and malignant diseases such as cancer. One of the most common mechanisms of activation and/or inactivation of signaling transduction pathways is phosphorylation and de-phosphorylation at serine, threonine and tyrosine residues. Phosphoproteomics is playing an important role in our understanding of how phosphorylation participates in translating distinct signals into the normal and or abnormal physiological responses, and has shifted research towards screening for potential therapies for diseases and in-depth analysis of phosphoproteomes. Given the importance of phosphoproteomics in translational research we aim at outlining phosphoproteomic approaches based on mass spectrometry (MS. This review focuses on (1b the role of phospho signaling in immunology, (2a current phosphopeptide enrichment methods based on IMAC and titanium dioxide, (2b phosphopeptide analysis by MS, and (2c issues concerned with interpretation of phospho spectra by database dependent search. Finally, quantitative methods used in phosphoproteomics such as Stable Isotope labeling with Amino acid in cell Culture (SILAC, isobaric Tag for Relative and Absolute Quantitation (iTRAQ and Absolute Quantification (AQUA is discussed in section 3.

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

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    Ilyas Singec

    2016-09-01

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

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

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    Miaomiao Tian

    2015-02-01

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

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

    Science.gov (United States)

    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

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

    KAUST Repository

    Alqurashi, May M.

    2013-11-01

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

  15. Quantitative phosphoproteomics using acetone-based peptide labeling: Method evaluation and application to a cardiac ischemia/reperfusion model

    OpenAIRE

    Wijeratne, Aruna B.; Manning, Janet R.; Schultz, Jo El J.; Greis, Kenneth D.

    2013-01-01

    Mass spectrometry (MS) techniques to globally profile protein phosphorylation in cellular systems that are relevant to physiological or pathological changes have been of significant interest in biological research. In this report, an MS-based strategy utilizing an inexpensive acetone-based peptide labeling technique known as reductive alkylation by acetone (RABA) for quantitative phosphoproteomics was explored to evaluate its capacity. Since the chemistry for RABA-labeling for phosphorylation...

  16. Progress in Quantitative Analysis Strategies of Phosphoproteomics%磷酸化蛋白质组定量研究策略

    Institute of Scientific and Technical Information of China (English)

    甄艳; 李春映; 施季森

    2014-01-01

    Protein phosphorylation is an important post-translational modification in organisms for the regulation of diverse biological processes. Quantitative analysis of the phosphorylated proteins is important for understanding the biological functions under internal and external factors. The early studies focus on the qualitative analysis, identification and location of protein phosphorylation sites. In recent years, with the rapid development of mass spectrometry, the quantitative analysis strategies on phosphoproteomics have been made great progress. Monitoring changes in phosphorylated proteins is capable of clarifying the processes of biological functions. In this paper, we mainly present an overview of several strategies of quantitative phosphoproteomics, such as strategies of quantitative phosphoproteomics based on 2-DE, quantitative phosphoproteomics based on gel-free MS/MS labelling and label free, etc. Quantitative analysis of protein phosphorylation would be help to better elucidate the signal transduction mechanisms of biological processes.%蛋白质磷酸化修饰是调控多样种生物过程的一种重要的翻译后修饰,定量分析内部和外部因子作用下磷酸化蛋白质的分子调控机制对理解生物功能非常重要。早期磷酸化蛋白质组研究的重点是定性检测、鉴定磷酸化蛋白质及磷酸化位点的定位。近年来,随着质谱技术的快速发展使得定量磷酸化蛋白质组研究有了很大的发展。利用磷酸化蛋白质组的定量分析监测蛋白质磷酸化的动态变化有助于阐明生物过程及其功能的实现机制。本文综述了磷酸化蛋白质组定量分析策略:基于2-DE的磷酸化蛋白质组定量策略,基于无胶的质谱磷酸化蛋白质组标记定量策略及无标记定量分析策略。磷酸化蛋白质组的定量分析将有助于更好的阐明生物的信号传导机制。

  17. Quantitative phosphoproteomics using acetone-based peptide labeling: method evaluation and application to a cardiac ischemia/reperfusion model.

    Science.gov (United States)

    Wijeratne, Aruna B; Manning, Janet R; Schultz, Jo El J; Greis, Kenneth D

    2013-10-01

    Mass spectrometry (MS) techniques to globally profile protein phosphorylation in cellular systems that are relevant to physiological or pathological changes have been of significant interest in biological research. An MS-based strategy utilizing an inexpensive acetone-based peptide-labeling technique known as reductive alkylation by acetone (RABA) for quantitative phosphoproteomics was explored to evaluate its capacity. Because the chemistry for RABA labeling for phosphorylation profiling had not been previously reported, it was first validated using a standard phosphoprotein and identical phosphoproteomes from cardiac tissue extracts. A workflow was then utilized to compare cardiac tissue phosphoproteomes from mouse hearts not expressing FGF2 versus hearts expressing low-molecular-weight fibroblast growth factor-2 (LMW FGF2) to relate low-molecular-weight fibroblast growth factor-2 (LMW FGF2)-mediated cardioprotective phenomena induced by ischemia/reperfusion injury of hearts, with downstream phosphorylation changes in LMW FGF2 signaling cascades. Statistically significant phosphorylation changes were identified at 14 different sites on 10 distinct proteins, including some with mechanisms already established for LMW FGF2-mediated cardioprotective signaling (e.g., connexin-43), some with new details linking LMW FGF2 to the cardioprotective mechanisms (e.g., cardiac myosin binding protein C or cMyBPC), and also several new downstream effectors not previously recognized for cardio-protective signaling by LMW FGF2. Additionally, one of the phosphopeptides, cMyBPC/pSer-282, identified was further verified with site-specific quantification using an SRM (selected reaction monitoring)-based approach that also relies on isotope labeling of a synthetic phosphopeptide with deuterated acetone as an internal standard. Overall, this study confirms that the inexpensive acetone-based peptide labeling can be used in both exploratory and targeted quantification phosphoproteomic

  18. Technologies and challenges in large-scale phosphoproteomics

    DEFF Research Database (Denmark)

    Engholm-Keller, Kasper; Larsen, Martin Røssel

    2013-01-01

    apoptosis, rely on phosphorylation. This PTM is thus involved in many diseases, rendering localization and assessment of extent of phosphorylation of major scientific interest. MS-based phosphoproteomics, which aims at describing all phosphorylation sites in a specific type of cell, tissue, or organism, has...... become the main technique for discovery and characterization of phosphoproteins in a nonhypothesis driven fashion. In this review, we describe methods for state-of-the-art MS-based analysis of protein phosphorylation as well as the strategies employed in large-scale phosphoproteomic experiments with...

  19. Large-Scale Phosphoproteomics Analysis of Whole Saliva Reveals a Distinct Phosphorylation Pattern

    OpenAIRE

    Stone, Matthew D.; Chen, Xiaobing; McGowan, Thomas; Bandhakavi, Sricharan; Bin CHENG; Rhodus, Nelson L.; Griffin, Timothy J.

    2011-01-01

    In-depth knowledge of bodily fluid phosphoproteomes, such as whole saliva, is limited. To better understand the whole saliva phosphoproteome, we generated a large-scale catalog of phosphorylated proteins. To circumvent the wide dynamic range of phosphoprotein abundance in whole saliva, we combined dynamic range compression using hexapeptide beads, strong cation exchange HPLC peptide fractionation, and immobilized metal affinity chromatography prior to mass spectrometry. In total, 217 unique p...

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

    Science.gov (United States)

    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

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

    DEFF Research Database (Denmark)

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

    2016-01-01

    The traditional sample preparation workflow for mass spectrometry (MS)-based phosphoproteomics is time consuming and usually requires multiple steps, e.g., lysis, protein precipitation, reduction, alkylation, digestion, fractionation, and phosphopeptide enrichment. Each step can introduce chemical...... artifacts, in vitro protein and peptide modifications, and contaminations. Those often result in sample loss and affect the sensitivity, dynamic range and accuracy of the mass spectrometric analysis. Here we describe a simple and reproducible phosphoproteomics protocol, where lysis, denaturation, reduction...

  2. Phosphoproteomic analysis reveals major default phosphorylation sites outside long intrinsically disordered regions of Arabidopsis plasma membrane proteins

    Directory of Open Access Journals (Sweden)

    Nespoulous Claude

    2012-10-01

    Full Text Available Abstract Background Genome-wide statistics established that long intrinsically disordered regions (over 30 residues are predicted in a large part of proteins in all eukaryotes, with a higher ratio in trans-membrane proteins. At functional level, such unstructured and flexible regions were suggested for years to favour phosphorylation events. In plants, despite increasing evidence of the regulation of transport and signalling processes by phosphorylation events, only few data are available without specific information regarding plasma membrane proteins, especially at proteome scale. Results Using a dedicated phosphoproteomic workflow, 75 novel and unambiguous phosphorylation sites were identified in Arabidopsis plasma membrane. Bioinformatics analysis showed that this new dataset concerned mostly integral proteins involved in key functions of the plasma membrane (such as transport and signal transduction, including protein phosphorylation. It thus expanded by 15% the directory of phosphosites previously characterized in signalling and transport proteins. Unexpectedly, 66% of phosphorylation sites were predicted to be located outside long intrinsically disordered regions. This result was further corroborated by analysis of publicly available data for the plasma membrane. Conclusions The new phosphoproteomics data presented here, with published datasets and functional annotation, suggest a previously unexpected topology of phosphorylation in the plant plasma membrane proteins. The significance of these new insights into the so far overlooked properties of the plant plasma membrane phosphoproteome and the long disordered regions is discussed.

  3. Quantitative analysis of a phosphoproteome readily altered by the protein kinase CK2 inhibitor quinalizarin in HEK-293T cells.

    Science.gov (United States)

    Franchin, Cinzia; Cesaro, Luca; Salvi, Mauro; Millioni, Renato; Iori, Elisabetta; Cifani, Paolo; James, Peter; Arrigoni, Giorgio; Pinna, Lorenzo

    2015-06-01

    CK2 is an extremely pleiotropic Ser/Thr protein kinase, responsible for the generation of a large proportion of the human phosphoproteome and implicated in a wide variety of biological functions. CK2 plays a global role as an anti-apoptotic agent, a property which is believed to partially account for the addiction of many cancer cells to high CK2 levels. To gain information about the CK2 targets whose phosphorylation is primarily implicated in its pro-survival signaling advantage has been taken of quinalizarin (QZ) a cell permeable fairly specific CK2 inhibitor, previously shown to be able to block endogenous CK2 triggering an apoptotic response. HEK-293T cells either treated or not for 3h with 50μM QZ were exploited to perform a quantitative SILAC phosphoproteomic analysis of phosphosites readily responsive to QZ treatment. Our analysis led to the identification of 4883 phosphosites, belonging to 1693 phosphoproteins. 71 phosphosites (belonging to 47 proteins) underwent a 50% or more decreased occupancy upon QZ treatment. Almost 50% of these fulfilled the typical consensus sequence recognized by CK2 (S/T-x-x-E/D/pS) and in several cases were validated as bona fide substrates of CK2 either based on data in the literature or by performing in vitro phosphorylation experiments with purified proteins. The majority of the remaining phosphosites drastically decreased upon QZ treatment display the pS/T-P motif typical of proline directed protein kinases and a web logo extracted from them differentiates from the web logo extracted from all the proline directed phosphosites quantified during our analysis (1151 altogether). A paradoxical outcome of our study was the detection of 116 phosphosites (belonging to 92 proteins altogether) whose occupancy is substantially increased (50% or more), rather than decreased by QZ treatment: 40% of these display the typical motif recognized by proline directed kinases, while about 25% fulfill the CK2 consensus. Collectively taken our

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

    Institute of Scientific and Technical Information of China (English)

    Yanmei Chen

    2012-01-01

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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  7. PAPE (Prefractionation-Assisted Phosphoprotein Enrichment: A Novel Approach for Phosphoproteomic Analysis of Green Tissues from Plants

    Directory of Open Access Journals (Sweden)

    Ines Lassowskat

    2013-12-01

    Full Text Available Phosphorylation is an important post-translational protein modification with regulatory roles in diverse cellular signaling pathways. Despite recent advances in mass spectrometry, the detection of phosphoproteins involved in signaling is still challenging, as protein phosphorylation is typically transient and/or occurs at low levels. In green plant tissues, the presence of highly abundant proteins, such as the subunits of the RuBisCO complex, further complicates phosphoprotein analysis. Here, we describe a simple, but powerful, method, which we named prefractionation-assisted phosphoprotein enrichment (PAPE, to increase the yield of phosphoproteins from Arabidopsis thaliana leaf material. The first step, a prefractionation via ammonium sulfate precipitation, not only depleted RuBisCO almost completely, but, serendipitously, also served as an efficient phosphoprotein enrichment step. When coupled with a subsequent metal oxide affinity chromatography (MOAC step, the phosphoprotein content was highly enriched. The reproducibility and efficiency of phosphoprotein enrichment was verified by phospho-specific staining and, further, by mass spectrometry, where it could be shown that the final PAPE fraction contained a significant number of known and additionally novel (potential phosphoproteins. Hence, this facile two-step procedure is a good prerequisite to probe the phosphoproteome and gain deeper insight into plant phosphorylation-based signaling events.

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

    Directory of Open Access Journals (Sweden)

    Kirti Sharma

    2014-09-01

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

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

    Science.gov (United States)

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

    2013-03-01

    The cell wall integrity (CWI) pathway of the model organism Saccharomyces cerevisiae has been thoroughly studied as a paradigm of the mitogen-activated protein kinase (MAPK) pathway. It consists of a classic MAPK module comprising the Bck1 MAPK kinase kinase, two redundant MAPK kinases (Mkk1 and Mkk2), and the Slt2 MAPK. This module is activated under a variety of stimuli related to cell wall homeostasis by Pkc1, the only member of the protein kinase C family in budding yeast. Quantitative phosphoproteomics based on stable isotope labeling of amino acids in cell culture is a powerful tool for globally studying protein phosphorylation. Here we report an analysis of the yeast phosphoproteome upon overexpression of a PKC1 hyperactive allele that specifically activates CWI MAPK signaling in the absence of external stimuli. We found 82 phosphopeptides originating from 43 proteins that showed enhanced phosphorylation in these conditions. The MAPK S/T-P target motif was significantly overrepresented in these phosphopeptides. Hyperphosphorylated proteins provide putative novel targets of the Pkc1-cell wall integrity pathway involved in diverse functions such as the control of gene expression, protein synthesis, cytoskeleton maintenance, DNA repair, and metabolism. Remarkably, five components of the plasma-membrane-associated protein complex known as eisosomes were found among the up-regulated proteins. We show here that Pkc1-induced phosphorylation of the eisosome core components Pil1 and Lsp1 was not exerted directly by Pkc1, but involved signaling through the Slt2 MAPK module. PMID:23221999

  10. Phosphoproteomic analysis provides novel insights into stress responses in Phaeodactylum tricornutum, a model diatom.

    Science.gov (United States)

    Chen, Zhuo; Yang, Ming-kun; Li, Chong-yang; Wang, Yan; Zhang, Jia; Wang, Dian-bing; Zhang, Xian-en; Ge, Feng

    2014-05-01

    Protein phosphorylation on serine, threonine, and tyrosine (Ser/Thr/Tyr) is well established as a key regulatory posttranslational modification used in signal transduction to control cell growth, proliferation, and stress responses. However, little is known about its extent and function in diatoms. Phaeodactylum tricornutum is a unicellular marine diatom that has been used as a model organism for research on diatom molecular biology. Although more than 1000 protein kinases and phosphatases with specificity for Ser/Thr/Tyr residues have been predicted in P. tricornutum, no phosphorylation event has so far been revealed by classical biochemical approaches. Here, we performed a global phosphoproteomic analysis combining protein/peptide fractionation, TiO(2) enrichment, and LC-MS/MS analyses. In total, we identified 264 unique phosphopeptides, including 434 in vivo phosphorylated sites on 245 phosphoproteins. The phosphorylated proteins were implicated in the regulation of diverse biological processes, including signaling, metabolic pathways, and stress responses. Six identified phosphoproteins were further validated by Western blotting using phospho-specific antibodies. The functions of these proteins are discussed in the context of signal transduction networks in P. tricornutum. Our results advance the current understanding of diatom biology and will be useful for elucidating the phosphor-relay signaling networks in this model diatom. PMID:24712722

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

    DEFF Research Database (Denmark)

    Manteca, Angel; Ye, Juanying; Sánchez, Jesús;

    2011-01-01

    bacteria encoding the largest number of eukaryotic type kinases, the biological role of protein phosphorylation in this bacterium has not been extensively studied before. In this issue, the variations of the phosphoproteome of S. coelicolor were characterized. Most distinct Ser/Thr/Tyr phosphorylation...

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

    DEFF Research Database (Denmark)

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

    2013-01-01

    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...

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    OpenAIRE

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

    2014-01-01

    Alzheimer's disease (AD) is the most common form of dementia, characterized by progressive loss of cognitive function. One of the pathological hallmarks of AD is the formation of neurofibrillary tangles composed of abnormally hyperphosphorylated tau protein, but global deregulation of protein phosphorylation in AD is not well analyzed. Here we report a pilot investigation of AD phosphoproteome by titanium dioxide enrichment coupled with high resolution liquid chromatography-tandem mass spectr...

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

    OpenAIRE

    Miaomiao Tian; Han Cheng; Zhiqiang Wang; Na Su; Zexian Liu; Changqing Sun; Bei Zhen; Xuechuan Hong; Yu Xue; Ping Xu

    2015-01-01

    Invasion and metastasis of hepatocellular carcinoma (HCC) is a major cause for lethal liver cancer. Signaling pathways associated with cancer progression are frequently reconfigured by aberrant phosphorylation of key proteins. To capture the key phosphorylation events in HCC metastasis, we established a methodology by an off-line high-pH HPLC separation strategy combined with multi-step IMAC and LC–MS/MS to study the phosphoproteome of a metastatic HCC cell line, MHCC97-H (high metastasis). I...

  17. Time-resolved quantitative phosphoproteomics

    DEFF Research Database (Denmark)

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

    2012-01-01

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

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

    Science.gov (United States)

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

    Directory of Open Access Journals (Sweden)

    Yunfeng Li

    2015-06-01

    Full Text Available Rice blast, caused by the fungal pathogen Magnaporthe oryzae, is the most destructive disease of rice and causes tremendous losses of rice yield worldwide. To explore the molecular mechanisms involved in the rice–M. oryzae interaction, we conducted a time-course phosphoproteomic analysis of leaf samples from resistant and susceptible rice cultivars infected with M. oryzae. This data article contains additional results and analysis of M. oryzae-regulated phosphoproteins in rice leaves [1]. We report the analysis of M. oryzae-regulated phosphoproteins at all time points, including Venn diagram analysis, close-up views, relative intensities, and functional category, and the MS spectra of representative phosphoprotein and representative phosphorylated peptides.

  20. Comparative phosphoproteomic analysis of the mouse testis reveals changes in phosphopeptide abundance in response to Ppp1cc deletion

    Directory of Open Access Journals (Sweden)

    Graham MacLeod

    2014-03-01

    Full Text Available Male Ppp1cc knockout mice are infertile due to failure of spermatogenesis, thought to arise from a deficiency of the predominant testis isoform PPP1CC2. We hypothesize that substrates of the PPP1CC2 will be hyperphosphorylated in Ppp1cc mutant testes. Quantitative RT-PCR and histological data suggest a role for PPP1CC2 by 3 weeks of age in the testis. Comparative phosphoproteomic analysis identified 828 proteins phosphorylated in the 3 week mouse testis, and confidently assigned 1026 unique phosphorylation sites. Thirty-two peptides corresponding to 30 proteins were found to be more abundant in Ppp1cc mutant samples than in wild-type, representing candidate substrates of PPP1CC2.

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

    Science.gov (United States)

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

    2016-02-01

    Rice (Oryza sativa L.) seed serves as a major food source for over half of the global population. Though it has been long recognized that phosphorylation plays an essential role in rice seed development, the phosphorylation events and dynamics in this process remain largely unknown so far. Here, we report the first large scale identification of rice seed phosphoproteins and phosphosites by using a quantitative phosphoproteomic approach. Thorough proteomic studies in pistils and seeds at 3, 7 days after pollination resulted in the successful identification of 3885, 4313 and 4135 phosphopeptides respectively. A total of 2487 proteins were differentially phosphorylated among the three stages, including Kip related protein 1, Rice basic leucine zipper factor 1, Rice prolamin box binding factor and numerous other master regulators of rice seed development. Moreover, differentially phosphorylated proteins may be extensively involved in the biosynthesis and signaling pathways of phytohormones such as auxin, gibberellin, abscisic acid and brassinosteroid. Our results strongly indicated that protein phosphorylation is a key mechanism regulating cell proliferation and enlargement, phytohormone biosynthesis and signaling, grain filling and grain quality during rice seed development. Overall, the current study enhanced our understanding of the rice phosphoproteome and shed novel insight into the regulatory mechanism of rice seed development. PMID:26613898

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

    Science.gov (United States)

    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

  3. Quantitative Measurement of Phosphoproteome Response to Osmotic Stress in Arabidopsis Based on Library-Assisted eXtracted Ion Chromatogram (LAXIC)*

    OpenAIRE

    XUE Liang; Wang, Pengcheng; Wang, Lianshui; Renzi, Emily; Radivojac, Predrag; Tang, Haixu; Arnold, Randy; Zhu, Jian-Kang; Tao, W. Andy

    2013-01-01

    Global phosphorylation changes in plants in response to environmental stress have been relatively poorly characterized to date. Here we introduce a novel mass spectrometry-based label-free quantitation method that facilitates systematic profiling plant phosphoproteome changes with high efficiency and accuracy. This method employs synthetic peptide libraries tailored specifically as internal standards for complex phosphopeptide samples and accordingly, a local normalization algorithm, LAXIC, w...

  4. Integrating phosphoproteomics in systems biology

    Directory of Open Access Journals (Sweden)

    Yu Liu

    2014-07-01

    Full Text Available Phosphorylation of serine, threonine and tyrosine plays significant roles in cellular signal transduction and in modifying multiple protein functions. Phosphoproteins are coordinated and regulated by a network of kinases, phosphatases and phospho-binding proteins, which modify the phosphorylation states, recognize unique phosphopeptides, or target proteins for degradation. Detailed and complete information on the structure and dynamics of these networks is required to better understand fundamental mechanisms of cellular processes and diseases. High-throughput technologies have been developed to investigate phosphoproteomes in model organisms and human diseases. Among them, mass spectrometry (MS-based technologies are the major platforms and have been widely applied, which has led to explosive growth of phosphoproteomic data in recent years. New bioinformatics tools are needed to analyze and make sense of these data. Moreover, most research has focused on individual phosphoproteins and kinases. To gain a more complete knowledge of cellular processes, systems biology approaches, including pathways and networks modeling, have to be applied to integrate all components of the phosphorylation machinery, including kinases, phosphatases, their substrates, and phospho-binding proteins. This review presents the latest developments of bioinformatics methods and attempts to apply systems biology to analyze phosphoproteomics data generated by MS-based technologies. Challenges and future directions in this field will be also discussed.

  5. Phosphoproteomics and Lung Cancer Research

    Directory of Open Access Journals (Sweden)

    William C. S. Cho

    2012-09-01

    Full Text Available 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.

  6. Elucidating the CXCL12/CXCR4 signaling network in chronic lymphocytic leukemia through phosphoproteomics analysis.

    Directory of Open Access Journals (Sweden)

    Morgan O'Hayre

    Full Text Available BACKGROUND: Chronic Lymphocytic Leukemia (CLL pathogenesis has been linked to the prolonged survival and/or apoptotic resistance of leukemic B cells in vivo, and is thought to be due to enhanced survival signaling responses to environmental factors that protect CLL cells from spontaneous and chemotherapy-induced death. Although normally associated with cell migration, the chemokine, CXCL12, is one of the factors known to support the survival of CLL cells. Thus, the signaling pathways activated by CXCL12 and its receptor, CXCR4, were investigated as components of these pathways and may represent targets that if inhibited, could render resistant CLL cells more susceptible to chemotherapy. METHODOLOGY/PRINCIPAL FINDINGS: To determine the downstream signaling targets that contribute to the survival effects of CXCL12 in CLL, we took a phosphoproteomics approach to identify and compare phosphopeptides in unstimulated and CXCL12-stimulated primary CLL cells. While some of the survival pathways activated by CXCL12 in CLL are known, including Akt and ERK1/2, this approach enabled the identification of additional signaling targets and novel phosphoproteins that could have implications in CLL disease and therapy. In addition to the phosphoproteomics results, we provide evidence from western blot validation that the tumor suppressor, programmed cell death factor 4 (PDCD4, is a previously unidentified phosphorylation target of CXCL12 signaling in all CLL cells probed. Additionally, heat shock protein 27 (HSP27, which mediates anti-apoptotic signaling and has previously been linked to chemotherapeutic resistance, was detected in a subset (approximately 25% of CLL patients cells examined. CONCLUSIONS/SIGNIFICANCE: Since PDCD4 and HSP27 have previously been associated with cancer and regulation of cell growth and apoptosis, these proteins may have novel implications in CLL cell survival and represent potential therapeutic targets. PDCD4 also represents a

  7. Analytical strategies for phosphoproteomics

    DEFF Research Database (Denmark)

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

    2009-01-01

    highly sensitive and specific strategies. Today, most phosphoproteomic studies are conducted by mass spectrometric strategies in combination with phospho-specific enrichment methods. This review presents an overview of different analytical strategies for the characterization of phosphoproteins. Emphasis...

  8. Phosphoproteomics in translational research: a sarcoma perspective.

    Science.gov (United States)

    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

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

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    Frithjof eGlowinski

    2014-07-01

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

  10. Analysis of the Rana catesbeiana tadpole tail fin proteome and phosphoproteome during T3-induced apoptosis: identification of a novel type I keratin

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    Helbing Caren C

    2007-08-01

    Full Text Available Abstract Background Thyroid hormones (THs are vital in the maintenance of homeostasis and in the control of development. One postembryonic developmental process that is principally regulated by THs is amphibian metamorphosis. This process has been intensively studied at the genomic level yet very little information at the proteomic level exists. In addition, there is increasing evidence that changes in the phosphoproteome influence TH action. Results Here we identify components of the proteome and phosphoproteome in the tail fin that changed within 48 h of exposure of premetamorphic Rana catesbeiana tadpoles to 10 nM 3,5,3'-triiodothyronine (T3. To this end, we developed a cell and protein fractionation method combined with two-dimensional gel electrophoresis and phosphoprotein-specific staining. Altered proteins were identified using mass spectrometry (MS. We identified and cloned a novel Rana larval type I keratin, RLK I, which may be a target for caspase-mediated proteolysis upon exposure to T3. In addition, the RLK I transcript is reduced during T3-induced and natural metamorphosis which is consistent with a larval keratin. Furthermore, GILT, a protein involved in the immune system, is changed in phosphorylation state which is linked to its activation. Using a complementary MS technique for the analysis of differentially-expressed proteins, isobaric tags for relative and absolute quantitation (iTRAQ revealed 15 additional proteins whose levels were altered upon T3 treatment. The success of identifying proteins whose levels changed upon T3 treatment with iTRAQ was enhanced through de novo sequencing of MS data and homology database searching. These proteins are involved in apoptosis, extracellular matrix structure, immune system, metabolism, mechanical function, and oxygen transport. Conclusion We have demonstrated the ability to derive proteomics-based information from a model species for postembryonic development for which no genome

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

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

    2016-04-01

    Elucidating the androgen-independent growth mechanism is critical for developing effective treatment strategies to combat androgen-independent prostate cancer. We performed a comparative phosphoproteome analysis using a prostate cancer cell line, LNCaP, and an LNCaP-derived androgen-independent cell line, LNCaP-AI, to identify phosphoproteins involved in this mechanism. We performed quantitative comparisons of the phosphopeptide levels in tryptic digests of protein extracts from these cell lines using MS. We found that the levels of 69 phosphopeptides in 66 proteins significantly differed between LNCaP and LNCaP-AI. In particular, we focused on thyroid hormone receptor associated protein 3 (THRAP3), which is a known transcriptional coactivator of the androgen receptor. The phosphorylation level of THRAP3 was significantly lower at S248 and S253 in LNCaP-AI cells. Furthermore, pull-down assays showed that 32 proteins uniquely bound to the nonphosphorylatable mutant form of THRAP3, whereas 31 other proteins uniquely bound to the phosphorylation-mimic form. Many of the differentially interacting proteins were identified as being involved with RNA splicing and processing. These results suggest that the phosphorylation state of THRAP3 at S248 and S253 might be involved in the mechanism of androgen-independent prostate cancer cell growth by changing the interaction partners. PMID:26841317

  14. Quantitative phosphoproteomics to characterize signaling networks

    DEFF Research Database (Denmark)

    Rigbolt, Kristoffer T G; Blagoev, Blagoy

    2012-01-01

    Reversible protein phosphorylation is involved in the regulation of most, if not all, major cellular processes via dynamic signal transduction pathways. During the last decade quantitative phosphoproteomics have evolved from a highly specialized area to a powerful and versatile platform for...... analyzing protein phosphorylation at a system-wide scale and has become the intuitive strategy for comprehensive characterization of signaling networks. Contemporary phosphoproteomics use highly optimized procedures for sample preparation, mass spectrometry and data analysis algorithms to identify and...... quantify thousands of phosphorylations, thus providing extensive overviews of the cellular signaling networks. As a result of these developments quantitative phosphoproteomics have been applied to study processes as diverse as immunology, stem cell biology and DNA damage. Here we review the developments in...

  15. Quantitative phosphoproteome analysis unveils LAT as a modulator of CD3ζ and ZAP-70 tyrosine phosphorylation.

    Directory of Open Access Journals (Sweden)

    Mogjiborahman Salek

    Full Text Available Signaling through the T cell receptor (TCR initiates adaptive immunity and its perturbation may results in autoimmunity. The plasma membrane scaffolding protein LAT acts as a central organizer of the TCR signaling machinery to activate many functional pathways. LAT-deficient mice develop an autoimmune syndrome but the mechanism of this pathology is unknown. In this work we have compared global dynamics of TCR signaling by MS-based quantitative phosphoproteomics in LAT-sufficient and LAT-defective Jurkat T cells. Surprisingly, we found that many TCR-induced phosphorylation events persist in the absence of LAT, despite ERK and PLCγ1 phosphorylation being repressed. Most importantly, the absence of LAT resulted in augmented and persistent tyrosine phosphorylation of CD3ζ and ZAP70. This indicates that LAT signaling hub is also implicated in negative feedback signals to modulate upstream phosphorylation events. Phosphorylation kinetics data resulting from this investigation is documented in a database (phosphoTCR accessible online. The MS data have been deposited to the ProteomeXchange with identifier PXD000341.

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

    DEFF Research Database (Denmark)

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

    2015-01-01

    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...

  17. Phosphoproteomics technologies and applications in plant biology research

    Directory of Open Access Journals (Sweden)

    Jinna eLi

    2015-06-01

    Full Text Available 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.

  18. Signaling network dynamics investigated by quantitative phosphoproteomics

    NARCIS (Netherlands)

    Giansanti, Piero

    2014-01-01

    This thesis describes the application of proteomics technologies to get insight into several aspects of phosphorylation signaling dynamics. The core tool in all performed experiments is mass spectrometry (MS)-based phosphoproteomics. In Chapter 1, a general introduction is given into proteomics and

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

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    Denis Avey

    2015-07-01

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

  20. Identifying differentially regulated subnetworks from phosphoproteomic data

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    Tebbe Andreas

    2010-06-01

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

  1. Evaluation and Properties of the Budding Yeast Phosphoproteome

    OpenAIRE

    Amoutzias, G. D.; He, Y.; Lilley, K. S.; Van de Peer, Y.; Oliver, S G

    2012-01-01

    We have assembled a reliable phosphoproteomic data set for budding yeast Saccharomyces cerevisiae and have investigated its properties. Twelve publicly available phosphoproteome data sets were triaged to obtain a subset of high-confidence phosphorylation sites (p-sites), free of "noisy" phosphorylations. Analysis of this combined data set suggests that the inventory of phosphoproteins in yeast is close to completion, but that these proteins may have many undiscovered p-sites. Proteins involve...

  2. Quantitative and Functional Phosphoproteomic Analysis Reveals that Ethylene Regulates Water Transport via the C-Terminal Phosphorylation of Aquaporin PIP2;1 in Arabidopsis.

    Science.gov (United States)

    Qing, Dongjin; Yang, Zhu; Li, Mingzhe; Wong, Wai Shing; Guo, Guangyu; Liu, Shichang; Guo, Hongwei; Li, Ning

    2016-01-01

    Ethylene participates in the regulation of numerous cellular events and biological processes, including water loss, during leaf and flower petal wilting. The diverse ethylene responses may be regulated via dynamic interplays between protein phosphorylation/dephosphorylation and ubiquitin/26S proteasome-mediated protein degradation and protease cleavage. To address how ethylene alters protein phosphorylation through multi-furcated signaling pathways, we performed a (15)N stable isotope labelling-based, differential, and quantitative phosphoproteomics study on air- and ethylene-treated ethylene-insensitive Arabidopsis double loss-of-function mutant ein3-1/eil1-1. Among 535 non-redundant phosphopeptides identified, two and four phosphopeptides were up- and downregulated by ethylene, respectively. Ethylene-regulated phosphorylation of aquaporin PIP2;1 is positively correlated with the water flux rate and water loss in leaf. Genetic studies in combination with quantitative proteomics, immunoblot analysis, protoplast swelling/shrinking experiments, and leaf water loss assays on the transgenic plants expressing both the wild-type and S280A/S283A-mutated PIP2;1 in the both Col-0 and ein3eil1 genetic backgrounds suggest that ethylene increases water transport rate in Arabidopsis cells by enhancing S280/S283 phosphorylation at the C terminus of PIP2;1. Unknown kinase and/or phosphatase activities may participate in the initial up-regulation independent of the cellular functions of EIN3/EIL1. This finding contributes to our understanding of ethylene-regulated leaf wilting that is commonly observed during post-harvest storage of plant organs. PMID:26476206

  3. Phosphoproteome Analysis of Functional Mitochondria Isolated from Resting Human Muscle Reveals Extensive Phosphorylation of Inner Membrane Protein Complexes and Enzymes*

    OpenAIRE

    Zhao, Xiaolu; León, Ileana R.; Bak, Steffen; Mogensen, Martin; Wrzesinski, Krzysztof; Højlund, Kurt; Jensen, Ole Nørregaard

    2010-01-01

    Mitochondria play a central role in energy metabolism and cellular survival, and consequently mitochondrial dysfunction is associated with a number of human pathologies. Reversible protein phosphorylation emerges as a central mechanism in the regulation of several mitochondrial processes. In skeletal muscle, mitochondrial dysfunction is linked to insulin resistance in humans with obesity and type 2 diabetes. We performed a phosphoproteomics study of functional mitochondria isolated from human...

  4. Feasibility of large-scale phosphoproteomics with higher energy collisional dissociation fragmentation

    DEFF Research Database (Denmark)

    Nagaraj, Nagarjuna; D'Souza, Rochelle C J; Cox, Juergen;

    2010-01-01

    Mass spectrometry (MS)-based proteomics now enables the analysis of thousands of phosphorylation sites in single projects. Among a wide range of analytical approaches, the combination of high resolution MS scans in an Orbitrap analyzer with low resolution MS/MS scans in a linear ion trap has prov...... large-scale phosphoproteome analysis alongside collisional induced dissociation, (CID) and electron capture/transfer dissociation (ECD/ETD)....

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-10-01

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

  6. Improving the Phosphoproteome Coverage for Limited Sample Amounts Using TiO2-SIMAC-HILIC (TiSH) Phosphopeptide Enrichment and Fractionation.

    Science.gov (United States)

    Engholm-Keller, Kasper; Larsen, Martin R

    2016-01-01

    Obtaining high phosphoproteome coverage requires specific enrichment of phosphorylated peptides from the often extremely complex peptide mixtures generated by proteolytic digestion of biological samples, as well as extensive chromatographic fractionation prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Due to the sample loss resulting from fractionation, this procedure is mainly performed when large quantities of sample are available. To make large-scale phosphoproteomics applicable to smaller amounts of protein we have recently combined highly specific TiO2-based phosphopeptide enrichment with sequential elution from immobilized metal affinity chromatography (SIMAC) for fractionation of mono- and multi-phosphorylated peptides prior to capillary scale hydrophilic interaction liquid chromatography (HILIC) based fractionation of monophosphorylated peptides. In the following protocol we describe the procedure step by step to allow for comprehensive coverage of the phosphoproteome utilizing only a few hundred micrograms of protein. PMID:26584925

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

    Directory of Open Access Journals (Sweden)

    Jason A Wojcechowskyj

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

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

    OpenAIRE

    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...

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

    DEFF Research Database (Denmark)

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

    2010-01-01

    , we performed a global quantitative phosphoproteomics analysis of the AT(1)R signaling network. We analyzed ligand-stimulated SILAC (stable isotope labeling by amino acids in cell culture) cells by high resolution (LTQ-Orbitrap) MS and compared the phosphoproteomes of the AT(1)R agonist angiotensin II...

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

    DEFF Research Database (Denmark)

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

    2010-01-01

    performed a global quantitative phosphoproteomics analysis of the AT1R signaling network. We analyzed ligand-stimulated SILAC cells by high-resolution mass spectrometry (LTQ Orbitrap MS) and compared the phosphoproteomes of the AT1R agonist Angiotensin II and the biased agonist SII Angiotensin II, which...

  11. Phosphoproteomic analysis of the human pathogen Trypanosoma cruzi at the epimastigote stage

    OpenAIRE

    Nakayasu, Ernesto S.; Gaynor, Matthew R.; Sobreira, Tiago J.P.; ROSS, JEREMY A.; Almeida, Igor C

    2009-01-01

    Trypanosoma cruzi is the etiologic agent of Chagas disease, which affects millions of people in Latin America and has become a public health concern in the United States and areas of Europe. The possibility that kinase inhibitors represent novel anti-parasitic agents is currently being explored. However, fundamental understanding of the cell signaling networks requires the detailed analysis of the involved phosphorylated proteins. Here, we have performed a comprehensive mass spectrometry (MS)...

  12. Phosphoproteomic analysis of cells treated with longevity-related autophagy inducers

    DEFF Research Database (Denmark)

    Bennetzen, Martin V; Mariño, Guillermo; Pultz, Dennis;

    2012-01-01

    Macroautophagy is a self-cannibalistic process that enables cells to adapt to various stresses and maintain energy homeostasis. Additionally, autophagy is an important route for turnover of misfolded proteins and damaged organelles, with important implications in cancer, neurodegenerative diseases...... crosstalk between distinct networks of post-translational modifications and provide a resource for future analysis of autophagy and cell death....... to resveratrol and spermidine and that key proteins modulating the acetylation, phosphorylation, methylation and ubiquitination status were affected by changes in phosphorylation during the autophagic response. Essential parts of the apoptotic signaling network were subjected to post-translational...

  13. Phosphoproteome analysis during larval development and metamorphosis in the spionid polychaete Pseudopolydora vexillosa

    Directory of Open Access Journals (Sweden)

    Qian Pei-Yuan

    2011-05-01

    Full Text Available Abstract Background The metamorphosis of the spionid polychaete Pseudopolydora vexillosa includes spontaneous settlement onto soft-bottom habitats and morphogenesis that can be completed in a very short time. A previous study on the total changes to the proteome during the various developmental stages of P. vexillosa suggested that little or no de novo protein synthesis occurs during metamorphosis. In this study, we used multicolor fluorescence detection of proteins in 2-D gels for differential analysis of proteins and phosphoproteins to reveal the dynamics of post-translational modification proteins in this species. A combination of affinity chromatography, 2D-PAGE, and mass spectrometry was used to identify the phosphoproteins in pre-competent larvae, competent larvae, and newly metamorphosed juveniles. Results We reproducibly detected 210, 492, and 172 phosphoproteins in pre-competent larvae, competent larvae, and newly metamorphosed juveniles, respectively. The highest percentage of phosphorylation was observed during the competent larval stage. About 64 stage-specific phosphoprotein spots were detected in the competent stage, and 32 phosphoproteins were found to be significantly differentially expressed in the three stages. We identified 38 phosphoproteins, 10 of which were differentially expressed during metamorphosis. These phosphoproteins belonged to six categories of biological processes: (1 development, (2 cell differentiation and integrity, (3 transcription and translation, (4 metabolism, (5 protein-protein interaction and proteolysis, and (6 receptors and enzymes. Conclusion This is the first study to report changes in phosphoprotein expression patterns during the metamorphosis of the marine polychaete P. vexillosa. The higher degree of phosphorylation during the process of attaining competence to settle and metamorphose may be due to fast morphological transitions regulated by various mechanisms. Our data are consistent with

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

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

    2016-01-01

    The traditional sample preparation workflow for mass spectrometry (MS)-based phosphoproteomics is time consuming and usually requires multiple steps, e.g., lysis, protein precipitation, reduction, alkylation, digestion, fractionation, and phosphopeptide enrichment. Each step can introduce chemical artifacts, in vitro protein and peptide modifications, and contaminations. Those often result in sample loss and affect the sensitivity, dynamic range and accuracy of the mass spectrometric analysis. Here we describe a simple and reproducible phosphoproteomics protocol, where lysis, denaturation, reduction, and alkylation are performed in a single step, thus reducing sample loss and increasing reproducibility. Moreover, unlike standard cell lysis procedures the cell harvesting is performed at high temperatures (99 °C) and without detergents and subsequent need for protein precipitation. Phosphopeptides are enriched using TiO2 beads and the orbitrap mass spectrometer is operated in a sensitive mode with higher energy collisional dissociation (HCD). PMID:26584931

  16. Phosphoproteome analysis during larval development and metamorphosis in the spionid polychaete Pseudopolydora vexillosa

    KAUST Repository

    Chandramouli, Kondethimmanahalli

    2011-05-25

    Background: The metamorphosis of the spionid polychaete Pseudopolydora vexillosa includes spontaneous settlement onto soft-bottom habitats and morphogenesis that can be completed in a very short time. A previous study on the total changes to the proteome during the various developmental stages of P. vexillosa suggested that little or no de novo protein synthesis occurs during metamorphosis. In this study, we used multicolor fluorescence detection of proteins in 2-D gels for differential analysis of proteins and phosphoproteins to reveal the dynamics of post-translational modification proteins in this species. A combination of affinity chromatography, 2D-PAGE, and mass spectrometry was used to identify the phosphoproteins in pre-competent larvae, competent larvae, and newly metamorphosed juveniles. Results: We reproducibly detected 210, 492, and 172 phosphoproteins in pre-competent larvae, competent larvae, and newly metamorphosed juveniles, respectively. The highest percentage of phosphorylation was observed during the competent larval stage. About 64 stage-specific phosphoprotein spots were detected in the competent stage, and 32 phosphoproteins were found to be significantly differentially expressed in the three stages. We identified 38 phosphoproteins, 10 of which were differentially expressed during metamorphosis. These phosphoproteins belonged to six categories of biological processes: (1) development, (2) cell differentiation and integrity, (3) transcription and translation, (4) metabolism, (5) protein-protein interaction and proteolysis, and (6) receptors and enzymes. Conclusion: This is the first study to report changes in phosphoprotein expression patterns during the metamorphosis of the marine polychaete P. vexillosa. The higher degree of phosphorylation during the process of attaining competence to settle and metamorphose may be due to fast morphological transitions regulated by various mechanisms. Our data are consistent with previous studies showing a

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

    DEFF Research Database (Denmark)

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

    2010-01-01

    Recognition of microbial danger signals by toll-like receptors (TLR) causes re-programming of macrophages. To investigate kinase cascades triggered by the TLR4 ligand lipopolysaccharide (LPS) on systems level, we performed a global, quantitative and kinetic analysis of the phosphoproteome of...... identified 1850 phosphoproteins with 6956 phosphorylation sites, two thirds of which were not reported earlier. LPS caused major dynamic changes in the phosphoproteome (24% up-regulation and 9% down-regulation). Functional bioinformatic analyses confirmed canonical players of the TLR pathway and highlighted...... other signalling modules (e.g. mTOR, ATM/ATR kinases) and the cytoskeleton as hotspots of LPS-regulated phosphorylation. Finally, weaving together phosphoproteome and nascent transcriptome data by in silico promoter analysis, we implicated several phosphorylated TFs in primary LPS-controlled gene...

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

    Science.gov (United States)

    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

  19. Tissue phosphoproteomics with PolyMAC identifies potential therapeutic targets in a transgenic mouse model of HER2 positive breast cancer.

    Science.gov (United States)

    Searleman, Adam C; Iliuk, Anton B; Collier, Timothy S; Chodosh, Lewis A; Tao, W Andy; Bose, Ron

    2014-12-01

    Altered protein phosphorylation is a feature of many human cancers that can be targeted therapeutically. Phosphopeptide enrichment is a critical step for maximizing the depth of phosphoproteome coverage by MS, but remains challenging for tissue specimens because of their high complexity. We describe the first analysis of a tissue phosphoproteome using polymer-based metal ion affinity capture (PolyMAC), a nanopolymer that has excellent yield and specificity for phosphopeptide enrichment, on a transgenic mouse model of HER2-driven breast cancer. By combining phosphotyrosine immunoprecipitation with PolyMAC, 411 unique peptides with 139 phosphotyrosine, 45 phosphoserine, and 29 phosphothreonine sites were identified from five LC-MS/MS runs. Combining reverse phase liquid chromatography fractionation at pH 8.0 with PolyMAC identified 1571 unique peptides with 1279 phosphoserine, 213 phosphothreonine, and 21 phosphotyrosine sites from eight LC-MS/MS runs. Linear motif analysis indicated that many of the phosphosites correspond to well-known phosphorylation motifs. Analysis of the tyrosine phosphoproteome with the Drug Gene Interaction database uncovered a network of potential therapeutic targets centered on Src family kinases with inhibitors that are either FDA-approved or in clinical development. These results demonstrate that PolyMAC is well suited for phosphoproteomic analysis of tissue specimens. PMID:24723360

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

    Science.gov (United States)

    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).

  1. Enrichment Strategies in Phosphoproteomics.

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  4. Phosphoproteomic analysis reveals the effects of PilF phosphorylation on type IV pilus and biofilm formation in Thermus thermophilus HB27.

    Science.gov (United States)

    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

    2013-10-01

    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

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

    OpenAIRE

    Drake, JM; Paull, EO; Graham, NA; Lee, JK; Smith, BA; Titz, B; Stoyanova, T; Faltermeier, CM; Uzunangelov, V; Carlin,, R.; Fleming, DT; Wong, CK; Newton, Y; Sudha, S; Vashisht, AA

    2016-01-01

    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 drugga...

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

    Science.gov (United States)

    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

  7. Offline High pH Reversed-Phase Peptide Fractionation for Deep Phosphoproteome Coverage

    DEFF Research Database (Denmark)

    Batth, Tanveer S; Olsen, Jesper V

    2016-01-01

    metabolic stimuli, and are thus highly regulated. Therefore, studying the mechanism of regulation by phosphorylation, and pinpointing the exact site of phosphorylation on proteins is of high importance. This protocol describes in detail a phosphoproteomics workflow for ultra-deep coverage by fractionating...... peptide mixtures based on high pH (basic) reversed-phase chromatography prior to phosphopeptide enrichment and mass spectrometric analysis. Peptides are separated on a C18 reversed-phase column under basic conditions and fractions collected in timed intervals followed by concatenation of the fractions...

  8. Integrative Features of the Yeast Phosphoproteome and Protein–Protein Interaction Map

    OpenAIRE

    Yachie, Nozomu; Saito, Rintaro; Sugiyama, Naoyuki; Tomita, Masaru; Ishihama, Yasushi

    2011-01-01

    Following recent advances in high-throughput mass spectrometry (MS)–based proteomics, the numbers of identified phosphoproteins and their phosphosites have greatly increased in a wide variety of organisms. Although a critical role of phosphorylation is control of protein signaling, our understanding of the phosphoproteome remains limited. Here, we report unexpected, large-scale connections revealed between the phosphoproteome and protein interactome by integrative data-mining of yeast multi-o...

  9. An Acetone-based Peptide Labeling and Mass Spectrometry Phosphoproteomics Workflow Enables Identification of Biomolecular Targets Relevant to a Fibroblast Growth Factor Induced Post-ischemic Cardiac Recovery

    OpenAIRE

    Manning, Janet; El Schultz, Jo; Greis, Kenneth D.; Wijeratne, Aruna

    2013-01-01

    Protein phosphorylation modulates normal cellular functions, and disease initiations/progressions, thus understanding phosphoproteomic changes of biological systems in response to external stimuli is essential for therapeutic interventions. Mass spectrometry (MS) methods are currently popular to study such changes, but have yet to become common in solving biological problems; partly due to expensive differential peptide tagging chemistries like iTRAQ (Isobaric Tags for Relative/Absolute Quant...

  10. Enrichment techniques employed in phosphoproteomics

    Czech Academy of Sciences Publication Activity Database

    Fíla, Jan; Honys, David

    2012-01-01

    Roč. 43, č. 3 (2012), s. 1025-1047. ISSN 0939-4451 R&D Projects: GA ČR(CZ) GAP501/11/1462; GA ČR GA522/09/0858; GA ČR GA525/09/0994; GA MŠk OC08011 Institutional research plan: CEZ:AV0Z50380511 Keywords : Phosphoproteomics * Enrichment * IMAC Subject RIV: ED - Physiology Impact factor: 3.914, year: 2012

  11. Taking Aim at Shotgun Phosphoproteomics

    OpenAIRE

    Hoffert, Jason D.; Knepper, Mark A.

    2007-01-01

    Shotgun phosphoproteomics employs liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) to analyze phosphopeptides from complex protein mixtures, allowing detection and quantification of phosphorylation events on a global scale. Within the past few years, this powerful technique has been used to uncover novel phosphorylation sites as well as explore changes in protein phosphorylation during cellular signaling in various systems. This review presents a general overview of current p...

  12. Phosphoproteomics: new insights into cellular signaling

    OpenAIRE

    Mumby, Marc; Brekken, Deirdre

    2005-01-01

    Developments in the field of phosphoproteomics have been fueled by the need simultaneously to monitor many different phosphoproteins within the signaling networks that coordinate responses to changes in the cellular environment. This article presents a brief review of phosphoproteomics with an emphasis on the biological insights that have been derived so far.

  13. Strategies for quantitation of phosphoproteomic data

    DEFF Research Database (Denmark)

    Palmisano, Giuseppe; Thingholm, Tine Engberg

    2010-01-01

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

  14. The phosphoproteome analysis in PBMCs of SLE patients%系统性红斑狼疮外周血单个核细胞磷酸化蛋白质组学研究

    Institute of Scientific and Technical Information of China (English)

    程娟; 马华林; 戴勇

    2014-01-01

    Objective To investigate the aberrant expression of phosphoproteome analysis of peripheral blood mononuclear cells( PBMCs) in patients with systemic lupus erythematosus( SLE) . Lay the foundation for further re-search of mechanism and treatment in patients with SLE. Methods Phosphopeptides were enriched using TiO2 from PBMCs of patients and healthy subjects, then analyzed by automated LC-MS/MS analysis. Phosphorylation sites were identified and quantitated by MASCOT and MaxQuant. Differential expressed proteins and peptides were screened based on the bioinformatics analysis. Results 1 035 phosphorylation sites were identified from SLE com-prared with normal subjects, 618 corresponding genes were screened out in annotation proteins. Pathway analyses showed 12 signaling pathways were identified. There were the most difference phosphorylation sites in mitogen acti-vated protein kinases( MAPK) signaling pathway. Conclusion Differently phosphorylated proteins and peptides can be detected in patients with SLE, which can be used as a mechanism of reference and supplement combined with metabolic pathway, and might be used as a potential target for treatment and research of SLE.%目的:通过系统性红斑狼疮( SLE)患者外周血单个核细胞( PBMCs)的磷蛋白组学分析,为SLE的进一步机制研究及治疗奠定基础。方法收集15例SLE患者和15例健康受试者的外周血,使用 TiO2富集 PBMCs 的磷酸化肽段,进行质谱分析,然后进行磷酸化肽段和磷酸化位点鉴定,并进行生物信息学分析。结果 SLE患者与正常人存在有差异的1035个磷酸化位点,与标注蛋白对应的基因有618个。共筛选出12条代谢通路,其中丝裂原活化蛋白激酶( MAPKs)通路含差异的磷酸化位点最多。结论 SLE患者PBMCs具有差异的磷酸化蛋白质及肽段,与代谢通路一起可作为SLE发病机制研究参考和补充,并可作为治疗靶点研究。

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

    DEFF Research Database (Denmark)

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

    2013-01-01

    A comprehensive quantitative analysis of changes in protein phosphorylation preceding or accompanying transcriptional activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in 5L rat hepatoma cells was performed using the SILAC approach. Following exposure of the cells to DMSO or 1 nM TCDD for 0...

  16. Systems-wide Analysis of K-Ras, Cdc42, and PAK4 Signaling by Quantitative Phosphoproteomics*

    OpenAIRE

    Gnad, Florian; Young, Amy; Zhou, Wei; Lyle, Karen; Ong, Christy C.; Matthew P. Stokes; Silva, Jeffrey C.; Belvin, Marcia; Friedman, Lori S.; Koeppen, Hartmut; Minden, Audrey; Hoeflich, Klaus P.

    2013-01-01

    Although K-Ras, Cdc42, and PAK4 signaling are commonly deregulated in cancer, only a few studies have sought to comprehensively examine the spectrum of phosphorylation-mediated signaling downstream of each of these key signaling nodes. In this study, we completed a label-free quantitative analysis of oncogenic K-Ras, activated Cdc42, and PAK4-mediated phosphorylation signaling, and report relative quantitation of 2152 phosphorylated peptides on 1062 proteins. We define the overlap in phosphop...

  17. Characterization of the Phosphoproteome in Human Bronchoalveolar Lavage Fluid

    Directory of Open Access Journals (Sweden)

    Francesco Giorgianni

    2012-01-01

    Full Text Available Global-scale examination of protein phosphorylation in human biological fluids by phosphoproteomics approaches is an emerging area of research with potential for significant contributions towards discovery of novel biomarkers. In this pilot work, we analyzed the phosphoproteome in human bronchoalveolar lavage fluid (BAL from nondiseased subjects. The main objectives were to assess the feasibility to probe phosphorylated proteins in human BAL and to obtain the initial catalog of BAL phosphoproteins, including protein identities and exact description of their phosphorylation sites. We used a gel-free bioanalytical workflow that included whole-proteome digestion of depleted BAL proteins, enrichment of phosphopeptides by immobilized metal ion affinity chromatography (IMAC, LC-MS/MS analyses with a linear ion trap mass spectrometer, and searches of a protein sequence database to generate a panel of BAL phosphoproteins and their sites of phosphorylation. Based on sequence-diagnostic MS/MS fragmentation patterns, we identified a collection of 36 phosphopeptides that contained 26 different phosphorylation sites. These phosphopeptides mapped to 21 phosphoproteins including, for example, vimentin, plastin-2, ferritin heavy chain, kininogen-1, and others. The characterized phosphoproteins have diverse characteristics in terms of cellular origin and biological function. To the best of our knowledge, results of this study represent the first description of the human BAL phosphoproteome.

  18. Research Progress on Plant Phosphoproteome%植物磷酸化蛋白质组研究进展

    Institute of Scientific and Technical Information of China (English)

    安春鹏; 聂玉哲; 李玉花

    2011-01-01

    Based on introducing phosphoproteome enrichment technique and mass spectrometry, the research progress of phosphoproteome in recent years was summarized, and the prospect was forecasted.%在介绍磷酸化蛋白富集技术和质谱技术的基础上,综述了近年来磷酸化蛋白质组的研究进展,并对其研究前景进行了展望.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Kehilwe Confidence Nakedi

    2015-04-01

    Full Text Available 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.

  1. Offline High pH Reversed-Phase Peptide Fractionation for Deep Phosphoproteome Coverage.

    Science.gov (United States)

    Batth, Tanveer S; Olsen, Jesper V

    2016-01-01

    Protein phosphorylation, a process in which kinases modify serines, threonines, and tyrosines with phosphoryl groups is of major importance in eukaryotic biology. Protein phosphorylation events are key initiators of signaling responses which determine cellular outcomes after environmental and metabolic stimuli, and are thus highly regulated. Therefore, studying the mechanism of regulation by phosphorylation, and pinpointing the exact site of phosphorylation on proteins is of high importance. This protocol describes in detail a phosphoproteomics workflow for ultra-deep coverage by fractionating peptide mixtures based on high pH (basic) reversed-phase chromatography prior to phosphopeptide enrichment and mass spectrometric analysis. Peptides are separated on a C18 reversed-phase column under basic conditions and fractions collected in timed intervals followed by concatenation of the fractions. Each Fraction is subsequently enriched for phosphopeptides using TiO2 followed by LC/MS analysis. PMID:26584926

  2. Investigation of a phosphoproteome qualitative and quantitative analysis method%一种磷酸化蛋白质组定性定量分析方法的考察

    Institute of Scientific and Technical Information of China (English)

    赵丽艳; 刘金凤; 蔡芸; 钱小红; 马守栋; 程艳芹; 纪松岗; 李明春

    2012-01-01

    目的 对用于磷酸化蛋白质的相对定量分析的一种基于双向差示凝胶电泳(2-D DIGE)技术和Pro-Q Diamond 荧光染色的方法进行考察.方法 采用DIGE技术,从Pro-Q Diamond染色后对Cy2、Cy5荧光强度的影响、Pro-QDiamond染色过程中采用的试剂对CyDye标记的蛋白质的影响、去离子水对DIGE扫描结果的影响、CyDye染料扫描的稳定性影响等方面,考察了该方法的有效性和特异性.结果 在Pro-Q Diamond染色前对DIGE凝胶进行处理的过程中,有多种因素影响荧光强度变化,以致在进行pro-Q Diamond染色后,凝胶点的荧光强度变化无法区分是染色造成的灰度值变化还是CyDye染料自身的变化,从而使通过荧光强度变化来定量检测磷酸化蛋白质变得困难.结论 不宜采用对同一DIGE凝胶进行Pro-Q Diamond再染色检测磷酸化蛋白质的方法进行比较蛋白质组学的定量分析,为能同时展示出胶上的磷酸化蛋白质和总蛋白质,可通过DIGE实验与Pro-Q Diamond分开运行,再通过DIGE扫描图谱与Pro-Q Diamond染色图谱进行匹配的方法进行改进.%Objective To investigate a phosphoproteome relative quantitative analysis method based on 2-D DIGE and fluorescence staining. Methods By the DIGE technique, the effect of Pro-Q Diamond on fluorescence intensity of Cy2 and Cy5, the effect of some reagents during the staining, the effect of deionized water on the scanning, and the scanning stability of CyDye staining were all investigated. Results Before the Pro-Q Diamond staining, the intensity fluorescence was changed during the procedure of DIGE could be affected by many factors. So the reason of the intensity changes could be due to the staining or the CyDye itself, therefore increased the difficulty of the subsequent quantitative analysis of phosphoproteins. Conclusion A relative quantitative analysis method based on 2-D DIGE following fluorescence staining was not suitable for phosphoprotein

  3. Off-Line High-pH Reversed-Phase Fractionation for In-Depth Phosphoproteomics

    DEFF Research Database (Denmark)

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

    2014-01-01

    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 (Hp...... 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...

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

    Energy Technology Data Exchange (ETDEWEB)

    Qu, Yi; Wu, Si; Zhao, Rui; Zink, Erika M.; Orton, Daniel J.; Moore, Ronald J.; Meng, Da; Clauss, Therese RW; Aldrich, Joshua T.; Lipton, Mary S.; Pasa-Tolic, Ljiljana

    2013-06-05

    Enrichment of bacterial phosphopeptides is an essential step prior to bottom-up mass spectrometry-based analysis of the phosphoproteome, which is fundamental to understanding the role of phosphoproteins in cell signaling and regulation of protein activity. We developed an automated IMAC system to enrich strong cation exchange-fractionated phosphopeptides from the soluble proteome of Escherichia coli MG1655 grown on minimal medium. Initial demonstration of the system resulted in identification of 75 phosphopeptides covering 52 phosphoproteins. Consistent with previous studies, many of these phosphoproteins are involved in the carbohydrate portion of central metabolism. The automated system utilizes a large capacity IMAC column that can effectively enrich phosphopeptides from a bacterial sample by increasing peptide loading and reducing the wash time. An additional benefit of the automated IMAC system is reduced labor and associated costs.

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

    DEFF Research Database (Denmark)

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

    2015-01-01

    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...

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

    Science.gov (United States)

    Le Bihan, Thierry; Hindle, Matthew; Martin, Sarah F.; Barrios‐Llerena, Martin E.; Krahmer, Johanna; Kis, Katalin; Millar, Andrew J.

    2015-01-01

    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

  7. Technical phosphoproteomic and bioinformatic tools useful in cancer research

    Directory of Open Access Journals (Sweden)

    López Elena

    2011-10-01

    Full Text Available Abstract 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.

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

    Science.gov (United States)

    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

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

    DEFF Research Database (Denmark)

    Engholm-Keller, Kasper; Birck, Pernille; Størling, Joachim;

    2012-01-01

    Large scale quantitative phosphoproteomics depends upon multidimensional strategies for peptide fractionation, phosphopeptide enrichment, and mass spectrometric analysis. Previously, most robust comprehensive large-scale phosphoproteomics strategies have relied on milligram amounts of protein. We...... have set up a multi-dimensional phosphoproteomics strategy combining a number of well-established enrichment and fraction methods: An initial TiO(2) phosphopeptide pre-enrichment step is followed by post-fractionation using sequential elution from IMAC (SIMAC) to separate multi- and mono......, with an enrichment specificity>94%. When doing network analysis of putative phosphorylation changes it could be noted that the identified protein interaction network centered upon proteins known to be affected by the interferon-¿ pathway, thereby supporting the utility of this global phosphoproteomics...

  10. Clinical and Technical Phosphoproteomic Research

    Directory of Open Access Journals (Sweden)

    Ferreira Antonio

    2011-06-01

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

  11. Protein kinases associated with the yeast phosphoproteome

    Directory of Open Access Journals (Sweden)

    Munn Alan L

    2006-01-01

    Full Text Available Abstract Background Protein phosphorylation is an extremely important mechanism of cellular regulation. A large-scale study of phosphoproteins in a whole-cell lysate of Saccharomyces cerevisiae has previously identified 383 phosphorylation sites in 216 peptide sequences. However, the protein kinases responsible for the phosphorylation of the identified proteins have not previously been assigned. Results We used Predikin in combination with other bioinformatic tools, to predict which of 116 unique protein kinases in yeast phosphorylates each experimentally determined site in the phosphoproteome. The prediction was based on the match between the phosphorylated 7-residue sequence and the predicted substrate specificity of each kinase, with the highest weight applied to the residues or positions that contribute most to the substrate specificity. We estimated the reliability of the predictions by performing a parallel prediction on phosphopeptides for which the kinase has been experimentally determined. Conclusion The results reveal that the functions of the protein kinases and their predicted phosphoprotein substrates are often correlated, for example in endocytosis, cytokinesis, transcription, replication, carbohydrate metabolism and stress response. The predictions link phosphoproteins of unknown function with protein kinases with known functions and vice versa, suggesting functions for the uncharacterized proteins. The study indicates that the phosphoproteins and the associated protein kinases represented in our dataset have housekeeping cellular roles; certain kinases are not represented because they may only be activated during specific cellular responses. Our results demonstrate the utility of our previously reported protein kinase substrate prediction approach (Predikin as a tool for establishing links between kinases and phosphoproteins that can subsequently be tested experimentally.

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

    Directory of Open Access Journals (Sweden)

    María Cristina Romero-Rodriguez

    2015-08-01

    Full Text Available 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 spp. 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, defence, protein folding and oxidation-reduction processes. With the exception of a putative cyclase, the other nineteen proteins had at least one orthologous phosphoprotein in A. thaliana, M. truncatula, N. tabacum and G. max. Out of the twenty 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.

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

    DEFF Research Database (Denmark)

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

    reorganization in β-cells by which this cytokine can modulate cell-matrix interactions during inflammation, a regulation shown in other cell types. Further data analysis is currently ongoing, and the collective results of the experiments will hopefully facilitate additional insights into the effect of IL-1β on......Novel Aspect: Global phosphoproteomic analysis of cytokine signaling in primary β-cells Introduction The insulin-producing β-cells of the pancreatic islets of Langerhans are targeted by aberrant immune system responses in diabetes mellitus involving cytokines, especially interleukin-1β (IL-1 β......), which initiate apoptosis of the β-cells. As only limited amounts of primary β-cells can be isolated from model organisms like mouse and rat, global phosphoproteomic analysis of these signaling events by mass spectrometry has generally been unfeasible. We have therefore developed a strategy for...

  14. 小鼠肝脏磷酸化蛋白质组鉴定及磷酸化修饰激酶的分析%Mouse liver phosphoproteome methodology optimization and kinase analysis

    Institute of Scientific and Technical Information of China (English)

    林丛; 任亮亮; 姜颖; 贺福初

    2015-01-01

    Objective To analyze the construction of mouse liver phosphoproteome and phosphorylated kinases to provide useful information for integrating mouse kinase phosphorylation regulatory networks.Methods A new method was established to identify phosphoproteome from the mouse liver.First of all, liver protein was digested with trypsin before the resulting peptides were subjected to a two-step phosphopeptide enrichment and separation procedure consisting of TiO2 chro-maphy enrichment combined with high pHHPLC separation.Samples were injected onto aNanolC-Ultra-2Dplus system cou-pled to an AB-Sciex 5600 Triple TOF mass spectrometer instrument.Then data analysis was performed to provide information of new identified phosphorylation sites of kinase.Results and Conclusion Using our efficient and high-throughput platform, we reported the identification of 5386 phosophorylation sites and 4553 phosphopeptides from 1533 proteins of the mouse liver.126 new phosphorylation sites were identified from 116 kinases, which provides valuable infor-mation for phosphorylation networks in the mouse liver.%目的:构建小鼠肝脏磷酸化蛋白质组并对磷酸化激酶进行分析,为完善小鼠激酶磷酸化调控网络提供有价值的信息。方法对正常小鼠肝组织总蛋白提取液进行FASP酶切,用TiO2富集磷酸化肽段,为降低样本的复杂度,对富集到的磷酸化肽段进行反相色谱分离后,质谱鉴定样本中的磷酸化蛋白质组,对鉴定到的磷酸化修饰的激酶进行分析,提供新鉴定到磷酸化修饰位点的信息。结果与结论成功构建了高效的鉴定小鼠肝磷酸化蛋白质组的方法,共鉴定到1533个磷酸化蛋白质,从中确认5386个磷酸化位点和4553个磷酸化肽段,其中包含116磷酸化修饰的激酶,并于发生磷酸化修饰的激酶中成功鉴定到126个新的磷酸化修饰位点,为完善小鼠肝磷酸化信号调控网络提供了有价值的信息。

  15. Comparison of Phosphoproteomic Separation Strategies Based on Strong Cation Exchange Chromatography-Isoelectric Focusing Techniques%基于强阳离子交换色谱与等电聚焦的磷酸化蛋白质组学分离策略比较

    Institute of Scientific and Technical Information of China (English)

    隋少卉; 董俊军; 王京兰; 蔡耘; 钱小红

    2012-01-01

    Efficient pre-purification steps for the enrichment of phosphorylated proteins or phos-phopeptides are necessary for better detection of phosphorylation sites in phosphoproteomic analysis. Currently, the most common first-dimensional separation technique used is strong cation exchange (SCX). A potential alternative to SCX-based separation is to use isoelectric focusing (IEF) as a first-dimensional separation technique, which has been demonstrated recently. In this study, we present a direct comparison between SCX and IEF based on IPG strips (IPG-IEF) for the phosphoproteomic separation. The comparison experiments discussed in this study utilized standard phosphoproteins and a real sample of HepG2 cell. Then the comparison of 18O labeling phosphopeptides' stability under immobilized pH gradient gel (IPG)-IEF with under SCX was made. Fractions from both technique (SCX and IPG-IEF) were analyzed using the High Mass Accuracy LTQ-FTICR-MS/MS. The results demonstrate that SCX-LTQ-FT and IPG-IEF-LTQ-FT are useful in the phosphopeptides enrichment analysis on a large scale. And SCX-LTQ-FT is relative superior to IPG-IEF-LTQ-FT, whereas the 18 O labeling phosphopeptides'stability under SCX-LTQ-FT is relatively poor with that under IPG-IEF-LTQ-FT.%比较分析了强阳离子交换(SCX)与等电聚焦(IPG-IEF)技术在磷酸化蛋白质组学中的应用.采用3种标准磷酸化蛋白对SCX与IPG-IEF两种技术对磷酸化肽段富集的有效性进行考察.以HepG2细胞为复杂样本,考察SCX与IPG-IEF在实际样本中的应用情况.对SCX与IPG-IEF技术在18O标记的磷酸化蛋白质组定量研究中的应用情况进行考察.蛋白鉴定采用高准确度、高灵敏度、高分辨率的LTQ-FTICR-MS/MS质谱仪.实验表明:SCX和IPG- IEF在大规模磷酸化肽段分离过程中均有效;在复杂样本中,SCX技术的分离效果优于IPG- IEF; IPG- IEF的重复性好于SCX;在磷酸化蛋白质组定量分析结果表明,IPG-IEF技术的稳定性优

  16. Confident and sensitive phosphoproteomics using combinations of collision induced dissociation and electron transfer dissociation ☆

    OpenAIRE

    Collins, Mark O.; Wright, James C.; Jones, Matthew; Rayner, Julian C; Choudhary, Jyoti S

    2014-01-01

    We present a workflow using an ETD-optimised version of Mascot Percolator and a modified version of SLoMo (turbo-SLoMo) for analysis of phosphoproteomic data. We have benchmarked this against several database searching algorithms and phosphorylation site localisation tools and show that it offers highly sensitive and confident phosphopeptide identification and site assignment with PSM-level statistics, enabling rigorous comparison of data acquisition methods. We analysed the Plasmodium falcip...

  17. Early cytokinin response proteins and phosphoproteins of Arabidopsis thaliana identified by proteome and phosphoproteome profiling

    Czech Academy of Sciences Publication Activity Database

    Černý, M.; Dyčka, Filip; Bobálová, Janette; Brzobohatý, B.

    2009-01-01

    Roč. 276, s1 (2009), s. 231. ISSN 1742-464X. [FEBS Congress /34./. 04.07.2009-09.07.2009, Prague] R&D Projects: GA MŠk 1M06030; GA AV ČR IAA600040701 Institutional research plan: CEZ:AV0Z40310501 Keywords : proteomic analysis * phosphoproteome * cytokinin Subject RIV: CB - Analytical Chemistry, Separation http://www3.interscience.wiley.com/cgi-bin/fulltext/122457210/PDFSTART

  18. Large-scale Arabidopsis phosphoproteome profiling reveals novel chloroplast kinase substrates and phosphorylation networks

    OpenAIRE

    Reiland, S; Messerli, G.; Baerenfaller, K.; Gerrits, B.; Endler, A; Grossmann, J.; Gruissem, W; Baginsky, S

    2009-01-01

    We have characterized the phosphoproteome of Arabidopsis (Arabidopsis thaliana) seedlings using high-accuracy mass spectrometry and report the identification of 1,429 phosphoproteins and 3,029 unique phosphopeptides. Among these, 174 proteins were chloroplast phosphoproteins. Motif-X (motif extractor) analysis of the phosphorylation sites in chloroplast proteins identified four significantly enriched kinase motifs, which include casein kinase II (CKII) and proline-directed kinase motifs, as w...

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

    DEFF Research Database (Denmark)

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

    2008-01-01

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

  20. Characterization of a TiO2 enrichment method for label-free quantitative phosphoproteomics

    OpenAIRE

    Montoya, Alex; Beltran, Luisa; Casado, Pedro; Rodríguez-Prados, Juan-Carlos; Cutillas, Pedro R.

    2011-01-01

    Phosphorylation is a protein post-translational modification with key roles in the regulation of cell biochemistry and signaling. In-depth analysis of phosphorylation using mass spectrometry is permitting the investigation of processes controlled by phosphorylation at the system level. A critical step of these phosphoproteomics methods involves the isolation of phosphorylated peptides from the more abundant unmodified peptides produced by the digestion of cell lysates. Although different tech...

  1. Integrative Network Analysis of Signaling in Human CD34+ Hematopoietic Progenitor Cells by Global Phosphoproteomic Profiling Using TiO2 Enrichment Combined with 2D LC-MS/MS and Pathway Mapping

    OpenAIRE

    Guo, Hongbo; Isserlin, Ruth; Chen, Xiaoji; Wang, Weijia; Phanse, Sadhna; Zandstra, Peter W.; Bader, Gary; Paddison, Patrick; Emili, Andrew

    2013-01-01

    Protein kinase signaling regulates human hematopoietic stem/progenitor cell (HSPC) fate, yet little is known about critical pathway substrates. To address this, we have developed and applied a large-scale, empirically-optimized phosphopeptide affinity enrichment strategy with high-throughput 2D LC-MS/MS screening to evaluate the phosphoproteome of an isolated human CD34+ HSPC population. We first used hydrophilic interaction chromatography (HILIC) as a first dimension separation to separate a...

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

    DEFF Research Database (Denmark)

    Osinalde, Nerea; Mitxelena, Jone; Sánchez-Quiles, Virginia;

    2016-01-01

    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...

  3. Quantitative phosphoproteomics reveals new roles for the protein phosphatase PP6 in mitotic cells.

    Science.gov (United States)

    Rusin, Scott F; Schlosser, Kate A; Adamo, Mark E; Kettenbach, Arminja N

    2015-10-13

    Protein phosphorylation is an important regulatory mechanism controlling mitotic progression. Protein phosphatase 6 (PP6) is an essential enzyme with conserved roles in chromosome segregation and spindle assembly from yeast to humans. We applied a baculovirus-mediated gene silencing approach to deplete HeLa cells of the catalytic subunit of PP6 (PP6c) and analyzed changes in the phosphoproteome and proteome in mitotic cells by quantitative mass spectrometry-based proteomics. We identified 408 phosphopeptides on 272 proteins that increased and 298 phosphopeptides on 220 proteins that decreased in phosphorylation upon PP6c depletion in mitotic cells. Motif analysis of the phosphorylated sites combined with bioinformatics pathway analysis revealed previously unknown PP6c-dependent regulatory pathways. Biochemical assays demonstrated that PP6c opposed casein kinase 2-dependent phosphorylation of the condensin I subunit NCAP-G, and cellular analysis showed that depletion of PP6c resulted in defects in chromosome condensation and segregation in anaphase, consistent with dysregulation of condensin I function in the absence of PP6 activity. PMID:26462736

  4. Characterization of the Phosphoproteome in Human Bronchoalveolar Lavage Fluid

    OpenAIRE

    Francesco Giorgianni; Valentina Mileo; Dominic M. Desiderio; Silvia Catinella; Sarka Beranova-Giorgianni

    2012-01-01

    Global-scale examination of protein phosphorylation in human biological fluids by phosphoproteomics approaches is an emerging area of research with potential for significant contributions towards discovery of novel biomarkers. In this pilot work, we analyzed the phosphoproteome in human bronchoalveolar lavage fluid (BAL) from nondiseased subjects. The main objectives were to assess the feasibility to probe phosphorylated proteins in human BAL and to obtain the initial catalog of BAL phosphopr...

  5. Dissection of the insulin signaling pathway via quantitative phosphoproteomics

    DEFF Research Database (Denmark)

    Krüger, Marcus; Kratchmarova, Irina; Blagoev, Blagoy;

    2008-01-01

    spectrum of the tyrosine phosphorylation cascade, we have defined the tyrosine-phosphoproteome of the insulin signaling pathway, using high resolution mass spectrometry in combination with phosphotyrosine immunoprecipitation and stable isotope labeling by amino acids in cell culture (SILAC) in...... calcium transporting ATPase SERCA2, supporting a connection to calcium signaling. The combination of quantitative phosphoproteomics with cell culture models provides a powerful strategy to dissect the insulin signaling pathways in intact cells....

  6. 王浆高产蜜蜂咽下腺磷酸化蛋白质组分析%Phosphoproteome Analysis of Hypopharyngeal Glands of High Royal Jelly Producing Bee (Apis mellifera L.)

    Institute of Scientific and Technical Information of China (English)

    鲁小山; 韩宾; 张兰; 冯毛; 房宇; 李荣丽; 周天娥; 李建科

    2013-01-01

    [Objective]High royal jelly producing bee (Apis mellifera L.) is the unique honeybee resource in China. However, the mechanism of high royal jelly producing has not been clearly addressed. In order to reveal the significance of protein phosphorylation in hypopharyngeal gland for royal jelly synthesis and secretion, the phosphoproteome of hypopharyngeal gland of nurse bee (6-12 day) was analyzed.[Method]IMAC (immobilized metal-affinity chromatography) phosphoprotein enrichment, SCX (strong cation exchange) separation, LC-MS/MS (liquid chromatography-mass/mass) identification and bioinformatics analysis were applied to analyze phosphoproteome of hypopharyngeal gland of nurse bee.[Result]Of the identified 117 proteins in the hypopharyngeal gland, 6 of them were phosphorylated on 6 phosphopeptides and assigned 8 phosphorylated sites. They were related to protein translation and synthesis, such as 60S acidic ribosomal proteins P0, P1, P2 and 60S ribosomal protein L15, and major royal jelly proteins 1 and 7 precursor.[Conclusion]The phosphorylation modifications occurred on ribosome proteins of hypopharyngeal gland mainly contribute to the high efficiency of synthesizing and secreting of royal jelly protein. The phosphorylation of royal jelly proteins 1 and 7 maintain the reasonable ratio of calcium to phosphorus of royal jelly with the increasing yield, thus meeting the nutrition demand of fertile egg-laying queens and developing larvae. Hence, the data obtained in this study will provide new knowledge to deeply understand the mechanism how high royal jelly producing bees could produce higher amount of royal jelly at the level of protein phosphorylation.%[目的]通过对王浆高产蜜蜂(Apis mellifera L.)(浙江浆蜂)哺育蜂(6-12 d)咽下腺的磷酸化蛋白质组分析,以期探明蛋白质磷酸化修饰对王浆分泌的生物学意义。[方法]将哺育蜂咽下腺蛋白质液内酶切后,用固相金属离子亲和层析色谱法(IMAC

  7. Phosphoproteomics and targeted anti-tumor therapies

    International Nuclear Information System (INIS)

    We have developed procedures to acquire the phosphoproteomic profiles of cell lines before and after stimulation with activators or treatment with kinase inhibitors. The profiles were obtained combining affinity chromatography, electrophoretic separation and MALDI-TOF mass spectrometry for protein identification. Studies were performed using a collection of cell lines from melanoma, hepatoma and other tumor types derived from thyroid, colon, breast and ovary tissues. Stimulations were performed using ligands for receptor tyrosine kinases and inhibitions using drugs specific for molecular targets, including Iressa, RPI-1, Erlotinib, PLX4032 and Sorafenib. This work has identified several new targets now being validated on clinical specimens. This second line of studies prompted to establish adequate methods of processing and storing of tissues to preserve protein phosphorylation status. Remarkably these activities, taken together, have contributed to the development of new interdisciplinary programs in 'Fondazione'

  8. Phosphoproteomic analysis of adhesion receptor signalling

    OpenAIRE

    Robertson, Joseph

    2014-01-01

    The binding of integrin adhesion receptors to their extracellular matrix (ECM) ligands activates intracellular signalling pathways that control diverse and fundamental aspects of cell behaviour. While it is clear that protein kinases and phosphatases play an integral role in such adhesion-mediated signalling, current knowledge of the phosphorylation events regulated downstream of integrin ligation is limited and prohibits a systems-level understanding of the molecular mechanisms through which...

  9. Quantitative phosphoproteomic analysis of postmortem muscle development

    DEFF Research Database (Denmark)

    Huang, Honggang

    Meat quality development is highly dependent on postmortem (PM) metabolism and rigor mortis development in PM muscle. PM glycometabolism and rigor mortis fundamentally determine most of the important qualities of raw meat, such as ultimate pH, tenderness, color and water-holding capacity. Protein...... proteins was significantly related to the synergy effects of pH and time, whereas that of myofibrillar proteins was mainly changed with PM time. In total, 72 unique proteins were identified in PM porcine muscle. In addition, electrical stimulation was also confirmed to affect protein phosphorylation level...... may contribute to the higher phosphorylation level of heat shock proteins (HSPs) in earlier PM, such as the putative Serine 84 on HSP27. These results indicated the involvement of protein phosphorylation in regulating meat quality development. PM muscle proteins underwent significant changes at...

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

    Directory of Open Access Journals (Sweden)

    Feng Ge

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

  11. Rapid and reproducible single-stage phosphopeptide enrichment of complex peptide mixtures: Application to general and phosphotyrosine-specific phosphoproteomics experiments

    OpenAIRE

    Kettenbach, Arminja N.; Gerber, Scott A.

    2011-01-01

    Reversible protein phosphorylation is an essential regulatory component of virtually every cellular process, and is frequently dysregulated in cancer. However, significant analytical barriers persist that hamper the routine application of phosphoproteomics in translational settings. Here, we present a straightforward and reproducible approach for the broadscale analysis of protein phosphorylation that relies on a single phosphopeptide enrichment step using titanium dioxide microspheres from w...

  12. Phosphoproteomic and its application in cellular signaling network analysis%磷酸化蛋白质组学方法在信号网络解析中的应用

    Institute of Scientific and Technical Information of China (English)

    段朝军; 陈主初

    2008-01-01

    信号转导是细胞对各种外界刺激的应答反应,蛋白磷酸化或去磷酸化是信号从胞外流向胞内并导致细胞效应过程中的关键机制.磷酸化蛋白质组学(phosphoproteomics)是采用蛋白质组学的分析方法,研究细胞中所有磷酸化蛋白质及其修饰过程,从整体上观察细胞中被修饰的磷酸化蛋白质的状态及其变化,进而分析特定磷酸化修饰对生命过程的调控作用及其分子机制.

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

    OpenAIRE

    Kauko, O.; Laajala, T. D.; Jumppanen, M.; Hintsanen, P; Suni, V.; Haapaniemi, P.; Corthals, G.; Aittokallio, T.; Westermarck, J; Imanishi, S.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 quantificat...

  14. Development of a Label-Free Quantitative Phosphoproteomics Platform Applicable to Non-Cell Culture Biological Matrices

    OpenAIRE

    Foster, M W; Thompson, J. W.; Moseley, M.A.; Soderblom, E.

    2011-01-01

    The most commonly employed LC-MS based quantitative phosphoproteomics experiments are performed using cultured cell lines with incorporated stable-isotope labeled amino acids. Although these systems simplify experimental manipulation and provide relatively large amounts of soluble protein, they do not account for the complexity of signal regulation from other factors or cell types present in the cell's native physiological environment. Here we describe the general analytical and informatic me...

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

    Science.gov (United States)

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

    2016-06-01

    Maize is unique since it is both monoecious and diclinous (separate male and female flowers on the same plant). We investigated the proteome and phosphoproteome of maize pollen containing modified proteins and here we provide a comprehensive pollen proteome and phosphoproteome which contain 100,990 peptides from 6750 proteins and 5292 phosphorylated sites corresponding to 2257 maize phosphoproteins, respectively. Interestingly, among the total 27 overrepresented phosphosite motifs we identified here, 11 were novel motifs, which suggested different modification mechanisms in plants compared to those of animals. Enrichment analysis of pollen phosphoproteins showed that pathways including DNA synthesis/chromatin structure, regulation of RNA transcription, protein modification, cell organization, signal transduction, cell cycle, vesicle transport, transport of ions and metabolisms, which were involved in pollen development, the following germination and pollen tube growth, were regulated by phosphorylation. In this study, we also found 430 kinases and 105 phosphatases in the maize pollen phosphoproteome, among which calcium dependent protein kinases (CDPKs), leucine rich repeat kinase, SNF1 related protein kinases and MAPK family proteins were heavily enriched and further analyzed. From our research, we also uncovered hundreds of male sterility-associated proteins and phosphoproteins that might influence maize productivity and serve as targets for hybrid maize seed production. At last, a putative complex signaling pathway involving CDPKs, MAPKs, ubiquitin ligases and multiple fertility proteins was constructed. Overall, our data provides new insight for further investigation of protein phosphorylation status in mature maize pollen and construction of maize male sterile mutants in the future. PMID:26969016

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

    Science.gov (United States)

    Wang, Jiaxi; Li, Jie; Wang, Yanan; Gao, Mingxia; Zhang, Xiangmin; Deng, Chunhui

    2016-07-01

    The enrichment and separation of phosphopeptides from mixed biological samples is a technologically very significance, but highly challenging work. Current designed materials are mainly based on the broad and effective adsorptive character of metal oxide affinity chromatography (MOAC). Though significant progress has been made in the enrichment of phosphopeptides with MOAC material, there are chances for further development. In this study, a novel pollen-based MOAC honeycomb material was firstly explored in which the suitable hydrophilic channels preferentially enrich much more endogenous phosphopeptides than nonphosphopeptides or proteins while doping binary metal oxides at the atomic level and the ultra-high specific surface area have further allowed it to possess more effective active sites. Based on these unique features, the pollen-based material exhibited high selectivity for β-casein (mass ratio of β-casein/BSA, 1:1500), ultra-low detection limit (0.1fmol), desirable reusability. Moreover, the bionics MOAC composites were investigated in the enrichment of phosphopeptides from nonfat milk, human serum (male and female at the same age) and mice liver, results of which indicate the great potential of the composite for the phosphoproteome analysis of complex biological samples through the cheap and environmentally friendly process. PMID:27154659

  17. Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway

    DEFF Research Database (Denmark)

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

    2005-01-01

    /MS/MS) for identification. This integrated phosphoproteomic technology identified and quantified phosphorylation in key regulator and effector proteins of a prototypical G-protein-coupled receptor signaling pathway, the yeast pheromone response. SILAC encoding of yeast proteomes was achieved by incorporation...

  18. Phosphoproteomics identifies oncogenic Ras signaling targets and their involvement in lung adenocarcinomas.

    Directory of Open Access Journals (Sweden)

    Putty-Reddy Sudhir

    Full Text Available BACKGROUND: Ras is frequently mutated in a variety of human cancers, including lung cancer, leading to constitutive activation of MAPK signaling. Despite decades of research focused on the Ras oncogene, Ras-targeted phosphorylation events and signaling pathways have not been described on a proteome-wide scale. METHODOLOGY/PRINCIPAL FINDINGS: By functional phosphoproteomics, we studied the molecular mechanics of oncogenic Ras signaling using a pathway-based approach. We identified Ras-regulated phosphorylation events (n = 77 using label-free comparative proteomics analysis of immortalized human bronchial epithelial cells with and without the expression of oncogenic Ras. Many were newly identified as potential targets of the Ras signaling pathway. A majority (∼60% of the Ras-targeted events consisted of a [pSer/Thr]-Pro motif, indicating the involvement of proline-directed kinases. By integrating the phosphorylated signatures into the Pathway Interaction Database, we further inferred Ras-regulated pathways, including MAPK signaling and other novel cascades, in governing diverse functions such as gene expression, apoptosis, cell growth, and RNA processing. Comparisons of Ras-regulated phosphorylation events, pathways, and related kinases in lung cancer-derived cells supported a role of oncogenic Ras signaling in lung adenocarcinoma A549 and H322 cells, but not in large cell carcinoma H1299 cells. CONCLUSIONS/SIGNIFICANCE: This study reveals phosphorylation events, signaling networks, and molecular functions that are regulated by oncogenic Ras. The results observed in this study may aid to extend our knowledge on Ras signaling in lung cancer.

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

    Directory of Open Access Journals (Sweden)

    Ping Chen

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

  20. Phosphoproteomic studies in Arabidopsis and tobacco male gametophytes

    OpenAIRE

    Fíla, J. (Jan); Čapková, V. (Věra); Honys, D. (David)

    2014-01-01

    Mature pollen represents an extremely resistant quiescent structure surrounded by a tough cell wall. After its hydration on stigma papillary cells, pollen tube growth starts rapidly. Massive metabolic changes are likely to be accompanied by changes in protein phosphorylation. Protein phosphorylation belongs among the most rapid post-translational modifications. To date, only Arabidopsis thaliana and tobacco (Nicotiana tabacum) mature pollen have been subjected to phosphoproteomic studies in o...

  1. Towards single-cell LC-MS phosphoproteomics

    OpenAIRE

    Polat, Ayşe Nur; Özlü, Nurhan

    2014-01-01

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

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

    Science.gov (United States)

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

    2010-02-01

    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

  3. Phosphoproteomics and new onco-bio technologies in the study of the pathogenesis and therapy of malignant gliomas

    International Nuclear Information System (INIS)

    Characterization and crio-conservation and of glioblastoma stem cells. Phosphoproteomic analysis of tumor stem cells and their progeny. The possibility to use in research stem cells isolated from glioblastoma (GSCs) is an indispensable tool for the development of new and more effective therapeutic strategies for this type of cancer, whose prognosis is still poor. The research conducted for the attainment of the first objective has led to isolation of 18 stem cells lines from cancer patients with glioblastoma who have undergone surgery at the Institute of Neurosurgery of the Catholic University of Rome. The resulting cell lines were phenotypically characterized for the expression of stem cell markers and differentiation capabilities

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Westa Domanova

    Full Text Available 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

  7. Sample preparation and analytical strategies for large-scale phosphoproteomics experiments.

    Science.gov (United States)

    Kanshin, Evgeny; Michnick, Stephen; Thibault, Pierre

    2012-10-01

    Reversible protein phosphorylation is an important post-translational modification that controls a wide range of protein functions including enzyme activity, subcellular localisation, protein degradation, intra- and inter-molecular protein interactions. Significant advances in both phosphopeptide enrichment methods and sensitive mass spectrometry instrumentation have been achieved over the past decade to facilitate the large-scale identification of protein phosphorylation in humans and different animal and microbial model systems. While mass spectrometry provides the ability to identify thousands of phosphorylation sites in a single experiment, the further understanding of the functional significance of this modification on protein substrates requires detailed information on the changes in phosphorylation stoichiometry and protein abundance across experimental paradigms. This review presents different sample preparation methods and analytical strategies used in mass spectrometry-based phosphoproteomics to profile protein phosphorylation and unravel the regulation of this modification on protein function. PMID:22683502

  8. Characterization of the human cerebrospinal fluid phosphoproteome by titanium dioxide affinity chromatography and mass spectrometry

    DEFF Research Database (Denmark)

    Bahl, Justyna Maria Czarna; Jensen, Søren Skov; Larsen, Martin R;

    2008-01-01

    investigations based on knowledge of the molecular pathology of the disease in question. In Alzheimer's disease, hyperphosphorylation of the tau protein is a characteristic feature and thus a comprehensive characterization of the phosphoproteome of the CSF may be pursued to obtain a complete picture of...... phosphorylation aberrations in health and disease. Toward that goal we here describe a method for a comprehensive isolation and identification of phosphorylated tryptic peptides derived from CSF proteins using a simple sample preparation step and titanium dioxide-affinity chromatography followed by MALDI-TOF or...... LC-MS/MS linear ion-trap-FT mass spectrometry. Whereas not all previously reported phosphoproteins were found in normal CSF, we detected 56 putative novel phosphorylation sites in 38 proteins in addition to known sites. The approach seems to be a promising foundation for the discovery of new...

  9. Online Nanoflow Multidimensional Fractionation for High Efficiency Phosphopeptide Analysis*

    OpenAIRE

    Ficarro, Scott B.; ZHANG Yi; Carrasco-Alfonso, Marlene J.; Garg, Brijesh; Adelmant, Guillaume; Webber, James T.; Luckey, C. John; Marto, Jarrod A.

    2011-01-01

    Despite intense, continued interest in global analyses of signaling cascades through mass spectrometry-based studies, the large-scale, systematic production of phosphoproteomics data has been hampered in-part by inefficient fractionation strategies subsequent to phosphopeptide enrichment. Here we explore two novel multidimensional fractionation strategies for analysis of phosphopeptides. In the first technique we utilize aliphatic ion pairing agents to improve retention of phosphopeptides at ...

  10. 巨噬细胞应激的铜绿假单胞菌磷酸化蛋白质组分析%Phosphoproteomic analysis of Pseudomonas aeruginosa strains in response to stress induced by macrophages

    Institute of Scientific and Technical Information of China (English)

    潘建义; 王长一; 叶智鸧; 陈冉; 赵辅昆

    2014-01-01

    Objective To investigate the role of protein phosphorylation in Pseudomonas aeruginosa (P.aeruginosa) strains in response to stress triggered by mouse macrophages.Methods The strong cation exchange-immobilized metal affinity chromatography (SCX-IMAC) was performed to enrich phosphopeptides.The nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS) was carried out to identify and analyze phosphoproteome.Results Fourteen phosphopeptides from twelve proteins were identified within thirty-one phosphorylation sites on serine,threonine and tyrosine residues.Fifty percent of these phosphorylated proteins were membrane proteins,indicating that their phosphorylation modification was more critical for bacteria in response to the stress.In terms of biological process of Gene Ontology,these identified proteins were involved in stress response,iron transport,anaerobic respiration,response to hydrogen peroxide and signal transduction by phosphorylation,etc.Conclusion These phosphorylated proteins in P.aeruginosa strains are necessary for signal transduction and their response to harsh environment within the macrophages,such as iron limitation,hypoxia and oxidative stress.This study provides evidence for further investigation on virulence and pathogenesis of P.aeruginosa.%目的 鉴定巨噬细胞应激的铜绿假单胞菌(Pseudomonas aeruginosa)磷酸化蛋白质组,分析磷酸化修饰在该菌应答应激中的作用.方法 采用强阳离子交换层析和固相金属离子亲和层析(SCX-IMAC)富集应激细菌的磷酸化肽段,并利用纳升级液相色谱-质谱联用技术(nano LC-MS/MS)进行鉴定和分析其磷酸化蛋白质组.结果 在应激细菌中鉴定到14个磷酸化肽段,对应于12个磷酸化蛋白质,其中膜蛋白占50%,提示膜蛋白的磷酸化修饰在这一过程中具有关键作用.蛋白质功能分析显示这些磷酸化蛋白质主要涉及应激应答、铁摄取转运、厌氧呼吸、过氧化氢的应

  11. Celiac anti-type 2 transglutaminase antibodies induce phosphoproteome modification in intestinal epithelial Caco-2 cells.

    Directory of Open Access Journals (Sweden)

    Gaetana Paolella

    Full Text Available BACKGROUND: Celiac disease is an inflammatory condition of the small intestine that affects genetically predisposed individuals after dietary wheat gliadin ingestion. Type 2-transglutaminase (TG2 activity seems to be responsible for a strong autoimmune response in celiac disease, TG2 being the main autoantigen. Several studies support the concept that celiac anti-TG2 antibodies may contribute to disease pathogenesis. Our recent findings on the ability of anti-TG2 antibodies to induce a rapid intracellular mobilization of calcium ions, as well as extracellular signal-regulated kinase phosphorylation, suggest that they potentially act as signaling molecules. In line with this concept, we have investigated whether anti-TG2 antibodies can induce phosphoproteome modification in an intestinal epithelial cell line. METHODS AND PRINCIPAL FINDINGS: We studied phosphoproteome modification in Caco-2 cells treated with recombinant celiac anti-TG2 antibodies. We performed a two-dimensional electrophoresis followed by specific staining of phosphoproteins and mass spectrometry analysis of differentially phosphorylated proteins. Of 14 identified proteins (excluding two uncharacterized proteins, three were hypophosphorylated and nine were hyperphosphorylated. Bioinformatics analyses confirmed the presence of phosphorylation sites in all the identified proteins and highlighted their involvement in several fundamental biological processes, such as cell cycle progression, cell stress response, cytoskeletal organization and apoptosis. CONCLUSIONS: Identification of differentially phosphorylated proteins downstream of TG2-antibody stimulation suggests that in Caco-2 cells these antibodies perturb cell homeostasis by behaving as signaling molecules. We hypothesize that anti-TG2 autoantibodies may destabilize the integrity of the intestinal mucosa in celiac individuals, thus contributing to celiac disease establishment and progression. Since several proteins here

  12. Phosphoproteomic Profiling of Human Myocardial Tissues Distinguishes Ischemic from Non-Ischemic End Stage Heart Failure

    Science.gov (United States)

    Njoroge, Linda W.; Thompson, J. Will; Soderblom, Erik J.; Feger, Bryan J.; Troupes, Constantine D.; Hershberger, Kathleen A.; Ilkayeva, Olga R.; Nagel, Whitney L.; Landinez, Gina P.; Shah, Kishan M.; Burns, Virginia A.; Santacruz, Lucia; Hirschey, Matthew D.; Foster, Matthew W.; Milano, Carmelo A.; Moseley, M. Arthur; Piacentino, Valentino; Bowles, Dawn E.

    2014-01-01

    The molecular differences between ischemic (IF) and non-ischemic (NIF) heart failure are poorly defined. A better understanding of the molecular differences between these two heart failure etiologies may lead to the development of more effective heart failure therapeutics. In this study extensive proteomic and phosphoproteomic profiles of myocardial tissue from patients diagnosed with IF or NIF were assembled and compared. Proteins extracted from left ventricular sections were proteolyzed and phosphopeptides were enriched using titanium dioxide resin. Gel- and label-free nanoscale capillary liquid chromatography coupled to high resolution accuracy mass tandem mass spectrometry allowed for the quantification of 4,436 peptides (corresponding to 450 proteins) and 823 phosphopeptides (corresponding to 400 proteins) from the unenriched and phospho-enriched fractions, respectively. Protein abundance did not distinguish NIF from IF. In contrast, 37 peptides (corresponding to 26 proteins) exhibited a ≥2-fold alteration in phosphorylation state (p<0.05) when comparing IF and NIF. The degree of protein phosphorylation at these 37 sites was specifically dependent upon the heart failure etiology examined. Proteins exhibiting phosphorylation alterations were grouped into functional categories: transcriptional activation/RNA processing; cytoskeleton structure/function; molecular chaperones; cell adhesion/signaling; apoptosis; and energetic/metabolism. Phosphoproteomic analysis demonstrated profound post-translational differences in proteins that are involved in multiple cellular processes between different heart failure phenotypes. Understanding the roles these phosphorylation alterations play in the development of NIF and IF has the potential to generate etiology-specific heart failure therapeutics, which could be more effective than current therapeutics in addressing the growing concern of heart failure. PMID:25117565

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-11-30

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

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

    Science.gov (United States)

    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

  17. Effective Enrichment and Mass Spectrometry Analysis of Phosphopeptides Using Mesoporous Metal Oxide Nanomaterials

    OpenAIRE

    Nelson, Cory A.; Szczech, Jeannine R.; Dooley, Chad J.; Xu, Qingge; Lawrence, Matthew J.; Zhu, Haoyue; Jin, Song; Ge, Ying

    2010-01-01

    Mass spectrometry (MS)-based phosphoproteomics remains challenging due to the low abundance of phosphoproteins and substoichiometric phosphorylation. This demands better methods to effectively enrich phosphoproteins/peptides prior to MS analysis. We have previously communicated the first use of mesoporous zirconium oxide (ZrO2) nanomaterials for effective phosphopeptide enrichment. Here we present the full report including the synthesis, characterization, and application of mesoporous titaniu...

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

    Science.gov (United States)

    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

  19. Optimization of titanium dioxide enrichment of phosphopeptides and application in the Thermoanaerobacter tengcongensis phosphoproteome analysis%二氧化钛富集磷酸肽方法优化及在腾冲嗜热厌氧菌磷酸化蛋白质组分析中的应用

    Institute of Scientific and Technical Information of China (English)

    林威; 王京兰; 应万涛; 钱小红

    2012-01-01

    Using titanium dioxide is a very good strategy for the phosphopeptide enrichment. There are many other factors can affect the enrichment efficiency, and the optimization of parameters was needed for better enrichment results. In this study, the peptide mixtures of six standard proteins were used as the model samples to evaluate and optimize the parameters such as the proportion of acetonitrile and trifluoroacetic acid in loading buffer and the TiO2-to-pep-tide ratio. The results showed that 80% (v/v) acetonitrile, 1% (v/v) trifluoroacetic acid and 40 :1 (m/m) TiO2-to-peptide ratio were the optimum parameters to obtain the best enrichment selectivity and maximum phosphopeptides identification. For the first time, the optimum enrichment conditions were applied for the phosphoproteome analysis of the Thermoanaerobacter tengcongensis, an anaerobic, saccharolytic, thermophilic bacterium isolated from a hot spring in Tengchong, China, and 25 phosphorylated proteins were identified in the preliminary experiment. The results provided a reference for further study on this organism survived under extreme environment.%为了提高二氧化钛富集磷酸肽法对磷酸肽的富集效率,以6种标准蛋白酶切肽段混合物为研究对象,对二氧化钛富集磷酸肽过程中的乙腈比例、三氟乙酸比例、二氧化钛用量等条件分别进行了优化.结果表明在乙腈含量为80% (v/v),三氟乙酸含量为1%(v/v),二氧化钛用量与需要富集肽段的质量比为40∶1的条件下,可以取得较好的富集效果.将优化后的富集方法应用于腾冲嗜热厌氧菌磷酸化蛋白质的分析,初步鉴定到25个磷酸化蛋白质,为进一步研究这种极端环境下生存的低等生物的生命活动提供了参考信息.

  20. Comprehensive analysis of signal transduction in three-dimensional ECM-based tumor cell cultures

    Directory of Open Access Journals (Sweden)

    Iris Eke

    2015-11-01

    Full Text Available Analysis of signal transduction and protein phosphorylation is fundamental to understand physiological and pathological cell behavior as well as identification of novel therapeutic targets. Despite the fact that more physiological three-dimensional cell culture assays are increasingly used, particularly proteomics and phosphoproteomics remain challenging due to easy, robust and reproducible sample preparation. Here, we present an easy-to-perform, reliable and time-efficient method for the production of 3D cell lysates without compromising cell adhesion before cell lysis. The samples can be used for Western blotting as well as phosphoproteome array technology. This technique would be of interest for researchers working in all fields of biology and drug development.

  1. From Phosphoproteome to Modeling of Plant Signaling Pathways.

    Science.gov (United States)

    Zakhartsev, Maksim; Pertl-Obermeyer, Heidi; Schulze, Waltraud X

    2016-01-01

    Quantitative proteomic experiments in recent years became almost routine in many aspects of biology. Particularly the quantification of peptides and corresponding phosphorylated counterparts from a single experiment is highly important for understanding of dynamics of signaling pathways. We developed an analytical method to quantify phosphopeptides (pP) in relation to the quantity of the corresponding non-phosphorylated parent peptides (P). We used mixed-mode solid-phase extraction to purify total peptides from tryptic digest and separated them from most of the phosphorous-containing compounds (e.g., phospholipids, nucleotides) which enhances pP enrichment on TiO2 beads. Phosphoproteomic data derived with this designed method allows quantifying pP/P stoichiometry, and qualifying experimental data for mathematical modeling. PMID:26700054

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

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

    2016-02-01

    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

  4. Implications of tyrosine phosphoproteomics in cervical carcinogenesis

    Directory of Open Access Journals (Sweden)

    DeFord James

    2008-01-01

    Full Text Available Abstract Background Worldwide cervical cancer remains a leading cause of mortality from gynecologic malignancies. The link between cervical cancer and persistent infection with HPV has been established. At a molecular level little is known about the transition from the precancerous state to invasive cancer. To elucidate this process, cervical biopsies from human specimens were obtained from precancerous state to stage III disease. Methods Cervical biopsies were obtained from patients with a diagnosis of cervical cancer undergoing definitive surgery or staging operation. Biopsies were obtained from patients with precancerous lesions at the time of their excisional procedure. Control samples were obtained from patients undergoing hysterectomy for benign conditions such as fibroids. Samples were subjected to proteomic profiling using two dimensional gel electrophoresis with subsequent trypsin digestion followed by MALDI-TOF protein identification. Candidate proteins were then further studied using western blotting, immunoprecipitation and immunohistochemistry. Results Annexin A1 and DNA-PKcs were found to be differentially expressed. Phosphorylated annexin A1 was up regulated in diseased states in comparison to control and its level was strongly detected in the serum of cervical cancer patients compared to controls. DNA-PKcs was noted to be hyperphosphorylated and fragmented in cancer when compared to controls. By immunohistochemistry annexin A1 was noted in the vascular environment in cancer and certain precancerous samples. Conclusion This study suggests a probable role for protein tyrosine phosphorylation in cervical carcinogenesis. Annexin A1 and DNA-PK cs may have synergistic effects with HPV infection. Precancerous lesions that may progress to cervical cancer may be differentiated from lesions that will not base on similar immunohistochemical profile to invasive squamous cell carcinoma.

  5. Trajectory Based Traffic Analysis

    DEFF Research Database (Denmark)

    Krogh, Benjamin Bjerre; Andersen, Ove; Lewis-Kelham, Edwin;

    2013-01-01

    We present the INTRA system for interactive path-based traffic analysis. The analyses are developed in collaboration with traffic researchers and provide novel insights into conditions such as congestion, travel-time, choice of route, and traffic-flow. INTRA supports interactive point-and-click a......We present the INTRA system for interactive path-based traffic analysis. The analyses are developed in collaboration with traffic researchers and provide novel insights into conditions such as congestion, travel-time, choice of route, and traffic-flow. INTRA supports interactive point......-and-click analysis, due to a novel and efficient indexing structure. With the web-site daisy.aau.dk/its/spqdemo/we will demonstrate several analyses, using a very large real-world data set consisting of 1.9 billion GPS records (1.5 million trajectories) recorded from more than 13000 vehicles, and touching most of...

  6. Newly fabricated magnetic lanthanide oxides core-shell nanoparticles in phosphoproteomics.

    Science.gov (United States)

    Jabeen, Fahmida; Najam-Ul-Haq, Muhammad; Rainer, Matthias; Güzel, Yüksel; Huck, Christian W; Bonn, Guenther K

    2015-01-01

    Metal oxides show high selectivity and sensitivity toward mass spectrometry based enrichment strategies. Phosphopeptides/phosphoproteins enrichment from biological samples is cumbersome because of their low abundance. Phosphopeptides are of interest in enzymes and phosphorylation pathways which lead to the clinical links of a disease. Magnetic core-shell lanthanide oxide nanoparticles (Fe3O4@SiO2-La2O3 and Fe3O4@SiO2-Sm2O3) are fabricated, characterized by SEM, FTIR, and EDX and employed in the enrichment of phosphopeptides. The nanoparticles enrich phosphopeptides from casein variants, nonfat milk, egg yolk, human serum and HeLa cell extract. The materials and enrichment protocols are designed in a way that there are almost no nonspecific bindings. The selectivity is achieved up to 1:8500 using β-casein/BSA mixture and sensitivity down to 1 atto-mole. Batch-to-batch reproducibility is high with the reuse of core-shell nanoparticles up to four cycles. The enrichment followed by MALDI-MS analyses is carried out for the identification of phosphopeptides from serum digest and HeLa cell extract. Characteristic phosphopeptides of phosphoproteins are identified from human serum after the enrichment, which have the diagnostic potential toward prostate cancer. Thus, the lanthanide based magnetic core-shell materials offer a highly selective and sensitive workflow in phosphoproteomics. PMID:25859614

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

    Science.gov (United States)

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

    2015-08-01

    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

  8. Comparative Proteome and Phosphoproteome Analyses during Cyprid Development of the Barnacle Balanus ( =Amphibalanus ) amphitrite

    KAUST Repository

    Zhang, Yu

    2010-06-04

    The barnacle Balanus amphitrite (=Amphibalanus amphitrite) is a major marine biofouling invertebrate worldwide. It has a complex life cycle during which the larva (called a nauplius) molts six times before transforming into the cyprid stage. The cyprid stage in B. amphitrite is the critical stage for the larval decision to attach and metamorphose. In this study, proteome and phosphoproteome alterations during cyprid development/aging and upon treatment with the antifouling agent butenolide were examined with a two-dimensional electrophoresis (2-DE) multiplexed fluorescent staining approach. Optimized protein separation strategies, including solution-phase isoelectric fractionation and narrow-pH-range 2-DE, were used in a proteomic analysis. Our results show that the differential regulation of the target proteins is highly dynamic on the levels of both protein expression and posttranslational modification. Two groups of proteins, stress-associated and energy metabolism-related proteins, are differentially expressed during cyprid development. Comparison of the control and treatment groups suggests that butenolide exerts its effects by sustaining the expression levels of these proteins. Altogether, our data suggest that proteins involved in stress regulation and energy metabolism play crucial roles in regulating larval attachment and metamorphosis of B. amphitrite. © 2010 American Chemical Society.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Wen-Jinn Liaw

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

  11. Proteome and phosphoproteome characterization reveals new response and defense mechanisms of Brachypodium distachyon leaves under salt stress.

    Science.gov (United States)

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

    2014-02-01

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Science.gov (United States)

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

    2016-04-01

    Tobacco mature pollen has extremely desiccated cytoplasm, and is metabolically quiescent. Upon re-hydration it becomes metabolically active and that results in later emergence of rapidly growing pollen tube. These changes in cytoplasm hydration and metabolic activity are accompanied by protein phosphorylation. In this study, we subjected mature pollen, 5-min-activated pollen, and 30-min-activated pollen to TCA/acetone protein extraction, trypsin digestion and phosphopeptide enrichment by titanium dioxide. The enriched fraction was subjected to nLC-MS/MS. We identified 471 phosphopeptides that carried 432 phosphorylation sites, position of which was exactly matched by mass spectrometry. These 471 phosphopeptides were assigned to 301 phosphoproteins, because some proteins carried more phosphorylation sites. Of the 13 functional groups, the majority of proteins were put into these categories: transcription, protein synthesis, protein destination and storage, and signal transduction. Many proteins were of unknown function, reflecting the fact that male gametophyte contains many specific proteins that have not been fully functionally annotated. The quantitative data highlighted the dynamics of protein phosphorylation during pollen activation; the identified phosphopeptides were divided into seven groups based on the regulatory trends. The major group comprised mature pollen-specific phosphopeptides that were dephosphorylated during pollen activation. Several phosphopeptides representing the same phosphoprotein had different regulation, which pinpointed the complexity of protein phosphorylation and its clear functional context. Collectively, we showed the first phosphoproteomics data on activated pollen where the position of phosphorylation sites was clearly demonstrated and regulatory kinetics was resolved. PMID:26792808

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

    DEFF Research Database (Denmark)

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

    2011-01-01

    Investigating stemporal changes in the yeast phosphoproteome upon fatty acid starvation Dennis Pultz*, Martin Bennetzen*, Jens S. Andersen and Nils J.Færgeman. Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark, 5230 Reducing food intake to induce...... changes in the phosphoproteome coupling fatty acid starvation to autophagy and starvation related cellular responses in general. By use of this approach we have identified approximately 2000 phosphorylation sites of which more than 400 have been identified as being regulated. Here, we present results...

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

    Science.gov (United States)

    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

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

    DEFF Research Database (Denmark)

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

    2014-01-01

    UNLABELLED: Protein phosphorylation can regulate most of the important processes in muscle, such as metabolism and contraction. The postmortem (PM) metabolism and rigor mortis have essential effects on meat quality. In order to identify and characterize the protein phosphorylation events involved...... phosphoproteins with 784 phosphorylation sites. Among these, 184 phosphorylation sites on 93 proteins had their phosphorylation levels significantly changed. The proteins involved in glucose metabolism and muscle contraction were the two largest clusters of phosphoproteins with significantly changed...... changes at the phosphorylation level but were relatively stable at the total protein level, suggesting that protein phosphorylation may have important roles in meat quality development through the regulation of proteins involved in glucose metabolism and muscle contraction, thereby affecting glycolysis...

  17. Quantitative Phosphoproteomics Analysis of ERBB3/ERBB4 Signaling

    OpenAIRE

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

    2016-01-01

    The four members of the epidermal growth factor receptor (EGFR/ERBB) family form homo- and heterodimers which mediate ligand-specific regulation of many key cellular processes in normal and cancer tissues. While signaling through the EGFR has been extensively studied on the molecular level, signal transduction through ERBB3/ERBB4 heterodimers is less well understood. Here, we generated isogenic mouse Ba/F3 cells that express full-length and functional membrane-integrated ERBB3 and ERBB4 or ER...

  18. Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome

    DEFF Research Database (Denmark)

    Mann, Matthias; Ong, Shao En; Grønborg, Mads;

    2002-01-01

    In signal transduction in eukaryotes, protein phosphorylation is a key event. To understand signaling processes, we must first acquire an inventory of phosphoproteins and their phosphorylation sites under different conditions. Because phosphorylation is a dynamic process, elucidation of signaling...

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

    DEFF Research Database (Denmark)

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

    2014-01-01

    knockdown by feeding the nematode on pre-labeled lysine auxotroph Escherichia coli. In this chapter, we describe in details the generation of the E. coli strain, incorporation of heavy isotope-labeled lysine in C. elegans, and the procedure for a comprehensive global phosphoproteomic experiment....

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

    DEFF Research Database (Denmark)

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

    2016-01-01

    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 (OSR1, HDGF and ccdc82...

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

    DEFF Research Database (Denmark)

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

    2011-01-01

    To elucidate cellular events underlying the pluripotency of human embryonic stem cells (hESCs), we performed parallel quantitative proteomic and phosphoproteomic analyses of hESCs during differentiation initiated by a diacylglycerol analog or transfer to media that had not been conditioned by...

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

    Science.gov (United States)

    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

  3. Identification of BCAP-{sub L} as a negative regulator of the TLR signaling-induced production of IL-6 and IL-10 in macrophages by tyrosine phosphoproteomics

    Energy Technology Data Exchange (ETDEWEB)

    Matsumura, Takayuki [Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, Shinjuku-ku, Tokyo 162-0041 (Japan); Department of Life Science and Medical Bio-Science, Waseda University, Shinjuku-ku, Tokyo 162-8480 (Japan); Oyama, Masaaki; Kozuka-Hata, Hiroko [Medical Proteomics Laboratory, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639 (Japan); Ishikawa, Kosuke; Inoue, Takafumi [Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, Shinjuku-ku, Tokyo 162-0041 (Japan); Department of Life Science and Medical Bio-Science, Waseda University, Shinjuku-ku, Tokyo 162-8480 (Japan); Muta, Tatsushi [Laboratory of Cell Recognition and Response, Graduate School of Life Sciences, Tohoku University, Sendai 980-8578 (Japan); Semba, Kentaro, E-mail: ksemba@waseda.jp [Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, Shinjuku-ku, Tokyo 162-0041 (Japan); Department of Life Science and Medical Bio-Science, Waseda University, Shinjuku-ku, Tokyo 162-8480 (Japan); Inoue, Jun-ichiro, E-mail: jun-i@ims.u-tokyo.ac.jp [Medical Proteomics Laboratory, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639 (Japan); Division of Cellular and Molecular Biology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639 (Japan)

    2010-09-17

    Research highlights: {yields} Twenty five tyrosine-phosphorylated proteins in LPS-stimulated macrophages were determined. {yields} BCAP is a novel tyrosine-phosphorylated protein in LPS-stimulated macrophages. {yields} BCAP-{sub L} inhibits IL-6 and IL-10 production in LPS-stimulated macrophages. -- Abstract: Toll-like receptor (TLR) signaling in macrophages is essential for anti-pathogen responses such as cytokine production and antigen presentation. Although numerous reports suggest that protein tyrosine kinases (PTKs) are involved in cytokine induction in response to lipopolysaccharides (LPS; TLR4 ligand) in macrophages, the PTK-mediated signal transduction pathway has yet to be analyzed in detail. Here, we carried out a comprehensive and quantitative dynamic tyrosine phosphoproteomic analysis on the TLR4-mediated host defense system in RAW264.7 macrophages using stable isotope labeling by amino acids in cell culture (SILAC). We determined the temporal profiles of 25 proteins based on SILAC-encoded peptide(s). Of these, we focused on the tyrosine phosphorylation of B-cell adaptor for phosphatidylinositol 3-kinase (BCAP) because the function of BCAP remains unknown in TLR signaling in macrophages. Furthermore, Bcap has two distinct transcripts, a full-length (Bcap-{sub L}) and an alternatively initiated or spliced (Bcap-{sub S}) mRNA, and little is known about the differential functions of the BCAP-{sub L} and BCAP-{sub S} proteins. Our study showed, for the first time, that RNAi-mediated selective depletion of BCAP-{sub L} enhanced IL-6 and IL-10 production but not TNF-{alpha} production in TLR ligand-stimulated macrophages. We propose that BCAP-{sub L} (but not BCAP-{sub S}) is a negative regulator of the TLR-mediated host defense system in macrophages.

  4. Quantitative Phospho-proteomics to Investigate the Polo-like Kinase 1-Dependent Phospho-proteome*

    OpenAIRE

    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

    2011-01-01

    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 ...

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

    DEFF Research Database (Denmark)

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

    2009-01-01

    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...... skeletal muscle phosphoproteins in health and disease and demonstrate feasibility of phosphoproteomics research of human skeletal muscle in vivo....

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

    Directory of Open Access Journals (Sweden)

    Zhengxiu Su

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-09-22

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

  8. An Augmented Multiple-Protease-Based Human Phosphopeptide Atlas

    OpenAIRE

    Piero Giansanti; Thin Thin Aye; Henk van den Toorn; Mao Peng; Bas van Breukelen; Albert J.R. Heck

    2015-01-01

    Although mass-spectrometry-based screens enable thousands of protein phosphorylation sites to be monitored simultaneously, they often do not cover important regulatory sites. Here, we hypothesized that this is due to the fact that nearly all large-scale phosphoproteome studies are initiated by trypsin digestion. We tested this hypothesis using multiple proteases for protein digestion prior to Ti4+-IMAC-based enrichment. This approach increases the size of the detectable phosphoproteome substa...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-01-15

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

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

    International Nuclear Information System (INIS)

    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 (Mn2+)-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 Mn2+-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 of GRP78, 14

  11. SILAC-Based Quantitative Proteomic Analysis of Human Lung Cell Response to Copper Oxide Nanoparticles

    OpenAIRE

    Edelmann, Mariola J.; Shack, Leslie A; Caitlin D Naske; Walters, Keisha B.; Nanduri, Bindu

    2014-01-01

    Copper (II) oxide (CuO) nanoparticles (NP) are widely used in industry and medicine. In our study we evaluated the response of BEAS-2B human lung cells to CuO NP, using Stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics and phosphoproteomics. Pathway modeling of the protein differential expression showed that CuO NP affect proteins relevant in cellular function and maintenance, protein synthesis, cell death and survival, cell cycle and cell morphology. Some of the...

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

    Directory of Open Access Journals (Sweden)

    Philipp eSpät

    2015-03-01

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

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

    OpenAIRE

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

    2012-01-01

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

  14. 15N-labeled brain enables quantification of proteome and phosphoproteome in cultured primary neurons.

    Science.gov (United States)

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

    2012-02-01

    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 (15)N-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

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

    Science.gov (United States)

    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

  16. Development and Application of Aphosphoproteomic Strategy Based Ontitanium Dioxideaffinity Chromatography

    Institute of Scientific and Technical Information of China (English)

    Mingquan Guo

    2012-01-01

    A sensitive and highly selective phosphoproteomic strategy,which allows simultaneous genome and transcriptome profiling from a single sample,has been developed by a combination of a highly selective titanium dioxide (TiO2)-based phosphopeptide enrichment method and a TRIzol-based protein extraction method.To establish more efficient phosphopeptide enrichment methods for complex cellular proteins,TiO2 method was comparedwith the most widely used IMAC method using model peptide mixturesbased on two criteria:one is the efficacy of the phosphopeptide enrichment (how many phosphopeptides each method can capture),and the other is the extent of exclusion of nonphosphopeptides from the pool of enriched phosphopeptides.The results indicate that the TiO2 enrichment method appears to be more effective than the IMAC method.This methodology was then further applied to the phosphoproteomic analysis of a small number of HeLa cells with and without calyculin A (CL-A) treatment.Significant increases in the number of identified phosphopeptides and phosphosites were observed in the CL-A-treated samples compared with nontreated samples as expected in the biological studies.Meanwhile,some novel phosphorylation sites,such as T19 and S20 of the myosin regulatory light chain and S116 and S379 in the heterogeneous nuclear ribonucleoprotein K transcript variant in the CL-A-treated samples,were revealed as well.Thus,the robustness,generality,and simplicity of this strategy should enable this methodology to find broad applications for phosphoproteomic analyses in systems biology,especially in cases of limited starting material requiring a systems-level analysis such as small clinical specimens.

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

    Science.gov (United States)

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

    2016-03-01

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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

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

    Science.gov (United States)

    Osinalde, Nerea; Mitxelena, Jone; Sánchez-Quiles, Virginia; Akimov, Vyacheslav; Aloria, Kerman; Arizmendi, Jesus M; Zubiaga, Ana M; Blagoev, Blagoy; Kratchmarova, Irina

    2016-06-01

    Anti-cancer immunotherapies commonly rely on the use of interleukin-2 (IL-2) to promote the expansion of T lymphocytes. IL-2- dependent proliferation is the culmination of a complex network of phosphorylation-driven signaling events that impact on gene transcription through mechanisms that are not clearly understood. To study the role of IL-2 in the regulation of nuclear protein function we have performed an unbiased mass spectrometry-based study of the nuclear phosphoproteome of resting and IL-2-treated CD4(+) T lymphocytes. We detected 8521distinct phosphosites including many that are not yet reported in curated phosphorylation databases. Although most phosphorylation sites remained unaffected upon IL-2 treatment, 391 sites corresponding to 288 gene products showed robust IL-2-dependent regulation. Importantly, we show that ATP-citrate lyase (ACLY) is a key phosphoprotein effector of IL-2-mediated T-cell responses. ACLY becomes phosphorylated on serine 455 in T lymphocytes upon IL-2-driven activation of AKT, and depletion or inactivation of ACLY compromises IL-2-promoted T-cell growth. Mechanistically, we demonstrate that ACLY is required for enhancing histone acetylation levels and inducing the expression of cell cycle regulating genes in response to IL-2. Thus, the metabolic enzyme ACLY emerges as a bridge between cytokine signaling and proliferation of T lymphocytes, and may be an attractive candidate target for the development of more efficient anti-cancer immunotherapies. PMID:27067055

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

    Science.gov (United States)

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

    2014-10-01

    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

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

    DEFF Research Database (Denmark)

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

    2013-01-01

    in resistant and susceptible wheat using quantitative proteomics and phosphoproteomics, with special emphasis on the initial biotrophic phase of interactions. Our study revealed an accumulation of defense and stress-related proteins, suppression of photosynthesis, and changes in sugar metabolism...... transduction cascades resulting in a multiple-level activation of transcription and translation processes of defense responses. Our sensitive approaches and model provide a comprehensive (phospho)proteomics resource for studying signaling from the point of view of both host and pathogen during a plant...

  4. Early cytokinin response proteins and phosphoproteins of Arabidopsis thaliana identified by proteome and phosphoproteome profiling

    Czech Academy of Sciences Publication Activity Database

    Černý, M.; Dyčka, Filip; Bobálová, Janette; Brzobohatý, Břetislav

    2011-01-01

    Roč. 62, č. 3 (2011), s. 921-937. ISSN 0022-0957 R&D Projects: GA MŠk(CZ) LC06034; GA MŠk(CZ) 1M06030; GA AV ČR IAA600040701; GA ČR(CZ) GA206/09/2062 Institutional research plan: CEZ:AV0Z40310501; CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : Arabidopsis thaliana * cytokinin * phosphoproteome * proteome Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 5.364, year: 2011

  5. Hand-Based Biometric Analysis

    Science.gov (United States)

    Bebis, George (Inventor); Amayeh, Gholamreza (Inventor)

    2015-01-01

    Hand-based biometric analysis systems and techniques are described which provide robust hand-based identification and verification. An image of a hand is obtained, which is then segmented into a palm region and separate finger regions. Acquisition of the image is performed without requiring particular orientation or placement restrictions. Segmentation is performed without the use of reference points on the images. Each segment is analyzed by calculating a set of Zernike moment descriptors for the segment. The feature parameters thus obtained are then fused and compared to stored sets of descriptors in enrollment templates to arrive at an identity decision. By using Zernike moments, and through additional manipulation, the biometric analysis is invariant to rotation, scale, or translation or an in put image. Additionally, the analysis utilizes re-use of commonly-seen terms in Zernike calculations to achieve additional efficiencies over traditional Zernike moment calculation.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  8. Proteomic and phosphoproteomic analyses of chromatin-associated proteins from Arabidopsis thaliana

    KAUST Repository

    Bigeard, Jean

    2014-07-10

    The nucleus is the organelle where basically all DNA-related processes take place in eukaryotes, such as replication, transcription, and splicing as well as epigenetic regulation. The identification and description of the nuclear proteins is one of the requisites toward a comprehensive understanding of the biological functions accomplished in the nucleus. Many of the regulatory mechanisms of protein functions rely on their PTMs among which phosphorylation is probably one of the most important properties affecting enzymatic activity, interaction with other molecules, localization, or stability. So far, the nuclear and subnuclear proteome and phosphoproteome of the model plant Arabidopsis thaliana have been the subject of very few studies. In this work, we developed a purification protocol of Arabidopsis chromatin-associated proteins and performed proteomic and phosphoproteomic analyses identifying a total of 879 proteins of which 198 were phosphoproteins that were mainly involved in chromatin remodeling, transcriptional regulation, and RNA processing. From 230 precisely localized phosphorylation sites (phosphosites), 52 correspond to hitherto unidentified sites. This protocol and data thereby obtained should be a valuable resource for many domains of plant research.

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

    Science.gov (United States)

    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. Cardiac Phosphoproteomics during Remote Ischemic Preconditioning: A Role for the Sarcomeric Z-Disk Proteins

    Directory of Open Access Journals (Sweden)

    Safa Abdul-Ghani

    2014-01-01

    Full Text Available Remote ischemic preconditioning (RIPC induced by brief ischemia/reperfusion cycles of remote organ (e.g., limb is cardioprotective. The myocardial cellular changes during RIPC responsible for this phenomenon are not currently known. The aim of this work was to identify the activation by phosphorylation of cardiac proteins following RIPC. To achieve our aim we used isobaric tandem mass tagging (TMT and reverse phase nanoliquid chromatography tandem spectrometry using a Linear Trap Quadropole (LTQ Orbitrap Velos mass spectrometer. Male C57/Bl6 mice were anesthetized by an intraperitoneal injection of Tribromoethanol. A cuff was placed around the hind limb and inflated at 200 mmHg to prevent blood flow as confirmed by Laser Doppler Flowmetry. RIPC was induced by 4 cycles of 5 min of limb ischemia followed by 5 min of reperfusion. Hearts were extracted for phosphoproteomics. We identified approximately 30 phosphoproteins that were differentially expressed in response to RIPC protocol. The levels of several phosphoproteins in the Z-disk of the sarcomere including phospho-myozenin-2 were significantly higher than control. This study describes and validates a novel approach to monitor the changes in the cardiac phosphoproteome following the cardioprotective intervention of RIPC and prior to index ischemia. The increased level of phosphorylated sarcomeric proteins suggests they may have a role in cardiac signaling during RIPC.

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

    Directory of Open Access Journals (Sweden)

    Raymond T Suhandynata

    Full Text Available 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.

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

    Science.gov (United States)

    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

  13. Phosphoproteomics of Klebsiella pneumoniae NTUH-K2044 reveals a tight link between tyrosine phosphorylation and virulence.

    Science.gov (United States)

    Lin, Miao-Hsia; Hsu, Tung-Li; Lin, Shu-Yu; Pan, Yi-Jiun; Jan, Jia-Tsrong; Wang, Jin-Town; Khoo, Kay-Hooi; Wu, Shih-Hsiung

    2009-12-01

    Encapsulated Klebsiella pneumoniae is the predominant causative agent of pyogenic liver abscess, an emerging infectious disease that often complicates metastatic meningitis or endophthalmitis. The capsular polysaccharide on K. pneumoniae surface was determined as the key to virulence. Although the regulation of capsular polysaccharide biosynthesis is largely unclear, it was found that protein-tyrosine kinases and phosphatases are involved. Therefore, the identification and characterization of such kinases, phosphatases, and their substrates would advance our knowledge of the underlying mechanism in capsule formation and could contribute to the development of new therapeutic strategies. Here, we analyzed the phosphoproteome of K. pneumoniae NTUH-K2044 with a shotgun approach and identified 117 unique phosphopeptides along with 93 in vivo phosphorylated sites corresponding to 81 proteins. Interestingly, three of the identified tyrosine phosphorylated proteins, namely protein-tyrosine kinase (Wzc), phosphomannomutase (ManB), and undecaprenyl-phosphate glycosyltransferase (WcaJ), were found to be distributed in the cps locus and thus were speculated to be involved in the converging signal transduction of capsule biosynthesis. Consequently, we decided to focus on the lesser studied ManB and WcaJ for mutation analysis. The capsular polysaccharides of WcaJ mutant (WcaJY5F) were dramatically reduced quantitatively, and the LD(50) increased by 200-fold in a mouse peritonitis model compared with the wild-type strain. However, the capsular polysaccharides of ManB mutant (ManBY26F) showed no difference in quantity, and the LD(50) increased by merely 6-fold in mice test. Our study provided a clear trend that WcaJ tyrosine phosphorylation can regulate the biosynthesis of capsular polysaccharides and result in the pathogenicity of K. pneumoniae NTUH-K2044. PMID:19696081

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

    Science.gov (United States)

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

    2014-05-01

    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

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

    Directory of Open Access Journals (Sweden)

    Feng Yang

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

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

    Directory of Open Access Journals (Sweden)

    Erick Contreras-Vallejos

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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  19. Certification-Based Process Analysis

    Science.gov (United States)

    Knight, Russell L.

    2013-01-01

    Space mission architects are often challenged with knowing which investment in technology infusion will have the highest return. Certification-based analysis (CBA) gives architects and technologists a means to communicate the risks and advantages of infusing technologies at various points in a process. Various alternatives can be compared, and requirements based on supporting streamlining or automation can be derived and levied on candidate technologies. CBA is a technique for analyzing a process and identifying potential areas of improvement. The process and analysis products are used to communicate between technologists and architects. Process means any of the standard representations of a production flow; in this case, any individual steps leading to products, which feed into other steps, until the final product is produced at the end. This sort of process is common for space mission operations, where a set of goals is reduced eventually to a fully vetted command sequence to be sent to the spacecraft. Fully vetting a product is synonymous with certification. For some types of products, this is referred to as verification and validation, and for others it is referred to as checking. Fundamentally, certification is the step in the process where one insures that a product works as intended, and contains no flaws.

  20. SILAC-based quantitative proteomic analysis of human lung cell response to copper oxide nanoparticles.

    Directory of Open Access Journals (Sweden)

    Mariola J Edelmann

    Full Text Available Copper (II oxide (CuO nanoparticles (NP are widely used in industry and medicine. In our study we evaluated the response of BEAS-2B human lung cells to CuO NP, using Stable isotope labeling by amino acids in cell culture (SILAC-based proteomics and phosphoproteomics. Pathway modeling of the protein differential expression showed that CuO NP affect proteins relevant in cellular function and maintenance, protein synthesis, cell death and survival, cell cycle and cell morphology. Some of the signaling pathways represented by BEAS-2B proteins responsive to the NP included mTOR signaling, protein ubiquitination pathway, actin cytoskeleton signaling and epithelial adherens junction signaling. Follow-up experiments showed that CuO NP altered actin cytoskeleton, protein phosphorylation and protein ubiquitination level.

  1. Developmentally-Dynamic Murine Brain Proteomes and Phosphoproteomes Revealed by Quantitative Proteomics

    Directory of Open Access Journals (Sweden)

    Peter F. Doubleday

    2014-04-01

    Full Text Available Developmental processes are governed by a diverse suite of signaling pathways employing reversible phosphorylation. Recent advances in large-scale phosphoproteomic methodologies have made possible the identification and quantification of hundreds to thousands of phosphorylation sites from primary tissues. Towards a global characterization of proteomic changes across brain development, we present the results of a large-scale quantitative mass spectrometry study comparing embryonic, newborn and adult murine brain. Using anti-phosphotyrosine immuno-affinity chromatography and strong cation exchange (SCX chromatography, coupled to immobilized metal affinity chromatography (IMAC, we identified and quantified over 1,750 phosphorylation sites and over 1,300 proteins between three developmental states. Bioinformatic analyses highlight functions associated with the identified proteins and phosphoproteins and their enrichment at distinct developmental stages. These results serve as a primary reference resource and reveal dynamic developmental profiles of proteins and phosphoproteins from the developing murine brain.

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

    Science.gov (United States)

    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

  3. A phosphoproteomics view at human pluripotent stem cells

    NARCIS (Netherlands)

    Zoumaro-Djayoon, A.D.

    2013-01-01

    Reversible protein phosphorylation has been one of the most investigated post-translational modifications (PTM) in the decade, made possible by advances in enrichment technologies and MS-based proteomics. PTMs are generally involved in the regulation of many biological processes, including processes

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

    Science.gov (United States)

    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

  5. Quantitative Label-Free Phosphoproteomics Strategy for Multifaceted Experimental Designs

    OpenAIRE

    Soderblom, Erik J.; Philipp, Melanie; Thompson, J. Will; Caron, Marc G.; Moseley, M Arthur

    2011-01-01

    Protein phosphorylation is a critical regulator of signaling in nearly all eukaryotic cellular pathways and dysregulated phosphorylation has been implicated in an array of diseases. The majority of MS-based quantitative phosphorylation studies are currently performed from transformed cell lines because of the ability to generate large amounts of starting material with incorporated isotopically labeled amino acids during cell culture. Here we describe a general label-free quantitative phosphop...

  6. Sequential Enrichment with Titania-coated Magnetic Mesoporous Hollow Silica Microspheres and Zirconium Arsenate-modified Magnetic Nanoparticles for the Study of Phosphoproteome of HL60 Cells

    Science.gov (United States)

    Yu, Qiong-Wei; Li, Xiao-Shui; Xiao, Yongsheng; Guo, Lei; Zhang, Fan; Cai, Qian; Feng, Yu-Qi; Yuan, Bi-Feng; Wang, Yinsheng

    2014-01-01

    As one of the most important types of post-translational modifications, reversible phosphorylation of proteins plays crucial roles in a large number of biological processes. However, owing to the relatively low abundance and dynamic nature of phosphorylation and the presence of the unphosphorylated peptides in large excess, phosphopeptide enrichment is indispensable in large-scale phosphoproteomic analysis. Metal oxides including titanium dioxide have become prominent affinity materials to enrich phosphopeptides prior to their analysis using liquid chromatography-mass spectrometry (LC-MS). In the current study, we established a novel strategy, which encompassed strong cation exchange chromatography, sequential enrichment of phosphopeptides using titania-coated magnetic mesoporous hollow silica microspheres (TiO2/MHMSS) and zirconium arsenate-modified magnetic nanoparticles (ZrAs-Fe3O4@SiO2), and LC-MS/MS analysis, for the proteome-wide identification of phosphosites of proteins in HL60 cells. In total, we were able to identify 11579 unique phosphorylation sites in 3432 unique proteins. Additionally, our results suggested that TiO2/MHMSS and ZrAs-Fe3O4@SiO2 are complementary in phosphopeptide enrichment, where the two types of materials displayed preferential binding of peptides carrying multiple and single phosphorylation sites, respectively. PMID:25262027

  7. ANALYSIS-BASED SPARSE RECONSTRUCTION WITH SYNTHESIS-BASED SOLVERS

    OpenAIRE

    Cleju, Nicolae; Jafari, Maria,; Plumbley, Mark D.

    2012-01-01

    Analysis based reconstruction has recently been introduced as an alternative to the well-known synthesis sparsity model used in a variety of signal processing areas. In this paper we convert the analysis exact-sparse reconstruction problem to an equivalent synthesis recovery problem with a set of additional constraints. We are therefore able to use existing synthesis-based algorithms for analysis-based exact-sparse recovery. We call this the Analysis-By-Synthesis (ABS) approach. We evaluate o...

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

    OpenAIRE

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

    2013-01-01

    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...

  9. The phosphoproteome of human Jurkat T cell clones upon costimulation with anti-CD3/anti-CD28 antibodies.

    Science.gov (United States)

    Nguyen, Tien Dung; Carrascal, Montserrat; Vidal-Cortes, Oriol; Gallardo, Oscar; Casas, Vanessa; Gay, Marina; Phan, Van Chi; Abian, Joaquin

    2016-01-10

    Phosphorylation is a reversible post-translational modification, playing a vital role in protein function. In T cells, protein phosphorylation is the key mechanism regulating T cell receptor-driven signaling pathways. In order to gain insights into the phosphoproteome evolution of T cell activation, we performed a large-scale quantitative phosphoproteomics study of Jurkat E6.1 (wild type) and Jurkat gamma1 (Phospholipase gamma1 null) cell clones upon costimulation with anti-CD3 and anti-CD28 antibodies at times ranging from 15min to as long as 120min. In total, we identified 5585 phosphopeptides belonging to 2008 phosphoproteins from both cell clones. We detected 130 and 114 novel phosphopeptides in Jurkat E6.1 and Jurkat gamma1 clones, respectively. A significantly lower number of proteins containing regulated phosphorylation sites were identified in Jurkat gamma1 in comparison to Jurkat E6.1, reflecting the vital role of Phospholipase gamma1 in T cell signaling. Several new phosphorylation sites from lymphocyte-specific protein tyrosine kinase (Lck) were identified. Of these, serine-121 showed significant changes in JE6.1 while only small changes in the Jgamma1 clone. Our data may contribute to the current human T cell phosphoproteome and provide a better understanding on T cell receptor signaling. Data are available via ProteomeXchange with identifier PXD002871. PMID:26546556

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

    Institute of Scientific and Technical Information of China (English)

    张媛媛; 胡启蒙; 王亮亮; 李新红

    2012-01-01

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

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

    Science.gov (United States)

    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

  12. Phosphoproteomic Analysis Based on Liquid Chromatography-Tandem Mass Spectrometry%基于液相色谱-串联质谱技术的磷酸化蛋白质组学分析

    Institute of Scientific and Technical Information of China (English)

    朱秀娟; 李好雨; 郭林; 赵晓璐

    2016-01-01

    本文运用稳定同位素双甲基化标记技术、固定相金属离子螯合层析(IMAC)技术并结合液相色谱-串联质谱法研究了仙台病毒感染引起的宿主细胞磷酸化蛋白质组变化.实验发现定量的4 289个磷酸化肽段有~20%的蛋白质磷酸化位点发生了显著变化;通路富集分析表明哺乳动物雷帕毒素靶蛋白(mTOR)通路和剪接体(Spliceosome)通路可能参与宿主细胞对病毒的应答;通过对显著变化磷酸化肽段进行基序分析,发现了9个代表性的磷酸化修饰位点基序.本研究方法对于深入解析抗病毒天然免疫信号通路提供了新线索.

  13. Probabilistic Model-Based Safety Analysis

    CERN Document Server

    Güdemann, Matthias; 10.4204/EPTCS.28.8

    2010-01-01

    Model-based safety analysis approaches aim at finding critical failure combinations by analysis of models of the whole system (i.e. software, hardware, failure modes and environment). The advantage of these methods compared to traditional approaches is that the analysis of the whole system gives more precise results. Only few model-based approaches have been applied to answer quantitative questions in safety analysis, often limited to analysis of specific failure propagation models, limited types of failure modes or without system dynamics and behavior, as direct quantitative analysis is uses large amounts of computing resources. New achievements in the domain of (probabilistic) model-checking now allow for overcoming this problem. This paper shows how functional models based on synchronous parallel semantics, which can be used for system design, implementation and qualitative safety analysis, can be directly re-used for (model-based) quantitative safety analysis. Accurate modeling of different types of proba...

  14. Feature-based sentiment analysis with ontologies

    OpenAIRE

    Taner, Berk

    2011-01-01

    Sentiment analysis is a topic that many researchers work on. In recent years, new research directions under sentiment analysis appeared. Feature-based sentiment analysis is one such topic that deals not only with finding sentiment in a sentence but providing a more detailed analysis on a given domain. In the beginning researchers focused on commercial products and manually generated list of features for a product. Then they tried to generate a feature-based approach to attach sentiments to th...

  15. Phosphoproteome Profiling of SH-SY5y Neuroblastoma Cells Treated with Anesthetics: Sevoflurane and Isoflurane Affect the Phosphorylation of Proteins Involved in Cytoskeletal Regulation.

    Science.gov (United States)

    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

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

    KAUST Repository

    Dash, Swagatika

    2012-04-01

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

  17. Multiple imputations applied to the DREAM3 phosphoproteomics challenge: a winning strategy.

    Directory of Open Access Journals (Sweden)

    Nicolas Guex

    Full Text Available DREAM is an initiative that allows researchers to assess how well their methods or approaches can describe and predict networks of interacting molecules [1]. Each year, recently acquired datasets are released to predictors ahead of publication. Researchers typically have about three months to predict the masked data or network of interactions, using any predictive method. Predictions are assessed prior to an annual conference where the best predictions are unveiled and discussed. Here we present the strategy we used to make a winning prediction for the DREAM3 phosphoproteomics challenge. We used Amelia II, a multiple imputation software method developed by Gary King, James Honaker and Matthew Blackwell[2] in the context of social sciences to predict the 476 out of 4624 measurements that had been masked for the challenge. To chose the best possible multiple imputation parameters to apply for the challenge, we evaluated how transforming the data and varying the imputation parameters affected the ability to predict additionally masked data. We discuss the accuracy of our findings and show that multiple imputations applied to this dataset is a powerful method to accurately estimate the missing data. We postulate that multiple imputations methods might become an integral part of experimental design as a mean to achieve cost savings in experimental design or to increase the quantity of samples that could be handled for a given cost.

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

    Directory of Open Access Journals (Sweden)

    Christof eRampitsch

    2012-07-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-04-17

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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  2. FAIMS and Phosphoproteomics of Fibroblast Growth Factor Signaling: Enhanced Identification of Multiply Phosphorylated Peptides.

    Science.gov (United States)

    Zhao, Hongyan; Cunningham, Debbie L; Creese, Andrew J; Heath, John K; Cooper, Helen J

    2015-12-01

    We have applied liquid chromatography high-field asymmetric waveform ion mobility spectrometry tandem mass spectrometry (LC-FAIMS-MS/MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS) to the investigation of site-specific phosphorylation in fibroblast growth factor (FGF) signaling. We have combined a SILAC approach with chemical inhibition by SU5402 (an FGF receptor tyrosine kinase inhibitor) and dasatinib (a Src family kinase inhibitor). The results show that incorporation of FAIMS within the workflow results in (a) an increase in the relative proportion of phosphothreonine and phosphotyrosine sites identified, (b) an increase in phosphopeptide identifications from precursors with charge states ≥ +3 (with an associated increase in peptide length), and (c) an increase in the identification of multiply phosphorylated peptides. Approximately 20% of the phosphorylation sites identified via the FAIMS workflow had not been reported previously, and over 80% of those were from multiply phosphorylated peptides. Moreover, FAIMS provided access to a distinct set of phosphorylation sites regulated in response to SU5402 and dasatinib. The enhanced identification of multiply phosphorylated peptides was particularly striking in the case of sites regulated by SU5402. In addition to providing a compelling example of the complementarity of FAIMS in phosphoproteomics, the results provide a valuable resource of phosphorylation sites for further investigation of FGF signaling and trafficking. PMID:26503514

  3. Pareto analysis based on records

    CERN Document Server

    Doostparast, M

    2012-01-01

    Estimation of the parameters of an exponential distribution based on record data has been treated by Samaniego and Whitaker (1986) and Doostparast (2009). Recently, Doostparast and Balakrishnan (2011) obtained optimal confidence intervals as well as uniformly most powerful tests for one- and two-sided hypotheses concerning location and scale parameters based on record data from a two-parameter exponential model. In this paper, we derive optimal statistical procedures including point and interval estimation as well as most powerful tests based on record data from a two-parameter Pareto model. For illustrative purpose, a data set on annual wages of a sample production-line workers in a large industrial firm is analyzed using the proposed procedures.

  4. TEXTURE ANALYSIS BASED IRIS RECOGNITION

    OpenAIRE

    GÜRKAN, Güray; AKAN, Aydın

    2012-01-01

    In this paper, we present a new method for personal identification, based on iris patterns. The method composed of iris image acquisition, image preprocessing, feature extraction and finally decision stages. Normalized iris images are vertically log-sampled and filtered by circular symmetric Gabor filters. The output of filters are windowed and mean absolute deviation of pixels in the window are calculated as the feature vectors. The proposed  method has the desired properties of an iris reco...

  5. ROAn, a ROOT based Analysis Framework

    CERN Document Server

    Lauf, Thomas

    2013-01-01

    The ROOT based Offline and Online Analysis (ROAn) framework was developed to perform data analysis on data from Depleted P-channel Field Effect Transistor (DePFET) detectors, a type of active pixel sensors developed at the MPI Halbleiterlabor (HLL). ROAn is highly flexible and extensible, thanks to ROOT's features like run-time type information and reflection. ROAn provides an analysis program which allows to perform configurable step-by-step analysis on arbitrary data, an associated suite of algorithms focused on DePFET data analysis, and a viewer program for displaying and processing online or offline detector data streams. The analysis program encapsulates the applied algorithms in objects called steps which produce analysis results. The dependency between results and thus the order of calculation is resolved automatically by the program. To optimize algorithms for studying detector effects, analysis parameters are often changed. Such changes of input parameters are detected in subsequent analysis runs and...

  6. Excel-Based Business Analysis

    CERN Document Server

    Anari, Ali

    2012-01-01

    ai"The trend is your friend"is a practical principle often used by business managers, who seek to forecast future sales, expenditures, and profitability in order to make production and other operational decisions. The problem is how best to identify and discover business trends and utilize trend information for attaining objectives of firms.This book contains an Excel-based solution to this problem, applying principles of the authors' "profit system model" of the firm that enables forecasts of trends in sales, expenditures, profits and other business variables. The program,

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

    NARCIS (Netherlands)

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

    2014-01-01

    The oomycete Phytophthora infestans is the causal agent of late blight in potato and tomato. Since the underlying processes that govern pathogenicity and development in P. infestans are largely unknown, we have performed a large-scale phosphoproteomics study of six different P. infestans life stages

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

    NARCIS (Netherlands)

    Resjö, S.; Ali, A.; Meijer, H.J.G.; Seidl, M.F.; Snel, B.; Sandin, M.; Levander, F.; Govers, F.; Andreasson, E.

    2014-01-01

    The oomycete Phytophthora infestans is the causal agent of late blight in potato and tomato. Since the underlying processes that govern pathogenicity and development in P. infestans are largely unknown, we have performed a large-scale phosphoproteomics study of six different P. infestans life stages

  9. JAVA based LCD Reconstruction and Analysis Tools

    International Nuclear Information System (INIS)

    We summarize the current status and future developments of the North American Group's Java-based system for studying physics and detector design issues at a linear collider. The system is built around Java Analysis Studio (JAS) an experiment-independent Java-based utility for data analysis. Although the system is an integrated package running in JAS, many parts of it are also standalone Java utilities

  10. Java based LCD reconstruction and analysis tools

    International Nuclear Information System (INIS)

    We summarize the current status and future developments of the North American Group's Java-based system for studying physics and detector design issues at a linear collider. The system is built around Java Analysis Studio (JAS) an experiment-independent Java-based utility for data analysis. Although the system is an integrated package running in JAS, many parts of it are also standalone Java utilities

  11. Reliability analysis of software based safety functions

    International Nuclear Information System (INIS)

    The methods applicable in the reliability analysis of software based safety functions are described in the report. Although the safety functions also include other components, the main emphasis in the report is on the reliability analysis of software. The check list type qualitative reliability analysis methods, such as failure mode and effects analysis (FMEA), are described, as well as the software fault tree analysis. The safety analysis based on the Petri nets is discussed. The most essential concepts and models of quantitative software reliability analysis are described. The most common software metrics and their combined use with software reliability models are discussed. The application of software reliability models in PSA is evaluated; it is observed that the recent software reliability models do not produce the estimates needed in PSA directly. As a result from the study some recommendations and conclusions are drawn. The need of formal methods in the analysis and development of software based systems, the applicability of qualitative reliability engineering methods in connection to PSA and the need to make more precise the requirements for software based systems and their analyses in the regulatory guides should be mentioned. (orig.). (46 refs., 13 figs., 1 tab.)

  12. Curvelet Based Offline Analysis of SEM Images

    OpenAIRE

    Shirazi, Syed Hamad; Haq, Nuhman ul; Hayat, Khizar; Naz, Saeeda; Haque, Ihsan ul

    2014-01-01

    Manual offline analysis, of a scanning electron microscopy (SEM) image, is a time consuming process and requires continuous human intervention and efforts. This paper presents an image processing based method for automated offline analyses of SEM images. To this end, our strategy relies on a two-stage process, viz. texture analysis and quantification. The method involves a preprocessing step, aimed at the noise removal, in order to avoid false edges. For texture analysis, the proposed method ...

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  15. Analysis of a Chaotic Memristor Based Oscillator

    Directory of Open Access Journals (Sweden)

    F. Setoudeh

    2014-01-01

    Full Text Available A chaotic oscillator based on the memristor is analyzed from a chaos theory viewpoint. Sensitivity to initial conditions is studied by considering a nonlinear model of the system, and also a new chaos analysis methodology based on the energy distribution is presented using the Discrete Wavelet Transform (DWT. Then, using Advance Design System (ADS software, implementation of chaotic oscillator based on the memristor is considered. Simulation results are provided to show the main points of the paper.

  16. Analysis of a Chaotic Memristor Based Oscillator

    OpenAIRE

    F. Setoudeh; Khaki Sedigh, A.; Dousti, M

    2014-01-01

    A chaotic oscillator based on the memristor is analyzed from a chaos theory viewpoint. Sensitivity to initial conditions is studied by considering a nonlinear model of the system, and also a new chaos analysis methodology based on the energy distribution is presented using the Discrete Wavelet Transform (DWT). Then, using Advance Design System (ADS) software, implementation of chaotic oscillator based on the memristor is considered. Simulation results are provided to show the main points of t...

  17. Proteomic and Phosphoproteomic Insights into a Signaling Hub Role for Cdc14 in Asexual Development and Multiple Stress Responses in Beauveria bassiana

    Science.gov (United States)

    Wang, Zhi-Kang; Wang, Jie; Liu, Jing; Ying, Sheng-Hua; Peng, Xiao-Jun; Feng, Ming-Guang

    2016-01-01

    Cdc14 is a dual-specificity phosphatase that regulates nuclear behavior by dephosphorylating phosphotyrosine and phosphoserine/phosphothreonine in fungi. Previously, Cdc14 was shown to act as a positive regulator of cytokinesis, asexual development and multiple stress responses in Beauveria bassiana, a fungal insect pathogen. This study seeks to gain deep insight into a pivotal role of Cdc14 in the signaling network of B. bassiana by analyzing the Cdc14-specific proteome and phosphoproteome generated by the 8-plex iTRAQ labeling and MS/MS analysis of peptides and phosphopeptides. Under normal conditions, 154 proteins and 86 phosphorylation sites in 67 phosphoproteins were upregulated in Δcdc14 versus wild-type, whereas 117 proteins and 85 phosphorylation sites in 58 phosphoproteins were significantly downregulated. Co-cultivation of Δcdc14 with NaCl (1 M), H2O2 (3 mM) and Congo red (0.15 mg/ml) resulted in the upregulation / downregulation of 23/63, 41/39 and 79/79 proteins and of 127/112, 52/47 and 105/226 phosphorylation sites in 85/92, 45/36 and 79/146 phosphoproteins, respectively. Bioinformatic analyses revealed that Cdc14 could participate in many biological and cellular processes, such as carbohydrate metabolism, glycerophospholipid metabolism, the MAP Kinase signaling pathway, and DNA conformation, by regulating protein expression and key kinase phosphorylation in response to different environmental cues. These indicate that in B. bassiana, Cdc14 is a vital regulator of not only protein expression but also many phosphorylation events involved in developmental and stress-responsive pathways. Fourteen conserved and novel motifs were identified in the fungal phosphorylation events. PMID:27055109

  18. 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.

    Science.gov (United States)

    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

  19. Proteomic and Phosphoproteomic Insights into a Signaling Hub Role for Cdc14 in Asexual Development and Multiple Stress Responses in Beauveria bassiana.

    Science.gov (United States)

    Wang, Zhi-Kang; Wang, Jie; Liu, Jing; Ying, Sheng-Hua; Peng, Xiao-Jun; Feng, Ming-Guang

    2016-01-01

    Cdc14 is a dual-specificity phosphatase that regulates nuclear behavior by dephosphorylating phosphotyrosine and phosphoserine/phosphothreonine in fungi. Previously, Cdc14 was shown to act as a positive regulator of cytokinesis, asexual development and multiple stress responses in Beauveria bassiana, a fungal insect pathogen. This study seeks to gain deep insight into a pivotal role of Cdc14 in the signaling network of B. bassiana by analyzing the Cdc14-specific proteome and phosphoproteome generated by the 8-plex iTRAQ labeling and MS/MS analysis of peptides and phosphopeptides. Under normal conditions, 154 proteins and 86 phosphorylation sites in 67 phosphoproteins were upregulated in Δcdc14 versus wild-type, whereas 117 proteins and 85 phosphorylation sites in 58 phosphoproteins were significantly downregulated. Co-cultivation of Δcdc14 with NaCl (1 M), H2O2 (3 mM) and Congo red (0.15 mg/ml) resulted in the upregulation / downregulation of 23/63, 41/39 and 79/79 proteins and of 127/112, 52/47 and 105/226 phosphorylation sites in 85/92, 45/36 and 79/146 phosphoproteins, respectively. Bioinformatic analyses revealed that Cdc14 could participate in many biological and cellular processes, such as carbohydrate metabolism, glycerophospholipid metabolism, the MAP Kinase signaling pathway, and DNA conformation, by regulating protein expression and key kinase phosphorylation in response to different environmental cues. These indicate that in B. bassiana, Cdc14 is a vital regulator of not only protein expression but also many phosphorylation events involved in developmental and stress-responsive pathways. Fourteen conserved and novel motifs were identified in the fungal phosphorylation events. PMID:27055109

  20. Epoch-based analysis of speech signals

    Indian Academy of Sciences (India)

    B Yegnanarayana; Suryakanth V Gangashetty

    2011-10-01

    Speech analysis is traditionally performed using short-time analysis to extract features in time and frequency domains. The window size for the analysis is fixed somewhat arbitrarily, mainly to account for the time varying vocal tract system during production. However, speech in its primary mode of excitation is produced due to impulse-like excitation in each glottal cycle. Anchoring the speech analysis around the glottal closure instants (epochs) yields significant benefits for speech analysis. Epoch-based analysis of speech helps not only to segment the speech signals based on speech production characteristics, but also helps in accurate analysis of speech. It enables extraction of important acoustic-phonetic features such as glottal vibrations, formants, instantaneous fundamental frequency, etc. Epoch sequence is useful to manipulate prosody in speech synthesis applications. Accurate estimation of epochs helps in characterizing voice quality features. Epoch extraction also helps in speech enhancement and multispeaker separation. In this tutorial article, the importance of epochs for speech analysis is discussed, and methods to extract the epoch information are reviewed. Applications of epoch extraction for some speech applications are demonstrated.

  1. Texture-based analysis of COPD

    DEFF Research Database (Denmark)

    Sørensen, Lauge Emil Borch Laurs; Nielsen, Mads; Lo, Pechin Chien Pau;

    2012-01-01

    This study presents a fully automatic, data-driven approach for texture-based quantitative analysis of chronic obstructive pulmonary disease (COPD) in pulmonary computed tomography (CT) images. The approach uses supervised learning where the class labels are, in contrast to previous work, based on...... subsequently applied to classify 200 independent images from the same screening trial. The texture-based measure was significantly better at discriminating between subjects with and without COPD than were the two most common quantitative measures of COPD in the literature, which are based on density. The...

  2. Phosphoproteomic analysis of differential expression of AGS cellular proteins in response to Helicobanter pyiori infection%幽门螺杆菌感染后人胃腺癌上皮细胞微量磷酸化蛋白质差异分析

    Institute of Scientific and Technical Information of China (English)

    肖迪; 宋衍燕; 赵飞; 何利华; 孟凡亮; 张慧芳; 张建中

    2009-01-01

    Objective To research the differential expression of trace phosphorylated proteins in human gastric adenocarcinoma epithelial (AGS) cells infected by Helicobacter pylori. Methods H. pylori 26695 strain infected AGS cells 4 h and AGS cells was cultivated for 4 h as a comparison. The proteins of AGS and comparison AGS cells were extracted. Their phosphorylated proteins were enriched by metal ion af-finity adsorption enrichment techniques. After desalinated and purified the phosphorylated proteins samples were separated by 2-dimensional polyacrylamide gel electrophoresis (2-DE) technique. Computer assisted image analysis was used to analyze the differential proteomic expression. The significantly differentially ex-pressed proteins were unambiguously assigned identities by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/TOF). Results Fifteen kinds of proteins were down-regulated, 4 kinds of new proteins were observed, 1 kind of proteins were up-regulated, 1 kind of proteins unexpression. The 21 proteins that were significantly differentially expressed , including cellular calcium ion homeostasis, transcription, interpretation, protein folding and transport, ribosomal assembly, centrosome replication, chromosome stability, cellular structure, cellular proliferation and apoptosis. Conclusion H. priori can cause a wide range change to human gastric adenocarcinoma epithelial cell protein pheshorylation. This change character has great significance to further comprehensive understanding of the pathogenesis of H. pylori.%目的 研究幽门螺杆菌(Hdicobacterpylori)作用后人胃腺癌上皮细胞(AGS)中微量磷酸化蛋白的变化情况.方法 采用金属离子亲和吸附富集技术富集幽门螺杆菌与AGS细胞相互作用以及AGS细胞的磷酸化蛋白,除盐纯化后利用二维凝胶电泳技术分离磷酸化蛋白,MALDI-TOF/TOF质谱答定确认蛋白.结果 幽门螺杆菌作用后AGS细胞有21种

  3. Cloud Based Development Issues: A Methodical Analysis

    Directory of Open Access Journals (Sweden)

    Sukhpal Singh

    2012-11-01

    Full Text Available Cloud based development is a challenging task for various software engineering projects, especifically for those which demand extraordinary quality, reusability and security along with general architecture. In this paper we present a report on a methodical analysis of cloud based development problems published in major computer science and software engineering journals and conferences organized by various researchers. Research papers were collected from different scholarly databases using search engines within a particular period of time. A total of 89 research papers were analyzed in this methodical study and we categorized into four classes according to the problems addressed by them. The majority of the research papers focused on quality (24 papers associated with cloud based development and 16 papers focused on analysis and design. By considering the areas focused by existing authors and their gaps, untouched areas of cloud based development can be discovered for future research works.

  4. Polyphase Order Analysis Based on Convolutional Approach

    Directory of Open Access Journals (Sweden)

    M. Drutarovsky

    1999-06-01

    Full Text Available The condition of rotating machines can be determined by measuring of periodic frequency components in the vibration signal which are directly related to the (typically changing rotational speed. Classical spectrum analysis with a constant sampling frequency is not an appropriate analysis method because of spectral smearing. Spectral analysis of vibration signal sampled synchronously with the angle of rotation, known as order analysis, suppress spectral smearing even with variable rotational speed. The paper presents optimised algorithm for polyphase order analysis based on non power of two DFT algorithm efficiently implemented by chirp FFT algorithm. Proposed algorithm decreases complexity of digital resampling algorithm, which is the most complex part of complete spectral order algorithm.

  5. Security Analysis of Discrete Logarithm Based Cryptosystems

    Institute of Scientific and Technical Information of China (English)

    WANG Yuzhu; LIAO Xiaofeng

    2006-01-01

    Discrete logarithm based cryptosystems have subtle problems that make the schemes vulnerable. This paper gives a comprehensive listing of security issues in the systems and analyzes three classes of attacks which are based on mathematical structure of the group which is used in the schemes, the disclosed information of the subgroup and implementation details respectively. The analysis will, in turn, allow us to motivate protocol design and implementation decisions.

  6. Social Network Analysis Based on Network Motifs

    OpenAIRE

    Xu Hong-lin; Yan Han-bing; Gao Cui-fang; Zhu Ping

    2014-01-01

    Based on the community structure characteristics, theory, and methods of frequent subgraph mining, network motifs findings are firstly introduced into social network analysis; the tendentiousness evaluation function and the importance evaluation function are proposed for effectiveness assessment. Compared with the traditional way based on nodes centrality degree, the new approach can be used to analyze the properties of social network more fully and judge the roles of the nodes effectively. I...

  7. Swarm Intelligence Based Algorithms: A Critical Analysis

    OpenAIRE

    Yang, Xin-She

    2014-01-01

    Many optimization algorithms have been developed by drawing inspiration from swarm intelligence (SI). These SI-based algorithms can have some advantages over traditional algorithms. In this paper, we carry out a critical analysis of these SI-based algorithms by analyzing their ways to mimic evolutionary operators. We also analyze the ways of achieving exploration and exploitation in algorithms by using mutation, crossover and selection. In addition, we also look at algorithms using dynamic sy...

  8. Unraveling the dynamics of sphingolipid metabolism by mass spectrometry based (phospho)proteomics

    NARCIS (Netherlands)

    Lebesgue, N.F.M.

    2016-01-01

    Sphingolipids (SLs) are essential components of cell membranes and are broad-range bioactive signaling molecules with imbalances affecting cellular function and contributing to pathologies ranging from neurodegenerative and metabolic disorders to cancer and aging. Deciphering how SL homeostasis is m

  9. Abstraction based Analysis and Arbiter Synthesis

    DEFF Research Database (Denmark)

    Ernits, Juhan-Peep; Yi, Wang

    2004-01-01

    The work focuses on the analysis of an example of synchronous systems containing FIFO buffers, registers and memory interconnected by several private and shared busses. The example used in this work is based on a Terma radar system memory interface case study from the IST AMETIST project....

  10. Node-based analysis of species distributions

    DEFF Research Database (Denmark)

    Borregaard, Michael Krabbe; Rahbek, Carsten; Fjeldså, Jon;

    2014-01-01

    with case studies on two groups with well-described biogeographical histories: a local-scale community data set of hummingbirds in the North Andes, and a large-scale data set of the distribution of all species of New World flycatchers. The node-based analysis of these two groups generates a set...

  11. Modeling Mass Spectrometry Based Protein Analysis

    OpenAIRE

    Eriksson, Jan; Fenyö, David

    2011-01-01

    The success of mass spectrometry based proteomics depends on efficient methods for data analysis. These methods require a detailed understanding of the information value of the data. Here, we describe how the information value can be elucidated by performing simulations using synthetic data.

  12. Desalting of phosphopeptides by tandem polypyrrole-c18 reverse phase micropipette tip (TMTip{sub PPY-C18}) based on hybrid electrostatic, {Pi}-{Pi} stacking and hydrophobic interactions for mass spectrometric analysis

    Energy Technology Data Exchange (ETDEWEB)

    Zheng Shi; Wang Xiaoli; Fu Jieying; Hu Xuejiao; Xiao Xiao; Huang Lulu; Zhou Youe [Key Laboratory of Pesticides and Chemical Biology, Ministry of Education, College of Chemistry, Central China Normal University, Wuhan, Hubei 430079 (China); Zhong Hongying, E-mail: hyzhong@mail.ccnu.edu.cn [Key Laboratory of Pesticides and Chemical Biology, Ministry of Education, College of Chemistry, Central China Normal University, Wuhan, Hubei 430079 (China)

    2012-04-29

    Highlights: Black-Right-Pointing-Pointer A new micropipette tip TMTip{sub PPY-C18} was developed for desalting of phosphopeptides. Black-Right-Pointing-Pointer TMTip{sub PPY-C18} is based on polypyrrole in tandem with C18 chromatographic material. Black-Right-Pointing-Pointer TMTip{sub PPY-C18} combines electrostatic, {Pi}-{Pi} stacking and hydrophobic interactions. Black-Right-Pointing-Pointer TMTip{sub PPY-C18} can be used in both acidic and basic experimental conditions. - Abstract: Desalting and concentration of peptides using reverse phase (RP) C18 chromatographic material based on hydrophobic interaction is a routine approach used in mass spectrometry (MS)-based proteomics. However, MS detection of small hydrophilic peptides, in particular, phosphopeptides that bear multiple negative charges, is challenging due to the insufficient binding to C18 stationary phase. We described here the development of a new desalting method that takes the unique properties of polypyrrole (PPY). The presence of positively charged nitrogen atoms under acidic conditions and polyunsaturated bonds in polypyrrole provide a prospect for enhanced adsorption of phosphopeptides or hydrophilic peptides through extra electrostatic and {Pi}-{Pi} stacking interactions in addition to hydrophobic interactions. In tandem with reversed phase C18 chromatographic material, the new type of desalting method termed as TMTip{sub PPY-C18} can significantly improve the MS detection of phosphopeptides with multiple phosphate groups and other small hydrophilic peptides. It has been applied to not only tryptic digest of model proteins but also the analysis of complex lysates of zebrafish eggs. The number of detected phosphate groups on a peptide ranged from 1 to 6. Particularly, polypyrrole based method can also be used in basic condition. Thus it provides a useful means to handle peptides that may not be detectable in acidic condition. It can be envisioned that the TMTip{sub PPY-C18} should be able to

  13. Desalting of phosphopeptides by tandem polypyrrole-c18 reverse phase micropipette tip (TMTipPPY-C18) based on hybrid electrostatic, Π–Π stacking and hydrophobic interactions for mass spectrometric analysis

    International Nuclear Information System (INIS)

    Highlights: ► A new micropipette tip TMTipPPY-C18 was developed for desalting of phosphopeptides. ► TMTipPPY-C18 is based on polypyrrole in tandem with C18 chromatographic material. ► TMTipPPY-C18 combines electrostatic, Π–Π stacking and hydrophobic interactions. ► TMTipPPY-C18 can be used in both acidic and basic experimental conditions. - Abstract: Desalting and concentration of peptides using reverse phase (RP) C18 chromatographic material based on hydrophobic interaction is a routine approach used in mass spectrometry (MS)-based proteomics. However, MS detection of small hydrophilic peptides, in particular, phosphopeptides that bear multiple negative charges, is challenging due to the insufficient binding to C18 stationary phase. We described here the development of a new desalting method that takes the unique properties of polypyrrole (PPY). The presence of positively charged nitrogen atoms under acidic conditions and polyunsaturated bonds in polypyrrole provide a prospect for enhanced adsorption of phosphopeptides or hydrophilic peptides through extra electrostatic and Π–Π stacking interactions in addition to hydrophobic interactions. In tandem with reversed phase C18 chromatographic material, the new type of desalting method termed as TMTipPPY-C18 can significantly improve the MS detection of phosphopeptides with multiple phosphate groups and other small hydrophilic peptides. It has been applied to not only tryptic digest of model proteins but also the analysis of complex lysates of zebrafish eggs. The number of detected phosphate groups on a peptide ranged from 1 to 6. Particularly, polypyrrole based method can also be used in basic condition. Thus it provides a useful means to handle peptides that may not be detectable in acidic condition. It can be envisioned that the TMTipPPY-C18 should be able to facilitate the exploration of large scale phosphoproteome.

  14. Phosphoproteome analysis during larval development and metamorphosis in the spionid polychaete Pseudopolydora vexillosa

    OpenAIRE

    Qian Pei-Yuan; Wang Hao; Mok Flora SY; Chandramouli Kondethimmanahalli H

    2011-01-01

    Abstract Background The metamorphosis of the spionid polychaete Pseudopolydora vexillosa includes spontaneous settlement onto soft-bottom habitats and morphogenesis that can be completed in a very short time. A previous study on the total changes to the proteome during the various developmental stages of P. vexillosa suggested that little or no de novo protein synthesis occurs during metamorphosis. In this study, we used multicolor fluorescence detection of proteins in 2-D gels for differenti...

  15. Large-scale phosphoproteome analysis in seedling leaves of Brachypodium distachyon L.

    OpenAIRE

    Lv, Dong-Wen; Li, Xin; Zhang, Ming; Gu, Ai-Qin; Zhen, Shou-Min; Wang, Chang; Li, Xiao-hui; Yan, Yue-Ming

    2014-01-01

    Background Protein phosphorylation is one of the most important post-translational modifications involved in the regulation of plant growth and development as well as diverse stress response. As a member of the Poaceae, Brachypodium distachyon L. is a new model plant for wheat and barley as well as several potential biofuel grasses such as switchgrass. Vegetative growth is vital for biomass accumulation of plants, but knowledge regarding the role of protein phosphorylation modification during...

  16. Large scale phosphoproteome analysis of LNCaP human prostate cancer cells

    OpenAIRE

    Myung, Jae-Kyung; Sadar, Marianne D

    2012-01-01

    Prostate cancer is the most frequently diagnosed cancer among men in the western world. The androgen receptor, a phosphoprotein, is suspected to be involved in all stages of the prostate cancer. Androgen receptor activity can be modulated by various kinases such as PKA, MAPK, AKT, and Src. Phosphorylation is an important post-translational modification and serves as a molecular on/off switch to regulate signaling. Disruptions of cellular phosphorylation are associated with various diseases su...

  17. Phosphoproteomic profiling analysis in pediatric acute leukemias and in solid tumors of the adult

    International Nuclear Information System (INIS)

    We analyzed 120 pediatric patients affected with B-cell AL (B-Acute Lymphoblastic Leukemia) by Reverse Phase Protein Arrays (RPPA). Leukemia cells from bone marrow aspirates were stored in liquid nitrogen in the Bio Bank of the Laboratory of Pediatric Onco hematology in Padova. Clinical data, such as immuno phenotype, outcome, response to therapy and chromosomal translocations, were collected for all the patients

  18. Proteomic and phosphoproteomic analysis of signalling by adhesion and growth factor receptors in mammary epithelial cells

    OpenAIRE

    Paul, Nikki

    2014-01-01

    Cell adhesion and communication are essential for tissue morphogenesis and repair in healthy multicellular organisms. However, dysregulation of these processes can drive disease progression in conditions such as cancer. Selective cell adhesion to the extracellular matrix is mediated by integrins, a family of transmembrane receptors that compartmentalise signalling and organise the cytoskeleton. Adhesion receptors provide spatial cues to cells to allow them to respond to growth factor and cyto...

  19. Online automated in vivo zebrafish phosphoproteomics: from large-scale analysis down to a single embryo.

    NARCIS (Netherlands)

    Lemeer, S.; Pinkse, M.W.H.; Mohammed, S.; van Breukelen, B.; den Hertog, J.; Slijper, M.; Heck, A.

    2008-01-01

    In the developing embryo, as in many other biological processes, complex signaling pathways are under tight control of reversible phosphorylation, guiding cell proliferation, differentiation, and growth. Therefore the large-scale identification of signaling proteins and their post-translational modi

  20. Phosphoproteome analysis of the MAPK pathway reveals previously undetected feedback mechanisms.

    Science.gov (United States)

    Gnad, Florian; Doll, Sophia; Song, Kyung; Stokes, Matthew P; Moffat, John; Liu, Bonnie; Arnott, David; Wallin, Jeffrey; Friedman, Lori S; Hatzivassiliou, Georgia; Belvin, Marcia

    2016-07-01

    The RAS-RAF-MEK-ERK (MAPK) pathway is prevalently perturbed in cancer. Recent large-scale sequencing initiatives profiled thousands of tumors providing insight into alterations at the DNA and RNA levels. These efforts confirmed that key nodes of the MAPK pathway, in particular KRAS and BRAF, are among the most frequently altered proteins in cancer. The establishment of targeted therapies, however, has proven difficult. To decipher the underlying challenges, it is essential to decrypt the phosphorylation network spanned by the MAPK core axis. Using mass spectrometry we identified 2241 phosphorylation sites on 1020 proteins, and measured their responses to inhibition of MEK or ERK. Multiple phosphorylation patterns revealed previously undetected feedback, as upstream signaling nodes, including receptor kinases, showed changes at the phosphorylation level. We provide a dataset rich in potential therapeutic targets downstream of the MAPK cascade. By integrating TCGA (The Cancer Genome Atlas) data, we highlight some downstream phosphoproteins that are frequently altered in cancer. All MS data have been deposited in the ProteomeXchange with identifier PXD003908 (http://proteomecentral.proteomexchange.org/dataset/PXD003908). PMID:27273156

  1. 2D-HPLC-MS phosphoproteomics analysis of lung and breast carcinomas

    International Nuclear Information System (INIS)

    The project was conceived as two phases. In the first phase, we planned to validate markers, or expression patterns, previously described in the literature as being characteristic of specific tumors. In a second phase, a search for new markers associated with different tumors was to be carried on, developing new 2D-HPLC methods coupled with API-ESI mass spectrometry, with the ultimate goal of their rapid identification in biological specimens. For what concerns the first phase, the most relevant data are described in (Ceccarelli et al., 2008) and further analyzed in (Rubartelli et al., 2007). In these studies, we have compared the synthesis and intracellular accumulation levels of two oxido-reductases having also an extracellular cytokine function, the macrophage inhibitory factor (MIF) and thioredoxin (Trx)

  2. Canonical analysis based on mutual information

    DEFF Research Database (Denmark)

    Nielsen, Allan Aasbjerg; Vestergaard, Jacob Schack

    2015-01-01

    Canonical correlation analysis (CCA) is an established multi-variate statistical method for finding similarities between linear combinations of (normally two) sets of multivariate observations. In this contribution we replace (linear) correlation as the measure of association between the linear...... combinations with the information theoretical measure mutual information (MI). We term this type of analysis canonical information analysis (CIA). MI allows for the actual joint distribution of the variables involved and not just second order statistics. While CCA is ideal for Gaussian data, CIA facilitates...... analysis of variables with different genesis and therefore different statistical distributions and different modalities. As a proof of concept we give a toy example. We also give an example with one (weather radar based) variable in the one set and eight spectral bands of optical satellite data in the...

  3. Network-based analysis of proteomic profiles

    KAUST Repository

    Wong, Limsoon

    2016-01-26

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

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

    Directory of Open Access Journals (Sweden)

    Mann Matthias

    2010-02-01

    Full Text Available Abstract 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

  5. TEST COVERAGE ANALYSIS BASED ON PROGRAM SLICING

    Institute of Scientific and Technical Information of China (English)

    Chen Zhenqiang; Xu Baowen; Guanjie

    2003-01-01

    Coverage analysis is a structural testing technique that helps to eliminate gaps in atest suite and determines when to stop testing. To compute test coverage, this letter proposes anew concept coverage about variables, based on program slicing. By adding powers accordingto their importance, the users can focus on the important variables to obtain higher test coverage.The letter presents methods to compute basic coverage based on program structure graphs. Inmost cases, the coverage obtained in the letter is bigger than that obtained by a traditionalmeasure, because the coverage about a variable takes only the related codes into account.

  6. SILAC-based quantification of changes in protein tyrosine phosphorylation induced by Interleukin-2 (IL-2 and IL-15 in T-lymphocytes

    Directory of Open Access Journals (Sweden)

    Nerea Osinalde

    2015-12-01

    Full Text Available This data article presents the first large-scale quantitative phosphoproteomics dataset generated to decipher the signaling networks initiated by IL-2 and IL-15 in T-lymphocytes. Data was collected by combining immunoprecipitation of tyrosine phosphorylated proteins and TiO2-based phosphopeptide enrichment with SILAC-based quantitative mass spectrometry. We report all the proteins and phosphotyrosine-containing peptides identified and quantified in IL-2- and IL-15-stimulated T-lymphocytes. The gene ontology analysis of IL-2 and IL-15 effector proteins detected in the present work is also included. The data supplied in this article is related to the research work entitled “Simultaneous dissection and comparison of IL-2 and IL-15 signaling pathways by global quantitative phosphoproteomics” [1]. All mass spectrometry data have been deposited in the ProteomeXchange with the identifier PXD001129.

  7. Structure-based analysis of Web sites

    OpenAIRE

    Yen, B

    2004-01-01

    The performance of information retrieval on the Web is heavily influenced by the organization of Web pages, user navigation patterns, and guidance-related functions. Having observed the lack of measures to reflect this factor, this paper focuses on an approach based on both structure properties and navigation data to analyze and improve the performance of Web site. Two types of indices are defined two major factors for analysis and improvement- "aaccessibility" reflects the structure property...

  8. Quantum entanglement analysis based on abstract interpretation

    OpenAIRE

    Perdrix, Simon

    2008-01-01

    Entanglement is a non local property of quantum states which has no classical counterpart and plays a decisive role in quantum information theory. Several protocols, like the teleportation, are based on quantum entangled states. Moreover, any quantum algorithm which does not create entanglement can be efficiently simulated on a classical computer. The exact role of the entanglement is nevertheless not well understood. Since an exact analysis of entanglement evolution induces an exponential sl...

  9. Particle Pollution Estimation Based on Image Analysis

    OpenAIRE

    Liu, Chenbin; Tsow, Francis; Zou, Yi; Tao, Nongjian

    2016-01-01

    Exposure to fine particles can cause various diseases, and an easily accessible method to monitor the particles can help raise public awareness and reduce harmful exposures. Here we report a method to estimate PM air pollution based on analysis of a large number of outdoor images available for Beijing, Shanghai (China) and Phoenix (US). Six image features were extracted from the images, which were used, together with other relevant data, such as the position of the sun, date, time, geographic...

  10. XML-based analysis interface for particle physics data analysis

    International Nuclear Information System (INIS)

    The letter emphasizes on an XML-based interface and its framework for particle physics data analysis. The interface uses a concise XML syntax to describe, in data analysis, the basic tasks: event-selection, kinematic fitting, particle identification, etc. and a basic processing logic: the next step goes on if and only if this step succeeds. The framework can perform an analysis without compiling by loading the XML-interface file, setting p in run-time and running dynamically. An analysis coding in XML instead of C++, easy-to-understood arid use, effectively reduces the work load, and enables users to carry out their analyses quickly. The framework has been developed on the BESⅢ offline software system (BOSS) with the object-oriented C++ programming. These functions, required by the regular tasks and the basic processing logic, are implemented with both standard modules or inherited from the modules in BOSS. The interface and its framework have been tested to perform physics analysis. (authors)

  11. Chapter 11. Community analysis-based methods

    Energy Technology Data Exchange (ETDEWEB)

    Cao, Y.; Wu, C.H.; Andersen, G.L.; Holden, P.A.

    2010-05-01

    Microbial communities are each a composite of populations whose presence and relative abundance in water or other environmental samples are a direct manifestation of environmental conditions, including the introduction of microbe-rich fecal material and factors promoting persistence of the microbes therein. As shown by culture-independent methods, different animal-host fecal microbial communities appear distinctive, suggesting that their community profiles can be used to differentiate fecal samples and to potentially reveal the presence of host fecal material in environmental waters. Cross-comparisons of microbial communities from different hosts also reveal relative abundances of genetic groups that can be used to distinguish sources. In increasing order of their information richness, several community analysis methods hold promise for MST applications: phospholipid fatty acid (PLFA) analysis, denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (TRFLP), cloning/sequencing, and PhyloChip. Specific case studies involving TRFLP and PhyloChip approaches demonstrate the ability of community-based analyses of contaminated waters to confirm a diagnosis of water quality based on host-specific marker(s). The success of community-based MST for comprehensively confirming fecal sources relies extensively upon using appropriate multivariate statistical approaches. While community-based MST is still under evaluation and development as a primary diagnostic tool, results presented herein demonstrate its promise. Coupled with its inherently comprehensive ability to capture an unprecedented amount of microbiological data that is relevant to water quality, the tools for microbial community analysis are increasingly accessible, and community-based approaches have unparalleled potential for translation into rapid, perhaps real-time, monitoring platforms.

  12. SILAC-based quantification of changes in protein tyrosine phosphorylation induced by Interleukin-2 (IL-2) and IL-15 in T-lymphocytes

    DEFF Research Database (Denmark)

    Osinalde, Nerea; Sánchez-Quiles, Virginia; Akimov, Vyacheslav;

    2015-01-01

    This data article presents the first large-scale quantitative phosphoproteomics dataset generated to decipher the signaling networks initiated by IL-2 and IL-15 in T-lymphocytes. Data was collected by combining immunoprecipitation of tyrosine phosphorylated proteins and TiO2-based phosphopeptide....... The data supplied in this article is related to the research work entitled "Simultaneous dissection and comparison of IL-2 and IL-15 signaling pathways by global quantitative phosphoproteomics" [1]. All mass spectrometry data have been deposited in the ProteomeXchange with the identifier PXD001129....

  13. Key Point Based Data Analysis Technique

    Science.gov (United States)

    Yang, Su; Zhang, Yong

    In this paper, a new framework for data analysis based on the "key points" in data distribution is proposed. Here, the key points contain three types of data points: bridge points, border points, and skeleton points, where our main contribution is the bridge points. For each type of key points, we have developed the corresponding detection algorithm and tested its effectiveness with several synthetic data sets. Meanwhile, we further developed a new hierarchical clustering algorithm SPHC (Skeleton Point based Hierarchical Clustering) to demonstrate the possible applications of the key points acquired. Based on some real-world data sets, we experimentally show that SPHC performs better compared with several classical clustering algorithms including Complete-Link Hierarchical Clustering, Single-Link Hierarchical Clustering, KMeans, Ncut, and DBSCAN.

  14. Chip based electroanalytical systems for cell analysis

    DEFF Research Database (Denmark)

    Spegel, C.; Heiskanen, A.; Skjolding, L.H.D.;

    2008-01-01

    ' measurements of processes related to living cells, i.e., systems without lysing the cells. The focus is on chip based amperometric and impedimetric cell analysis systems where measurements utilizing solely carbon fiber microelectrodes (CFME) and other nonchip electrode formats, such as CFME for exocytosis......This review with 239 references has as its aim to give the reader an introduction to the kinds of methods used for developing microchip based electrode systems as well as to cover the existing literature on electroanalytical systems where microchips play a crucial role for 'nondestructive...... studies and scanning electrochemical microscopy (SECM) studies of living cells have been omitted. Included is also a discussion about some future and emerging nano tools and considerations that might have an impact on the future of "nondestructive" chip based electroanalysis of living cells....

  15. Fault-based analysis of flexible ciphers

    Directory of Open Access Journals (Sweden)

    V.I.Korjik

    2002-07-01

    Full Text Available We consider security of some flexible ciphers against differential fault analysis (DFA. We present a description of the fault-based attack on two kinds of the flexible ciphers. The first kind is represented by the fast software-oriented cipher based on data-dependent subkey selection (DDSS, in which flexibility corresponds to the use of key-dependent operations. The second kind is represented by a DES-like cryptosystem GOST with secrete S-boxes. In general, the use of some secrete operations and procedures contributes to the security of the cryptosystem, however degree of this contribution depends significantly on the structure of the encryption mechanism. It is shown how to attack the DDSS-based flexible cipher using DFA though this cipher is secure against standard variants of the differential and linear cryptanalysis. We also give an outline of ciphers RC5 and GOST showing that they are also insecure against DFA-based attack. We suggest also a modification of the DDSS mechanism and a variant of the advanced DDSS-based flexible cipher that is secure against attacks based on random hardware faults.

  16. A Comprehensive Analysis of the Dynamic Response to Aphidicolin-Mediated Replication Stress Uncovers Targets for ATM and ATMIN

    Directory of Open Access Journals (Sweden)

    Abdelghani Mazouzi

    2016-04-01

    Full Text Available The cellular response to replication stress requires the DNA-damage-responsive kinase ATM and its cofactor ATMIN; however, the roles of this signaling pathway following replication stress are unclear. To identify the functions of ATM and ATMIN in response to replication stress, we utilized both transcriptomics and quantitative mass-spectrometry-based phosphoproteomics. We found that replication stress induced by aphidicolin triggered widespread changes in both gene expression and protein phosphorylation patterns. These changes gave rise to distinct early and late replication stress responses. Furthermore, our analysis revealed previously unknown targets of ATM and ATMIN downstream of replication stress. We demonstrate ATMIN-dependent phosphorylation of H2AX and of CRMP2, a protein previously implicated in Alzheimer’s disease but not in the DNA damage response. Overall, our dataset provides a comprehensive resource for discovering the cellular responses to replication stress and, potentially, associated pathologies.

  17. System based practice: a concept analysis

    Science.gov (United States)

    YAZDANI, SHAHRAM; HOSSEINI, FAKHROLSADAT; AHMADY, SOLEIMAN

    2016-01-01

    Introduction Systems-Based Practice (SBP) is one of the six competencies introduced by the ACGME for physicians to provide high quality of care and also the most challenging of them in performance, training, and evaluation of medical students. This concept analysis clarifies the concept of SBP by identifying its components to make it possible to differentiate it from other similar concepts. For proper training of SBP and to ensure these competencies in physicians, it is necessary to have an operational definition, and SBP’s components must be precisely defined in order to provide valid and reliable assessment tools. Methods Walker & Avant’s approach to concept analysis was performed in eight stages: choosing a concept, determining the purpose of analysis, identifying all uses of the concept, defining attributes, identifying a model case, identifying borderline, related, and contrary cases, identifying antecedents and consequences, and defining empirical referents. Results Based on the analysis undertaken, the attributes of SBP includes knowledge of the system, balanced decision between patients’ need and system goals, effective role playing in interprofessional health care team, system level of health advocacy, and acting for system improvement. System thinking and a functional system are antecedents and system goals are consequences. A case model, as well as border, and contrary cases of SBP, has been introduced. Conclusion he identification of SBP attributes in this study contributes to the body of knowledge in SBP and reduces the ambiguity of this concept to make it possible for applying it in training of different medical specialties. Also, it would be possible to develop and use more precise tools to evaluate SBP competency by using empirical referents of the analysis. PMID:27104198

  18. Integrative analysis of kinase networks in TRAIL-induced apoptosis provides a source of potential targets for combination therapy

    DEFF Research Database (Denmark)

    So, Jonathan; Pasculescu, Adrian; Dai, Anna Y.;

    2015-01-01

    phosphoproteomics. With these protein interaction maps, we modeled information flow through the networks and identified apoptosis-modifying kinases that are highly connected to regulated substrates downstream of TRAIL. The results of this analysis provide a resource of potential targets for the development of TRAIL...

  19. 小鼠肝脏磷酸化蛋白质组的二维液相色谱分离%Two dimensional liquid phase chromatographic fractionation of phosphoproteome of mouse liver

    Institute of Scientific and Technical Information of China (English)

    黎永明; 陈腾祥; 杨丽萍; 刘亚伟; 姜勇

    2005-01-01

    Objective To fractionate phosphoproteome of mouse liver by two-dimensional (2D) liquid phase chromatography fractionation. Methods Phosphoproteins were extracted from lysates of normal mice livers by phosphate metal affinity chromatography (PMAC) resin. The phosphoproteins were exchanged by start buffer and separated by chromatofocusing in the first dimension. Then the fractions between pH 8.5 and pH 4.0 were separated by non-porous silica (NPS) reverse-phase high performance liquid chromatography (RP-HPLC). Finally, the UV maps were converted into gel-like maps by ProteoVue software. Results Phosphoproteins of mouse liver were successfully extracted and fractionated by two dimensional liquid phase chromatographic fractionation after concentration and desalt. Then pI/UV map of mouse liver phosphoproteome was successfully set-up. There are 16 fractions between pH 8.5 and pH 4.0 after chromatofocusing in the first dimension and the UV maps of each fraction were converted into pI/UV gel-like maps. Conclusions Combination of technique of phosphoproteins enrichment and 2-D liquid phase chromatographic fractionation is an effective approach to research phosphoproteome and the key base for further identification and investigation of phosphoproteins.%目的利用二维液相色谱法分离小鼠肝脏磷酸化蛋白质组.方法取正常小鼠肝脏,裂解肝脏后利用磷酸盐金属亲和层析(PMAC)树脂提取磷酸化蛋白.将磷酸化蛋白用初始缓冲液置换后,进行一维色谱聚焦分离,再将一维收集的pH值在8.5至4.0之间的组分分别进行二维无孔硅胶反相高效液相色谱(RP-HPLC)分离.最后利用ProteoVue软件将二维UV图转换成胶图进行分析.结果成功提取了小鼠肝脏磷酸化蛋白,并在浓缩除盐后通过二维液相色谱分离成功建立小鼠肝脏磷酸化蛋白质组pI/UV图谱.其中,一维色谱聚焦分离pH值在8.5至4.0之间共收集16个组分,每个组分的二维UV图转换成p

  20. Dependent failure analysis of NPP data bases

    International Nuclear Information System (INIS)

    A technical approach for analyzing plant-specific data bases for vulnerabilities to dependent failures has been developed and applied. Since the focus of this work is to aid in the formulation of defenses to dependent failures, rather than to quantify dependent failure probabilities, the approach of this analysis is critically different. For instance, the determination of component failure dependencies has been based upon identical failure mechanisms related to component piecepart failures, rather than failure modes. Also, component failures involving all types of component function loss (e.g., catastrophic, degraded, incipient) are equally important to the predictive purposes of dependent failure defense development. Consequently, dependent component failures are identified with a different dependent failure definition which uses a component failure mechanism categorization scheme in this study. In this context, clusters of component failures which satisfy the revised dependent failure definition are termed common failure mechanism (CFM) events. Motor-operated valves (MOVs) in two nuclear power plant data bases have been analyzed with this approach. The analysis results include seven different failure mechanism categories; identified potential CFM events; an assessment of the risk-significance of the potential CFM events using existing probabilistic risk assessments (PRAs); and postulated defenses to the identified potential CFM events. (orig.)

  1. Gait correlation analysis based human identification.

    Science.gov (United States)

    Chen, Jinyan

    2014-01-01

    Human gait identification aims to identify people by a sequence of walking images. Comparing with fingerprint or iris based identification, the most important advantage of gait identification is that it can be done at a distance. In this paper, silhouette correlation analysis based human identification approach is proposed. By background subtracting algorithm, the moving silhouette figure can be extracted from the walking images sequence. Every pixel in the silhouette has three dimensions: horizontal axis (x), vertical axis (y), and temporal axis (t). By moving every pixel in the silhouette image along these three dimensions, we can get a new silhouette. The correlation result between the original silhouette and the new one can be used as the raw feature of human gait. Discrete Fourier transform is used to extract features from this correlation result. Then, these features are normalized to minimize the affection of noise. Primary component analysis method is used to reduce the features' dimensions. Experiment based on CASIA database shows that this method has an encouraging recognition performance. PMID:24592144

  2. Gait Correlation Analysis Based Human Identification

    Directory of Open Access Journals (Sweden)

    Jinyan Chen

    2014-01-01

    Full Text Available Human gait identification aims to identify people by a sequence of walking images. Comparing with fingerprint or iris based identification, the most important advantage of gait identification is that it can be done at a distance. In this paper, silhouette correlation analysis based human identification approach is proposed. By background subtracting algorithm, the moving silhouette figure can be extracted from the walking images sequence. Every pixel in the silhouette has three dimensions: horizontal axis (x, vertical axis (y, and temporal axis (t. By moving every pixel in the silhouette image along these three dimensions, we can get a new silhouette. The correlation result between the original silhouette and the new one can be used as the raw feature of human gait. Discrete Fourier transform is used to extract features from this correlation result. Then, these features are normalized to minimize the affection of noise. Primary component analysis method is used to reduce the features’ dimensions. Experiment based on CASIA database shows that this method has an encouraging recognition performance.

  3. Multifractal Time Series Analysis Based on Detrended Fluctuation Analysis

    Science.gov (United States)

    Kantelhardt, Jan; Stanley, H. Eugene; Zschiegner, Stephan; Bunde, Armin; Koscielny-Bunde, Eva; Havlin, Shlomo

    2002-03-01

    In order to develop an easily applicable method for the multifractal characterization of non-stationary time series, we generalize the detrended fluctuation analysis (DFA), which is a well-established method for the determination of the monofractal scaling properties and the detection of long-range correlations. We relate the new multifractal DFA method to the standard partition function-based multifractal formalism, and compare it to the wavelet transform modulus maxima (WTMM) method which is a well-established, but more difficult procedure for this purpose. We employ the multifractal DFA method to determine if the heartrhythm during different sleep stages is characterized by different multifractal properties.

  4. Rweb:Web-based Statistical Analysis

    Directory of Open Access Journals (Sweden)

    Jeff Banfield

    1999-03-01

    Full Text Available Rweb is a freely accessible statistical analysis environment that is delivered through the World Wide Web (WWW. It is based on R, a well known statistical analysis package. The only requirement to run the basic Rweb interface is a WWW browser that supports forms. If you want graphical output you must, of course, have a browser that supports graphics. The interface provides access to WWW accessible data sets, so you may run Rweb on your own data. Rweb can provide a four window statistical computing environment (code input, text output, graphical output, and error information through browsers that support Javascript. There is also a set of point and click modules under development for use in introductory statistics courses.

  5. Electric Equipment Diagnosis based on Wavelet Analysis

    Directory of Open Access Journals (Sweden)

    Stavitsky Sergey A.

    2016-01-01

    Full Text Available Due to electric equipment development and complication it is necessary to have a precise and intense diagnosis. Nowadays there are two basic ways of diagnosis: analog signal processing and digital signal processing. The latter is more preferable. The basic ways of digital signal processing (Fourier transform and Fast Fourier transform include one of the modern methods based on wavelet transform. This research is dedicated to analyzing characteristic features and advantages of wavelet transform. This article shows the ways of using wavelet analysis and the process of test signal converting. In order to carry out this analysis, computer software Mathcad was used and 2D wavelet spectrum for a complex function was created.

  6. Arabic Interface Analysis Based on Cultural Markers

    Directory of Open Access Journals (Sweden)

    Mohammadi Akheela Khanum

    2012-01-01

    Full Text Available This study examines the Arabic interface design elements that are largely influenced by the cultural values. Cultural markers are examined in websites from educational, business, and media. Cultural values analysis is based on Geert Hofstedes cultural dimensions. The findings show that there are cultural markers which are largely influenced by the culture and that the Hofstedes score for Arab countries is partially supported by the website design components examined in this study. Moderate support was also found for the long term orientation, for which Hoftsede has no score.

  7. Similarity-based pattern analysis and recognition

    CERN Document Server

    Pelillo, Marcello

    2013-01-01

    This accessible text/reference presents a coherent overview of the emerging field of non-Euclidean similarity learning. The book presents a broad range of perspectives on similarity-based pattern analysis and recognition methods, from purely theoretical challenges to practical, real-world applications. The coverage includes both supervised and unsupervised learning paradigms, as well as generative and discriminative models. Topics and features: explores the origination and causes of non-Euclidean (dis)similarity measures, and how they influence the performance of traditional classification alg

  8. Arabic Interface Analysis Based on Cultural Markers

    CERN Document Server

    Khanum, Mohammadi Akheela; Chaurasia, Mousmi A

    2012-01-01

    This study examines the Arabic interface design elements that are largely influenced by the cultural values. Cultural markers are examined in websites from educational, business, and media. Cultural values analysis is based on Geert Hofstede's cultural dimensions. The findings show that there are cultural markers which are largely influenced by the culture and that the Hofstede's score for Arab countries is partially supported by the website design components examined in this study. Moderate support was also found for the long term orientation, for which Hoftsede has no score.

  9. Video semantic content analysis based on ontology

    OpenAIRE

    Bai, Liang; Lao, Songyang; Jones, Gareth J.F.; Smeaton, Alan F.

    2007-01-01

    The rapid increase in the available amount of video data is creating a growing demand for efficient methods for understanding and managing it at the semantic level. New multimedia standards, such as MPEG-4 and MPEG-7, provide the basic functionalities in order to manipulate and transmit objects and metadata. But importantly, most of the content of video data at a semantic level is out of the scope of the standards. In this paper, a video semantic content analysis framework based on ontology i...

  10. Phosphoproteomics reveals extensive in vivo phosphorylation of Arabidopsis proteins involved in RNA metabolism

    OpenAIRE

    la Fuente van Bentem, Sergio de; Anrather, Dorothea; Roitinger, Elisabeth; Djamei, Armin; Hufnagl, Thomas; Barta, Andrea; Csaszar, Edina; Dohnal, Ilse; Lecourieux, David; Hirt, Heribert

    2006-01-01

    Most regulatory pathways are governed by the reversible phosphorylation of proteins. Recent developments in mass spectrometry-based technology allow the large-scale analysis of protein phosphorylation. Here, we show the application of immobilized metal affinity chromatography to purify phosphopeptides from Arabidopsis extracts. Phosphopeptide sequences were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS/MS). A total of 79 unique phosphorylation sites were determined in...

  11. Motion Analysis Based on Invertible Rapid Transform

    Directory of Open Access Journals (Sweden)

    J. Turan

    1999-06-01

    Full Text Available This paper presents the results of a study on the use of invertible rapid transform (IRT for the motion estimation in a sequence of images. Motion estimation algorithms based on the analysis of the matrix of states (produced in the IRT calculation are described. The new method was used experimentally to estimate crowd and traffic motion from the image data sequences captured at railway stations and at high ways in large cities. The motion vectors may be used to devise a polar plot (showing velocity magnitude and direction for moving objects where the dominant motion tendency can be seen. The experimental results of comparison of the new motion estimation methods with other well known block matching methods (full search, 2D-log, method based on conventional (cross correlation (CC function or phase correlation (PC function for application of crowd motion estimation are also presented.

  12. Visibility Graph Based Time Series Analysis.

    Directory of Open Access Journals (Sweden)

    Mutua Stephen

    Full Text Available Network based time series analysis has made considerable achievements in the recent years. By mapping mono/multivariate time series into networks, one can investigate both it's microscopic and macroscopic behaviors. However, most proposed approaches lead to the construction of static networks consequently providing limited information on evolutionary behaviors. In the present paper we propose a method called visibility graph based time series analysis, in which series segments are mapped to visibility graphs as being descriptions of the corresponding states and the successively occurring states are linked. This procedure converts a time series to a temporal network and at the same time a network of networks. Findings from empirical records for stock markets in USA (S&P500 and Nasdaq and artificial series generated by means of fractional Gaussian motions show that the method can provide us rich information benefiting short-term and long-term predictions. Theoretically, we propose a method to investigate time series from the viewpoint of network of networks.

  13. Curvelet based offline analysis of SEM images.

    Directory of Open Access Journals (Sweden)

    Syed Hamad Shirazi

    Full Text Available Manual offline analysis, of a scanning electron microscopy (SEM image, is a time consuming process and requires continuous human intervention and efforts. This paper presents an image processing based method for automated offline analyses of SEM images. To this end, our strategy relies on a two-stage process, viz. texture analysis and quantification. The method involves a preprocessing step, aimed at the noise removal, in order to avoid false edges. For texture analysis, the proposed method employs a state of the art Curvelet transform followed by segmentation through a combination of entropy filtering, thresholding and mathematical morphology (MM. The quantification is carried out by the application of a box-counting algorithm, for fractal dimension (FD calculations, with the ultimate goal of measuring the parameters, like surface area and perimeter. The perimeter is estimated indirectly by counting the boundary boxes of the filled shapes. The proposed method, when applied to a representative set of SEM images, not only showed better results in image segmentation but also exhibited a good accuracy in the calculation of surface area and perimeter. The proposed method outperforms the well-known Watershed segmentation algorithm.

  14. Analysis of Protein Phosphorylation Using Mass Spectrometry

    Directory of Open Access Journals (Sweden)

    Pao-Chi Liao

    2008-06-01

    Full Text Available Protein phosphorylation has been known to be a pivotalmodification regulating many cellular activities and functions.Except for several conventional techniques, mass spectrometry-based strategies are increasingly considered as vital toolsthat can be utilized to characterize phosphorylated peptides orproteins. In this article, we summarized currently availablemass spectrometry-based techniques for the analysis of phosphorylation.Due to the low abundance of phosphopeptides,enrichment steps such as specific antibodies, immobilizedmetal affinity chromatography, and specific tags are crucial fortheir use in detection. Since the non-specific binding of theenrichment techniques are constantly of major concerns, phosphatasetreatment, neutral loss scan, or precursor ion scanenable the recognition of the phosphopeptide signals. In addition,quantitative methods including isotope labeling and masstags are also discussed. Phosphoproteome analysis seems to provide elucidation of signalingnetworks and global decipherment of cell activities, which require powerful analytical methodsfor complete and routine identification of the phosphorylation event. Despite thatnumerous approaches have been exploited, comprehensive analysis of protein phosphorylationremains a challenging task. With the progressively more improvements of instrumentsand methodologies, we can foresee the implementation of a comprehensive approach for theanalysis of phosphorylation states of proteins.

  15. Visual Similarity Based Document Layout Analysis

    Institute of Scientific and Technical Information of China (English)

    Di Wen; Xiao-Qing Ding

    2006-01-01

    In this paper, a visual similarity based document layout analysis (DLA) scheme is proposed, which by using clustering strategy can adaptively deal with documents in different languages, with different layout structures and skew angles. Aiming at a robust and adaptive DLA approach, the authors first manage to find a set of representative filters and statistics to characterize typical texture patterns in document images, which is through a visual similarity testing process.Texture features are then extracted from these filters and passed into a dynamic clustering procedure, which is called visual similarity clustering. Finally, text contents are located from the clustered results. Benefit from this scheme, the algorithm demonstrates strong robustness and adaptability in a wide variety of documents, which previous traditional DLA approaches do not possess.

  16. Watermark Resistance Analysis Based On Linear Transformation

    Directory of Open Access Journals (Sweden)

    N.Karthika Devi

    2012-06-01

    Full Text Available Generally, digital watermark can be embedded in any copyright image whose size is not larger than it. The watermarking schemes can be classified into two categories: spatial domain approach or transform domain approach. Previous works have shown that the transform domain scheme is typically more robust to noise, common image processing, and compression when compared with the spatial transform scheme. Improvements in performance of watermarking schemes can be obtained by exploiting the characteristics of the human visual system (HVS in the watermarking process. We propose a linear transformation based watermarking algorithm. The watermarking bits are embedded into cover image to produce watermarked image. The efficiency of watermark is checked using pre-defined attacks. Attack resistance analysis is done using BER (Bit Error Rate calculation. Finally, the Quality of the watermarked image can be obtained.

  17. Voxel-Based LIDAR Analysis and Applications

    Science.gov (United States)

    Hagstrom, Shea T.

    One of the greatest recent changes in the field of remote sensing is the addition of high-quality Light Detection and Ranging (LIDAR) instruments. In particular, the past few decades have been greatly beneficial to these systems because of increases in data collection speed and accuracy, as well as a reduction in the costs of components. These improvements allow modern airborne instruments to resolve sub-meter details, making them ideal for a wide variety of applications. Because LIDAR uses active illumination to capture 3D information, its output is fundamentally different from other modalities. Despite this difference, LIDAR datasets are often processed using methods appropriate for 2D images and that do not take advantage of its primary virtue of 3-dimensional data. It is this problem we explore by using volumetric voxel modeling. Voxel-based analysis has been used in many applications, especially medical imaging, but rarely in traditional remote sensing. In part this is because the memory requirements are substantial when handling large areas, but with modern computing and storage this is no longer a significant impediment. Our reason for using voxels to model scenes from LIDAR data is that there are several advantages over standard triangle-based models, including better handling of overlapping surfaces and complex shapes. We show how incorporating system position information from early in the LIDAR point cloud generation process allows radiometrically-correct transmission and other novel voxel properties to be recovered. This voxelization technique is validated on simulated data using the Digital Imaging and Remote Sensing Image Generation (DIRSIG) software, a first-principles based ray-tracer developed at the Rochester Institute of Technology. Voxel-based modeling of LIDAR can be useful on its own, but we believe its primary advantage is when applied to problems where simpler surface-based 3D models conflict with the requirement of realistic geometry. To

  18. Cognitive fusion analysis based on context

    Science.gov (United States)

    Blasch, Erik P.; Plano, Susan

    2004-04-01

    The standard fusion model includes active and passive user interaction in level 5 - "User Refinement". User refinement is more than just details of passive automation partitioning - it is the active management of information. While a fusion system can explore many operational conditions over myopic changes, the user has the ability to reason about the hyperopic "big picture." Blasch and Plano developed cognitive-fusion models that address user constraints including: intent, attention, trust, workload, and throughput to facilitate hyperopic analysis. To enhance user-fusion performance modeling (i.e. confidence, timeliness, and accuracy); we seek to explore the nature of context. Context, the interrelated conditions of which something exists, can be modeled in many ways including geographic, sensor, object, and environmental conditioning. This paper highlights user refinement actions based on context to constrain the fusion analysis for accurately representing the trade space in the real world. As an example, we explore a target identification task in which contextual information from the user"s cognitive model is imparted to a fusion belief filter.

  19. Interactive analysis of geodata based intelligence

    Science.gov (United States)

    Wagner, Boris; Eck, Ralf; Unmüessig, Gabriel; Peinsipp-Byma, Elisabeth

    2016-05-01

    When a spatiotemporal events happens, multi-source intelligence data is gathered to understand the problem, and strategies for solving the problem are investigated. The difficulties arising from handling spatial and temporal intelligence data represent the main problem. The map might be the bridge to visualize the data and to get the most understand model for all stakeholders. For the analysis of geodata based intelligence data, a software was developed as a working environment that combines geodata with optimized ergonomics. The interaction with the common operational picture (COP) is so essentially facilitated. The composition of the COP is based on geodata services, which are normalized by international standards of the Open Geospatial Consortium (OGC). The basic geodata are combined with intelligence data from images (IMINT) and humans (HUMINT), stored in a NATO Coalition Shared Data Server (CSD). These intelligence data can be combined with further information sources, i.e., live sensors. As a result a COP is generated and an interaction suitable for the specific workspace is added. This allows the users to work interactively with the COP, i.e., searching with an on board CSD client for suitable intelligence data and integrate them into the COP. Furthermore, users can enrich the scenario with findings out of the data of interactive live sensors and add data from other sources. This allows intelligence services to contribute effectively to the process by what military and disaster management are organized.

  20. Operating cost analysis of anaesthesia: Activity based costing (ABC analysis

    Directory of Open Access Journals (Sweden)

    Majstorović Branislava M.

    2011-01-01

    Full Text Available Introduction. Cost of anaesthesiology represent defined measures to determine a precise profile of expenditure estimation of surgical treatment, which is important regarding planning of healthcare activities, prices and budget. Objective. In order to determine the actual value of anaestesiological services, we started with the analysis of activity based costing (ABC analysis. Methods. Retrospectively, in 2005 and 2006, we estimated the direct costs of anestesiological services (salaries, drugs, supplying materials and other: analyses and equipment. of the Institute of Anaesthesia and Resuscitation of the Clinical Centre of Serbia. The group included all anesthetized patients of both sexes and all ages. We compared direct costs with direct expenditure, “each cost object (service or unit” of the Republican Health-care Insurance. The Summary data of the Departments of Anaesthesia documented in the database of the Clinical Centre of Serbia. Numerical data were utilized and the numerical data were estimated and analyzed by computer programs Microsoft Office Excel 2003 and SPSS for Windows. We compared using the linear model of direct costs and unit costs of anaesthesiological services from the Costs List of the Republican Health-care Insurance. Results. Direct costs showed 40% of costs were spent on salaries, (32% on drugs and supplies, and 28% on other costs, such as analyses and equipment. The correlation of the direct costs of anaestesiological services showed a linear correlation with the unit costs of the Republican Healthcare Insurance. Conclusion. During surgery, costs of anaesthesia would increase by 10% the surgical treatment cost of patients. Regarding the actual costs of drugs and supplies, we do not see any possibility of costs reduction. Fixed elements of direct costs provide the possibility of rationalization of resources in anaesthesia.

  1. Exploring the phosphoproteome profiles during Xenopus egg activation by calcium stimulation using a fully automated phosphopeptide purification system.

    Science.gov (United States)

    Kanno, Takuma; Furukawa, Kazuhiro; Horigome, Tsuneyoshi

    2016-04-01

    To explore the phosphoproteome profiles duringXenopusegg activation by Ca(2+)-stimulation, an automated phosphopeptide purification system involving a titania column was improved by introducing 4-step elution with phosphate buffers. The number of detected phosphopeptides in the tryptic digest of aXenopusegg cytosol fraction on mass spectrometry (MS) was increased 1.5-fold and the percentage of multiply phosphorylated peptides increased from 17 to 24% with introduction of the 4-step elution method. Phosphopeptides were purified by the improved method from tryptic digests of cytosol fractions ofXenopuseggs without and with a Ca(2+)-stimulus, and then, analysed by MS. One thousand three hundred and seventy-five and 994 phosphopeptides were reproducibly detected on duplicate MS, respectively. They included 818 and 437 phosphopeptides specific to each digest, respectively. A method involving isobaric tags for relative and absolute quantitation (iTRAQ) was also applied to compare the phosphorylation levels inXenopuseggs without and with a Ca(2+)-stimulus, the ratios for 112 phosphopeptides in tryptic digests of these egg cytosol fractions being obtained. It was suggested from all the results that the phosphorylation sites and levels change duringXenopusegg activation for many known and unknown sites on structural proteins, signalling related proteins, cell cycle-related proteins and others. PMID:26530081

  2. Changes in the proteome and phosphoproteome expression in the bryozoan Bugula neritina larvae in response to the antifouling agent butenolide

    KAUST Repository

    Qian, Pei Yuan

    2010-09-08

    Larval attachment and metamorphosis, commonly referred to as larval settlement, of marine sessile invertebrates can be triggered or blocked by chemical cues and affected by changes in overall protein expression pattern and phosphorylation dynamics. This study focuses on the effects of butenolide, an effective larval settlement inhibitor, on larval settlement at the proteome level in the bryozoan Bugula neritina. Liquid-phase IEF sample prefractionation combined with 2-DE and MALDI-TOF MS was used to identify the differentially expressed proteins. Substantial changes occurred both in protein abundance and in phosphorylation status during larval settlement and when settling larvae were challenged with butenolide. The proteins that responded to treatment were identified as structural proteins, molecular chaperones, mitochondrial peptidases and calcium-binding proteins. Compared with our earlier results, both genistein and butenolide inhibited larval settlement of B. neritina primarily by changes in protein abundance and the phosphorylation status of proteins but have different protein targets in the same species. Clearly, to design potent antifouling compounds and to understand the mode of action of compounds, more studies on the effects of different compounds on proteome and phosphoproteome of different larval species are required. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.

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

    Science.gov (United States)

    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

  4. The phosphoproteome of bloodstream form Trypanosoma brucei, causative agent of African sleeping sickness.

    Science.gov (United States)

    Nett, Isabelle R E; Martin, David M A; Miranda-Saavedra, Diego; Lamont, Douglas; Barber, Jonathan D; Mehlert, Angela; Ferguson, Michael A J

    2009-07-01

    The protozoan parasite Trypanosoma brucei is the causative agent of human African sleeping sickness and related animal diseases, and it has over 170 predicted protein kinases. Protein phosphorylation is a key regulatory mechanism for cellular function that, thus far, has been studied in T.brucei principally through putative kinase mRNA knockdown and observation of the resulting phenotype. However, despite the relatively large kinome of this organism and the demonstrated essentiality of several T. brucei kinases, very few specific phosphorylation sites have been determined in this organism. Using a gel-free, phosphopeptide enrichment-based proteomics approach we performed the first large scale phosphorylation site analyses for T.brucei. Serine, threonine, and tyrosine phosphorylation sites were determined for a cytosolic protein fraction of the bloodstream form of the parasite, resulting in the identification of 491 phosphoproteins based on the identification of 852 unique phosphopeptides and 1204 phosphorylation sites. The phosphoproteins detected in this study are predicted from their genome annotations to participate in a wide variety of biological processes, including signal transduction, processing of DNA and RNA, protein synthesis, and degradation and to a minor extent in metabolic pathways. The analysis of phosphopeptides and phosphorylation sites was facilitated by in-house developed software, and this automated approach was validated by manual annotation of spectra of the kinase subset of proteins. Analysis of the cytosolic bloodstream form T. brucei kinome revealed the presence of 44 phosphorylated protein kinases in our data set that could be classified into the major eukaryotic protein kinase groups by applying a multilevel hidden Markov model library of the kinase catalytic domain. Identification of the kinase phosphorylation sites showed conserved phosphorylation sequence motifs in several kinase activation segments, supporting the view that

  5. Automatic malware analysis an emulator based approach

    CERN Document Server

    Yin, Heng

    2012-01-01

    Malicious software (i.e., malware) has become a severe threat to interconnected computer systems for decades and has caused billions of dollars damages each year. A large volume of new malware samples are discovered daily. Even worse, malware is rapidly evolving becoming more sophisticated and evasive to strike against current malware analysis and defense systems. Automatic Malware Analysis presents a virtualized malware analysis framework that addresses common challenges in malware analysis. In regards to this new analysis framework, a series of analysis techniques for automatic malware analy

  6. Phosphoproteomic identification of targets of the Arabidopsis sucrose nonfermenting-like kinase SnRK2.8 reveals a connection to metabolic processes

    OpenAIRE

    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 ...

  7. Polyphase Order Analysis Based on Convolutional Approach

    OpenAIRE

    M. Drutarovsky

    1999-01-01

    The condition of rotating machines can be determined by measuring of periodic frequency components in the vibration signal which are directly related to the (typically changing) rotational speed. Classical spectrum analysis with a constant sampling frequency is not an appropriate analysis method because of spectral smearing. Spectral analysis of vibration signal sampled synchronously with the angle of rotation, known as order analysis, suppress spectral smearing even with variable rotational ...

  8. Ovarian Cancer Proteomic, Phosphoproteomic, and Glycoproteomic Data Released - Office of Cancer Clinical Proteomics Research

    Science.gov (United States)

    National Cancer Institute (NCI) Clinical Proteomic Tumor Analysis Consortium (CPTAC) scientists have just released a comprehensive dataset of the proteomic analysis of high grade serous ovarian tumor samples,

  9. ANALYSIS OF CIRCUIT TOLERANCE BASED ON RANDOM SET THEORY

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Monte Carlo Analysis has been an accepted method for circuit tolerance analysis,but the heavy computational complexity has always prevented its applications.Based on random set theory,this paper presents a simple and flexible tolerance analysis method to estimate circuit yield.It is the alternative to Monte Carlo analysis,but reduces the number of calculations dramatically.

  10. An Augmented Multiple-Protease-Based Human Phosphopeptide Atlas

    Directory of Open Access Journals (Sweden)

    Piero Giansanti

    2015-06-01

    Full Text Available Although mass-spectrometry-based screens enable thousands of protein phosphorylation sites to be monitored simultaneously, they often do not cover important regulatory sites. Here, we hypothesized that this is due to the fact that nearly all large-scale phosphoproteome studies are initiated by trypsin digestion. We tested this hypothesis using multiple proteases for protein digestion prior to Ti4+-IMAC-based enrichment. This approach increases the size of the detectable phosphoproteome substantially and confirms the considerable tryptic bias in public repositories. We define and make available a less biased human phosphopeptide atlas of 37,771 unique phosphopeptides, correlating to 18,430 unique phosphosites, of which fewer than 1/3 were identified in more than one protease data set. We demonstrate that each protein phosphorylation site can be linked to a preferred protease, enhancing its detection by mass spectrometry (MS. For specific sites, this approach increases their detectability by more than 1,000-fold.

  11. Scope-Based Method Cache Analysis

    DEFF Research Database (Denmark)

    Huber, Benedikt; Hepp, Stefan; Schoeberl, Martin

    requests memory transfers at well-defined instructions only. In this article, we present a new cache analysis framework that generalizes and improves work on cache persistence analysis. The analysis demonstrates that a global view on the cache behavior permits the precise analyses of caches which are hard......The quest for time-predictable systems has led to the exploration of new hardware architectures that simplify analysis and reasoning in the temporal domain, while still providing competitive performance. For the instruction memory, the method cache is a conceptually attractive solution, as it...

  12. A Requirements Analysis Model Based on QFD

    Institute of Scientific and Technical Information of China (English)

    TANG Zhi-wei; Nelson K.H.Tang

    2004-01-01

    The enterprise resource planning (ERP) system has emerged to offer an integrated IT solution and more and more enterprises are increasing by adopting this system and regarding it as an important innovation. However, there is already evidence of high failure risks in ERP project implementation, one major reason is poor analysis of the requirements for system implementation. In this paper, the importance of requirements analysis for ERP project implementation is highlighted, and a requirements analysis model by applying quality function deployment (QFD) is presented, which will support to conduct requirements analysis for ERP project.

  13. Location-based Modeling and Analysis: Tropos-based Approach

    OpenAIRE

    Ali, Raian; Dalpiaz, Fabiano; Giorgini, Paolo

    2008-01-01

    The continuous growth of interest in mobile applications makes the concept of location essential to design and develop software systems. Location-based software is supposed to be able to monitor the location and choose accordingly the most appropriate behavior. In this paper, we propose a novel conceptual framework to model and analyze location-based software. We mainly focus on the social facets of locations adopting concepts such as social actor, resource, and location-based behavior. Our a...

  14. Statistical Analysis of Nonlinear Processes Based on Penalty Factor

    OpenAIRE

    Zhang, Yingwei; Zhang, Chuanfang; Zhang, Wei

    2014-01-01

    A new process monitoring approach is proposed for handling the nonlinear monitoring problem in the electrofused magnesia furnace (EFMF). Compared to conventional method, the contributions are as follows: (1) a new kernel principal component analysis is proposed based on loss function in the feature space; (2) the model of kernel principal component analysis based on forgetting factor is updated; (3) a new iterative kernel principal component analysis algorithm is proposed based on penalty fac...

  15. Web Based Distributed Coastal Image Analysis System Project

    Data.gov (United States)

    National Aeronautics and Space Administration — This project develops Web based distributed image analysis system processing the Moderate Resolution Imaging Spectroradiometer (MODIS) data to provide decision...

  16. Integrative transcriptome, proteome, phosphoproteome and genetic mapping reveals new aspects in a fiberless mutant of cotton.

    Science.gov (United States)

    Ma, Qi-Feng; Wu, Chun-Hui; Wu, Man; Pei, Wen-Feng; Li, Xing-Li; Wang, Wen-Kui; Zhang, Jinfa; Yu, Ji-Wen; Yu, Shu-Xun

    2016-01-01

    To investigate the molecular mechanisms of fiber initiation in cotton (Gossypium spp.), an integrated approach combining transcriptome, iTRAQ-based proteome and genetic mapping was taken to compare the ovules of the Xuzhou 142 wild type (WT) with its fuzzless-lintless (fl) mutant at -3 and 0 day post-anthesis. A total of 1,953 mRNAs, 187 proteins, and 131 phosphoproteins were differentially expressed (DE) between WT and fl, and the levels of transcripts and their encoded proteins and phosphoproteins were highly congruent. A functional analysis suggested that the abundance of proteins were mainly involved in amino sugar, nucleotide sugar and fatty acid metabolism, one carbon pool for folate metabolism and flavonoid biosynthesis. qRT-PCR, Western blotting, and enzymatic assays were performed to confirm the regulation of these transcripts and proteins. A molecular mapping located the lintless gene li3 in the fl mutant on chromosome 26 for the first time. A further in-silico physical mapping of DE genes with sequence variations between fl and WT identified one and four candidate genes in the li3 and n2 regions, respectively. Taken together, the transcript abundance, phosphorylation status of proteins at the fiber initiation stage and candidate genes have provided insights into regulatory processes underlying cotton fiber initiation. PMID:27075604

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

    Science.gov (United States)

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

    2016-02-01

    The filamentous fungus Trichoderma reesei is used for industrial production of secreted enzymes including carbohydrate active enzymes, such as cellulases and hemicellulases. The production of many of these enzymes by T. reesei is influenced by the carbon source it grows on, where the regulation system controlling hydrolase genes involves various signaling pathways. T. reesei was cultivated in the presence of sorbitol, a carbon source that does not induce the production of cellulases and hemicellulases, and then exposed to either sophorose or spent-grain extract, which are efficient inducers of the enzyme production. Specific changes at phosphorylation sites were investigated in relation to the production of cellulases and hemicellulases using an MS-based framework. Proteome-wide phosphorylation following carbon source exchange was investigated in the early stages of induction: 0, 2, 5, and 10 min. The workflow involved sequential trypsin digestion, TiO2 enrichment, and MS analysis using a Q Exactive mass spectrometer. We report on the identification and quantitation of 1721 phosphorylation sites. Investigation of the data revealed a complex signaling network activated upon induction involving components related to light-mediated cellulase induction, osmoregulation, and carbon sensing. Changes in protein phosphorylation were detected in the glycolytic pathway, suggesting an inhibition of glucose catabolism at 10 min after the addition of sophorose and as early as 2 min after the addition of spent-grain extract. Differential phosphorylation of factors related to carbon storage, intracellular trafficking, cytoskeleton, and cellulase gene regulation were also observed. PMID:26689635

  18. Combined Phosphoproteomics and Bioinformatics Strategy in Deciphering Drug Resistant Related Pathways in Triple Negative Breast Cancer

    Directory of Open Access Journals (Sweden)

    Xinyu Deng

    2014-01-01

    Full Text Available Because of the absence of a clear therapeutic target for triple negative breast cancer (TNBC, conventional chemotherapy is the only available systemic treatment option for these patients. Despite chemotherapy treatment, TNBC patients still have worse prognosis when compared with other breast cancer patients. The study is to investigate unique phosphorylated proteins expressed in chemoresistant TNBC cell lines. In the current study, twelve TNBC cell lines were subjected to drug sensitivity assays against chemotherapy drugs docetaxel, doxorubicin, gemcitabine, and cisplatin. Based on their half maximal inhibitory concentrations, four resistant and two sensitive cell lines were selected for further analysis. The phosphopeptides from these cells were enriched with TiO2 beads and fractionated using strong cation exchange. 1,645 phosphoprotein groups and 9,585 unique phosphopeptides were identified by a high throughput LC-MS/MS system LTQ-Orbitrap. The phosphopeptides were further filtered with Ascore system and 1,340 phosphoprotein groups, 2,760 unique phosphopeptides, and 4,549 unique phosphosites were identified. Our study suggested that differentially phosphorylated Cdk5, PML, AP-1, and HSF-1 might work together to promote vimentin induced epithelial to mesenchymal transition (EMT in the drug resistant cells. EGFR and HGF were also shown to be involved in this process.

  19. Pathway-Based Functional Analysis of Metagenomes

    Science.gov (United States)

    Bercovici, Sivan; Sharon, Itai; Pinter, Ron Y.; Shlomi, Tomer

    Metagenomic data enables the study of microbes and viruses through their DNA as retrieved directly from the environment in which they live. Functional analysis of metagenomes explores the abundance of gene families, pathways, and systems, rather than their taxonomy. Through such analysis researchers are able to identify those functional capabilities most important to organisms in the examined environment. Recently, a statistical framework for the functional analysis of metagenomes was described that focuses on gene families. Here we describe two pathway level computational models for functional analysis that take into account important, yet unaddressed issues such as pathway size, gene length and overlap in gene content among pathways. We test our models over carefully designed simulated data and propose novel approaches for performance evaluation. Our models significantly improve over current approach with respect to pathway ranking and the computations of relative abundance of pathways in environments.

  20. Analysis of Task-based Syllabus

    Institute of Scientific and Technical Information of China (English)

    马进胜

    2011-01-01

    Task-based language teaching is very popular in the modem English teaching.It is based on the Task-based Syllabus.Taskbased Syllabus focuses on the learners' communicative competence,which stresses learning by doing.From the theoretical assumption and definitions of the task,the paper analysizes the components of the task,then points out the merits and demerits of the syllabus.By this means the paper may give some tips to teachers and students when they use the tsk-based language teaching.

  1. Market Based Analysis of Power System Interconnections

    OpenAIRE

    Obuševs, A; Turcik, M; Oļeiņikova, I; Junghāns, G

    2011-01-01

    Analysis in this Article is focused on usage of transmission grid under liberalized market with implicit transmission capacity allocation method, e.g. Nordic market. Attention is paid on fundamental changes in transmission utilization and its economical effective operation. For interconnection and power flow analysis and losses calculation model of Nordic grid was developed and transmission losses calculation method was created. Given approach will improve economical efficiency of system oper...

  2. Description-based and experience-based decisions: individual analysis

    Directory of Open Access Journals (Sweden)

    Andrey Kudryavtsev

    2012-05-01

    Full Text Available We analyze behavior in two basic classes of decision tasks: description-based and experience-based. In particular, we compare the prediction power of a number of decision learning models in both kinds of tasks. Unlike most previous studies, we focus on individual, rather than aggregate, behavioral characteristics. We carry out an experiment involving a battery of both description- and experience-based choices between two mixed binary prospects made by each of the participants, and employ a number of formal models for explaining and predicting participants' choices: Prospect theory (PT (Kahneman and Tversky, 1979; Expectancy-Valence model (EVL (Busemeyer and Stout, 2002; and three combinations of these well-established models. We document that the PT and the EVL models are best for predicting people's decisions in description- and experience-based tasks, respectively, which is not surprising as these two models are designed specially for these kinds of tasks. Furthermore, we find that models involving linear weighting of gains and losses perform better in both kinds of tasks, from the point of view of generalizability and individual parameter consistency. We therefore, conclude that, overall, when both prospects are mixed, the assumption of diminishing sensitivity does not improve models' prediction power for individual decision-makers. Finally, for some of the models' parameters, we document consistency at the individual level between description- and experience-based tasks.

  3. Iris recognition based on subspace analysis

    Directory of Open Access Journals (Sweden)

    Pravin S.Patil

    2014-10-01

    Full Text Available Biometrics deals with the uniqueness of an individual arising from their physiological or behavioral characteristics for the purpose of personal identification. Among many biometrics techniques, iris recognition is one of the most promising approache. This paper presents traditional subspace analysis method for iris recognition. Initially the eye images have been localized in circular form by using Daugman’s grid method and circular Hough transform method. The algorithms for subspace analysis methods namely PCA and LDA are implemented and experimental results are reported. The comparative performance for both the algorithms has been observed in term of recognition rate. The comprehensive experiments completed on UPOL and CASIA V1 iris databases.

  4. Performance Analysis Based on Timing Simulation

    DEFF Research Database (Denmark)

    Nielsen, Christian Dalsgaard; Kishinevsky, Michael

    Determining the cycle time and a critical cycle is a fundamental problem in the analysis of concurrent systems. We solve this problemusing timing simulation of an underlying Signal Graph (an extension of Marked Graphs). For a Signal Graph with n vertices and m arcs our algorithm has the polynomial...... time complexity O(b2m), where b is the number of vertices with initially marked in-arcs (typically b≪n). The algorithm has a clear semantic and a low descriptive complexity. We illustrate the use of the algorithm by applying it to performance analysis of asynchronous circuits....

  5. Environmentally based Cost-Benefit Analysis

    International Nuclear Information System (INIS)

    The fundamentals of the basic elements of a new comprehensive economic assessment, MILA, developed in Sweden with inspiration from the Total Cost Assessment-model are presented. The core of the MILA approach is an expanded cost and benefit inventory. But MILA also includes a complementary addition of an internal waste stream analysis, a tool for evaluation of environmental conflicts in monetary terms, an extended time horizon and direct allocation of costs and revenues to products and processes. However, MILA does not ensure profitability for environmentally sound projects. Essentially, MILA is an approach of refining investment and profitability analysis of a project, investment or product. 109 refs., 38 figs

  6. Gender-Based Analysis On-Line Dialogue. Final Report.

    Science.gov (United States)

    2001

    An online dialogue on gender-based analysis (GBA) was held from February 15 to March 7, 2001. Invitations and a background paper titled "Why Gender-Based Analysis?" were sent to 350 women's organizations and individuals throughout Canada. Efforts were made to ensure that aboriginal and Metis women, visible minority women, and women with special…

  7. Science Based Governance? EU Food Regulation Submitted to Risk Analysis

    NARCIS (Netherlands)

    Szajkowska, A.; Meulen, van der B.M.J.

    2014-01-01

    Anna Szajkowska and Bernd van der Meulen analyse in their contribution, Science Based Governance? EU Food Regulation Submitted to Risk Analysis, the scope of application of risk analysis and the precautionary principle in EU food safety regulation. To what extent does this technocratic, science-base

  8. SSI Analysis for Base-Isolated Nuclear Power Plants

    International Nuclear Information System (INIS)

    Safety of NPPs much higher than other structures is required. An earthquake is one of the most important parameters which govern safety of NPPs among external events. Application of base isolation system for NPPs can reduce the risk for earthquakes. At present, a soil structure interaction(SSI) analysis is essential in seismic design of NPPs in consideration of ground structure interaction. In the seismic analysis of the base-isolated NPP, it is restrictive to consider nonlinear properties of seismic isolation bearings due to linear analysis of SSI analysis programs such as SASSI. Thus, in this study, SSI analyses are performed using an iterative approach considering material nonlinearity of isolators. By performing the SSI analysis using an iterative approach, nonlinear properties of isolators can be considered. The results of the SSI analysis show that the response of the base-isolated NPP with base isolation systems is significantly reduced horizontally

  9. Analysis of Financial Position Based on the Balance Sheet

    OpenAIRE

    Spineanu-Georgescu Luciana

    2011-01-01

    Analysis of financial position based on the balance sheet is mainly aimed at assessing the extent to which financial structure chosen by the firm, namely, financial resources, covering the needs reflected in the balance sheet financed. This is done through an analysis known as horizontal analysis balance sheet financial imbalances.

  10. Transportation Mode Choice Analysis Based on Classification Methods

    OpenAIRE

    Zeņina, N; Borisovs, A

    2011-01-01

    Mode choice analysis has received the most attention among discrete choice problems in travel behavior literature. Most traditional mode choice models are based on the principle of random utility maximization derived from econometric theory. This paper investigates performance of mode choice analysis with classification methods - decision trees, discriminant analysis and multinomial logit. Experimental results have demonstrated satisfactory quality of classification.

  11. Encounter-based worms: Analysis and Defense

    CERN Document Server

    Tanachaiwiwat, Sapon

    2007-01-01

    Encounter-based network is a frequently-disconnected wireless ad-hoc network requiring immediate neighbors to store and forward aggregated data for information disseminations. Using traditional approaches such as gateways or firewalls for deterring worm propagation in encounter-based networks is inappropriate. We propose the worm interaction approach that relies upon automated beneficial worm generation aiming to alleviate problems of worm propagations in such networks. To understand the dynamic of worm interactions and its performance, we mathematically model worm interactions based on major worm interaction factors including worm interaction types, network characteristics, and node characteristics using ordinary differential equations and analyze their effects on our proposed metrics. We validate our proposed model using extensive synthetic and trace-driven simulations. We find that, all worm interaction factors significantly affect the pattern of worm propagations. For example, immunization linearly decrea...

  12. Accelerator based techniques for aerosol analysis

    International Nuclear Information System (INIS)

    At the 3 MV Tandetron accelerator of the LABEC laboratory of INFN (Florence, Italy) an external beam facility is fully dedicated to PIXE-PIGE measurements of elemental composition of atmospheric aerosols. Examples regarding recent monitoring campaigns, performed in urban and remote areas, both on a daily basis and with high time resolution, as well as with size selection, will be presented. It will be evidenced how PIXE can provide unique information in aerosol studies or can play a complementary role to traditional chemical analysis. Finally a short presentation of 14C analysis of the atmospheric aerosol by Accelerator Mass Spectrometry (AMS) for the evaluation of the contributions from either fossil fuel combustion or modern sources (wood burning, biogenic activity) will be given. (author)

  13. Structural Analysis of Plate Based Tensegrity Structures

    DEFF Research Database (Denmark)

    Hald, Frederik; Kirkegaard, Poul Henning; Damkilde, Lars

    2013-01-01

    Plate tensegrity structures combine tension cables with a cross laminated timber plate and can then form e.g. a roof structure. The topology of plate tensegrity structures is investigated through a parametric investigation. Plate tensegrity structures are investigated, and a method for...... determination of the structures pre-stresses is used. A parametric investigation is performed to determine a more optimized form of the plate based tensegrity structure. Conclusions of the use of plate based tensegrity in civil engineering and further research areas are discussed....

  14. Symbolic Analysis of OTRAs-Based Circuits

    Directory of Open Access Journals (Sweden)

    C. Sánchez-López

    2011-04-01

    Full Text Available A new nullor-based model to describe the behavior of Operational Transresistance Amplifiers (OTRAs is introduced.The new model is composed of four nullors and three grounded resistors. As a consequence, standard nodal analysiscan be applied to compute fully-symbolic small-signal characteristics of OTRA-based analog circuits, and the nullorbasedOTRAs model can be used in CAD tools. In this manner, the fully-symbolic transfer functions of severalapplication circuits, such as filters and oscillators can easily be approximated.

  15. Iris recognition based on subspace analysis

    OpenAIRE

    Pravin S.Patil

    2014-01-01

    Biometrics deals with the uniqueness of an individual arising from their physiological or behavioral characteristics for the purpose of personal identification. Among many biometrics techniques, iris recognition is one of the most promising approache. This paper presents traditional subspace analysis method for iris recognition. Initially the eye images have been localized in circular form by using Daugman’s grid method and circular Hough transform method. The algorithms for subspace analy...

  16. Texton-based analysis of paintings

    Science.gov (United States)

    van der Maaten, Laurens J. P.; Postma, Eric O.

    2010-08-01

    The visual examination of paintings is traditionally performed by skilled art historians using their eyes. Recent advances in intelligent systems may support art historians in determining the authenticity or date of creation of paintings. In this paper, we propose a technique for the examination of brushstroke structure that views the wildly overlapping brushstrokes as texture. The analysis of the painting texture is performed with the help of a texton codebook, i.e., a codebook of small prototypical textural patches. The texton codebook can be learned from a collection of paintings. Our textural analysis technique represents paintings in terms of histograms that measure the frequency by which the textons in the codebook occur in the painting (so-called texton histograms). We present experiments that show the validity and effectiveness of our technique for textural analysis on a collection of digitized high-resolution reproductions of paintings by Van Gogh and his contemporaries. As texton histograms cannot be easily be interpreted by art experts, the paper proposes to approaches to visualize the results on the textural analysis. The first approach visualizes the similarities between the histogram representations of paintings by employing a recently proposed dimensionality reduction technique, called t-SNE. We show that t-SNE reveals a clear separation of paintings created by Van Gogh and those created by other painters. In addition, the period of creation is faithfully reflected in the t-SNE visualizations. The second approach visualizes the similarities and differences between paintings by highlighting regions in a painting in which the textural structure of the painting is unusual. We illustrate the validity of this approach by means of an experiment in which we highlight regions in a painting by Monet that are not very "Van Gogh-like". Taken together, we believe the tools developed in this study are well capable of assisting for art historians in support of

  17. CLUSTERING-BASED ANALYSIS OF TEXT SIMILARITY

    OpenAIRE

    Bovcon , Borja

    2013-01-01

    The focus of this thesis is comparison of analysis of text-document similarity using clustering algorithms. We begin by defining main problem and then, we proceed to describe the two most used text-document representation techniques, where we present words filtering methods and their importance, Porter's algorithm and tf-idf term weighting algorithm. We then proceed to apply all previously described algorithms on selected data-sets, which vary in size and compactness. Fallowing this, we ...

  18. Wavelet Based Fractal Analysis of Airborne Pollen

    OpenAIRE

    Degaudenzi, M. E.; Arizmendi, C. M.

    1998-01-01

    The most abundant biological particles in the atmosphere are pollen grains and spores. Self protection of pollen allergy is possible through the information of future pollen contents in the air. In spite of the importance of airborne pol len concentration forecasting, it has not been possible to predict the pollen concentrations with great accuracy, and about 25% of the daily pollen forecasts have resulted in failures. Previous analysis of the dynamic characteristics of atmospheric pollen tim...

  19. Thanatophoric dysplasia: case-based bioethical analysis

    Directory of Open Access Journals (Sweden)

    Edgar Abarca López

    2014-04-01

    Full Text Available This paper presents a case report of thanatophoric displasia diagnosed in the prenatal period using ultrasound standards. The course of the case pregnancy, birth process, and postnatal period is described. This report invites bioethical analysis using its principles, appealing to human dignity, diversity and otherness, particularly in the mother-child dyad and their family. An early diagnosis allows parental support as they face the course of this condition and its potentially fatal outcome.

  20. Thanatophoric dysplasia: case-based bioethical analysis

    OpenAIRE

    Edgar Abarca López; Alejandra Rodríguez Torres; Donovan Casas Patiño; Esteban Espíndola Benítez

    2014-01-01

    This paper presents a case report of thanatophoric displasia diagnosed in the prenatal period using ultrasound standards. The course of the case pregnancy, birth process, and postnatal period is described. This report invites bioethical analysis using its principles, appealing to human dignity, diversity and otherness, particularly in the mother-child dyad and their family. An early diagnosis allows parental support as they face the course of this condition and its potentially fatal outcome.

  1. Movement Pattern Analysis Based on Sequence Signatures

    Directory of Open Access Journals (Sweden)

    Seyed Hossein Chavoshi

    2015-09-01

    Full Text Available Increased affordability and deployment of advanced tracking technologies have led researchers from various domains to analyze the resulting spatio-temporal movement data sets for the purpose of knowledge discovery. Two different approaches can be considered in the analysis of moving objects: quantitative analysis and qualitative analysis. This research focuses on the latter and uses the qualitative trajectory calculus (QTC, a type of calculus that represents qualitative data on moving point objects (MPOs, and establishes a framework to analyze the relative movement of multiple MPOs. A visualization technique called sequence signature (SESI is used, which enables to map QTC patterns in a 2D indexed rasterized space in order to evaluate the similarity of relative movement patterns of multiple MPOs. The applicability of the proposed methodology is illustrated by means of two practical examples of interacting MPOs: cars on a highway and body parts of a samba dancer. The results show that the proposed method can be effectively used to analyze interactions of multiple MPOs in different domains.

  2. Remote sensing based on hyperspectral data analysis

    Science.gov (United States)

    Sharifahmadian, Ershad

    In remote sensing, accurate identification of far objects, especially concealed objects is difficult. In this study, to improve object detection from a distance, the hyperspecral imaging and wideband technology are employed with the emphasis on wideband radar. As the wideband data includes a broad range of frequencies, it can reveal information about both the surface of the object and its content. Two main contributions are made in this study: 1) Developing concept of return loss for target detection: Unlike typical radar detection methods which uses radar cross section to detect an object, it is possible to enhance the process of detection and identification of concealed targets using the wideband radar based on the electromagnetic characteristics --conductivity, permeability, permittivity, and return loss-- of materials. During the identification process, collected wideband data is evaluated with information from wideband signature library which has already been built. In fact, several classes (e.g. metal, wood, etc.) and subclasses (ex. metals with high conductivity) have been defined based on their electromagnetic characteristics. Materials in a scene are then classified based on these classes. As an example, materials with high electrical conductivity can be conveniently detected. In fact, increasing relative conductivity leads to a reduction in the return loss. Therefore, metals with high conductivity (ex. copper) shows stronger radar reflections compared with metals with low conductivity (ex. stainless steel). Thus, it is possible to appropriately discriminate copper from stainless steel. 2) Target recognition techniques: To detect and identify targets, several techniques have been proposed, in particular the Multi-Spectral Wideband Radar Image (MSWRI) which is able to localize and identify concealed targets. The MSWRI is based on the theory of robust capon beamformer. During identification process, information from wideband signature library is utilized

  3. Google glass based immunochromatographic diagnostic test analysis

    Science.gov (United States)

    Feng, Steve; Caire, Romain; Cortazar, Bingen; Turan, Mehmet; Wong, Andrew; Ozcan, Aydogan

    2015-03-01

    Integration of optical imagers and sensors into recently emerging wearable computational devices allows for simpler and more intuitive methods of integrating biomedical imaging and medical diagnostics tasks into existing infrastructures. Here we demonstrate the ability of one such device, the Google Glass, to perform qualitative and quantitative analysis of immunochromatographic rapid diagnostic tests (RDTs) using a voice-commandable hands-free software-only interface, as an alternative to larger and more bulky desktop or handheld units. Using the built-in camera of Glass to image one or more RDTs (labeled with Quick Response (QR) codes), our Glass software application uploads the captured image and related information (e.g., user name, GPS, etc.) to our servers for remote analysis and storage. After digital analysis of the RDT images, the results are transmitted back to the originating Glass device, and made available through a website in geospatial and tabular representations. We tested this system on qualitative human immunodeficiency virus (HIV) and quantitative prostate-specific antigen (PSA) RDTs. For qualitative HIV tests, we demonstrate successful detection and labeling (i.e., yes/no decisions) for up to 6-fold dilution of HIV samples. For quantitative measurements, we activated and imaged PSA concentrations ranging from 0 to 200 ng/mL and generated calibration curves relating the RDT line intensity values to PSA concentration. By providing automated digitization of both qualitative and quantitative test results, this wearable colorimetric diagnostic test reader platform on Google Glass can reduce operator errors caused by poor training, provide real-time spatiotemporal mapping of test results, and assist with remote monitoring of various biomedical conditions.

  4. Knowledge-based analysis of phenotypes

    KAUST Repository

    Hoendorf, Robert

    2016-01-27

    Phenotypes are the observable characteristics of an organism, and they are widely recorded in biology and medicine. To facilitate data integration, ontologies that formally describe phenotypes are being developed in several domains. I will describe a formal framework to describe phenotypes. A formalized theory of phenotypes is not only useful for domain analysis, but can also be applied to assist in the diagnosis of rare genetic diseases, and I will show how our results on the ontology of phenotypes is now applied in biomedical research.

  5. A Goal based methodology for HAZOP analysis

    DEFF Research Database (Denmark)

    Rossing, Netta Liin; Lind, Morten; Jensen, Niels;

    2010-01-01

    directly for implementation into a computer aided reasoning tool for HAZOP studies to perform root cause and consequence analysis. Such a tool will facilitate finding causes far away from the site of the deviation. A Functional HAZOP Assistant is proposed and investigated in a HAZOP study of an industrial...... to nodes with simple functions such as liquid transport, gas transport, liquid storage, gas-liquid contacting etc. From the functions of the nodes the selection of relevant process variables and deviation variables follows directly. The knowledge required to perform the pre-meeting HAZOP task of...

  6. Communication Error Analysis Method based on CREAM

    International Nuclear Information System (INIS)

    Communication error has been considered as a primary reason of many incidents and accidents in nuclear industry. In order to prevent these accidents, an analysis method of communication errors is proposed. This study presents a qualitative method to analyze communication errors. The qualitative method focuses on finding a root cause of the communication error and predicting the type of communication error which could happen in nuclear power plants. We develop context conditions and antecedent-consequent links of influential factors related to communication error. A case study has been conducted to validate the applicability of the proposed methods

  7. Web Application Comprehension Based on Dependence Analysis

    Institute of Scientific and Technical Information of China (English)

    WU Jun-hua; XU Bao-wen; JIANG Ji-xiang

    2004-01-01

    Many research indicate a lot of money and time are spent on maintaining and modifying program delivered.So the policies to support program comprehension are very important.Program comprehension is a crucial and difficult task.Insufficient design, illogical code structure, short documents will enhance the comprehensive difficulty.Developing Web application is usually a process with quick implementation and delivery.In addition, generally a Web application is coded by combining mark language statements with some embedded applets.Such programming mode affects comprehension of Web applications disadvantageously.This paper proposes a method to improving understanding Web by dependence analysis and slice technology.

  8. Analysis and Protection of SIP based Services

    OpenAIRE

    Ferdous, Raihana

    2014-01-01

    Multimedia communications over IP are booming as they offer higher flexibility and more features than traditional voice and video services. IP telephony known as Voice over IP (VoIP) is one of the commercially most important emerging trends in multimedia communications over IP. Due to the flexibility and descriptive power, the Session Initiation Protocol (SIP) is becoming the root of many sessions-based applications such as VoIP and media streaming that are used by a growing number of use...

  9. Analysis of Hashrate-Based Double Spending

    OpenAIRE

    Rosenfeld, Meni

    2014-01-01

    Bitcoin is the world's first decentralized digital currency. Its main technical innovation is the use of a blockchain and hash-based proof of work to synchronize transactions and prevent double-spending the currency. While the qualitative nature of this system is well understood, there is widespread confusion about its quantitative aspects and how they relate to attack vectors and their countermeasures. In this paper we take a look at the stochastic processes underlying typical attacks and th...

  10. Interest Based Financial Intermediation: Analysis and Solutions

    OpenAIRE

    Shaikh, Salman

    2012-01-01

    Interest is prohibited in all monotheist religions. Apart from religion, interest is also regarded as unjust price of money capital by pioneer secular philosophers as well as some renowned economists. However, it is argued by some economists that modern day, market driven interest rate in a competitive financial market is different from usury and that the interest based financial intermediation has served a useful purpose in allocation of resources as well as in allocation of risk, given the ...

  11. Value-Based Analysis of Mobile Tagging

    OpenAIRE

    Oguzhan Aygoren; Kaan Varnali

    2011-01-01

    Innovative use of the mobile medium in delivering customer value presents unprecedented opportunities for marketers. Various types of mobile applications have evolved to provide ubiquitous and instant customer service to capitalize on this opportunity. One application is mobile tagging, a mobile-based innovative tool for convergence marketing. The accumulated academic knowledge on mobile marketing lacks consumer-centric information about this phenomenon. This paper addresses this issue and co...

  12. Confidence-Based Learning in Investment Analysis

    Science.gov (United States)

    Serradell-Lopez, Enric; Lara-Navarra, Pablo; Castillo-Merino, David; González-González, Inés

    The aim of this study is to determine the effectiveness of using multiple choice tests in subjects related to the administration and business management. To this end we used a multiple-choice test with specific questions to verify the extent of knowledge gained and the confidence and trust in the answers. The tests were performed in a group of 200 students at the bachelor's degree in Business Administration and Management. The analysis made have been implemented in one subject of the scope of investment analysis and measured the level of knowledge gained and the degree of trust and security in the responses at two different times of the course. The measurements have been taken into account different levels of difficulty in the questions asked and the time spent by students to complete the test. The results confirm that students are generally able to obtain more knowledge along the way and get increases in the degree of trust and confidence in the answers. It is confirmed as the difficulty level of the questions set a priori by the heads of the subjects are related to levels of security and confidence in the answers. It is estimated that the improvement in the skills learned is viewed favourably by businesses and are especially important for job placement of students.

  13. Dynamic Chest Image Analysis: Model-Based Perfusion Analysis in Dynamic Pulmonary Imaging

    OpenAIRE

    Kiuru Aaro; Kormano Martti; Svedström Erkki; Liang Jianming; Järvi Timo

    2003-01-01

    The "Dynamic Chest Image Analysis" project aims to develop model-based computer analysis and visualization methods for showing focal and general abnormalities of lung ventilation and perfusion based on a sequence of digital chest fluoroscopy frames collected with the dynamic pulmonary imaging technique. We have proposed and evaluated a multiresolutional method with an explicit ventilation model for ventilation analysis. This paper presents a new model-based method for pulmonary perfusion ana...

  14. Engineering Analysis Using a Web-based Protocol

    Science.gov (United States)

    Schoeffler, James D.; Claus, Russell W.

    2002-01-01

    This paper reviews the development of a web-based framework for engineering analysis. A one-dimensional, high-speed analysis code called LAPIN was used in this study, but the approach can be generalized to any engineering analysis tool. The web-based framework enables users to store, retrieve, and execute an engineering analysis from a standard web-browser. We review the encapsulation of the engineering data into the eXtensible Markup Language (XML) and various design considerations in the storage and retrieval of application data.

  15. Network Anomaly Detection Based on Wavelet Analysis

    Directory of Open Access Journals (Sweden)

    Ali A. Ghorbani

    2008-11-01

    Full Text Available Signal processing techniques have been applied recently for analyzing and detecting network anomalies due to their potential to find novel or unknown intrusions. In this paper, we propose a new network signal modelling technique for detecting network anomalies, combining the wavelet approximation and system identification theory. In order to characterize network traffic behaviors, we present fifteen features and use them as the input signals in our system. We then evaluate our approach with the 1999 DARPA intrusion detection dataset and conduct a comprehensive analysis of the intrusions in the dataset. Evaluation results show that the approach achieves high-detection rates in terms of both attack instances and attack types. Furthermore, we conduct a full day's evaluation in a real large-scale WiFi ISP network where five attack types are successfully detected from over 30 millions flows.

  16. Network Anomaly Detection Based on Wavelet Analysis

    Science.gov (United States)

    Lu, Wei; Ghorbani, Ali A.

    2008-12-01

    Signal processing techniques have been applied recently for analyzing and detecting network anomalies due to their potential to find novel or unknown intrusions. In this paper, we propose a new network signal modelling technique for detecting network anomalies, combining the wavelet approximation and system identification theory. In order to characterize network traffic behaviors, we present fifteen features and use them as the input signals in our system. We then evaluate our approach with the 1999 DARPA intrusion detection dataset and conduct a comprehensive analysis of the intrusions in the dataset. Evaluation results show that the approach achieves high-detection rates in terms of both attack instances and attack types. Furthermore, we conduct a full day's evaluation in a real large-scale WiFi ISP network where five attack types are successfully detected from over 30 millions flows.

  17. Sandia National Laboratories analysis code data base

    Energy Technology Data Exchange (ETDEWEB)

    Peterson, C.W.

    1994-11-01

    Sandia National Laboratories, mission is to solve important problems in the areas of national defense, energy security, environmental integrity, and industrial technology. The Laboratories` strategy for accomplishing this mission is to conduct research to provide an understanding of the important physical phenomena underlying any problem, and then to construct validated computational models of the phenomena which can be used as tools to solve the problem. In the course of implementing this strategy, Sandia`s technical staff has produced a wide variety of numerical problem-solving tools which they use regularly in the design, analysis, performance prediction, and optimization of Sandia components, systems and manufacturing processes. This report provides the relevant technical and accessibility data on the numerical codes used at Sandia, including information on the technical competency or capability area that each code addresses, code ``ownership`` and release status, and references describing the physical models and numerical implementation.

  18. Mental EEG Analysis Based on Infomax Algorithm

    Institute of Scientific and Technical Information of China (English)

    WUXiao-pei; GuoXiao-jing; ZANGDao-xin; SHENQian

    2004-01-01

    The patterns of EEG will change with mental tasks performed by the subject. In the field of EEG signal analysis and application, the study to get the patterns of mental EEG and then to use them to classify mental tasks has the significant scientific meaning and great application value. But for the reasons of different artifacts existing in EEG, the pattern detection of EEG under normal mental states is a very difficult problem. In this paper, Independent Component Analysisis applied to EEG signals collected from performing different mental tasks. The experiment results show that when one subject performs a single mental task in different trials, the independent components of EEG are very similar. It means that the independent components can be used as the mental EEG patterns to classify the different mental tasks.

  19. Wavelet Based Fractal Analysis of Airborne Pollen

    CERN Document Server

    Degaudenzi, M E

    1999-01-01

    The most abundant biological particles in the atmosphere are pollen grains and spores. Self protection of pollen allergy is possible through the information of future pollen contents in the air. In spite of the importance of airborne pol len concentration forecasting, it has not been possible to predict the pollen concentrations with great accuracy, and about 25% of the daily pollen forecasts have resulted in failures. Previous analysis of the dynamic characteristics of atmospheric pollen time series indicate that the system can be described by a low dimensional chaotic map. We apply the wavelet transform to study the multifractal characteristics of an a irborne pollen time series. We find the persistence behaviour associated to low pollen concentration values and to the most rare events of highest pollen co ncentration values. The information and the correlation dimensions correspond to a chaotic system showing loss of information with time evolution.

  20. Computational based functional analysis of Bacillus phytases.

    Science.gov (United States)

    Verma, Anukriti; Singh, Vinay Kumar; Gaur, Smriti

    2016-02-01

    Phytase is an enzyme which catalyzes the total hydrolysis of phytate to less phosphorylated myo-inositol derivatives and inorganic phosphate and digests the undigestable phytate part present in seeds and grains and therefore provides digestible phosphorus, calcium and other mineral nutrients. Phytases are frequently added to the feed of monogastric animals so that bioavailability of phytic acid-bound phosphate increases, ultimately enhancing the nutritional value of diets. The Bacillus phytase is very suitable to be used in animal feed because of its optimum pH with excellent thermal stability. Present study is aimed to perform an in silico comparative characterization and functional analysis of phytases from Bacillus amyloliquefaciens to explore physico-chemical properties using various bio-computational tools. All proteins are acidic and thermostable and can be used as suitable candidates in the feed industry. PMID:26672917

  1. Face Recognition Based on Principal Component Analysis

    Directory of Open Access Journals (Sweden)

    Ali Javed

    2013-02-01

    Full Text Available The purpose of the proposed research work is to develop a computer system that can recognize a person by comparing the characteristics of face to those of known individuals. The main focus is on frontal two dimensional images that are taken in a controlled environment i.e. the illumination and the background will be constant. All the other methods of person’s identification and verification like iris scan or finger print scan require high quality and costly equipment’s but in face recognition we only require a normal camera giving us a 2-D frontal image of the person that will be used for the process of the person’s recognition. Principal Component Analysis technique has been used in the proposed system of face recognition. The purpose is to compare the results of the technique under the different conditions and to find the most efficient approach for developing a facial recognition system

  2. Building Extraction from LIDAR Based Semantic Analysis

    Institute of Scientific and Technical Information of China (English)

    YU Jie; YANG Haiquan; TAN Ming; ZHANG Guoning

    2006-01-01

    Extraction of buildings from LIDAR data has been an active research field in recent years. A scheme for building detection and reconstruction from LIDAR data is presented with an object-oriented method which is based on the buildings' semantic rules. Two key steps are discussed: how to group the discrete LIDAR points into single objects and how to establish the buildings' semantic rules. In the end, the buildings are reconstructed in 3D form and three common parametric building models (flat, gabled, hipped) are implemented.

  3. Trajectory Based Behavior Analysis for User Verification

    Science.gov (United States)

    Pao, Hsing-Kuo; Lin, Hong-Yi; Chen, Kuan-Ta; Fadlil, Junaidillah

    Many of our activities on computer need a verification step for authorized access. The goal of verification is to tell apart the true account owner from intruders. We propose a general approach for user verification based on user trajectory inputs. The approach is labor-free for users and is likely to avoid the possible copy or simulation from other non-authorized users or even automatic programs like bots. Our study focuses on finding the hidden patterns embedded in the trajectories produced by account users. We employ a Markov chain model with Gaussian distribution in its transitions to describe the behavior in the trajectory. To distinguish between two trajectories, we propose a novel dissimilarity measure combined with a manifold learnt tuning for catching the pairwise relationship. Based on the pairwise relationship, we plug-in any effective classification or clustering methods for the detection of unauthorized access. The method can also be applied for the task of recognition, predicting the trajectory type without pre-defined identity. Given a trajectory input, the results show that the proposed method can accurately verify the user identity, or suggest whom owns the trajectory if the input identity is not provided.

  4. IMAGE ANALYSIS BASED ON EDGE DETECTION TECHNIQUES

    Institute of Scientific and Technical Information of China (English)

    纳瑟; 刘重庆

    2002-01-01

    A method that incorporates edge detection technique, Markov Random field (MRF), watershed segmentation and merging techniques was presented for performing image segmentation and edge detection tasks. It first applies edge detection technique to obtain a Difference In Strength (DIS) map. An initial segmented result is obtained based on K-means clustering technique and the minimum distance. Then the region process is modeled by MRF to obtain an image that contains different intensity regions. The gradient values are calculated and then the watershed technique is used. DIS calculation is used for each pixel to define all the edges (weak or strong) in the image. The DIS map is obtained. This help as priority knowledge to know the possibility of the region segmentation by the next step (MRF), which gives an image that has all the edges and regions information. In MRF model,gray level l, at pixel location i, in an image X, depends on the gray levels of neighboring pixels. The segmentation results are improved by using watershed algorithm. After all pixels of the segmented regions are processed, a map of primitive region with edges is generated. The edge map is obtained using a merge process based on averaged intensity mean values. A common edge detectors that work on (MRF) segmented image are used and the results are compared. The segmentation and edge detection result is one closed boundary per actual region in the image.

  5. The Route Analysis Based On Flight Plan

    Science.gov (United States)

    Feriyanto, Nur; Saleh, Chairul; Fauzi, Achmad; Rachman Dzakiyullah, Nur; Riza Iwaputra, Kahfi

    2016-02-01

    Economic development effects use of air transportation since the business process in every aspect was increased. Many people these days was prefer using airplane because it can save time and money. This situation also effects flight routes, many airlines offer new routes to deal with competition. Managing flight routes is one of the problems that must be faced in order to find the efficient and effective routes. This paper investigates the best routes based on flight performance by determining the amount of block fuel for the Jakarta-Denpasar flight route. Moreover, in this work compares a two kinds of aircraft and tracks by calculating flight distance, flight time and block fuel. The result shows Jakarta-Denpasar in the Track II has effective and efficient block fuel that can be performed by Airbus 320-200 aircraft. This study can contribute to practice in making an effective decision, especially helping executive management of company due to selecting appropriate aircraft and the track in the flight plan based on the block fuel consumption for business operation.

  6. Network reliability analysis based on percolation theory

    International Nuclear Information System (INIS)

    In this paper, we propose a new way of looking at the reliability of a network using percolation theory. In this new view, a network failure can be regarded as a percolation process and the critical threshold of percolation can be used as network failure criterion linked to the operational settings under control. To demonstrate our approach, we consider both random network models and real networks with different nodes and/or edges lifetime distributions. We study numerically and theoretically the network reliability and find that the network reliability can be solved as a voting system with threshold given by percolation theory. Then we find that the average lifetime of random network increases linearly with the average lifetime of its nodes with uniform life distributions. Furthermore, the average lifetime of the network becomes saturated when system size is increased. Finally, we demonstrate our method on the transmission network system of IEEE 14 bus. - Highlights: • Based on percolation theory, we address questions of practical interest such as “how many failed nodes/edges will break down the whole network?” • The percolation threshold naturally gives a network failure criterion. • The approach based on percolation theory is suited for calculations of large-scale networks

  7. SENTIMENT ANALYSIS OF DOCUMENT BASED ON ANNOTATION

    Directory of Open Access Journals (Sweden)

    Archana Shukla

    2011-11-01

    Full Text Available I present a tool which tells the quality of document or its usefulness based on annotations. Annotation mayinclude comments, notes, observation, highlights, underline, explanation, question or help etc. commentsare used for evaluative purpose while others are used for summarization or for expansion also. Furtherthese comments may be on another annotation. Such annotations are referred as meta-annotation. Allannotation may not get equal weightage. My tool considered highlights, underline as well as comments toinfer the collective sentiment of annotators. Collective sentiments of annotators are classified as positive,negative, objectivity. My tool computes collective sentiment of annotations in two manners. It counts all theannotation present on the documents as well as it also computes sentiment scores of all annotation whichincludes comments to obtain the collective sentiments about the document or to judge the quality ofdocument. I demonstrate the use of tool on research paper.

  8. Analysis of Vehicle-Based Security Operations

    Energy Technology Data Exchange (ETDEWEB)

    Carter, Jason M [ORNL; Paul, Nate R [ORNL

    2015-01-01

    Vehicle-to-vehicle (V2V) communications promises to increase roadway safety by providing each vehicle with 360 degree situational awareness of other vehicles in proximity, and by complementing onboard sensors such as radar or camera in detecting imminent crash scenarios. In the United States, approximately three hundred million automobiles could participate in a fully deployed V2V system if Dedicated Short-Range Communication (DSRC) device use becomes mandatory. The system s reliance on continuous communication, however, provides a potential means for unscrupulous persons to transmit false data in an attempt to cause crashes, create traffic congestion, or simply render the system useless. V2V communications must be highly scalable while retaining robust security and privacy preserving features to meet the intra-vehicle and vehicle-to-infrastructure communication requirements for a growing vehicle population. Oakridge National Research Laboratory is investigating a Vehicle-Based Security System (VBSS) to provide security and privacy for a fully deployed V2V and V2I system. In the VBSS an On-board Unit (OBU) generates short-term certificates and signs Basic Safety Messages (BSM) to preserve privacy and enhance security. This work outlines a potential VBSS structure and its operational concepts; it examines how a vehicle-based system might feasibly provide security and privacy, highlights remaining challenges, and explores potential mitigations to address those challenges. Certificate management alternatives that attempt to meet V2V security and privacy requirements have been examined previously by the research community including privacy-preserving group certificates, shared certificates, and functional encryption. Due to real-world operational constraints, adopting one of these approaches for VBSS V2V communication is difficult. Timely misbehavior detection and revocation are still open problems for any V2V system. We explore the alternative approaches that may be

  9. Simulation based analysis of laser beam brazing

    Science.gov (United States)

    Dobler, Michael; Wiethop, Philipp; Schmid, Daniel; Schmidt, Michael

    2016-03-01

    Laser beam brazing is a well-established joining technology in car body manufacturing with main applications in the joining of divided tailgates and the joining of roof and side panels. A key advantage of laser brazed joints is the seam's visual quality which satisfies highest requirements. However, the laser beam brazing process is very complex and process dynamics are only partially understood. In order to gain deeper knowledge of the laser beam brazing process, to determine optimal process parameters and to test process variants, a transient three-dimensional simulation model of laser beam brazing is developed. This model takes into account energy input, heat transfer as well as fluid and wetting dynamics that lead to the formation of the brazing seam. A validation of the simulation model is performed by metallographic analysis and thermocouple measurements for different parameter sets of the brazing process. These results show that the multi-physical simulation model not only can be used to gain insight into the laser brazing process but also offers the possibility of process optimization in industrial applications. The model's capabilities in determining optimal process parameters are exemplarily shown for the laser power. Small deviations in the energy input can affect the brazing results significantly. Therefore, the simulation model is used to analyze the effect of the lateral laser beam position on the energy input and the resulting brazing seam.

  10. Structural Analysis Using Computer Based Methods

    Science.gov (United States)

    Dietz, Matthew R.

    2013-01-01

    The stiffness of a flex hose that will be used in the umbilical arms of the Space Launch Systems mobile launcher needed to be determined in order to properly qualify ground umbilical plate behavior during vehicle separation post T-0. This data is also necessary to properly size and design the motors used to retract the umbilical arms. Therefore an experiment was created to determine the stiffness of the hose. Before the test apparatus for the experiment could be built, the structure had to be analyzed to ensure it would not fail under given loading conditions. The design model was imported into the analysis software and optimized to decrease runtime while still providing accurate restlts and allow for seamless meshing. Areas exceeding the allowable stresses in the structure were located and modified before submitting the design for fabrication. In addition, a mock up of a deep space habitat and the support frame was designed and needed to be analyzed for structural integrity under different loading conditions. The load cases were provided by the customer and were applied to the structure after optimizing the geometry. Once again, weak points in the structure were located and recommended design changes were made to the customer and the process was repeated until the load conditions were met without exceeding the allowable stresses. After the stresses met the required factors of safety the designs were released for fabrication.

  11. Mammogram-based discriminant fusion analysis for breast cancer diagnosis.

    Science.gov (United States)

    Li, Jun-Bao; Wang, Yun-Heng; Tang, Lin-Lin

    2012-01-01

    Mammogram-based classification is an important and effective way for computer-aided diagnosis (CAD)-based breast cancer diagnosis. In this paper, we present a novel discriminant fusing analysis (DFA)-based mammogram classification CAD-based breast cancer diagnosis. The discriminative breast tissue features are exacted and fused by DFA, and DFA achieves the optimal fusion coefficients. The largest class discriminant in the fused feature space is achieved by DFA for classification. Beside the detailed theory derivation, many experimental evaluations are implemented on Mammography Image Analysis Society mammogram database for breast cancer diagnosis. PMID:23153999

  12. Informatics tools for the analysis and assignment of phosphorylation status in proteomics

    OpenAIRE

    Lee, Dave

    2015-01-01

    Presently, progress in the field of phosphoproteomics has been accelerated by mass spectrometry. This is not a surprise owing to not only the accuracy, precision and high-throughput capabilities of MS but also due to the support it receives from informaticians whom allow the automated analysis; making the task of going from a complex sample to a statistically satisfactory set of phosphopeptides and corresponding site positions with relative ease. However, the process of identifying and subseq...

  13. Bayesian analysis for EMP damaged function based on Weibull distribution

    International Nuclear Information System (INIS)

    Weibull distribution is one of the most commonly used statistical distribution in EMP vulnerability analysis. In the paper, the EMP damage function based on Weibull distribution of solid state relays was solved by bayesian computation using gibbs sampling algorithm. (authors)

  14. Indoor air quality analysis based on Hadoop

    International Nuclear Information System (INIS)

    The air of the office environment is our research object. The data of temperature, humidity, concentrations of carbon dioxide, carbon monoxide and ammonia are collected peer one to eight seconds by the sensor monitoring system. And all the data are stored in the Hbase database of Hadoop platform. With the help of HBase feature of column-oriented store and versioned (automatically add the time column), the time-series data sets are bulit based on the primary key Row-key and timestamp. The parallel computing programming model MapReduce is used to process millions of data collected by sensors. By analysing the changing trend of parameters' value at different time of the same day and at the same time of various dates, the impact of human factor and other factors on the room microenvironment is achieved according to the liquidity of the office staff. Moreover, the effective way to improve indoor air quality is proposed in the end of this paper

  15. Indoor air quality analysis based on Hadoop

    Science.gov (United States)

    Tuo, Wang; Yunhua, Sun; Song, Tian; Liang, Yu; Weihong, Cui

    2014-03-01

    The air of the office environment is our research object. The data of temperature, humidity, concentrations of carbon dioxide, carbon monoxide and ammonia are collected peer one to eight seconds by the sensor monitoring system. And all the data are stored in the Hbase database of Hadoop platform. With the help of HBase feature of column-oriented store and versioned (automatically add the time column), the time-series data sets are bulit based on the primary key Row-key and timestamp. The parallel computing programming model MapReduce is used to process millions of data collected by sensors. By analysing the changing trend of parameters' value at different time of the same day and at the same time of various dates, the impact of human factor and other factors on the room microenvironment is achieved according to the liquidity of the office staff. Moreover, the effective way to improve indoor air quality is proposed in the end of this paper.

  16. Surveillance data bases, analysis, and standardization program

    Energy Technology Data Exchange (ETDEWEB)

    Kam, F.B.K.

    1990-09-26

    The traveler presented a paper at the Seventh ASTM-EURATOM Symposium on Reactor Dosimetry and co-chaired an oral session on Computer Codes and Methods. Papers of considerable interest to the NRC Surveillance Dosimetry Program involved statistically based adjustment procedures and uncertainties. The information exchange meetings with Czechoslovakia and Hungary were very enlightening. Lack of large computers have hindered their surveillance program. They depended very highly on information from their measurement programs which were somewhat limited because of the lack of sophisticated electronics. The Nuclear Research Institute at Rez had to rely on expensive mockups of power reactor configurations to test their fluence exposures. Computers, computer codes, and updated nuclear data would advance their technology rapidly, and they were not hesitant to admit this fact. Both eastern-bloc countries said that IBM is providing an IBM 3090 for educational purposes but research and development studies would have very limited access. They were very apologetic that their currencies were not convertible, and any exchange means that they could provide services or pay for US scientists in their respective countries, but funding for their scientists in the United States, or expenses that involved payment in dollars, must come from us.

  17. Activation analysis based on secondary nuclear reactions

    International Nuclear Information System (INIS)

    Various types of analytical techniques founded on achievements of nuclear physics are used. There are two directions of the using of the main sources of the nuclear projectiles at development of the nuclear methods. In the first, the particles from the source are used directly for the excitation of nuclear reactions. In the second, the particles from the source are used for the generating of intermediate particles of other types which are used in turn for excitation of secondary nuclear reactions. In our research the neutrons are used for the generating of secondary charged particles which serve for excitation of nuclear reactions on elements with small atomic numbers. There are two variants in which both types of neutrons, as thermal, so and fast neutrons are used: 1) The triton flow is produced by thermal neutrons flux, which excites the nuclear reaction 6Li(n, α)T on lithium; 2) The recoil protons are produced as the result of (n, p) elastic or inelastic scattering interaction of fast neutrons with nucleus of light elements, for example, hydrogen. In this work the theoretical base of the application of secondary nuclear reactions excited by recoil protons was investigated

  18. On spectral methods for variance based sensitivity analysis

    OpenAIRE

    Alexanderian, Alen

    2013-01-01

    Consider a mathematical model with a finite number of random parameters. Variance based sensitivity analysis provides a framework to characterize the contribution of the individual parameters to the total variance of the model response. We consider the spectral methods for variance based sensitivity analysis which utilize representations of square integrable random variables in a generalized polynomial chaos basis. Taking a measure theoretic point of view, we provide a rigorous and at the sam...

  19. Architecture Level Dependency Analysis of SOA Based System through ?-Adl

    OpenAIRE

    Pawan Kumar; Ratneshwer

    2016-01-01

    A formal Architecture Description Language (ADL) provides an effective way to dependency analysis at early stage of development. ?-ADL is an ADL that represents the static and dynamic features of software services. In this paper, we describe an approach of dependency analysis of SOA (Service Oriented Architecture) based system, at architecture level, through ?-ADL. A set of algorithms are also proposed for identification of dependency relationships from a SOA based system. The proposed algori...

  20. Toward farm-based policy analysis: concepts applied in Haiti

    OpenAIRE

    Martinez, Juan Carlos; Sain, Gustavo; Yates, Michael

    1991-01-01

    Many policies - on the delivery of inputs or on marketing systems, credit, or extension - influence the potential utilization of new technologies. Through 'farm-based policy analysis' it is possible to use data generated in on-farm research (OFR) to identify policy constraints to the use of new technologies, and to effectively communicate that information to policy makers. This paper describes a tentative framework for farm-based policy analysis and suggests a sequence of five steps for the a...

  1. Multilevel Solvers with Aggregations for Voxel Based Analysis of Geomaterials

    OpenAIRE

    Blaheta, R. (Radim); V. Sokol

    2012-01-01

    Our motivation for voxel based analysis comes from the investigation of geomaterials (geocomposites) arising from rock grouting or sealing. We use finite element analysis based on voxel data from tomography. The arising finite element systems are large scale, which motivates the use of multilevel iterative solvers or preconditioners. Among others we concentrate on multilevel Schwarz preconditioners with aggregations. The aggregations are efficient even in the case of problems with hete...

  2. Product Profitability Analysis Based on EVA and ABC

    OpenAIRE

    Chen Lin; Shuangyuan Wang; Zhilin Qiao

    2013-01-01

    On the purpose of maximizing shareholders’ value, the profitability analysis established on the basis oftraditional accounting earnings cannot meet the demands of providing accurate decision-making information forenterprises. Therefore, this paper implements the Activity Based Costing (ABC) and the Economic Value Added(EVA) into the traditional profitability analysis system, sets up an improved EVA-ABC based profitabilityanalysis system as well as its relative indexes, and applies it to the s...

  3. Empirical validation and comparison of models for customer base analysis

    OpenAIRE

    Persentili Batislam, Emine; Denizel, Meltem; Filiztekin, Alpay

    2007-01-01

    The benefits of retaining customers lead companies to search for means to profile their customers individually and track their retention and defection behaviors. To this end, the main issues addressed in customer base analysis are identification of customer active/inactive status and prediction of future purchase levels. We compare the predictive performance of Pareto/NBD and BG/NBD models from the customer base analysis literature — in terms of repeat purchase levels and active status — usi...

  4. Discrete Discriminant analysis based on tree-structured graphical models

    DEFF Research Database (Denmark)

    Perez de la Cruz, Gonzalo; Eslava, Guillermina

    The purpose of this paper is to illustrate the potential use of discriminant analysis based on tree{structured graphical models for discrete variables. This is done by comparing its empirical performance using estimated error rates for real and simulated data. The results show that discriminant a...... analysis based on tree{structured graphical models is a simple nonlinear method competitive with, and sometimes superior to, other well{known linear methods like those assuming mutual independence between variables and linear logistic regression....

  5. Method for detecting software anomalies based on recurrence plot analysis

    Directory of Open Access Journals (Sweden)

    Michał Mosdorf

    2012-03-01

    Full Text Available Presented paper evaluates method for detecting software anomalies based on recurrence plot analysis of trace log generated by software execution. Described method for detecting software anomalies is based on windowed recurrence quantification analysis for selected measures (e.g. Recurrence rate - RR or Determinism - DET. Initial results show that proposed method is useful in detecting silent software anomalies that do not result in typical crashes (e.g. exceptions.

  6. Fast and Efficient IMAC Protocol for Phosphopeptide enrichment for phosphoproteomic Studies via LC-MS/MS

    OpenAIRE

    McKennan, C.; Spruce, L.; Seeholzer, S; Ding, H.

    2014-01-01

    Recent developments in first dimension High-Performance Liquid Chromatography (HPLC) separation of complex peptide mixtures, followed by a subsequent immobilized metal ion affinity chromatography (IMAC) for phosphopeptide enrichment have shown great promise in both selectivity and quantification of phosphopeptides via LC-MS/MS analysis. The first dimension HPLC, such as hydrophilic interaction chromatography (HILIC) or high pH Reverse Phase chromatography, was employed for its being orthogona...

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

    DEFF Research Database (Denmark)

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

    2009-01-01

    An important mechanism by which plants defend themselves against pathogens is the rapid execution of a hypersensitive response (HR). Tomato plants containing the Cf-4 resistance gene mount an HR that relies on the activation of phosphorylation cascades, when challenged with the Avr4 elicitor....... We show that label-free relative quantification of the phosphoproteome of complex samples is feasible, allowing extension of our knowledge on the general physiology and defense signaling of plants mounting the HR.......-dependent way during the very early stages of HR development. In addition, phosphopeptides originating from four Hsp90 isoforms exhibited altered abundances in Cf-4/Avr4 seedlings compared to control seedlings, suggesting that the isoforms of this chaperone protein have a different function in defense signaling...

  8. PYTHON-based Physics Analysis Environment for LHCb

    CERN Document Server

    Belyaev, I; Mato, P; Barrand, G; Tsaregorodtsev, A; de Oliveira, E

    2004-01-01

    BENDER is the PYTHON based physics analysis application for LHCb. It combines the best features of the underlying GAUDI software architecture with the flexibility of the PYTHON scripting language and provides end-users with a friendly physics analysis oriented environment.

  9. Product Lifecycle Management Architecture: A Model Based Systems Engineering Analysis.

    Energy Technology Data Exchange (ETDEWEB)

    Noonan, Nicholas James [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)

    2015-07-01

    This report is an analysis of the Product Lifecycle Management (PLM) program. The analysis is centered on a need statement generated by a Nuclear Weapons (NW) customer. The need statement captured in this report creates an opportunity for the PLM to provide a robust service as a solution. Lifecycles for both the NW and PLM are analyzed using Model Based System Engineering (MBSE).

  10. An IBM-PC based reactor neutronics analysis package

    International Nuclear Information System (INIS)

    The development of a comprehensive system of microcomputer-based codes suitable for neutronics and shielding analysis of nuclear reactors has been undertaken by EGandG Idaho, Inc., at the Idaho National Engineering Laboratory (INEL). This system has been designed for cross section generation, one-dimensional discrete-ordinates analysis, one- two- and three-dimensional diffusion theory analysis, and various other radiation transport applications of interest

  11. Benefits of Computer Based Content Analysis to Foresight

    OpenAIRE

    Kováříková, Ludmila; Grosová, Stanislava

    2014-01-01

    Purpose of the article: The present manuscript summarizes benefits of the use of computer-based content analysis in a generation phase of foresight initiatives. Possible advantages, disadvantages and limitations of the content analysis for the foresight projects are discussed as well. Methodology/methods: In order to specify the benefits and identify the limitations of the content analysis within the foresight, results of the generation phase of a particular foresight project perf...

  12. Some Linguistic-based and temporal analysis on Wikipedia

    International Nuclear Information System (INIS)

    Wikipedia as a web-based, collaborative, multilingual encyclopaedia project is a very suitable field to carry out research on social dynamics and to investigate the complex concepts of conflict, collaboration, competition, dispute, etc in a large community (∼26 Million) of Wikipedia users. The other face of Wikipedia as a productive society, is its output, consisting of (∼17) Millions of articles written unsupervised by unprofessional editors in more than 270 different languages. In this talk we report some analysis performed on Wikipedia in two different approaches: temporal analysis to characterize disputes and controversies among users and linguistic-based analysis to characterize linguistic features of English texts in Wikipedia. (author)

  13. Alignment analysis of urban railways based on passenger travel demand

    DEFF Research Database (Denmark)

    Andersen, Jonas Lohmann Elkjær; Landex, Alex

    2010-01-01

    , this article presents a computerised GIS based methodology that can be used as decision support for selecting the best alignment. The methodology calculates travel potential within defined buffers surrounding the alignment. The methodology has three different approaches depending on the desired level of detail......: the simple but straight-forward to implement line potential approach that perform corridor analysis, the detailed catchment area analysis based on stops on the alignment and the refined service area analysis that uses search distances in street networks. All three approaches produce trustworthy results...

  14. Chemical Cytometry: Fluorescence-Based Single-Cell Analysis

    Science.gov (United States)

    Cohen, Daniella; Dickerson, Jane A.; Whitmore, Colin D.; Turner, Emily H.; Palcic, Monica M.; Hindsgaul, Ole; Dovichi, Norman J.

    2008-07-01

    Cytometry deals with the analysis of the composition of single cells. Flow and image cytometry employ antibody-based stains to characterize a handful of components in single cells. Chemical cytometry, in contrast, employs a suite of powerful analytical tools to characterize a large number of components. Tools have been developed to characterize nucleic acids, proteins, and metabolites in single cells. Whereas nucleic acid analysis employs powerful polymerase chain reaction-based amplification techniques, protein and metabolite analysis tends to employ capillary electrophoresis separation and ultrasensitive laser-induced fluorescence detection. It is now possible to detect yoctomole amounts of many analytes in single cells.

  15. Fatigue analysis of steam generator cassette parts based on CAE

    International Nuclear Information System (INIS)

    Fatigue analysis has been performed for steam generator nozzle header and tube based on CAE. Three dimensional model was produced using the commercial CAD program, IDEAS and the geometry and boundary condition information have been transformed into input format of ABAQUS for thermal analysis, stress analysis, and fatigue analysis. Cassette nozzle, which has a complex geometry, has been analysed by using the three dimensional model. But steam generator tube has been analysed according to ASME procedure since it can be modelled as a two dimensional finite element model. S-N curve for the titanium alloy of the steam generator tube material was obtained from the material tests. From the analysis, it has been confirmed that these parts of the steam generator cassette satisfy the lifetime of the steam generator cassette. Three dimensional modelling strategy from the thermal analysis to fatigue analysis should be implemented into the design of reactor major components to enhance the efficiency of design procedure

  16. Statistical analysis of MRI-only based dose planning

    DEFF Research Database (Denmark)

    Korsholm, M. E.; Waring, L. W.; Paulsen, Rasmus Reinhold; Edmund, J. M.

    2 %. Conclusions: The investigated DVH points show that MRIonly based RT seems to be a feasible alternative to CT based RT. However, the analysis only describes similarities in DVH points and not in the shape of the DVH. Even though the mean differences are nonsignificant there might be...

  17. Agent-based analysis of organizations : formalization and simulation

    OpenAIRE

    Dignum, M.V.; Tick, C.

    2008-01-01

    Organizational effectiveness depends on many factors, including individual excellence, efficient structures, effective planning and capability to understand and match context requirements. We propose a way to model organizational performance based on a combination of formal models and agent-based simulation that supports the analysis of the congruence of different organizational structures to changing environments

  18. Reliability-Based Robustness Analysis for a Croatian Sports Hall

    DEFF Research Database (Denmark)

    Čizmar, Dean; Kirkegaard, Poul Henning; Sørensen, John Dalsgaard;

    2011-01-01

    complex timber structure with a large number of failure modes is modelled with only a few dominant failure modes. First, a component based robustness analysis is performed based on the reliability indices of the remaining elements after the removal of selected critical elements. The robustness is...

  19. An Analysis of an Improved Bus-Based Multiprocessor Architecture

    Science.gov (United States)

    Ricks, Kenneth G.; Wells, B. Earl

    1998-01-01

    This paper analyses the effectiveness of a hybrid multiprocessing/multicomputing architecture that is based upon a single-board-computer multiprocessor (SBCM) architecture. Based upon empirical analysis using discrete event simulations and Monte Carlo techniques, this hybrid architecture, called the enhanced single-board-computer multiprocessor (ESBCM), is shown to have improved performance and scalability characteristics over current SBCM designs.

  20. Differential Regulatory Analysis Based on Coexpression Network in Cancer Research.

    Science.gov (United States)

    Li, Junyi; Li, Yi-Xue; Li, Yuan-Yuan

    2016-01-01

    With rapid development of high-throughput techniques and accumulation of big transcriptomic data, plenty of computational methods and algorithms such as differential analysis and network analysis have been proposed to explore genome-wide gene expression characteristics. These efforts are aiming to transform underlying genomic information into valuable knowledges in biological and medical research fields. Recently, tremendous integrative research methods are dedicated to interpret the development and progress of neoplastic diseases, whereas differential regulatory analysis (DRA) based on gene coexpression network (GCN) increasingly plays a robust complement to regular differential expression analysis in revealing regulatory functions of cancer related genes such as evading growth suppressors and resisting cell death. Differential regulatory analysis based on GCN is prospective and shows its essential role in discovering the system properties of carcinogenesis features. Here we briefly review the paradigm of differential regulatory analysis based on GCN. We also focus on the applications of differential regulatory analysis based on GCN in cancer research and point out that DRA is necessary and extraordinary to reveal underlying molecular mechanism in large-scale carcinogenesis studies. PMID:27597964

  1. Differential Regulatory Analysis Based on Coexpression Network in Cancer Research

    Directory of Open Access Journals (Sweden)

    Junyi Li

    2016-01-01

    Full Text Available With rapid development of high-throughput techniques and accumulation of big transcriptomic data, plenty of computational methods and algorithms such as differential analysis and network analysis have been proposed to explore genome-wide gene expression characteristics. These efforts are aiming to transform underlying genomic information into valuable knowledges in biological and medical research fields. Recently, tremendous integrative research methods are dedicated to interpret the development and progress of neoplastic diseases, whereas differential regulatory analysis (DRA based on gene coexpression network (GCN increasingly plays a robust complement to regular differential expression analysis in revealing regulatory functions of cancer related genes such as evading growth suppressors and resisting cell death. Differential regulatory analysis based on GCN is prospective and shows its essential role in discovering the system properties of carcinogenesis features. Here we briefly review the paradigm of differential regulatory analysis based on GCN. We also focus on the applications of differential regulatory analysis based on GCN in cancer research and point out that DRA is necessary and extraordinary to reveal underlying molecular mechanism in large-scale carcinogenesis studies.

  2. Kernel-Based Nonlinear Discriminant Analysis for Face Recognition

    Institute of Scientific and Technical Information of China (English)

    LIU QingShan (刘青山); HUANG Rui (黄锐); LU HanQing (卢汉清); MA SongDe (马颂德)

    2003-01-01

    Linear subspace analysis methods have been successfully applied to extract features for face recognition. But they are inadequate to represent the complex and nonlinear variations of real face images, such as illumination, facial expression and pose variations, because of their linear properties. In this paper, a nonlinear subspace analysis method, Kernel-based Nonlinear Discriminant Analysis (KNDA), is presented for face recognition, which combines the nonlinear kernel trick with the linear subspace analysis method - Fisher Linear Discriminant Analysis (FLDA).First, the kernel trick is used to project the input data into an implicit feature space, then FLDA is performed in this feature space. Thus nonlinear discriminant features of the input data are yielded. In addition, in order to reduce the computational complexity, a geometry-based feature vectors selection scheme is adopted. Another similar nonlinear subspace analysis is Kernel-based Principal Component Analysis (KPCA), which combines the kernel trick with linear Principal Component Analysis (PCA). Experiments are performed with the polynomial kernel, and KNDA is compared with KPCA and FLDA. Extensive experimental results show that KNDA can give a higher recognition rate than KPCA and FLDA.

  3. Phosphoproteomics study based on in vivo inhibition reveals sites of calmodulin-dependent protein kinase II regulation in the heart

    NARCIS (Netherlands)

    Scholten, A.; Preisinger, C.; Corradini, E.; Bourgonje, V.J.A.; Hennrich, M.L.; van Veen, T.A.B.; Swaminathan, P.D.; Joiner, M.L.; Vos, M.A.; Anderson, M.E.; Heck, A.J.R.

    2013-01-01

    Background The multifunctional Ca2+‐ and calmodulin‐dependent protein kinase II (CaMKII) is a crucial mediator of cardiac physiology and pathology. Increased expression and activation of CaMKII has been linked to elevated risk for arrhythmic events and is a hallmark of human heart failure. A useful

  4. Affine invariant texture analysis based on structural properties

    OpenAIRE

    Zhang, Jianguo; Tan, Tieniu

    2002-01-01

    This paper presents a new texture analysis method based on structural properties. The texture features extracted using this algorithm are invariant to affine transform (including rotation, translation, scaling, and skewing). Affine invariant structural properties are derived based on texel areas. An area-ratio map utilizing these properties is introduced to characterize texture images. Histogram based on this map is constructed for texture classification. Efficiency of this algorithm for affi...

  5. Computer-based modelling and analysis in engineering geology

    OpenAIRE

    Giles, David

    2014-01-01

    This body of work presents the research and publications undertaken under a general theme of computer-based modelling and analysis in engineering geology. Papers are presented on geotechnical data management, data interchange, Geographical Information Systems, surface modelling, geostatistical methods, risk-based modelling, knowledge-based systems, remote sensing in engineering geology and on the integration of computer applications into applied geoscience teaching. The work highlights my...

  6. Earthquake Analysis of Structure by Base Isolation Technique in SAP

    OpenAIRE

    T. Subramani; J. Jothi

    2014-01-01

    This paper presents an overview of the present state of base isolation techniques with special emphasis and a brief on other techniques developed world over for mitigating earthquake forces on the structures. The dynamic analysis procedure for isolated structures is briefly explained. The provisions of FEMA 450 for base isolated structures are highlighted. The effects of base isolation on structures located on soft soils and near active faults are given in brief. Simple case s...

  7. Abnormal traffic flow data detection based on wavelet analysis

    Directory of Open Access Journals (Sweden)

    Xiao Qian

    2016-01-01

    Full Text Available In view of the traffic flow data of non-stationary, the abnormal data detection is difficult.proposed basing on the wavelet analysis and least squares method of abnormal traffic flow data detection in this paper.First using wavelet analysis to make the traffic flow data of high frequency and low frequency component and separation, and then, combined with least square method to find abnormal points in the reconstructed signal data.Wavelet analysis and least square method, the simulation results show that using wavelet analysis of abnormal traffic flow data detection, effectively reduce the detection results of misjudgment rate and false negative rate.

  8. Adaptive Fourier Decomposition Based Time-Frequency Analysis

    Institute of Scientific and Technical Information of China (English)

    Li-Ming Zhang

    2014-01-01

    The attempt to represent a signal simultaneously in time and frequency domains is full of challenges. The recently proposed adaptive Fourier decomposition (AFD) offers a practical approach to solve this problem. This paper presents the principles of the AFD based time-frequency analysis in three aspects: instantaneous frequency analysis, frequency spectrum analysis, and the spectrogram analysis. An experiment is conducted and compared with the Fourier transform in convergence rate and short-time Fourier transform in time-frequency distribution. The proposed approach performs better than both the Fourier transform and short-time Fourier transform.

  9. A scenario-based procedure for seismic risk analysis

    International Nuclear Information System (INIS)

    A new methodology for seismic risk analysis based on probabilistic interpretation of deterministic or scenario-based hazard analysis, in full compliance with the likelihood principle and therefore meeting the requirements of modern risk analysis, has been developed. The proposed methodology can easily be adjusted to deliver its output in a format required for safety analysts and civil engineers. The scenario-based approach allows the incorporation of all available information collected in a geological, seismotectonic and geotechnical database of the site of interest as well as advanced physical modelling techniques to provide a reliable and robust deterministic design basis for civil infrastructures. The robustness of this approach is of special importance for critical infrastructures. At the same time a scenario-based seismic hazard analysis allows the development of the required input for probabilistic risk assessment (PRA) as required by safety analysts and insurance companies. The scenario-based approach removes the ambiguity in the results of probabilistic seismic hazard analysis (PSHA) which relies on the projections of Gutenberg-Richter (G-R) equation. The problems in the validity of G-R projections, because of incomplete to total absence of data for making the projections, are still unresolved. Consequently, the information from G-R must not be used in decisions for design of critical structures or critical elements in a structure. The scenario-based methodology is strictly based on observable facts and data and complemented by physical modelling techniques, which can be submitted to a formalised validation process. By means of sensitivity analysis, knowledge gaps related to lack of data can be dealt with easily, due to the limited amount of scenarios to be investigated. The proposed seismic risk analysis can be used with confidence for planning, insurance and engineering applications. (author)

  10. Identification of novel PAMP-triggered phosphorylation and dephosphorylation events in arabidopsis thaliana by quantitative phosphoproteomic analysis

    KAUST Repository

    Rayapuram, Naganand

    2014-04-04

    Signaling cascades rely strongly on protein kinase-mediated substrate phosphorylation. Currently a major challenge in signal transduction research is to obtain high confidence substrate phosphorylation sites and assign them to specific kinases. In response to bacterial flagellin, a pathogen-associated molecular pattern (PAMP), we searched for rapidly phosphorylated proteins in Arabidopsis thaliana by combining multistage activation (MSA) and electron transfer dissociation (ETD) fragmentation modes, which generate complementary spectra and identify phosphopeptide sites with increased reliability. Of a total of 825 phosphopeptides, we identified 58 to be differentially phosphorylated. These peptides harbor kinase motifs of mitogen-activated protein kinases (MAPKs) and calcium-dependent protein kinases (CDPKs), as well as yet unknown protein kinases. Importantly, 12 of the phosphopeptides show reduced phosphorylation upon flagellin treatment. Since protein abundance levels did not change, these results indicate that flagellin induces not only various protein kinases but also protein phosphatases, even though a scenario of inhibited kinase activity may also be possible. © 2014 American Chemical Society.

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

    DEFF Research Database (Denmark)

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

    2011-01-01

    Mitochondria play a central role in energy metabolism and cellular survival, and consequently mitochondrial dysfunction is associated with a number of human pathologies. Reversible protein phosphorylation emerges as a central mechanism in the regulation of several mitochondrial processes. In skel...

  12. 磷蛋白组的研究技术及其进展%Advances in Analysis Techniques of Phosphoproteome

    Institute of Scientific and Technical Information of China (English)

    杨珺; 邹全明; 蔡绍皙; 郭刚; 朱永红

    2003-01-01

    真核细胞中蛋白质磷酸化是一个重要事件.真核细胞利用可逆的蛋白磷酸化来控制许多细胞过程包括信号转换、基因表达、细胞周期等.磷蛋白组的研究涉及磷蛋白的分离和鉴定,磷酸化残基定位和定量分析.由于蛋白质磷酸化是一个动态过程,在细胞中磷蛋白含量低,磷酸化位点可变,且磷酸肽的质谱信号常常会受到抑制,所以磷蛋白的分析存在更多的困难.本文介绍了国内外在磷酸蛋白的分离鉴定及定量分析方面的研究技术以及进展情况.目前,质谱仍然是核心的鉴定技术,寻找更好富集方法是最大的挑战.定量蛋白组学是对蛋白质的差异表达进行精确的定量分析.目前还不存在一种独立的方法可以完成磷蛋白的分离、鉴定,以及磷酸位点的定位和定量分析.随着样品分离技术和相关仪器的发展,磷酸蛋白快速、准确、全面分析鉴定将能够实现.

  13. Phosphoproteomic analysis for the identification of predictive molecular factors and new molecular targets in breast, colorectal, ovary, and lung cancer

    International Nuclear Information System (INIS)

    Neoadjuvant chemotherapy is a standard therapeutic approach for several types of locally advanced and metastatic tumors. Molecular factors predictive of response to therapy are highly needed for exploiting the potential benefit of properative chemotherapy. Moreover, neoadjuvant chemotherapy represents an ideal clinical model for studying predictive factors since it allows to obtain pre-treatment tissue biopsies which can be analyzed and the results compared to clinical and pathological response. Protein kinases represent some of the most important drug targets in medicine today and their aberrant activation are involved in many disease processes including cancer development and progression

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

    Science.gov (United States)

    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

  15. Implementation of Ultraviolet Photodissociation on a Benchtop Q Exactive Mass Spectrometer and Its Application to Phosphoproteomics.

    Science.gov (United States)

    Fort, Kyle L; Dyachenko, Andrey; Potel, Clement M; Corradini, Eleonora; Marino, Fabio; Barendregt, Arjan; Makarov, Alexander A; Scheltema, Richard A; Heck, Albert J R

    2016-02-16

    Proteomics applications performed on the popular benchtop Q Exactive Orbitrap mass spectrometer have so far relied exclusively on higher collision-energy dissociation (HCD) fragmentation for peptide sequencing. While this fragmentation technique is applicable to a wide range of biological questions, it also has limitations, and all questions cannot be addressed equally well. Here, we demonstrate that the fragmentation capabilities of the Q Exactive mass spectrometer can be extended with ultraviolet photodissociation (UVPD) fragmentation, complete with synchronization triggering to make it compatible with liquid chromatography (LC)/tandem mass spectrometry (MS/MS) workflows. We show that UVPD not only is directly compatible with LC/MS workflows but also, when combined with these workflows, can result in higher database scores and increased identification rates for complex samples as compared to HCD methods. UVPD as a fragmentation technique offers prompt, high-energy fragmentation, which can potentially lead to improved analyses of labile post-translational modifications. Techniques like HCD result in substantial amounts of modification losses, competing with fragmentation pathways that provide information-rich ion fragments. We investigate here the utility of UVPD for identification of phosphorylated peptides and find that UVPD fragmentation reduces the extent of labile modification loss by up to ∼60%. Collectively, when integrated into a complete workflow on the Q Exactive Orbitrap, UVPD provides distinct advantages to the analysis of post-translational modifications and is a powerful and complementary addition to the proteomic toolbox. PMID:26760441

  16. Analysis of security protocols based on challenge-response

    Institute of Scientific and Technical Information of China (English)

    LUO JunZhou; YANG Ming

    2007-01-01

    Security protocol is specified as the procedure of challenge-response, which uses applied cryptography to confirm the existence of other principals and fulfill some data negotiation such as session keys. Most of the existing analysis methods,which either adopt theorem proving techniques such as state exploration or logic reasoning techniques such as authentication logic, face the conflicts between analysis power and operability. To solve the problem, a new efficient method is proposed that provides SSM semantics-based definition of secrecy and authentication goals and applies authentication logic as fundamental analysis techniques,in which secrecy analysis is split into two parts: Explicit-Information-Leakage and Implicit-Information-Leakage, and correspondence analysis is concluded as the analysis of the existence relationship of Strands and the agreement of Strand parameters. This new method owns both the power of the Strand Space Model and concision of authentication logic.

  17. Virtual stress amplitude-based low cycle fatigue reliability analysis

    International Nuclear Information System (INIS)

    A method for virtual stress amplitude-based low cycle fatigue reliability analysis is developed. Different from existent methods, probability-based modified Ramberg-Osgood stress-strain relations (P-ε-σ curves) are newly introduced to take into account the scatter of stress-strain responses, where the metallurgical quality of material is not enough good i.e. weld metal to show a same stress-strain response for different specimens under same loading level. In addition, a virtual stress amplitude-based analysis is used to be in agreement with the existent codes for nuclear components. i.e. ASME section III. The analysis is performed by a principle of the stochastic analysis system in same safety level concurrently. Combined the probability-based modified Ramberg-Osgood stress-strain relations, the probability-based Langer S-N curves (P-S-N curves) and the Neuber's local stress-strain rule, the method can be applied to predict the fatigue life at specified reliability and loading history and to estimate the reliability at specified loading history and expectation fatigue life. Applicability of the method has been indicated by a test analysis of 1Cr18Ni9ti steel-weld metal, which was used for machining the pipes of some nuclear reactors, during low cycle fatigue

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

    Science.gov (United States)

    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

  19. Bayesian-network-based safety risk analysis in construction projects

    International Nuclear Information System (INIS)

    This paper presents a systemic decision support approach for safety risk analysis under uncertainty in tunnel construction. Fuzzy Bayesian Networks (FBN) is used to investigate causal relationships between tunnel-induced damage and its influential variables based upon the risk/hazard mechanism analysis. Aiming to overcome limitations on the current probability estimation, an expert confidence indicator is proposed to ensure the reliability of the surveyed data for fuzzy probability assessment of basic risk factors. A detailed fuzzy-based inference procedure is developed, which has a capacity of implementing deductive reasoning, sensitivity analysis and abductive reasoning. The “3σ criterion” is adopted to calculate the characteristic values of a triangular fuzzy number in the probability fuzzification process, and the α-weighted valuation method is adopted for defuzzification. The construction safety analysis progress is extended to the entire life cycle of risk-prone events, including the pre-accident, during-construction continuous and post-accident control. A typical hazard concerning the tunnel leakage in the construction of Wuhan Yangtze Metro Tunnel in China is presented as a case study, in order to verify the applicability of the proposed approach. The results demonstrate the feasibility of the proposed approach and its application potential. A comparison of advantages and disadvantages between FBN and fuzzy fault tree analysis (FFTA) as risk analysis tools is also conducted. The proposed approach can be used to provide guidelines for safety analysis and management in construction projects, and thus increase the likelihood of a successful project in a complex environment. - Highlights: • A systemic Bayesian network based approach for safety risk analysis is developed. • An expert confidence indicator for probability fuzzification is proposed. • Safety risk analysis progress is extended to entire life cycle of risk-prone events. • A typical

  20. A Framework for Web-Based Mechanical Design and Analysis

    Institute of Scientific and Technical Information of China (English)

    Chiaming; Yen; Wujeng; Li

    2002-01-01

    In this paper, a Web-based Mechanical Design and A na lysis Framework (WMDAF) is proposed. This WMADF allows designers to develop web -based computer aided programs in a systematic way during the collaborative mec hanical system design and analysis process. This system is based on an emerg ing web-based Content Management System (CMS) called eXtended Object Oriented P ortal System (XOOPS). Due to the Open Source Status of the XOOPS CMS, programs d eveloped with this framework can be further customized to ...

  1. Tikhonov regularization-based operational transfer path analysis

    Science.gov (United States)

    Cheng, Wei; Lu, Yingying; Zhang, Zhousuo

    2016-06-01

    To overcome ill-posed problems in operational transfer path analysis (OTPA), and improve the stability of solutions, this paper proposes a novel OTPA based on Tikhonov regularization, which considers both fitting degrees and stability of solutions. Firstly, fundamental theory of Tikhonov regularization-based OTPA is presented, and comparative studies are provided to validate the effectiveness on ill-posed problems. Secondly, transfer path analysis and source contribution evaluations for numerical cases studies on spherical radiating acoustical sources are comparatively studied. Finally, transfer path analysis and source contribution evaluations for experimental case studies on a test bed with thin shell structures are provided. This study provides more accurate transfer path analysis for mechanical systems, which can benefit for vibration reduction by structural path optimization. Furthermore, with accurate evaluation of source contributions, vibration monitoring and control by active controlling vibration sources can be effectively carried out.

  2. NONLINEAR DATA RECONCILIATION METHOD BASED ON KERNEL PRINCIPAL COMPONENT ANALYSIS

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    In the industrial process situation, principal component analysis (PCA) is a general method in data reconciliation.However, PCA sometime is unfeasible to nonlinear feature analysis and limited in application to nonlinear industrial process.Kernel PCA (KPCA) is extension of PCA and can be used for nonlinear feature analysis.A nonlinear data reconciliation method based on KPCA is proposed.The basic idea of this method is that firstly original data are mapped to high dimensional feature space by nonlinear function, and PCA is implemented in the feature space.Then nonlinear feature analysis is implemented and data are reconstructed by using the kernel.The data reconciliation method based on KPCA is applied to ternary distillation column.Simulation results show that this method can filter the noise in measurements of nonlinear process and reconciliated data can represent the true information of nonlinear process.

  3. Automatic Video-based Analysis of Human Motion

    DEFF Research Database (Denmark)

    Fihl, Preben

    . A multi-view approach to pose estimation is also presented that integrates low level information from different cameras to generate better pose estimates during heavy occlusions. The works presented in this thesis contribute in these different areas of video-based analysis of human motion and altogether......The human motion contains valuable information in many situations and people frequently perform an unconscious analysis of the motion of other people to understand their actions, intentions, and state of mind. An automatic analysis of human motion will facilitate many applications and thus has...... received great interest from both industry and research communities. The focus of this thesis is on video-based analysis of human motion and the thesis presents work within three overall topics, namely foreground segmentation, action recognition, and human pose estimation. Foreground segmentation is often...

  4. Data Warehouse Requirements Analysis Framework: Business-Object Based Approach

    Directory of Open Access Journals (Sweden)

    Anirban Sarkar

    2012-01-01

    Full Text Available Detailed requirements analysis plays a key role towards the design of successful Data Warehouse (DW system. The requirements analysis specifications are used as the prime input for the construction of conceptual level multidimensional data model. This paper has proposed a Business Object based requirements analysis framework for DW system which is supported with abstraction mechanism and reuse capability. It also facilitate the stepwise mapping of requirements descriptions into high level design components of graph semantic based conceptual level object oriented multidimensional data model. The proposed framework starts with the identification of the analytical requirements using business process driven approach and finally refine the requirements in further detail to map into the conceptual level DW design model using either Demand-driven of Mixed-driven approach for DW requirements analysi

  5. Similar words analysis based on POS-CBOW language model

    Directory of Open Access Journals (Sweden)

    Dongru RUAN

    2015-10-01

    Full Text Available Similar words analysis is one of the important aspects in the field of natural language processing, and it has important research and application values in text classification, machine translation and information recommendation. Focusing on the features of Sina Weibo's short text, this paper presents a language model named as POS-CBOW, which is a kind of continuous bag-of-words language model with the filtering layer and part-of-speech tagging layer. The proposed approach can adjust the word vectors' similarity according to the cosine similarity and the word vectors' part-of-speech metrics. It can also filter those similar words set on the base of the statistical analysis model. The experimental result shows that the similar words analysis algorithm based on the proposed POS-CBOW language model is better than that based on the traditional CBOW language model.

  6. Applying measurement-based probabilistic timing analysis to buffer resources

    OpenAIRE

    Kosmidis L.; Vardanega T.; Abella J.; Quinones E.; Cazorla F.J.

    2013-01-01

    The use of complex hardware makes it difficult for current timing analysis techniques to compute trustworthy and tight worst-case execution time (WCET) bounds. Those techniques require detailed knowledge of the internal operation and state of the platform, at both the software and hardware level. Obtaining that information for modern hardware platforms is increasingly difficult. Measurement-Based Probabilistic Timing Analysis (MBPTA) reduces the cost of acquiring the knowledge needed for comp...

  7. UML based risk analysis - Application to a medical robot

    OpenAIRE

    Guiochet, Jérémie; Baron, Claude

    2004-01-01

    Medical robots perform complex tasks and share their working area with humans. Therefore , they belong to safety critical systems. In nowadays development process, safety is often managed by the way of dependability techniques. We propose a new global approach , based on the risk concept in order to guide designers along the safety analysis of such complex systems. Safety depends on risk management activity, which core is risk analysis. This one consists in three steps: system definition, haz...

  8. Study of engine noise based on independent component analysis

    Institute of Scientific and Technical Information of China (English)

    HAO Zhi-yong; JIN Yan; YANG Chen

    2007-01-01

    Independent component analysis was applied to analyze the acoustic signals from diesel engine. First the basic principle of independent component analysis (ICA) was reviewed. Diesel engine acoustic signal was decomposed into several independent components (Ics); Fourier transform and continuous wavelet transform (CWT) were applied to analyze the independent components. Different noise sources of the diesel engine were separated, based on the characteristics of different component in time-frequency domain.

  9. FDTD method based electromagnetic solver for printed-circuit analysis

    OpenAIRE

    Gnilenko, Alexey B.; Paliy, Oleg V.

    2003-01-01

    An electromagnetic solver for printed-circuit analysis is presented. The electromagnetic simulator is based on the finite-difference time-domain method with first-order Mur's absorbing boundary conditions. The solver environment comprises a layout graphic editor for circuit topology preparation and a data postprocessor for presenting the calculation results. The solver has been applied to the analysis of printed-circuit components such as printed antenna, microstrip discontinuities, etc.

  10. A Contrastive Analysis on Web-based Intercultural Peer Feedback

    OpenAIRE

    Qin Wang

    2013-01-01

    This paper made a contrastive analysis on peer feedback generated by Chinese EFL learners and Native Speakers of English, who participated in a web-based Cross-Pacific Writing Exchange program. The analysis mainly focused on differences in terms of commenting size, nature and function; the pragmatic differences between two groups of learners were investigated as well. The present study afforded us lessons for peer review training program and provided pedagogical implications for L2 writing.Ke...

  11. Reliable analysis for pressure vessel based on ANSYS

    International Nuclear Information System (INIS)

    With the PDS of ANSYS procedure, the ramdomicity of the actually structure design parameters is simulated, by taking the wall thickness, pressure load and elastic module as input random variables. Based on the reliability analysis of the pressure vessel by Monte-Carlo procedure, the stress probability distribution of this finite element analysis model and the sensitivity of the design parameters such as the pressure load and wall thickness to the stress distribution are obtained. (authors)

  12. iBarcode.org: web-based molecular biodiversity analysis

    OpenAIRE

    Hajibabaei Mehrdad; Singer Gregory AC

    2009-01-01

    Abstract Background DNA sequences have become a primary source of information in biodiversity analysis. For example, short standardized species-specific genomic regions, DNA barcodes, are being used as a global standard for species identification and biodiversity studies. Most DNA barcodes are being generated by laboratories that have an expertise in DNA sequencing but not in bioinformatics data analysis. Therefore, we have developed a web-based suite of tools to help the DNA barcode research...

  13. European Climate - Energy Security Nexus. A model based scenario analysis

    International Nuclear Information System (INIS)

    In this research, we have provided an overview of the climate-security nexus in the European sector through a model based scenario analysis with POLES model. The analysis underline that under stringent climate policies, Europe take advantage of a double dividend in its capacity to develop a new cleaner energy model and in lower vulnerability to potential shocks on the international energy markets. (authors)

  14. Phosphopeptide enrichment: Development of magnetic solid phase extraction method based on polydopamine coating and Ti(4+)-IMAC.

    Science.gov (United States)

    Piovesana, Susy; Capriotti, Anna Laura; Cavaliere, Chiara; Ferraris, Francesca; Samperi, Roberto; Ventura, Salvatore; Laganà, Aldo

    2016-02-25

    Protein post translational modifications currently represent one of the main challenges with proteomic analysis, due to the important biological role they play within cells. Protein phosphorylation is one of the most important, with several approaches developed for phosphopeptides enrichment and analysis, essential for comprehensive phosphoproteomic analysis. However, the development of new materials for phosphopeptides enrichment may overcome previous drawbacks and improve enrichment of such peptides. In this regard, new magnetic stationary phases based on polydopamine coating and Ti(4+) immobilization exploit the potential of IMAC enrichment and couple it with the versatility of magnetic solid phase extraction. In this work the use of such stationary phase was extended from the MALDI proof of concept stage with the development of an optimized method for phosphopeptides enrichment compatible with typical shotgun proteomics experimental workflows. Different loading and elution buffers were tested to improve phosphopeptides recovery and enrichment selectivity. Finally, the analysis of isolated peptides pointed out that polydopamine alone is an ideal support matrix for polar post translational modifications because it enables to reduce unspecific binding and preferentially binds hydrophilic peptides. PMID:26851086

  15. Using the DOE Knowledge Base for Special Event Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Armstrong, H.M.; Harris, J.M.; Young, C.J.

    1998-10-20

    The DOE Knowledge Base is a library of detailed information whose purpose is to support the United States National Data Center (USNDC) in its mission to monitor compliance with the Comprehensive Test Ban Treaty (CTBT). One of the important tasks which the USNDC must accomplish is to periodically perform detailed analysis of events of high interest, so-called "Special Events", to provide the national authority with information needed to make policy decisions. In this paper we investigate some possible uses of the Knowledge Base for Special Event Analysis (SEA), and make recommendations for improving Knowledge Base support for SEA. To analyze an event in detail, there are two basic types of data which must be used sensor-derived data (wave- forms, arrivals, events, etc.) and regiohalized contextual data (known sources, geological characteristics, etc.). Cur- rently there is no single package which can provide full access to both types of data, so for our study we use a separate package for each MatSeis, the Sandia Labs-developed MATLAB-based seismic analysis package, for wave- form data analysis, and ArcView, an ESRI product, for contextual data analysis. Both packages are well-suited to pro- totyping because they provide a rich set of currently available functionality and yet are also flexible and easily extensible, . Using these tools and Phase I Knowledge Base data sets, we show how the Knowledge Base can improve both the speed and the quality of SEA. Empirically-derived interpolated correction information can be accessed to improve both location estimates and associated error estimates. This information can in turn be used to identi~ any known nearby sources (e.g. mines, volcanos), which may then trigger specialized processing of the sensor data. Based on the location estimate, preferred magnitude formulas and discriminants can be retrieved, and any known blockages can be identified to prevent miscalculations. Relevant historic events can be identilled either by

  16. AN HMM BASED ANALYSIS FRAMEWORK FOR SEMANTIC VIDEO EVENTS

    Institute of Scientific and Technical Information of China (English)

    You Junyong; Liu Guizhong; Zhang Yaxin

    2007-01-01

    Semantic video analysis plays an important role in the field of machine intelligence and pattern recognition. In this paper, based on the Hidden Markov Model (HMM), a semantic recognition framework on compressed videos is proposed to analyze the video events according to six low-level features. After the detailed analysis of video events, the pattern of global motion and five features in foreground--the principal parts of videos, are employed as the observations of the Hidden Markov Model to classify events in videos. The applications of the proposed framework in some video event detections demonstrate the promising success of the proposed framework on semantic video analysis.

  17. RULE-BASED SENTIMENT ANALYSIS OF UKRAINIAN REVIEWS

    Directory of Open Access Journals (Sweden)

    Mariana Romanyshyn

    2013-07-01

    Full Text Available Last decade witnessed a lot of research in the field of sentiment analysis. Understanding the attitude and the emotions that people express in written text proved to be really important and helpful in sociology, political science, psychology, market research, and, of course, artificial intelligence. This paper demonstrates a rule-based approach to clause-level sentiment analysis of reviews in Ukrainian. The general architecture of the implemented sentiment analysis system is presented, the current stage of research is described and further work is explained. The main emphasis is made on the design of rules for computing sentiments.

  18. Open access for ALICE analysis based on virtualization technology

    CERN Document Server

    Buncic, P; Schutz, Y

    2015-01-01

    Open access is one of the important leverages for long-term data preservation for a HEP experiment. To guarantee the usability of data analysis tools beyond the experiment lifetime it is crucial that third party users from the scientific community have access to the data and associated software. The ALICE Collaboration has developed a layer of lightweight components built on top of virtualization technology to hide the complexity and details of the experiment-specific software. Users can perform basic analysis tasks within CernVM, a lightweight generic virtual machine, paired with an ALICE specific contextualization. Once the virtual machine is launched, a graphical user interface is automatically started without any additional configuration. This interface allows downloading the base ALICE analysis software and running a set of ALICE analysis modules. Currently the available tools include fully documented tutorials for ALICE analysis, such as the measurement of strange particle production or the nuclear modi...

  19. A Comprehensive Transcriptomic and Proteomic Analysis of Hydra Head Regeneration.

    Science.gov (United States)

    Petersen, Hendrik O; Höger, Stefanie K; Looso, Mario; Lengfeld, Tobias; Kuhn, Anne; Warnken, Uwe; Nishimiya-Fujisawa, Chiemi; Schnölzer, Martina; Krüger, Marcus; Özbek, Suat; Simakov, Oleg; Holstein, Thomas W

    2015-08-01

    The cnidarian freshwater polyp Hydra sp. exhibits an unparalleled regeneration capacity in the animal kingdom. Using an integrative transcriptomic and stable isotope labeling by amino acids in cell culture proteomic/phosphoproteomic approach, we studied stem cell-based regeneration in Hydra polyps. As major contributors to head regeneration, we identified diverse signaling pathways adopted for the regeneration response as well as enriched novel genes. Our global analysis reveals two distinct molecular cascades: an early injury response and a subsequent, signaling driven patterning of the regenerating tissue. A key factor of the initial injury response is a general stabilization of proteins and a net upregulation of transcripts, which is followed by a subsequent activation cascade of signaling molecules including Wnts and transforming growth factor (TGF) beta-related factors. We observed moderate overlap between the factors contributing to proteomic and transcriptomic responses suggesting a decoupled regulation between the transcriptional and translational levels. Our data also indicate that interstitial stem cells and their derivatives (e.g., neurons) have no major role in Hydra head regeneration. Remarkably, we found an enrichment of evolutionarily more recent genes in the early regeneration response, whereas conserved genes are more enriched in the late phase. In addition, genes specific to the early injury response were enriched in transposon insertions. Genetic dynamicity and taxon-specific factors might therefore play a hitherto underestimated role in Hydra regeneration. PMID:25841488

  20. A New Customer Segmentation Framework Based on Biclustering Analysis

    OpenAIRE

    Xiaohui Hu; Haolan Zhang; Xiaosheng Wu; Jianlin Chen; Yu Xiao; Yun Xue; Tiechen Li; Hongya Zhao

    2014-01-01

    The paper presents a novel approach for customer segmentation which is the basic issue for an effective CRM ( Customer Relationship Management ). Firstly, the chi-square statistical analysis is applied to choose set of attributes and K-means algorithm is employed to quantize the value of each attribute. Then DBSCAN algorithm based on density is introduced to classify the customers into three groups (the first, the second and the third class). Finally biclustering based on improved Apriori alg...