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Sample records for quantitative phosphoproteome analysis

  1. Quantitative Phosphoproteomics Analysis of ERBB3/ERBB4 Signaling

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed

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

    2016-02-01

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

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

    SciTech Connect

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

    2012-11-11

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2014-03-01

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

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

    PubMed Central

    Ji, Qinqin; Salomon, Arthur R.

    2015-01-01

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

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

    PubMed

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

    2016-07-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-03-01

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

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

    PubMed Central

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

    2014-01-01

    Helicobacter pylori is a Gram-negative bacterial pathogen colonizing the human stomach. Infection with H. pylori causes chronic inflammation of the gastric mucosa and may lead to peptic ulceration and/or gastric cancer. A major virulence determinant of H. pylori is the type IV secretion system (T4SS), which is used to inject the virulence factor CagA into the host cell, triggering a wide range of cellular signaling events. Here, we used a phosphoproteomic approach to investigate tyrosine signaling in response to host-pathogen interaction, using stable isotope labeling in cell culture (SILAC) of AGS cells to obtain a differential picture between multiple infection conditions. Cells were infected with wild type H. pylori P12, a P12Δ CagA deletion mutant, and a P12Δ PAI deletion mutant to compare signaling changes over time and in the absence of CagA or the T4SS. Tryptic peptides were enriched for tyrosine (Tyr) phosphopeptides and analyzed by nano-LC-Orbitrap MS. In total, 85 different phosphosites were found to be regulated following infection. The majority of phosphosites identified were kinases of the MAPK family. CagA and the T4SS were found to be key regulators of Tyr phosphosites. Our findings indicate that CagA primarily induces activation of ERK1 and integrin-linked factors, whereas the T4SS primarily modulates JNK and p38 activation. PMID:25101063

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

    SciTech Connect

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

    2008-10-01

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

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

    PubMed Central

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

    2012-01-01

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

  15. Sample Collection Method Bias Effects in Quantitative Phosphoproteomics.

    PubMed

    Kanshin, Evgeny; Tyers, Michael; Thibault, Pierre

    2015-07-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    Adachi, Jun; Narumi, Ryohei; Tomonaga, Takeshi

    2016-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-12-01

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

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

    PubMed

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

    2015-11-01

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

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

    PubMed

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

    2015-10-01

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

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

    PubMed

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

    2014-11-01

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

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

    PubMed Central

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

    2015-01-01

    Reversible protein phosphorylation, regulated by protein kinases and phosphatases, mediates a switch between protein activity and cellular pathways that contribute to a large number of cellular processes. The Mycobacterium tuberculosis genome encodes 11 Serine/Threonine kinases (STPKs) which show close homology to eukaryotic kinases. This study aimed to elucidate the phosphoproteomic landscape of a clinical isolate of M. tuberculosis. We performed a high throughput mass spectrometric analysis of proteins extracted from an early-logarithmic phase culture. Whole cell lysate proteins were processed using the filter-aided sample preparation method, followed by phosphopeptide enrichment of tryptic peptides by strong cation exchange (SCX) and Titanium dioxide (TiO2) chromatography. The MaxQuant quantitative proteomics software package was used for protein identification. Our analysis identified 414 serine/threonine/tyrosine phosphorylated sites, with a distribution of S/T/Y sites; 38% on serine, 59% on threonine and 3% on tyrosine; present on 303 unique peptides mapping to 214 M. tuberculosis proteins. Only 45 of the S/T/Y phosphorylated proteins identified in our study had been previously described in the laboratory strain H37Rv, confirming previous reports. The remaining 169 phosphorylated proteins were newly identified in this clinical M. tuberculosis Beijing strain. We identified 5 novel tyrosine phosphorylated proteins. These findings not only expand upon our current understanding of the protein phosphorylation network in clinical M. tuberculosis but the data set also further extends and complements previous knowledge regarding phosphorylated peptides and phosphorylation sites in M. tuberculosis. PMID:25713560

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

    PubMed

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

    2016-07-01

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

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

    PubMed Central

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

    2015-01-01

    Background: The molecular mechanisms underlying the endometriosis are still not completely understood. In order to test the hypothesis that the approaches in phosphoproteomics might contribute to the identification of key biomarkers to assess disease pathogenesis and drug targets, we carried out a phosphoproteomics analysis of human endometrium. Methods: A large-scale differential phosphoproteome analysis, using peptide enrichment of titanium dioxide purify and sequential elution from immobilized metal affinity chromatography with linear trap quadrupole-tandem mass spectrometry, was performed in endometrium tissues from 8 women with or without endometriosis. Results: The phosphorylation profiling of endometrium from endometriosis patients had been obtained, and found that identified 516 proteins were modified at phosphorylation level during endometriosis. Gene ontology annotation analysis showed that these proteins were enriched in cellular processes of binding and catalytic activity. Further pathway analysis showed that ribosome pathway and focal adhesion pathway were the top two pathways, which might be deregulated during the development of endometriosis. Conclusions: That large-scale phosphoproteome quantification has been successfully identified in endometrium tissues of women with or without endometriosis will provide new insights to understand the molecular mechanisms of the development of endometriosis. PMID:26415800

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2011-11-01

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

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

    PubMed

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

    2016-02-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2015-08-01

    Trees adjust their growth following forced changes in orientation to re-establish a vertical position. In angiosperms, this adjustment involves the differential regulation of vascular cambial activity between the lower (opposite wood) and upper (tension wood) sides of the leaning stem. We investigated the molecular mechanisms leading to the formation of differential wood types through a quantitative proteomic and phosphoproteomic analysis on poplar subjected to a gravitropic stimulus. We identified and quantified 675 phosphopeptides, corresponding to 468 phosphoproteins, and 3 763 nonphosphorylated peptides, corresponding to 1 155 proteins, in the differentiating xylem of straight-growing trees (control) and trees subjected to a gravitational stimulus during 8 weeks. About 1% of the peptides were specific to a wood type (straight, opposite, or tension wood). Proteins quantified in more than one type of wood were more numerous: a mixed linear model showed 389 phosphopeptides and 556 proteins to differ in abundance between tension wood and opposite wood. Twenty-one percent of the phosphoproteins identified here were described in their phosphorylated form for the first time. Our analyses revealed remarkable developmental molecular plasticity, with wood type-specific phosphorylation events, and highlighted the involvement of different proteins in the biosynthesis of cell wall components during the formation of the three types of wood. PMID:26112267

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2016-07-01

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

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

    PubMed Central

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

    2015-01-01

    Drought and heat stress, especially their combination, greatly affect crop production. Many studies have described transcriptome, proteome and phosphoproteome changes in response of plants to drought or heat stress. However, the study about the phosphoproteomic changes in response of crops to the combination stress is scare. To understand the mechanism of maize responses to the drought and heat combination stress, phosphoproteomic analysis was performed on maize leaves by using multiplex iTRAQ-based quantitative proteomic and LC-MS/MS methods. Five-leaf-stage maize was subjected to drought, heat or their combination, and the leaves were collected. Globally, heat, drought and the combined stress significantly changed the phosphorylation levels of 172, 149, and 144 phosphopeptides, respectively. These phosphopeptides corresponded to 282 proteins. Among them, 23 only responded to the combined stress and could not be predicted from their responses to single stressors; 30 and 75 only responded to drought and heat, respectively. Notably, 19 proteins were phosphorylated on different sites in response to the single and combination stresses. Of the seven significantly enriched phosphorylation motifs identified, two were common for all stresses, two were common for heat and the combined stress, and one was specific to the combined stress. The signaling pathways in which the phosphoproteins were involved clearly differed among the three stresses. Functional characterization of the phosphoproteins and the pathways identified here could lead to new targets for the enhancement of crop stress tolerance, which will be particularly important in the face of climate change and the increasing prevalence of abiotic stressors. PMID:25999967

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

    PubMed

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

    2015-07-01

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

  18. Phosphoproteomics in Cancer

    PubMed Central

    Harsha, H. C.; Pandey, Akhilesh

    2010-01-01

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

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

    PubMed Central

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

    2012-01-01

    Vasopressin regulates transport across the collecting duct epithelium in part via effects on gene transcription. Transcriptional regulation occurs partially via changes in phosphorylation of transcription factors, transcriptional coactivators, and protein kinases in the nucleus. To test whether vasopressin alters the nuclear phosphoproteome of vasopressin-sensitive cultured mouse mpkCCD cells, we used stable isotope labeling and mass spectrometry to quantify thousands of phosphorylation sites in nuclear extracts and nuclear pellet fractions. Measurements were made in the presence and absence of the vasopressin analog dDAVP. Of the 1,251 sites quantified, 39 changed significantly in response to dDAVP. Network analysis of the regulated proteins revealed two major clusters (“cell-cell adhesion” and “transcriptional regulation”) that were connected to known elements of the vasopressin signaling pathway. The hub proteins for these two clusters were the transcriptional coactivator β-catenin and the transcription factor c-Jun. Phosphorylation of β-catenin at Ser552 was increased by dDAVP [log2(dDAVP/vehicle) = 1.79], and phosphorylation of c-Jun at Ser73 was decreased [log2(dDAVP/vehicle) = −0.53]. The β-catenin site is known to be targeted by either protein kinase A or Akt, both of which are activated in response to vasopressin. The c-Jun site is a canonical target for the MAP kinase Jnk2, which is downregulated in response to vasopressin in the collecting duct. The data support the idea that vasopressin-mediated control of transcription in collecting duct cells involves selective changes in the nuclear phosphoproteome. All data are available to users at http://helixweb.nih.gov/ESBL/Database/mNPPD/. PMID:22992673

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

    PubMed Central

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

    2015-01-01

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

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

    SciTech Connect

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

    2012-04-17

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2015-12-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2013-06-01

    β-Blockers are widely used to prevent cardiac arrhythmias and to treat hypertension by inhibiting β-adrenergic receptors (βARs) and thus decreasing contractility and heart rate. βARs initiate phosphorylation-dependent signaling cascades, but only a small number of the target proteins are known. We used quantitative in vivo phosphoproteomics to identify 670 site-specific phosphorylation changes in murine hearts in response to acute treatment with specific βAR agonists. The residues adjacent to the regulated phosphorylation sites exhibited a sequence-specific preference (R-X-X-pS/T), and integrative analysis of sequence motifs and interaction networks suggested that the kinases AMPK (adenosine 5'-monophosphate-activated protein kinase), Akt, and mTOR (mammalian target of rapamycin) mediate βAR signaling, in addition to the well-established pathways mediated by PKA (cyclic adenosine monophosphate-dependent protein kinase) and CaMKII (calcium/calmodulin-dependent protein kinase type II). We found specific regulation of phosphorylation sites on six ion channels and transporters that mediate increased ion fluxes at higher heart rates, and we showed that phosphorylation of one of these, Ser(92) of the potassium channel KV7.1, increased current amplitude. Our data set represents a quantitative analysis of phosphorylated proteins regulated in vivo upon stimulation of seven-transmembrane receptors, and our findings reveal previously unknown phosphorylation sites that regulate myocardial contractility, suggesting new potential targets for the treatment of heart disease and hypertension. PMID:23737553

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-04-01

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

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

    PubMed

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

    2016-06-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2014-05-01

    The serotonin 5-HT(2A) receptor is a primary target of psychedelic hallucinogens such as lysergic acid diethylamine, mescaline, and psilocybin, which reproduce some of the core symptoms of schizophrenia. An incompletely resolved paradox is that only some 5-HT(2A) receptor agonists exhibit hallucinogenic activity, whereas structurally related agonists with comparable affinity and activity lack such a psychoactive activity. Using a strategy combining stable isotope labeling by amino acids in cell culture with enrichment in phosphorylated peptides by means of hydrophilic interaction liquid chromatography followed by immobilized metal affinity chromatography, we compared the phosphoproteome in HEK-293 cells transiently expressing the 5-HT(2A) receptor and exposed to either vehicle or the synthetic hallucinogen 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI) or the nonhallucinogenic 5-HT(2A) agonist lisuride. Among the 5995 identified phosphorylated peptides, 16 sites were differentially phosphorylated upon exposure of cells to DOI versus lisuride. These include a serine (Ser(280)) located in the third intracellular loop of the 5-HT(2A) receptor, a region important for its desensitization. The specific phosphorylation of Ser(280) by hallucinogens was further validated by quantitative mass spectrometry analysis of immunopurified receptor digests and by Western blotting using a phosphosite specific antibody. The administration of DOI, but not of lisuride, to mice, enhanced the phosphorylation of 5-HT(2A) receptors at Ser(280) in the prefrontal cortex. Moreover, hallucinogens induced a less pronounced desensitization of receptor-operated signaling in HEK-293 cells and neurons than did nonhallucinogenic agonists. The mutation of Ser(280) to aspartic acid (to mimic phosphorylation) reduced receptor desensitization by nonhallucinogenic agonists, whereas its mutation to alanine increased the ability of hallucinogens to desensitize the receptor. This study reveals a biased

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2008-11-01

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

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

    PubMed

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

    2015-12-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-05-20

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

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

    PubMed

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

    2014-01-01

    Stable isotope labeling by amino acids combined with mass spectrometry is a widely used methodology for measuring relative changes in protein and phosphorylation levels at a global level. We have applied this method to the model organism Caenorhabditis elegans in combination with RNAi-mediated gene knockdown by feeding the nematode on pre-labeled lysine auxotroph Escherichia coli. In this chapter, we describe in details the generation of the E. coli strain, incorporation of heavy isotope-labeled lysine in C. elegans, and the procedure for a comprehensive global phosphoproteomic experiment. PMID:25059608

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed Central

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

    2015-01-01

    Hyperactivated RAS drives progression of many human malignancies. However, oncogenic activity of RAS is dependent on simultaneous inactivation of protein phosphatase 2A (PP2A) activity. Although PP2A is known to regulate some of the RAS effector pathways, it has not been systematically assessed how these proteins functionally interact. Here we have analyzed phosphoproteomes regulated by either RAS or PP2A, by phosphopeptide enrichment followed by mass-spectrometry-based label-free quantification. To allow data normalization in situations where depletion of RAS or PP2A inhibitor CIP2A causes a large uni-directional change in the phosphopeptide abundance, we developed a novel normalization strategy, named pairwise normalization. This normalization is based on adjusting phosphopeptide abundances measured before and after the enrichment. The superior performance of the pairwise normalization was verified by various independent methods. Additionally, we demonstrate how the selected normalization method influences the downstream analyses and interpretation of pathway activities. Consequently, bioinformatics analysis of RAS and CIP2A regulated phosphoproteomes revealed a significant overlap in their functional pathways. This is most likely biologically meaningful as we observed a synergistic survival effect between CIP2A and RAS expression as well as KRAS activating mutations in TCGA pan-cancer data set, and synergistic relationship between CIP2A and KRAS depletion in colony growth assays. PMID:26278961

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

    PubMed

    Teets, Nicholas M; Denlinger, David L

    2016-08-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2014-01-01

    DNA damaging agents such as ionizing radiation or chemotherapy are frequently used in oncology. DNA damage response (DDR)—triggered by radiation-induced double strand breaks—is orchestrated mainly by three Phosphatidylinositol 3-kinase-related kinases (PIKKs): Ataxia teleangiectasia mutated (ATM), DNA-dependent protein kinase (DNA-PK) and ATM and Rad3-related kinase (ATR). Their activation promotes cell-cycle arrest and facilitates DNA damage repair, resulting in radioresistance. Recently developed specific ATR inhibitor, VE-821 (3-amino-6-(4-(methylsulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide), has been reported to have a significant radio- and chemo-sensitizing effect delimited to cancer cells (largely p53-deficient) without affecting normal cells. In this study, we employed SILAC-based quantitative phosphoproteomics to describe the mechanism of the radiosensitizing effect of VE-821 in human promyelocytic leukemic cells HL-60 (p53-negative). Hydrophilic interaction liquid chromatography (HILIC)-prefractionation with TiO2-enrichment and nano-liquid chromatography—tandem mass spectrometry (LC-MS/MS) analysis revealed 9834 phosphorylation sites. Proteins with differentially up-/down-regulated phosphorylation were mostly localized in the nucleus and were involved in cellular processes such as DDR, all phases of the cell cycle, and cell division. Moreover, sequence motif analysis revealed significant changes in the activities of kinases involved in these processes. Taken together, our data indicates that ATR kinase has multiple roles in response to DNA damage throughout the cell cycle and that its inhibitor VE-821 is a potent radiosensitizing agent for p53-negative HL-60 cells. PMID:25003641

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

    Adams, Nancy E.; Maurelli, Anthony T.

    2015-01-01

    Chlamydia are Gram-negative, obligate intracellular bacteria responsible for significant diseases in humans and economically important domestic animals. These pathogens undergo a unique biphasic developmental cycle transitioning between the environmentally stable elementary body (EB) and the replicative intracellular reticulate body (RB), a conversion that appears to require extensive regulation of protein synthesis and function. However, Chlamydia possess a limited number of canonical mechanisms of transcriptional regulation. Ser/Thr/Tyr phosphorylation of proteins in bacteria has been increasingly recognized as an important mechanism of post-translational control of protein function. We utilized 2D gel electrophoresis coupled with phosphoprotein staining and MALDI-TOF/TOF analysis to map the phosphoproteome of the EB and RB forms of Chlamydia caviae. Forty-two non-redundant phosphorylated proteins were identified (some proteins were present in multiple locations within the gels). Thirty-four phosphorylated proteins were identified in EBs, including proteins found in central metabolism and protein synthesis, Chlamydia-specific hypothetical proteins and virulence-related proteins. Eleven phosphorylated proteins were identified in RBs, mostly involved in protein synthesis and folding and a single virulence-related protein. Only three phosphoproteins were found in both EB and RB phosphoproteomes. Collectively, 41 of 42 C. caviae phosphoproteins were present across Chlamydia species, consistent with the existence of a conserved chlamydial phosphoproteome. The abundance of stage-specific phosphoproteins suggests that protein phosphorylation may play a role in regulating the function of developmental-stage-specific proteins and/or may function in concert with other factors in directing EB–RB transitions. PMID:25998263

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

    PubMed

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

    2016-06-01

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

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

    PubMed

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

    2014-10-01

    Fragile X mental retardation protein (FMRP) is an RNA-binding protein that has a major effect on neuronal protein synthesis. Transcriptional silencing of the FMR1 gene leads to loss of FMRP and development of Fragile X syndrome (FXS), the most common known hereditary cause of intellectual impairment and autism. Here we utilize SILAC-based quantitative phosphoproteomics to analyze murine FMR1(-) and FMR1(+) fibroblastic cell lines derived from FMR1-KO embryos to identify proteins and phosphorylation sites dysregulated as a consequence of FMRP loss. We quantify FMRP-related changes in the levels of 5,023 proteins and 6,133 phosphorylation events and map them onto major signal transduction pathways. Our study confirms global downregulation of the MAPK/ERK pathway and decrease in phosphorylation level of ERK1/2 in the absence of FMRP, which is connected to attenuation of long-term potentiation. We detect differential expression of several key proteins from the p53 pathway, pointing to the involvement of p53 signaling in dysregulated cell cycle control in FXS. Finally, we detect differential expression and phosphorylation of proteins involved in pre-mRNA processing and nuclear transport, as well as Wnt and calcium signaling, such as PLC, PKC, NFAT, and cPLA2. We postulate that calcium homeostasis is likely affected in molecular pathogenesis of FXS. PMID:25168779

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

    PubMed

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

    2011-08-25

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

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

    PubMed Central

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

    2008-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-03-01

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

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

    PubMed

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

    2015-04-29

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

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

    PubMed

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

    2016-02-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    Narumi, Ryohei; Tomonaga, Takeshi

    2016-01-01

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

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

    PubMed

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

    2016-04-01

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

  3. Phosphoproteomic analysis reveals regulatory mechanisms at the kidney filtration barrier.

    PubMed

    Rinschen, Markus M; Wu, Xiongwu; König, Tim; Pisitkun, Trairak; Hagmann, Henning; Pahmeyer, Caroline; Lamkemeyer, Tobias; Kohli, Priyanka; Schnell, Nicole; Schermer, Bernhard; Dryer, Stuart; Brooks, Bernard R; Beltrao, Pedro; Krueger, Marcus; Brinkkoetter, Paul T; Benzing, Thomas

    2014-07-01

    Diseases of the kidney filtration barrier are a leading cause of ESRD. Most disorders affect the podocytes, polarized cells with a limited capacity for self-renewal that require tightly controlled signaling to maintain their integrity, viability, and function. Here, we provide an atlas of in vivo phosphorylated, glomerulus-expressed proteins, including podocyte-specific gene products, identified in an unbiased tandem mass spectrometry-based approach. We discovered 2449 phosphorylated proteins corresponding to 4079 identified high-confidence phosphorylated residues and performed a systematic bioinformatics analysis of this dataset. We discovered 146 phosphorylation sites on proteins abundantly expressed in podocytes. The prohibitin homology domain of the slit diaphragm protein podocin contained one such site, threonine 234 (T234), located within a phosphorylation motif that is mutated in human genetic forms of proteinuria. The T234 site resides at the interface of podocin dimers. Free energy calculation through molecular dynamic simulations revealed a role for T234 in regulating podocin dimerization. We show that phosphorylation critically regulates formation of high molecular weight complexes and that this may represent a general principle for the assembly of proteins containing prohibitin homology domains. PMID:24511133

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

    PubMed Central

    Horst, Walter Johannes

    2013-01-01

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

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

    PubMed

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

    2016-05-01

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

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

    PubMed

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

    2016-04-12

    Cortistatin A (CA) is a highly selective inhibitor of the Mediator kinases CDK8 and CDK19. Using CA, we now report a large-scale identification of Mediator kinase substrates in human cells (HCT116). We identified over 16,000 quantified phosphosites including 78 high-confidence Mediator kinase targets within 64 proteins, including DNA-binding transcription factors and proteins associated with chromatin, DNA repair, and RNA polymerase II. Although RNA-seq data correlated with Mediator kinase targets, the effects of CA on gene expression were limited and distinct from CDK8 or CDK19 knockdown. Quantitative proteome analyses, tracking around 7,000 proteins across six time points (0-24 hr), revealed that CA selectively affected pathways implicated in inflammation, growth, and metabolic regulation. Contrary to expectations, increased turnover of Mediator kinase targets was not generally observed. Collectively, these data support Mediator kinases as regulators of chromatin and RNA polymerase II activity and suggest their roles extend beyond transcription to metabolism and DNA repair. PMID:27050516

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

    PubMed Central

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

    2009-01-01

    Aberrant signaling causes many diseases, and manipulating signaling pathways with kinase inhibitors has emerged as a promising area of drug research. Most kinase inhibitors target the conserved ATP-binding pocket; therefore specificity is a major concern. Proteomics has previously been used to identify the direct targets of kinase inhibitors upon affinity purification from cellular extracts. Here we introduce a complementary approach to evaluate the effects of kinase inhibitors on the entire cell signaling network. We used triple labeling SILAC (stable isotope labeling by amino acids in cell culture) to compare cellular phosphorylation levels for control, epidermal growth factor stimulus, and growth factor combined with kinase inhibitors. Of thousands of phosphopeptides, less than 10% had a response pattern indicative of targets of U0126 and SB202190, two widely used MAPK inhibitors. Interestingly, 83% of the growth factor-induced phosphorylation events were affected by either or both inhibitors, showing quantitatively that early signaling processes are predominantly transmitted through the MAPK cascades. In contrast to MAPK inhibitors, dasatinib, a clinical drug directed against BCR-ABL, which is the cause of chronic myelogenous leukemia, affected nearly 1,000 phosphopeptides. In addition to the proximal effects on ABL and its immediate targets, dasatinib broadly affected the downstream MAPK pathways. Pathway mapping of regulated sites implicated a variety of cellular functions, such as chromosome remodeling, RNA splicing, and cytoskeletal organization, some of which have been described in the literature before. Our assay is streamlined and generic and could become a useful tool in kinase drug development. PMID:19651622

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

    PubMed Central

    Dephoure, Noah; Gygi, Steven P.

    2011-01-01

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

  9. Challenges in plasma membrane phosphoproteomics

    PubMed Central

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

    2011-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2016-05-17

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

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

    PubMed Central

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

    2015-01-01

    Rice blast, caused by the fungal pathogen Magnaporthe oryzae, is the most destructive disease of rice and causes tremendous losses of rice yield worldwide. To explore the molecular mechanisms involved in the rice–M. oryzae interaction, we conducted a time-course phosphoproteomic analysis of leaf samples from resistant and susceptible rice cultivars infected with M. oryzae. This data article contains additional results and analysis of M. oryzae-regulated phosphoproteins in rice leaves [1]. We report the analysis of M. oryzae-regulated phosphoproteins at all time points, including Venn diagram analysis, close-up views, relative intensities, and functional category, and the MS spectra of representative phosphoprotein and representative phosphorylated peptides. PMID:26217708

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

    PubMed

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

    2016-08-01

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

  15. Quantitative Hydrocarbon Surface Analysis

    NASA Technical Reports Server (NTRS)

    Douglas, Vonnie M.

    2000-01-01

    The elimination of ozone depleting substances, such as carbon tetrachloride, has resulted in the use of new analytical techniques for cleanliness verification and contamination sampling. The last remaining application at Rocketdyne which required a replacement technique was the quantitative analysis of hydrocarbons by infrared spectrometry. This application, which previously utilized carbon tetrachloride, was successfully modified using the SOC-400, a compact portable FTIR manufactured by Surface Optics Corporation. This instrument can quantitatively measure and identify hydrocarbons from solvent flush of hardware as well as directly analyze the surface of metallic components without the use of ozone depleting chemicals. Several sampling accessories are utilized to perform analysis for various applications.

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

    PubMed

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

    2014-10-01

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

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

    PubMed Central

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

    2014-01-01

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

  18. Phosphoproteomics and Lung Cancer Research

    PubMed Central

    López, Elena; Cho, William C. S.

    2012-01-01

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

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

    PubMed

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

    2016-05-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-11-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  5. Quantitative environmental risk analysis

    SciTech Connect

    Klovning, J.; Nilsen, E.F.

    1995-12-31

    According to regulations relating to implementation and rise of risk analysis in the petroleum activities issued by the Norwegian Petroleum Directorate, it is mandatory for an operator on the Norwegian Continental Shelf to establish acceptance criteria for environmental risk in the activities and carry out environmental risk analysis. This paper presents a {open_quotes}new{close_quotes} method for environmental risk analysis developed by the company. The objective has been to assist the company to meet rules and regulations and to assess and describe the environmental risk in a systematic manner. In the environmental risk analysis the most sensitive biological resource in the affected area is used to assess the environmental damage. The analytical method is based on the methodology for quantitative risk analysis related to loss of life. In addition it incorporates the effect of seasonal fluctuations in the environmental risk evaluations. The paper is describing the function of the main analytical sequences exemplified through an analysis of environmental risk related to exploration drilling in an environmental sensitive area on the Norwegian Continental Shelf.

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

    PubMed

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

    2011-01-01

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

  7. Technical phosphoproteomic and bioinformatic tools useful in cancer research

    PubMed Central

    2011-01-01

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

  8. Sperm phosphoproteomics: historical perspectives and current methodologies

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

    2013-01-01

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

  10. Toward defining the phosphoproteome of Xenopus laevis embryos

    PubMed Central

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

    2010-01-01

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

  11. Computational phosphoproteomics: From identification to localization

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2013-09-17

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

  13. Analysis of dynamic tyrosine phosphoproteome in LFA-1 triggered migrating T-cells.

    PubMed

    Verma, Navin K; Dempsey, Eugene; Freeley, Michael; Botting, Catherine H; Long, Aideen; Kelleher, Dermot; Volkov, Yuri

    2011-06-01

    The ordered, directional migration of T-lymphocytes is a key process during immune surveillance and response. This requires cell adhesion to the high endothelial venules or to the extracellular matrix by a series of surface receptor/ligand interactions involving adhesion molecules of the integrin family including lymphocyte function associated molecule-1 (LFA-1) and intercellular adhesion molecules (ICAMs). Reversible protein phosphorylation is emerging as a key player in the regulation of biological functions with tyrosine phosphorylation playing a crucial role in signal transduction. Thus, the study of this type of post-translational modification at the proteomic level has great biological significance. In this work, phospho-enriched cell lysates from LFA-1-triggered migrating human T-cells were subjected to immunoaffinity purification of tyrosine phosphorylated proteins, mass spectrometric, and bioinformatic analysis. In addition to the identification of several well-documented proteins, the analysis suggested involvement of a number of new and novel proteins in LFA-1 induced T-cell migration. This dataset expands the list of the signaling components of the LFA-1 induced phosphotyrosine protein complexes in migrating T-cells that will be extremely useful in the study of their specific roles within LFA-1 associated signaling pathways. Identification of proteins previously not reported in the context of LFA-1 stimulated signal transduction might provide new insights into understanding the LFA-1 signaling networks and aid in the search for new potential therapeutic targets. PMID:20945386

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

    2011-01-01

    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

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

    PubMed

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

    2015-08-01

    Labeling-based proteomics is a powerful method for detection of differentially expressed proteins (DEPs). The current data analysis platform typically relies on protein-level ratios, which is obtained by summarizing peptide-level ratios for each protein. In shotgun proteomics, however, some proteins are quantified with more peptides than others, and this reproducibility information is not incorporated into the differential expression (DE) analysis. Here, we propose a novel probabilistic framework EBprot that directly models the peptide-protein hierarchy and rewards the proteins with reproducible evidence of DE over multiple peptides. To evaluate its performance with known DE states, we conducted a simulation study to show that the peptide-level analysis of EBprot provides better receiver-operating characteristic and more accurate estimation of the false discovery rates than the methods based on protein-level ratios. We also demonstrate superior classification performance of peptide-level EBprot analysis in a spike-in dataset. To illustrate the wide applicability of EBprot in different experimental designs, we applied EBprot to a dataset for lung cancer subtype analysis with biological replicates and another dataset for time course phosphoproteome analysis of EGF-stimulated HeLa cells with multiplexed labeling. Through these examples, we show that the peptide-level analysis of EBprot is a robust alternative to the existing statistical methods for the DE analysis of labeling-based quantitative datasets. The software suite is freely available on the Sourceforge website http://ebprot.sourceforge.net/. All MS data have been deposited in the ProteomeXchange with identifier PXD001426 (http://proteomecentral.proteomexchange.org/dataset/PXD001426/). PMID:25913743

  17. Characterization of the Phosphoproteome in SLE Patients

    PubMed Central

    Huang, Jianrong; Dai, Yong

    2012-01-01

    Protein phosphorylation is a complex regulatory event that is involved in the signaling networks that affect virtually every cellular process. The protein phosphorylation may be a novel source for discovering biomarkers and drug targets. However, a systematic analysis of the phosphoproteome in patients with SLE has not been performed. To clarify the pathogenesis of systemic lupus erythematosus (SLE), we compared phosphoprotein expression in PBMCs from SLE patients and normal subjects using proteomics analyses. Phosphopeptides were enriched using TiO2 from PBMCs isolated from 15 SLE patients and 15 healthy subjects and then analyzed by automated LC-MS/MS analysis. Phosphorylation sites were identified and quantitated by MASCOT and MaxQuant. A total of 1035 phosphorylation sites corresponding to 618 NCBI-annotated genes were identified in SLE patients compared with normal subjects. Differentially expressed proteins, peptides and phosphorylation sites were then subjected to bioinformatics analyses. Gene ontology(GO) and pathway analyses showed that nucleic acid metabolism, cellular component organization, transport and multicellular organismal development pathways made up the largest proportions of the differentially expressed genes. Pathway analyses showed that the mitogen-activated protein kinase (MAPK) signaling pathway and actin cytoskeleton regulators made up the largest proportions of the metabolic pathways. Network analysis showed that rous sarcoma oncogene (SRC), v-rel reticuloendotheliosis viral oncogene homolog A (RELA), histone deacetylase (HDA1C) and protein kinase C, delta (PRKCD) play important roles in the stability of the network. These data suggest that aberrant protein phosphorylation may contribute to SLE pathogenesis. PMID:23285258

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  20. Quantitative intracerebral brain hemorrhage analysis

    NASA Astrophysics Data System (ADS)

    Loncaric, Sven; Dhawan, Atam P.; Cosic, Dubravko; Kovacevic, Domagoj; Broderick, Joseph; Brott, Thomas

    1999-05-01

    In this paper a system for 3-D quantitative analysis of human spontaneous intracerebral brain hemorrhage (ICH) is described. The purpose of the developed system is to perform quantitative 3-D measurements of the parameters of ICH region and from computed tomography (CT) images. The measured parameter in this phase of the system development is volume of the hemorrhage region. The goal of the project is to measure parameters for a large number of patients having ICH and to correlate measured parameters to patient morbidity and mortality.

  1. Software for quantitative trait analysis

    PubMed Central

    2005-01-01

    This paper provides a brief overview of software currently available for the genetic analysis of quantitative traits in humans. Programs that implement variance components, Markov Chain Monte Carlo (MCMC), Haseman-Elston (H-E) and penetrance model-based linkage analyses are discussed, as are programs for measured genotype association analyses and quantitative trait transmission disequilibrium tests. The software compared includes LINKAGE, FASTLINK, PAP, SOLAR, SEGPATH, ACT, Mx, MERLIN, GENEHUNTER, Loki, Mendel, SAGE, QTDT and FBAT. Where possible, the paper provides URLs for acquiring these programs through the internet, details of the platforms for which the software is available and the types of analyses performed. PMID:16197737

  2. Image analysis and quantitative morphology.

    PubMed

    Mandarim-de-Lacerda, Carlos Alberto; Fernandes-Santos, Caroline; Aguila, Marcia Barbosa

    2010-01-01

    Quantitative studies are increasingly found in the literature, particularly in the fields of development/evolution, pathology, and neurosciences. Image digitalization converts tissue images into a numeric form by dividing them into very small regions termed picture elements or pixels. Image analysis allows automatic morphometry of digitalized images, and stereology aims to understand the structural inner three-dimensional arrangement based on the analysis of slices showing two-dimensional information. To quantify morphological structures in an unbiased and reproducible manner, appropriate isotropic and uniform random sampling of sections, and updated stereological tools are needed. Through the correct use of stereology, a quantitative study can be performed with little effort; efficiency in stereology means as little counting as possible (little work), low cost (section preparation), but still good accuracy. This short text provides a background guide for non-expert morphologists. PMID:19960334

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

    Cancer.gov

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

  4. Enrichment Strategies in Phosphoproteomics.

    PubMed

    Leitner, Alexander

    2016-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  6. Bioimaging for quantitative phenotype analysis.

    PubMed

    Chen, Weiyang; Xia, Xian; Huang, Yi; Chen, Xingwei; Han, Jing-Dong J

    2016-06-01

    With the development of bio-imaging techniques, an increasing number of studies apply these techniques to generate a myriad of image data. Its applications range from quantification of cellular, tissue, organismal and behavioral phenotypes of model organisms, to human facial phenotypes. The bio-imaging approaches to automatically detect, quantify, and profile phenotypic changes related to specific biological questions open new doors to studying phenotype-genotype associations and to precisely evaluating molecular changes associated with quantitative phenotypes. Here, we review major applications of bioimage-based quantitative phenotype analysis. Specifically, we describe the biological questions and experimental needs addressable by these analyses, computational techniques and tools that are available in these contexts, and the new perspectives on phenotype-genotype association uncovered by such analyses. PMID:26850283

  7. Optimization of quantitative infrared analysis

    NASA Astrophysics Data System (ADS)

    Duerst, Richard W.; Breneman, W. E.; Dittmar, Rebecca M.; Drugge, Richard E.; Gagnon, Jim E.; Pranis, Robert A.; Spicer, Colleen K.; Stebbings, William L.; Westberg, J. W.; Duerst, Marilyn D.

    1994-01-01

    A number of industrial processes, especially quality assurance procedures, accept information on relative quantities of components in mixtures, whenever absolute values for the quantitative analysis are unavailable. These relative quantities may be determined from infrared intensity ratios even though known standards are unavailable. Repeatability [vs precisionhl in quantitative analysis is a critical parameter for meaningful results. In any given analysis, multiple runs provide "answers" with a certain standard deviation. Obviously, the lower the standard deviation, the better the precision. In attempting to minimize the standard deviation and thus improve precision, we need to delineate which contributing factors we have control over (such as sample preparation techniques, data analysis methodology) and which factors we have little control over (environmental and instrument noise, for example). For a given set of conditions, the best instrumental precision achievable on an IR instrument should be determinable. Traditionally, the term "signal-to-noise" (S/N) has been used for a single spectrum, realizing that S/N improves with an increase in number of scans coadded for generation of that single spectrum. However, the S/N ratio does not directly reflect the precision achievable for an absorbing band. We prefer to use the phrase "maximum achievable instrument precision" (MAIP), which is equivalent to the minimum relative standard deviation for a given peak (either height or area) in spectra. For a specific analysis, the analyst should have in mind the desired precision. Only if the desired precision is less than the MA1P will the analysis be feasible. Once the MAIP is established, other experimental procedures may be modified to improve the analytical precision, if it is below that which is expected (the MAIP).

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

    PubMed Central

    2015-01-01

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

  9. Phosphoproteomic Analysis Reveals the Effects of PilF Phosphorylation on Type IV Pilus and Biofilm Formation in Thermus thermophilus HB27*

    PubMed Central

    Wu, Wan-Ling; Liao, Jiahn-Haur; Lin, Guang-Huey; Lin, Miao-Hsia; Chang, Ying-Che; Liang, Suh-Yuen; Yang, Feng-Ling; Khoo, Kay-Hooi; Wu, Shih-Hsiung

    2013-01-01

    Thermus thermophilus HB27 is an extremely thermophilic eubacteria with a high frequency of natural competence. This organism is therefore often used as a thermophilic model to investigate the molecular basis of type IV pili–mediated functions, such as the uptake of free DNA, adhesion, twitching motility, and biofilm formation, in hot environments. In this study, the phosphoproteome of T. thermophilus HB27 was analyzed via a shotgun approach and high-accuracy mass spectrometry. Ninety-three unique phosphopeptides, including 67 in vivo phosphorylated sites on 53 phosphoproteins, were identified. The distribution of Ser/Thr/Tyr phosphorylation sites was 57%/36%/7%. The phosphoproteins were mostly involved in central metabolic pathways and protein/cell envelope biosynthesis. According to this analysis, the ATPase motor PilF, a type IV pili–related component, was first found to be phosphorylated on Thr-368 and Ser-372. Through the point mutation of PilF, mimic phosphorylated mutants T368D and S372E resulted in nonpiliated and nontwitching phenotypes, whereas nonphosphorylated mutants T368V and S372A displayed piliation and twitching motility. In addition, mimic phosphorylated mutants showed elevated biofilm-forming abilities with a higher initial attachment rate, caused by increasing exopolysaccharide production. In summary, the phosphorylation of PilF might regulate the pili and biofilm formation associated with exopolysaccharide production. PMID:23828892

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

    2013-01-01

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

  12. Automated quantitative analysis for pneumoconiosis

    NASA Astrophysics Data System (ADS)

    Kondo, Hiroshi; Zhao, Bin; Mino, Masako

    1998-09-01

    Automated quantitative analysis for pneumoconiosis is presented. In this paper Japanese standard radiographs of pneumoconiosis are categorized by measuring the area density and the number density of small rounded opacities. And furthermore the classification of the size and shape of the opacities is made from the measuring of the equivalent radiuses of each opacity. The proposed method includes a bi- level unsharp masking filter with a 1D uniform impulse response in order to eliminate the undesired parts such as the images of blood vessels and ribs in the chest x-ray photo. The fuzzy contrast enhancement is also introduced in this method for easy and exact detection of small rounded opacities. Many simulation examples show that the proposed method is more reliable than the former method.

  13. A quantitative fitness analysis workflow.

    PubMed

    Banks, A P; Lawless, C; Lydall, D A

    2012-01-01

    Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel(1,2,3,4). QFA can be applied to focused observations of single cultures but is most useful for genome-wide genetic interaction or drug screens investigating up to thousands of independent cultures. The central experimental method is the inoculation of independent, dilute liquid microbial cultures onto solid agar plates which are incubated and regularly photographed. Photographs from each time-point are analyzed, producing quantitative cell density estimates, which are used to construct growth curves, allowing quantitative fitness measures to be derived. Culture fitnesses can be compared to quantify and rank genetic interaction strengths or drug sensitivities. The effect on culture fitness of any treatments added into substrate agar (e.g. small molecules, antibiotics or nutrients) or applied to plates externally (e.g. UV irradiation, temperature) can be quantified by QFA. The QFA workflow produces growth rate estimates analogous to those obtained by spectrophotometric measurement of parallel liquid cultures in 96-well or 200-well plate readers. Importantly, QFA has significantly higher throughput compared with such methods. QFA cultures grow on a solid agar surface and are therefore well aerated during growth without the need for stirring or shaking. QFA throughput is not as high as that of some Synthetic Genetic Array (SGA) screening methods(5,6). However, since QFA cultures are heavily diluted before being inoculated onto agar, QFA can capture more complete growth curves, including exponential and saturation phases(3). For example, growth curve observations allow culture doubling times to be estimated directly with high precision, as discussed previously(1). Here we present a specific QFA protocol applied to thousands of S. cerevisiae cultures which are automatically handled by robots during inoculation, incubation and

  14. A Quantitative Fitness Analysis Workflow

    PubMed Central

    Lydall, D.A.

    2012-01-01

    Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. QFA can be applied to focused observations of single cultures but is most useful for genome-wide genetic interaction or drug screens investigating up to thousands of independent cultures. The central experimental method is the inoculation of independent, dilute liquid microbial cultures onto solid agar plates which are incubated and regularly photographed. Photographs from each time-point are analyzed, producing quantitative cell density estimates, which are used to construct growth curves, allowing quantitative fitness measures to be derived. Culture fitnesses can be compared to quantify and rank genetic interaction strengths or drug sensitivities. The effect on culture fitness of any treatments added into substrate agar (e.g. small molecules, antibiotics or nutrients) or applied to plates externally (e.g. UV irradiation, temperature) can be quantified by QFA. The QFA workflow produces growth rate estimates analogous to those obtained by spectrophotometric measurement of parallel liquid cultures in 96-well or 200-well plate readers. Importantly, QFA has significantly higher throughput compared with such methods. QFA cultures grow on a solid agar surface and are therefore well aerated during growth without the need for stirring or shaking. QFA throughput is not as high as that of some Synthetic Genetic Array (SGA) screening methods5,6. However, since QFA cultures are heavily diluted before being inoculated onto agar, QFA can capture more complete growth curves, including exponential and saturation phases3. For example, growth curve observations allow culture doubling times to be estimated directly with high precision, as discussed previously1. Here we present a specific QFA protocol applied to thousands of S. cerevisiae cultures which are automatically handled by robots during inoculation, incubation and imaging

  15. Quantitative analysis of endogenous compounds.

    PubMed

    Thakare, Rhishikesh; Chhonker, Yashpal S; Gautam, Nagsen; Alamoudi, Jawaher Abdullah; Alnouti, Yazen

    2016-09-01

    Accurate quantitative analysis of endogenous analytes is essential for several clinical and non-clinical applications. LC-MS/MS is the technique of choice for quantitative analyses. Absolute quantification by LC/MS requires preparing standard curves in the same matrix as the study samples so that the matrix effect and the extraction efficiency for analytes are the same in both the standard and study samples. However, by definition, analyte-free biological matrices do not exist for endogenous compounds. To address the lack of blank matrices for the quantification of endogenous compounds by LC-MS/MS, four approaches are used including the standard addition, the background subtraction, the surrogate matrix, and the surrogate analyte methods. This review article presents an overview these approaches, cite and summarize their applications, and compare their advantages and disadvantages. In addition, we discuss in details, validation requirements and compatibility with FDA guidelines to ensure method reliability in quantifying endogenous compounds. The standard addition, background subtraction, and the surrogate analyte approaches allow the use of the same matrix for the calibration curve as the one to be analyzed in the test samples. However, in the surrogate matrix approach, various matrices such as artificial, stripped, and neat matrices are used as surrogate matrices for the actual matrix of study samples. For the surrogate analyte approach, it is required to demonstrate similarity in matrix effect and recovery between surrogate and authentic endogenous analytes. Similarly, for the surrogate matrix approach, it is required to demonstrate similar matrix effect and extraction recovery in both the surrogate and original matrices. All these methods represent indirect approaches to quantify endogenous compounds and regardless of what approach is followed, it has to be shown that none of the validation criteria have been compromised due to the indirect analyses. PMID

  16. Quantitative analysis of sandstone porosity

    SciTech Connect

    Ferrell, R.E. Jr.; Carpenter, P.K.

    1988-01-01

    A quantitative analysis of changes in porosity associated with sandstone diagenesis was accomplished with digital back-scattered electron image analysis techniques. The volume percent (vol. %) of macroporosity, quartz, clay minerals, feldspar, and other constituents combined with stereological parameters, such as the size and shape of the analyzed features, permitted the determination of cement volumes, the ratio of primary to secondary porosity, and the relative abundance of detrital and authigenic clay minerals. The analyses were produced with a JEOL 733 Superprobe and a TRACOR/NORTHERN 5700 Image Analyzer System. The results provided a numerical evaluation of sedimentological facies controls and diagenetic effects on the permeabilities of potential reservoirs. In a typical application, subtle differences in the diagnetic development of porosity were detected in Wilcox sandstones from central Louisiana. Mechanical compaction of these shoreface sandstones has reduced the porosity to approximately 20%. In most samples with permeabilities greater than 10 md, the measured ratio of macroporosity to microporosity associated with pore-filling kaolinite was 3:1. In other sandstones with lower permeabilities, the measured ratio was higher, but the volume of pore-filling clay was essentially the same. An analysis of the frequency distribution of pore diameters and shapes revealed that the latter samples contained 2-3 vol% of grain-dissolution or moldic porosity. Fluid entry to these large pores was restricted and the clays produced from the grain dissolution products reduced the observed permeability. The image analysis technique provided valuable data for the distinction of productive and nonproductive intervals in this reservoir.

  17. Quantitative Analysis of Glaciated Landscapes

    NASA Astrophysics Data System (ADS)

    Huerta, A. D.

    2005-12-01

    The evolution of glaciated mountains is at the heart of the debate over Late Cenozoic linkages between climate and tectonics. Traditionally, the development of high summit elevations is attributed to tectonic processes. However, much of the high elevation of the Transantarctic Mountains can be attributed solely to uplift in response to glacial erosion (Stern et al., 2005). The Transantarctic Mountains (TAM) provide an unparalleled opportunity to study glacial erosion. The mountain range has experienced glacial conditions since Oligocene time. In the higher and dryer regions of the TAM there is only a thin veneer of ice and snow draping the topography. In these regions landforms that were shaped during earlier climatic conditions are preserved. In fact, both glacial and fluvial landforms dating as far back as 18 Ma are preserved locally. In addition, the TAM are ideal for studying glacial erosion since the range has experienced minimal tectonic uplift since late Oligocene time, thus isolating the erosion signal from any tectonic signal. With the advent of digital data sets and GIS methodologies, quantitative analysis can identify key aspects of glaciated landscape morphology, and thus develop powerful analytical techniques for objective study of glaciation. Inspection of USGS topographic maps of the TAM reveals that mountain tops display an extreme range of glacial modification. For example, in the Mt. Rabot region (83°-84° S), mountain peaks are strongly affected by glaciation; cirque development is advanced with cirque diameters on the range of several kilometers, and cirque confluence has resulted in the formation of ``knife-edge'' arêtes up to 10 km long. In contrast, in the Mt. Murchison area (73°-74° S) cirque development is youthful, and there is minimal development of arêtes. Preliminary work indicates that analysis of DEM's and contour lines can be used to distinguish degree of glaciation. In particular, slope, curvature, and power spectrum analysis

  18. Quantitative analysis of retinal OCT.

    PubMed

    Sonka, Milan; Abràmoff, Michael D

    2016-10-01

    Clinical acceptance of 3-D OCT retinal imaging brought rapid development of quantitative 3-D analysis of retinal layers, vasculature, retinal lesions as well as facilitated new research in retinal diseases. One of the cornerstones of many such analyses is segmentation and thickness quantification of retinal layers and the choroid, with an inherently 3-D simultaneous multi-layer LOGISMOS (Layered Optimal Graph Image Segmentation for Multiple Objects and Surfaces) segmentation approach being extremely well suited for the task. Once retinal layers are segmented, regional thickness, brightness, or texture-based indices of individual layers can be easily determined and thus contribute to our understanding of retinal or optic nerve head (ONH) disease processes and can be employed for determination of disease status, treatment responses, visual function, etc. Out of many applications, examples provided in this paper focus on image-guided therapy and outcome prediction in age-related macular degeneration and on assessing visual function from retinal layer structure in glaucoma. PMID:27503080

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2012-02-01

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

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

    PubMed Central

    2014-01-01

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

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

    PubMed

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

    2016-08-11

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

  4. Quantitative Analysis of Face Symmetry.

    PubMed

    Tamir, Abraham

    2015-06-01

    The major objective of this article was to report quantitatively the degree of human face symmetry for reported images taken from the Internet. From the original image of a certain person that appears in the center of each triplet, 2 symmetric combinations were constructed that are based on the left part of the image and its mirror image (left-left) and on the right part of the image and its mirror image (right-right). By applying a computer software that enables to determine length, surface area, and perimeter of any geometric shape, the following measurements were obtained for each triplet: face perimeter and area; distance between the pupils; mouth length; its perimeter and area; nose length and face length, usually below the ears; as well as the area and perimeter of the pupils. Then, for each of the above measurements, the value C, which characterizes the degree of symmetry of the real image with respect to the combinations right-right and left-left, was calculated. C appears on the right-hand side below each image. A high value of C indicates a low symmetry, and as the value is decreasing, the symmetry is increasing. The magnitude on the left relates to the pupils and compares the difference between the area and perimeter of the 2 pupils. The major conclusion arrived at here is that the human face is asymmetric to some degree; the degree of asymmetry is reported quantitatively under each portrait. PMID:26080172

  5. Quantitative analysis of digital microscope images.

    PubMed

    Wolf, David E; Samarasekera, Champika; Swedlow, Jason R

    2013-01-01

    This chapter discusses quantitative analysis of digital microscope images and presents several exercises to provide examples to explain the concept. This chapter also presents the basic concepts in quantitative analysis for imaging, but these concepts rest on a well-established foundation of signal theory and quantitative data analysis. This chapter presents several examples for understanding the imaging process as a transformation from sample to image and the limits and considerations of quantitative analysis. This chapter introduces to the concept of digitally correcting the images and also focuses on some of the more critical types of data transformation and some of the frequently encountered issues in quantization. Image processing represents a form of data processing. There are many examples of data processing such as fitting the data to a theoretical curve. In all these cases, it is critical that care is taken during all steps of transformation, processing, and quantization. PMID:23931513

  6. Quantitative analysis of qualitative images

    NASA Astrophysics Data System (ADS)

    Hockney, David; Falco, Charles M.

    2005-03-01

    We show optical evidence that demonstrates artists as early as Jan van Eyck and Robert Campin (c1425) used optical projections as aids for producing their paintings. We also have found optical evidence within works by later artists, including Bermejo (c1475), Lotto (c1525), Caravaggio (c1600), de la Tour (c1650), Chardin (c1750) and Ingres (c1825), demonstrating a continuum in the use of optical projections by artists, along with an evolution in the sophistication of that use. However, even for paintings where we have been able to extract unambiguous, quantitative evidence of the direct use of optical projections for producing certain of the features, this does not mean that paintings are effectively photographs. Because the hand and mind of the artist are intimately involved in the creation process, understanding these complex images requires more than can be obtained from only applying the equations of geometrical optics.

  7. Phosphoproteomics for the masses.

    PubMed

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

    2010-01-15

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

  8. Phosphoproteomics for the masses

    PubMed Central

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

    2010-01-01

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

  9. Qualitative and Quantitative Analysis: Interpretation of Electropherograms

    NASA Astrophysics Data System (ADS)

    Szumski, Michał; Buszewski, Bogusław

    In this chapter the basic information on qualitative and quantitative analysis in CE is provided. Migration time and spectral data are described as the most important parameters used for identification of compounds. The parameters that negatively influence qualitative analysis are briefly mentioned. In the quantitative analysis section the external standard and internal standard calibration methods are described. Variables influencing peak height and peak area in capillary electrophoresis are briefly summarized. Also, a discussion on electrodisperssion and its influence on a observed peak shape is provided.

  10. Quantitative histogram analysis of images

    NASA Astrophysics Data System (ADS)

    Holub, Oliver; Ferreira, Sérgio T.

    2006-11-01

    A routine for histogram analysis of images has been written in the object-oriented, graphical development environment LabVIEW. The program converts an RGB bitmap image into an intensity-linear greyscale image according to selectable conversion coefficients. This greyscale image is subsequently analysed by plots of the intensity histogram and probability distribution of brightness, and by calculation of various parameters, including average brightness, standard deviation, variance, minimal and maximal brightness, mode, skewness and kurtosis of the histogram and the median of the probability distribution. The program allows interactive selection of specific regions of interest (ROI) in the image and definition of lower and upper threshold levels (e.g., to permit the removal of a constant background signal). The results of the analysis of multiple images can be conveniently saved and exported for plotting in other programs, which allows fast analysis of relatively large sets of image data. The program file accompanies this manuscript together with a detailed description of two application examples: The analysis of fluorescence microscopy images, specifically of tau-immunofluorescence in primary cultures of rat cortical and hippocampal neurons, and the quantification of protein bands by Western-blot. The possibilities and limitations of this kind of analysis are discussed. Program summaryTitle of program: HAWGC Catalogue identifier: ADXG_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/ADXG_v1_0 Program obtainable from: CPC Program Library, Queen's University of Belfast, N. Ireland Computers: Mobile Intel Pentium III, AMD Duron Installations: No installation necessary—Executable file together with necessary files for LabVIEW Run-time engine Operating systems or monitors under which the program has been tested: WindowsME/2000/XP Programming language used: LabVIEW 7.0 Memory required to execute with typical data:˜16MB for starting and ˜160MB used for

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

    PubMed Central

    Yang, Chenxi; Zhong, Xuefei; Li, Lingjun

    2016-01-01

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

  12. Mobile app-based quantitative scanometric analysis.

    PubMed

    Wong, Jessica X H; Liu, Frank S F; Yu, Hua-Zhong

    2014-12-16

    The feasibility of using smartphones and other mobile devices as the detection platform for quantitative scanometric assays is demonstrated. The different scanning modes (color, grayscale, black/white) and grayscale converting protocols (average, weighted average/luminosity, and software specific) have been compared in determining the optical darkness ratio (ODR) values, a conventional quantitation measure for scanometric assays. A mobile app was developed to image and analyze scanometric assays, as demonstrated by paper-printed tests and a biotin-streptavidin assay on a plastic substrate. Primarily for ODR analysis, the app has been shown to perform as well as a traditional desktop scanner, augmenting that smartphones (and other mobile devices) promise to be a practical platform for accurate, quantitative chemical analysis and medical diagnostics. PMID:25420202

  13. Quantitative WDS analysis using electron probe microanalyzer

    SciTech Connect

    Ul-Hamid, Anwar . E-mail: anwar@kfupm.edu.sa; Tawancy, Hani M.; Mohammed, Abdul-Rashid I.; Al-Jaroudi, Said S.; Abbas, Nureddin M.

    2006-04-15

    In this paper, the procedure for conducting quantitative elemental analysis by ZAF correction method using wavelength dispersive X-ray spectroscopy (WDS) in an electron probe microanalyzer (EPMA) is elaborated. Analysis of a thermal barrier coating (TBC) system formed on a Ni-based single crystal superalloy is presented as an example to illustrate the analysis of samples consisting of a large number of major and minor elements. The analysis was performed by known standards and measured peak-to-background intensity ratios. The procedure for using separate set of acquisition conditions for major and minor element analysis is explained and its importance is stressed.

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

    PubMed Central

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

    2014-01-01

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

  15. Seniors' Online Communities: A Quantitative Content Analysis

    ERIC Educational Resources Information Center

    Nimrod, Galit

    2010-01-01

    Purpose: To examine the contents and characteristics of seniors' online communities and to explore their potential benefits to older adults. Design and Methods: Quantitative content analysis of a full year's data from 14 leading online communities using a novel computerized system. The overall database included 686,283 messages. Results: There was…

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

    PubMed Central

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

    2013-01-01

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

  17. Quantitative Proteomics Analysis of Leukemia Cells.

    PubMed

    Halbach, Sebastian; Dengjel, Jörn; Brummer, Tilman

    2016-01-01

    Chronic myeloid leukemia (CML) is driven by the oncogenic fusion kinase Bcr-Abl, which organizes its own signaling network with various proteins. These proteins, their interactions, and their role in relevant signaling pathways can be analyzed by quantitative mass spectrometry (MS) approaches in various models systems, e.g., in cell culture models. In this chapter, we describe in detail immunoprecipitations and quantitative proteomics analysis using stable isotope labeling by amino acids in cell culture (SILAC) of components of the Bcr-Abl signaling pathway in the human CML cell line K562. PMID:27581145

  18. Method and apparatus for chromatographic quantitative analysis

    DOEpatents

    Fritz, James S.; Gjerde, Douglas T.; Schmuckler, Gabriella

    1981-06-09

    An improved apparatus and method for the quantitative analysis of a solution containing a plurality of anion species by ion exchange chromatography which utilizes a single eluent and a single ion exchange bed which does not require periodic regeneration. The solution containing the anions is added to an anion exchange resin bed which is a low capacity macroreticular polystyrene-divinylbenzene resin containing quarternary ammonium functional groups, and is eluted therefrom with a dilute solution of a low electrical conductance organic acid salt. As each anion species is eluted from the bed, it is quantitatively sensed by conventional detection means such as a conductivity cell.

  19. Quantitative analysis of blood vessel geometry

    NASA Astrophysics Data System (ADS)

    Fuhrman, Michael G.; Abdul-Karim, Othman; Shah, Sujal; Gilbert, Steven G.; Van Bibber, Richard

    2001-07-01

    Re-narrowing or restenosis of a human coronary artery occurs within six months in one third of balloon angioplasty procedures. Accurate and repeatable quantitative analysis of vessel shape is important to characterize the progression and type of restenosis, and to evaluate effects new therapies might have. A combination of complicated geometry and image variability, and the need for high resolution and large image size makes visual/manual analysis slow, difficult, and prone to error. The image processing and analysis described here was developed to automate feature extraction of the lumen, internal elastic lamina, neointima, external elastic lamina, and tunica adventitia and to enable an objective, quantitative definition of blood vessel geometry. The quantitative geometrical analysis enables the measurement of several features including perimeter, area, and other metrics of vessel damage. Automation of feature extraction creates a high throughput capability that enables analysis of serial sections for more accurate measurement of restenosis dimensions. Measurement results are input into a relational database where they can be statistically analyzed compared across studies. As part of the integrated process, results are also imprinted on the images themselves to facilitate auditing of the results. The analysis is fast, repeatable and accurate while allowing the pathologist to control the measurement process.

  20. Comprehensive Quantitative Analysis on Privacy Leak Behavior

    PubMed Central

    Fan, Lejun; Wang, Yuanzhuo; Jin, Xiaolong; Li, Jingyuan; Cheng, Xueqi; Jin, Shuyuan

    2013-01-01

    Privacy information is prone to be leaked by illegal software providers with various motivations. Privacy leak behavior has thus become an important research issue of cyber security. However, existing approaches can only qualitatively analyze privacy leak behavior of software applications. No quantitative approach, to the best of our knowledge, has been developed in the open literature. To fill this gap, in this paper we propose for the first time four quantitative metrics, namely, possibility, severity, crypticity, and manipulability, for privacy leak behavior analysis based on Privacy Petri Net (PPN). In order to compare the privacy leak behavior among different software, we further propose a comprehensive metric, namely, overall leak degree, based on these four metrics. Finally, we validate the effectiveness of the proposed approach using real-world software applications. The experimental results demonstrate that our approach can quantitatively analyze the privacy leak behaviors of various software types and reveal their characteristics from different aspects. PMID:24066046

  1. Comprehensive quantitative analysis on privacy leak behavior.

    PubMed

    Fan, Lejun; Wang, Yuanzhuo; Jin, Xiaolong; Li, Jingyuan; Cheng, Xueqi; Jin, Shuyuan

    2013-01-01

    Privacy information is prone to be leaked by illegal software providers with various motivations. Privacy leak behavior has thus become an important research issue of cyber security. However, existing approaches can only qualitatively analyze privacy leak behavior of software applications. No quantitative approach, to the best of our knowledge, has been developed in the open literature. To fill this gap, in this paper we propose for the first time four quantitative metrics, namely, possibility, severity, crypticity, and manipulability, for privacy leak behavior analysis based on Privacy Petri Net (PPN). In order to compare the privacy leak behavior among different software, we further propose a comprehensive metric, namely, overall leak degree, based on these four metrics. Finally, we validate the effectiveness of the proposed approach using real-world software applications. The experimental results demonstrate that our approach can quantitatively analyze the privacy leak behaviors of various software types and reveal their characteristics from different aspects. PMID:24066046

  2. Quantitative Proteomic Analysis of the Human Nucleolus.

    PubMed

    Bensaddek, Dalila; Nicolas, Armel; Lamond, Angus I

    2016-01-01

    Recent years have witnessed spectacular progress in the field of mass spectrometry (MS)-based quantitative proteomics, including advances in instrumentation, chromatography, sample preparation methods, and experimental design for multidimensional analyses. It is now possible not only to identify most of the protein components of a cell proteome in a single experiment, but also to describe additional proteome dimensions, such as protein turnover rates, posttranslational modifications, and subcellular localization. Furthermore, by comparing the proteome at different time points, it is possible to create a "time-lapse" view of proteome dynamics. By combining high-throughput quantitative proteomics with detailed subcellular fractionation protocols and data analysis techniques it is also now possible to characterize in detail the proteomes of specific subcellular organelles, providing important insights into cell regulatory mechanisms and physiological responses. In this chapter we present a reliable workflow and protocol for MS-based analysis and quantitation of the proteome of nucleoli isolated from human cells. The protocol presented is based on a SILAC analysis of human MCF10A-Src-ER cells with analysis performed on a Q-Exactive Plus Orbitrap MS instrument (Thermo Fisher Scientific). The subsequent chapter describes how to process the resulting raw MS files from this experiment using MaxQuant software and data analysis procedures to evaluate the nucleolar proteome using customized R scripts. PMID:27576725

  3. Phosphoproteomics in translational research: a sarcoma perspective.

    PubMed

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

    2016-05-01

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

  4. Quantitative image analysis of celiac disease

    PubMed Central

    Ciaccio, Edward J; Bhagat, Govind; Lewis, Suzanne K; Green, Peter H

    2015-01-01

    We outline the use of quantitative techniques that are currently used for analysis of celiac disease. Image processing techniques can be useful to statistically analyze the pixular data of endoscopic images that is acquired with standard or videocapsule endoscopy. It is shown how current techniques have evolved to become more useful for gastroenterologists who seek to understand celiac disease and to screen for it in suspected patients. New directions for focus in the development of methodology for diagnosis and treatment of this disease are suggested. It is evident that there are yet broad areas where there is potential to expand the use of quantitative techniques for improved analysis in suspected or known celiac disease patients. PMID:25759524

  5. Using Qualitative Hazard Analysis to Guide Quantitative Safety Analysis

    NASA Technical Reports Server (NTRS)

    Shortle, J. F.; Allocco, M.

    2005-01-01

    Quantitative methods can be beneficial in many types of safety investigations. However, there are many difficulties in using quantitative m ethods. Far example, there may be little relevant data available. This paper proposes a framework for using quantitative hazard analysis to prioritize hazard scenarios most suitable for quantitative mziysis. The framework first categorizes hazard scenarios by severity and likelihood. We then propose another metric "modeling difficulty" that desc ribes the complexity in modeling a given hazard scenario quantitatively. The combined metrics of severity, likelihood, and modeling difficu lty help to prioritize hazard scenarios for which quantitative analys is should be applied. We have applied this methodology to proposed concepts of operations for reduced wake separation for airplane operatio ns at closely spaced parallel runways.

  6. Quantitative resilience analysis through control design.

    SciTech Connect

    Sunderland, Daniel; Vugrin, Eric D.; Camphouse, Russell Chris

    2009-09-01

    Critical infrastructure resilience has become a national priority for the U. S. Department of Homeland Security. System resilience has been studied for several decades in many different disciplines, but no standards or unifying methods exist for critical infrastructure resilience analysis. Few quantitative resilience methods exist, and those existing approaches tend to be rather simplistic and, hence, not capable of sufficiently assessing all aspects of critical infrastructure resilience. This report documents the results of a late-start Laboratory Directed Research and Development (LDRD) project that investigated the development of quantitative resilience through application of control design methods. Specifically, we conducted a survey of infrastructure models to assess what types of control design might be applicable for critical infrastructure resilience assessment. As a result of this survey, we developed a decision process that directs the resilience analyst to the control method that is most likely applicable to the system under consideration. Furthermore, we developed optimal control strategies for two sets of representative infrastructure systems to demonstrate how control methods could be used to assess the resilience of the systems to catastrophic disruptions. We present recommendations for future work to continue the development of quantitative resilience analysis methods.

  7. Quantitative Bias Analysis in Regulatory Settings.

    PubMed

    Lash, Timothy L; Fox, Matthew P; Cooney, Darryl; Lu, Yun; Forshee, Richard A

    2016-07-01

    Nonrandomized studies are essential in the postmarket activities of the US Food and Drug Administration, which, however, must often act on the basis of imperfect data. Systematic errors can lead to inaccurate inferences, so it is critical to develop analytic methods that quantify uncertainty and bias and ensure that these methods are implemented when needed. "Quantitative bias analysis" is an overarching term for methods that estimate quantitatively the direction, magnitude, and uncertainty associated with systematic errors influencing measures of associations. The Food and Drug Administration sponsored a collaborative project to develop tools to better quantify the uncertainties associated with postmarket surveillance studies used in regulatory decision making. We have described the rationale, progress, and future directions of this project. PMID:27196652

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

    PubMed Central

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

    2012-01-01

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

  9. Accuracy in Quantitative 3D Image Analysis

    PubMed Central

    Bassel, George W.

    2015-01-01

    Quantitative 3D imaging is becoming an increasingly popular and powerful approach to investigate plant growth and development. With the increased use of 3D image analysis, standards to ensure the accuracy and reproducibility of these data are required. This commentary highlights how image acquisition and postprocessing can introduce artifacts into 3D image data and proposes steps to increase both the accuracy and reproducibility of these analyses. It is intended to aid researchers entering the field of 3D image processing of plant cells and tissues and to help general readers in understanding and evaluating such data. PMID:25804539

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

    PubMed

    Yu, Li-Rong; Veenstra, Timothy

    2013-01-01

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

  11. Phosphoproteomic analysis of anaplastic lymphoma kinase (ALK) downstream signaling pathways identifies signal transducer and activator of transcription 3 as a functional target of activated ALK in neuroblastoma cells

    PubMed Central

    Sattu, Kamaraj; Hochgräfe, Falko; Wu, Jianmin; Umapathy, Ganesh; Schönherr, Christina; Ruuth, Kristina; Chand, Damini; Witek, Barbara; Fuchs, James; Li, Pui-Kai; Hugosson, Fredrik; Daly, Roger J; Palmer, Ruth H; Hallberg, Bengt

    2013-01-01

    Activation of the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase is a key oncogenic mechanism in a growing number of tumor types. In the majority of cases, ALK is activated by fusion with a dimerizing partner protein as a result of chromosomal translocation events, most studied in the case of the nucleophosmin–ALK and echinoderm microtubule-associated protein-like 4–ALK oncoproteins. It is now also appreciated that the full-length ALK receptor can be activated by point mutations and by deletions within the extracellular domain, such as those observed in neuroblastoma. Several studies have employed phosphoproteomics approaches to find substrates of ALK fusion proteins. In this study, we used MS-based phosphotyrosine profiling to characterize phosphotyrosine signaling events associated with the full-length ALK receptor. A number of previously identified and novel targets were identified. One of these, signal transducer and activator of transcription 3 (STAT3), has previously been observed to be activated in response to oncogenic ALK signaling, but the significance of this in signaling from the full-length ALK receptor has not been explored further. We show here that activated ALK robustly activates STAT3 on Tyr705 in a number of independent neuroblastoma cell lines. Furthermore, knockdown of STAT3 by RNA interference resulted in a reduction in myelocytomatosis neuroblastom (MYCN) protein levels downstream of ALK signaling. These observations, together with a decreased level of MYCN and inhibition of neuroblastoma cell growth in the presence of STAT3 inhibitors, suggest that activation of STAT3 is important for ALK signaling activity in neuroblastoma. PMID:23889739

  12. Quantitative architectural analysis of bronchial intraepithelial neoplasia

    NASA Astrophysics Data System (ADS)

    Guillaud, Martial; MacAulay, Calum E.; Le Riche, Jean C.; Dawe, Chris; Korbelik, Jagoda; Lam, Stephen

    2000-04-01

    Considerable variation exists among pathologist in the interpretation of intraepithelial neoplasia making it difficult to determine the natural history of these lesion and to establish management guidelines for chemoprevention. The aim of the study is to evaluate architectural features of pre-neoplastic progression in lung cancer, and to search for a correlation between architectural index and conventional pathology. Quantitative architectural analysis was performed on a series of normal lung biopsies and Carcinoma In Situ (CIS). Centers of gravity of the nuclei within a pre-defined region of interest were used as seeds to generate a Voronoi Diagram. About 30 features derived from the Voronoi diagram, its dual the Delaunay tessellation, and the Minimum Spanning Tree were extracted. A discriminant analysis was performed to separate between the two groups. The architectural Index was calculated for each of the bronchial biopsies that were interpreted as hyperplasia, metaplasia, mild, moderate or severe dysplasia by conventional histopathology criteria. As a group, lesions classified as CIS by conventional histopathology criteria could be distinguished from dysplasia using the architectural Index. Metaplasia was distinct from hyperplasia and hyperplasia from normal. There was overlap between severe and moderate dysplasia but mild dysplasia could be distinguished form moderate dysplasia. Bronchial intraepithelial neoplastic lesions can be degraded objectively by architectural features. Combination of architectural features and nuclear morphometric features may improve the quantitation of the changes occurring during the intra-epithelial neoplastic process.

  13. Quantitative interactome analysis reveals a chemoresistant edgotype

    PubMed Central

    Chavez, Juan D.; Schweppe, Devin K.; Eng, Jimmy K.; Zheng, Chunxiang; Taipale, Alex; Zhang, Yiyi; Takara, Kohji; Bruce, James E.

    2015-01-01

    Chemoresistance is a common mode of therapy failure for many cancers. Tumours develop resistance to chemotherapeutics through a variety of mechanisms, with proteins serving pivotal roles. Changes in protein conformations and interactions affect the cellular response to environmental conditions contributing to the development of new phenotypes. The ability to understand how protein interaction networks adapt to yield new function or alter phenotype is limited by the inability to determine structural and protein interaction changes on a proteomic scale. Here, chemical crosslinking and mass spectrometry were employed to quantify changes in protein structures and interactions in multidrug-resistant human carcinoma cells. Quantitative analysis of the largest crosslinking-derived, protein interaction network comprising 1,391 crosslinked peptides allows for ‘edgotype' analysis in a cell model of chemoresistance. We detect consistent changes to protein interactions and structures, including those involving cytokeratins, topoisomerase-2-alpha, and post-translationally modified histones, which correlate with a chemoresistant phenotype. PMID:26235782

  14. Quantitative interactome analysis reveals a chemoresistant edgotype.

    PubMed

    Chavez, Juan D; Schweppe, Devin K; Eng, Jimmy K; Zheng, Chunxiang; Taipale, Alex; Zhang, Yiyi; Takara, Kohji; Bruce, James E

    2015-01-01

    Chemoresistance is a common mode of therapy failure for many cancers. Tumours develop resistance to chemotherapeutics through a variety of mechanisms, with proteins serving pivotal roles. Changes in protein conformations and interactions affect the cellular response to environmental conditions contributing to the development of new phenotypes. The ability to understand how protein interaction networks adapt to yield new function or alter phenotype is limited by the inability to determine structural and protein interaction changes on a proteomic scale. Here, chemical crosslinking and mass spectrometry were employed to quantify changes in protein structures and interactions in multidrug-resistant human carcinoma cells. Quantitative analysis of the largest crosslinking-derived, protein interaction network comprising 1,391 crosslinked peptides allows for 'edgotype' analysis in a cell model of chemoresistance. We detect consistent changes to protein interactions and structures, including those involving cytokeratins, topoisomerase-2-alpha, and post-translationally modified histones, which correlate with a chemoresistant phenotype. PMID:26235782

  15. Quantitative analysis of NMR spectra with chemometrics

    NASA Astrophysics Data System (ADS)

    Winning, H.; Larsen, F. H.; Bro, R.; Engelsen, S. B.

    2008-01-01

    The number of applications of chemometrics to series of NMR spectra is rapidly increasing due to an emerging interest for quantitative NMR spectroscopy e.g. in the pharmaceutical and food industries. This paper gives an analysis of advantages and limitations of applying the two most common chemometric procedures, Principal Component Analysis (PCA) and Multivariate Curve Resolution (MCR), to a designed set of 231 simple alcohol mixture (propanol, butanol and pentanol) 1H 400 MHz spectra. The study clearly demonstrates that the major advantage of chemometrics is the visualisation of larger data structures which adds a new exploratory dimension to NMR research. While robustness and powerful data visualisation and exploration are the main qualities of the PCA method, the study demonstrates that the bilinear MCR method is an even more powerful method for resolving pure component NMR spectra from mixtures when certain conditions are met.

  16. 15N labeled brain enables quantification of proteome and phosphoproteome in cultured primary neurons

    PubMed Central

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

    2011-01-01

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

  17. Materials characterization through quantitative digital image analysis

    SciTech Connect

    J. Philliber; B. Antoun; B. Somerday; N. Yang

    2000-07-01

    A digital image analysis system has been developed to allow advanced quantitative measurement of microstructural features. This capability is maintained as part of the microscopy facility at Sandia, Livermore. The system records images digitally, eliminating the use of film. Images obtained from other sources may also be imported into the system. Subsequent digital image processing enhances image appearance through the contrast and brightness adjustments. The system measures a variety of user-defined microstructural features--including area fraction, particle size and spatial distributions, grain sizes and orientations of elongated particles. These measurements are made in a semi-automatic mode through the use of macro programs and a computer controlled translation stage. A routine has been developed to create large montages of 50+ separate images. Individual image frames are matched to the nearest pixel to create seamless montages. Results from three different studies are presented to illustrate the capabilities of the system.

  18. Quantitative proteomic analysis of single pancreatic islets

    PubMed Central

    Waanders, Leonie F.; Chwalek, Karolina; Monetti, Mara; Kumar, Chanchal; Lammert, Eckhard; Mann, Matthias

    2009-01-01

    Technological developments make mass spectrometry (MS)-based proteomics a central pillar of biochemical research. MS has been very successful in cell culture systems, where sample amounts are not limiting. To extend its capabilities to extremely small, physiologically distinct cell types isolated from tissue, we developed a high sensitivity chromatographic system that measures nanogram protein mixtures for 8 h with very high resolution. This technology is based on splitting gradient effluents into a capture capillary and provides an inherent technical replicate. In a single analysis, this allowed us to characterize kidney glomeruli isolated by laser capture microdissection to a depth of more than 2,400 proteins. From pooled pancreatic islets of Langerhans, another type of “miniorgan,” we obtained an in-depth proteome of 6,873 proteins, many of them involved in diabetes. We quantitatively compared the proteome of single islets, containing 2,000–4,000 cells, treated with high or low glucose levels, and covered most of the characteristic functions of beta cells. Our ultrasensitive analysis recapitulated known hyperglycemic changes but we also find components up-regulated such as the mitochondrial stress regulator Park7. Direct proteomic analysis of functionally distinct cellular structures opens up perspectives in physiology and pathology. PMID:19846766

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  3. Applying Knowledge of Quantitative Design and Analysis

    ERIC Educational Resources Information Center

    Baskas, Richard S.

    2011-01-01

    This study compared and contrasted two quantitative scholarly articles in relation to their research designs. Their designs were analyzed by the comparison of research references and research specific vocabulary to describe how various research methods were used. When researching and analyzing quantitative scholarly articles, it is imperative to…

  4. Error Propagation Analysis for Quantitative Intracellular Metabolomics

    PubMed Central

    Tillack, Jana; Paczia, Nicole; Nöh, Katharina; Wiechert, Wolfgang; Noack, Stephan

    2012-01-01

    Model-based analyses have become an integral part of modern metabolic engineering and systems biology in order to gain knowledge about complex and not directly observable cellular processes. For quantitative analyses, not only experimental data, but also measurement errors, play a crucial role. The total measurement error of any analytical protocol is the result of an accumulation of single errors introduced by several processing steps. Here, we present a framework for the quantification of intracellular metabolites, including error propagation during metabolome sample processing. Focusing on one specific protocol, we comprehensively investigate all currently known and accessible factors that ultimately impact the accuracy of intracellular metabolite concentration data. All intermediate steps are modeled, and their uncertainty with respect to the final concentration data is rigorously quantified. Finally, on the basis of a comprehensive metabolome dataset of Corynebacterium glutamicum, an integrated error propagation analysis for all parts of the model is conducted, and the most critical steps for intracellular metabolite quantification are detected. PMID:24957773

  5. Quantitative methods for ecological network analysis.

    PubMed

    Ulanowicz, Robert E

    2004-12-01

    The analysis of networks of ecological trophic transfers is a useful complement to simulation modeling in the quest for understanding whole-ecosystem dynamics. Trophic networks can be studied in quantitative and systematic fashion at several levels. Indirect relationships between any two individual taxa in an ecosystem, which often differ in either nature or magnitude from their direct influences, can be assayed using techniques from linear algebra. The same mathematics can also be employed to ascertain where along the trophic continuum any individual taxon is operating, or to map the web of connections into a virtual linear chain that summarizes trophodynamic performance by the system. Backtracking algorithms with pruning have been written which identify pathways for the recycle of materials and energy within the system. The pattern of such cycling often reveals modes of control or types of functions exhibited by various groups of taxa. The performance of the system as a whole at processing material and energy can be quantified using information theory. In particular, the complexity of process interactions can be parsed into separate terms that distinguish organized, efficient performance from the capacity for further development and recovery from disturbance. Finally, the sensitivities of the information-theoretic system indices appear to identify the dynamical bottlenecks in ecosystem functioning. PMID:15556474

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-07-01

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

  9. Some Epistemological Considerations Concerning Quantitative Analysis

    ERIC Educational Resources Information Center

    Dobrescu, Emilian

    2008-01-01

    This article presents the author's address at the 2007 "Journal of Applied Quantitative Methods" ("JAQM") prize awarding festivity. The festivity was included in the opening of the 4th International Conference on Applied Statistics, November 22, 2008, Bucharest, Romania. In the address, the author reflects on three theses that question the…

  10. Quantitative Analysis of Radar Returns from Insects

    NASA Technical Reports Server (NTRS)

    Riley, J. R.

    1979-01-01

    When a number of flying insects is low enough to permit their resolution as individual radar targets, quantitative estimates of their aerial density are developed. Accurate measurements of heading distribution using a rotating polarization radar to enhance the wingbeat frequency method of identification are presented.

  11. Phosphoproteomics technologies and applications in plant biology research

    PubMed Central

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

    2015-01-01

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

  12. Quantitative analysis of comparative genomic hybridization

    SciTech Connect

    Manoir, S. du; Bentz, M.; Joos, S. |

    1995-01-01

    Comparative genomic hybridization (CGH) is a new molecular cytogenetic method for the detection of chromosomal imbalances. Following cohybridization of DNA prepared from a sample to be studied and control DNA to normal metaphase spreads, probes are detected via different fluorochromes. The ratio of the test and control fluorescence intensities along a chromosome reflects the relative copy number of segments of a chromosome in the test genome. Quantitative evaluation of CGH experiments is required for the determination of low copy changes, e.g., monosomy or trisomy, and for the definition of the breakpoints involved in unbalanced rearrangements. In this study, a program for quantitation of CGH preparations is presented. This program is based on the extraction of the fluorescence ratio profile along each chromosome, followed by averaging of individual profiles from several metaphase spreads. Objective parameters critical for quantitative evaluations were tested, and the criteria for selection of suitable CGH preparations are described. The granularity of the chromosome painting and the regional inhomogeneity of fluorescence intensities in metaphase spreads proved to be crucial parameters. The coefficient of variation of the ratio value for chromosomes in balanced state (CVBS) provides a general quality criterion for CGH experiments. Different cutoff levels (thresholds) of average fluorescence ratio values were compared for their specificity and sensitivity with regard to the detection of chromosomal imbalances. 27 refs., 15 figs., 1 tab.

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

    PubMed Central

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

    2012-01-01

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

  14. Energy Dispersive Spectrometry and Quantitative Analysis Short Course. Introduction to X-ray Energy Dispersive Spectrometry and Quantitative Analysis

    NASA Technical Reports Server (NTRS)

    Carpenter, Paul; Curreri, Peter A. (Technical Monitor)

    2002-01-01

    This course will cover practical applications of the energy-dispersive spectrometer (EDS) to x-ray microanalysis. Topics covered will include detector technology, advances in pulse processing, resolution and performance monitoring, detector modeling, peak deconvolution and fitting, qualitative and quantitative analysis, compositional mapping, and standards. An emphasis will be placed on use of the EDS for quantitative analysis, with discussion of typical problems encountered in the analysis of a wide range of materials and sample geometries.

  15. Structural and quantitative analysis of Equisetum alkaloids.

    PubMed

    Cramer, Luise; Ernst, Ludger; Lubienski, Marcus; Papke, Uli; Schiebel, Hans-Martin; Jerz, Gerold; Beuerle, Till

    2015-08-01

    Equisetum palustre L. is known for its toxicity for livestock. Several studies in the past addressed the isolation and identification of the responsible alkaloids. So far, palustrine (1) and N(5)-formylpalustrine (2) are known alkaloids of E. palustre. A HPLC-ESI-MS/MS method in combination with simple sample work-up was developed to identify and quantitate Equisetum alkaloids. Besides the two known alkaloids six related alkaloids were detected in different Equisetum samples. The structure of the alkaloid palustridiene (3) was derived by comprehensive 1D and 2D NMR experiments. N(5)-Acetylpalustrine (4) was also thoroughly characterized by NMR for the first time. The structure of N(5)-formylpalustridiene (5) is proposed based on mass spectrometry results. Twenty-two E. palustre samples were screened by a HPLC-ESI-MS/MS method after development of a simple sample work-up and in most cases the set of all eight alkaloids were detected in all parts of the plant. A high variability of the alkaloid content and distribution was found depending on plant organ, plant origin and season ranging from 88 to 597mg/kg dried weight. However, palustrine (1) and the alkaloid palustridiene (3) always represented the main alkaloids. For the first time, a comprehensive identification, quantitation and distribution of Equisetum alkaloids was achieved. PMID:25823584

  16. Joint association analysis of bivariate quantitative and qualitative traits.

    PubMed

    Yuan, Mengdie; Diao, Guoqing

    2011-01-01

    Univariate genome-wide association analysis of quantitative and qualitative traits has been investigated extensively in the literature. In the presence of correlated phenotypes, it is more intuitive to analyze all phenotypes simultaneously. We describe an efficient likelihood-based approach for the joint association analysis of quantitative and qualitative traits in unrelated individuals. We assume a probit model for the qualitative trait, under which an unobserved latent variable and a prespecified threshold determine the value of the qualitative trait. To jointly model the quantitative and qualitative traits, we assume that the quantitative trait and the latent variable follow a bivariate normal distribution. The latent variable is allowed to be correlated with the quantitative phenotype. Simultaneous modeling of the quantitative and qualitative traits allows us to make more precise inference on the pleiotropic genetic effects. We derive likelihood ratio tests for the testing of genetic effects. An application to the Genetic Analysis Workshop 17 data is provided. The new method yields reasonable power and meaningful results for the joint association analysis of the quantitative trait Q1 and the qualitative trait disease status at SNPs with not too small MAF. PMID:22373162

  17. Quantitative data analysis of ESAR data

    NASA Astrophysics Data System (ADS)

    Phruksahiran, N.; Chandra, M.

    2013-07-01

    A synthetic aperture radar (SAR) data processing uses the backscattered electromagnetic wave to map radar reflectivity of the ground surface. The polarization property in radar remote sensing was used successfully in many applications, especially in target decomposition. This paper presents a case study to the experiments which are performed on ESAR L-Band full polarized data sets from German Aerospace Center (DLR) to demonstrate the potential of coherent target decomposition and the possibility of using the weather radar measurement parameter, such as the differential reflectivity and the linear depolarization ratio to obtain the quantitative information of the ground surface. The raw data of ESAR has been processed by the SAR simulator developed using MATLAB program code with Range-Doppler algorithm.

  18. Qualitative and quantitative analysis of endocytic recycling.

    PubMed

    Reineke, James B; Xie, Shuwei; Naslavsky, Naava; Caplan, Steve

    2015-01-01

    Endocytosis, which encompasses the internalization and sorting of plasma membrane (PM) lipids and proteins to distinct membrane-bound intracellular compartments, is a highly regulated and fundamental cellular process by which eukaryotic cells dynamically regulate their PM composition. Indeed, endocytosis is implicated in crucial cellular processes that include proliferation, migration, and cell division as well as maintenance of tissue homeostasis such as apical-basal polarity. Once PM constituents have been taken up into the cell, either via clathrin-dependent endocytosis (CDE) or clathrin-independent endocytosis (CIE), they typically have two fates: degradation through the late-endosomal/lysosomal pathway or returning to the PM via endocytic recycling pathways. In this review, we will detail experimental procedures that allow for both qualitative and quantitative assessment of endocytic recycling of transmembrane proteins internalized by CDE and CIE, using the HeLa cervical cancer cell line as a model system. PMID:26360033

  19. Towards a Quantitative OCT Image Analysis

    PubMed Central

    Garcia Garrido, Marina; Beck, Susanne C.; Mühlfriedel, Regine; Julien, Sylvie; Schraermeyer, Ulrich; Seeliger, Mathias W.

    2014-01-01

    Background Optical coherence tomography (OCT) is an invaluable diagnostic tool for the detection and follow-up of retinal pathology in patients and experimental disease models. However, as morphological structures and layering in health as well as their alterations in disease are complex, segmentation procedures have not yet reached a satisfactory level of performance. Therefore, raw images and qualitative data are commonly used in clinical and scientific reports. Here, we assess the value of OCT reflectivity profiles as a basis for a quantitative characterization of the retinal status in a cross-species comparative study. Methods Spectral-Domain Optical Coherence Tomography (OCT), confocal Scanning-La­ser Ophthalmoscopy (SLO), and Fluorescein Angiography (FA) were performed in mice (Mus musculus), gerbils (Gerbillus perpadillus), and cynomolgus monkeys (Macaca fascicularis) using the Heidelberg Engineering Spectralis system, and additional SLOs and FAs were obtained with the HRA I (same manufacturer). Reflectivity profiles were extracted from 8-bit greyscale OCT images using the ImageJ software package (http://rsb.info.nih.gov/ij/). Results Reflectivity profiles obtained from OCT scans of all three animal species correlated well with ex vivo histomorphometric data. Each of the retinal layers showed a typical pattern that varied in relative size and degree of reflectivity across species. In general, plexiform layers showed a higher level of reflectivity than nuclear layers. A comparison of reflectivity profiles from specialized retinal regions (e.g. visual streak in gerbils, fovea in non-human primates) with respective regions of human retina revealed multiple similarities. In a model of Retinitis Pigmentosa (RP), the value of reflectivity profiles for the follow-up of therapeutic interventions was demonstrated. Conclusions OCT reflectivity profiles provide a detailed, quantitative description of retinal layers and structures including specialized retinal regions

  20. Multiple quantitative trait analysis using bayesian networks.

    PubMed

    Scutari, Marco; Howell, Phil; Balding, David J; Mackay, Ian

    2014-09-01

    Models for genome-wide prediction and association studies usually target a single phenotypic trait. However, in animal and plant genetics it is common to record information on multiple phenotypes for each individual that will be genotyped. Modeling traits individually disregards the fact that they are most likely associated due to pleiotropy and shared biological basis, thus providing only a partial, confounded view of genetic effects and phenotypic interactions. In this article we use data from a Multiparent Advanced Generation Inter-Cross (MAGIC) winter wheat population to explore Bayesian networks as a convenient and interpretable framework for the simultaneous modeling of multiple quantitative traits. We show that they are equivalent to multivariate genetic best linear unbiased prediction (GBLUP) and that they are competitive with single-trait elastic net and single-trait GBLUP in predictive performance. Finally, we discuss their relationship with other additive-effects models and their advantages in inference and interpretation. MAGIC populations provide an ideal setting for this kind of investigation because the very low population structure and large sample size result in predictive models with good power and limited confounding due to relatedness. PMID:25236454

  1. Quantitative infrared analysis of hydrogen fluoride

    SciTech Connect

    Manuta, D.M.

    1997-04-01

    This work was performed at the Portsmouth Gaseous Diffusion Plant where hydrogen fluoride is produced upon the hydrolysis of UF{sub 6}. This poses a problem for in this setting and a method for determining the mole percent concentration was desired. HF has been considered to be a non-ideal gas for many years. D. F. Smith utilized complex equations in his HF studies in the 1950s. We have evaluated HF behavior as a function of pressure from three different perspectives. (1) Absorbance at 3877 cm{sup -1} as a function of pressure for 100% HF. (2) Absorbance at 3877 cm{sup -1} as a function of increasing partial pressure HF. Total pressure = 300 mm HgA maintained with nitrogen. (3) Absorbance at 3877 cm{sup -1} for constant partial pressure HF. Total pressure is increased to greater than 800 mm HgA with nitrogen. These experiments have shown that at partial pressures up to 35mm HgA, HIF follows the ideal gas law. The absorbance at 3877 cm{sup -1} can be quantitatively analyzed via infrared methods.

  2. Quantitative multi-modal NDT data analysis

    SciTech Connect

    Heideklang, René; Shokouhi, Parisa

    2014-02-18

    A single NDT technique is often not adequate to provide assessments about the integrity of test objects with the required coverage or accuracy. In such situations, it is often resorted to multi-modal testing, where complementary and overlapping information from different NDT techniques are combined for a more comprehensive evaluation. Multi-modal material and defect characterization is an interesting task which involves several diverse fields of research, including signal and image processing, statistics and data mining. The fusion of different modalities may improve quantitative nondestructive evaluation by effectively exploiting the augmented set of multi-sensor information about the material. It is the redundant information in particular, whose quantification is expected to lead to increased reliability and robustness of the inspection results. There are different systematic approaches to data fusion, each with its specific advantages and drawbacks. In our contribution, these will be discussed in the context of nondestructive materials testing. A practical study adopting a high-level scheme for the fusion of Eddy Current, GMR and Thermography measurements on a reference metallic specimen with built-in grooves will be presented. Results show that fusion is able to outperform the best single sensor regarding detection specificity, while retaining the same level of sensitivity.

  3. Quantitative analysis of myocardial tissue with digital autofluorescence microscopy

    PubMed Central

    Jensen, Thomas; Holten-Rossing, Henrik; Svendsen, Ida M H; Jacobsen, Christina; Vainer, Ben

    2016-01-01

    Background: The opportunity offered by whole slide scanners of automated histological analysis implies an ever increasing importance of digital pathology. To go beyond the importance of conventional pathology, however, digital pathology may need a basic histological starting point similar to that of hematoxylin and eosin staining in conventional pathology. This study presents an automated fluorescence-based microscopy approach providing highly detailed morphological data from unstained microsections. This data may provide a basic histological starting point from which further digital analysis including staining may benefit. Methods: This study explores the inherent tissue fluorescence, also known as autofluorescence, as a mean to quantitate cardiac tissue components in histological microsections. Data acquisition using a commercially available whole slide scanner and an image-based quantitation algorithm are presented. Results: It is shown that the autofluorescence intensity of unstained microsections at two different wavelengths is a suitable starting point for automated digital analysis of myocytes, fibrous tissue, lipofuscin, and the extracellular compartment. The output of the method is absolute quantitation along with accurate outlines of above-mentioned components. The digital quantitations are verified by comparison to point grid quantitations performed on the microsections after Van Gieson staining. Conclusion: The presented method is amply described as a prestain multicomponent quantitation and outlining tool for histological sections of cardiac tissue. The main perspective is the opportunity for combination with digital analysis of stained microsections, for which the method may provide an accurate digital framework. PMID:27141321

  4. The quantitative failure of human reliability analysis

    SciTech Connect

    Bennett, C.T.

    1995-07-01

    This philosophical treatise argues the merits of Human Reliability Analysis (HRA) in the context of the nuclear power industry. Actually, the author attacks historic and current HRA as having failed in informing policy makers who make decisions based on risk that humans contribute to systems performance. He argues for an HRA based on Bayesian (fact-based) inferential statistics, which advocates a systems analysis process that employs cogent heuristics when using opinion, and tempers itself with a rational debate over the weight given subjective and empirical probabilities.

  5. A Quantitative Analysis of Countries' Research Strengths

    ERIC Educational Resources Information Center

    Saxena, Anurag; Brazer, S. David; Gupta, B. M.

    2009-01-01

    This study employed a multidimensional analysis to evaluate transnational patterns of scientific research to determine relative research strengths among widely varying nations. Findings from this study may inform national policy with regard to the most efficient use of scarce national research resources, including government and private funding.…

  6. A Comparative Assessment of Greek Universities' Efficiency Using Quantitative Analysis

    ERIC Educational Resources Information Center

    Katharaki, Maria; Katharakis, George

    2010-01-01

    In part due to the increased demand for higher education, typical evaluation frameworks for universities often address the key issue of available resource utilisation. This study seeks to estimate the efficiency of 20 public universities in Greece through quantitative analysis (including performance indicators, data envelopment analysis (DEA) and…

  7. Influence of corrosion layers on quantitative analysis

    NASA Astrophysics Data System (ADS)

    Denker, A.; Bohne, W.; Opitz-Coutureau, J.; Rauschenberg, J.; Röhrich, J.; Strub, E.

    2005-09-01

    Art historians and restorers in charge of ancient metal objects are often reluctant to remove the corrosion layer evolved over time, as this would change the appearance of the artefact dramatically. Therefore, when an elemental analysis of the objects is required, this has to be done by penetrating the corrosion layer. In this work the influence of corrosion was studied on Chinese and Roman coins, where removal of oxidized material was possible. Measurements on spots with and without corrosion are presented and the results discussed.

  8. Functional phosphoproteomic analysis reveals cold-shock domain protein A to be a Bcr-Abl effector-regulating proliferation and transformation in chronic myeloid leukemia

    PubMed Central

    Sears, D; Luong, P; Yuan, M; Nteliopoulos, G; Man, Y K S; Melo, J V; Basu, S

    2010-01-01

    One proposed strategy to suppress the proliferation of imatinib-resistant cells in chronic myeloid leukemia (CML) is to inhibit key proteins downstream of Bcr-Abl. The PI3K/Akt pathway is activated by Bcr-Abl and is specifically required for the growth of CML cells. To identify targets of this pathway, we undertook a proteomic screen and identified several proteins that differentially bind 14-3-3, dependent on Bcr-Abl kinase activity. An siRNA screen of candidates selected by bioinformatics analysis reveals cold-shock domain protein A (CSDA), shown previously to regulate cell cycle progression in epithelial cells, to be a positive regulator of proliferation in a CML cell line. We show that Akt can phosphorylate the serine 134 residue of CSDA but, downstream of Bcr-Abl activity, this modification is mediated through the activation of MEK/p90 ribosomal S6 kinase (RSK) signaling. Inhibition of RSK, similarly to treatment with imatinib, blocked proliferation specifically in Bcr-Abl-positive leukemia cell lines, as well as cells from CML patients. Furthermore, these primary CML cells showed an increase in CSDA phosphorylation. Expression of a CSDA phospho-deficient mutant resulted in the decrease of Bcr-Abl-dependent transformation in Rat1 cells. Our results support a model whereby phosphorylation of CSDA downstream of Bcr-Abl enhances proliferation in CML cells to drive leukemogenesis. PMID:21368869

  9. Quantitative transcriptome analysis using RNA-seq.

    PubMed

    Külahoglu, Canan; Bräutigam, Andrea

    2014-01-01

    RNA-seq has emerged as the technology of choice to quantify gene expression. This technology is a convenient accurate tool to quantify diurnal changes in gene expression, gene discovery, differential use of promoters, and splice variants for all genes expressed in a single tissue. Thus, RNA-seq experiments provide sequence information and absolute expression values about transcripts in addition to relative quantification available with microarrays or qRT-PCR. The depth of information by sequencing requires careful assessment of RNA intactness and DNA contamination. Although the RNA-seq is comparatively recent, a standard analysis framework has emerged with the packages of Bowtie2, TopHat, and Cufflinks. With rising popularity of RNA-seq tools have become manageable for researchers without much bioinformatical knowledge or programming skills. Here, we present a workflow for a RNA-seq experiment from experimental planning to biological data extraction. PMID:24792045

  10. Uncertainty of quantitative microbiological methods of pharmaceutical analysis.

    PubMed

    Gunar, O V; Sakhno, N G

    2015-12-30

    The total uncertainty of quantitative microbiological methods, used in pharmaceutical analysis, consists of several components. The analysis of the most important sources of the quantitative microbiological methods variability demonstrated no effect of culture media and plate-count techniques in the estimation of microbial count while the highly significant effect of other factors (type of microorganism, pharmaceutical product and individual reading and interpreting errors) was established. The most appropriate method of statistical analysis of such data was ANOVA which enabled not only the effect of individual factors to be estimated but also their interactions. Considering all the elements of uncertainty and combining them mathematically the combined relative uncertainty of the test results was estimated both for method of quantitative examination of non-sterile pharmaceuticals and microbial count technique without any product. These data did not exceed 35%, appropriated for a traditional plate count methods. PMID:26456251

  11. Quantitative surface spectroscopic analysis of multicomponent polymers

    NASA Astrophysics Data System (ADS)

    Zhuang, Hengzhong

    Angle-dependent electron spectroscopy for chemical analysis (ESCA) has been successfully used to examine the surface compositional gradient of a multicomponent polymer. However, photoelectron intensities detected at each take-off angle of ESCA measurements are convoluted signals. The convoluted nature of the signal distorts depth profiles for samples having compositional gradients. To recover the true concentration profiles for the samples, a deconvolution program has been described in Chapter 2. The compositional profiles of two classes of important multicomponent polymers, i.e., poly(dimethysiloxane urethane) (PU-DMS) segmented copolymers and fluorinated poly(amide urethane) block copolymers, are achieved using this program. The effects of the polymer molecular structure and the processing variation on its surface compositional profile have been studied. Besides surface composition, it is desirable to know whether the distribution of segment or block lengths at the surface is different than in the bulk, because this aspect of surface structure may lead to properties different than that predicted simply by knowledge of the surface composition and the bulk structure. In Chapter 3, we pioneered the direct determination of the distribution of polydimethylsiloxane (PDMS) segment lengths at the surface of PU-DMS using time-of-flight secondary ion mass spectrometry (SUMS). Exciting preliminary results are provided: for the thick film of PU-DMS with nominal MW of PDMS = 1000, the distribution of the PDMS segment lengths at the surface is nearly identical to that in the bulk, whereas in the case of the thick films of PU-DMS with nominal MW of PDMS = 2400, only those PDMS segments with MW of ca. 1000 preferentially segregated at the surface. As a potential minimal fouling coating or biocompatible cardio-vascular materials, PU-DMS copolymers eventually come into contact with water once in use. Could such an environmental change (from air to aqueous) induce any undesirable

  12. Quantitative analysis of in vivo cell proliferation.

    PubMed

    Cameron, Heather A

    2006-11-01

    Injection and immunohistochemical detection of 5-bromo-2'-deoxyuridine (BrdU) has become the standard method for studying the birth and survival of neurons, glia, and other cell types in the nervous system. BrdU, a thymidine analog, becomes stably incorporated into DNA during the S-phase of mitosis. Because DNA containing BrdU can be specifically recognized by antibodies, this method allows dividing cells to be marked at any given time and then identified at time points from a few minutes to several years later. BrdU immunohistochemistry is suitable for cell counting to examine the regulation of cell proliferation and cell fate. It can be combined with labeling by other antibodies, allowing confocal analysis of cell phenotype or expression of other proteins. The potential for nonspecific labeling and toxicity are discussed. Although BrdU immunohistochemistry has almost completely replaced tritiated thymidine autoradiography for labeling dividing cells, this method and situations in which it is still useful are also described. PMID:18428635

  13. [Qualitative and quantitative gamma-hydroxybutyrate analysis].

    PubMed

    Petek, Maja Jelena; Vrdoljak, Ana Lucić

    2006-12-01

    Gamma-hydroxybutyrate (GHB) is a naturally occurring compound present in the brain and peripheral tissues of mammals. It is a minor metabolite and precursor of gamma-aminobutyric acid (GABA). Just as GABA, GHB is believed to play a role in neurotransmission. GHB was first synthesized in vitro in 1960, when it revealed depressive and hypnotic effects on the central nervous system. In 1960s it was used as an anaesthetic and later as an alternative to anabolic steroids, in order to enhance muscle growth. However, after it was shown that it caused strong physical dependence and severe side effects, GHB was banned. For the last fifteen years, GHB has been abused for its intoxicating effects such as euphoria, reduced inhibitions and sedation. Illicitly it is available as white powder or as clear liquid. Paradoxically GHB can easily be manufactured from its precursor gamma-butyrolactone (GBL), which has not yet been banned. Because of many car accidents and criminal acts in which it is involved, GHB has become an important object of forensic laboratory analysis. This paper describes gas and liquid chromatography, infrared spectroscopy, microscopy, colourimetry and nuclear magnetic resonance as methods for detection and quantification of GHB in urine and illicit products. PMID:17265679

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

    PubMed

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

    2016-03-01

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

  15. Impact of TGF-b on breast cancer from a quantitative proteomic analysis.

    PubMed

    Ahn, Jaegyoon; Yoon, Youngmi; Yeu, Yunku; Lee, Hookuen; Park, Sanghyun

    2013-12-01

    There has been much active research in bioinformatics to support our understanding of oncogenesis and tumor progression. Most research relies on mRNA gene expression data to identify marker genes or cancer specific gene networks. However, considering that proteins are functional molecules that carry out the biological tasks of genes, they can be direct markers of biological functions. Protein abundance data on a genome scale have not been investigated in depth due to the limited availability of high throughput protein assays. This hindrance is chiefly caused by a lack of robust techniques such as RT-PCR (real-time polymerase chain reaction). In this study, we quantified phospho-proteomes of breast cancer cell lines treated with TGF-beta (transforming growth factor beta). To discover biomarkers and observe changes in the signaling pathways related to breast cancer, we applied a protein network-based approach to generate a classifier of subnet markers. The accuracy of that classifier outperformed other network-based classification algorithms, and current feature selection and classification algorithms. Moreover, many cancer-related proteins were identified in those sub-networks. Each sub-network provides functional insights and can serve as a potential marker for TGF-beta treatments. After interpreting the roles of proteins in sub-networks with various signaling pathways, we found strong candidate proteins and various related interactions that are expected to affect breast cancer outcomes. These results demonstrate the high quality of the quantified phospho-proteomes data and show that our network construction and classification method is appropriate for an analysis of this type of data. PMID:24290926

  16. Control of separation and quantitative analysis by GC-FTIR

    NASA Astrophysics Data System (ADS)

    Semmoud, A.; Huvenne, Jean P.; Legrand, P.

    1992-03-01

    Software for 3-D representations of the 'Absorbance-Wavenumber-Retention time' is used to control the quality of the GC separation. Spectral information given by the FTIR detection allows the user to be sure that a chromatographic peak is 'pure.' The analysis of peppermint essential oil is presented as an example. This assurance is absolutely required for quantitative applications. In these conditions, we have worked out a quantitative analysis of caffeine. Correlation coefficients between integrated absorbance measurements and concentration of caffeine are discussed at two steps of the data treatment.

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

    PubMed Central

    He, Yikun

    2014-01-01

    The moss Physcomitrella patens is an ideal model plant to study plant developmental processes. To better understand the mechanism of protoplast regeneration, a phosphoproteome analysis was performed. Protoplasts were prepared from protonemata. By 4 d of protoplast regeneration, the first cell divisions had ensued. Through a highly selective titanium dioxide (TiO2)-based phosphopeptide enrichment method and mass spectrometric technology, more than 300 phosphoproteins were identified as protoplast regeneration responsive. Of these, 108 phosphoproteins were present on day 4 but not in fresh protoplasts or those cultured for 2 d. These proteins are catalogued here. They were involved in cell-wall metabolism, transcription, signal transduction, cell growth/division, and cell structure. These protein functions are related to cell morphogenesis, organogenesis, and development adjustment. This study presents a comprehensive analysis of phosphoproteome involved in protoplast regeneration and indicates that the mechanism of plant protoplast regeneration is similar to that of postembryonic development. PMID:24700621

  18. A quantitative analysis of the F18 flight control system

    NASA Technical Reports Server (NTRS)

    Doyle, Stacy A.; Dugan, Joanne B.; Patterson-Hine, Ann

    1993-01-01

    This paper presents an informal quantitative analysis of the F18 flight control system (FCS). The analysis technique combines a coverage model with a fault tree model. To demonstrate the method's extensive capabilities, we replace the fault tree with a digraph model of the F18 FCS, the only model available to us. The substitution shows that while digraphs have primarily been used for qualitative analysis, they can also be used for quantitative analysis. Based on our assumptions and the particular failure rates assigned to the F18 FCS components, we show that coverage does have a significant effect on the system's reliability and thus it is important to include coverage in the reliability analysis.

  19. Dynamic Adipocyte Phosphoproteome Reveals that Akt Directly Regulates mTORC2

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2016-01-01

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

  1. Quantitative transverse flow measurement using OCT speckle decorrelation analysis

    PubMed Central

    Liu, Xuan; Huang, Yong; Ramella-Roman, Jessica C.; Mathews, Scott A.; Kang, Jin U.

    2014-01-01

    We propose an inter-Ascan speckle decorrelation based method that can quantitatively assess blood flow normal to the direction of the OCT imaging beam. To validate this method, we performed a systematic study using both phantom and in vivo animal models. Results show that our speckle analysis method can accurately extract transverse flow speed with high spatial and temporal resolution. PMID:23455305

  2. Early Child Grammars: Qualitative and Quantitative Analysis of Morphosyntactic Production

    ERIC Educational Resources Information Center

    Legendre, Geraldine

    2006-01-01

    This article reports on a series of 5 analyses of spontaneous production of verbal inflection (tense and person-number agreement) by 2-year-olds acquiring French as a native language. A formal analysis of the qualitative and quantitative results is developed using the unique resources of Optimality Theory (OT; Prince & Smolensky, 2004). It is…

  3. Quantitating the subtleties of microglial morphology with fractal analysis

    PubMed Central

    Karperien, Audrey; Ahammer, Helmut; Jelinek, Herbert F.

    2013-01-01

    It is well established that microglial form and function are inextricably linked. In recent years, the traditional view that microglial form ranges between “ramified resting” and “activated amoeboid” has been emphasized through advancing imaging techniques that point to microglial form being highly dynamic even within the currently accepted morphological categories. Moreover, microglia adopt meaningful intermediate forms between categories, with considerable crossover in function and varying morphologies as they cycle, migrate, wave, phagocytose, and extend and retract fine and gross processes. From a quantitative perspective, it is problematic to measure such variability using traditional methods, but one way of quantitating such detail is through fractal analysis. The techniques of fractal analysis have been used for quantitating microglial morphology, to categorize gross differences but also to differentiate subtle differences (e.g., amongst ramified cells). Multifractal analysis in particular is one technique of fractal analysis that may be useful for identifying intermediate forms. Here we review current trends and methods of fractal analysis, focusing on box counting analysis, including lacunarity and multifractal analysis, as applied to microglial morphology. PMID:23386810

  4. Development of a quantitative autoradiography image analysis system

    SciTech Connect

    Hoffman, T.J.; Volkert, W.A.; Holmes R.A.

    1986-03-01

    A low cost image analysis system suitable for quantitative autoradiography (QAR) analysis has been developed. Autoradiographs can be digitized using a conventional Newvicon television camera interfaced to an IBM-XT microcomputer. Software routines for image digitization and capture permit the acquisition of thresholded or windowed images with graphic overlays that can be stored on storage devices. Image analysis software performs all background and non-linearity corrections prior to display as black/white or pseudocolor images. The relationship of pixel intensity to a standard radionuclide concentration allows the production of quantitative maps of tissue radiotracer concentrations. An easily modified subroutine is provided for adaptation to use appropriate operational equations when parameters such as regional cerebral blood flow or regional cerebral glucose metabolism are under investigation. This system could provide smaller research laboratories with the capability of QAR analysis at relatively low cost.

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

    PubMed

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

    2015-01-01

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

  6. Quantitative analysis of regional myocardial performance in coronary artery disease

    NASA Technical Reports Server (NTRS)

    Stewart, D. K.; Dodge, H. T.; Frimer, M.

    1975-01-01

    Findings from a group of subjects with significant coronary artery stenosis are given. A group of controls determined by use of a quantitative method for the study of regional myocardial performance based on the frame-by-frame analysis of biplane left ventricular angiograms are presented. Particular emphasis was placed upon the analysis of wall motion in terms of normalized segment dimensions, timing and velocity of contraction. The results were compared with the method of subjective assessment used clinically.

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

    PubMed

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

    2016-04-22

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

  8. Improved method and apparatus for chromatographic quantitative analysis

    DOEpatents

    Fritz, J.S.; Gjerde, D.T.; Schmuckler, G.

    An improved apparatus and method are described for the quantitative analysis of a solution containing a plurality of anion species by ion exchange chromatography which utilizes a single element and a single ion exchange bed which does not require periodic regeneration. The solution containing the anions is added to an anion exchange resin bed which is a low capacity macroreticular polystyrene-divinylbenzene resin containing quarternary ammonium functional groups, and is eluted therefrom with a dilute solution of a low electrical conductance organic acid salt. As each anion species is eluted from the bed, it is quantitatively sensed by conventional detection means such as a conductivity cell.

  9. Quantitative numerical analysis of transient IR-experiments on buildings

    NASA Astrophysics Data System (ADS)

    Maierhofer, Ch.; Wiggenhauser, H.; Brink, A.; Röllig, M.

    2004-12-01

    Impulse-thermography has been established as a fast and reliable tool in many areas of non-destructive testing. In recent years several investigations have been done to apply active thermography to civil engineering. For quantitative investigations in this area of application, finite difference calculations have been performed for systematic studies on the influence of environmental conditions, heating power and time, defect depth and size and thermal properties of the bulk material (concrete). The comparison of simulated and experimental data enables the quantitative analysis of defects.

  10. Scanning tunneling microscopy on rough surfaces-quantitative image analysis

    NASA Astrophysics Data System (ADS)

    Reiss, G.; Brückl, H.; Vancea, J.; Lecheler, R.; Hastreiter, E.

    1991-07-01

    In this communication, the application of scanning tunneling microscopy (STM) for a quantitative evaluation of roughnesses and mean island sizes of polycrystalline thin films is discussed. Provided strong conditions concerning the resolution are satisfied, the results are in good agreement with standard techniques as, for example, transmission electron microscopy. Owing to its high resolution, STM can supply a better characterization of surfaces than established methods, especially concerning the roughness. Microscopic interpretations of surface dependent physical properties thus can be considerably improved by a quantitative analysis of STM images.

  11. Quantitative analysis of culture using millions of digitized books.

    PubMed

    Michel, Jean-Baptiste; Shen, Yuan Kui; Aiden, Aviva Presser; Veres, Adrian; Gray, Matthew K; Pickett, Joseph P; Hoiberg, Dale; Clancy, Dan; Norvig, Peter; Orwant, Jon; Pinker, Steven; Nowak, Martin A; Aiden, Erez Lieberman

    2011-01-14

    We constructed a corpus of digitized texts containing about 4% of all books ever printed. Analysis of this corpus enables us to investigate cultural trends quantitatively. We survey the vast terrain of 'culturomics,' focusing on linguistic and cultural phenomena that were reflected in the English language between 1800 and 2000. We show how this approach can provide insights about fields as diverse as lexicography, the evolution of grammar, collective memory, the adoption of technology, the pursuit of fame, censorship, and historical epidemiology. Culturomics extends the boundaries of rigorous quantitative inquiry to a wide array of new phenomena spanning the social sciences and the humanities. PMID:21163965

  12. Quantitative analysis of culture using millions of digitized books

    PubMed Central

    Michel, Jean-Baptiste; Shen, Yuan Kui; Aiden, Aviva P.; Veres, Adrian; Gray, Matthew K.; Pickett, Joseph P.; Hoiberg, Dale; Clancy, Dan; Norvig, Peter; Orwant, Jon; Pinker, Steven; Nowak, Martin A.; Aiden, Erez Lieberman

    2011-01-01

    We constructed a corpus of digitized texts containing about 4% of all books ever printed. Analysis of this corpus enables us to investigate cultural trends quantitatively. We survey the vast terrain of ‘culturomics’, focusing on linguistic and cultural phenomena that were reflected in the English language between 1800 and 2000. We show how this approach can provide insights about fields as diverse as lexicography, the evolution of grammar, collective memory, the adoption of technology, the pursuit of fame, censorship, and historical epidemiology. ‘Culturomics’ extends the boundaries of rigorous quantitative inquiry to a wide array of new phenomena spanning the social sciences and the humanities. PMID:21163965

  13. An Initial Characterization of the Serum Phosphoproteome

    PubMed Central

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

    2009-01-01

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

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

    PubMed

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

    2016-06-01

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

  15. Quantitative mass spectrometric analysis of glycoproteins combined with enrichment methods.

    PubMed

    Ahn, Yeong Hee; Kim, Jin Young; Yoo, Jong Shin

    2015-01-01

    Mass spectrometry (MS) has been a core technology for high sensitive and high-throughput analysis of the enriched glycoproteome in aspects of quantitative assays as well as qualitative profiling of glycoproteins. Because it has been widely recognized that aberrant glycosylation in a glycoprotein may involve in progression of a certain disease, the development of efficient analysis tool for the aberrant glycoproteins is very important for deep understanding about pathological function of the glycoprotein and new biomarker development. This review first describes the protein glycosylation-targeting enrichment technologies mainly employing solid-phase extraction methods such as hydrizide-capturing, lectin-specific capturing, and affinity separation techniques based on porous graphitized carbon, hydrophilic interaction chromatography, or immobilized boronic acid. Second, MS-based quantitative analysis strategies coupled with the protein glycosylation-targeting enrichment technologies, by using a label-free MS, stable isotope-labeling, or targeted multiple reaction monitoring (MRM) MS, are summarized with recent published studies. PMID:24889823

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

    PubMed

    Contreras-Vallejos, Erick; Utreras, Elías; Bórquez, Daniel A; Prochazkova, Michaela; Terse, Anita; Jaffe, Howard; Toledo, Andrea; Arruti, Cristina; Pant, Harish C; Kulkarni, Ashok B; González-Billault, Christian

    2014-01-01

    Protein phosphorylation is the most common post-translational modification that regulates several pivotal functions in cells. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase which is mostly active in the nervous system. It regulates several biological processes such as neuronal migration, cytoskeletal dynamics, axonal guidance and synaptic plasticity among others. In search for novel substrates of Cdk5 in the brain we performed quantitative phosphoproteomics analysis, isolating phosphoproteins from whole brain derived from E18.5 Cdk5+/+ and Cdk5-/- embryos, using an Immobilized Metal-Ion Affinity Chromatography (IMAC), which specifically binds to phosphorylated proteins. The isolated phosphoproteins were eluted and isotopically labeled for relative and absolute quantitation (iTRAQ) and mass spectrometry identification. We found 40 proteins that showed decreased phosphorylation at Cdk5-/- brains. In addition, out of these 40 hypophosphorylated proteins we characterized two proteins, :MARCKS (Myristoylated Alanine-Rich protein Kinase C substrate) and Grin1 (G protein regulated inducer of neurite outgrowth 1). MARCKS is known to be phosphorylated by Cdk5 in chick neural cells while Grin1 has not been reported to be phosphorylated by Cdk5. When these proteins were overexpressed in N2A neuroblastoma cell line along with p35, serine phosphorylation in their Cdk5 motifs was found to be increased. In contrast, treatments with roscovitine, the Cdk5 inhibitor, resulted in an opposite effect on serine phosphorylation in N2A cells and primary hippocampal neurons transfected with MARCKS. In summary, the results presented here identify Grin 1 as novel Cdk5 substrate and confirm previously identified MARCKS as a a bona fide Cdk5 substrate. PMID:24658276

  17. An Analysis of Critical Factors for Quantitative Immunoblotting

    PubMed Central

    Janes, Kevin A.

    2015-01-01

    Immunoblotting (also known as Western blotting) combined with digital image analysis can be a reliable method for analyzing the abundance of proteins and protein modifications, but not every immunoblot-analysis combination produces an accurate result. Here, I illustrate how sample preparation, protocol implementation, detection scheme, and normalization approach profoundly affect the quantitative performance of immunoblotting. This study implemented diagnostic experiments that assess an immunoblot-analysis workflow for accuracy and precision. The results showed that ignoring such diagnostics can lead to pseudoquantitative immunoblot data that dramatically overestimate or underestimate true differences in protein abundance. PMID:25852189

  18. Some selected quantitative methods of thermal image analysis in Matlab.

    PubMed

    Koprowski, Robert

    2016-05-01

    The paper presents a new algorithm based on some selected automatic quantitative methods for analysing thermal images. It shows the practical implementation of these image analysis methods in Matlab. It enables to perform fully automated and reproducible measurements of selected parameters in thermal images. The paper also shows two examples of the use of the proposed image analysis methods for the area of ​​the skin of a human foot and face. The full source code of the developed application is also provided as an attachment. The main window of the program during dynamic analysis of the foot thermal image. PMID:26556680

  19. An Improved Quantitative Analysis Method for Plant Cortical Microtubules

    PubMed Central

    Lu, Yi; Huang, Chenyang; Wang, Jia; Shang, Peng

    2014-01-01

    The arrangement of plant cortical microtubules can reflect the physiological state of cells. However, little attention has been paid to the image quantitative analysis of plant cortical microtubules so far. In this paper, Bidimensional Empirical Mode Decomposition (BEMD) algorithm was applied in the image preprocessing of the original microtubule image. And then Intrinsic Mode Function 1 (IMF1) image obtained by decomposition was selected to do the texture analysis based on Grey-Level Cooccurrence Matrix (GLCM) algorithm. Meanwhile, in order to further verify its reliability, the proposed texture analysis method was utilized to distinguish different images of Arabidopsis microtubules. The results showed that the effect of BEMD algorithm on edge preserving accompanied with noise reduction was positive, and the geometrical characteristic of the texture was obvious. Four texture parameters extracted by GLCM perfectly reflected the different arrangements between the two images of cortical microtubules. In summary, the results indicate that this method is feasible and effective for the image quantitative analysis of plant cortical microtubules. It not only provides a new quantitative approach for the comprehensive study of the role played by microtubules in cell life activities but also supplies references for other similar studies. PMID:24744684

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

    PubMed

    Dudekula, Khadar; Le Bihan, Thierry

    2016-09-01

    The dataset presented in this work has been obtained using a label-free quantitative proteomic analysis of rat spleen. A robust method for extraction of proteins from rat spleen tissue and LC-MS-MS analysis was developed using a urea and SDS-based buffer. Different fractionation methods were compared. A total of 3484 different proteins were identified from the pool of all experiments run in this study (a total of 2460 proteins with at least two peptides). A total of 1822 proteins were identified from nine non-fractionated pulse gels, 2288 proteins and 2864 proteins were identified by SDS-PAGE fractionation into three and five fractions respectively. The proteomics data are deposited in ProteomeXchange Consortium via PRIDE PXD003520, Progenesis and Maxquant output are presented in the supported information. The generated list of proteins under different regimes of fractionation allow assessing the nature of the identified proteins; variability in the quantitative analysis associated with the different sampling strategy and allow defining a proper number of replicates for future quantitative analysis. PMID:27358910

  1. Quantitative risk analysis of oil storage facilities in seismic areas.

    PubMed

    Fabbrocino, Giovanni; Iervolino, Iunio; Orlando, Francesca; Salzano, Ernesto

    2005-08-31

    Quantitative risk analysis (QRA) of industrial facilities has to take into account multiple hazards threatening critical equipment. Nevertheless, engineering procedures able to evaluate quantitatively the effect of seismic action are not well established. Indeed, relevant industrial accidents may be triggered by loss of containment following ground shaking or other relevant natural hazards, either directly or through cascade effects ('domino effects'). The issue of integrating structural seismic risk into quantitative probabilistic seismic risk analysis (QpsRA) is addressed in this paper by a representative study case regarding an oil storage plant with a number of atmospheric steel tanks containing flammable substances. Empirical seismic fragility curves and probit functions, properly defined both for building-like and non building-like industrial components, have been crossed with outcomes of probabilistic seismic hazard analysis (PSHA) for a test site located in south Italy. Once the seismic failure probabilities have been quantified, consequence analysis has been performed for those events which may be triggered by the loss of containment following seismic action. Results are combined by means of a specific developed code in terms of local risk contour plots, i.e. the contour line for the probability of fatal injures at any point (x, y) in the analysed area. Finally, a comparison with QRA obtained by considering only process-related top events is reported for reference. PMID:15908107

  2. Single-Molecule Sensors: Challenges and Opportunities for Quantitative Analysis.

    PubMed

    Gooding, J Justin; Gaus, Katharina

    2016-09-12

    Measurement science has been converging to smaller and smaller samples, such that it is now possible to detect single molecules. This Review focuses on the next generation of analytical tools that combine single-molecule detection with the ability to measure many single molecules simultaneously and/or process larger and more complex samples. Such single-molecule sensors constitute a new type of quantitative analytical tool, as they perform analysis by molecular counting and thus potentially capture the heterogeneity of the sample. This Review outlines the advantages and potential of these new, quantitative single-molecule sensors, the measurement challenges in making single-molecule devices suitable for analysis, the inspiration biology provides for overcoming these challenges, and some of the solutions currently being explored. PMID:27444661

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

    PubMed Central

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

    2010-01-01

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

  4. Quantitative NMR Analysis of Partially Substituted Biodiesel Glycerols

    SciTech Connect

    Nagy, M.; Alleman, T. L.; Dyer, T.; Ragauskas, A. J.

    2009-01-01

    Phosphitylation of hydroxyl groups in biodiesel samples with 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane followed by 31P-NMR analysis provides a rapid quantitative analytical technique for the determination of substitution patterns on partially esterified glycerols. The unique 31P-NMR chemical shift data was established with a series mono and di-substituted fatty acid esters of glycerol and then utilized to characterize an industrial sample of partially processed biodiesel.

  5. Quantitative analysis of chaotic synchronization by means of coherence

    NASA Astrophysics Data System (ADS)

    Shabunin, A.; Astakhov, V.; Kurths, J.

    2005-07-01

    We use an index of chaotic synchronization based on the averaged coherence function for the quantitative analysis of the process of the complete synchronization loss in unidirectionally coupled oscillators and maps. We demonstrate that this value manifests different stages of the synchronization breaking. It is invariant to time delay and insensitive to small noise and distortions, which can influence the accessible signals at measurements. Peculiarities of the synchronization destruction in maps and oscillators are investigated.

  6. A quantitative analysis of coupled oscillations using mobile accelerometer sensors

    NASA Astrophysics Data System (ADS)

    Castro-Palacio, Juan Carlos; Velázquez-Abad, Luisberis; Giménez, Fernando; Monsoriu, Juan A.

    2013-05-01

    In this paper, smartphone acceleration sensors were used to perform a quantitative analysis of mechanical coupled oscillations. Symmetric and asymmetric normal modes were studied separately in the first two experiments. In the third, a coupled oscillation was studied as a combination of the normal modes. Results indicate that acceleration sensors of smartphones, which are very familiar to students, represent valuable measurement instruments for introductory and first-year physics courses.

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

    PubMed

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

    2014-12-01

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

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

    SciTech Connect

    Huang, Zhenlie; Ichihara, Sahoko; Oikawa, Shinji; Chang, Jie; Zhang, Lingyi; Hu, Shijie; Huang, Hanlin; Ichihara, Gaku

    2015-01-15

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

  9. Quantitation of glycerophosphorylcholine by flow injection analysis using immobilized enzymes.

    PubMed

    Mancini, A; Del Rosso, F; Roberti, R; Caligiana, P; Vecchini, A; Binaglia, L

    1996-09-20

    A method for quantitating glycerophosphorylcholine by flow injection analysis is reported in the present paper. Glycerophosphorylcholine phosphodiesterase and choline oxidase, immobilized on controlled porosity glass beads, are packed in a small reactor inserted in a flow injection manifold. When samples containing glycerophosphorylcholine are injected, glycerophosphorylcholine is hydrolyzed into choline and sn-glycerol-3-phosphate. The free choline produced in this reaction is oxidized to betain and hydrogen peroxide. Hydrogen peroxide is detected amperometrically. Quantitation of glycerophosphorylcholine in samples containing choline and phosphorylcholine is obtained inserting ahead of the reactor a small column packed with a mixed bed ion exchange resin. The time needed for each determination does not exceed one minute. The present method, applied to quantitate glycerophosphorylcholine in samples of seminal plasma, gave results comparable with those obtained using the standard enzymatic-spectrophotometric procedure. An alternative procedure, making use of co-immobilized glycerophosphorylcholine phosphodiesterase and glycerol-3-phosphate oxidase for quantitating glycerophosphorylcholine, glycerophosphorylethanolamine and glycerophosphorylserine is also described. PMID:8905629

  10. Quantitative Motion Analysis in Two and Three Dimensions.

    PubMed

    Wessels, Deborah J; Lusche, Daniel F; Kuhl, Spencer; Scherer, Amanda; Voss, Edward; Soll, David R

    2016-01-01

    This chapter describes 2D quantitative methods for motion analysis as well as 3D motion analysis and reconstruction methods. Emphasis is placed on the analysis of dynamic cell shape changes that occur through extension and retraction of force generating structures such as pseudopodia and lamellipodia. Quantitative analysis of these structures is an underutilized tool in the field of cell migration. Our intent, therefore, is to present methods that we developed in an effort to elucidate mechanisms of basic cell motility, directed cell motion during chemotaxis, and metastasis. We hope to demonstrate how application of these methods can more clearly define alterations in motility that arise due to specific mutations or disease and hence, suggest mechanisms or pathways involved in normal cell crawling and treatment strategies in the case of disease. In addition, we present a 4D tumorigenesis model for high-resolution analysis of cancer cells from cell lines and human cancer tissue in a 3D matrix. Use of this model led to the discovery of the coalescence of cancer cell aggregates and unique cell behaviors not seen in normal cells or normal tissue. Graphic illustrations to visually display and quantify cell shape are presented along with algorithms and formulae for calculating select 2D and 3D motion analysis parameters. PMID:26498790