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Identification of Novel PAMP-Triggered Phosphorylation and Dephosphorylation Events in Arabidopsis thaliana by Quantitative Phosphoproteomic Analysis

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    Identification of Novel PAMP-Triggered Phosphorylation and Dephosphorylation Events in Arabidopsis thaliana by Quantitative Phosphoproteomic Analysis
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    † CNRS, UMR 8587, boulevard François Mitterrand, 91025 Evry, France
    ‡ Université Evry Val d’Essonne (UEVE), LAMBE, boulevard François Mitterrand, 91025 Evry, France
    § URGV Plant Genomics, INRA/CNRS/Université d’Evry Val d’Essonne, 2 rue Gaston Crémieux, 91057 Evry, France
    ∥ Center for Desert Agriculture, KAUST, 4700 Thuwal, Saudi Arabia
    *Phone: +33 1 60 87 45 08. Fax: +33 1 60 87 45 10. E-mail: [email protected]
    *Phone: +33 1 69 47 76 54. Fax: +33 1 69 47 76 55. E-mail: [email protected]
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    Journal of Proteome Research

    Cite this: J. Proteome Res. 2014, 13, 4, 2137–2151
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    https://doi.org/10.1021/pr401268v
    Published March 6, 2014
    Copyright © 2014 American Chemical Society
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    Abstract

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    Signaling cascades rely strongly on protein kinase-mediated substrate phosphorylation. Currently a major challenge in signal transduction research is to obtain high confidence substrate phosphorylation sites and assign them to specific kinases. In response to bacterial flagellin, a pathogen-associated molecular pattern (PAMP), we searched for rapidly phosphorylated proteins in Arabidopsis thaliana by combining multistage activation (MSA) and electron transfer dissociation (ETD) fragmentation modes, which generate complementary spectra and identify phosphopeptide sites with increased reliability. Of a total of 825 phosphopeptides, we identified 58 to be differentially phosphorylated. These peptides harbor kinase motifs of mitogen-activated protein kinases (MAPKs) and calcium-dependent protein kinases (CDPKs), as well as yet unknown protein kinases. Importantly, 12 of the phosphopeptides show reduced phosphorylation upon flagellin treatment. Since protein abundance levels did not change, these results indicate that flagellin induces not only various protein kinases but also protein phosphatases, even though a scenario of inhibited kinase activity may also be possible.

    Copyright © 2014 American Chemical Society

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    MassChropQ_parameters.txt. All_FragMixer_outputs_Fsamples_analyses. All_FragMixer_outputs_Msamples_analyses. Supplementary and figures. We also provide ZIP files containing all the annotated MS/MS spectra leading to phosphopeptide identification (at an FDR of 1% estimated by FragMixer), with names such as: F_02_23_B_ETD (for Flg-treated sample, biological replicate “02”, ions of charge states 2 + 3+ selected for MS/MS, B = Blind analysis, ETD fragmentation mode); or M_03_S4_E2_MSA (for Mock-treated sample, biological replicate “03”, ions of charge states ≥4 selected for MS/MS, analysis performed with a 2nd Exclusion list combining the m/z ratios of peptides identified in the two former analyses of the same sample; MSA fragmentation mode). This material is available free of charge via the Internet at http://pubs.acs.org.

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    Journal of Proteome Research

    Cite this: J. Proteome Res. 2014, 13, 4, 2137–2151
    Click to copy citationCitation copied!
    https://doi.org/10.1021/pr401268v
    Published March 6, 2014
    Copyright © 2014 American Chemical Society
    Request reuse permissions

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