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Probing the global kinome and phosphoproteome in Chlamydomonas reinhardtii via sequential enrichment and quantitative proteomics
Author(s) -
Werth Emily G.,
McConnell Evan W.,
Gilbert Thomas S. Karim,
Couso Lianez Inmaculada,
Perez Carlos A.,
Manley Cherrel K.,
Graves Lee M.,
Umen James G.,
Hicks Leslie M.
Publication year - 2017
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/tpj.13384
Subject(s) - chlamydomonas reinhardtii , kinome , quantitative proteomics , phosphoproteomics , proteomics , chlamydomonas , phosphorylation , biology , protein phosphorylation , kinase , biochemistry , computational biology , microbiology and biotechnology , chemistry , protein kinase a , gene , mutant
Summary The identification of dynamic protein phosphorylation events is critical for understanding kinase/phosphatase‐regulated signaling pathways. To date, protein phosphorylation and kinase expression have been examined independently in photosynthetic organisms. Here we present a method to study the global kinome and phosphoproteome in tandem in a model photosynthetic organism, the alga Chlamydomonas reinhardtii (Chlamydomonas), using mass spectrometry‐based label‐free proteomics. A dual enrichment strategy targets intact protein kinases via capture on immobilized multiplexed inhibitor beads with subsequent proteolytic digestion of unbound proteins and peptide‐based phosphorylation enrichment. To increase depth of coverage, both data‐dependent and data‐independent (via SWATH , Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra) mass spectrometric acquisitions were performed to obtain a more than 50% increase in coverage of the enriched Chlamydomonas kinome over coverage found with no enrichment. The quantitative phosphoproteomic dataset yielded 2250 phosphopeptides and 1314 localized phosphosites with excellent reproducibility across biological replicates (90% of quantified sites with coefficient of variation below 11%). This approach enables simultaneous investigation of kinases and phosphorylation events at the global level to facilitate understanding of kinase networks and their influence in cell signaling events.