Probing the global kinome and phosphoproteome in Chlamydomonas reinhardtii via sequential enrichment and quantitative proteomics. (17th January 2017)

• Record Type: Journal Article
• Authors: 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.
• Is Part Of: Plant journal. Volume 89:Number 2(2017)
• Page Start: 416
• Page End: 426
• Publication Date: 2017-01-17
• Subjects: mass spectrometry -- quantitative label‐free proteomics -- kinome -- phosphoproteomics -- Chlamydomonas reinhardtii -- technical advance; Plant molecular biology -- Periodicals; Plant cells and tissues -- Periodicals; Botany -- Periodicals; 580
• DOI: 10.1111/tpj.13384
• Languages: English
• ISSNs: 0960-7412

Abstract

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. Significance Statement: Identifying dynamic protein phosphorylation events is critical for understanding kinase/phosphatase regulated signaling pathways. Here we used tandem label‐free quantitative proteomics and phoshoproteomics to globally and simultaneously investigate kinases and phosphorylation events in Chlamydomonas.