Probing the global kinome and phosphoproteome in Chlamydomonas reinhardtii via sequential enrichment and quantitative proteomics

ABSTRACT: Identifying 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. Presented here is a method to study the global kinome and phosphoproteome in tandem for the first time in a photosynthetic organism, the model alga Chlamydomonas reinhardtii (Chlamydomonas), using mass spectrometry-based label-free proteomics. A dual enrichment strategy first targets intact protein kinases via capture on immobilized multiplexed inhibitor beads (MIBs) 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) mass spectrometric acquisitions were performed to obtain a >50% increase in coverage of the enriched Chlamydomonas kinome over coverage found with no enrichment. The quantitative phosphoproteomic dataset yielded 2,304 phosphopeptides and 1,765 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.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Chlamydomonas Reinhardtii

SUBMITTER: Evan McConnell  

LAB HEAD: Leslie M. Hicks

PROVIDER: PXD004681 | Pride | 2016-10-05

REPOSITORIES: Pride