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. Research aimed at dissecting the roles of kinases on a global “‐omics” scale has grown within the past decade with the potential to become more comprehensive for discovery‐based, untargeted kinome analyses. Utilization of immobilized kinase inhibitors [e.g., multiplexed inhibitor beads (MIBs) or kinobeads] broadly captures protein kinases through binding to the ATP‐binding domain of intact native kinases. 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 in the 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 MIBs columns 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 kinome and phosphoproteomic datasets yield 115 protein kinases and 2,250 phosphopeptides with 1,314 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. For example, through the induction of stress in Chlamydomonas and the use of differential proteomic studies of the kinome and phosphoproteome, future studies using this tandem enrichment have the potential to target specific pathways and investigate global kinome and phosphoproteome changes in a quantitative fashion.Support or Funding InformationThis research was supported by a National Science Foundation CAREER award (MCB‐1552522) to L.M.H. Authors would like to acknowledge Dr. Gary Johnson for providing the MIBs beads.