Probing the global kinome and phosphoproteome in Chlamydomonas reinhardtii via sequential enrichment and quantitative proteomics.
Emily G WerthEvan W McConnellThomas S Karim GilbertInmaculada Couso LianezCarlos A PerezCherrel K ManleyLee M GravesJames G UmenLeslie M HicksPublished in: The Plant journal : for cell and molecular biology (2017)
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.
Keyphrases
- protein kinase
- label free
- mass spectrometry
- high resolution
- protein protein
- binding protein
- electronic health record
- signaling pathway
- affordable care act
- poor prognosis
- tyrosine kinase
- big data
- capillary electrophoresis
- optical coherence tomography
- liquid chromatography
- artificial intelligence
- healthcare
- small molecule
- magnetic resonance imaging
- machine learning
- epithelial mesenchymal transition
- computed tomography
- anaerobic digestion
- simultaneous determination
- density functional theory