Probing the global kinome and phosphoproteome in Chlamydomonas reinhardtii via sequential enrichment and quantitative proteomics.
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.
Department of Chemistry, University of North Carolina at Chapel Hill, 125 South Road, CB#3290, Chapel Hill, NC, 2759934, USA.
Emily G
EG
Werth
Department of Chemistry, University of North Carolina at Chapel Hill, 125 South Road, CB#3290, Chapel Hill, NC, 2759934, USA.
Evan W
EW
McConnell
The Department of Pharmacology, The University of North Carolina at Chapel Hill, NC 27599, USA.
Thomas S Karim
TS
Gilbert
Donald Danforth Plant Science Center, St. Louis, MO 63132, USA.
Inmaculada
I
Couso Lianez
Department of Chemistry, University of North Carolina at Chapel Hill, 125 South Road, CB#3290, Chapel Hill, NC, 2759934, USA.
Carlos A
CA
Perez
Department of Chemistry, University of North Carolina at Chapel Hill, 125 South Road, CB#3290, Chapel Hill, NC, 2759934, USA.
Cherrel K
CK
Manley
The Department of Pharmacology, The University of North Carolina at Chapel Hill, NC 27599, USA.
Lee M
LM
Graves
Donald Danforth Plant Science Center, St. Louis, MO 63132, USA.
James G
JG
Umen
Department of Chemistry, University of North Carolina at Chapel Hill, 125 South Road, CB#3290, Chapel Hill, NC, 2759934, USA.
Leslie M
0000-0002-8008-3998
LM
Hicks
D002473:Cell Wall / Q000737:chemistry; D005591:Chemical Fractionation; D016825:Chlamydomonas reinhardtii / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods; D010750:Phosphoproteins / Q000032:analysis / Q000378:metabolism*; D010940:Plant Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D011494:Protein Kinases / Q000032:analysis / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results
D010750:Phosphoproteins; D010940:Plant Proteins; D011494:Protein Kinases

Chlamydomonas reinhardtii
; kinome; mass spectrometry; phosphoproteomics; quantitative label-free proteomics; technical advance