KiC assay: a quantitative mass spectrometry-based approach for kinase client screening and activity analysis [corrected].
Protein phosphorylation is one of the most important posttranslational modifications (PTMs) involved in the transduction of cellular signals. The number of kinases in eukaryotic genomes ranges from several hundred to over one thousand. And with rapidly evolving mass spectrometry (MS)-based approaches, thousands to tens of thousands of phosphorylation sites (phosphosites) have been reported from various eukaryotic organisms, from man to plants. In this relative context, few bona fide kinase-client relationships have been identified to date. To merge the gap between these phosphosites and the cognate kinases that beget these events, comparable large-scale methodologies are required. We describe in detail a MS-based method for identifying kinase-client interactions and quantifying kinase activity. We term this novel Kinase-Client assay, the KiC assay. The KiC assay relies upon the fact that substrate specificities of many kinases are largely determined by primary amino acid sequence or phosphorylation motifs, which consist of key amino acids surrounding the phosphorylation sites. The workflow for detecting kinase-substrate interactions includes four major steps: (1) preparation of purified kinases and synthetic peptide library, (2) in vitro kinase peptide library assay, (3) liquid chromatography (LC)-tandem MS (MS/MS) analysis, and (4) data processing and interpretation. Kinase activity is quantified with the KiC assay by monitoring spectral counts of phosphorylated and unphosphorylated peptides as the readout from LC-tandem mass spectrometry. The KiC assay can be applied as a discovery assay to screen kinases against a synthetic peptide library to find kinase-client relationships or as a targeted assay to characterize kinase kinetics.
Interdisciplinary Plant Group, Department of Biochemistry, University of Missouri-Columbia, Columbia, MO, USA.
Yadong
Y
Huang
Jay J
JJ
Thelen
D020816:Amino Acid Motifs; D000818:Animals; D002850:Chromatography, Gel; D057075:Enzyme Assays; D006801:Humans; D013058:Mass Spectrometry; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D019151:Peptide Library; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification; D011494:Protein Kinases / Q000737:chemistry*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
D010446:Peptide Fragments; D019151:Peptide Library; D010750:Phosphoproteins; D011994:Recombinant Proteins; D011494:Protein Kinases