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We developed a capillary LC/MS/MS-based approach to monitor intracellular kinase activities. Selected reaction monitoring (SRM) mode was employed to quantitate a kinase substrate-digested peptide phosphorylated by kinase or kinase-digested peptides containing phosphosites which regulate the kinase activities. Ten kinases in EGFR-MAPK signaling pathway were targeted for the SRM assay and the experimental conditions such as the selection of target phosphopeptides, SRM transitions, LC parameters and sample pre-treatment steps were optimized. The validation study on accuracy, precision, linearity, limit of detection and limit of quantitation was carried out to confirm the capability for measuring the kinase activities through the phosphopeptide quantities in the biological samples. Finally, we applied this SRM assay to kinase activation dynamics induced by pervanadate, a tyrosine phosphatase inhibitor, in HeLa cells. As a result, it was found that 6 kinases out of 10 were activated, which were consistent with those by conventional Western blotting using phosphosite-specific antibodies. Since this SRM assay can be extended to kinome-wide analysis, it will be useful to unveil the entire signaling network in cells.

Selected Reaction Monitoring of Kinase Activity-Targeted Phosphopeptides