Kinobead/LC-MS Phosphokinome Profiling Enables Rapid Analyses of Kinase-Dependent Cell Signaling Networks.

Kinase-catalyzed protein phosphorylation is fundamental to eukaryotic signal transduction, regulating most cellular processes. Kinases are frequently dysregulated in cancer, inflammation and degenerative diseases, and because they can be inhibited with small molecules, they became important drug targets. Accordingly, analytical approaches that determine kinase activation states are critically important to understand kinase-dependent signal transduction, and to identify novel drug targets and predictive biomarkers. Multiplexed inhibitor beads (MIBs or kinobeads) efficiently enrich kinases from cell lysates for LC-MS analysis. When combined with phosphopeptide enrichment, kinobead/LC-MS can also quantify the phosphorylation state of kinases, which determines their activation state. However, an efficient kinobead/LC-MS kinase phospho-profiling protocol that allows routine analyses of cell lines and tissues has not yet been developed. Here, we present a facile workflow that quantifies the global phosphorylati...