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
liquid chromatography-mass spectrometry (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 phosphorylation state of kinases with unprecedented sensitivity.
We also found that our kinobead/LC-MS protocol can measure changes
in kinase complex composition and show how these changes can indicate
kinase activity. We demonstrate the utility of our approach in specifying
kinase signaling pathways that control the acute steroidogenic response
in Leydig cells; this analysis establishes the first comprehensive
framework for the post-translational control of steroid biosynthesis.