ChemRxiv
These are preliminary reports that have not been peer-reviewed. They should not be regarded as conclusive, guide clinical practice/health-related behavior, or be reported in news media as established information. For more information, please see our FAQs.
1/1
2 files

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

preprint
submitted on 26.10.2019 and posted on 30.10.2019 by Martin Golkowski, Venkata Narayana Vidadala, Ho-Tak Lau, Anna Shoemaker, Masami Shimizu-Albergine, Joseph A. Beavo, Dustin J. Maly, Shao-En Ong

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 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.

History

Email Address of Submitting Author

golkom@uw.edu

Institution

University of Washington

Country

United States

ORCID For Submitting Author

0000-0002-0996-1655

Declaration of Conflict of Interest

The authors declare that there are no competing financial interests.

Exports

Logo branding

Exports