Journal
JOURNAL OF PROTEOME RESEARCH
Volume 16, Issue 3, Pages 1216-1227Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.jproteome.6b00817
Keywords
kinobeads; SILAC; kinase inhibitor; protein kinase D; target identification; proteomics
Categories
Funding
- National Institutes of Health [RO1GM086858, R01GM083926, R01AI111341, R21EB018384, R21CA177402]
- DFG (German Research Foundation) [GO 2358/1-1]
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ATP-competitive protein kinase inhibitors are important research tools and therapeutic agents. Because there are >500 Inhibitors human kinases that contain highly conserved active sites, the development of selective inhibitors is extremely challenging. Methods to rapidly and efficiently profile kinase inhibitor targets in cell lysates are urgently needed to discover selective compounds and to elucidate the mechanisms of action for polypharmacological inhibitors. Here, we describe a protocol for microgram-scale chemoproteomic profiling of ATP-competitive kinase inhibitors using kinobeads. We employed a gel-free in situ digestion protocol coupled to nanoflow liquid chromatography-mass spectrometry to profile similar to 200 kinases in single analytical runs using as little as 5 mu L of kinobeads and 300 mu g of protein. With our kinobead reagents, we obtained broad coverage of the kinome, monitoring the relative expression levels of 312 kinases in a diverse panel of 11 cancer cell lines. Further, we profiled a set of pyrrolopyrimidine- and pyrazolopyrimidine-based kinase inhibitors in competition-binding experiments with label-free quantification, leading to the discovery of a novel selective and potent inhibitor of protein kinase D (PKD) 1, 2, and 3. Our protocol is useful for rapid and sensitive profiling of kinase expression levels and ATP-competitive kinase inhibitor selectivity in native proteomes.
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