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Anal Chem. 2017 Apr 18;89(8):4550-4558. doi: 10.1021/acs.analchem.6b05002. Epub 2017 Apr 4.

Multiplex Substrate Profiling by Mass Spectrometry for Kinases as a Method for Revealing Quantitative Substrate Motifs.

Author information

1
Department of Pharmaceutical Chemistry, University of California San Francisco , San Francisco, California 94158, United States.
2
Department of Molecular and Cell Biology, University of California Berkeley , Berkeley, California 94720, United States.

Abstract

The more than 500 protein kinases comprising the human kinome catalyze hundreds of thousands of phosphorylation events to regulate a diversity of cellular functions; however, the extended substrate specificity is still unknown for many of these kinases. We report here a method for quantitatively describing kinase substrate specificity using an unbiased peptide library-based approach with direct measurement of phosphorylation by tandem liquid chromatography-tandem mass spectrometry (LC-MS/MS) peptide sequencing (multiplex substrate profiling by mass spectrometry, MSP-MS). This method can be deployed with as low as 10 nM enzyme to determine activity against S/T/Y-containing peptides; additionally, label-free quantitation is used to ascertain catalytic efficiency values for individual peptide substrates in the multiplex assay. Using this approach we developed quantitative motifs for a selection of kinases from each branch of the kinome, with and without known substrates, highlighting the applicability of the method. The sensitivity of this approach is evidenced by its ability to detect phosphorylation events from nanogram quantities of immunoprecipitated material, which allows for wider applicability of this method. To increase the information content of the quantitative kinase motifs, a sublibrary approach was used to expand the testable sequence space within a peptide library of approximately 100 members for CDK1, CDK7, and CDK9. Kinetic analysis of the HIV-1 Tat (transactivator of transcription)-positive transcription elongation factor b (P-TEFb) interaction allowed for localization of the P-TEFb phosphorylation site as well as characterization of the stimulatory effect of Tat on P-TEFb catalytic efficiency.

PMID:
28322550
PMCID:
PMC5500290
DOI:
10.1021/acs.analchem.6b05002
[Indexed for MEDLINE]
Free PMC Article

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