A quantitative mass spectrometry-based approach for identifying protein kinase clients and quantifying kinase activity

Yadong Huang, Norma L. Houston, Alejandro Tovar-Mendez, Severin E. Stevenson, Jan A. Miernyk, Douglas D. Randall, Jay J. Thelen

Research output: Contribution to journalArticlepeer-review

23 Scopus citations

Abstract

The Homo sapiens and Arabidopsis thaliana genomes are believed to encode more than 500 and 1000 protein kinases, respectively. Despite this abundance, few bona fide kinase-client relationships have been described in detail. Here we describe a quantitative mass spectrometry (MS)-based approach for identifying kinase-client proteins. During method development, we used the dedicated kinase pyruvate dehydrogenase kinase (PDK) for the in vitro assays. As kinase substrate, we used synthetic peptide cocktails and, in the process, demonstrated that the assay is both sensitive and specific. The method is also useful for characterizing protein kinase-substrate kinetics once the peptide substrate is detected. Applying a label-free spectral counting method, the activity of PDK was determined using the peptide substrate YHGH292SMSDPGSTYR derived from the pyruvate dehydrogenase E1α subunit sequence. The utility of spectral counting was further validated by studying the negative effect of Met oxidation on peptide phosphorylation. We also measured the activity of the unrelated calcium-dependent protein kinase 3 (CPK3), demonstrating the utility of the method in protein kinase screening applications.

Original languageEnglish (US)
Pages (from-to)69-76
Number of pages8
JournalAnalytical Biochemistry
Volume402
Issue number1
DOIs
StatePublished - Jul 2010

Keywords

  • CPK3
  • Mass spectrometry
  • Method
  • PDK
  • Peptide kinase assay
  • Protein phosphorylation

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