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A quantitative mass spectrometry-based approach for identifying protein kinase clients and quantifying kinase activity.
- Source :
-
Analytical biochemistry [Anal Biochem] 2010 Jul 01; Vol. 402 (1), pp. 69-76. Date of Electronic Publication: 2010 Mar 25. - Publication Year :
- 2010
-
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 YHGH(292)SMSDPGSTYR derived from the pyruvate dehydrogenase E1alpha 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.<br /> (2010 Elsevier Inc. All rights reserved.)
- Subjects :
- Amino Acid Sequence
Peptides chemistry
Phosphorylation
Pyruvate Dehydrogenase Acetyl-Transferring Kinase
Sensitivity and Specificity
Substrate Specificity
Arabidopsis enzymology
Arabidopsis Proteins metabolism
Calcium-Calmodulin-Dependent Protein Kinases metabolism
Peptides metabolism
Protein Serine-Threonine Kinases metabolism
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods
Subjects
Details
- Language :
- English
- ISSN :
- 1096-0309
- Volume :
- 402
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Analytical biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 20346904
- Full Text :
- https://doi.org/10.1016/j.ab.2010.03.028