A quantitative mass spectrometry-based approach for identifying protein kinase clients and quantifying kinase activity.
Anal Biochem
; 402(1): 69-76, 2010 Jul 01.
Article
em En
| MEDLINE
| ID: mdl-20346904
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Peptídeos
/
Proteínas Serina-Treonina Quinases
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Arabidopsis
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Proteínas Quinases Dependentes de Cálcio-Calmodulina
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Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
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Proteínas de Arabidopsis
Tipo de estudo:
Diagnostic_studies
/
Evaluation_studies
Idioma:
En
Revista:
Anal Biochem
Ano de publicação:
2010
Tipo de documento:
Article
País de afiliação:
Estados Unidos