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J Am Chem Soc. 2005 Apr 20;127(15):5288-9.

Bio-orthogonal affinity purification of direct kinase substrates.

Author information

1
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143, USA.

Abstract

Protein phosphorylation is a major mechanism of post-translational protein modification used to control cellular signaling. A challenge in phosphoproteomics is to identify the direct substrates of each protein kinase. Herein, we describe a chemical strategy for delivery of a bio-orthogonal affinity tag to the substrates of an individual protein kinase. The kinase of interest is engineered to transfer a phosphorothioate moiety to phosphoacceptor hydroxyl groups on direct substrates. In a second nonenzymatic step, the introduced phosphorothioate is alkylated with p-nitrobenzylmesylate (PNBM). Antibodies directed against the alkylated phosphorothioate epitope recognize these labeled substrates, but not alkylation products of other cellular nucleophiles. This strategy is demonstrated with Cdk1/cyclinB substrates using ELISA, western blotting, and immunoprecipitation in the context of whole cell lysates.

PMID:
15826144
PMCID:
PMC2943827
DOI:
10.1021/ja050727t
[Indexed for MEDLINE]
Free PMC Article

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