Bio-orthogonal Affinity Purification of Direct Kinase Substrates
- 24 March 2005
- journal article
- research article
- Published by American Chemical Society (ACS) in Journal of the American Chemical Society
- Vol. 127 (15), 5288-5289
- https://doi.org/10.1021/ja050727t
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.This publication has 23 references indexed in Scilit:
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