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Chemical Immunological Identification of Direct Kinase Substrates

Protein phosphorylation is a major mechanism of post‐translational protein modification used to direct cellular signalling. A challenge in phosphoproteomics is to identify the direct substrates of each protein kinase. Here 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 protein substrates. In a second non‐enzymatic step, the introduced phosphorothioate is alkylated with p‐nitrobenzylmesylate (PNBM). Antibodies directed against the PNBM modified phosphorothioate epitope recognize these labelled substrates, but not alkylation products of other cellular nucleophiles. Further, the elicited monoclonal and polyclonal antibodies selectively bind the substrates of several divergent kinases, irrespective of the primary amino acid sequence surrounding the site of phosphorylation. Immunoprecipitation and subsequent mass spectrometric identification of the selectively labelled proteins is likely to provide a general route for mapping kinase substrates.