a smaller residue (Fig. 2). ATP analogs (*ATP) possessing a side group “bump” designed to fit into the hole can be used to identify substrates specific to the analog-sensitive (as) kinase mutant. Specificity for the mutant kinase is achieved because wild-type kinases cannot efficiently use the ATP analogs as phosphate donors. To facilitate the identification and purification of substrates, *ATP-γS can be used instead of *ATP to label kinase substrates. This results in the transfer of a thiophosphate group onto the target substrate residue instead of a phosphate group. Thiophosphorylated residues can then be alkylated by para-nitrobenzylmesylate to create an epitope that is recognized by specific antibodies. The tagged substrates can be detected by western blot analysis and isolated by immunoprecipitation or immunoaffinity purification for detection by mass spectrometry. The approach has been successfully used to identify direct substrates of kinases.283 In our view, as-kinases could be extremely useful in isolating distinct kinase substrates phosphorylated in response to drugs of abuse. The hope is that some of these substrates will prove to be targets for the development of useful agents to treat substance abuse in the future.
