Thiol-ene enabled detection of thiophosphorylated kinase substrates.

Protein phosphorylation is a ubiquitous posttranslational modification that regulates cell signaling in both prokaryotes and eukaryotes. Although the study of phosphorylation has made great progress, several major hurdles remain, including the difficulty of the assignment of endogenous substrates to a discrete kinase and of global phosphoproteomics investigations. We have developed a novel chemical strategy for detecting phosphorylated proteins. This method utilizes adenosine 5'-O-(3-thiotriphosphate) (ATPγS), which results in the transfer of a thiophosphate moiety by a kinase to its substrate(s). This group can subsequently be employed as a nucleophilic handle to promote protein detection. To selectively label thiophosphorylated proteins, cellular thiols (e.g., cysteine-containing proteins) must first be blocked. Most common cysteine-capping strategies rely upon the nucleophilicity of the sulfur group and would therefore also modify the thiophosphate moiety. We hypothesized that the radical-mediated thiol-ene reaction, however, would be selective for cysteine over thiophosphorylated amino acids due to the differences in the electronics and pKa values between these groups. Here, we report rapid and specific tagging of thiophosphorylated proteins in vitro following chemoselective thiol capping using the thiol-ene reaction.