Posttranslational modifications (PTMs) are important in the regulation of protein function, trafficking, localization, and marking for degradation. Here, we describe development of peptide activity-based probes for the discovery of proteins that recognize novel acyl-based PTMs on lysine residues in the proteome. The probes contain surrogates of epsilon-<i>N</i>-acyllysine by introduction of either hydrazide or thioamide functionalities to circumvent hydrolysis of the modification during the experiments. In addition to the modified PTMs, the developed chemotypes were analyzed with respect to effect of peptide sequence. The photo cross-linking conditions and subsequent functionalization of the covalent adducts were systematically optimized by applying fluorophore labeling and gel electrophoresis (in-gel fluorescence measurements). Finally, selected probes, containing the epsilon-<i>N</i>-glutaryllysine and epsilon<i>-N</i>-myristoyllysine analogues, were successfully applied to enrichment of native, endogenous proteins from cell lysate, recapitulating the expected interactions of SIRT5 and SIRT2, respectively. Interestingly, the latter mentioned was able to pull down two different splice variants of SIRT2, which has not been achieved with a covalent probe before. Based on this elaborate proof-of-concept study, we expect that the technology will have broad future applications for pairing of novel PTMs with the proteins that target them in the cell.
Mechanism of inhibition of adenylate cyclase by phospholipase C-catalyzed hydrolysis of phosphatidylcholine. Involvement of a pertussis toxin-sensitive G protein and protein kinase C.