Postproline Cleaving Enzymes also Show Specificity to Reduced Cysteine

In proteomics, postproline cleaving enzymes (PPCEs), such as <i>Aspergillus niger</i> prolyl endopeptidase (<i>An</i>PEP) and neprosin, complement proteolytic tools because proline is a stop site for many proteases. But while aiming at using <i>An</i>PEP in online proteolysis, we found that this enzyme also displayed specificity to reduced cysteine. By LC-MS/MS, we systematically analyzed <i>An</i>PEP sources and conditions that could affect this cleavage preference. Postcysteine cleavage was blocked by cysteine modifications, including disulfide bond formation, oxidation, and alkylation. The last modification explains why this activity has remained undetected so far. In the same experimental paradigm, neprosin mimicked this cleavage specificity. Based on these findings, PPCEs cleavage preferences should be redefined from post-Pro/Ala to post-Pro/Ala/Cys. Moreover, this evidence demands reconsidering PPCEs applications, whether cleaving Cys-rich proteins or assessing Cys status in proteins, and calls for revisiting the proposed enzymatic mechanism of these proteases.