Postproline Cleaving Enzymes also Show Specificity\nto Reduced Cysteine

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