Post-proline cleaving enzymes also show specificity to reduced cysteine.

In proteomics, post-proline cleaving enzymes (PPCEs) like Aspergillus niger prolyl endopeptidase and neprosin complement proteolytic tools because proline is a stop site for many proteases. However, our systematic analysis of cleavage preferences showed that both PPCEs also display specificity to reduced cysteine. Post-cysteine cleavage was blocked by Cys alkylation, explaining why this activity has remained undetected. Our findings redefine their applicability and how we study and interpret their cleavage mechanism.