Beyond the known cuts: trypsin specificity in native proteins

Trypsin is a serine protease that plays a pivotal role in protein digestion, being extensively used in various proteomics workflows due to its cleavage profile and specificity. Understanding trypsin’s enzymatic behavior is thus critical. We have employed Above-Filter Digestion Proteomics (AFDIP) to investigate trypsin’s cleavage preferences in HeLa cell lysates, preserving the proteins’ native state. We quantified over 18,000 unique peptides and correlated their emergence rates with cleavage window sequence motifs and physicochemical properties. Contrary to previous studies performed on denatured proteomes, we found that in native proteins cleavages at lysine residues were more abundant and faster than at arginine residues, and that physicochemical properties of the peptides affected their emergence times. These findings may need to be taken into account when interpreting the results of limited proteolysis experiments as well as when designing a food protein with extremely fast digestion times.