Yoshikatsu Kanai

Proteases play crucial roles in numerous biological processes through specific protein cleavage, and their dysregulation has been implicated in various diseases. To better understand protease specificity, we developed a lauroylation-assisted proteomic identification of protease cleavage sites (PICS) workflow that labels and enriches targeted protease-generated neo-N-termini using economical reagents and standard laboratory equipment. The lauroylation enables both discrimination of the neo-N-termini in LC-MS/MS and efficient enrichment on a C18 StageTip by exploiting its hydrophobicity. Among tested acylations, we found lauroylation to be optimal for PICS and improved enrichment and fractionation conditions. We demonstrated that this method can profile specificities of multiple proteases with high sensitivity. Furthermore, we extended this concept to N-terminomics to examine proteolysis at the protein level. Protein N-terminal dimethylation is used for labeling, and tryptic internal peptides are lauroylated for removal. This approach identified over 1500 cleavages induced by etoposide, including 912 Asp-cleaved sites consistent with caspase-3 motifs and sensitive to inhibition by Z-DEVD-FMK. Additionally, 2286 protein N-termini were identified in untreated cells, including 1794 non-ORF N-termini with 665 previously annotated processing sites. These results demonstrate that our workflow provides a simple, economical, and widely applicable method for characterizing protease cleavage at both peptide and protein levels.