Protease Activity Profiling via Programmable Phage Display of Comprehensive Proteome-Scale Peptide Libraries.

Endopeptidases catalyze the internal cleavage of proteins, playing pivotal roles in protein turnover, substrate maturation, and the activation of signaling cascades. A broad range of biological functions in health and disease are controlled by proteases, yet assays to characterize their activities at a proteomic scale do not exist. To address this unmet need, we developed Sensing EndoPeptidase Activity via Release and recapture using flAnking Tag Epitopes (SEPARATE), which uses a monovalent phage display of the human proteome at a 90-aa peptide resolution. We demonstrate that SEPARATE is compatible with several human proteases from distinct catalytic classes, including caspase-1, ADAM17, and thrombin. Both well-characterized and newly identified substrates of these enzymes were detected in the assay. SEPARATE was used to discover a non-canonical caspase-1 substrate, the E3 ubiquitin ligase HUWE1, a key mediator of apoptotic cell death. SEPARATE enables efficient, unbiased assessment of endopeptidase activity by using a phage-displayed proteome. A record of this paper's Transparent Peer Review process is included in the Supplemental Information.