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

Gabriel D. Román-Meléndez, Thiagarajan Venkataraman, Daniel R. Monaco, H. Benjamin Larman

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish (US)
Pages (from-to)375-381.e4
JournalCell Systems
Issue number4
StatePublished - Oct 21 2020


  • next generation DNA sequencing
  • programmable phage display
  • protease activity profiling
  • proteomic analysis

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Histology
  • Cell Biology


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