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.

内肽酶催化蛋白质的内部裂解，在蛋白质周转、底物成熟和信号级联激活中起关键作用。蛋白酶在健康和疾病中的广泛生物学功能受蛋白酶控制，但尚不存在在蛋白质组学尺度上表征其活性的分析方法。为了解决这一未满足的需求，我们开发了通过释放和重新捕获使用侧翼标签表位 (SEPARATE) 来感知内肽酶活性，该表位使用人类蛋白质组的单价噬菌体展示，分辨率为 90-aa。我们证明 SEPARATE 与来自不同催化类别的几种人类蛋白酶兼容，包括 caspase-1、ADAM17 和凝血酶。在测定中检测到这些酶的充分表征和新鉴定的底物。SEPARATE 用于发现非经典 caspase-1 底物，即 E3 泛素连接酶 HUWE1，它是凋亡细胞死亡的关键介质。SEPARATE 通过使用噬菌体展示的蛋白质组，能够对内肽酶活性进行有效、无偏见的评估。本文的透明同行评审过程的记录包含在补充信息中。