TY - JOUR T1 - ABPP-HT - high-throughput activity-based profiling of deubiquitylating enzyme inhibitors in a cellular context JF - bioRxiv DO - 10.1101/2020.12.10.419499 SP - 2020.12.10.419499 AU - Hannah Jones AU - Raphael Heilig AU - Roman Fischer AU - Benedikt M Kessler AU - Adan Pinto-Fernandez Y1 - 2020/01/01 UR - http://biorxiv.org/content/early/2020/12/10/2020.12.10.419499.abstract N2 - The potency and selectivity of a small molecule inhibitor are key parameters to assess during the early stages of drug discovery. In particular, it is very informative for characterizing compounds in a relevant cellular context in order to reveal potential off-target effects and drug efficacy. Activity-based probes (ABPs) are valuable tools for that purpose, however, obtaining cellular target engagement data in a high-throughput format has been particularly challenging. Here, we describe a new methodology named ABPP-HT (high-throughput-compatible activity-based protein profiling), implementing a semi-automated proteomic sample preparation workflow that increases the throughput capabilities of the classical ABPP workflow approximately ten times while preserving its enzyme profiling characteristics. Using a panel of deubiquitylating enzyme (DUB) inhibitors, we demonstrate the feasibility of ABPP-HT to provide compound selectivity profiles of endogenous DUBs in a cellular context at a fraction of time as compared to previous methodologies.Competing Interest StatementThe authors have declared no competing interest. ER -