The Proteome‐Wide Potential for Reversible Covalency at Cysteine

Abstract Reversible covalency, achieved with, for instance, highly electron‐deficient olefins, offers a compelling strategy to design chemical probes and drugs that benefit from the sustained target engagement afforded by irreversible compounds, while avoiding permanent protein modification. Reversible covalency has mainly been evaluated for cysteine residues in individual kinases and the broader potential for this strategy to engage cysteines across the proteome remains unexplored. Herein, we describe a mass‐spectrometry‐based platform that integrates gel filtration with activity‐based protein profiling to assess cysteine residues across the human proteome for both irreversible and reversible interactions with small‐molecule electrophiles. Using this method, we identify numerous cysteine residues from diverse protein classes that are reversibly engaged by cyanoacrylamide fragment electrophiles, revealing the broad potential for reversible covalency as a strategy for chemical‐probe discovery.