Acrolein as an Alternative Broadly Reactive Activity-Based Probe for Activity-Based Protein Profiling of Cysteinome

Iodoacetamide (IAM)-based probes have established the activity-based protein profiling (ABPP) paradigm; however, their reliance on the SN2 mechanism limits the labeling of all cysteine residues, highlighting the need for complementary probes based on alternative reactions. Additionally, probe design must balance between minimizing probe size and simplifying workflow complexity. As a solution to the above issues, we propose acrolein (ACR), a simple α, β-unsaturated carbonyl compound, as an alternative broadly reactive ABPP probe. ACR possesses sp²-hybridized electrophilic groups that react with nucleophilic amino acids via Michael addition, and its aldehyde group facilitates efficient enrichment. We developed a user-friendly chemoproteomic platform for ACR, enabling the identification of over 28,000 nucleophilic residues across three cell lines. ACR demonstrated its ability to detect reactivity changes in nucleophilic residues under native and denatured conditions and to identify known targets of entacapone. Overall, ACR represents a versatile and efficient tool for studying functional nucleophilic sites, with potential applications in drug discovery.