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.