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

Area of Science:

• Chemical Biology
• Proteomics
• Drug Discovery

Background:

• Traditional iodoacetamide (IAM)-based probes for activity-based protein profiling (ABPP) rely on SN2 reactions, limiting cysteine residue labeling.
• Existing probes face challenges in probe size and workflow complexity, necessitating alternative approaches.

Purpose of the Study:

• To introduce acrolein (ACR), a novel α, β-unsaturated carbonyl compound, as a broadly reactive probe for ABPP.
• To develop a user-friendly chemoproteomic platform utilizing ACR for comprehensive nucleophilic site identification.

Main Methods:

• ACR, a Michael acceptor, reacts with nucleophilic amino acids via Michael addition.
• An aldehyde group on ACR facilitates efficient enrichment of labeled residues.
• A chemoproteomic platform was developed and applied across three cell lines.

Main Results:

• Identification of over 28,000 nucleophilic residues across three cell lines.
• ACR detected reactivity changes in nucleophilic residues under native and denatured conditions.
• ACR successfully identified known targets of entacapone.

Conclusions:

• ACR is a versatile and efficient probe for activity-based protein profiling, complementing existing methods.
• The ACR-based platform enables comprehensive analysis of functional nucleophilic sites.
• ACR shows promise for applications in drug discovery and chemical biology research.