Development of a maleimide-based chemical proteomic method for quantitative analysis of cysteine reactivity

Cysteine is the most intrinsically nucleophilic residue in proteins and serves as a sentinel against increasing reactive oxygen species (ROS) via reversible thiol oxidation. Despite the importance of Cys oxidation in understanding biological stress response, it remains largely unknown and a major analytical challenge to determine which protein Cys-sites are the most reactive toward ROS. Herein, we describe initial coverage and sensitivity of a chemical proteomic method to quantify site-specific Cys reactivity using a maleimide-activated, thiol-specific probe (N-propargylmaleimide, NPM).