Cysteine is the most intrinsically nucleophilic residue in proteins and serves as a mediator against increasing reactive oxygen species (ROS) via reversible thiol oxidation. Despite the importance of cysteine oxidation in understanding biological stress response, cysteine sites most reactive toward ROS remain largely unknown and are a major analytical challenge. Herein, a chemical proteomic method to quantify site-specific cysteine reactivity using a maleimide-activated, thiol-reactive probe (-propargylmaleimide, NPM) is described. Implementation of a gel-based approach via conjugation of rhodamine-azide to NPM-labeled cysteine residues by copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry allowed simple and highly sensitive fluorescence profiling. Relative quantification of >1500 unique cysteine sites from greater than 800 proteins was achieved by conjugating dialkoxydiphenylsilane (DADPS) biotin-azide by the CuAAC reaction and subsequently performing biotin-streptavidin affinity purification and mass-spectrometry-based proteomics. Taken together, this work defines a novel role for the NPM probe in chemical proteomics and presents a robust method for determination of cysteine reactivity during oxidative stress response.
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