Maleimide-Based Chemical Proteomics for Quantitative Analysis of Cysteine Reactivity

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
(N-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.