biostudies-literatureYang JNICHD NIH HHSNCI NIH HHSNIGMS NIH HHS1022-37https://www.ebi.ac.uk/biostudies/studies/S-EPMC4608369biostudies-literatureUnknown10(7)Protein S-sulfenylation is the reversible oxidative modification of cysteine thiol groups to form cysteine S-sulfenic acids. Mapping the specific sites of protein S-sulfenylation onto complex proteomes is crucial to understanding the molecular mechanisms controlling redox signaling and regulation. This protocol describes global, in situ, site-specific analysis of protein S-sulfenylation using sulfenic acid-specific chemical probes and mass spectrometry (MS)-based proteomics. The major steps in this protocol are as follows: (i) optimization of conditions for selective labeling of cysteine S-sulfenic acids in intact cells with the commercially available dimedone-based probe, DYn-2; (ii) tagging the modified cysteines with a functionalized biotin reagent containing a cleavable linker via Cu(INature protocolsGlobal, in situ, site-specific analysis of protein S-sulfenylation.PMC4608369R01 GM102187R01 CA174864R01GM102187R01 HD064727U24CA159988U24 CA159988Yang JGupta VPorter NACarroll KSLiebler DCTallman KAfalseGlobal, in situ, site-specific analysis of protein S-sulfenylation.Protein S-sulfenylation is the reversible oxidative modification of cysteine thiol groups to form cysteine S-sulfenic acids. Mapping the specific sites of protein S-sulfenylation onto complex proteomes is crucial to understanding the molecular mechanisms controlling redox signaling and regulation. This protocol describes global, in situ, site-specific analysis of protein S-sulfenylation using sulfenic acid-specific chemical probes and mass spectrometry (MS)-based proteomics. The major steps in this protocol are as follows: (i) optimization of conditions for selective labeling of cysteine S-sulfenic acids in intact cells with the commercially available dimedone-based probe, DYn-2; (ii) tagging the modified cysteines with a functionalized biotin reagent containing a cleavable linker via Cu(I2015-01-01T00:00:00Z2015 Jul2025-04-04T13:25:38.009Z2019-03-27T02:00:11ZS-EPMC46083692608640510.1038/nprot.2015.062