Site-specific mapping and quantification of protein S-sulfenylation in cells

Cysteine S-sulfenylation provides redox regulation of protein functions, but the global cellular impact of this transient post-translational modification remains unexplored. We describe a chemoproteomic workflow to map and quantify over 1,000 S-sulfenylation sites on more than 700 proteins in intact cells. Quantitative analysis of human cells stimulated with hydrogen peroxide or epidermal growth factor measured hundreds of site selective redox changes. Different cysteines in the same proteins displayed dramatic differences in susceptibility to S-sulfenylation. Newly discovered S-sulfenylations provided mechanistic support for proposed cysteine redox reactions and suggested novel redox mechanisms, including S-sulfenyl-mediated redox regulation of the transcription factor HIF1A by SIRT6. S-sulfenylation is favored at solvent-exposed protein surfaces and is associated with sequence motifs that are distinct from those for other thiol modifications. S-sulfenylations affect regulators of phosphorylation, acetylation and ubiquitylation, which suggests regulatory crosstalk between redox control and signaling pathways.