Measuring reactive oxygen, nitrogen and sulfur species in cells is established technology, but turn-on fluorescence tools for detecting the products of their reaction with protein cysteines remain essentially unknown. Toward this goal, here we describe fluorogenic probes for sulfenic acid, a redox modification of protein cysteines inextricably linked to signaling and oxidative stress. The probes, called CysOx1 and CysOx2, are reaction-based, exhibit excellent cell permeability, rapid

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... and high selectivity with minimal cytotoxicity. We applied CysOx2 in a cell-based 96-well plate assay to determine whether kinase inhibitors modulate protein S-sulfenylation as well as O-phosphorylation. Analysis of these data revealed an unexpected positive association of S-sulfenylation and inhibition of select kinases within the TK, AGC, and CMGC families including GSK3, a multitasking Ser/Thr kinase and emerging therapeutic target for neurodegenerative and mood disorders. Chemoproteomic mapping of sulfenic acid-modified cysteines in GSK3 inhibitor-treated cells shows that sites of S-oxidation localize to regulatory cysteines within key components of antioxidant defense systems. Our studies with CysOx probes offer up new insights into kinase-inhibitor dependent modulation of sulfenylome dynamics and should accelerate future efforts in the modern era of translational redox medicine.