Cysteine residues on proteins fulfill diverse functions in catalysis and protein regulation and are subject to a variety of posttranslational modifications (PTMs) including oxidation, nitrosation and lipidation. Cysteine oxidation, in particular, is known to play important roles in mediating signaling pathways, akin to phosphorylation. For example, the binding of the epidermal growth factor (EGF) to the EGF receptor (EGFR) triggers the production of a burst of hydrogen peroxide that is critical to the observed increase in cell growth proliferation and differentiation that is characteristic of growth factor stimulation. This generatd hydrogen peroxide mediates downstream processes through reversible oxidation of cysteine residues on cellular proteins. Recently, due to the advent of dimedone probes that selectively modify sulfenic acids, a deeper understanding cysteine oxidation during EGF signaling has emerged. However, other oxidative cysteine modifications, in particular, redox-active disulfide formation, are poorly characterized with respect to relevance to EGF signaling. Here, we will develop a chemical-proteomic platform that allows for monitoring diverse cysteine PTMs, including sulfenic-acid and disulfide-bond formation, directly within the context of living cells wth enrichment of targets in subcellular compartments such as the mitochondria and endoplasmic reticulum. This will be achieved with the use of a caged cysteine-reactive probe that can be activated in situ to covalently modify reactive (non-oxidized) cysteines. Appending these caged probes to organelle targeting sequences allows for selective localization in subcellular compartments. A loss in cysteine reactivity upon EGF stimulation is indicative of a variety of modes of cysteine oxidation, including redox-active disulfide formation. In this proposal, we will develop and optimize our chemical-proteomic platform, whilst performing targeted biochemical studies on oxidized cysteines we identify from our global proteomic data. In early studies, we identified a redox-active disulfide bond in a fatty-acid binding protein, FABP5, which is formed upon EGF stimulation of A431 cells. We will investigate the effects of disulfide-bond formation on the lipid-binding properties of FABP5, as well as on the downstream signaling pathways mediated by EGF stimulation. Together, our studies will provide a generalizable platform for profiling cysteine reactivity in living cells with subcellular resolution and identify novel modes f protein regulation through cysteine PTMs, particularly with relevance to the EGF signaling pathway.