Hydrogen peroxide (H(2)O(2)) sensing and signaling involves the reversible oxidation of particular thiols on particular proteins to modulate protein function in a dynamic manner. H(2)O(2) can be generated from various intracellular sources, but their identities and relative contributions are often unknown. To identify endogenous “hotspots” of H(2)O(2) generation on the scale of individual proteins and protein complexes, we generated a yeast library in which the H(2)O(2) sensor HyPer7 was …