Major intracellular disulfide formation is prevented in the cytosol by potent reducing systems. However, protein thiols can be oxidized as a consequence of redox-mediated physiological reactions or due to the unwanted toxicity of reactive oxygen species. In addition, the reactivity of cysteine residues towards peroxides is used by H(2)O(2) sensors in signal transduction pathways in a gain-of-function process to induce transcriptional antioxidant responses. Thus, the Schizosaccharomyces pombe peroxiredoxin Tpx1 and the transcription factor Pap1 are sensors of H(2)O(2) meant to promote cell survival. In an attempt to compare signaling events versus global thiol oxidation, we have optimized thiol-labeling approaches to characterize the disulfide proteome of fission yeast in response to added H(2)O(2). We propose a method based on (i) freezing the redox state of thiols with strong acids prior to cell lysis; (ii) blocking thiol groups with iodoacetamide, and reversibly oxidized thiols with heavy and light isotope-coded affinity tags (ICAT) reagents; and (iii) quantifying individual relative protein concentrations with stable-isotope dimethyl labeling. We have applied this highly sensitive strategy to provide a map of H(2)O(2)-dependent oxidized thiols in fission yeast, and found Tpx1 and Pap1 as some of the major targets.