FIG. 2.

Protein site-specific relative quantification of sulfhydryls using the OxICAT technique. In general, the ICAT reagent consists of a thiol-reactive probe (typically iodoacetamide—though conceptually any of the thiol probes in Table 1 could be utilized), a light or heavy labeled linker, and a biotin group for affinity purification (33, 91). OxICAT may be employed for (a) within-sample determination of site-specific sulfhydryl status relative to a reversible cysteine modification or, in modified form, for (b) relative quantification of the degree to which specific cysteine residues are modified after oxidative treatment. Only one sulfhydryl group per protein is shown for the sake of simplicity; in practice, additional sulfhydryl groups would generate tryptic fragments with distinct masses, and each protein sulfhydryl group would be analyzed independently by the mass spectrometer. Information about modification of multiple cysteines within a single protein molecule can only be derived from analysis of the intact protein in a separate experiment. OxICAT, oxidation-based isotope-coded affinity tag.