Fig. 1.

Determining the oxidation state of protein thiol using ICAT technology (OxICAT). A hypothetical cellular protein, which exists in either the reduced (Upper) or disulfide-linked form (Lower), is incubated under denaturing conditions to expose all of its cysteine side chains. In step I, isotopically light ¹²C- ICAT reagent (green ovals) is added, which irreversibly modifies all reduced cysteines in the protein. In step II, all oxidized cysteines are reduced with Tris(2-carboxyethyl)phosphine (TCEP) and subsequently modified with isotopically heavy ¹³C- ICAT reagent (red ovals). In step III, the protein mixture is digested and ICAT-labeled peptides are purified by using the biotin-affinity tag. In step IV, quantitative MS of the protein mixture reveals the extent of thiol modification in any given peptide. Peptide sequence and identity of the modified cysteine are determined by MS/MS.