Fig. 2.

Quantification of thiol-oxidation using OxICAT. First, proteins of interest are solubilized and denatured, which allows the reaction of the isotopically light ¹²C-ICAT reagent (LICAT, green) with all free cysteines. Second, reversibly oxidized cysteines are reduced using Tris(2-carboxyethyl)phosphine and labeled with the isotopically heavy ¹³C-ICAT (HICAT, red). Then, the protein mixture is digested by trypsin and the ICAT-tagged peptides are purified using the biotin tag. Finally, the peptide mixture is analyzed using mass spectrometry. Partially thiol-oxidized proteins are labeled with both the LICAT and the HICAT. Fully oxidized thiol-oxidized proteins are labeled with HICAT only. The relative oxidation of a cysteine is reflected by the proportion of its respective LICAT- and HICAT-labeling.