Identification of Thioredoxin Target Disulfides Using Isotope-Coded Affinity Tags

Thioredoxins (Trx) are small redox proteins that reduce disulfide bonds in various target proteins and maintain cellular thiol redox control. Here, a thiol-specific labeling and affinity enrichment approach for identification and relative quantification of Trx target disulfides in complex protein extracts is described. The procedure utilizes the isotope-coded affinity tag (ICAT) reagents containing a thiol reactive iodoacetamide group and a biotin affinity tag to target peptides containing reduced cysteine residues. The identification of substrates for Trx and the extent of target disulfide reduction is determined by LC-MS/MS-based quantification of tryptic peptides labeled with “light” (¹² C) and “heavy” (¹³ C) ICAT reagents. The methodology can be adapted to monitor the effect of different reductants or oxidants on the redox status of thiol/disulfide proteomes in biological systems.