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. © 2014 Springer Science+Business Media, LLC.
CITATION STYLE
Hägglund, P., Bunkenborg, J., Maeda, K., Finnie, C., & Svensson, B. (2014). Identification of thioredoxin target disulfides using isotope-coded affinity tags. Methods in Molecular Biology, 1072, 677–685. https://doi.org/10.1007/978-1-62703-631-3_47
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