Identification of thioredoxin target disulfides using isotope-coded affinity tags

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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" (12C) and "heavy" (13C) 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.

Original languageEnglish
Title of host publicationPlant Proteomics : Methods and Protocols
Number of pages9
PublisherHumana Press
Publication date2014
Pages677-685
ISBN (Print)9781627036306
DOIs
Publication statusPublished - 2014
SeriesMethods in Molecular Biology
Volume1072
ISSN1064-3745

    Research areas

  • Cysteine, Disulfide, Iodoacetamide, Isotope-coded affinity tag, Redox proteomics, Thiol, Thioredoxin

ID: 240157984