title: Identification of thioredoxin target disulfides using isotope-coded affinity tags.
abstract: 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.
authors.affiliation: Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Kongens Lyngby, Denmark.
authors.forename: Per
authors.initials: P
authors.lastname: Hägglund
authors.forename: Jakob
authors.initials: J
authors.lastname: Bunkenborg
authors.forename: Kenji
authors.initials: K
authors.lastname: Maeda
authors.forename: Christine
authors.initials: C
authors.lastname: Finnie
authors.forename: Birte
authors.initials: B
authors.lastname: Svensson
mesh_terms: D001360:Avidin / Q000378:metabolism; D002412:Cations; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D004220:Disulfides / Q000378:metabolism*; D007202:Indicators and Reagents; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry; D010455:Peptides / Q000302:isolation & purification; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification; D013879:Thioredoxins / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
chemical_list: D002412:Cations; D004220:Disulfides; D007202:Indicators and Reagents; D010455:Peptides; D010940:Plant Proteins; D001360:Avidin; D013879:Thioredoxins; D014357:Trypsin