Identification of thioredoxin disulfide targets using a quantitative proteomics approach based on isotope-coded affinity tags.
Thioredoxin (Trx) is a ubiquitous protein disulfide reductase involved in a wide range of cellular redox processes. A large number of putative target proteins have been identified using proteomics approaches, but insight into target specificity at the molecular level is lacking since the reactivity of Trx toward individual disulfides has not been quantified. Here, a novel proteomics procedure is described for quantification of Trx-mediated target disulfide reduction based on thiol-specific differential labeling with the iodoacetamide-based isotope-coded affinity tag (ICAT) reagents. Briefly, protein extract of embryos from germinated barley seeds was treated +/- Trx, and thiols released from target protein disulfides were irreversibly blocked with iodoacetamide. The remaining cysteine residues in the Trx-treated and the control (-Trx) samples were then chemically reduced and labeled with the "light" (12C) and "heavy" (13C) ICAT reagent, respectively. The extent of Trx-mediated reduction was thus quantified for individual cysteine residues based on ratios of tryptic peptides labeled with the two ICAT reagents as measured by liquid chromatography coupled with mass spectrometry (LC-MS). A threshold for significant target reduction was set and disulfide targets were identified in 104 among a total of 199 identified ICAT-labeled peptides. Trx-reduced disulfides were found in several previously identified target proteins, for example, peroxiredoxin and cyclophilin, as well as from a wide range of new targets including several ribosomal proteins that point to a link between Trx h and translation. The catalytic cysteine in dehydroascorbate reductase constituted the most extensively reduced target suggesting that Trx h has an important role in the ascorbate-glutathione cycle.
Enzyme and Protein Chemistry, Department of Systems Biology, Søltofts Plads, Building 224, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark.
Per
P
Hägglund
Jakob
J
Bunkenborg
Kenji
K
Maeda
Birte
B
Svensson
D001205:Ascorbic Acid / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods; D004220:Disulfides / Q000737:chemistry*; D005978:Glutathione / Q000378:metabolism; D001467:Hordeum; D006801:Humans; D007554:Isotopes; D013058:Mass Spectrometry / Q000379:methods; D008956:Models, Chemical; D010084:Oxidation-Reduction; D020543:Proteome / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry; D013879:Thioredoxins / Q000737:chemistry*
D004220:Disulfides; D007554:Isotopes; D020543:Proteome; D013879:Thioredoxins; D001710:Biotin; D013880:Thioredoxin-Disulfide Reductase; D005978:Glutathione; D001205:Ascorbic Acid