Hägglund Per, Bunkenborg Jakob, Maeda Kenji, Svensson Birte
Enzyme and Protein Chemistry, Department of Systems Biology, Søltofts Plads, Building 224, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark.
J Proteome Res. 2008 Dec;7(12):5270-6. doi: 10.1021/pr800633y.
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.
硫氧还蛋白（Trx）是一种广泛存在的蛋白质二硫键还原酶，参与多种细胞氧化还原过程。使用蛋白质组学方法已鉴定出大量假定的靶蛋白，但由于尚未对Trx与单个二硫键的反应性进行量化，因此缺乏对分子水平靶标特异性的深入了解。在此，描述了一种基于用基于碘乙酰胺的同位素编码亲和标签（ICAT）试剂进行硫醇特异性差异标记来定量Trx介导的靶标二硫键还原的新型蛋白质组学方法。简要地说，将发芽大麦种子胚的蛋白质提取物用+/-Trx处理，靶蛋白二硫键释放的硫醇用碘乙酰胺不可逆地封闭。然后将Trx处理的样品和对照（-Trx）样品中剩余的半胱氨酸残基进行化学还原，分别用“轻”（12C）和“重”（13C）ICAT试剂标记。因此，基于通过液相色谱-质谱联用（LC-MS）测量的用两种ICAT试剂标记的胰蛋白酶肽的比率，对单个半胱氨酸残基的Trx介导的还原程度进行了定量。设定了显著靶标还原的阈值，在总共199个鉴定出的ICAT标记肽中，有104个鉴定出了二硫键靶标。在几个先前鉴定的靶蛋白中发现了Trx还原的二硫键，例如过氧化物酶和亲环蛋白，以及来自广泛新靶标的二硫键，包括几种核糖体蛋白，这表明Trx h与翻译之间存在联系。脱氢抗坏血酸还原酶中的催化半胱氨酸是还原程度最高的靶标，表明Trx h在抗坏血酸-谷胱甘肽循环中起重要作用。
