Identification of Thioredoxin Disulfide Targets Using a Quantitative Proteomics Approach Based on Isotope-Coded Affinity Tags
Identification of Thioredoxin Disulfide Targets Using a Quantitative Proteomics Approach Based on Isotope-Coded Affinity Tags
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DOI：
10.1021/pr800633y
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发表时间：
2008-12-01
影响因子：
4.4
通讯作者：
Svensson, Birte
中科院分区：
文献类型：
--
作者：
Haegglund, Per;Bunkenborg, Jakob;Svensson, Birte
硫氧还蛋白（Trx）是一种普遍存在的蛋白质二硫键还原酶，参与广泛的细胞氧化还原过程。大量的推定的靶蛋白已被确定使用蛋白质组学方法，但在分子水平上的目标特异性的洞察是缺乏的，因为对个别二硫化物的Trx的反应性尚未被量化。在这里，描述了一种新的蛋白质组学方法，用于定量Trx介导的目标二硫键还原的基础上，巯基特异性差异标记与碘乙酰胺为基础的同位素编码的亲和标签（ICAT）试剂。简言之，将来自发芽的大麦种子的胚的蛋白质提取物用+/- Trx处理，并且用碘乙酰胺不可逆地阻断从靶蛋白二硫化物释放的硫醇。然后将Trx处理和对照（-Trx）样品中剩余的半胱氨酸残基进行化学还原，并分别用“轻”（C-12）和“重”（C-13）ICAT试剂标记。因此，基于用两种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定量，如通过液相色谱-质谱联用（LC-MS）测量的。设定了显著靶标减少的阈值，并在总共199个鉴定的ICAT标记肽中的104个中鉴定了二硫键靶标。Trx还原的二硫化物在几种先前鉴定的靶蛋白中发现，例如过氧化物氧还蛋白和亲环蛋白，以及来自广泛的新靶点，包括几种核糖体蛋白，其指向Trx h和翻译之间的联系。脱氢抗坏血酸还原酶中的催化半胱氨酸构成了最广泛的还原目标，这表明Trx h在抗坏血酸-谷胱甘肽循环中具有重要作用。
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" (C-12) and "heavy" (C-13) 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.