Hägglund Per, Bunkenborg Jakob, Maeda Kenji, Finnie Christine, Svensson Birte
Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Kongens Lyngby, Denmark.
Methods Mol Biol. 2014;1072:677-85. doi: 10.1007/978-1-62703-631-3_47.
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.
硫氧还蛋白（Trx）是一类小的氧化还原蛋白，可还原各种靶蛋白中的二硫键并维持细胞内硫醇的氧化还原调控。本文描述了一种用于在复杂蛋白质提取物中鉴定和相对定量Trx靶标二硫键的硫醇特异性标记和亲和富集方法。该方法利用含有硫醇反应性碘乙酰胺基团和生物素亲和标签的同位素编码亲和标签（ICAT）试剂来靶向含有还原型半胱氨酸残基的肽段。通过基于液相色谱-串联质谱（LC-MS/MS）对用“轻”（（12）C）和“重”（（13）C）ICAT试剂标记的胰蛋白酶肽段进行定量，来确定Trx的底物以及靶标二硫键的还原程度。该方法可用于监测不同还原剂或氧化剂对生物系统中硫醇/二硫键蛋白质组氧化还原状态的影响。
