Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, USA.
J Proteome Res. 2010 Mar 5;9(3):1330-43. doi: 10.1021/pr900890k.
This study reports the first proteomic-based identification and characterization of oxidized proteins in human plasma. The study was conducted by isolating carbonylated proteins from the plasma of male subjects (age 32-36) with avidin affinity chromatography subsequent to biotinylation of carbonyl groups with biotin hydrazide and sodium cyanoborohydride reduction of the resulting Schiff's bases. Avidin selected proteins were digested with trypsin, and the peptide fragments were separated by C18 reversed phase chromatography and identified and characterized by both electrospray ionization and matrix assisted laser desorption ionization mass spectrometry. Approximately 0.2% of the total protein in plasma was selected with this method. Sixty-five high, medium, and low abundance proteins were identified, the majority appearing in all subjects. An interesting feature of the oxidized proteins isolated was that in addition to carbonylation they often bore other types of oxidative modification. Twenty-four oxidative modifications were mapped in 14 proteins. Fifteen carbonylation sites carried on 7 proteins were detected. Methionine oxidation was the most frequent single type of oxidative modification followed by tryptophan oxidation. Apolipoprotein B-100 had 20 oxidative modifications, the largest number for any protein observed in this study. Among the organs contributing oxidized proteins to plasma, kidney, liver, and soft tissues were the most frequent donors. One of the more important outcomes of this work was that mass spectral analysis allowed differentiation between different biological mechanisms of oxidation in individual proteins. For the first time, oxidation products arising from direct ROS oxidation of amino acid side chains in proteins, formation of advanced glycation endproducts (AGEs) adducts, and formation of adducts with lipid peroxidation products were simultaneously recognized and assigned to specific sites in proteins.
本研究报告了首次基于蛋白质组学的人血浆中氧化蛋白质的鉴定和特性描述。该研究通过亲和层析从男性受试者（年龄 32-36 岁）的血浆中分离羰基化蛋白，随后用生物素酰肼将羰基基团生物素化，并将生成的希夫碱用氰基硼氢化钠还原。亲和素选择的蛋白质用胰蛋白酶消化，肽片段用 C18 反相色谱分离，并通过电喷雾电离和基质辅助激光解吸电离质谱进行鉴定和特性描述。用这种方法大约可以选择血浆中总蛋白的 0.2%。鉴定出 65 种高、中、低丰度的蛋白质，大多数蛋白质在所有受试者中都有出现。分离出的氧化蛋白质的一个有趣特征是，除了羰基化外，它们通常还带有其他类型的氧化修饰。在 14 种蛋白质中发现了 24 种氧化修饰。在 7 种蛋白质上检测到 15 个羰基化位点。蛋氨酸氧化是最常见的单一类型氧化修饰，其次是色氨酸氧化。载脂蛋白 B-100 有 20 种氧化修饰，是本研究中观察到的任何蛋白质中数量最多的。在向血浆中提供氧化蛋白质的器官中，肾脏、肝脏和软组织是最常见的供体。这项工作的一个重要成果是，质谱分析允许区分个体蛋白质中不同的氧化生物学机制。这是首次同时识别和分配蛋白质中氨基酸侧链直接由 ROS 氧化、形成晚期糖基化终产物（AGEs）加合物以及与脂质过氧化产物形成加合物的氧化产物，并将其分配到特定的蛋白质位点上。
