Profiling Carbonylated Proteins in Human Plasma
Profiling Carbonylated Proteins in Human Plasma
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DOI：
10.1021/pr900890k
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发表时间：
2010-03-01
影响因子：
4.4
通讯作者：
Regnier, Fred E.
中科院分区：
文献类型：
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作者：
Madian, Ashraf G.;Regnier, Fred E.
本研究首次报道了基于蛋白质组学对人血浆中氧化蛋白质的鉴定和表征。该研究通过用生物素酰肼对羰基进行生物素化，并用氰基硼氢化钠还原生成的席夫碱，然后用亲和素亲和色谱从男性受试者（年龄32 - 36岁）的血浆中分离羰基化蛋白质。亲和素选择的蛋白质用胰蛋白酶消化，肽片段通过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表征。用这种方法大约选择了血浆中总蛋白质的0.2%。鉴定出65种高、中、低丰度蛋白质，大多数在所有受试者中都出现。所分离的氧化蛋白质的一个有趣特征是，除了羰基化之外，它们通常还带有其他类型的氧化修饰。在14种蛋白质中定位了24种氧化修饰。检测到7种蛋白质上的15个羰基化位点。蛋氨酸氧化是最常见的单一氧化修饰类型，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。这项工作的一个更重要的成果是，质谱分析能够区分单个蛋白质中不同的氧化生物学机制。首次同时识别了由蛋白质中氨基酸侧链的直接活性氧氧化、晚期糖基化终产物（AGES）加合物的形成以及与脂质过氧化产物形成加合物所产生的氧化产物，并将其定位到蛋白质中的特定位点。
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.