ISOTOPE DILUTION MASS SPECTROMETRIC QUANTIIFCATION OF 3 NITROTYROSEINE

3-硝基酪氨酸的同位素稀释质谱定量

• 批准号: 6336729
• 负责人: JAN R CROWLEY
• 金额: $ 1.09万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2001-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6336729
• 关键词: biomedical resource; cardiovascular system; enzymes; gas; human tissue; minerals; proteins; spectrometry; technology /technique

Oxidative damage by reactive nitrogen species has been implicated in the pathogenesis of atherosclerosis and other inflammatory diseases. The mechanims of tissue damage are poorly understoond, however, because the toxic intermediates are short-lived. Previous in vitro studies have suggested that 3-nitrotyrosine represents a specific marker of protein oxidation by reactive nitrogen species. The detection of this nitrated aromatic amino acid may thus serve as an indciator of tissue injury by nitrogen species in vivo. Here we describe a highly sensitive ans specific analytical method for quantifying free and protein-bound 3-nitrotyrosine. The assay involves acid hydrolysis of proteins, isolation of 3-nitrotyrosine by ion exchange chromatography, and reduction of 3-nitrotyrosine to 3-aminotyrosine with dithionite. The reduced amino acid is then converted to its n-propyl, per-heptafluorobutyryl derivative and quanitfied by isotope dilution gas chromagtography negative-io n chemical ionization mass spectrometry. Attomole levels of 3-nitrotyrosine can be reproducibly measured in this manner. Quantifying 3-nitrotyrosine levels of tissues by stable isotope dilution gas chromtogrpahy/mass spectrometry should provide a powerful tool for exploriong the impact of reactive nitrogen species on oxidative readtions in vivo.

由活性氮物种引起的氧化损伤已被牵连 在动脉粥样硬化和其他炎症的发病机制中， 疾病 组织损伤的机制还不清楚， 然而，因为有毒的中间体是短暂的。 先前的 体外研究表明，3-硝基酪氨酸代表了 活性氮类蛋白质氧化的特异性标志物。 因此，这种硝化的芳香族氨基酸的检测可以用作 体内氮类物质对组织损伤的指示剂。 这里我们 描述了一种高度灵敏的ANS特异性分析方法， 定量游离和蛋白结合的3-硝基酪氨酸。 测定 包括蛋白质的酸水解，3-硝基酪氨酸的分离， 离子交换色谱法，并将3-硝基酪氨酸还原为 3-氨基酪氨酸与连二亚硫酸盐。 然后还原的氨基酸是 转化成其正丙基全七氟丁酰基衍生物， 同位素稀释气相色谱负离子定量 化学 电离质谱法 阿托摩尔水平的3-硝基酪氨酸可以 以这种方式进行重复测量。 定量3-硝基酪氨酸 用稳定同位素稀释气体色谱/质谱法测定的组织水平 光谱学应该为探索撞击提供一个强有力的工具 活性氮对体内氧化反应的影响。