Mass Spectrometry And Oxidative Stress

对组织的累积性氧化损伤与许多疾病状态有关，例如衰老过程、癌症和缺血再灌注。线粒体中氧化应激的研究表明，过氧化氢是通过氧化磷酸化过程中氧的不完全还原产生的。过氧化氢水平在正常生理条件下保持相对较低。在某些情况下，如炎症，过量的过氧化氢被认为是在几种情况之前发生的，如脂质过氧化，DNA和/或蛋白质损伤，以及谷胱甘肽耗尽，这些都是氧化应激的特征。自由基在氧化应激中的作用引起了人们对自由基及其反应的研究兴趣。活性氧代谢物可以与细胞成分相互作用，包括DNA/RNA、蛋白质和不饱和脂质。以前的研究表明，血红素蛋白可能参与氧化还原反应，通过与过氧化氢反应导致组织和/或器官损伤。此外，最近的一项研究报告了硝基酪氨酸水平和冠状动脉疾病之间的关联的证据。因此，蛋白质自由基中间体的测定和表征对于理解这些反应的机制及其对人类疾病的贡献是重要的。质谱工作组与罗纳德梅森有着长期的合作关系。自由基工作组（LPC）。这项合作的目标是深入了解氧化应激的机制，使用氧化诱导的自由基的自旋捕获，然后使用质谱进行结构表征的组合。 马肌红蛋白、抹香鲸肌红蛋白和血红蛋白上自由基的形成是通过在存在自旋捕获剂5，5-二甲基-1-吡咯啉N-氧化物（DMPO）的情况下用过氧化氢处理每种蛋白质的溶液而开始的。当肌红蛋白或血红蛋白与过氧化氢在DMPO的存在下反应，DMPO硝酮加合物可以通过免疫染色检测。为了验证已经形成蛋白质自由基的DMPO加合物，通过MS分析反应混合物。这些数据显示肌红蛋白的加合物，其对应于添加一个DMPO分子。消化和MS/MS分析后，我们确定DMPO共价结合到马肌红蛋白的Tyr-103残基。 我们已经开始了初步工作，将单克隆抗DMPO抗体固定到琼脂糖珠上，以特异性地拉出蛋白质髓过氧化物酶和乳过氧化物酶上的蛋白质-DMPO和/或肽-DMPO加合物。该技术在测定非常大的蛋白质和/或来自生物样品的蛋白质上的DMPO加合物中应该是有用的。

Cumulative oxidative damage to tissues has been implicated in a number of disease states, e.g. the aging process, cancer, and ischemia reperfusion. The study of oxidative stress in the mitochondria has shown that hydrogen peroxide is produced via the incomplete reduction of oxygen during oxidative phosphorylation. Hydrogen peroxide levels are kept relatively low under normal physiological conditions. Under certain conditions, such as inflammation, excessive amounts of hydrogen peroxide is thought to precede several occurrences, such as lipid peroxidation, DNA and/or protein damage, and glutathione depletion, that are characteristic of oxidative stress. The role of free radicals in oxidative stress has led to an increased interest in the study of free radicals and their reactions. Reactive oxygen metabolites can interact with cellular constituents, including DNA/RNA, proteins, and unsaturated lipids. Previous studies have suggested that hemoproteins may be involved in redox reactions which contribute to tissue and/or organ damage via reaction with hydrogen peroxide. In addition, a recent study has reported evidence for the association between nitrotyrosine levels and coronary artery disease. Thus, the determination and characterization of protein radical intermediates is important in understanding the mechanisms of these reactions and their contributions to human diseases. The Mass Spectrometry Workgroup has had a long-term collaboration with Ronald Mason?s Free Radical Workgroup (LPC). The goal of this collaboration is to gain insight on the mechanisms of oxidative stress using a combination of spin trapping of oxidatively-induced free-radicals followed by structural characterization using mass spectrometry. The formation of radicals on horse myoglobin, sperm whale myoglobin, and hemoglobin was initiated by treating a solution of each protein with hydrogen peroxide in the presence of the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). When either myoglobin or hemoglobin was reacted with hydrogen peroxide in the presence of DMPO, a DMPO nitrone adduct could be detected by immunostaining. To verify that DMPO adducts of the protein free-radicals had been formed, the reaction mixtures were analyzed by MS. These data showed an adduct of myoglobin which corresponds to the addition of one DMPO molecule. After digestion and MS/MS analysis, we determined that the DMPO was covalently bound to the Tyr-103 residue of the horse myoglobin. We have begun preliminary work to immobilize a monoclonal anti-DMPO antibody to sepharose beads to specifically pull out protein-DMPO and/or peptide-DMPO adducts on the proteins myeloperoxidase and lactoperoxidase. This technique should be useful in determining DMPO adducts on very large proteins and/or proteins from biological samples.