Reactive Species in Vascular Disease-Injury Mechanisms

描述（由申请人提供）：一氧化氮（NO）已被证明可调节多种细胞系统中的生物过程。NO的多方面生物学功能部分通过将蛋白质半胱氨酸残基修饰为S-亚硝基半胱氨酸来促进。直到最近，我们的能力，询问复杂的生物系统，这些新的蛋白质靶点的NO已受到限制，缺乏互补的，有效的蛋白质组学方法。尽管生物素转换方法的发展在一定程度上促进了蛋白质靶点的发现，但对蛋白质靶点验证的担忧和灵敏度问题限制了该方法的应用。因此，我们已经开发并建议进一步完善一种方法，可以特异性地识别复杂生物样品中的S-亚硝基半胱氨酸残基。该方法采用选择性肽捕获和位点特异性加合物液相色谱-串联质谱图。在人主动脉平滑肌细胞中，从每mg蛋白质总共4纳摩尔蛋白质S-亚硝基半胱氨酸开始，该策略鉴定了属于16种蛋白质的18种含S-亚硝基半胱氨酸的肽。在这个应用程序中，我们建议开发互补的方法直接标记和捕获的S-亚硝基半胱氨酸加合物。此外，使用同位素标记的细胞蛋白质或S-亚硝基半胱氨酸加合物，我们建议开发和实施的S-亚硝基半胱氨酸蛋白质组的定量方法。将选择性S-亚硝基半胱氨酸肽鉴定与定量蛋白质组学相结合，将首次对活细胞中特定S-亚硝基蛋白水平的动态变化进行全面评估。这些方法结合免疫组织化学和高分辨率免疫电子显微镜将被应用于定义的时间和空间变化的三个蛋白质的S-亚硝基化，黏着斑蛋白，14-3- 3 θ和线粒体热休克蛋白70，调节血管平滑肌生物学的关键方面。总体而言，所提出的全球和有针对性的蛋白质组学方法将探索S-亚硝基半胱氨酸在心血管系统的信号通路和蛋白质网络中的生物学意义。此外，全球性的S-亚硝基半胱氨酸蛋白质组的表征将显着推进我们的了解NO的生物学功能，揭示以前未被识别的分子靶点，这个分子。

DESCRIPTION (provided by applicant): Nitric oxide (NO) has been shown to modulate biological processes in diverse cellular systems. In part the multifaceted biological functions of NO are facilitated through the modification of protein cysteine residues to S-nitrosocysteine. Until recently our ability to interrogate complex biological systems for these novel protein targets of NO has been restricted by the lack of complementary, validated proteomic methodologies. Although the development of the biotin-switch approach has in part advanced the discovery of protein targets, concerns with validation of the protein targets and issues with sensitivity limit the application of this methodology. Therefore, we have developed and propose to further refine a methodology that can specifically identify the S-nitrosocysteine residues in complex biological samples. The method employs selective peptide capturing and site-specific adduct mapping by liquid chromatography-tandem mass spectrometry. In human aortic smooth muscle cells, starting with a total of 4 nmoles protein S- nitrosocysteine per mg of protein this strategy identified 18 S-nitrosocysteine-containing peptides belonging to 16 proteins. In this application we propose to develop complementary approaches for direct labeling and capture of the S-nitrosocysteine adducts. Moreover, using isotopic labeling of cellular proteins or of S- nitrosocysteine adducts we propose to develop and implement a quantitative method for the S- nitrosocysteine proteome. Combining the power of selective S-nitrosocysteine peptide identification with quantitative proteomics will provide for the first time a global evaluation of the dynamic changes in levels of specific S-nitrosoproteins in living cells. These methodologies in conjunction with immunohistochemical and high resolution immuno-electron microscopy will be applied to define the temporal and spatial changes of three protein targets of S-nitrosylation, vinculin, 14-3-3theta and mitochondrial heat shock protein 70, which regulate critical aspects of vascular smooth muscle biology. Overall the proposed global and targeted proteomic approaches will explore the biological significance of S-nitrosocysteine in signaling pathways and protein networks in the cardiovascular system. Moreover the global characterization of the S-nitrosocysteine proteome will significantly advance our understanding of the biological functions of NO by uncovering previously unrecognized molecular targets for this molecule.