Reactive Species in Vascular Disease-Injury Mechanisms

说明(申请人提供)：一氧化氮(NO)已被证明在不同的细胞系统中调节生物过程。在一定程度上，通过将蛋白质半胱氨酸残基修饰为S亚硝基半胱氨酸，促进了NO的多方面生物学功能。直到最近，由于缺乏补充的、有效的蛋白质组学方法，我们在复杂的生物系统中询问这些NO的新蛋白质靶标的能力一直受到限制。尽管生物素开关方法的发展在一定程度上促进了蛋白质靶标的发现，但对蛋白质靶标的确认和敏感性问题的担忧限制了这一方法学的应用。因此，我们发展并建议进一步完善一种能够特异鉴定复杂生物样品中S-亚硝基半胱氨酸残留量的方法。该方法采用选择性多肽捕获和液相色谱-串联质谱仪定位的加合物图谱。在人主动脉平滑肌细胞中，从每毫克含4摩尔S-亚硝半胱氨酸的蛋白质开始，这一策略鉴定出了18个含有S-亚硝半胱氨酸的多肽，分属于16种蛋白质。在本申请中，我们建议开发互补的方法来直接标记和捕获S-亚硝基半胱氨酸加合物。此外，利用细胞蛋白质或S-亚硝基半胱氨酸加合物的同位素标记，我们提出并实现了S-亚硝基半胱氨酸蛋白质组的定量方法。将选择性S亚硝基半胱氨酸多肽鉴定与定量蛋白质组学相结合，将首次提供活细胞中特定S亚硝基蛋白水平的动态变化的全球评估。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用于确定S的三个蛋白质靶标-硝酸化、纽蛋白、14-3-3theta和线粒体热休克蛋白70的时空变化，这些蛋白质调控血管平滑肌生物学的关键方面。总体而言，所提出的全球和靶向蛋白质组学方法将探索S-亚硝半胱氨酸在心血管系统中的信号通路和蛋白质网络中的生物学意义。此外，S亚硝基半胱氨酸蛋白质组的全球特征将通过揭示该分子先前未被识别的分子靶点，显着地促进我们对该分子生物学功能的理解。

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.