PROTEOMICS, METABONOMICS OF EFFECTS OF NITRITE TREATMENT ON THE HEART

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 亚硝酸盐在体内稳定而丰富，长期以来被认为在生理浓度下是生物惰性的。它由一氧化氮(一种普遍存在的信号分子，已知可引起广泛的生物学效应)内源性形成，广泛来自饮食来源，最近被证明是一种信号分子，影响可溶性鸟苷酸环化酶和细胞色素P450的活性，热休克蛋白70和血红素加氧酶-1的表达，并对心肌缺血再灌注损伤具有保护作用。因此，我们致力于利用2D凝胶差异显示、LC-MS/MS或MALDI-TOF MS和多肽质量指纹图谱分析来表征全身亚硝酸盐可获得性变化对心肌蛋白质组的影响，并将这些数据与代谢组学研究相关联。雄性Wistar大鼠一次性腹腔注射亚硝酸钠(0.1、1、10 mg/kg)，24 h后麻醉，无血灌流，取心脏组织。匀浆后即刻用气相化学发光法和高效液相色谱法测定NO相关代谢物的浓度。组织匀浆在IEF缓冲液中变性，进行2D-PAGE分析，考马斯亮蓝或银染。蛋白质斑点用PDQuest软件成像和定量，切下后经凝胶内胰酶消化，用MALDI-TOF MS或LC-MS和MS/MS洗脱、脱盐和分析，用MoverZ“or MassLynx”和ProteinLynx软件分析Spectra，并进行PMF分析。我们已经创建了对照和亚硝酸盐管理的Wistar大鼠心脏蛋白质组的初步2D-PAGE参考图。通过PMF和/或LC-MS/MS分析，我们已经鉴定了100多个分离的蛋白质点。定量比较分析揭示了亚硝酸盐处理后心脏蛋白表达的显著变化，包括稳态蛋白水平的上调和下调，以及翻译后蛋白修饰的明显变化。这些蛋白质涉及细胞结构、能量代谢、氧化还原平衡和伴侣活性等。我们还测量了代谢组学变化和细胞氧化还原状态的变化，并将这些变化与蛋白质组学数据相关联。检测组织中还原型/氧化型抗坏血酸和谷胱甘肽的比值，组织中S亚硝基和N亚硝基以及亚铁血红素种类的变化。结果表明，亚硝酸盐触发的细胞应激反应蛋白表达的特定变化可能赋予对进一步氧化损伤的抵抗力。我们的数据表明，亚硝酸盐可能能够以一种远程内分泌的方式在心脏中建立这种保护性基调。我们的方法是向阐明心脏保护的范围和机制以及NO的潜在活性迈出了一步，新发现的NO在人类健康和医学中具有重要意义。我们的数据可能对目前亚硝酸盐的实验性治疗用途有直接和直接的影响，并可能引发对每天饮食摄入亚硝酸盐的影响的重新评估。这项工作被列入2009年9月在多伦多举行的HuPo会议和2009年5月在费城举行的ASMS会议的讲座中。描述这些结果的手稿发表在《循环研究》，《利用整合的代谢组学-蛋白质组学方法对亚硝酸盐诱导的心脏保护的机制洞察》。中国保监会。2009年，104,796-804)。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Nitrite, stable and abundant in vivo, was long thought to be biologically inert at physiological concentrations. Formed endogenously through oxidation of nitric oxide (an ubiquitous signaling molecule known to elicit broad biological effects) and derived extensively from dietary sources, it has itself recently proven to be a signaling molecule, affecting soluble guanylyl cyclase and cytochrome P450 activities, heat shock protein 70 and heme oxygenase-1 expression, and protecting against cardiac ischemia-reperfusion injury. We are therefore engaged in characterizing the impact of changes in systemic nitrite availability on the cardiac proteome, using 2D gel-based differential display followed by LC-MS/MS or MALDI-TOF MS and peptide mass fingerprinting analyses, and correlate these data to metabonomic studies. Male Wistar rats administered a single intraperitoneal injection of sodium nitrite (0.1, 1 and 10 mg/kg) were anesthetized after 24 h, perfused free of blood and their cardiac tissue harvested. Immediately after homogenization the concentrations of NO-related metabolites were determined by gas phase chemiluminescence and HPLC. Tissue aliquots were denatured in IEF buffer and subjected to 2D-PAGE analysis, with Coomassie or silver staining. Protein spots were imaged and quantitated using PDQuest" software, excised and subjected to in-gel trypsin digestion. The resulting peptides were eluted, de-salted and analyzed by MALDI-TOF MS or by LC-MS and MS/MS. Spectra were analyzed with MoverZ" or MassLynx" and ProteinLynx" software, and PMF analysis was conducted. We have created preliminary 2D-PAGE reference maps of cardiac proteomes of control and nitrite-administered Wistar rats. We have identified more than 100 isolated protein spots by PMF and/or LC-MS/MS analyses. Quantitative comparative analyses have revealed significant changes to cardiac protein expression upon treatment with nitrite, including both up- and down-regulation of steady-state protein levels, and apparent alterations in post-translational protein modification. The proteins are involved in cell structure, energy metabolism, redox balance, and chaperone activity, etc. We also measured metabonomic changes and changes to the cellular redox status and correlated these to the proteomics data. We detected changes in the ratio of reduced/oxidized ascorbate and glutathione, and in the levels of S- and N-nitroso, and heme-nitroso species in the tissues. The results indicate specific changes in expression of cellular stress response proteins triggered by nitrite that may confer resistance to further oxidative insults. Our data suggest that nitrite may be able to act in a long-range endocrine fashion to establish this protective tone in the heart. Our approach is a step toward elucidating the scope and mechanism of cardioprotection and the potential activity of NO with newly discovered importance in human health and medicine. Our data may have direct and immediate implications for current experimental therapeutic uses of nitrite and may provoke a reassessment of the impact of daily dietary intake of nitrite. The work was included in lectures at the HUPO meeting in Totonto in September 2009 and the ASMS meeting in Philadelphia in May 2009. A manuscript describing these results was published in Circulation Research DH Perlman et al., Mechanistic Insights into Nitrite-Induced Cardioprotection Using an Integrated Metabonomic-Proteomic Approach. Circ. Res., 2009, 104, 796-804).