PROTEOMICS, METABONOMICS OF EFFECTS OF NITRITE TREATMENT ON THE HEART

亚硝酸盐治疗对心脏影响的蛋白质组学、代谢组学

• 批准号: 7602062
• 负责人: MARTIN FEELISCH
• 金额: $ 0.86万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-03 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7602062
• 关键词: Affect; Aliquot; Biological; Blood; Buffers; Cardiac; Cellular Stress Response; Cellular Structures; Computer Retrieval of Information on Scientific Projects Database; Computer software; Cytochrome P450; Daily; Data; Dietary intake; Digestion; Down-Regulation; Endocrine; Energy Metabolism; Equilibrium; Fingerprint; Funding; Gases; Gel; Glutathione; Grant; Health; Heart; Heat-Shock Proteins 70; Heme; High Pressure Liquid Chromatography; Hour; Human; Image; Institution; Intraperitoneal Injections; Manuscripts; Maps; Measures; Medicine; Molecular Chaperones; Nitric Oxide; Nitrites; Oxidation-Reduction; Peptides; Phase; Physiological; Post-Translational Protein Processing; Proteins; Proteome; Proteomics; Range; Reperfusion Injury; Research; Research Personnel; Resistance; Resources; Signaling Molecule; Silver Staining; Sodium Nitrite; Soluble Guanylate Cyclase; Source; Spottings; Therapeutic; Therapeutic Uses; Thinking; Tissue Harvesting; Tissues; Trypsin; Two-Dimensional Gel Electrophoresis; United States National Institutes of Health; Wistar Rats; ascorbate; base; comparative; heme oxygenase-1; in vivo; mRNA Differential Displays; male; oxidation; protein expression

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 thought for decades 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 very recently proven to be a signaling molecule in its own right, affecting soluble guanylyl cyclase and cytochrome P450 activities, heat shock protein 70 and heme oxygenase-1 expression, and protecting against cardiac ischemia-reperfusion injury. Based on this recent evidence, we are seeking to characterize 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, then 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 24 hours later, 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, followed by Coomassie or silver staining. Protein spots were imaged and quantitated using PDQuest" software, excised and subjected to in-gel trypsin digestion. 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 peptide mass fingerprinting analysis was conducted by submission of peak lists to MASCOT. Using 2D gel electrophoresis, we have created preliminary 2D reference maps of cardiac proteomes of control Wistar rats and those systemically administered physiologic and therapeutic levels of nitrite. We have confirmed the identity of over a hundred isolated protein spots through in-gel digestion followed by MALDI-TOF MS and peptide mass fingerprinting or by LC-MS/MS analyses. Quantitative comparative analyses have revealed significant changes to cardiac protein expression upon treatment with nitrite. These changes consisted of both up- and down-regulation of steady-state protein levels, as well as apparent alterations in post-translational protein modification and have included proteins involved in cell structure, energy metabolism, redox balance, and chaperone activity, among others. Additionally, we have measured metabonomic changes and changes to the cellular redox status that we have correlated to these proteomics data. Specifically, we have detected changes in the ratio of reduced to oxidized ascorbate and glutathione, as well as changes in the levels of S-nitroso, N-nitroso, and heme-nitroso species in the tissues. The results obtained using this combined proteomics/metabonomics approach 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, due to its stability in vivo, may be able to act in a long-range endocrine fashion to establish this protective tone in the heart. Our integrated proteomic and metabonomic approach is a step toward elucidating the scope and mechanism of cardioprotection and the potential activity of this biological compound 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. A manuscript describing our results is being prepared.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 亚硝酸盐在体内稳定且丰富，几十年来被认为在生理浓度下是生物惰性的。通过氧化一氧化氮（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，并广泛来源于饮食来源，最近已证明其本身是一种信号分子，影响可溶性鸟苷酸环化酶和细胞色素P450活性，热休克蛋白70和血红素加氧酶-1表达，并保护心脏缺血-再灌注损伤。 基于最近的证据，我们正在寻求表征全身亚硝酸盐可用性变化对心脏蛋白质组的影响，使用基于2D凝胶的差异显示，然后进行LC-MS/MS或MALDI-TOF MS和肽质量指纹分析，然后将这些数据与代谢组学研究相关联。 单次腹腔注射亚硝酸钠（0.1、1和10 mg/kg）给药的雄性Wistar大鼠在24小时后麻醉，无血灌注并收获其心脏组织。匀浆后立即通过气相化学发光和HPLC测定NO相关代谢物的浓度。将组织等分试样在IEF缓冲液中变性并进行2D-PAGE分析，然后进行考马斯或银染色。 使用PDQuest软件对蛋白质点进行成像和定量，切除并进行凝胶内胰蛋白酶消化。将肽洗脱、脱盐并通过MALDI-TOF MS或通过LC-MS和MS/MS分析。用MoverZ”或MassLynx”和ProteinLynx”软件分析光谱，并通过向MASCOT提交峰列表进行肽质量指纹分析。 使用二维凝胶电泳，我们已经创建了初步的二维参考地图的心脏蛋白质组的控制Wistar大鼠和那些全身管理的生理和治疗水平的亚硝酸盐。 我们已经通过凝胶内消化，然后通过MALDI-TOF MS和肽质量指纹图谱或LC-MS/MS分析确认了一百多个分离的蛋白质点的身份。定量比较分析显示，亚硝酸盐处理后，心脏蛋白表达的显着变化。这些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改变，包括参与细胞结构、能量代谢、氧化还原平衡和伴侣活性等的蛋白质。此外，我们还测量了代谢组学变化和细胞氧化还原状态的变化，这些变化与蛋白质组学数据相关。具体来说，我们已经检测到的比例的变化，还原氧化抗坏血酸和谷胱甘肽，以及在组织中的S-亚硝基，N-亚硝基，血红素亚硝基物种的水平的变化。 使用这种组合的蛋白质组学/代谢组学方法获得的结果表明亚硝酸盐引发的细胞应激反应蛋白表达的特定变化，可能赋予进一步的氧化损伤的抗性。我们的数据表明，亚硝酸盐，由于其在体内的稳定性，可能能够发挥作用，在一个长期的内分泌方式，以建立这种保护性的基调在心脏。我们的综合蛋白质组学和代谢组学方法是朝着阐明心脏保护的范围和机制以及这种生物化合物的潜在活性迈出的一步，这种生物化合物在人类健康和医学中具有新发现的重要性。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，并可能引起对每日膳食摄入亚硝酸盐的影响的重新评估。描述我们研究结果的手稿正在编写中。