PROTEOMICS

蛋白质化学和质谱（“蛋白质组学”）核心将由 巴克研究所的化学项目主任是吉布森博士。吉布森医生有20多年的经验 在质谱分析和蛋白质及其他大分子的分析中。在过去的七年里， 该小组在线粒体蛋白质组学方面进行了广泛的研究，并开发了新的方法， 研究翻译后修饰，如3-硝基酪氨酸，磷酸化和半胱氨酸氧化。的 核心区的蛋白质组学设施包括广泛的质谱分析、色谱分析和蛋白质组学。 化学成分，非常适合提供所需的专业知识和方法，以执行 蛋白质化学实验在项目1-3中描述。核心工作人员的资源和知识 包括线粒体蛋白质组学、蛋白质鉴定、化学和定量方法，此外， 已经描述了快速分离和分析大复合物的方法（例如，线粒体 电子传递链，或ETC），和蛋白质翻译后修饰（PTM）的分析， 包括氧化损伤、蛋白质磷酸化和亚基化学计量的变化。因此，我们认为， 我们的核心目标是与各个项目的PL密切合作， 蛋白质的分析，即，线粒体OXPHOS复合物I-V、p53和tau以及其他蛋白质，和 它们响应于影响整体氧化还原状态的基因/酶的操纵而经历的变化， 或各种年龄相关疾病细胞和/或动物模型系统的“氧化应激”。 蛋白质组学核心与本计划项目中的三个项目相关的具体目标是： Andersen，项目1：我们将详细研究氧化损伤（可逆和不可逆）， 线粒体复合物I（以及可能的其他ETC）的46个亚基之间的磷酸化变化 复合物和线粒体蛋白质），伴随谷胱甘肽库的急性和/或慢性消耗 在转基因抗GCL小鼠和大鼠多巴胺能N27细胞系中。 Campisi和Benz，项目2：我们将研究p53磷酸化和半胱氨酸氧化还原状态的变化 (i.e.,谷胱甘肽化、磺基丙氨酸等）在人和小鼠上皮成纤维细胞（MEF）中， 谷胱甘肽和p53表达水平已经被操纵，并与p53水平的变化相关， 或DNA结合。 Melov项目3：我们将研究tau蛋白磷酸化状态的变化， 阶段，作为年龄和氧化应激的结果的诱导型sod 2缺失小鼠模型。

The Protein Chemistry and Mass Spectrometry ('Proteomics') Core will be under the administration of the Buck Institute's Chemistry Program whose director is Dr. Gibson. Dr. Gibson has over 20 years experience in mass spectrometry and the analysis of proteins and other macromolecules. In the last seven years, this group has carried out extensive studies in mitochondrial proteomics as well as developing new methods to study posttranslational modifications such as 3-nitrotyrosine, phosphorylation and cysteine oxidation. The proteomics facilities in the Core include extensive mass spectrometry, chromatography, and protein chemistry components that are well suited to provide the expertise and methodology needed to carry out the protein chemistry experiments described in Projects 1-3. The resources and knowledge of the Core staff include methods for mitochondrial proteomics, protein identification, chemistry and quantitation, Moreover, methods have been described for the rapid isolation and analysis of large complexes (e.g., mitochondrial electron transport chain, or ETC), and the analysis of protein posttranslational modifications (PTMs), including oxidative damage, protein phosphorylation, and changes in subunit stoichiometries. Therefore, our aims for this Core are to work closely with the PL's of the individual Projects to provide detailed analysis of proteins, i.e., mitochondrial OXPHOS complexes I-V, p53 and tau as well as other proteins, and changes they undergo in response to the manipulation of genes/enzymes that affect the overall redox status, or 'oxidative stress' of the various age-related disease cell and/or animal model systems. The specific aims of the Proteomics Core in relation to the three Projects in this Program Project are: Andersen, Project 1: We will examine in detail both oxidative damage (both reversible and irreversible) and changes in phosphorylation among the 46 subunits of mitochondrial Complex I (and possibly other ETC complexes and mitochondrial proteins) that accompany acute and/or chronic depletion of glutathione pools in an transgenic anti-GCLmouse and a rat dopaminergic N27 cell line. Campisi and Benz, Project 2: We will examine changes in p53 phosphorylation and cysteinyl redox status (i.e., glutathionylation, cysteic acid, etc.) in human and mouse epithelial fibroblasts (MEFs) when SOD2, glutathione and p53 expression levels have been manipulated and correlated to changes in either p53 levels or DNA binding. Melov Project 3: We will examine changes in the phosphorylation state of tau in a constituitive and, at later stages, an inducible sod2 nullizygous mouse model as a consequence of age and oxidative stress.