MONITORING PROTEIN OXIDATIVE DAMAGE IN AGING AND ALZHEIMER?S DISEASE

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 由活性氧诱导的氧化应激，如蛋白质的羰化和亚硝化的形成，已被认为是阿尔茨海默病(AD)的一个促成因素。它们被认为是衰老和阿尔茨海默病氧化应激的标志。在蛋白质组学之前，一种“微流控芯片”被开发出来，用来浓缩微量受损的蛋白质。微芯片的这个表面经过化学修饰，专门结合到特定的亚蛋白质组上。分别用荧光成像仪、原子力显微镜(AFM)和X射线光电子能谱(XPS)对改性过程中的表面官能团、拓扑结构和元素分析进行了表征。事实证明，这种微芯片是一种非常灵敏和特异的亲和装置，可以从极小的样品中富集蛋白质羰基。蛋白质组反应器用于通过组合多个蛋白质组步骤来简化复杂蛋白质组样本的处理，并帮助了解氧化损伤特定蛋白质靶标的特性和相对比例的纵向变化。此外，我们利用定量蛋白质组学的方法，从体外氧化应激模型HT29人结肠腺癌细胞HT29中，以剂量依赖的方式鉴定了易受羰化和亚硝化影响的线粒体蛋白质。从细胞骨架、信号、氧化还原到细胞膜蛋白，广泛的蛋白质都被发现亚硝化。此外，还建立了高灵敏标记和毛细管电泳激光诱导荧光检测(CE-LIF)技术，用于检测和定量亚硝化和谷胱甘肽蛋白以及DNA损伤。另外，在AD转基因小鼠模型(B6Cg-TG)中，谷胱甘肽(GSH)和谷胱甘肽二硫化物(GSSG)的氧化还原状态也表明GSSG在海马区显著增加，这表明氧化损伤开始在记忆中枢-海马区积累。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Oxidative stress such as protein carbonylation and nitrosylation formation, induced by reactive oxygen species has been implicated as a contributing factor to Alzheimer Disease (AD). They are considered markers of oxidative stress in aging and AD. A "micro-fluidic chip" was developed to enrich a minute amount of damaged proteins prior to proteomics. This surface of microchip was chemically modified to specifically bind to specific subproteomes. The surface functional group, topology and element analysis at each modification steps were evaluated using fluorescence imaging, Atomic Force Microscopy (AFM) and X-ray Photoelectron Spectroscopy (XPS), respectively. This microchip proves to be a very sensitive and specific affinity device to enrich protein carbonyls from minuscule samples. A proteomic reactor was used to simplify the processing of complex proteomic samples by combining multiple proteomic steps and to help understand the longitudinal change in the identities and relative ratios of specific protein targets of oxidative damage. Additionally, we identified mitochondrial proteins susceptible to carbonylation and nitrosylation in a dose-dependent manner from In vitro oxidative stress model-HT29 human colon adenocarcinoma cell with menadione treatment using quantitative proteomics. A wide range of proteins from cytoskeleton, signaling, redox, to cell membrane proteins were found nitrosylated. Furthermore, the detection and quantitation of nitrosylated and glutathionylated proteins as well as DNA damage, hydroxydeoxygunasine (8-OHdG) using highly sensitive labeling and capillary electrophoresis with laser induced fluorescence detection (CE-LIF) were developed to study protein damage. Also, Glutathione (GSH) and Glutathione disulfide (GSSG) redox status in a AD transgenic mice model (B6Cg-Tg), demonstrated the significant increase of GSSG in hippocampus, which suggested the oxidative damage accumulation start to build up at memory center, hippocampus.