THE STUDY OF PROTEIN DAMAGE IN AGING AND AGE-RELATED DISEASES IN BRAIN USING BI

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。 由活性氧诱导的氧化应激，如蛋白质的羰化、亚硝化和谷胱甘肽的形成，被认为是阿尔茨海默病(AD)的一个重要因素。它们被认为是衰老和阿尔茨海默病氧化应激的标志。这项研究的总体目标是确定衰老大脑中参与AD发病机制的主要碳化和硝化蛋白，并确定这些氧化损伤蛋白的存在会影响哪些神经通路。 我们的第一个具体目标是开发一种“微流控芯片”，在蛋白质组学之前丰富微量的受损蛋白质。微芯片是从镍模具母版上复制出来的，采用热压花法。微芯片的这个表面经过化学修饰，专门结合到羰基上。通过芯片内蛋白质标记和荧光显微镜进一步验证了对蛋白质羰基的捕获。这种基于微芯片的蛋白质羰基富集器被证明是一种非常灵敏和特异的亲和装置，可以从微量样品中富含蛋白质羰基。它可以探测和捕获40毫微克分子的碳。 我们的第二个具体目标是开发一种蛋白质组反应器，通过结合多个蛋白质组步骤来简化复杂蛋白质组样本的处理，并允许识别羰化、亚硝化和谷胱甘肽基化的蛋白质。这将有助于理解氧化损伤的特定蛋白质靶标的身份和相对比例的纵向变化。以过氧化氢氧化后的SD大鼠肝脏线粒体蛋白质为研究对象，验证了蛋白质组学反应器和iTRAQ标记技术定量鉴定碳化蛋白质的可行性。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。 在未来的研究中，为了了解AD发生和发展过程中早期和进行性的细胞变化，使用定量蛋白质组学分析，本实验室将研究AD转基因小鼠模型在疾病进展的三个阶段：疾病进展之前很长时间(2个月大)、紧接之前(6个月大)和之后(12个月大)，淀粉样蛋白病理和认知损害的出现。

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. Oxidative stress such as protein carbonylation, nitrosylation and glutathionylation 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. The overall goal of this research is to identify the major carbonylated and nitorsylated proteins in the aging brain that contribute to the pathogenesis of AD and determine which neurological pathways are affected by the presence of these oxidative damaged proteins. Our first specific aim is to develop a "micro-fluidic chip" to enrich a minute amount of damaged proteins prior to proteomics. The microchip is replicated from a nickel mold master using hot embossing. This surface of microchip is chemically modified to specifically bind to carbonyls. The capture of the protein carbonyls is further validated with in-chip protein labeling and fluorescence microscopy. This microchip based protein carbonyl enrichment proves to be a very sensitive and specific affinity device to enrich protein carbonyls from minuscule samples. It can detect and capture 40 femtomole of carbonlys. Our second specific aim is to develop a proteomic reactor to simplify the processing of complex proteomic samples by combining multiple proteomic steps and allow for the identification of carbonylated, nitrosylated and glutathionylated proteins. This will help to understand the longitudinal change in the identities and relative ratios of specific protein targets of oxidative damage. Liver mitochondrial proteins from Sprague Dawley rats subjected to hydrogen peroxide oxidation are used to confirm the feasibility of quantitative identification of carbonylated proteins using the proteomic reactor and iTRAQ labeling. Additionally, we identify mitochondrial proteins susceptible to carbonylation and nitrosylation in a dose-dependent manner from In vitro oxidative stress model-HT29 human colon adenocarcinoma cell with menodione treatment using quantitative proteomics. In the future study, to understand the early and progressive cellular changes in AD development and progression, using quantitative proteomic profiling, this laboratory will investigate protein carbonylation in the AD transgenic mouse model at three stages of disease progression: long before (2-months old), immediately before (6-months old) and after (12-months old), the appearance of amyloid pathology and cognitive impairment.