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.
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