Oxidative stress may be a major causal factor underlying the aging process. Protein carbonylation is a potential marker for age-related deleterious changes. The present study applies a proteomic approach to facilitate the elucidation of the biochemical mechanisms that cause decrements in brain function, as reflected by behavioral performance of aged mice in cognitive and psychomotor tasks. The main methodological focus of the studies is the development of an isotope-coded affinity-tag (ICAT) strategy to enable mechanistic and quantitative studies on protein carbonylation. The proposed studies will determine appropriate parameters for carbonyl-directed identification and differential quantification of aging-associated oxidation-sensitive proteins based on this ICAT strategy and using mass spectrometry. Results of proteome-wide survey of oxidatively induced carbonylation in the aging mouse brain and the identification of age-associated oxidation-sensitive proteins will be related to the severity of the age-associated attenuations in specific cognitive and psychomotor functions. To this end, specific aims include the synthesis of reagent(s) that enable differential proteomics studies on protein carbonylation and the development of methods for the determination of oxidatively induced carbonylation of brain proteins. Oxidative stress on a synaptosomal fraction from mouse brain will then be used to study the occurrence, pathways and extent of protein carbonylation in vitro. Ultimately, a correlation of in vivo carbonylation of specific aging-associated oxidation-sensitive proteins with age and behavioral impairment will be sought. Experimental design of this study will entail scoring groups of aged mice on a specific cognitive or psychomotor task for the proteomics survey with young mice used as a control. Specific brain regions and subcellular fractions to be targeted for identification of carbonylated proteins will be those showing age-associated oxidative stress as measured by various methods. The degree of association will be determined between performance in each task and the degree of specific carbonylation associated with individual aging-associated oxidation-sensitive proteins.
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