Survey of Age-Associated Carbonylation of Brain Proteins

描述（由申请人提供）：氧化应激可能是导致衰老过程的主要因素。蛋白质羰基化是与年龄相关的有害变化的潜在标志。本研究应用蛋白质组学方法来阐明导致脑功能下降的生化机制，这反映在老年小鼠在认知和精神运动任务中的行为表现上。这些研究的主要方法学重点是发展同位素编码亲和标签策略，以便对蛋白质羰基化进行机制和定量研究。拟议的研究将确定适当的参数羰基定向鉴定和基于该ICAT策略和使用质谱的衰老相关氧化敏感蛋白的差异定量。衰老小鼠大脑中氧化诱导羰基化的蛋白质组调查结果和年龄相关氧化敏感蛋白的鉴定将与特定认知和精神运动功能中年龄相关衰减的严重程度有关。为此，具体目标包括合成能够对蛋白质羰基化进行差异蛋白质组学研究的试剂，以及开发测定氧化诱导的脑蛋白质羰基化的方法。氧化应激对小鼠大脑突触体部分的影响将用于体外研究蛋白质羰基化的发生、途径和程度。最终，将寻求体内特定衰老相关氧化敏感蛋白的羰基化与年龄和行为障碍的相关性。本研究的实验设计将需要对老年小鼠进行特定认知或精神运动任务的评分，以进行蛋白质组学调查，年轻小鼠作为对照。鉴定羰基化蛋白的特定大脑区域和亚细胞部分将是那些通过各种方法测量出与年龄相关的氧化应激的区域。这种关联的程度将取决于每项任务的表现和与个体衰老相关氧化敏感蛋白相关的特定羰基化程度。

DESCRIPTION (provided by applicant): 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.