Survey of Age-Associated Carbonylation of Brain Proteins

描述(由申请人提供)：氧化应激可能是衰老过程中的一个主要原因。蛋白质羰化是与年龄相关的有害变化的潜在标志物。本研究应用蛋白质组学方法来帮助阐明导致脑功能下降的生化机制，如老年小鼠在认知和精神运动任务中的行为表现所反映的。研究的主要方法学重点是开发一种同位素编码的亲和标记(ICAT)策略，以实现对蛋白质羰化的机械性和定量研究。拟议的研究将确定合适的参数，用于基于这一ICAT策略和使用质谱学的与衰老相关的氧化敏感蛋白的羰基定向鉴定和差异定量。对衰老小鼠大脑中氧化诱导的羰化作用的蛋白质组研究结果以及与年龄相关的氧化敏感蛋白的鉴定将与特定认知和精神运动功能中与年龄相关的衰退的严重程度有关。为此，具体的目标包括合成试剂(S)，使蛋白质甲基化的差异蛋白质组学研究成为可能，并开发测定大脑蛋白质氧化诱导的甲基化的方法。然后，从小鼠脑中提取的突触体组分的氧化应激将被用来研究蛋白质在体外发生甲基化的情况、途径和程度。最终，将寻求特定衰老相关氧化敏感蛋白在体内的羰基化与年龄和行为损害的相关性。这项研究的实验设计将需要为蛋白质组学调查的特定认知或精神运动任务的老年小鼠分组评分，并以年轻小鼠为对照。特定的大脑区域和亚细胞部分将被用来识别碳化蛋白，这些区域和亚细胞部分将显示出与年龄相关的氧化应激，这是通过各种方法测量的。关联程度将在每项任务中的表现和与单个老化相关的氧化敏感蛋白相关的特定羰化程度之间确定。

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.