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的主要原因。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，以促进阐明的生化机制，导致递减的大脑功能，反映了老年小鼠在认知和心理任务的行为表现。这些研究的主要方法重点是开发同位素编码的亲和标签（ICAT）策略，以实现对蛋白质羰基化的机理和定量研究。拟议的研究将确定适当的参数羰基定向识别和差异定量的衰老相关的氧化敏感蛋白的基础上，这种ICAT策略和使用质谱。衰老小鼠大脑中氧化诱导羰基化的蛋白质组范围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功能的年龄相关衰减的严重程度相关。为此，具体的目标包括试剂的合成，使差异蛋白质组学研究的蛋白质羰基化和方法的发展，用于确定氧化诱导的羰基化的脑蛋白质。氧化应激对小鼠脑突触体部分，然后将用于研究蛋白质羰基化的发生，途径和程度在体外。最终，将寻求特定衰老相关氧化敏感蛋白体内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组进行特定认知或心理任务的评分，以进行蛋白质组学调查，并将年轻小鼠用作对照。用于鉴定羰基化蛋白质的特定脑区域和亚细胞级分将是通过各种方法测量的显示与年龄相关的氧化应激的那些。将确定每项任务中的表现与与个体衰老相关氧化敏感蛋白相关的特定羰基化程度之间的关联程度。

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.