Mitochondrial Proteomics of the Aging Heart

描述（由申请人提供）：心力衰竭仍然是美国65岁以上人群死亡的主要原因。线粒体蛋白的氧化损伤被认为在衰老过程中线粒体功能下降和心肌衰退中起重要作用，但线粒体蛋白因衰老而损伤的性质和程度在很大程度上是未知的。要评估的总体假设是，由于脂质过氧化产物和谷胱甘肽化内聚导致线粒体蛋白损伤，导致老化心脏中线粒体功能丧失。为了验证这一假设，将使用功能蛋白质组学方法，通过质谱法对蛋白质进行鉴定和定量，并通过多元方法对数据进行分析。

DESCRIPTION (provided by applicant): Heart failure remains the leading cause of death for people over the age of 65 in the US. Oxidative damage to mitochondrial proteins is thought to play an important role in decline of mitochondrial function and myocardial decay during the aging process, but the nature and extent of damage to mitochondrial proteins as a result of aging is largely unknown. The overall hypothesis to be evaluated is that damage to mitochondrial proteins as a result of adduction of lipid peroxidation products and glutathionylation contributes to loss of mitochondrial function in the aging heart. To test this hypothesis, a functional proteomics approach will be used based on identification and quantification of proteins by mass spectrometry and analysis of the data by multivariate methods. Specific Aims: (1) Selective detection of mitochondrial proteins that contain free, exposed cysteines by using a thiol-specific probe with the aim to identify thiol proteins that are potential targets of oxidative damage. (2) To characterize and quantify adduction of lipid peroxidation products to thiol proteins. (3) To identify and quantify glutathionylated proteins. (4) To determine the activity of mitochondrial enzymes that are highly modified by lipid peroxidation products or by glutathionylation during aging, with the aim to correlate the proteomic data with changes in enzyme activity. The levels of oxylipid adduction and glutathionylation of mitochondrial thiol proteins are anticipated to be low, and therefore specific isotope-coded affinity tags will be used for enrichment of these modified proteins. Protein quantification will be achieved with isotope-dilution mass spectrometry by comparing mitochondria from young and old rat hearts. Significance: These studies will provide unique insights relating how specific protein modification may impact mitochondrial enzyme function by using novel mass spectrometry-based functional proteomics approaches. The results will be the basis for our long-term goals to identify unique mitochondrial protein targets for oxidative modification that affect overall organelle and cardiac function in the elderly.