Mitochondrial Proteomics of the Aging Heart

描述(申请人提供)：心力衰竭仍然是美国65岁以上老年人的主要死因。线粒体蛋白质的氧化损伤被认为是衰老过程中线粒体功能下降和心肌衰退的重要原因，但衰老对线粒体蛋白质造成的损伤的性质和程度在很大程度上尚不清楚。有待评估的总体假设是，由于脂质过氧化产物和谷胱甘肽基化对线粒体蛋白质的损伤，导致了老化心脏线粒体功能的丧失。为了验证这一假设，将使用一种功能蛋白质组学方法，基于质谱学对蛋白质的鉴定和定量，并使用多变量方法对数据进行分析。 具体目的：(1)通过使用硫醇特异的探针选择性地检测含有游离暴露的半胱氨酸的线粒体蛋白，目的是识别潜在的氧化损伤的硫醇蛋白。(2)表征和定量脂质过氧化产物对硫醇蛋白的加合作用。(3)鉴定和定量谷胱甘肽基化蛋白。(4)测定在衰老过程中被脂质过氧化产物或谷胱甘肽高度修饰的线粒体酶的活性，目的是将蛋白质组学数据与酶活性的变化联系起来。预计线粒体硫醇蛋白的氧化脂质加合和谷胱甘肽基化水平较低，因此将使用特定的同位素编码的亲和标记来浓缩这些修饰的蛋白质。通过比较年轻和老年大鼠心脏的线粒体，将使用同位素稀释质谱仪实现蛋白质的定量。 意义：这些研究将通过使用新的基于质谱学的功能蛋白质组学方法，提供关于特定蛋白质修饰如何影响线粒体酶功能的独特见解。这一结果将成为我们长期目标的基础，以确定影响老年人整体细胞器和心脏功能的独特线粒体蛋白质氧化修饰靶点。

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.