Mitochondrial Proteomics of the Aging Heart

描述（由申请人提供）：心力衰竭仍然是美国65岁以上人群的主要死因。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，但由于衰老导致的线粒体蛋白损伤的性质和程度在很大程度上是未知的。待评价的总体假设是，由于脂质过氧化产物和谷胱甘肽化的加合导致线粒体蛋白的损伤导致衰老心脏中线粒体功能的丧失。为了验证这一假设，将使用功能蛋白质组学方法，该方法基于通过质谱法对蛋白质进行鉴定和定量以及通过多变量方法对数据进行分析。 具体目标：（1）通过使用巯基特异性探针选择性检测含有游离暴露的半胱氨酸的线粒体蛋白，目的是鉴定作为氧化损伤的潜在靶标的巯基蛋白。(2)表征和定量脂质过氧化产物与巯基蛋白的加合。(3)识别和定量谷胱甘肽化蛋白质。(4)确定衰老过程中脂质过氧化产物或谷胱甘肽化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变化相关联。线粒体巯基蛋白的氧化脂质加合和谷胱甘肽化的水平预计是低的，因此特定的同位素编码的亲和标签将用于富集这些修饰的蛋白质。通过比较年轻和老年大鼠心脏的线粒体，采用同位素稀释质谱法实现蛋白质定量。 重要性：这些研究将提供独特的见解，涉及特定的蛋白质修饰如何通过使用新的基于质谱的功能蛋白质组学方法影响线粒体酶功能。这些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氧化修饰的独特线粒体蛋白靶点。

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.