Mitochondrial Proteomics of the Aging Heart

衰老心脏的线粒体蛋白质组学

• 批准号: 7092144
• 负责人: CLAUDIA S MAIER
• 金额: $ 27.64万
• 依托单位: OREGON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-30 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7092144
• 关键词: adduct; aging; carnitine palmitoyltransferase 1; clinical research; cytochrome oxidase; electrospray ionization mass spectrometry; enzyme activity; heart; laboratory rat; liquid chromatography mass spectrometry; mitochondria; oxidative stress; peroxidation; posttranslational modifications; protein quantitation /detection; proteomics; pyruvate dehydrogenase

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.

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发挥着重要作用，但衰老导致的线粒体蛋白损伤的性质和程度在很大程度上尚不清楚。待评估的总体假设是，由于脂质过氧化产物和谷胱甘肽加合而导致的线粒体蛋白质损伤导致衰老心脏中线粒体功能的丧失。为了检验这一假设，将使用功能蛋白质组学方法，该方法基于质谱法对蛋白质的识别和定量以及多变量方法的数据分析。 具体目标：（1）使用硫醇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，旨在识别作为氧化损伤潜在目标的硫醇蛋白。 (2) 表征和量化脂质过氧化产物与硫醇蛋白的加合。 (3) 鉴定和定量谷胱甘肽化蛋白质。 (4) 测定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，旨在将蛋白质组数据与酶活性的变化联系起来。线粒体硫醇蛋白的氧脂加合和谷胱甘肽化水平预计较低，因此特定同位素编码的亲和标签将用于富集这些修饰的蛋白。通过比较年轻和年老大鼠心脏的线粒体，利用同位素稀释质谱法实现蛋白质定量。 意义：这些研究将通过使用基于质谱的新型功能蛋白质组学方法，提供有关特定蛋白质修饰如何影响线粒体酶功能的独特见解。这些结果将成为我们长期目标的基础，即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标。