CHARACTERIZING THE AGING MUSCLE MITOCHONDRIAL PROTEOME

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心机构，不一定是研究者机构。骨骼肌线粒体（mt）蛋白表达的紊乱可能是老年人代谢紊乱的发病机制之一。骨骼肌是人体内最大的富含mt的组织，也是葡萄糖储存的主要场所。正常肌肉mt蛋白表达是正常葡萄糖和脂肪酸代谢所必需的。然而，我们缺乏灵敏，特异性和全面的分析工具来检查人类肌肉mt蛋白质组。我们建议开发灵敏的，mt特异性的，基于质谱（MS）的比较蛋白质组学工具和方法，可用于识别，表征和定量的人肌肉mt蛋白质组的小肌肉样本（10- 100毫克）从转基因小鼠，以及良好的特征年轻（18-35岁）和老年男性和女性（65- 80岁）与正常或受损的葡萄糖耐量。我们假设，新的策略将提供更全面的细胞器特异性覆盖的肌肉mt蛋白质组，并允许更简化，比较（年轻与老年），和可解释的结果，在低丰度肌肉mt蛋白质的变化，在年轻与老年人，和转基因与野生型小鼠的特征。我们假设，通过靶向肌肉mt蛋白，并采用MS技术来检测单个肌肉样本中的许多mt蛋白，我们将识别和表征肌肉mt蛋白表达和翻译后修饰的全局变化，这些变化与衰老和胰岛素抵抗相关。具体而言，我们将提取和分离肌肉mt蛋白使用定制的亚细胞分级分离，蛋白富集和免疫沉淀。我们将使用二维差分荧光凝胶电泳、一维和二维液相色谱分离和同位素编码亲和标记来鉴定和表征肌肉mt蛋白/肽，随后分别使用MALDI-TOF-TOF-MS和nano-LC-FT-串联MS进行精确的质量测量和氨基酸测序。我们将发现新的和重要的mt蛋白的形式，并产生新的方法和新的假说的发病机制的肌肉mt为基础的代谢紊乱的老年人。这些策略已用于检查小生物体中的蛋白质组，但它们需要应用于涉及人类底物代谢紊乱的复杂人体组织（肌肉），这可能最终导致新的治疗方法。为此，我们将利用华盛顿大学的专业知识和MS仪器。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Perturbations in skeletal muscle mitochondrial (mt) protein expression may contribute to the pathogenesis of metabolic disorders in the elderly. Skeletal muscle is the largest, mt-rich tissue in the body and the primary site for glucose storage. Normal muscle mt protein expression is required for normal glucose and fatty acid metabolism. However, we lack sensitive, specific and comprehensive analytical tools for examining the human muscle mt proteome. We propose to develop sensitive, mt-specific, mass spectrometry (MS)-based comparative proteomics tools and approaches that can be used to identify, characterize and quantify the human muscle mt proteome in small muscle samples (10-100mg) obtained from genetically modified mice, and well-characterized young (18-35 yr) and elderly men and women (65-80yr) with normal or impaired glucose tolerance. We hypothesize that novel strategies will provide more comprehensive organelle-specific coverage of the muscle mt proteome, and allow for more simplified, comparative (young vs old), and interpretable outcomes for characterizing alterations in low abundance muscle mt proteins in young vs elderly adults, and genetically modified vs wild-type mice. We hypothesize that by targeting muscle mt proteins and by employing MS techniques to detect many mt proteins in a single muscle sample, we will identify and characterize global alterations in muscle mt protein expression and post-translational modifications that are associated with aging and insulin resistance. Specifically, we will extract and separate muscle mt proteins using customized sub-cellular fractionation, protein enrichment and immunoprecipitation. We will identify and characterize muscle mt proteins/peptides using 2D-differential fluorescence gel electrophoresis, 1D-and 2D-liquid chromatographic separation, and isotope coded affinity tagging, each followed by MALDI-TOF-TOF-MS and nano-LC-FT-tandem MS for accurate mass measurements and amino acid sequencing. We will discover new and important mt protein forms, and generate novel approaches and new hypotheses about the pathogenesis of muscle mt-based metabolic disorders in the elderly. These strategies have been used to examine proteomes in small organisms, but they need to be applied to complex human tissues (muscle) involved in disorders of human substrate metabolism, and that might ultimately lead to novel treatments. To do this, we will take advantage of the expertise and MS instrumentation available at Washington University.