CHARACTERIZING THE AGING MUSCLE MITOCHONDRIAL PROTEOME

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。骨骼肌线粒体(MT)蛋白表达的紊乱可能参与了老年人代谢紊乱的发病机制。骨骼肌是体内最大的富含mt的组织，也是储存葡萄糖的主要部位。正常的肌肉MT蛋白表达是正常的葡萄糖和脂肪酸代谢所必需的。然而，我们缺乏灵敏、特异和全面的分析工具来研究人类肌肉mt蛋白质组。我们建议开发灵敏的、mt特异的、基于质谱学(MS)的比较蛋白质组学工具和方法，这些工具和方法可用于从转基因小鼠的小肌肉样本(10-100 mg)以及糖耐量正常或受损的青年(18-35岁)和老年男性和女性(65-80岁)中识别、表征和量化人类肌肉的mt蛋白质组。我们假设，新的策略将提供更全面的肌肉mt蛋白质组的细胞器特异性覆盖，并允许更简单、更具可比性(年轻人与老年人)和可解释的结果来表征年轻与老年以及转基因与野生型小鼠的低丰度肌肉mt蛋白的变化。我们假设，通过靶向肌肉MT蛋白和使用MS技术检测单个肌肉样本中的许多MT蛋白，我们将识别和表征与衰老和胰岛素抵抗相关的肌肉MT蛋白表达和翻译后修饰的全球变化。具体地说，我们将使用定制的亚细胞分离、蛋白质浓缩和免疫沉淀来提取和分离肌肉mt蛋白。我们将使用2D差示荧光凝胶电泳法、一维和二维液相色谱分离以及同位素编码的亲和标记，分别使用MALDI-TOF-TOF-MS和Nano-LC-FT-Tandem MS进行准确的质量测量和氨基酸测序，从而鉴定和表征肌肉MT蛋白/肽。我们将发现新的和重要的mt蛋白形式，并产生新的方法和新的假设，以肌肉mt为基础的老年人代谢紊乱的发病机制。这些策略已被用于研究小型生物的蛋白质组，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)，这最终可能导致新的治疗方法。要做到这一点，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.