CHARACTERIZING THE AGING MUSCLE MITOCHONDRIAL PROTEOME

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 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，表征和定量人肌肉mt蛋白表达的小肌肉样本（10- 100 mg），这些样本来自具有正常或受损葡萄糖耐量的年轻（18-35岁）和老年男性和女性（65- 80岁）。 我们假设，新的策略将提供更全面的细胞器特异性的肌肉mt蛋白表达模式的覆盖面，并允许更简化，比较（男性与女性，年轻与老年），和解释的结果，在年轻与老年男性和女性的低丰度肌肉mt蛋白的特征改变。 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几种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特定改变，这些改变与性别、衰老和胰岛素抵抗相关。 具体来说，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，免疫沉淀和mt蛋白富集，和二维差异荧光凝胶电泳。我们将使用各种质谱仪（MALDI-TOF-、LC-ESI-、nano-LC-FT-串联MS）鉴定和表征肌肉蛋白/肽，以进行准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。这些策略已被用于检查小生物体中的蛋白质组，但它们需要被改进，完善，并应用于涉及底物代谢紊乱的复杂人体组织（肌肉）。这些工具可以用于生物标记物的发现，开发疾病特异性肌肉mt蛋白检测阵列，并确定新的治疗靶点。

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 protein expression in small muscle samples (10-100mg) obtained from 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 muscle mt protein expression patterns, and allow for more simplified, comparative (men vs women, young vs old), and interpretable outcomes for characterizing alterations in low abundance muscle mt proteins in young vs elderly men and women. We hypothesize that by targeting muscle mt proteins and by employing MS techniques to detect several mt proteins in a single muscle sample, we will identify and characterize specific alterations in muscle mt protein expression and post-translational modifications that are associated with gender, aging and insulin resistance. Specifically, we will extract and separate muscle mt proteins using customized sub-cellular fractionation, immunoprecipitation and mt protein enrichment, and 2D-differential fluorescence gel electrophoresis. We will identify and characterize muscle protein/peptides using a variety of mass spectrometers (MALDI-TOF-, LC-ESI-, 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 advanced, refined, and applied to complex human tissues (muscle) involved in disorders of substrate metabolism. These tools can then be utilized for biomarker discovery, to develop disease-specific muscle mt protein detection arrays, and to identify novel therapeutic targets.