CHARACTERIZING THE AGING MUSCLE MITOCHONDRIAL PROTEOME

表征衰老肌肉线粒体蛋白质组

• 批准号: 7721481
• 负责人: KEVIN E YARASHESKI
• 金额: $ 0.8万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7721481
• 关键词: Aging; Amino Acid Sequence; Biological Markers; Complex; Computer Retrieval of Information on Scientific Projects Database; Detection; Disease; Elderly; Elderly man; Fluorescence; Fractionation; Funding; Gender; Glucose; Grant; Human; Immunoprecipitation; Institution; Insulin Resistance; Mass Spectrum Analysis; Measurement; Metabolic Diseases; Metabolism; Mitochondria; Muscle; Muscle Proteins; Organelles; Organism; Outcome; Pathogenesis; Pattern; Peptides; Post-Translational Protein Processing; Proteins; Proteome; Proteomics; Research; Research Personnel; Resources; Sampling; Site; Skeletal Muscle; Source; Techniques; Tissues; United States National Institutes of Health; Woman; analytical tool; base; comparative; fatty acid metabolism; gel electrophoresis; human tissue; impaired glucose tolerance; mass spectrometer; men; nano; novel strategies; novel therapeutics; protein expression; tandem mass spectrometry; therapeutic target; tool

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 骨骼肌线粒体（mt）蛋白表达的扰动可能导致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，也是葡萄糖储存的主要场所。正常的肌肉 mt 蛋白表达是正常葡萄糖和脂肪酸代谢所必需的。 然而，我们缺乏敏感、特异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、mt特异性、基于质谱（MS）的比较蛋白质组学工具和方法，可用于识别、表征和量化小肌肉样本（10-100mg）中人类肌肉mt蛋白的表达，这些样本取自具有良好特征的年轻人（18-35岁）和老年男性和女性（65-80岁），糖耐量正常或受损。 我们假设新的策略将提供更全面的肌肉 mt 蛋白表达模式的细胞器特异性覆盖，并允许更简化、比较（男性与女性、年轻与老年）和可解释的结果来表征年轻与老年男性和女性中低丰度肌肉 mt 蛋白的变化。 我们假设，通过靶向肌肉 mt 蛋白并采用 MS 技术检测单个肌肉样本中的几种 mt 蛋白，我们将识别和表征与性别、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特定变化。 具体来说，我们将使用定制的亚细胞分级分离、免疫沉淀和 mt 蛋白富集以及 2D 差异荧光凝胶电泳来提取和分离肌肉 mt 蛋白。我们将使用各种质谱仪（MALDI-TOF-、LC-ESI-、nano-LC-FT-tandem MS）来识别和表征肌肉蛋白/肽，以进行精确的质量测量和氨基酸测序。 我们将发现新的重要的 mt 蛋白形式，并产生关于老年人肌肉 mt 代谢紊乱发病机制的新方法和新假设。这些策略已用于检查小型生物体中的蛋白质组，但它们需要先进、完善并应用于涉及底物代谢紊乱的复杂人体组织（肌肉）。然后，这些工具可用于发现生物标志物、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。