Characterizing the HIV-Muscle Mitochondrial Proteme

描述（由申请人提供）：骨骼肌线粒体（mt）蛋白表达的扰动可能与接受胸腺嘧啶类似物抗逆转录病毒疗法（AZT-或d4T-ARV）治疗的hiv感染者代谢紊乱的发病机制有关。骨骼肌是人体最大的富含mt的组织，也是葡萄糖储存的主要部位。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检测人类肌肉蛋白质组。我们建议开发敏感的，mt特异性的，基于质谱的比较蛋白质组学工具和方法，可用于鉴定，表征和量化人类肌肉mt蛋白质组，这些样品先前从四组已被充分表征的受试者中获得：hiv血清阴性，正常葡萄糖耐量（NGT）；HIV+对ARV和NGT的初次接触，HIV+接受AZT或基于d4t的ARV和NGT；以及接受基于AZT或d4的ARV治疗并伴有胰岛素抵抗的HIV阳性患者。我们假设基于AZT或d4T的方案损害肌肉mt蛋白表达并诱导与hiv相关胰岛素抵抗相关的mt蛋白的翻译后修饰。具体来说，我们将使用定制的亚细胞分离、蛋白质富集和耗尽方法、2D-差示荧光凝胶电泳和1d -液相色谱分离肌肉mt蛋白。我们将使用多种质谱仪（MALDI-TOF, LC-ESI-， nano- lc - ft -串联质谱）进行精确的质量测量和氨基酸测序，以鉴定和表征肌肉蛋白/肽。我们将发现新的和重要的mt蛋白形式，这将为hiv感染者肌肉mt代谢紊乱的发病机制提供新的方法和新的假设。这些分析方法和工具已经被用于检测小生物中的蛋白质组，但我们需要推进和促进它们在复杂的人体组织（肌肉）中的应用，这些组织与人体底物代谢紊乱密切相关，这可能最终导致新的治疗策略。为了实现这一目标，我们将利用华盛顿大学的专业知识和各种高分辨率质谱仪器。

DESCRIPTION (provided by applicant): Perturbations in skeletal muscle mitochondrial (mt) protein expression may contribute to the pathogenesis of metabolic disorders in HIV-infected people treated with thymidine-analog-based antiretroviral therapy (AZT- or d4T-ARV). 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-based comparative proteomics tools and approaches that can be used to identify, characterize, and quantify the human muscle mt proteome in specimens previously obtained from 4 groups of well characterized subjects: HIV-seronegative with normal glucose tolerance (NGT); HIV+ naive to ARV with NGT, HIV+ receiving AZT- or d4T-based ARV with NGT; and HIV+ receiving AZT- or d4T-based ARV with insulin resistance. We hypothesize that AZT- or d4T- based regimens impair muscle mt protein expression and induce post-translational modifications to mt proteins that are associated with HIV-related insulin resistance. Specifically, we will separate muscle mt proteins using customized sub-cellular fractionation, protein enrichment and depletion methods, 2D- differential fluorescence gel electrophoresis, and 1D-liquid chromatography. 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 that will generate novel approaches and new hypotheses about the pathogenesis of muscle mt-based metabolic disorders in HIV-infected people. These analytical approaches and tools have been used to examine proteomes in small organisms, but we need to advance and facilitate their application to complex human tissues (muscle) that are critically involved in disorders of human substrate metabolism, and that might ultimately lead to novel treatment strategies. To accomplish this, we will take advantage of the expertise and the wide variety of high resolution mass spectrometry instrumentation available at Washington University.