Oxidized Proteome of Normal/Sideroblastic Erythroid Cell

描述(由申请人提供)：我们建议对具有特定类型的翻译后修饰蛋白羰基的蛋白质组的子集进行表征，该蛋白质与多种病理相关，包括炎症条件、神经退行性疾病、衰老和贫血。我们的目标是开发改进的试剂和技术，以研究在正常和病理状态下蛋白质羰化在细胞生理中的作用。技术能力的提高将有助于研究一些与蛋白质氧化有关的基本问题：蛋白质氧化的层次是什么--是否有特定的蛋白质首先被碳化，作为抵抗氧化侮辱的缓冲？在蛋白酶体中，碳化蛋白容易降解--“羰基感应器”蛋白的降解是否会激活细胞对氧化损伤的反应？蛋白质氧化是否有明显的标志或模式是疾病特有的？如果是这样的话，这些模式能否提供有用的诊断或预后信息？在这项提案中，我们将使用已建立的铁粒母细胞性贫血(SA)小鼠模型作为底物，用于开发和验证富集、定量和鉴定具有这种类型氧化损伤的蛋白质的新技术。然后我们将使用这些方法分析从SA患者的临床标本中分离出的纯化的铁母细胞(这种疾病的特征是铁负载细胞)。我们提出了一种从患者样本中纯化这些铁负载细胞的新方法，这将有助于这些研究。氧化蛋白质的分析将使用亲和纯化、差示凝胶分析(DGE)和质谱仪来确定小鼠模型和临床标本中的氧化蛋白质组。

DESCRIPTION (provided by applicant): We propose to characterize that subset of the proteome possessing a particular type of post-translational modification protein carbonyls that has been associated with diverse pathologies including inflammatory conditions, neurodegenerative diseases, aging and anemia. Our goal is to develop improved reagents and techniques for study of the role of protein carbonylation in cellular physiology in both normal and pathologic states. Improved technical ability will facilitate investigation of a number of basic questions regarding protein oxidation: What is the hierarchy of protein oxidation -- are there specific proteins that are carbonylated first, which serve as a buffer against oxidative insult? Carbonylated proteins are subject to degradation in the proteasome --does degradation of `carbonyl sensor' proteins activate cellular responses to oxidative damage? Are there distinct signatures or patterns of protein oxidation that are disease specific? If so, can these patterns provide useful diagnostic or prognostic information? In this proposal we will use an established mouse model of Sideroblastic Anemia (SA), in which protein carbonyls are significantly elevated, as substrate for the development and validation of novel techniques for the enrichment, quantification and identification of proteins possessing this type of oxidative lesion. We will then use these methods in the analysis of purified sideroblasts (iron loaded cells characteristic of this disorder) isolated from clinical specimens of patients with SA. We present a novel method for purification of these iron loaded cells from patient samples that will facilitate these studies. Analysis of oxidized proteins will employ affinity purification, differential gel analysis (DIGE) and mass spectrometry to define the oxidized proteome in both the mouse model and clinical specimens.