Oxidized Proteome of Normal/Sideroblastic Erythroid Cell

描述（由申请人提供）：我们建议表征具有特定类型的翻译后修饰蛋白羰基的蛋白质组的子集，其与多种病理学相关，包括炎症性疾病、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，用于研究蛋白质羰基化在正常和病理状态下的细胞生理学中的作用。提高技术能力将有助于研究一些关于蛋白质氧化的基本问题：蛋白质氧化的层次是什么--是否有特定的蛋白质首先被羰基化，作为抗氧化损伤的缓冲剂？羰基化蛋白在蛋白酶体中易降解-“羰基传感器”蛋白的降解是否激活细胞对氧化损伤的反应？是否存在疾病特异性的蛋白质氧化的不同特征或模式？如果是这样，这些模式能否提供有用的诊断或预后信息？在这项提案中，我们将使用一个已建立的小鼠模型铁幼细胞贫血（SA），其中蛋白质羰基显着升高，作为基板的开发和验证的新技术的富集，定量和鉴定具有这种类型的氧化损伤的蛋白质。然后，我们将使用这些方法在分析纯化的铁粒幼细胞（铁负载细胞的特点，这种疾病）分离与SA患者的临床标本。我们提出了一种新的方法纯化这些铁负载细胞从患者样本，将促进这些研究。氧化蛋白的分析将采用亲和纯化、差异凝胶分析（DIGE）和质谱法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.