QUALITATIVE AND QUANTITATIVE ANALYSIS OF THE 2SC PROTEOME IN DIABETES

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 S-(2-琥珀酰基)半胱氨酸(2SC)是蛋白质中Krebs环中间体富马酸和硫醇基团发生Michael加成反应的产物，这一过程被称为蛋白质琥珀酸化。2D-PAGE和免疫印迹检测到脂肪细胞和脂肪组织中有60多个2SC蛋白斑点，包括脂联素、收缩蛋白、热休克蛋白、酶和调节蛋白。该项目将利用PNNL的资源进行多肽的高分辨率二维LC分离，并结合串联质谱仪(MS/MS)进行鉴定，以开发一个数据库，其中包括所有可能的富马酸浓度高的脂肪细胞蛋白孵育的2SC多肽。随后的高分辨率LC-FTICR MS实验将与双同位素标记技术一起用于对比分析对照组和糖尿病小鼠的脂肪组织。在这些研究中获得的信息和开发的方法将被用作NIH关于琥珀酸化在糖尿病及其视网膜、肾脏和血管并发症发展中的作用的研究建议的初步数据。平行应用可包括分析糖尿病中蛋白质的其他化学修饰的范围和程度，包括对蛋白质的氧化、糖氧化和脂氧化损伤。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. S-(2-succinyl)cysteine (2SC) is the product of a Michael addition reaction between the Krebs cycle intermediate, fumarate, and thiol groups in proteins - a process termed succination of protein. This novel post-translational modification increases >10 fold in adipocytes grown in high glucose medium, in muscle of type 1 diabetic rats, and in adipose tissue of type 2 diabetic (db/db) mice. Over 60 2SC-protein spots have been detected by 2D-PAGE and western blotting with anti-2SC antibody in adipocyte and adipose tissue proteins, including adiponectin, contractile proteins, heat shock proteins, enzymes and regulatory proteins. 2SC is considered an early biomarker of mitochondrial and oxidative stress in diabetes and may also have a role in signal transduction in response to glucotoxicity in diabetes or pre-diabetic states, such as obesity and metabolic syndrome. The goal of this project is to define the 2SC proteome in adipose tissue of db/db mice and thereby to gain insight into the role of this chemical modification of protein in the alteration of metabolism in diabetes. The project will employ resources at PNNL for high resolution two-dimensional LC separations of peptides in conjunction with identification by tandem mass spectrometry (MS/MS) in order to develop a database of all possible 2SC-peptides from incubation of adipocyte proteins with high fumarate concentration. Subsequent high resolution LC-FTICR MS experiments together with a double isotope tagging technique will be used for comparative analysis of adipose tissue from control and diabetic mice. The information obtained and methods developed in these studies will be used as preliminary data for NIH research proposals on the role of succination in development of diabetes and its retinal, renal and vascular complications. Parallel applications could include analysis of the scope and extent of other chemical modifications of protein in diabetes, including oxidative, glycoxidative and lipoxidative damage to proteins.