QUALITATIVE AND QUANTITATIVE ANALYSIS OF THE 2SC PROTEOME IN DIABETES

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH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 中心，不一定是研究者的机构。 S-（2-琥珀酰）半胱氨酸（2SC）是克雷布斯循环中间体、富马酸盐和蛋白质中巯基之间的迈克尔加成反应的产物-这一过程称为蛋白质的琥珀酰化。 这种新的翻译后修饰在高葡萄糖培养基中生长的脂肪细胞、1型糖尿病大鼠的肌肉和2型糖尿病（db/db）小鼠的脂肪组织中增加> 10倍。 用2D-PAGE和抗2SC抗体的Western blotting在脂肪细胞和脂肪组织蛋白中检测到60多个2SC蛋白点，包括脂联素、收缩蛋白、热休克蛋白、酶和调节蛋白。 2SC被认为是糖尿病中线粒体和氧化应激的早期生物标志物，并且还可能在响应糖尿病或糖尿病前期状态（例如肥胖和代谢综合征）中的葡萄糖毒性的信号转导中起作用。 本项目的目标是确定db/db小鼠脂肪组织中的2SC蛋白质组，从而深入了解这种蛋白质的化学修饰在糖尿病代谢改变中的作用。 该项目将利用PNNL的资源对肽进行高分辨率二维LC分离，并通过串联质谱法（MS/MS）进行鉴定，以开发来自高富马酸盐浓度脂肪细胞蛋白孵育的所有可能的2SC肽的数据库。 随后的高分辨率LC-FTICR MS实验以及双同位素标记技术将用于对照和糖尿病小鼠脂肪组织的比较分析。 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提案的初步数据。 平行应用可能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. 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. 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.