QUALITATIVE AND QUANTITATIVE ANALYSIS OF THE 2SC PROTEOME IN DIABETES

糖尿病 2SC 蛋白质组的定性和定量分析

• 批准号: 7957016
• 负责人: JOHN William BAYNES
• 金额: $ 3.25万
• 依托单位: BATTELLE PACIFIC NORTHWEST LABORATORIES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7957016
• 关键词: Adipocytes; Adipose tissue; Antibodies; Biological Markers; Biology; Chemicals; Citric Acid Cycle; Computer Retrieval of Information on Scientific Projects Database; Contractile Proteins; Cysteine; Data; Databases; Development; Diabetes Mellitus; Diabetic mouse; Enzymes; Fourier transform ion cyclotron resonance; Fumarates; Funding; Glucose; Goals; Grant; Heat shock proteins; Histocompatibility Testing; Institution; Isotopes; Metabolic syndrome; Metabolism; Methods; Mitochondria; Muscle; Obesity; Oxidative Stress; Peptides; Post-Translational Protein Processing; Process; Proteins; Proteome; Proteomics; Reaction; Research; Research Personnel; Research Proposals; Resolution; Resources; Retinal; Role; Signal Transduction; Source; Spottings; Sulfhydryl Compounds; Techniques; United States National Institutes of Health; Western Blotting; adiponectin; comparative; db/db mouse; diabetic; diabetic rat; genetic regulatory protein; insight; kidney vascular structure; novel; research study; response; tandem mass spectrometry; two-dimensional; type I diabetic

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.

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 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。这种新的翻译后修饰在高糖介质中生长的脂肪细胞、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了10倍。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。2SC被认为是糖尿病线粒体和氧化应激的早期生物标志物，也可能在糖尿病或糖尿病前期状态(如肥胖和代谢综合征)糖毒性反应的信号转导中发挥作用。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。