Adv.Proteomics & Metabolomics:Type 2 Diabet & Pre-Diabet

高级蛋白质组学

• 批准号: 7684251
• 负责人: RICHARD D SMITH
• 金额: $ 57.03万
• 依托单位: BATTELLE PACIFIC NORTHWEST LABORATORIES
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-06-20 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7684251
• 关键词: Age; Algorithms; American; Biological Assay; Biological Markers; Blood Cells; Blood Glucose; Caliber; Cells; Chemicals; Chromatography; Chronic Disease; Comparative Study; Complement; Complications of Diabetes Mellitus; Cysteine; Data Analyses; Databases; Development; Diabetes Mellitus; Diagnosis; Disease; Erythrocytes; Fasting; Feasibility Studies; Fourier transform ion cyclotron resonance; Generations; Glycopeptides; Glycoproteins; Goals; Housing; Human; Individual; Insulin; Insulin Resistance; Ions; Kidney Diseases; Label; Link; Liquid Chromatography; Liquid substance; Lymphocyte; Maillard Reaction; Mammalian Cell; Mass Spectrum Analysis; Measures; Methods; Modification; National Institute of Diabetes and Digestive and Kidney Diseases; Neuropathy; Newly Diagnosed; Non-Insulin-Dependent Diabetes Mellitus; OGTT; Pancreas; Patients; Pattern Recognition; Peptides; Peripheral; Phase; Pilot Projects; Plasma; Plasma Cells; Plasma Proteins; Prediabetes syndrome; Process; Production; Proteins; Proteome; Proteomics; Protocols documentation; Reproducibility; Research; Research Design; Research Personnel; Resistance; Resolution; Retinal Diseases; Sampling; Severities; Site; Solvents; South Carolina; Specific qualifier value; Stable Isotope Labeling; Techniques; Technology; Time; Tissues; United States; Universities; Validation; base; capillary liquid chromatography; comparative; diabetic; experience; glucose tolerance; glycation; impaired glucose tolerance; improved; in vivo; insulin secretion; ionization; macrovascular disease; metabolomics; novel; programs; protein metabolite

DESCRIPTION (provided by applicant): In this 5 year (R21/R33) project we will apply advanced proteomic and metabolomic nanoflow liquid chromatography-Fourier transform ion cyclotron resonance (FTICR) mass spectrometry technologies in the study of both plasma and blood cells from individuals with normal glucose tolerance (NOT), impaired glucose tolerance (IGT), and recently diagnosed type 2 diabetes (T2DM). The overall approach endeavors to advance the study of Type 2 diabetes and pre-diabetes by identifying biomarkers at the level of the proteome and metabolome that are predictive of Type 2 diabetes and pre-diabetes in vivo. Our approach will utilize proteome-wide stable isotope labeling of peptides, as well as quantitative cysteine-peptide enrichment technology (QCET) and N-linked glycopeptide enrichment strategies to obtain broad proteome coverage and enhance quantitation. We will also utilize very low nanoflow LC separations to minimize ionization suppression and eliminate background ions originating from the solvent, thereby improving normalization of metabolite peak intensities and improve quantitation. This approach will be capable of rapidly identifying and measuring expression levels for thousands of peptides or concentrations of metabolites in a single analysis. Phase 1 of this project will (a) Define the sample processing and LC separation conditions necessary for broad proteome and metabolome coverage in human plasma and blood cell samples, (b) Perform a pilot study to define specific differences in the plasma proteome and metabolome of 10 individuals each with NGT, IGT, and T2DM, (c) Establish accurate mass and time tag databases for both peptides and metabolites detected in plasma and blood cells from individuals with NGT, IGT, and T2DM, and (d) Perform a pilot study to determine the feasibility of applying the nanoflow FTICR approach in the identification of non-enzymatically glycated plasma proteins. The refinement of this technological approach will provide the basis for high throughput studies of large numbers of samples. The application of this technology in Phase 2 of the project will involve (a) High throughput studies of large numbers of plasma and blood cell samples from individuals with NGT, IGT, and T2DM, (b) Validation of proteomic and metabolomic biomarkers of T2DM and pre-diabetes identified in Phase 1, and (c) Refinement of the sample processing and data analysis approach used in the identification of non-enzymatically glycated plasma proteins (Phase 1), in order to identify plasma proteins containing advanced glycation and/or advanced lipoxidation end products (AGEs/ALEs).

描述（由申请人提供）：在这个为期5年（R21/R33）的项目中，我们将应用先进的蛋白质组学和代谢组学纳流液相色谱-傅里叶变换离子回旋共振（FTICR）质谱技术来研究个体的血浆和血细胞糖耐量正常 (NOT)、糖耐量受损 (IGT) 和最近诊断的 2 型糖尿病 (T2DM)。总体方法致力于通过识别蛋白质组和代谢组水平上可预测体内 2 型糖尿病和糖尿病前期的生物标志物来推进 2 型糖尿病和糖尿病前期的研究。我们的方法将利用全蛋白质组稳定同位素标记肽，以及定量半胱氨酸肽富集技术 (QCET) 和 N-连接糖肽富集策略，以获得广泛的蛋白质组覆盖并增强定量。我们还将利用非常低的纳流 LC 分离来最大限度地减少电离抑制并消除源自溶剂的背景离子，从而改善代谢物峰强度的标准化并改善定量。这种方法将能够在一次分析中快速识别和测量数千种肽的表达水平或代谢物的浓度。该项目的第一阶段将 (a) 确定样品处理和 LC 分离条件，以实现人血浆和血细胞样品中广泛的蛋白质组和代谢组覆盖，(b) 进行试点研究，以确定血浆蛋白质组和代谢组的具体差异10 名 NGT、IGT 和 T2DM 个体，(c) 建立 NGT、IGT 和 T2DM 个体血浆和血细胞中检测到的肽和代谢物的精确质量和时间标签数据库，以及(d) 进行试点研究以确定应用纳流 FTICR 方法鉴定非酶糖化血浆蛋白的可行性。这种技术方法的完善将为大量样品的高通量研究提供基础。该技术在该项目第二阶段的应用将涉及 (a) 对来自 NGT、IGT 和 T2DM 患者的大量血浆和血细胞样本进行高通量研究，(b) T2DM 和 T2DM 的蛋白质组和代谢组生物标志物的验证(c) 改进用于鉴定非酶糖化血浆蛋白（第一阶段）的样品处理和数据分析方法，以便鉴定含有晚期糖化和/或晚期脂氧化终产物 (AGE/ALE) 的血浆蛋白。