MULTIPLEX CHEMICAL TAGS FOR HIGH-THROUGHPUT GLYCAN AND GLYCOPEPTIDE QUANTITATION AND CHARACTERIZATION

用于高通量聚糖和糖肽定量和表征的多重化学标签

• 批准号: 9755397
• 负责人: LINGJUN LI
• 金额: $ 44.02万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-03 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9755397
• 关键词: Address; Amines; Benchmarking; Biological; Biological Process; Capillary Electrophoresis; Carbohydrates; Carbon; Cardiovascular system; Cell Adhesion; Chemicals; Communities; Complement; Complex; Coupled; Databases; Defect; Detection; Digestion; Disease; Disease Pathway; Dissociation; Electron Transport; Glycine; Glycopeptides; Glycoproteins; Goals; Immune System Diseases; Ions; Isomerism; Isotopes; Label; Leucine; Ligand Binding; Link; Liquid Chromatography; Malignant Neoplasms; Mass Spectrum Analysis; Methods; Natural graphite; Neurodegenerative Disorders; Ornithine; Pathway interactions; Peptide N-glycohydrolase F; Peptides; Performance; Play; Polysaccharides; Post-Translational Protein Processing; Protein Analysis; Protein Glycosylation; Proteins; Reagent; Reporter; Research; Research Personnel; Resolution; Role; Sampling; Site; Spectrometry, Mass, Electrospray Ionization; Structure; Techniques; base; clinically relevant; cost effective; design; experimental study; glycoproteomics; glycosylation; human disease; improved; innovation; instrument; instrumentation; intercellular communication; ion mobility; mass spectrometer; novel; pancreatic cancer cells; protein complex; protein transport; receptor; response; sialylation; tool; two-dimensional

ABSTRACT Gycosylation is one of the most important and most complex protein post-translational modifications. Studies have shown that the glycan moieties on glycoproteins play critical roles in structural modulation and function as specific binding ligands for endogenous receptors or exogenous agents in many biological processes such as protein trafficking, cell−cell signaling, and cellular adhesion. Alterations in glycomic profiles have been linked to various diseases, including cancer, neurodegenerative disorders, immunological diseases and cardiovascular problems. These implications urge researchers to develop innovative cutting-edge bioanalytical platforms for quantitative analysis of glycans to facilitate elucidation of the diverse biological roles of glycans and their roles in human diseases. Advances in mass spectrometry (MS)-based glycoproteomics and glycomics are increasingly enabling qualitative and quantitative approaches for site-specific structural analysis of protein glycosylation. However, quantitative analysis of native glycans remains extremely challenging due to high complexity and diversity of glycan structures, difficulty of synthesizing glycan standards, the relatively low response in MS detection, and the wide dynamic range of glycans in clinically relevant samples. The primary goal of this proposal is to develop several versatile mass defect-based multiplex tags for high- throughput quantification of glycans and glycopeptides in complex biological samples using high resolution mass spectrometry (MS) instrumentation and ion mobility (IM) MS coupled with multidimensional separation techniques. We propose the following specific aims: Specific Aim 1 – To develop and validate novel mass defect-based multiplex dimethyl pyrimidinyl ornithine (DiPyrO) tags for cost effective and high-throughput MS1-level relative quantification of N-glycans released from biological samples. Specific Aim 2 – To design and synthesize multiplex isobaric multiplex reagents for carbonyl containing compounds (SUGAR) tags for high-throughput MS2-level glycan characterization and relative quantitation. Specific Aim 3 – To develop and implement a novel capillary electrophoresis (CE)/porous graphite carbon (PGC)-LC-IM-MS platform for isomer-specific quantitative glycomics and glycoproteomics analysis, particularly α2,3-/α2,6-sialylation ratio analysis, and construction of intact N-glycopetide, N-glycan, and deglycosylated peptide collision cross section (CCS) database facilitated by electron-transfer high energy collision dissociation (EThcD)-enabled highly confident identification. Collectively, our proposed experiments will develop novel enabling tools and will generate cost-effective and novel mass defect-based labeling reagents for robust, sensitive and accurate glycan analysis with enhanced quantitative performance and structural elucidation capabilities. The performance of these tags will be cross validated within the glycoscience community.

抽象的 妇科基化是翻译后最重要，最复杂的蛋白质之一。研究 已经表明，糖蛋白上的聚糖部分在结构调制和功能中起关键作用 在许多生物学过程中，内源性受体或外源性剂的特定结合配体，例如 蛋白质运输，细胞信号传导和细胞粘合剂。胶质剖面的改变已链接到 各种疾病，包括癌症，神经退行性疾病，免疫疾病和心血管疾病 问题。这些含义促使研究人员开发创新的尖端生物分析平台 对聚糖的定量分析，以促进聚糖的潜水生物学作用及其作用 在人类疾病中。质谱（MS）基于糖蛋白质组学和糖果学的进步是 越来越有助于定性和定量方法，用于蛋白质的位点特异性结构分析 糖基化。但是，由于高的量子，对天然聚糖的定量分析仍然极为挑战 聚糖结构的复杂性和多样性，难以合成聚糖标准，相对较低 MS检测的反应，以及临床相关样品中的聚糖的广泛动态范围。主要 该提案的目标是为高 - 使用高的复杂生物样品中的糖量和糖肽的吞吐量数量 分辨率质谱（MS）仪器和离子迁移率（IM）MS与 多维分离技术。我们提出以下具体目标：特定目标1 - 开发和验证基于质量缺陷的新型多重多重二甲基吡啶基鸟氨酸（二皮酸）标签的成本 从生物样品释放的N-聚糖的有效和高通量MS1级相对定量。 特定目的2 - 设计和合成含羰基的多种同型多重试剂 高通量MS2级聚糖表征和相对定量的化合物（糖）标签。 特定目标3 - 开发和实施新型毛细管电泳（CE）/多孔石墨碳 （PGC）-LC-IM-MS平台，用于异构体特异性定量糖果学和糖蛋白质组学分析，尤其是 α2,3-/α2,6-溶解率分析，以及完整的N-聚酯，N-聚糖和脱脂糖基化的构建 通过电子转移高能碰撞解离制备的肽碰撞横截面（CCS）数据库 （ETHCD）支持高度自信的标识。总体而言，我们提出的实验将发展出新颖 启用工具，并将生成具有成本效益和新颖的基于质量缺陷的标签试剂，以实现鲁棒 具有增强的定量性能和结构阐明的敏感和准确的聚糖分析 功能。这些标签的性能将在聚糖社区中进行交叉验证。