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 糖基化是最重要和最复杂的蛋白质翻译后修饰之一。 研究表明，糖蛋白上的聚糖部分在结构调节和功能中发挥着关键作用 许多生物过程中内源性受体或外源性试剂的特异性结合配体，例如 蛋白质运输、细胞间信号传导和细胞粘附与糖组谱的改变有关。 各种疾病，包括癌症、神经退行性疾病、免疫性疾病和心血管疾病 这些问题促使研究人员开发创新的尖端生物分析平台。 聚糖分析有助于阐明聚糖的多种生物学作用及其作用 基于质谱 (MS) 的糖蛋白组学和糖组学的进展。 越来越多地采用定性和定量方法进行蛋白质位点特异性结构分析 然而，由于糖基化程度高，天然聚糖的定量分析仍然极具挑战性。 聚糖结构复杂多样，聚糖标准品合成困难，相对较低 MS 检测中的响应，以及临床相关样品中聚糖的宽动态范围。 该提案的目标是开发几种基于质量缺陷的多功能标签，用于高 使用高通量对复杂生物样品中的聚糖和糖肽进行通量定量 分辨率质谱 (MS) 仪器和离子淌度 (IM) MS 结合 我们提出以下具体目标： 具体目标 1 – 达到 开发和验证新型基于质量缺陷的多重二甲基嘧啶基鸟氨酸 (DiPyrO) 标签的成本 对生物样品中释放的 N-聚糖进行有效且高通量的 MS1 水平相对定量。 具体目标 2 – 设计和合成含羰基的多重同量异位多重试剂 用于高通量 MS2 水平聚糖表征和相对定量的化合物 (SUGAR) 标签。 具体目标 3 – 开发并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组学和糖蛋白质组学分析，特别是 α2,3-/α2,6-唾液酸化比率分析以及完整 N-糖肽、N-聚糖和去糖基化的构建 电子转移高能碰撞解离促进的肽碰撞截面（CCS）数据库 总的来说，我们提出的实验将开发出新颖的实验。 使能工具，并将产生具有成本效益和新颖的基于质量缺陷的标记试剂，以实现稳健、 灵敏、准确的聚糖分析，具有增强的定量性能和结构解析 这些标签的性能将在糖科学界进行交叉验证。