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.

摘要