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

摘要 糖基化是蛋白质翻译后修饰中最重要、最复杂的修饰之一。研究 已经表明，糖蛋白上的聚糖部分在结构调节中起着关键作用， 在许多生物学过程中， 蛋白质运输、细胞-细胞信号传导和细胞粘附。糖组学特征的改变与以下因素有关： 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 问题这些影响促使研究人员开发创新的尖端生物分析平台， 聚糖的定量分析，以便于阐明聚糖的各种生物学作用及其作用 在人类疾病中。介绍了基于质谱的糖蛋白质组学和糖组学的研究进展 越来越多的定性和定量方法用于蛋白质的位点特异性结构分析 糖基化然而，天然聚糖的定量分析仍然极具挑战性，这是由于高浓度的聚糖。 聚糖结构的复杂性和多样性，合成聚糖标准品的难度， MS检测中的响应，以及临床相关样品中聚糖的宽动态范围。主 该建议的目标是开发几种通用的基于质量缺陷的多重标签，用于高- 高通量定量复杂生物样品中的聚糖和糖肽 分辨率质谱（MS）仪器和离子迁移率（IM）MS， 多维分离技术我们提出以下具体目标： 开发和验证新型的基于质量缺陷的多重二甲基嘧啶基鸟氨酸（DiPyrO）标签， 有效和高通量的MS 1水平相对定量从生物样品中释放的N-聚糖。 具体目标2 -设计和合成用于含羰基化合物的多元同量异位试剂 用于高通量MS 2水平聚糖表征和相对定量的化合物（SUGAR）标签。 具体目标3 -开发和实现一种新型的毛细管电泳（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 α 2，3-/α 2，6-唾液酸化比率分析，以及完整N-糖肽、N-聚糖和去糖基化 电子转移高能碰撞解离促进的肽碰撞截面（CCS）数据库 （EThcD）启用的高度可信的识别。总的来说，我们提出的实验将开发新的 使工具，并将产生成本有效的和新的质量缺陷为基础的标记试剂， 具有增强的定量性能和结构解析的灵敏和准确的聚糖分析 能力的这些标签的性能将在糖科学界进行交叉验证。

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.