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

摘要 糖基化是蛋白质翻译后最重要、最复杂的修饰之一。研究 已经表明，糖蛋白上的糖链部分在结构调节中起着关键作用，并具有如下功能 内源性受体或外源性试剂在许多生物过程中的特定结合配体，如 蛋白质运输、细胞−、细胞信号和细胞黏附。血糖水平的改变与 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 有问题。这些影响促使研究人员开发创新的尖端生物分析平台 多聚糖的定量分析有助于阐明多聚糖的不同生物学作用及其作用 在人类疾病方面。基于MS的糖蛋白组学和糖组学研究进展 为蛋白质的特定部位结构分析提供越来越多的定性和定量方法 糖基化。然而，对天然多糖的定量分析仍然具有极大的挑战性，因为 多糖结构的复杂性和多样性，多糖标准品的合成难度相对较低 MS检测中的响应，以及临床相关样本中葡聚糖的广泛动态范围。初级阶段 该方案的目标是开发几个通用的基于质量缺陷的多路标签，用于高性能标签。 高效毛细管电泳法定量测定复杂生物样品中的糖链和糖肽 分辨率质谱仪和离子迁移率(IM)质谱仪与 多维分离技术。我们提出以下具体目标：具体目标1-- 开发和验证基于质量缺陷的新型复合二甲基嘧啶鸟氨酸(DiPyrO)标签的成本 从生物样品中释放的N-糖链的有效和高通量MS1水平的相对定量。 特定目标2-设计和合成含羰基的多元等压多重试剂 化合物(糖)标签，用于高通量MS2水平的糖链表征和相对定量。 具体目标3-开发和实现一种新型毛细管电泳法/多孔石墨炭 (PGC)-LC-IM-MS平台用于异构体特异性定量糖组和糖蛋白质组分析，特别是 α2，3-/α2，6-唾液酸化比率分析及完整N-糖肽、N-糖链和脱糖基的构建 电子转移高能碰撞解离促进的肽碰撞截面(CCS)数据库 (EThcD)支持高度自信的身份识别。总而言之，我们提议的实验将开发出新的 使能工具，并将产生成本效益和新颖的基于质量缺陷的标记试剂， 灵敏、准确的多糖分析，增强了定量性能和结构说明 能力。这些标签的性能将在糖科学社区内进行交叉验证。

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.