Hybrid structural mass spectrometry for rapid site-specific glycan structural elucidation

项目摘要 广泛的糖基化覆盖了更高水平生物体的细胞外环境。这些 聚糖结构附着在人类蛋白质组的一半上，并且对于它们在几乎所有蛋白质组中的功能至关重要。 生物过程。监测糖脂和糖蛋白上糖基化结构的改变 可以发现抑制感染的新方法，并确定检测和治疗自身免疫性疾病的新靶点， 疾病和癌症。尽管它们在生物学上很重要，但我们监测大量聚糖的能力 结构及其在疾病状态中的确切作用仍然有限。这主要是由于挑战 与分析复杂的碳水化合物结构相关，这提供了无与伦比的结构水平， 多样性用于大规模糖组学研究的工具不能提供必要的结构细节水平， 有效地解决聚糖结构中的细微差异。另一方面，技术能够详细 聚糖的结构解析是缓慢的、实验密集的，并且经常需要多种方法。这 一项提案旨在开发一个分析平台，该平台将实现快速聚糖表征， 现有技术无法提供的结构细节。 该提案旨在结合离子迁移率（IMS）和气相氢/氘交换 (HDX)用于聚糖结构分析，并整合到一个强大的质谱平台中。我们将利用这个平台， 新的内标物，用于分析单糖、寡糖、聚糖链库， 糖蛋白和糖脂来组装离子迁移率（Aim 1）和氢/氘的数据库 交换（目标2）生物相关的碳水化合物离子的性质。分子模型将被用来帮助 解释和合理化每个结构的不同行为，并扩展我们的结构参考数据库 计算机模拟（目标3）。离子迁移率和氢/氘交换的组合将被实施为 色谱时间标度“GlySECT”上聚糖结构解析的LC-MS平台（目的4）。在 与此同时，我们将开发软件，用于提取数据和检索已建立的数据库 在这个项目中，以帮助阐明聚糖链的结构。这项技术有可能使 快速、位点特异性、聚糖结构测定，并为糖生物学、生物标志物 发现和治疗性蛋白质表征。

PROJECT SUMMARY An extensive array of glycosylation covers the extracellular milieu of higher level organisms. These glycan structures are attached to half of the human proteome and are critical for their function in nearly all biological processes. Monitoring alterations in the structures of glycosylation on glycolipids and glycoproteins can identify new ways of inhibiting infections and pinpoint new targets for detecting and treating autoimmune diseases and cancer. Despite their biological importance, our ability to monitor the vast array of glycan structures and their exact role in disease states remains limited. This is primarily due to the challenges associated with analyzing complex carbohydrate structures, which present an unparalleled level of structural diversity. Tools for large-scale glycomic studies do not provide the level of structural detail necessary to effectively resolve subtle differences in glycan structures. On the other hand, techniques capable of detailed structural elucidation of glycans are slow, experimentally intensive, and often require multiple approaches. This proposal seeks to develop an analytical platform that will enable rapid glycan characterization with a level of structural detail that is unavailable with existing techniques. This proposal aims to incorporate ion mobility (IMS) and gas-phase hydrogen/deuterium exchange (HDX) for glycan structural analysis into a robust mass spectrometry platform. We will use this platform with novel internal standards to analyze a library of monosaccharides, oligosaccharides, glycan chains from glycoproteins, and glycolipids to assemble a database of the ion mobility (Aim 1) and hydrogen/deuterium exchange (Aim 2) properties of biologically relevant carbohydrate ions. Molecular modeling will be used to help interpret and rationalize the different behaviors of each structure and expand our structural reference database in silico (Aim 3). The combination of ion mobility and hydrogen/deuterium exchange will be implemented into a LC-MS platform for Glycan Structural Elucidation on a Chromatographic Timescale “GlySECT” (Aim 4). In parallel, we will develop the software for extracting the data and searching against the databases established in this project to aid in structural elucidation of the glycan chains. This technology has the potential to enable rapid, site-specific, glycan structural determination and provide a much-needed tool for glycobiology, biomarker discovery, and therapeutic protein characterization.