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

项目总结 一系列广泛的糖基化作用覆盖了较高水平生物体的细胞外环境。这些 糖链结构附着在人类蛋白质组的一半上，对几乎所有蛋白质组的功能都是至关重要的 生物过程。监测糖脂和糖蛋白糖基化结构的变化 可以确定抑制感染的新方法，并确定检测和治疗自身免疫的新靶点 疾病和癌症。尽管它们在生物学上很重要，但我们监测大量多糖的能力 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联，这呈现出无与伦比的结构 多样性。用于大规模血糖研究的工具不能提供必要的结构细节水平 有效地解决多糖结构中的细微差异。另一方面，能够详细描述的技术 葡聚糖的结构阐明是缓慢的，实验密集的，并且通常需要多种方法。这 Proposal寻求开发一个分析平台，以实现快速的葡聚糖表征 现有技术无法提供的结构详图。 这项提议旨在结合离子迁移率(IMS)和气相氢/氘交换 将用于多糖结构分析的(HDX)整合到一个强大的质谱学平台中。我们将通过以下方式使用此平台 用于分析单糖、低聚糖、糖链文库的新型内部标准 糖蛋白和糖脂组成一个离子迁移率数据库(目标1)和氢/重离子 交换(目标2)生物相关碳水化合物离子的性质。分子建模将被用来帮助 对每个结构的不同行为进行解释和合理化，并扩展我们的结构参考数据库 在Silico(目标3)中。离子迁移率和氢/氚交换的组合将被实施到一个 LC-MS平台，用于在层析时间刻度“GlySECT”上阐明多糖结构(目标4)。在……里面 同时，我们将开发提取数据和根据所建立的数据库进行搜索的软件 在这个项目中，以帮助阐明糖链的结构。这项技术有可能使 快速、定点、糖链结构测定，为糖生物学提供了急需的工具--生物标记物 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.