Mass Spectrometry Method for Carbohydrate Structure

描述（由申请人提供）：复合碳水化合物是基本上所有生命系统的组成部分，在各种各样的生物过程和识别事件中发挥关键作用。然而，它们的详细结构确定仍然很困难。虽然质谱法可以提供有关碳水化合物结构的重要信息，但通常不可能确定由较大碳水化合物结构断裂产生的单个单糖离子的立体化学。此外，通过质谱法分析异构寡糖的混合物仍然很困难，因为单独的基本电荷/质量比不能物理解析异构结构。因此，本项目的目标是开发一种分析仪器，通过解析异构体结构，显著提高复杂碳水化合物结构测定的能力。拟议的仪器将基于离子阱质谱和离子迁移谱的原理。在其最终形式中，该仪器将具有耦合到双门离子迁移谱仪（IMS）的电喷雾电离（ESI）源。双门IMS根据大小和形状而不是质量分离离子，将用于选择特定的寡糖异构体注入离子阱质谱仪（ITMS）。迁移率选择的寡糖的脉冲将在ITMS中浓缩，然后碰撞诱导解离以产生选择的糖单体的离子。然后将这些单体注入第二个串联IMS中，以鉴定单体离子的立体化学和端基异构体构型。通过将单一寡糖分离并引入ITMS中，解离并鉴定单糖，可以对复杂寡糖进行完整的结构分析。该计划中的关键步骤是通过离子阱中的碎片化生产单糖和通过IMS分离单糖。因此，该项目的R21阶段建议开发用于寡糖片段化的新型MS方法和用于分离寡糖以及单糖离子的立体异构体和端基异构体对的新型IMS方法。在R33阶段，将构建、表征和证明IMS-IT-IMS仪器，用于阐明寡糖结构。在这个补助金申请中提出的整体工具有可能产生以前从未有可能对这一重要和神秘的生物化合物类的数据，并将提供一个新的工具，为新兴领域的结构糖组学。迄今为止，大多数天然存在的寡糖的详细化学结构仅通过质谱法无法识别。该项目的成功完成将提供一种工具，不仅将质谱的分析能力扩展到分析同分异构的单糖和低聚糖，而且通常还可用于分析所有类型的分子，其中可能存在关于混合物中可能的同分异构变体或较大聚合物分子的同分异构单体成分的模糊性。

DESCRIPTION (provided by applicant): Complex carbohydrates are integral, functioning components of essentially all living systems, playing critical roles in an enormous variety of biological processes and recognition events. Yet, their detailed structural determination remains difficult. While mass spectrometry can provide important information about carbohydrate structures, a determination of the stereochemistry of individual monosaccharide ions that arise by fragmentation of larger carbohydrate structures is not generally possible. Also, analysis of mixtures of isomeric oligosaccharides by mass spectrometry remains difficult because the fundamental charge/mass ratio alone does not physically resolve isomeric structures. Thus, the goal of this project is to develop an analytical instrument that will markedly improve capabilities in the structural determination of complex carbohydrates by enabling isomeric structures to be resolved. The proposed instrument will be based on the principles of both ion trap mass spectrometry and ion mobility spectrometry. In its final form, the instrument will have an electrospray ionization (ESI) source coupled to a double gate ion mobility spectrometer (IMS). The double gate IMS, separating ions on the basis of size and shape rather than mass, will serve to select specific oligosaccharide isomers for injection into an ion trap mass spectrometer (ITMS). Pulses of the mobility-selected oligosaccharide will be concentrated in the ITMS followed by collision induced dissociation to generate ions of selected sugar monomers. These monomers will then be injected into a second, tandem IMS to identify the stereochemistry and anomeric configuration of monomer ions. By the separation and introduction of single oligosaccharides into the ITMS with dissociation and identification of the monomeric sugars, complete structural analysis of complex oligosaccharides may be possible. The critical steps in this plan are the production of monosaccharides by fragmentation in the ion trap and the separation of monosaccharides by IMS. Thus, the R21 phase of the project proposes to develop novel MS methods for the fragmentation of oligosaccharides and novel IMS methods for the separation of oligosaccharides as well as stereoisomeric and anomeric pairs of monosaccharide ions. In the R33 phase an IMS-IT-IMS instrument will be constructed, characterized and demonstrated for the elucidation of oligosaccharide structures. The overall instrument proposed in this grant application has the potential to generate data never before possible on this important and enigmatic class of biological compounds and will provide a novel tool for the emerging field of Structural Glycomics. To date, the detailed chemical structures of most naturally occurring oligosaccharides have eluded identification solely by mass spectrometry. Successful completion of this project will provide a tool which will not only extend the analytical power of mass spectrometry to analysis of isomeric mono- and oligosaccharides, but will also be generally useful for analysis of all types of molecules where ambiguity may exist concerning possible isomeric variants in mixtures or isomeric monomeric constituents of larger polymeric molecules.