Mass Spectrometry Method for Carbohydrate Structure

描述(由申请人提供)：复合碳水化合物是几乎所有生命系统不可或缺的功能组件，在各种生物过程和认知事件中发挥关键作用。然而，他们详细的结构性决定仍然很难。虽然质谱学可以提供关于碳水化合物结构的重要信息，但通常不可能确定由于较大的碳水化合物结构裂解而产生的单个单糖离子的立体化学。此外，用质谱仪分析低聚异构体混合物仍然很困难，因为单靠基本电荷/质量比本身并不能物理地解析异构体结构。因此，该项目的目标是开发一种分析仪器，通过能够拆分异构体结构，显著提高确定复杂碳水化合物结构的能力。建议的仪器将基于离子陷阱质谱仪和离子迁移率光谱的原理。在其最终形式，该仪器将有一个电喷雾电离(ESI)源耦合到一个双门离子迁移率光谱仪(IMS)。双门IMS根据离子的大小和形状而不是质量来分离离子，将用于选择特定的低聚糖异构体注入离子陷阱质谱仪(ITMS)。流动性选择的寡糖的脉冲将在ITMS中浓缩，然后碰撞诱导解离，生成选择的糖单体的离子。然后，这些单体将被注入第二个串联的IMS中，以确定单体离子的立体化学和异常构型。通过将单一低聚糖分离并引入到ITMS中，并对单体糖进行解离和鉴定，可以对复杂低聚糖进行完整的结构分析。该计划的关键步骤是通过离子捕捉器中的碎裂来生产单糖，并用IMS分离单糖。因此，该项目的R21阶段建议开发用于低聚糖裂解的新的MS方法和用于分离低聚糖以及单糖离子的立体异构体和异构体对的新的IMS方法。在R33阶段，将构建一个IMS-IT-IMS仪器，并对其进行表征和演示，以确定寡糖的结构。在这项赠款申请中提出的整个仪器有可能产生关于这一重要而神秘的生物化合物类别的数据，这是前所未有的，并将为新兴的结构糖类领域提供一个新的工具。到目前为止，大多数天然低聚糖的详细化学结构一直无法仅通过质谱学进行鉴定。这个项目的成功完成将提供一种工具，它不仅将把质谱学的分析能力扩展到同分异构体单糖和低聚糖的分析，而且还将普遍用于对可能存在关于混合物中可能的异构体变体或较大聚合物分子的同分异构体组成的所有类型的分子的分析。

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.