Mass Spectrometry Method for Carbohydrate Structure

描述（由申请人提供）：复合碳水化合物是所有生命系统不可或缺的功能组成部分，在各种生物过程和识别事件中发挥着关键作用。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破碎产生的单个单糖离子的立体化学。此外，用质谱法分析同分异构体低聚糖的混合物仍然很困难，因为单靠基本电荷/质量比不能物理地分辨同分异构体结构。因此，该项目的目标是开发一种分析仪器，通过能够分辨异构体结构来显著提高复杂碳水化合物结构测定的能力。提出的仪器将基于离子阱质谱法和离子迁移谱法的原理。在其最终形式中，该仪器将有一个电喷雾电离（ESI）源与一个双栅离子迁移谱仪（IMS）耦合。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，以注入离子阱质谱仪（ITMS）。迁移率选择的低聚糖脉冲将在ITMS中浓缩，然后通过碰撞诱导解离产生选定的糖单体离子。然后将这些单体注入到第二个串联IMS中，以确定单体离子的立体化学和异构结构。通过将单个低聚糖分离并引入到ITMS中，并对其进行解离和鉴定，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阱破碎生产单糖和用IMS分离单糖。因此，该项目的R21阶段提出了开发用于低聚糖片段化的新型质谱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对的新型IMS方法。在R33阶段，一个IMS-IT-IMS仪器将被构建，表征和演示用于阐释低聚糖结构。在这项拨款申请中提出的整体仪器有可能产生前所未有的关于这类重要而神秘的生物化合物的数据，并将为结构糖组学的新兴领域提供一种新的工具。迄今为止，大多数天然存在的低聚糖的详细化学结构都无法通过质谱法进行鉴定。该项目的成功完成将提供一种工具，不仅可以将质谱分析的分析能力扩展到异构体单糖和寡糖的分析，而且还可以用于分析所有类型的分子，其中可能存在混合物中可能的异构体变体或较大聚合物分子的异构体单体成分。

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.