Characterization of glycan isomers by trapped ion mobility spectrometry-electron activated dissociation tandem mass spectrometry

项目摘要/摘要 糖基化在许多细胞事件中起着至关重要的作用，包括蛋白质折叠、病原体识别和 癌症转移。多糖的结构复杂性和多样性与其不同的功能是平行的。鉴于 线性生物聚合物的一级结构，如蛋白质和寡核苷酸，由 它们的一维序列，完整的结构表征需要确定它的两个- 空间拓扑、连接和立体化学构型。进一步的分析挑战来自于 非模板驱动的葡聚糖生物合成的性质，导致糖类包含一系列紧密的- 相关结构，其中许多是结构异构体。 最近，大量发展了许多电子激活解离(EXD)方法 用于多糖分析的光谱分析实验室。电子捕获解离(ECD)，电子转移 解离(ETD)和电子激发解离(EED)可以产生丰富的结构信息片段 正电离模式下分析的葡聚糖离子。在负电离模式下，电子脱离 解离(EDD)和负ETD(NETD)是酸性物质测序的强有力的碎裂方法 糖胺聚糖。同时，离子迁移率光谱(IMS)被应用于多糖的分离。AS IMS是一种后电离、气相分离方法，它是对溶液相分离方法的补充，例如 如毛细管电泳法(CE)和液相色谱仪(LC)，并可实现基于 它们在气相构象上的差异。然而，传统的漂移时间IMS分离也发生在 较短的时间刻度，与较慢的EXD分析方法兼容。一种新的IMS技术，称为 囚禁离子迁移率谱(TIMS)是Bruker Daltonics最近提出的。我们已经证明了 TIMS与高性能傅里叶变换离子回旋共振(FTICR)MS的成功耦合 葡聚糖键合异构体分离鉴定仪器。在这里，我们建议修改TIM 设备及其控制软件，用于提高移动性分辨率、增加m/z工作范围，以及更好 与EXD-FTICR MS/MS分析集成。然后，我们将利用改进的TIMS-EXD方法进行详细的 多聚糖的结构表征。我们还将TIMS-EXD MS/MS与离线LC结合使用 分级以产生包含已鉴定的具有其碰撞截面的多糖结构的文库 价值观。这个图书馆将向公众开放。 最初的开发将在FTICR MS平台上进行，因为它提供了卓越的质量精度和 分辨率，以及最佳的EXD性能。我们在这里开发的技术可以稍后 随着替代ECD电池的发展，转移到其他更负担得起的MS仪器 将EXD功能引入非ICR仪器。

Project summary/abstract Glycosylation plays vital roles in many cellular events, including protein folding, pathogen recognition, and cancer metastasis. The structural complexity and diversity of glycans parallel their diverse functions. Whereas the primary structures of linear biopolymers, such as proteins and oligonucleotides, are uniquely defined by their one-dimensional sequence, full structural characterization of a glycan requires determination of its two- dimensional topology, linkage and stereochemical configurations. Further analytical challenges arise from the non-template-driven nature of glycan biosynthesis, resulting in glycomes comprising a repertoire of closely- related structures, many of which structural isomers. Recently, a number of electron activated dissociation (ExD) methods have been developed in mass spectrometry laboratories for glycan analysis. Electron capture dissociation (ECD), electron transfer dissociation (ETD), and electronic excitation dissociation (EED) can yield rich structurally informative fragment ions for glycans analyzed in the positive ionization mode. In the negative ionization mode, electron detachment dissociation (EDD) and negative ETD (NETD) are powerful fragmentation methods for sequencing of acidic glycosaminoglycans. Meanwhile, ion mobility spectrometry (IMS) has been applied to separation of glycans. As a post-ionization, gas-phase separation method, IMS complements solution-phase separation methods such as capillary electrophoresis (CE) and liquid chromatography (LC), and can achieve isomer resolution based on differences in their gas-phase conformations. However, conventional drift-time IMS separation occurs on too short a time-scale to be compatible with the slower ExD analysis methods. A new IMS technique, termed trapped ion mobility spectrometry (TIMS), was recently introduced by Bruker Daltonics. We have demonstrated successful coupling of TIMS to high-performance Fourier-transform ion cyclotron resonance (FTICR) MS instrument for separation and identification of glycan linkage isomers. Here, we propose to modify the TIMS device and its control software, for improved mobility resolution, increased m/z operating range, and better integration with ExD-FTICR MS/MS analysis. We will then utilize the improved TIMS-ExD method for detailed structural characterization of glycans. We will also use TIMS-ExD MS/MS in conjunction with off-line LC fractionation to produce a library that contains identified glycan structures with their collision cross section values. This library will be made available to public. The initial development will be carried out on the FTICR MS platform, as it offers superior mass accuracy and resolving power, as well as the best ExD performance. The technology we develop here can later be transferred to other, more affordable MS instruments, following the development of alternative ECD cells to bring the ExD capability to non-ICR instruments.