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

项目概要/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、病原体识别和免疫应答。 癌症转移聚糖的结构复杂性和多样性与其多种功能并行。而 线性生物聚合物（如蛋白质和寡核苷酸）的一级结构由以下唯一定义： 它们的一维序列，聚糖的完整结构表征需要确定其二维序列， 空间拓扑学、连接和立体化学构型。进一步的分析挑战来自于 聚糖生物合成的非模板驱动性质，导致糖组包含紧密结合的 相关结构，其中许多结构异构体。 近年来，大量的电子活化解离（ExD）方法得到了发展 用于聚糖分析的光谱实验室。电子捕获解离（ECD），电子转移 电子解离（ETD）和电子激发解离（EED）可以产生丰富的结构信息片段 正离子化模式下分析的聚糖离子。在负电离模式下，电子脱离 解离（EDD）和负ETD（NETD）是用于酸性蛋白质测序的强大片段化方法。 糖胺聚糖同时，离子迁移谱（IMS）已被应用于聚糖的分离。作为 作为一种电离后气相分离方法，IMS补充了溶液相分离方法， 如毛细管电泳（CE）和液相色谱（LC），并可以实现异构体的决议，根据 它们的气相构象的差异。然而，传统的漂移时间IMS分离也发生在 缩短时间尺度以与较慢的ExD分析方法兼容。一种新的IMS技术，称为 捕获离子迁移谱（TIMS）最近由Bruker Daltonics引入。我们已经证明 TIMS与高性能傅里叶变换离子回旋共振（FTICR）质谱成功耦合 用于分离和鉴定聚糖键异构体仪器。在这里，我们建议修改TIMS 设备及其控制软件，用于提高迁移率分辨率，增加m/z操作范围，以及更好地 与ExD-FTICR MS/MS分析集成。然后我们将利用改进的TIMS-ExD方法进行详细分析 聚糖的结构表征。我们还将使用TIMS-ExD MS/MS结合离线LC 分离以产生含有具有其碰撞横截面的鉴定的聚糖结构的文库 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，因为它提供了上级质量精度， 分辨率以及最佳ExD性能。我们在这里开发的技术 转移到其他，更负担得起的MS仪器，以下替代ECD细胞的发展， 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.