ESI ION MOBILITY SPECTROMETRY OF CARBOHYDRATE ISOMERS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 研究中心，而研究中心不一定是研究者所在的机构。 糖缀合物聚糖在共同的核心结构上由称为糖型的变体的混合物组成。 这些变体是在复杂调控下生物合成事件的结果。 糖缀合物聚糖的质谱分析中的挑战之一是对应于给定寡糖组成的离子信号可能由结构异构体的混合物产生。 离子迁移谱（IMS）需要使离子通过在相对于真空的高压下操作的迁移池。 对于一个给定的电荷状态，迁移时间增加的碰撞截面的离子。 这项工作的目标是确定在何种程度上可以使用离子迁移率解决碳水化合物异构体。使用配备有在1毫巴下操作的行波离子导向器的改进的沃茨QTOF Premier获得离子迁移谱。 离子引导由122个平行板组成，每个板具有2.5 mm的孔和1.5 mm的中心间距。L在10%异丙醇中，并注入到电喷雾源在5？每个15-ms离子迁移谱由一系列的200 75-？s TOF扫描。 将大约360个离子迁移率谱相加以产生在数据系统上显示的扫描，重复5.4秒，并将20-30个这样的扫描相加以产生最终谱。研究了以下化合物类别：天然糖胺聚糖二糖、天然和全甲基化乳低聚糖以及天然和全甲基化高甘露糖N-连接低聚糖。 [M-H]-离子产生的对？HexA（？1，3）GalNAc和？HexA（？1，4）GlcNAc产生相同的离子迁移率。 一系列的五个异构体的组合物（？HexA）（HexNAc）（SO 3）对[M-H]-离子的迁移率产生细微差异，对[M-2 H]2-离子没有差异。 一系列组成为（HexA）（HexNAc）（SO 3）2的三种异构体产生不同的迁移率曲线，其区分[M-H]-和[M（Na）-H]-、[M-2 H]2-和[M（Na）-2H 2-离子的异构体。 迁移率差异对应于一次或两次75 usec TOF扫描。 牛奶低聚糖LNT和LNnT，由Gal-GlcNAc键不同的四糖，产生的迁移率的痕迹不同的两个TOF扫描的天然[M-H]-，天然[M+Na]+和全甲基化[M+Na]+离子。 这些聚糖（LST-a和LST-d）的唾液酸化形式产生的迁移率迹线与天然[M-H]-、[M（Na）+H]+和[M（Na）+Na]+的两次TOF扫描不同。 LST聚糖的全甲基化形式产生相同的迁移率迹线。 刘易斯寡糖（LeX和莱亚）产生的迁移率迹线因一次TOF扫描而不同，用于天然[M-H]-和全甲基化[M+Na]+离子。 唾液酸化形式（唾液酸化LeX和唾液酸化莱亚）对天然[M-H]-和全甲基化[M+Na]+离子产生相同的迁移率迹线。 从核糖核酸酶B释放的高甘露糖寡糖由一系列含有5-9个甘露糖残基的糖型组成。 基于天然[M-H]-和[M+Na]+以及全甲基化[M+Na]+的迁移率迹线中的组成，糖型均得到明确解析。 （GlcNAc）2（Man）7糖型以位置异构体的混合物形式存在。 这些异构体在任何实验中均未分离。 正在进行进一步的实验，以探索可能使这些较大聚糖的异构体拆分的条件。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Glycoconjugate glycans consist of mixtures of variants, known as glycoforms, on a common core structure. These variants arise as a result of biosynthetic events under complex regulation. One of the challenges in mass spectral analysis of glycoconjugate glycans is that the ion signals corresponding to a given oligosaccharide composition may be produced by a mixture of structural isomers. Ion mobility spectrometry (IMS) entails passing ions through a mobility cell operated at elevated pressure, relative to vacuum. For a given charge state, the mobility time increases with the collisional cross section of the ions. The goal of this work is to determine the extent to which carbohydrate isomers may be resolved using ion mobility. Ion mobility spectra were acquired using a modified Waters QTOF Premier equipped with a traveling wave ion guide operated at 1 mbar. The ion guided consisted of 122 parallel plates, each with a 2.5 mm orifice and center-to-center spacing of 1.5 mm. Oligosaccharides were dissolved at 1 pmol/?L in 10% isopropanol and infused into the electrospray source at 5 ?L/min. Each 15-ms ion mobility spectrum consisted of a series of 200 75-?s TOF scans. Approximately 360 ion mobility spectra were summed to produce scans displayed on the data system with a 5.4 s repeat, and 20-30 such scans were summed to produce the final spectra. The following compound classes were studied: native glycosaminoglycan disaccharides, native and permethylated milk oligosaccharides, and native and permethylated high mannose N-linked oligosaccharides. The [M-H]- ions generated from the pair ?HexA(?1,3)GalNAc and ?HexA(?1,4)GlcNAc produced the same ion mobility. A series of five isomers of the composition (?HexA)(HexNAc)(SO3) produced subtle differences in mobility for [M-H]- ions and no differences for the [M-2H]2- ions. A series of three isomers of composition (HexA)(HexNAc)(SO3)2 produced distinct mobility profiles that differentiated isomers for [M-H]- and [M(Na)-H]- , [M-2H]2-, and [M(Na)-2H]2- ions. The mobility differences corresponded to one or two 75-usec TOF scans. Milk oligosaccharides LNT and LNnT, tetrasaccharides differing by the Gal-GlcNAc linkage, produced mobility traces differing by two TOF scans for native [M-H]-, native [M+Na]+ and permethylated [M+Na]+ ions. The sialylated forms of these glycans, LST-a and LST-d, produced mobility traces that differed by two TOF scans for native [M-H]-, [M(Na)+H]+, and [M(Na)+Na]+ . The permethylated forms of the LST glycans produced identical mobility traces. Lewis oligosaccharides (LeX and LeA) produced mobility traces differing by one TOF scan for native [M-H]- and permethylated [M+Na]+ ions. The sialylated forms (sialyl LeX and sialyl LeA) produced identical mobility traces for native [M-H]- and permethylated [M+Na]+ ions. High mannose oligosaccharides released from ribonuclease B consist of a series of glycoforms containing 5-9 mannose residues. The glycoforms were all clearly resolved on the basis of composition in the mobility traces for native [M-H]- and [M+Na]+ and permethylated [M+Na]+. The (GlcNAc)2(Man)7 glycoform exists as a mixture of positional isomers. These isomers were not resolved in any of the experiments. Further experiments are underway to explore conditions that may enable isomer resolution of these larger glycans.