ESI ION MOBILITY SPECTROMETRY OF CARBOHYDRATE ISOMERS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 糖共轭多聚糖由一种常见的核心结构上的变异体的混合物组成，称为糖型。这些变异是复杂调控下生物合成事件的结果。糖共轭多聚糖的质谱分析的挑战之一是，与给定的低聚糖组成相对应的离子信号可能是由结构异构体的混合物产生的。离子迁移率光谱(IMS)是指离子通过在相对于真空较高的压力下运行的流动池。对于给定的电荷态，迁移率时间随离子碰撞截面的增大而增大。这项工作的目标是确定碳水化合物异构体可以用离子迁移率来分解的程度。离子迁移率光谱是使用改装的Waters QTOF Premier获得的，该Premier配备了工作在1mbar的行波离子导轨。离子导向器由122个平行板组成，每个平行板的开口为2.5 mm，中心到中心的间距为1.5 mm。低聚糖以1pm ol/m in的速度溶解于10%异丙醇中，以5 m o l/m in的速度注入电喷雾源。每个15ms的离子迁移率谱由200次75ms的S飞行时间扫描组成。大约360个离子迁移率光谱被相加，以产生在数据系统上显示的具有5.4S重复的扫描，并且20-30个这样的扫描被相加以产生最终的光谱。研究了以下化合物类别：天然糖胺多糖二糖、天然和全甲基化牛奶低聚糖，以及天然和全甲基化高甘露糖N-连接低聚糖。由六(？1，3)GalNAc和？六(？1，4)GlcNAc生成的[M-H]-离子产生相同的离子迁移率。[H-H]-离子的迁移率略有不同，而[M-2H]2-离子的迁移率没有差别。一系列(六)(六钠)(SO3)2的三种异构体产生了不同的迁移率分布，区分了[M-H]-和[M(Na)-H]-，[M-2H]2-和[M(Na)-2H]2-离子。机动性差异相当于一次或两次75微秒的TOF扫描。牛奶低聚糖LNT和LNnT是通过Gal-GlcNAc连接方式不同的四糖，它们在两次TOF扫描中对天然[M-H]-、天然[M+Na]+和过甲基化[M+Na]+离子产生了不同的流动性痕迹。这些糖链的唾液酸化形式LST-a和LST-d产生的迁移率轨迹与两次TOF扫描对天然[M-H]-、[M(Na)+H]+和[M(Na)+Na]+的扫描不同。LST多糖的全甲基化形式产生了相同的流动性痕迹。Lewis寡糖(Lex和Lea)在一次TOF扫描中对天然的[M-H]-和全甲基化的[M+Na]+离子产生了不同的迁移率轨迹。唾液酸化形式(唾液酸化Lex和唾液酸化Lea)对天然的[M-H]-离子和全甲基化的[M+Na]+离子产生相同的迁移率轨迹。核糖核酸酶B释放的高甘露糖低聚糖由含有5-9个甘露糖残基的一系列糖型组成。根据天然的[M-H]-和[M+Na]+和全甲基化的[M+Na]+的迁移率轨迹中的组成，糖形式都被清楚地分解了。(GlcNAc)2(Man)7糖体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都没有被拆分。进一步的实验正在进行中，以探索可能使这些较大的多糖能够拆分异构体的条件。

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.