ESI ION MOBILITY SPECTROMETRY OF CARBOHYDRATE ISOMERS

碳水化合物异构体的 ESI 离子淌度谱测定

• 批准号: 7723046
• 负责人: JOSEPH ZAIA
• 金额: $ 0.07万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7723046
• 关键词: Base Composition; Carbohydrates; Cell Mobility; Charge; Class; Complex; Computer Retrieval of Information on Scientific Projects Database; Condition; Disaccharides; Event; Funding; Glycoconjugates; Glycosaminoglycans; Goals; Grant; Information Systems; Institution; Ions; Isomerism; Isopropanol; Link; Mannose; Milk; Oligosaccharides; Polysaccharides; Regulation; Relative (related person); Research; Research Personnel; Resolution; Resources; Scanning; Series; Signal Transduction; Source; Spectrometry; Structure; Sum; Time; Travel; United States National Institutes of Health; Vacuum; Variant; Water; Work; ion mobility; pressure; research study; ribonuclease B; sialyl-Lex

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 糖复合聚糖由位于共同核心结构上的变体混合物（称为糖型）组成。 这些变异是复杂调控下生物合成事件的结果。 复合糖聚糖质谱分析的挑战之一是与给定寡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。 离子迁移谱 (IMS) 需要使离子通过在相对于真空的高压下运行的迁移池。 对于给定的电荷状态，迁移时间随着离子的碰撞截面而增加。 这项工作的目标是确定使用离子淌度解析碳水化合物异构体的程度。使用改进的 Waters QTOF Premier 采集离子淌度谱，该 Waters QTOF Premier 配备在 1 mbar 下运行的行波离子导向器。 离子引导由 122 个平行板组成，每个平行板有 2.5 毫米的孔口，中心间距为 1.5 毫米。将低聚糖以 1 pmol/μL 的浓度溶解在 10% 异丙醇中，并以 5μL/min 的速度注入电喷雾源。 每个 15 毫秒离子淌度谱由一系列 200 个 75 秒 TOF 扫描组成。 对大约 360 个离子淌度谱进行求和，以产生在数据系统上显示的扫描，重复时间为 5.4 秒，对 20-30 个这样的扫描进行求和，以产生最终的谱。研究了以下化合物类别：天​​然糖胺聚糖二糖、天然和全甲基化乳寡糖以及天然和全甲基化高甘露糖N-连接寡糖。 由 δHexA(δ1,3)GalNAc 和 δHexA(δ1,4)GlcNAc 对产生的[M-H]-离子产生相同的离子迁移率。 组合物(?HexA)(HexNAc)(SO3)的一系列五种异构体在[M-H]-离子的迁移率方面产生细微差异，而对于[M-2H]2-离子则没有差异。 (HexA)(HexNAc)(SO3)2 的一系列三种异构体产生了不同的迁移率曲线，可区分 [M-H]- 和 [M(Na)-H]-、[M-2H]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 微秒的 TOF 扫描。 牛奶寡糖 LNT 和 LNnT（四糖的 Gal-GlcNAc 连接不同）产生的迁移率痕迹因天然 [M-H]-、天然 [M+Na]+ 和全甲基化 [M+Na]+ 离子的两次 TOF 扫描而不同。 这些聚糖的唾液酸化形式 LST-a 和 LST-d 产生的迁移率痕迹与天然 [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轨迹。 对于天然 [M-H]- 和全甲基化 [M+Na]+ 离子，路易斯寡糖（LeX 和 LeA）通过一次 TOF 扫描产生不同的迁移率迹线。 唾液酸化形式（唾液酸 LeX 和唾液酸 LeA）对天然 [M-H]- 和全甲基化 [M+Na]+ 离子产生相同的迁移率痕迹。 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。 根据天然 [M-H]- 和 [M+Na]+ 以及全甲基化 [M+Na]+ 迁移率迹线中的组成，糖型均得到清晰解析。 (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得到解析。 进一步的实验正在进行中，以探索可能实现这些较大聚糖异构体解析的条件。