LINKAGE ANALYSIS OF PMAAS BY GC-MS

通过 GC-MS 对 PMAAS 进行连锁分析

• 批准号: 8170778
• 负责人: Parastoo Azadi
• 金额: $ 0.13万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8170778
• 关键词: Acetates; Acetic Acids; Blood capillaries; Borates; Carbohydrates; Cleaved cell; Computer Retrieval of Information on Scientific Projects Database; Dialysis procedure; Dimethyl Sulfoxide; Electrons; Funding; Gases; Grant; Hour; Hydrolysis; Hydroxyl Radical; Incubated; Institution; Link; Mass Fragmentography; Mass Spectrum Analysis; Membrane; Methanol; Methylation; Methylene Chloride; Nitrogen; Phase; Plant Resins; Polysaccharides; Procedures; Reaction; Research; Research Personnel; Resources; Salts; Sampling; Silicon Dioxide; Source; Stream; Sugar Alcohols; Temperature; Time; Trifluoroacetic Acid; Tube; United States National Institutes of Health; Water; acetic anhydride; capillary; detector; genetic linkage analysis; ionization; methyl iodide; pyridine; sodium borohydride

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. Dialysis The samples were dialyzed using a Tube-O-Dialyzer (4.0 kDa cut-off membrane; G BioSciences) against nanopure water at 4oC for about 22 hours to remove salts and other contaminants. Nanopure water was replaced three times during the entire dialysis period. Release of O-linked glycans by ¿-elimination O-linked carbohydrates were cleaved from the samples by ¿-elimination procedure. Briefly, 250 ¿L of 50 mM NaOH was added to each of the samples and then checked for pH. Upon determination that the pH was basic, another 250 ¿L of 50 mM NaOH containing 19 mg of sodium borohydride was added to each of the samples, vortexed, and incubated overnight at 450C. The incubated samples then were neutralized with 10% acetic acid and desalted by passing through a packed column of Dowex resins and then were lyophilized. Dried samples were cleaned of borate with methanol:acetic acid (9:1) under a stream of nitrogen gas before permethylation. Per-O-methylation of O-linked glycans Released O-linked glycans were permethylated and were evaluated by mass spectrometry (Anumula and Taylor, 1992) to verify if the derivatization was carried to completion. The glycans were dissolved with dimethylsulfoxide and then methylated with NaOH and methyl iodide. The reaction was quenched with water and per-O-methylated carbohydrates were extracted with methylene chloride. Glycosyl Linkage Analysis For determination of glycosyl linkages, partially methylated alditol acetates (PMAAs) were prepared from the released permethylated O-linked glycans. Briefly, permethylated glycans were hydrolyzed with 2N trifluoroacetic acid (TFA) at 100oC for 4 h, followed by reduction with NaBD4. The latter-freed hydroxyls were acetylated with acetic anhydride/pyridine (1:1, v/v) at 100 ¿C for 15 min. Gas Chromatography-Mass Spectrometry (GC-MS) The PMAAs were analyzed on a Hewlett Packard 5890 GC interfaced to a 5970 MSD (mass selective detector, electron impact ionization mode). The separation was performed on a 30 m EC 1 bonded phase fused silica capillary column (Altech). Electron impact mass spectra were obtained under the following conditions: oven temperature, 80 ¿C (2 min) ¿ 180 ¿C (20 ¿C/min) ¿ 240 ¿C (4 ¿C/min); detector temperature, 280 ¿C; inlet temperature, 250 ¿C.

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。 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以出现在其他 CRISP 条目中 列出的机构是。 对于中心来说，它不一定是研究者的机构。 透析 使用 Tube-O-Dialyzer（4.0 kDa 截止膜；G BioSciences）在 4℃ 下对纳米纯水进行透析约 22 小时，以去除盐和其他污染物。在整个透析期间更换了 3 次纳米纯水。 通过 ¿ 释放 O-连接聚糖-消除 O-连接碳水化合物通过 ¿ 从样品中裂解下来- 简单地说，250 ¿将 L 50 mM NaOH 添加到每个样品中，然后检查 pH 值，确定 pH 值呈碱性后，再检查 250 ¿将含有 19 mg 硼氢化钠的 L 50 mM NaOH 添加到每个样品中，涡旋并在 450°C 下孵育过夜。然后用 10% 乙酸中和孵育的样品，并通过 Dowex 树脂填充柱脱盐。然后在全甲基化之前在氮气流下用甲醇：乙酸（9：1）将干燥的样品冻干。 O-连接聚糖的全-O-甲基化 将释放的 O-连接聚糖进行全甲基化并通过质谱法进行评估（Anumula 和 Taylor，1992）以验证衍生化是否完成。将聚糖用二甲亚砜溶解，然后用 NaOH 和碘甲烷进行甲基化。用二氯甲烷提取水和全O-甲基化碳水化合物。 糖基连接分析 为了测定糖基键，从释放的全甲基化 O-连接聚糖中制备部分甲基化糖醇乙酸酯 (PMAA)。 简而言之，将全甲基化聚糖用 2N 三氟乙酸 (TFA) 在 100℃ 下水解 4 小时，然后用 NaBD4 还原。后者释放的羟基用乙酸酐/吡啶（1:1， v/v) 在 100¿ C 15 分钟。 气相色谱-质谱联用 (GC-MS) PMAA 在与 5970 MSD 连接的 Hewlett Packard 5890 GC 上进行分析。分离在 30 m EC 1 键合相熔融石英毛细管柱 (Altech) 上进行。在以下条件下获得：烘箱温度，80 ¿ C (2 分钟) ¿ 180 ?? C (20°C/分钟)°第240章C（4℃/分钟）；检测器温度，280℃入口温度，250°C； C.