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.

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。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 透析 使用Tube-O-Dialyzer（4.0 kDa截留膜; G BioSciences）在4 ℃下对纳米纯水透析样品约22小时，以去除盐和其他污染物。 在整个透析期间，纳米纯水更换三次。 通过消除释放O-连接聚糖 O-连接的碳水化合物通过消除程序从样品中裂解。 简而言之，250 º向每个样品中加入250 μ L 50 mM NaOH，然后检查pH值。确定pH值为碱性后，向每个样品中加入另外250 μ L含有19 mg硼氢化钠的50 mM NaOH，涡旋，并在45 ℃下孵育过夜。 然后将孵育的样品用10%乙酸中和，并通过Dowex树脂的填充柱脱盐，然后冻干。 在全甲基化之前，在氮气流下用甲醇：乙酸（9：1）清洗干燥的样品的硼酸盐。 O-连接聚糖的全O-甲基化 将释放的O-连接聚糖全甲基化，并通过质谱法（Anumula和Taylor，1992）进行评价，以验证衍生化是否完成。 用二甲亚砜溶解聚糖，然后用NaOH和碘甲烷甲基化。 用水淬灭反应，用二氯甲烷萃取全-O-甲基化的碳水化合物。 糖基连接分析 为了测定糖基连接，从释放的全甲基化O-连接聚糖制备部分甲基化糖醇乙酸酯（PMAA）。 简言之，用2N三氟乙酸（TFA）在100 ℃下水解全甲基化聚糖4 h，然后用NaBD 4还原。 将后者游离的羟基用乙酸酐/吡啶（1：1，v/v）在100 ℃下乙酰化15 min。 气相色谱-质谱（GC-MS） 在与5970 MSD（质量选择检测器，电子碰撞电离模式）连接的Hewlett Packard 5890 GC上分析PMAA。 在30 μ m EC 1键合相熔融石英毛细管柱（Altech）上进行分离。 在以下条件下获得电子轰击质谱：烘箱温度，80 ℃（2 min）<$180 ℃（20 ℃/min）<$240 ℃（4 ℃/min）;检测器温度，280 ℃;入口温度，250 ℃。