LINKAGE ANALYSIS OF PMAAS BY GC-MS

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。 透析 样品用Tube-O-透析器(4.0 kDa截止膜；G BioSciences)与纳米水在4摄氏度下进行约22小时的透析，以去除盐和其他污染物。在整个透析期内，纳米纯水被更换了三次。 通过-消除释放O-连接的葡聚糖 通过消除过程从样品中分离出O-连接的碳水化合物。简单地说，向每个样品中加入250°L的50 mM氢氧化钠，然后检查pH值。在确定pH值为碱性后，向每个样品中再加入250°L的50 mM氢氧化钠，其中含有19毫克的硼氢化钠，旋转，并在450℃下孵育一夜。孵化后的样品用10%的冰醋酸中和，通过填充的Dowex树脂柱脱盐，然后冷冻干燥。干燥样品在全甲基化前用甲醇：冰醋酸(9：1)在氮气中清洗。 O-连接糖链的Per-O-甲基化 释放的O-连接的葡聚糖被全甲基化，并通过质谱学(Anumula和Taylor，1992)进行评估，以验证衍生化是否进行到完成。用二甲基亚砜溶解多糖，然后用氢氧化钠和碘甲烷甲基化。反应用水熄灭，用二氯甲烷提取全O-甲基化碳水化合物。 糖基连接分析 为了确定糖基键，从释放的全甲基化O-连接的多糖中制备了部分甲基化的糖醇乙酸酯(PMAA)。简而言之，用2N-三氟乙酸(TFA)在100oC下水解4h，然后用NaBD4还原。后者用乙酸酐/吡啶(1：1，v/v)在100℃下乙酰化15min。 气相色谱-质谱法(GC-MS) 在Hewlett Packard 5890 GC与5970 MSD(质量选择检测器，电子碰撞电离模式)接口上分析了PMA。色谱柱为30m EC1固定相熔融石英毛细管柱(ALTECH)。在烘箱温度80℃(2 Min)、180℃(20℃/min)、240℃(4℃/min)、检测器温度280℃、进口温度250℃的条件下获得了电子轰击质谱图。

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.