LINKED GLYCOSYLS ANALYSIS BY GC-MS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。次级项目的主要支助 子项目的主要研究者可能是由其他来源提供的， 包括其他NIH来源。 为子项目列出的总成本可能 代表子项目使用的中心基础设施的估计数量， NCRR赠款不直接向子项目或子项目工作人员提供资金。 研究方法： 连接糖基组成分析 部分甲基化糖醇乙酸酯（PMAA）的制备 部分甲基化糖醇乙酸酯由CA 215批次S15 K，s100425的全甲基化O-连接聚糖的剩余部分制备（参见CCRC代码GL 042810 Z）。 简而言之，在121 ° C下用2N三氟乙酸水解全甲基化聚糖2小时，然后用NaBD 4还原。 经过水解和还原步骤后，部分甲基化糖醇的游离羟基在100 ° C下用乙酸酐：吡啶（1：1，v/v）乙酰化1小时以产生PMAA。 用二氯甲烷萃取部分甲基化的糖醇乙酸酯。 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，140 ℃（2.0 ℃/min）<$220 ℃（20 ℃/min）<$300 ℃（7.5 min）;检测器温度，280 ℃;入口温度，250 ℃。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Methods: Linked Glycosyl Composition Analysis Preparation of Partially Methylated Alditol Acetates (PMAAs) Partially methylated alditol acetates were prepared from the remainder of permethylated O-linked glycans of CA215 Lot # S15K, s100425 (refer to CCRC code # GL042810Z). Briefly, permethylated glycans were hydrolyzed with 2N trifluoroacetic acid at 121oC for 2 hr, followed by reduction with NaBD4. After hydrolysis and reduction steps, the free hydroxyls of the partially methylated alditols were acetylated with acetic anhydride:pyridine (1:1, v/v) at 100oC for 1 hr to produce PMAAs. Partially methylated alditol acetates were extracted with methylene chloride. Gas Chromatograph-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, 140¿C (2.0¿C/min) ¿ 220¿C (20¿C/min) ¿ 300¿C (7.5 min); detector temperature, 280 ¿C; inlet temperature, 250 ¿C.