LINKED GLYCOSYLS ANALYSIS BY GC-MS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 方法： 键合糖基组成分析 部分甲基化糖醇乙酸酯(PMAAs)的制备 从CA215批号为S15K、S100425(参见CCRC代码#GL042810Z)的全甲基化O-连接多糖的剩余部分制备部分甲基化的阿迪醇乙酸酯。简单地说，用2N三氟乙酸在121oC下水解2小时，然后用NaBD4还原。经过水解和还原步骤，部分甲基化的醛二醇的游离羟基与乙酸酐：吡啶(1：1，v/v)在100℃下乙酰化1小时得到PMAA。用二氯甲烷提取部分甲基化的阿迪醇乙酸酯。 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)。在以下条件下获得电子轰击质谱图：烘箱温度140℃(2.0℃/min)220℃(20℃/min)300℃(7.5 min)，检测器温度280℃，进口温度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.