URONIC ACID/AMINO SUGAR LINKAGE AND 1-D NMR OF 2 SAMPLES

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。 糖基连接分析 对于糖基键分析，通过修改Hakomori的方法使样品甲基化，然后用超重氢预还原法。然后样品用NaOH方法重新甲基化、解聚、还原和乙酰化；生成的部分甲基化的糖醇乙酸酯(PMAA)用气相色谱-质谱仪(GC-MS)分析，如York等(1985)方法Enzyol所描述的那样。118：3-40。 简单地说，在冻干后的样品中取等分并溶解在0.4mLDMSO中，并加入0.6mLDMSO中的二磺酸钾(3.6M)。在室温下搅拌7h后，将反应混合物冷却至0℃，加入过量的碘甲烷(0.7mL)，并密封管子。然后在室温下继续孵化一夜。在样品处理之后，甲基化物质用超氢化氢还原，然后用氢氧化钠和甲基碘重新甲基化，以确保全甲基化完成。过甲基化后的样品用2M三氟乙酸水解液(在100℃密闭的管内反应2 h)，用NaBD4还原，用乙酸酐/吡啶进行乙酰化。所得的PMA在惠普5890 GC与5970 MSD(质量选择检测器，电子碰撞电离模式)接口上进行分析；糖醛酸的分离在30 m Supelco 2330键合相熔融石英毛细管柱上进行。生成的PMA也在惠普5890 GC与5970 MSD(质量选择检测器，电子碰撞电离模式)接口上进行分析；氨基糖的分离是在30m EC-1键合相熔融石英毛细管柱上进行的。

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. Glycosyl Linkage Analysis For glycosyl linkage analysis, the sample is methylated by a modification of the method of Hakomori, followed by pre-reduction by superdeuteride. Then the sample is repermethylated by the NaOH method, depolymerized, reduced, and acetylated; and the resultant partially methylated alditol acetates (PMAAs) are analyzed by gas chromatography-mass spectrometry (GC-MS) as described by York et al (1985) Methods Enzymol. 118:3-40. Briefly, an aliquot is taken from the sample after lyophilizing and dissolved in 0.4 mL DMSO, and 0.6 mL potassium dimsylate (3.6 M) in DMSO is added. After stirring for 7 hours at room temperature, the reaction mixture is cooled to 0 ¿C, excess methyl iodide (0.7 mL) is added, and the tube sealed. Incubation is then continued overnight at room temperature. Following sample workup, the methylated material is reduced by super-deuteride, followed by re-permethylation using NaOH base and methyl iodide to make sure that the permethylation is complete. Then the permethylated sample is hydrolyzed using 2 M trifluoroacetic acid (2 h in sealed tube at 100 ¿C), reduced with NaBD4, and acetylated using acetic anhydride/pyridine. The resulting PMAAs are analyzed on a Hewlett Packard 5890 GC interfaced to a 5970 MSD (mass selective detector, electron impact ionization mode); the separation of uronic acid is performed on a 30 m Supelco 2330 bonded phase fused silica capillary column. The resulting PMAAs are also analyzed on a Hewlett Packard 5890 GC interfaced to a 5970 MSD (mass selective detector, electron impact ionization mode); the separation of aminosugar is performed on a 30 m EC-1 bonded phase fused silica capillary column.