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

2 个样品的糖醛酸/氨基酸键和一维核磁共振

• 批准号: 8168889
• 负责人: Parastoo Azadi
• 金额: $ 0.17万
• 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: 2010-04-10 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8168889
• 关键词: Acetates; Aliquot; Amino Sugars; Blood capillaries; Computer Retrieval of Information on Scientific Projects Database; Dimethyl Sulfoxide; Electrons; Funding; Grant; Hour; Hydrolysis; Institution; Mass Fragmentography; Methods; Modification; Phase; Potassium; Reaction; Research; Research Personnel; Resources; Sampling; Silicon Dioxide; Source; Sugar Alcohols; Temperature; Trifluoroacetic Acid; Tube; United States National Institutes of Health; Uronic Acids; acetic anhydride; base; capillary; detector; genetic linkage analysis; ionization; methyl iodide; pyridine; seal

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.

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。 糖基连接分析 对于糖基连接分析，样品通过Hakomori方法的改进进行甲基化，然后通过超氘化物进行预还原。然后通过NaOH方法使样品再甲基化，解聚，还原和乙酰化;并且通过气相色谱-质谱法（GC-MS）分析所得的部分甲基化的糖醇乙酸酯（PMAA），如约克等人（1985）酶学方法（Methods Enzymol. 118：3-40。 简而言之，在冻干后从样品中取出等分试样并溶解于0.4 mL DMSO中，并加入0.6 mL二甲磺酸钾（3.6 M）的DMSO溶液。 在室温下搅拌7小时后，将反应混合物冷却至0 ℃，加入过量的碘甲烷（0.7 mL），并密封管。 然后在室温下继续孵育过夜。 样品处理后，用超氘化物还原甲基化材料，然后使用NaOH碱和碘甲烷进行再全甲基化，以确保全甲基化完成。然后，使用2 M三氟乙酸水解全甲基化样品（在密封管中于100 ℃下2 h），用NaBD 4还原，并使用乙酸酐/吡啶乙酰化。 在与5970 MSD（质量选择检测器，电子碰撞电离模式）接口的Hewlett Packard 5890 GC上分析所得PMAA;在30 m Supelco 2330键合相熔融石英毛细管柱上进行糖醛酸的分离。还在与5970 MSD（质量选择检测器，电子碰撞电离模式）连接的Hewlett Packard 5890 GC上分析所得PMAA;在30 μ m EC-1键合相熔融石英毛细管柱上进行氨基糖的分离。