GLYCOSYL COMPOSITION BY TWO DIFFERENT METHODS

两种不同方法的糖基组成

• 批准号: 8363093
• 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: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363093
• 关键词: Acetylation; Acids; Aliquot; Amino Sugars; Blood capillaries; Chitin; Detection; Funding; Glycosides; Grant; Hour; Inositol; Mass Fragmentography; Methanol; Methods; Monosaccharides; National Center for Research Resources; Principal Investigator; Procedures; Research; Research Infrastructure; Resources; Running; Sampling; Silicon Dioxide; Source; Technology; Tube; United States National Institutes of Health; acetic anhydride; capillary; cost; pyridine

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: Glycosyl composition Glycosyl composition analysis was performed by combined gas chromatography/mass spectrometry (GC/MS) of the per-O-trimethylsilyl (TMS) derivatives of the monosaccharide methyl glycosides produced from the sample by acidic methanolysis. A 500 and 700ug aliquot was taken from the sample and added to separate tubes with 20ug of inositol as the internal standard. For chitin analysis, the tube with 700ug was hydrolyzed in 4M HCl for 4 hours at 100 degrees Celsius. The sample was then dried down and run in parallel with the 500ug sample. Methyl glycosides were then prepared from the two dry samples following the mild acid treatment by methanolysis in 1 M HCl in methanol at 80¿C (18 hours), followed by re-N-acetylation with pyridine and acetic anhydride in methanol (for detection of amino sugars). The samples were then per-O-trimethylsilylated by treatment with Tri-Sil (Pierce) at 80¿C (0.5 hours). These procedures were carried out as previously described in Merkle and Poppe (1994) Methods Enzymol. 230: 1-15; York, et al. (1985) Methods Enzymol. 118:3-40. GC/MS analysis of the TMS methyl glycosides was performed on an AT 6890N GC interfaced to a 5975B MSD, using a Supelco EC-1 fused silica capillary column (30m ¿ 0.25 mm ID).

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供 该子项目的主要支持。 并且子项目的首席研究员可能是由其他来源提供的， 包括其他 NIH 来源的子项目可能列出的总成本。 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。 方法： 糖基组成 通过气相色谱/质谱联用 (GC/MS) 对通过酸性甲醇分解样品产生的单糖甲基糖苷的全 O-三甲基甲硅烷基 (TMS) 衍生物进行糖基组成分析。 从样品中取出 500 微克和 700 微克等分试样，分别加入装有 20 微克肌醇作为内标的试管中。为了进行几丁质分析，将装有 700 微克的试管在 4 M HCl 中于 100 摄氏度下水解 4 小时，然后干燥样品。然后，通过弱酸处理，从两个干燥样品中制备出 500ug 甲基糖苷。在 80° 的 1 M HCl 甲醇溶液中进行甲醇分解C（18小时），然后用吡啶和乙酸酐在甲醇中进行再N-乙酰化（用于检测氨基糖），然后通过在80°下用Tri-Sil（Pierce）处理来对样品进行全O-三甲基甲硅烷基化。 C（0.5小时）。这些程序如先前在Merkle和Poppe（1994）Methods Enzymol.230：1-15；York等人（1985）Methods Enzymol.118：3-40中所述进行。 TMS 甲基糖苷的分析是在连接 5975B MSD 的 AT 6890N GC 上进行的，使用Supelco EC-1 熔融石英毛细管柱（30m ¿ 0.25 mm ID）。