COMPOSITION ANALYSIS BY GC-MS

通过 GC-MS 进行成分分析

• 批准号: 8363054
• 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/8363054
• 关键词: Acetylation; Amino Sugars; Blood capillaries; Detection; Dialysis procedure; Dryness; Funding; Gases; Glycosides; Grant; Guanine + Cytosine Composition; Hour; Iduronic Acid; Inositol; Membrane; Methanol; Methods; Monosaccharides; National Center for Research Resources; Principal Investigator; Procedures; Reagent; Research; Research Infrastructure; Resources; Salts; Sampling; Series; Silicon Dioxide; Source; Technology; Time; Tube; United States National Institutes of Health; Vacuum; Water; 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: Dialysis The sample was dialyzed using a Tube-O-Dialyzer (4.0 kDa cut-off membrane; G BioSciences) against nanopure water at 4oC for about 24 hours to remove salts and other contaminants. Nanopure water was replaced four times during the entire dialysis period. After dialysis, the sample was transferred into a screw-cap tube and lyophilized. Glycosyl composition by GC-MS The sample was added with 5 ¿g inositol as internal standard. The dried sample, monosaccharides standard mixture, and iduronic acid standard were placed under vacuum to attain complete dryness before methanolysis. Methyl glycosides then were prepared from the dried sample by methanolysis with 3 M HCl in methanol at 100¿C for 2 h followed by re-N-acetylation with pyridine and acetic anhydride in methanol (for detection of amino sugars). The preceding methanolysis and re-N-acetylation steps were repeated two times. The samples then were per-O-trimethylsilylated (TMS) with a Tri-Sil reagent (Thermo Scientific) at 80¿C for 0.5 h. These procedures were carried out as described previously in Merkle and Poppe (1994) Methods Enzymol. 230: 1-15; York, et al. (1985) Methods Enzymol. 118:3-40. Analysis of the TMS methyl glycosides was performed on a Hewlett Packard Series II 5890 gas chromatograph equipped with a Supelco EC-1 fused silica capillary column (30m ¿ 0.25 mm ID) and interfaced to a Hewlett Packard 5970 MSD.

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。 并且子项目的主要研究者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： 透析 使用 Tube-O-Dialyzer（4.0 kDa 截止膜；G BioSciences）在 4℃ 下对纳米纯水进行透析约 24 小时，以去除盐和其他污染物。在整个透析期间更换四次纳米纯水。透析后，将样品转移至螺帽管中并冻干。 GC-MS 测定的糖基组成 样品中添加了 5 ¿ g 肌醇作为内标，在甲醇解之前将干燥的样品、单糖标准混合物和艾杜糖醛酸标准品置于真空下以达到完全干燥，然后通过在 100°C 下用 3 M HCl 的甲醇溶液进行甲醇解来制备甲基糖苷。 C 2小时，然后用甲醇中的吡啶和乙酸酐进行再N-乙酰化（用于检测氨基糖），然后将样品进行全O-三甲基甲硅烷基化。 (TMS) 使用 Tri-Sil 试剂 (Thermo Scientific) 在 80¿ C 0.5 小时，如先前 Merkle 和 Poppe (1994)Methods Enzymol.230:1-15；York 等人 (1985)Methods Enzymol.118:3-40 中所述进行。甲基糖苷分析是在配有 Supelco EC-1 融合的 Hewlett Packard Series II 5890 气相色谱仪上进行的硅胶毛细管柱（30m ¿ 0.25 mm ID）并连接到 Hewlett Packard 5970 MSD。