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.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 研究方法： 透析 使用Tube-O-Dialyzer（4.0 kDa截留膜; G BioSciences）在4 ℃下对纳米纯水透析样品约24小时，以去除盐和其他污染物。 在整个透析期间，纳米纯水更换了四次。 透析后，将样品转移至螺旋盖管中并冻干。 糖基组成（GC-MS法） 向样品中加入5 μ g肌醇作为内标。 在甲醇分解之前，将干燥样品、单糖标准品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 ℃下用3 M HCl的甲醇溶液甲醇分解2 h，然后用吡啶和乙酸酐的甲醇溶液进行再N-乙酰化（用于检测氨基糖），从干燥样品中制备甲基糖苷。 将前述甲醇分解和再-N-乙酰化步骤重复两次。 然后将样品用Tri-Sil试剂（Thermo Scientific）在80 ℃下全-O-三甲基甲硅烷基化（TMS）0.5小时。这些程序如先前在Merkle和Poppe（1994）Methods Enzymol. 230：1-15;约克等人（1985）酶学方法（Methods Enzymol. 118：3-40。 TMS甲基糖苷的分析在配备有Supelco EC-1熔融石英毛细管柱（30 m × 0.25 mm ID）并与Hewlett Packard 5970 MSD连接的Hewlett Packard Series II 5890气相色谱仪上进行。