COMPOSITION ANALYSIS BY GC-MS

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。 糖基组成分析进行了组合的气相色谱/质谱（GC/MS）的全-O-三甲基甲硅烷基（TMS）衍生物的单糖甲基糖苷的样品通过酸性甲醇分解。 将样品转移到螺旋盖管中，加入4 μ g肌醇作为内标，并干燥。 然后，通过用1 M HCl的甲醇溶液在80 ° C下甲醇分解18小时，从干燥的样品制备甲基糖苷。 在前一步骤之后，在甲醇中用吡啶和乙酸酐进行再N-乙酰化（用于检测氨基糖）。 然后将样品用Tri-Sil（Pierce）在80 ℃下全-O-三甲基甲硅烷基化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GC/MS分析在与Hewlett Packard 5970 MSD连接的Hewlett Packard Series II 5890上进行，使用Supelco EC-1熔融二氧化硅毛细管柱（30 m ² 0.25 mm ID）。

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 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. The sample was transferred to a screw-cap tube and added with 4 ¿g inositol as internal standard, and dried. Methyl glycosides then were prepared from the dried samples by methanolysis with 1 M HCl in methanol at 80¿C for 18 h. The preceding step was followed by re-N-acetylation with pyridine and acetic anhydride in methanol (for detection of amino sugars). The samples then were per-O-trimethylsilylated with Tri-Sil (Pierce) 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. GC/MS analysis of the TMS methyl glycosides was performed on a Hewlett Packard Series II 5890 interfaced to a Hewlett Packard 5970 MSD, using a Supelco EC-1 fused silica capillary column (30m ¿ 0.25 mm ID).