SEPARATION AND CHARACTERIZATION OF GLYCOPROTEIN AND GLYCOLIPID OLIGOSACCHARIDES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 在这门课程中，学员通过实践、讲座和演示相结合的方式学习了低聚糖分离和表征的基本技术。低聚糖或糖蛋白糖肽的混合物通过大小排除、高效液相色谱、凝集素亲和层析和/或聚丙烯酰胺凝胶电泳等层析程序被分解成单独的组分。用薄层层析法分离了鞘糖脂，并对检测方案进行了讨论。使用凝集素印迹技术表征低聚糖的结构特征。其他主题包括低聚糖与荧光标记的结合，毛细管电泳法和单糖组成分析。此外，还包括使用质谱学和/或核磁共振光谱学分析糖共轭化合物的模块(见下文描述)。基本的生化技术经验是参加的先决条件。 附加课程模块 糖结合物的质谱学分析：讨论了利用质谱学对糖结合物进行表征的方法。这一领域包括的主题包括用LC-MS确定占据的糖基化位点，并用顺序的外切糖苷酶消化对糖蛋白多糖进行测序，然后进行MS。就这两个主题进行了实验室演示。 碳水化合物的核磁共振：本模块介绍了多糖和糖共轭化合物的核磁共振波谱的应用，涵盖了i)核磁共振谱的基本特征，ii)用于解析一级结构的方案，iii)当前文献中的应用和例子，以及iv)信息资源。在本模块中，我们使用了四台核磁共振仪器(300 MHz、500 MHz、600 MHz和800 MHz)进行演示。

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. In this course participants learned basic techniques for the isolation and characterization of oligosaccharides via a combination of hands-on work, lectures and demonstrations. Mixtures of oligosaccharides or glycopeptides derived from glycoproteins were resolved into individual components by chromatographic procedures such as size-exclusion, HPLC, lectin affinity chromatography, and/or polyacrylamide gel electrophoresis. Glycosphingolipids were separated by thin-layer chromatography, and detection protocols were discussed. The use of lectin blotting techniques for characterization of oligosaccharide structural features were covered. Other topics which were included are conjugation of oligosaccharides with fluorescent tags, capillary electrophoresis and monosaccharide composition analysis. In addition, modules on using mass spectrometry and/or NMR spectroscopy for analysis of glycoconjugates were included (see descriptions below). Experience with basic biochemical techniques was a prerequisite for participation. Additional Course Modules Mass Spectrometric (MS) Analysis of Glycoconjugates: The use of mass spectrometry for the characterization of glycoconjugates was discussed. Topics included in this area included identifying occupied glycosylation sites by LC-MS and sequencing glycoprotein glycans with sequential exoglycosidase digestions followed by MS. Laboratory demonstrations were performed on these two topics. NMR of Carbohydrates: This module was an introduction to the application of NMR spectroscopy of polysaccharides and glycoconjugates covering i) essential features of NMR spectra, ii) protocols used to solve primary structures, iii) applications and examples from the current literature, and iv) resources for information. Our four NMR instruments (300-, 500-, 600- and 800 MHz) were used for demonstrations during this module.