SEPARATION AND CHARACTERIZATION OF GLYCOPROTEIN AND GLYCOLIPID OLIGOSACCHARIDES

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。 在本课程中，学员通过动手操作、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。 来自糖蛋白的寡糖或糖肽的混合物通过色谱程序如尺寸排阻、HPLC、凝集素亲和色谱和/或聚丙烯酰胺凝胶电泳被解析成单个组分。 鞘糖脂的薄层色谱分离，并讨论了检测方案。 使用凝集素印迹技术表征寡糖的结构特征。 其他主题包括寡糖与荧光标记的结合，毛细管电泳和单糖组成分析。 此外，还包括使用质谱和/或NMR光谱分析糖缀合物的模块（见下文描述）。 基本生物化学技术的经验是参加的先决条件。 附加课程模块 糖缀合物的质谱（MS）分析：讨论了质谱法用于糖缀合物表征的用途。 这一领域的主题包括通过LC-MS确定已占据的糖基化位点，并通过连续的外切糖苷酶diglase和MS对糖蛋白聚糖进行测序。对这两个主题进行了实验室演示。 碳水化合物的NMR：该模块介绍了多糖和糖缀合物的NMR光谱应用，涵盖i）NMR光谱的基本特征，ii）用于解决一级结构的协议，iii）当前文献中的应用和示例，以及iv）信息资源。 我们的四台NMR仪器（300-，500-，600-和800 MHz）在本模块中用于演示。

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.