SEPARATION AND CHARACTERIZATION OF GLYCOCONJUGATE 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。 糖共轭低聚糖的分离与表征(一周课程) 这门为期一周的课程涵盖了低聚糖的分离和表征的基本技术，包括糖的比色法定量，低聚糖混合物的高效液相色谱分离，用于单糖组成分析的Metrohm Peak高pH阴离子交换层析，低聚糖从糖偶联物中的酶释放，糖脂的薄层分离和重叠分析检测，以及低聚糖的内外糖苷酶处理。使用凝集素印迹技术来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。除了CCRC的教职员工外，其他讲者也被邀请帮助讲授这门课程。佐治亚理工学院的Alfred Merrill博士作了题为“糖脂的质谱学结构分析”的讲座。此外，Dionex Inc.的Val Hanko还作了题为《高pH阴离子交换层析分析碳水化合物》的讲座。佐治亚大学化学系的Jon Amster博士也作了题为《FT-MS分析寡糖》的讲座。 附加课程模块 糖共轭化合物的质谱学分析：讨论了利用质谱学对糖共轭化合物进行表征。这一领域的主题包括用LC-MS鉴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。 碳水化合物的核磁共振：核磁共振模块包括对核磁共振波谱在多糖和糖共轭化合物中的应用的介绍，包括(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。我们的四台核磁共振仪器(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. Separation and Characterization of Glycoconjugate Oligosaccharides (one-week course) This is a one-week course that covers basic techniques for the isolation and characterization of oligosaccharides, including colorimetric quantitation of sugars, chromatographic separation of oligosaccharide mixtures by HPLC, Metrohm peak high-pH anion-exchange chromatography for monosaccharide composition analysis, enzymatic release of oligosaccharides from a glycoconjugate, separation of glycolipids by thin-layer chromatography and detection by overlay assay, and endo- and exoglycosidase treatment of oligosaccharides. The use of lectin blotting techniques for characterization of oligosaccharide structural features are covered. Other topics 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 are included (see descriptions below). Experience with basic biochemical techniques is a prerequisite for participation. In addition to CCRC faculty, other speakers were invited to help teach the course. Dr. Alfred Merrill from Georgia Institute of Technology gave a lecture on "Structural Analysis of Glycolipids by Mass Spectrometry". In addition, Val Hanko from Dionex Inc. gave a lecture on "Carbohydrates analysis by High pH Anion Exchange Chromatography." Dr. Jon Amster from the Chemistry Department at University of Georgia also gave a lecture on "Analysis of oligosaccharides by FT-MS. Additional Course Modules Mass Spectrometric (MS) Analysis of Glycoconjugates: The use of mass spectrometry for the characterization of glycoconjugates is discussed. Topics in this area include identifying occupied glycosylation sites by LC-MS and sequencing glycoprotein glycans with sequential exoglycosidase digestions followed by MS. Laboratory demonstrations are performed on these two topics. NMR of Carbohydrates: The NMR module consists of an introduction to the application of NMR spectroscopy to 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) are used for demonstrations during this module.