ANALYTICAL TECHNIQUES FOR CARBOHYDRATE STRUCTURE DETERMINATION

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。 碳水化合物结构测定的分析技术(一周课程) 本课程为期一周，内容包括使用气-液色谱/质谱仪(GC/MS)进行糖基残基和糖键组成分析的技术。实际操作的工作包括糖样的衍生化以制备糖醇醋酸酯和三甲基硅基衍生物，以及生产部分甲基化的糖醇醋酸酯，学员通过GC/MS进行分析。讲座和演示涵盖结构分析技术、MALDI-MS ANS ESI-MS质谱仪、^1H-核磁共振波谱，以及使用Metrohm峰高效液相色谱和其他色谱技术分离和纯化单糖和低聚糖的方法。课程内容还包括低聚糖的化学合成和多糖的分离提纯等。 本课程包括使用气-液色谱/质谱仪进行糖基残基(组成)和糖基连接(甲基化)分析的技术。参与者对纯化的低聚糖进行分析，他们可以携带自己的样本之一。讲座和演示涵盖了结构的其他技术 分析。此外，还包括使用质谱学或核磁共振光谱学分析糖共轭化合物的模块选择(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。 碳水化合物构象的计算模拟：本课程讨论了低聚糖的构象偏好。重点转移到预测和验证低聚糖结构产生的计算与分子动力学模拟与琥珀/GLYCAM力场。

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. Analytical Techniques for Carbohydrate Structural Determination (one-week course) This is a one-week course which covers the techniques of glycosyl-residue and glycosyl-linkage composition analyses using gas-liquid chromatography/mass spectrometry (GC/MS). The hands-on work includes the derivatization of sugar samples to make alditol acetates and trimethylsilyl derivatives and the production of partially methylated alditol acetates that the trainees analyze by GC/MS. Lectures and demonstrations cover such techniques for structural analysis MALDI-MS ans ESI-MS mass spectrometry, ^1 H-NMR spectroscopy, and methods for the separation and purification of monosaccharides and oligosaccharides using Metrohm peak HPLC and other chromatographic techniques. Additional lectures are also given on specific topics such as Chemical synthesis of oligosaccharides and isolation and purification of polysaccharides This course covers the techniques of glycosyl residue (composition) and glycosyl linkage (methylation) analyses using gas-liquid chromatography/mass spectrometry. Participants perform analyses of purified oligosaccharides, and they may bring one of their own samples. Lectures and demonstrations cover other techniques for structural analysis. In addition, a choice of modules either on using mass spectrometry or NMR spectroscopy for analysis of glycoconjugates is included (see descriptions below). Experience with basic biochemical techniques is a prerequisite for participation. 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. Computational Simulations of Carbohydrate Conformation: The course presents a discussion of the conformational preferences of oligosaccharides. Focus shifts to the prediction and validation of oligosaccharide structures produced computationally with molecular dynamics simulations with the AMBER/GLYCAM force field.