SEPARATION AND CHARACTERIZATION OF GLYCOPROTEIN AND GLYCOLIPID OLIGOSACCHARIDES

糖蛋白和糖脂低聚糖的分离和表征

• 批准号: 8361869
• 负责人: Parastoo Azadi
• 金额: $ 0.44万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-02-01 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8361869
• 关键词: Area; Biochemical; Carbohydrate Conformation; Carbohydrates; Computer Simulation; Detection; Digestion; Exoglycosidases; Funding; Glycoconjugates; Glycolipids; Glycoproteins; Grant; Laboratories; Learning; Lectin; Link; Literature; Mass Spectrum Analysis; Monosaccharides; NMR Spectroscopy; National Center for Research Resources; Oligosaccharides; Participant; Polysaccharides; Principal Investigator; Protocols documentation; Research; Research Infrastructure; Resources; Site; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Structure; Techniques; Thin Layer Chromatography; United States National Institutes of Health; Validation; Work; cost; experience; glycosylation; instrument; interest; lectures; molecular dynamics; preference

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Participants will learn basic techniques for the isolation and characterization of glycoprotein and glycolipid oligosaccharides via a combination of hands-on work, lectures and demonstrations. Participants will release N- and O-linked glycans from a glycoprotein. The released glycans will be purified, derivertized and profiled by mass spectrometry. (MALDI/TOF-MS and ESI-MSn). The monosaccharide composition on a glycoprotein will be analyzed by HPEAC. Glycophingolipids will be separated by thin-layer chromatography and detection protocols will be discussed. The use of lectin blotting techniques for characterization of oligosaccharide structural features will be covered. In addition, modules on using mass spectrometry, computer modeling or NMR spectroscopy for analysis of glycoconjugates will be included. Experience with basic biochemical techniques is a prerequisite for participation. Additional Course Modules On the Friday of each of these two courses, participants can choose one of the following three modules, Mass Spectrometric (MS) Analysis of Glycoconjugates, Computational Simulations of Carbohydrate Conformation and NMR of Carbohydrates. The course participants should indicate on the course application form which module they would be interested in taking. Mass Spectrometric (MS) Analysis of Glycoconjugates: The use of mass spectrometry for the characterization of glycoconjugates will be discussed. Topics in this area will include identifying occupied glycosylation sites by LC-MS and sequencing glycoprotein glycans with sequential exoglycosidase digestions followed by MS. Laboratory demonstrations will also be performed on these two topics. Computational Simulations of Carbohydrate Conformation: The course will present a discussion of the conformational preferences of oligosaccharides. Focus will then shift to the prediction and validation of oligosaccharide structures produced computationally with molecular dynamics simulations with the AMBER/GLYCAM force field. NMR of Carbohydrates: This module will be an introduction to the application of NMR spectroscopy of polysaccharides and glycoconjugates covering: a) essential features of NMR spectra; b) protocols used to solve primary structures; c) applications and examples from the current literature, and; d) resources for information. Our four NMR instruments (300-, 500-, 600-, and 800 mHz) will be used for demonstrations during this module.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 参加者将通过动手操作、讲座和演示相结合的方式学习糖蛋白和糖脂寡糖的分离和表征的基本技术。 参与者将从糖蛋白中释放N-和O-连接聚糖。 将通过质谱法对释放的聚糖进行纯化、衍生化和分析。（MALDI/TOF-MS和ESI-MSn）。将通过HPEAC分析糖蛋白上的单糖组成。 糖鞘脂将被分离的薄层色谱和检测协议将进行讨论。 将包括使用凝集素印迹技术来表征寡糖的结构特征。 此外，将包括使用质谱，计算机建模或NMR光谱分析糖缀合物的模块。 具有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 在这两门课程的每一个星期五，参与者可以选择以下三个模块之一，糖缀合物的质谱（MS）分析，碳水化合物构象的计算模拟和碳水化合物的NMR。 参加者应在课程申请表上注明他们有兴趣参加哪个单元。 糖缀合物的质谱（MS）分析：将讨论质谱法用于糖缀合物的表征。 在这方面的主题将包括确定占领的糖基化位点的LC-MS和测序糖蛋白聚糖与顺序外切糖苷酶diglase随后由MS实验室演示也将在这两个主题进行。 碳水化合物构象的计算机模拟：本课程将讨论寡糖的构象偏好。然后，重点将转移到预测和验证寡糖结构的计算与分子动力学模拟与琥珀/GLYCAM力场。 碳水化合物的NMR：本模块将介绍多糖和糖缀合物的NMR光谱应用，涵盖：a）NMR光谱的基本特征; B）用于解析一级结构的协议; c）当前文献中的应用和示例; d）信息资源。 我们的四台NMR仪器（300-，500-，600-和800 mHz）将用于本模块的演示。