MASS SPECTRAL SEQUENCING OF CARBOHYDRATES

碳水化合物的质谱测序

• 批准号: 2023284
• 负责人: VERNON Nye REINHOLD
• 金额: $ 35.11万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-01 至 2000-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2023284
• 关键词: adduct; carbohydrate sequence; chloride ion; chromium; glycosphingolipids; high performance liquid chromatography; mass spectrometry; method development; oligosaccharides

DESCRIPTION: This proposal outlines methodology to define all components of a primary carbohydrate sequence at the sensitivities characteristic of contemporary mass spectrometry. Two studies are detailed: (i) completion of a recently initiated sequencing strategy using chemically modified and derivatized glycans; and (ii) investigation of a significantly new and promising area for sequencing underivatized samples. Aspects of the derivatization method have been described, but components of structure remain unresolved. To cope with these details, Dr. Reinhold discusses glycan depolymerization, (to augment CID linkage information), releasing labile termini (to localize sialic acid and fucosyl termini), and studies to closely evaluate chromium trioxide oxidation (to determination anomeric configuration). These objectives can be subdivided into four separate subprojects: first, extend linkage and branching details to complex and hybrid glycans by preparing smaller, overlapping oligomer subsets via partial depolymerization; second, for multibranched (antennae) structures, evaluate methodology for locating labile non-reducing termini, by mild chemical release and CD(3)-remethylation, and stablizing the neuraminyl linkage through carboxyl modification; third, commence a detailed study into the chemistry and applicability of chromium trioxide oxidation (CTO) for the characterization of anomeric configuration in larger and more complex glycans; and fourth, initiate an investigation of negative ion CID to capitalize on the extensive fragmentation observed for chloride adducts. Capillary HPLC-MS and CID will be utilized to understand the diverse and isomeric mixtures anticipated following depolymerization. Chromatographic protocols will be established for the methylated products under investigation, (e.g., modified and methylated glycans, oligosaccharides, and glycosphingolipids), using a new silica C-18 with improved alkane packing densities. Classical techniques of composition and linkage analysis (methanolysis, methylated alditol acetates) by GC-MS will support methods development projects. The overall goal of this research is to obtain sufficient sequence information to completely define high mannose, complex, and hybrid glycotypes with the trace amounts of sample frequently available.

描述：本提案概述了定义以下所有组件的方法 一种初级碳水化合物序列，具有以下特征的敏感性 当代质谱学。详细介绍了两项研究：(I)完成 一种最近启动的测序策略，使用化学修饰和 以及(Ii)对一种显著的新的和 未衍生样本测序的前景看好。 已经描述了衍生化方法的各个方面，但是 结构仍未解决。为了应对这些细节，莱因霍尔德博士 讨论多糖解聚，(以增加CID连接信息)， 释放不稳定末端(定位唾液酸和岩藻糖基末端)，以及 严密评价三氧化二铬氧化(测定)的研究 异常配置)。这些目标可以细分为四个 单独的子项目：首先，将链接和分支详细信息扩展到 通过制备更小的重叠低聚物来制备复杂和杂化的葡聚糖 通过部分解聚的子集；第二，用于多分支(天线) 结构，评价定位不稳定的非还原末端的方法， 通过温和的化学释放和Cd(3)-再甲基化，并稳定 通过羧基修饰的神经氨基连接；第三，开始详细的 三氧化二铬氧化的化学性质及适用性研究 (CTO)用于表征更大和更多的反常构型 复合多糖；第四，启动负离子CID的研究 以利用观察到的氯化物加合物的广泛碎裂。 将利用毛细管高效液相色谱-质谱仪和毛细管电泳联用来了解不同的和 解聚后预计会出现同分异构体混合物。色谱学 将为下列甲基化产品制定协议 研究(例如，修饰和甲基化的多糖、低聚糖和 鞘糖脂)，使用具有改进的烷烃填充的新的硅胶C-18 密度。 经典的组成和连锁分析技术(甲烷分解， 经GC-MS分析的甲基化阿迪醇乙酸酯)将支持方法的开发 项目。这项研究的总体目标是获得足够的 完整定义高甘露糖、复合体和杂交种的序列信息 具有微量样品的糖类经常可用。