MASS SPECTRAL SEQUENCING OF CARBOHYDRATES

碳水化合物的质谱测序

• 批准号: 6138514
• 负责人: VERNON Nye REINHOLD
• 金额: $ 29.76万
• 依托单位: UNIVERSITY OF NEW HAMPSHIRE
• 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/6138514
• 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：该提案概述了定义所有组件的方法 初级碳水化合物序列的敏感性特征 现代质谱分析法。 两项研究的详细内容如下：(i) 完成情况 最近启动的测序策略使用化学修饰和 衍生聚糖； (ii) 对一项重大新的和 未衍生样品测序的有前景的领域。 已经描述了衍生化方法的各个方面，但是衍生化方法的组成部分 结构仍未解决。 为了应对这些细节，Reinhold 博士 讨论聚糖解聚（以增强 CID 连接信息）， 释放不稳定末端（以定位唾液酸和岩藻糖基末端），以及 密切评估三氧化铬氧化的研究（测定 异头构型）。 这些目标可以细分为四个 单独的子项目：首先，将链接和分支细节扩展到 通过制备更小的、重叠的寡聚物来复杂和杂化聚糖 通过部分解聚形成子集；第二，对于多分支（天线） 结构，评估定位不稳定非还原末端的方法， 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酰基连接；第三，开始详细的 三氧化铬氧化的化学性质及其应用研究 （CTO）用于表征更大和更多的异头构型 复杂聚糖；四、发起负离子CID调查 利用观察到的氯化物加合物的广泛碎片。 毛细管 HPLC-MS 和 CID 将用于了解多样化和 预计解聚后会出现异构体混合物。 色谱法 将为甲基化产品制定协议 研究（例如，修饰和甲基化聚糖、寡糖和 鞘糖脂），使用改进烷烃填料的新型二氧化硅 C-18 密度。 组成和连锁分析的经典技术（甲醇分解， GC-MS 检测的甲基化糖醇乙酸酯）将支持方法开发 项目。 本研究的总体目标是获得足够的 序列信息可完整定义高甘露糖、复合糖和杂合糖 经常可获得微量样品的糖型。