MASS SPECTRAL SEQUENCING OF CARBOHYDRATES

描述：本提案概述了定义以下内容的方法： 一级碳水化合物序列，其敏感性特征为 现代质谱法 详细介绍了两项研究：（一）完成 最近启动的测序策略，使用化学修饰和 衍生聚糖;和（ii）研究一种新的， 对未衍生化样本进行测序的有前途的领域。 已经描述了衍生化方法的各个方面，但是衍生化方法的组分是： 结构问题仍未解决。 为了科普这些细节，莱因霍尔德博士 讨论了聚糖解聚，（以增加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），和 研究密切评估三氧化铬氧化（以确定 异头构型）。 这些目标可细分为四个 独立的子项目：首先，扩展链接和分支细节， 通过制备较小的重叠低聚物的复合和杂合聚糖 通过部分解聚的子集;第二，对于多分支（触角） 结构，评价定位不稳定非还原性末端的方法， 通过温和的化学释放和CD（3）-再甲基化， 通过羧基修饰的神经氨酰连接;第三，开始详细的 三氧化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异头构型 复合聚糖;第四，启动负离子CID调查 以利用对于氯化物加合物观察到的广泛碎片化。 毛细管HPLC-MS和CID将用于了解不同的和 预期解聚后的异构体混合物。 色谱 甲基化产物的方案将根据 调查，（例如，修饰的和甲基化的聚糖、寡糖和 鞘糖脂），使用具有改进的烷烃填充的新二氧化硅C-18 密度 组成和连接分析的经典技术（甲醇分解， 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 完整定义高甘露糖、复合物和杂合物的序列信息 糖型与微量的样品经常可用。

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.