Glycan linkage and sequence plus determination of site of glycosylation by permethylation of glycopeptides and MSn analysis in a one pot experiment

项目摘要/摘要 碳水化合物覆盖在每个活细胞的表面，是细胞与每个活细胞交流的方式 其他的。因此，它们对健康的身体和许多疾病过程都有巨大的影响， 包括癌症、糖尿病、心脏病和肺病、艾滋病等等。为了理解这门语言 在碳水化合物介导的细胞间通讯中，了解糖蛋白结构的细节是至关重要的。 然而，由于碳水化合物结构的巨大多样性，这是一项艰巨的任务，目前只有一个 美国有少数几个实验室有能力进行这项研究。详细的糖蛋白结构分析 必须能够识别连接多糖的肽序列以及糖链的结构 葡聚糖部分，包括低聚糖异构体、序列和糖基键。目前，质量 需要对释放的多糖和完整的糖肽进行光谱(MS)实验，以充分 阐明糖蛋白的结构。释放的多糖分析依赖于衍生化过程 称为“全甲基化”，即每个羟基质子都被一个甲基取代。单独实验 在完整的糖肽上是必需的，因为糖的释放取消了所有特定部位的信息。 还需要额外的工作流程来分离N-和O-连接的葡聚糖并确定 糖基化。本提案的第一个目标是通过以下方式将上述所有工作流程合并为一个工作流程 对完整的糖肽进行全甲基化和MS，而不是对释放的多糖进行全甲基化和MS。这将允许 与糖蛋白有关的所有必要信息，包括糖链、异构体、序列、 结合部位和糖基化程度将在一锅实验中获得。扩张的唯一途径 糖生物学领域是为了让其他科学家更容易接触到它，并结合糖的结构 在一次实验中用糖肽分析来阐明是朝着这一目标迈出的一步。胎球蛋白将被用作 以真核模型糖蛋白和空肠弯曲菌糖蛋白AcrA为模型原核糖蛋白。 涉及糖肽全甲基化和MSN分析的技术将被纳入 CCRC每年为糖业社区提供的实践培训课程。动手培训就是其中之一 使更大的科学界可以评估任何技术的最有效方法。为了 协助解释MS中产生的复杂数据，该项目的第二个目标是扩展 以前内部生成的软件，以包括全甲基化糖肽的自动注释 质谱图。现有软件是以模块化方式构建的，以便能够添加更多功能 通过“插件”。第三个目标是开发一种全甲基化试剂盒，以促进单一样本的分析 准备工作流程，以帮助不是碳水化合物分析专家的研究人员。

Project Summary/Abstract Carbohydrates cover the surface of every living cell and are the means by which cells communicate with each other. They have, therefore, enormous impact both in the healthy body as well as in many disease processes, including cancer, diabetes, heart and lung disease, AIDS, and many more. In order to understand the language of carbohydrate-mediated cell-cell communication, it is vital to know the details of glycoprotein structure. However, due to the huge diversity of carbohydrate structures this is a daunting task, and presently only a handful of laboratories in the U.S. are capable of doing this research. Detailed glycoprotein structural analysis has to be able to identify the peptide sequence where the glycans are attached, as well as the structure of the glycan portion, including oligosaccharide isomers, sequence and glycosyl linkages. Currently, mass spectrometry (MS) experiments on both released glycans as well as on intact glycopeptides are needed to fully elucidate the structure of glycoproteins. Released glycan analysis depends on a derivatization procedure called “permethylation”, in which every hydroxyl proton is replaced by a methyl group. Separate experiments on the intact glycopeptides are needed because glycan release abolishes all site-specific information. Additional workflows are also required to separate N- from O-linked glycans and to determine degree of glycosylation. The first aim of the present proposal is to consolidate all the above workflows into one by carrying out the permethylation and MS on intact glycopeptides instead of on released glycans. This will allow all the necessary information pertaining to the glycoprotein, including glycan linkage, isomers, sequence, attachment site, and degree of glycosylation to be obtained in a one-pot experiment. The only way to expand the field of glycobiology is to make it more accessible to other scientists, and combining glycan structure elucidation with glycopeptide analysis in a single experiment is a step towards that goal. Fetuin will be used as the eukaryotic model glycoprotein and C. jejuni glycoprotein AcrA as the model for prokaryotic glycoproteins. The technology involving permethylation of glycopeptides and MSn analysis will be incorporated into the CCRC's hands-on training courses that are annually offered to the glyco-community. Hands-on training is one of the most efficient ways to make any technology assessable to the larger scientific community. In order to assist the interpretation of the complex data generated in the MS, the second aim of this project is expansion of the software generated previously in-house to include automatic annotation of permethylated glycopeptide mass spectra. The existing software was built in a modular fashion to enable the addition of further functionality through “plugins”. The third aim is the development of a permethylation kit to facilitate the single sample preparation workflow to aid researchers who are not specialists in carbohydrate analysis.