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

项目总结/摘要 碳水化合物覆盖在每个活细胞的表面，是细胞与细胞之间相互交流的方式。 其他.因此，它们对健康的身体以及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。为了理解语言 糖蛋白是糖介导的细胞间通讯的重要组成部分，了解糖蛋白结构的细节至关重要。 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 美国只有少数几个实验室有能力进行这项研究。详细糖蛋白结构分析 必须能够识别聚糖连接的肽序列，以及聚糖的结构。 聚糖部分，包括寡糖异构体、序列和糖基键。目前，Mass 需要对释放的聚糖以及完整的糖肽进行质谱（MS）实验，以充分 阐明糖蛋白的结构。释放的聚糖分析取决于衍生化程序 称为“全甲基化”，其中每个羟基质子被甲基取代。单独的实验 因为聚糖的释放消除了所有的位点特异性信息。 还需要额外的工作流程来分离N-和O-连接的聚糖，并确定N-和O-连接的聚糖的程度。 糖基化本提案的第一个目的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： 对完整的糖肽而不是释放的聚糖进行全甲基化和MS。这将允许 与糖蛋白有关的所有必要信息，包括聚糖键，异构体，序列， 连接位点和糖基化程度。扩张的唯一途径 糖生物学领域是使它更容易为其他科学家所用， 在单个实验中用糖肽分析进行阐明是朝着该目标迈出的一步。胎球蛋白将用作 真核模式糖蛋白和C.空肠糖蛋白AcrA作为原核糖蛋白的模型。 涉及糖肽全甲基化和MSn分析的技术将被纳入 CCRC每年为糖社区提供的实践培训课程。实践训练是一种 最有效的方法，使任何技术评估更大的科学界。为了 帮助解释MS中生成的复杂数据，该项目的第二个目的是扩展 之前内部生成的软件，包括全甲基化糖肽的自动注释 质谱现有的软件是以模块化的方式建立的，以便能够增加更多的功能 通过“插件”。第三个目标是开发一种全甲基化试剂盒，以方便单样本 准备工作流程，以帮助研究人员谁不是专家在碳水化合物分析。

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.