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

一锅实验中的聚糖连接和序列以及通过糖肽全甲基化和 MSn 分析确定糖基化位点

• 批准号: 9337473
• 负责人: Parastoo Azadi
• 金额: $ 29.1万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9337473
• 关键词: Acquired Immunodeficiency Syndrome; Biomedical Research; Bos taurus structural-GP protein; Campylobacter jejuni; Carbohydrates; Cell Communication; Cells; Chemicals; Communities; Complex; Computer software; Core Facility; Data; Data Analyses; Development; Diabetes Mellitus; Disease; Glycobiology; Glycopeptides; Glycoproteins; Goals; HSV glycoprotein C; Heart Diseases; Hydroxyl Radical; Isomerism; Laboratories; Language; Link; Lung diseases; Malignant Neoplasms; Manuals; Mass Spectrum Analysis; Mediating; Methodology; Modeling; Molecular Biology; Oligosaccharides; Polysaccharides; Preparation; Procedures; Process; Proteins; Protocols documentation; Protons; Reaction; Research; Research Personnel; Retrieval; Sampling; Science; Scientist; Site; Specialist; Structure; Surface; Technology; Time; Training; alpha-Fetoproteins; analytical tool; carbohydrate structure; experimental study; glycoprotein structure; glycoproteomics; glycosylation; instrument; methyl group; protein aminoacid sequence; skills; success; tandem mass spectrometry; tool

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.

项目概要/摘要 碳水化合物覆盖每个活细胞的表面，是细胞相互沟通的手段 其他。因此，它们对健康身体以及许多疾病过程都有巨大影响， 包括癌症、糖尿病、心脏病和肺病、艾滋病等等。为了理解语言 对于碳水化合物介导的细胞间通讯，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，目前只有一个 美国只有少数实验室有能力进行这项研究。详细的糖蛋白结构分析 必须能够识别聚糖所附着的肽序列以及聚糖的结构 聚糖部分，包括寡糖异构体、序列和糖基连接。目前，大众 需要对释放的聚糖以及完整的糖肽进行光谱测定 (MS) 实验，以充分了解 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”，其中每个羟基质子都被甲基取代。单独的实验 需要对完整的糖肽进行分析，因为聚糖的释放消除了所有位点特异性信息。 还需要额外的工作流程来分离 N- 连接聚糖和 O-连接聚糖并确定 糖基化。本提案的第一个目标是将上述所有工作流程整合为一个 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、 一锅实验中获得的附着位点和糖基化程度。扩张的唯一途径 糖生物学领域的目标是让其他科学家更容易理解，并将聚糖结构结合起来 在一次实验中通过糖肽分析进行阐明是朝着这一目标迈出的一步。胎球蛋白将用作 真核模型糖蛋白和空肠弯曲菌糖蛋白AcrA作为原核糖蛋白模型。 涉及糖肽全甲基化和MSn分析的技术将被纳入 CCRC 每年向糖界提供实践培训课程。实践培训是其中之一 使任何技术都能被更广泛的科学界评估的最有效方法。为了 协助解释 MS 中生成的复杂数据，该项目的第二个目标是扩展 之前内部生成的软件，包括全甲基化糖肽的自动注释 质谱。现有软件以模块化方式构建，以便添加更多功能 通过“插件”。第三个目标是开发全甲基化试剂盒以方便单个样品 准备工作流程可帮助非碳水化合物分析专家的研究人员。