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.

项目摘要/摘要 碳水化合物覆盖了每个活细胞的表面，并且是细胞与每个生物的通信 其他。因此，它们在健康身体以及许多疾病过程中都产生了巨大影响， 包括癌症，糖尿病，心脏和肺部疾病，艾滋病等。为了理解语言 碳氢化物介导的细胞 - 细胞通信，了解糖蛋白结构的细节至关重要。 但是，由于碳水化合物结构的多样性，这是一项艰巨的任务，只提出 美国的少数实验室能够进行这项研究。详细的糖蛋白结构分析 必须能够识别固定聚糖的肽序列，以及 聚糖部分，包括寡糖异构体，序列和糖基键。目前，弥撒 在释放的聚糖以及完整的糖肽上进行的光谱法（MS）实验都需要充分 阐明糖蛋白的结构。释放的聚糖分析取决于衍生化过程 称为“苄苄氨基化”，其中每个羟基质子被甲基取代。单独的实验 需要在完整的糖肽上，因为聚糖释放废除了所有特定地点的信息。 还需要其他工作流程才能将N-与O连锁聚糖分开并确定程度 糖基化。本提案的第一个目的是将上述所有工作流程合并为一个 在完整的糖磷酸酯上进行苄苄苄化和MS，而不是在释放的聚糖上进行。这将允许 与糖蛋白有关的所有必要信息，包括聚糖链接，异构体，序列， 附着位点以及在一锅实验中获得的糖基化程度。扩展的唯一方法 糖生物学领域是使其更容易被其他科学家使用，并结合聚糖结构 在单个实验中用糖肽分析阐明是朝着该目标迈出的一步。 fetuin将被用作 真核生物模型糖蛋白和空肠糖蛋白糖蛋白ACRA作为原核糖蛋白的模型。 涉及糖肽和MSN分析的二甲基化的技术将纳入 CCRC每年提供给Glyco-Community的动手培训课程。动手训练是一个 最有效的方法是使任何技术都可以评估为更大的科学界。为了 协助解释MS中生成的复杂数据，该项目的第二个目的是扩展 以前在内部生成的软件，包括自动注释氯二糖肽的自动注释 质谱。现有软件是以模块化的方式构建的，可以添加进一步的功能 通过“插件”。第三个目的是开发苄氯化试剂盒以促进单个样品 准备工作流程，以帮助不是碳氢分析专家的研究人员。