North Simon J, Jang-Lee Jihye, Harrison Rebecca, Canis Kévin, Ismail Mohd Nazri, Trollope Alana, Antonopoulos Aristotelis, Pang Poh-Choo, Grassi Paola, Al-Chalabi Sara, Etienne A Tony, Dell Anne, Haslam Stuart M
Division of Molecular Biosciences, Faculty of Natural Sciences, Imperial College London, London, United Kingdom.
Methods Enzymol. 2010;478:27-77. doi: 10.1016/S0076-6879(10)78002-2.
Mass spectrometry (MS) has proven to be the preeminent tool for the rapid, high-sensitivity analysis of the primary structure of glycans derived from diverse biological sources including cells, fluids, secretions, tissues, and organs. These analyses are anchored by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) analysis of permethylated derivatives of glycan pools released from the samples, to produce glycomic mass fingerprints. The application of complimentary techniques, such as chemical and enzymatic digestions, GC-MS linkage analysis, and tandem mass spectrometry (MS/MS) utilizing both electrospray (ES) and MALDI-TOF/TOF, together with bioinformatic tools allows the elucidation of incrementally more detailed structural information from the sample(s) of interest. The mouse as a model organism offers many advantages in the study of human biology, health, and disease; it is a mammal, shares 99% genetic homology with humans and its genome supports targeted mutagenesis in specific genes to produce knockouts efficiently and precisely. Glycomic analyses of tissues and organs from mice genetically deficient in one or more glycosylation gene and comparison with data collected from wild-type samples enables the facile identification of changes and perturbations within the glycome. The Consortium for Functional Glycomics (CFG) has been applying such MS-based glycomic analyses to a range of murine tissues from both wild-type and glycosylation-knockout mice in order to provide a repository of structural data for the glycobiology community. In this chapter, we describe in detail the methodologies used to prepare, derivatize, purify, and analyze glycan pools from mouse organs and tissues by MS. We also present a summary of data produced from the CFG systematic structural analysis of wild-type and knockout mouse tissues, together with a detailed example of a glycomic analysis of the Mgat4a knockout mouse.
质谱（MS）已被证明是用于快速、高灵敏度分析源自多种生物来源（包括细胞、体液、分泌物、组织和器官）的聚糖一级结构的卓越工具。这些分析通过对从样品中释放的聚糖池的全甲基化衍生物进行基质辅助激光解吸电离飞行时间（MALDI-TOF）分析来实现，以产生糖组质谱指纹图谱。运用诸如化学和酶消化、气相色谱-质谱联用分析以及利用电喷雾（ES）和MALDI-TOF/TOF的串联质谱（MS/MS）等互补技术，再结合生物信息学工具，能够从感兴趣的样品中逐步阐明更详细的结构信息。小鼠作为一种模式生物，在人类生物学、健康和疾病研究中具有诸多优势；它是一种哺乳动物，与人类有99%的基因同源性，其基因组支持对特定基因进行靶向诱变，从而高效、精确地产生基因敲除模型。对一个或多个糖基化基因存在遗传缺陷的小鼠的组织和器官进行糖组分析，并与从野生型样品收集的数据进行比较，能够轻松识别糖组内的变化和扰动。功能糖组学协会（CFG）一直在将这种基于质谱的糖组分析应用于野生型和糖基化基因敲除小鼠的一系列鼠类组织，以便为糖生物学界提供一个结构数据储存库。在本章中，我们详细描述了用于通过质谱制备、衍生化、纯化和分析小鼠器官和组织中的聚糖池的方法。我们还展示了CFG对野生型和基因敲除小鼠组织进行系统结构分析所产生的数据总结，以及对Mgat4a基因敲除小鼠进行糖组分析的详细示例。
