Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, USA.
Department of Biotechnology, Institute of Graduate Studies and Research, University of Alexandria, Alexandria, Egypt.
Methods Mol Biol. 2021;2271:281-301. doi: 10.1007/978-1-0716-1241-5_20.
The existence of glycans in isomeric forms is responsible for the multifariousness of their properties and biological functions. Their altered expression has been associated with various diseases and cancers. Analysis of native glycans is not very sensitive due to the low ionization efficiency of glycans. These facts necessitate their comprehensive structural studies and establishes a high demand for sensitive and reliable techniques. In this chapter, we discuss the strategies for effective separation and identification of permethylated isomeric glycans. The sample preparation for permethylated glycans derived from model glycoproteins and complex biological samples, analyzed using LC-MS/MS, is delineated. We introduce protein extraction and release of glycans, followed by strategies to purify the released glycans, which are reduced and permethylated to improve ionization efficiency and stabilize sialic acid residues. High-temperature LC-based separation on PGC (porous graphitized carbon) column is conducive to isomeric separation of glycans and allows their sensitive identification and quantification using MS/MS.
糖链以同分异构形式存在，这是其具有多样化性质和生物学功能的原因。它们的表达发生改变与多种疾病和癌症有关。由于糖链的电离效率较低，因此对天然糖链的分析不是很灵敏。这些事实需要对其进行全面的结构研究，这就对灵敏可靠的技术提出了高要求。在本章中，我们讨论了有效分离和鉴定全甲基化同分异构糖链的策略。讨论了使用 LC-MS/MS 分析从模型糖蛋白和复杂生物样品中衍生的全甲基化糖链的样品制备。我们介绍了蛋白质的提取和糖链的释放，然后介绍了纯化释放的糖链的策略，包括还原和全甲基化以提高电离效率并稳定唾液酸残基。基于 PGC（多孔石墨化碳）柱的高温 LC 分离有利于糖链的同分异构分离，并允许使用 MS/MS 对其进行灵敏的鉴定和定量。
