Solid-Phase Permethylation for Glycomic Analysis

Abstract

This chapter discusses in detail a miniaturized version of the widely used permethylation technique which permits quantitative derivatization of oligosaccharides derived from minute quantities of glycoprotein. The approach involves packing of sodium hydroxide powder or beads in a microcolumn format, including spin columns, fused silica capillaries (500 microm i.d.) and plastic tubes (1 mm i.d.). The derivatization proceeds effectively in less than a minute time scale and it is applicable to glycans derived from femtomole quantities of glycoproteins. Prior to mass spectrometry (MS), methyl iodide is added to analytes suspended in dimethyl sulfoxide solution containing traces of water. The reaction mixture is then immediately infused through the microreactor. The packed sodium hydroxide powder or beads inside the microcolumns minimize oxidative degradation and peeling reactions which are otherwise commonly associated with the conventional permethylation technique. In addition, this solid-phase permethylation approach eliminates the need for excessive sample clean-up. As demonstrated below, picomole amounts of various types of glycans derived from model glycoproteins as well as real samples, including linear and branched, sialylated and neutral glycans were shown to become rapidly and efficiently permethylated through this approach.

Derivatization of carbohydrates for analysis by chromatography; electrophoresis and mass spectrometry

“…Reactions were said to take less than one minute and the procedure minimized oxidative degradation and peeling reactions and avoided the need of excessive clean-up. Picomole amounts of linear and branched, sialylated and neutral glycan samples were rapidly and efficiently permethylated by this approach [28,29] and a high-throughput extension of the method utilizing spin columns packed with sodium hydroxide beads has recently been described [30].…”

Toward a Platform for Comprehensive Glycan Sequencing

“…1D), methylation ( Fig. 1E) (8), and direct infusion for a detailed examination of structure as discussed below.…”

Novel Ionic Matrices for Enhanced Ionization of Oligosaccharides/Glycopeptides during MALDI-MS Analysis

“…近年来, 基质辅助 激光解析电离飞行时间质谱(MALDI-TOF-MS)以其极 DOI: 10.6023/A13030299 高的检测灵敏度和广泛的适用性, 为一系列生物大分子 的检测、鉴定与表征提供了有效的手段, 包括蛋白, 肽 段, 核糖核苷酸等 [3,4] . 但是, 由于寡糖具有较强的亲水 性, 加之其结构中缺乏与质子结合的碱性基团, 导致其 在 MALDI-TOF-MS 中的离子化效率极低, 因此, 寡糖 和糖肽的质谱分析依然面临着巨大的挑战 [5] . 为了应对这一挑战, 许多研究者们为此做出了不懈 努力.…”