Madson - Mass spectrometry: techniques for structural characterization of glycans

1. Figures in Mass Spectral Analysis of Carbohydrates

Michael A. Madson

BioLogistics LLC, Ames, IA, USA

Elsevier

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ISBN: 978-0-12-804129-1

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We dedicate this book to my Lord and Savior Jesus the Christ, His Father & mine and the Holy Spirit who have spent countless hours with me in writing this book so that many others may grasp the power of these techniques and apply them to their quests in science. We hope chemistry and medicine will be advanced and lives will be saved or lengthened because of Their intervention into my life.

We describe the use of mass spectral analysis for the qualitative analysis of glycans. We examine the spectra from Electrospray Ionization Mass Spectrometry (ESI MS), Matrix Assisted Laser Desorption Mass Spectrometry (MALDI MS) and Atmospheric Pressure Ionization Mass Spectrometry (API MS) of carbohydrates. The former are time-of-flight (TOF) instruments and the last a triple quadrapole instrument. We have one spectrum that is a single quadrapole instrument. We describe several methods that aid in the determination of oligosaccharide structure by MS. They are: N- and O-linked oligosaccharide enzymic/borohydride N-linkage cleavage and O-linked reductive -elimination, from glycoproteins, nonreductive -elimination of O-linked oligosaccharides from glycoproteins, two isolation methods for nonglycoprotein oligosaccharides, sulfate/phosphate substitution discernment of oligosaccharides and the isolation method for amino acid substituted O-linked oligosaccharides from glycoproteins. We provide a partial bibliography for the possible future participation of sialyl lactose-6 phosphate in disease treatment.

Glycan isolation; phosphate-sulfate discernment; elemental analysis-MS ions

In this book, we describe the methods required for isolation of intact oligosaccharides for analysis by mass spectrometry. There are two basic methods we use. One is done by passing ammonium salts through a cation exchange cartridge in the ammonium form []. The method is described below.

The monosaccharide composition of the glycan to be analyzed is accomplished by known methods. Generally, the glycan to be analyzed is hydrolyzed with strong acid by HCl solution at various concentrations of acid, various times and various temperatures due to the varied acid labilities and stabilities of the oligosaccharides and monosaccharides to be analyzed. Two different hydrolytic conditions [].

Often glycans can be in the reducing sugar or glycitol form. An example of glycan reducing sugar analysis and the corresponding glycan alditol analysis can be done and is shown below []. This work establishes which monosaccharide is the reducing sugar of the glycan. That is, the reducing glycan is hydrolyzed, possibly using more than one hydrolytic conditions, to give the complete monosaccharide composition. Then the reducing glycan is reduced with NaBH4 with standard conditions (given below) and the reducing monosaccharide will disappear from the monosaccharide composition, the alditol will have a different retention time. Most of the time monosaccharide alditols have a much lower retention time than the corresponding reducing monosaccharides. An example is given for bovine thyroglobulin O-linked oligosaccharide.

To help identify the reducing sugar of a glycan, as noted above, to provide a reactive site for further derivatization to allow further characterization by MS or chromatography, a method for the nonreductive -elimination of O-linked oligosaccharides from glycoprotein has been developed. The method involves first removing any N-linked oligosaccharides by the method described (N- and O-linked oligosaccharide isolation method) []. The O-linked oligosaccharide, removed from the ammonium form cation exchange cartridge is treated, as shown below, to release the glycan which was formerly linked to protein by an O-glycosidic linkage.

As an example of negative ion ESI-MS of glycans, we chose banana fruit extract ESI-MS [).