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Exoglycosidase Digestions

An important method for N- and O-glycan analysis, and one that provides information both on the nature of the monosaccharide constituents and on their linkage, is disassembly by use of exoglycosidase digestion [162-164]. Glycan mixtures are profiled, most commonly by HPLC, although MS can be used as well and then the mixture is digested with a series of enzymes (see Dwek et al. [165] for suitable enzymes) that remove successive monosaccharide residues from the reducing terminus. At each stage, the mixture is examined to determine the number of residues that have been removed. Although a popular method for glycan analysis, the technique suffers from several drawbacks, predominant of which is reliance on a supply of suitable enzymes and the absence of enzymes that reveal specific structural features such as the presence of bisecting GlcNAc residues. Also, there is no indication as to the site of attachment of the monosaccharide residues that are removed at each stage of the disassembly.

Several investigators have developed methods for glycan release [166] and exoglycosidase sequencing directly on a MALDI target. The method developed by Kuster et al. [167] for on-target sequencing involves sequential incubation of the glycans with the relevant enzyme, spectral recording, and removal of the matrix (2,5-dihydroxybenzoic acid (DHB)) prior to analysis of the next digest. Starting with 100 pmoles of glycan, it was possible to conduct three successive enzyme digestions before the amount of sample became insufficient to give a MALDI signal. Other investigators [166, 168] have developed similar methods using mixtures of exoglycosidase (exoglycosidase arrays) on different target spots in order to avoid removal of the matrix at each stage. The neutral matrix 6-aza-2-thiothymine (ATT), rather than the acidic DHB, has been used so that enzyme digests could be performed in the presence of a matrix that did not affect the enzyme activities [169, 170].

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