Linkage analysis in disaccharides by electrospray mass spectrometry

Abstract

Analysis of the structures of complex carbohydrates requires knowledge of the identity, anomeric configuration, and sequence of the sugar residues, and identification of the reducing terminus and the positions of the glycosidic linkages. Desorption-m.s. and f.a.b.-m.s. are powerful techniques for determining the sequence, the pattern of branching, and the molecular weight of oligosaccharides containing up to 30 sugar units, and the structure of the aglycon14. Negative-ion tandem-f.a.b.-m.s. can be used to discriminate between the linkage positions in underivatised oligosaccharides5,6. Electrospray (e.s.) ionisation has also been described7-9. Although most of the applications have been concerned with the determination of molecular weights and sequencing of proteins, some studies have shown that it can be applied to carbohydrates, and we now report its application in the linkage analysis of reducing disaccharides. The negative-ion e-S.-mass spectra of (1~2)(sophorose), (l-+3)(laminaribiose), (1+4)(cellobiose), and (l-+6)(gentiobiose), /I-linked glucodisaccharides shown in Figs. l-4, respectively, reflect the positions of the linkages. Each mass spectrum contains peaks at m/z 34 1 for (M H)and at m/z I79 and 16 1 associated with fragmentation which involves the glycosidic linkages. In addition to these peaks, there are peaks at m/z 323 (sophorose, Fig. l), 28 1 (cellobiose and gentiobiose, Figs. 3 and 4), 263 (sophorose and cellobiose Figs. 1 and 3), 251 (gentiobiose, Fig. 4), and 221 (sophorose, cellobiose, and gentiobiose, Figs. 1,3, and 4). For laminaribiose, only the peaks at m/z 341, 179, and 161 are present (Fig. 2). The peak at m/z 323 is due to loss of water from the (M H)ion, and those at m/z 281, 263, 251, and 221 are associated with fragments from the sugar rings which are diagnostic of the position of the linkage. These fragmentations are likely to involve the reducing moiety, since the non-reducing moieties are identical in the four disaccharides.