Emanuela Spina

Linkage analysis in disaccharides by electrospray mass spectrometry.

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.

Sequencing of oligosaccharides by collision-induced dissociation matrix-assisted laser desorption/ionization mass spectrometry.

A study of the collision-induced dissociation post-source decay (PSD) spectra of free oligosaccharides is presented. These spectra, when obtained with helium as collision gas, show (1,5)X fragments containing the reducing end sugar. The presence of these fragments permits Y ions and, consequently, B and C peaks to be identified. This is a common behaviour from which it has been possible to delineate a general method for the easy assignment of the peaks in PSD spectra of underivatized neutral sugars, allowing the sequence of a real unknown to be obtained.

Studies on the primary structure of short polysaccharides using SEC MALDI mass spectroscopy.

The introduction of size-exclusion chromatography (SEC) analysis of polysaccharides prior to MALDI mass spectroscopy accounts for the determination of the molecular mass of the repeating unit when neutral homopolymers are investigated. In the case of natural polysaccharides characterised by more complicated structural features (presence of non-carbohydrate substituents, charged groups, etc.), this mass value usually is in agreement with more than one sugar composition. Therefore, it is not sufficient to give the correct monosaccharidic composition of the polysaccharide investigated. To solve this problem, MALDI spectra were recorded on the permethylated sample and post-source decay experiments were performed on precursor ions. In this way, the composition (in terms of Hex, HexNAc, etc.), size and sequence of the repeating unit were determined.

The structure of the exocellular polysaccharide from the cyanobacterium Cyanospira capsulata.

The exocellular polysaccharide produced by the cyanobacterium Cyanospira capsulata has been subjected to partial acid hydrolysis and N-deacetylation-nitrous acid deamination. The oligosaccharides released have been isolated by weak anion exchange and aqueous size exclusion chromatography, and characterized by a combination of 1D and 2D nuclear magnetic resonance spectroscopy, mass spectrometry, sugar composition and linkage analyses. The polysaccharide has an octasaccharide repeating unit with the following structure: [formula: see text]

Discrimination of isomeric oligosaccharides and sequencing of unknowns by post source decay matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

The PSD spectra of some isomeric oligosaccharides are investigated in order to study the fragmentation mechanisms. Results show that intense peaks are formed from one or more glycosidic linkage cleavages, while ring fragmentation produces very weak signals. Two analytical applications of post source decay (PSD) to the structural analysis of oligosaccharides are also illustrated: the discrimination of isomeric structures and the identification of a real unknown by combining PSD data with the linkage analysis results.