High
-energy collision-induced dissociation (CID) matrix-assisted laser desorptionhonization mass spectra of Nlinked oligosaccharides bearing different, commonly encountered, reducing terminal modifications (hydroxyl, 2-aminobenzamide, asparagine and a tetrapeptide) were recorded on a magnetic sector instrument equipped with an orthogonal-acceleration time-of-flight (OA-TOF) analyser. All the compounds formed abundant molecular (MNa') and fragment ions, the latter corresponding to glycosidic and cross-ring cleavages as well as to internal fragment ions, all of which provided much insight into the oligosaccharide structure. The nature of the modification considerably influenced the CID behaviour. The strongest and most complete series of glycosidic cleavage ions (mainly Y and B-Domon and Costello nomenclature) was formed by the underivatized oligosaccharide whereas most cross-ring fragment ions, diagnostic of linkage, were found in the spectra of the glycopeptides. A-type cross-ring cleavage ions were particularly abundant in the spectrum of the asparagine derivative. Reductive amination using 2-aminobenzamide resulted in an opened reducing-terminal sugar ring and suppression of the cross-ring fragment ions carrying information associated with that ring. This information was present in the spectra of the free carbohydrate and the peptide derivatives.
Glycosylation is one of the major types of post-translational modification that many secreted or membrane-associated proteins undergo during biosynthesis and is implicated in many different biological functions as reviewed by Varki in 1993.' Naturally occurring glycoproteins usually exhibit a high degree of structural diversity as a result of both the range of glycans present, and the fact that these glycans may occur at multiple glycosylation sites. Structural elucidation of the glycans is usually performed following release of the sugars by either enzymatic or chemical means. The resulting carbohydrate mixtures can then be examined directly as native sugars by mass spectrometry or, more commonly, they may require fractionation by chromatographic techniques such as gel filtration, lectin-affinity chromatography or high-performance liquid chromatography (HPLC) prior to further structural analysis. Since oligosaccharides lack an inherent chromophore, UV active or fluorescent tags, such as the recently introduced 2-aminobenzamide (2-AB),* normally have to be attached to the glycan molecules to enhance chromatographic detection.
An alternative approach to the structural analysis of protein-linked carbohydrates is to examine them as tryptic glycopeptides or as the carbohydrate-asparagine conjugate resulting from pronase digestion. Thus, carbohydrates with a range of substituents at the reducing terminus may be encountered during subsequent mass spectrometric analysis and the nature of the substituent may affect the pattern of fragmentation that is needed for detailed structural determination.
Matrix-assisted laser desorptionhonization (MALDI) mass spectrometry3 is able to ionize all of these modified sugars, without further derivatization, but the spectra, when