EXAMINATION OF COMPLEX OLIGOSACCHARIDES BY MATRIX-ASSISTED LASER-DESORPTION IONIZATION MASS-SPECTROMETRY ON TIME-OF-FLIGHT AND MAGNETIC-SECTOR INSTRUMENTS

Matrix-assisted laser desorption/ionization (MALDI) spectra of underiv atized oligosaccharides of the type attached to asparagine in glycopro teins (N-linked oligosaccharides) were examined with linear time-of-fl ight (TOF) and magnetic sector instruments using 2,5-dihydroxybenzoic acid (2,5-DHB), alpha-cyano-4-hydroxycinnamic acid, sinapinic acid, 1, 4-dihydroxynaphthalene-2-carboxylic acid or 2-(4-hydroxypheaylazo)benz oic acid (HABA) as the matrices. All compounds formed abundant [M + Na ](+) ions with the strongest signals being obtained from 2,5-DHB after recrystallization of the initially dried sample spot from ethanol. On ly traces of fragmentation were detected from neutral oligosaccharides on the TOF system but more abundant fragment ions (about 5% relative abundance) were present in the spectra from the magnetic sector instru ment. Fragmentation was dominated by Y-type glycosidic cleavages (Domo n and Costello nomenclature) between all sugar residues yielding seque nce and branching information. Sialic acid-containing oligosaccharides generally produced the sodium adduct of the sodium salt and gave much weaker signals than the neutral sugars in the positive-ion mode. Ther e was also considerable loss of the sialic acid moieties as the result of fragmentation on the magnetic sector instrument. The least fragmen tation of both neutral and acidic sugars was caused by 2.5 DHB, which proved to be the most appropriate matrix for examination of oligosacch aride mixtures. Much better resolution of the oligosaccharides was obt ained than by traditional methods such as the use of Bio-Gel P-4 gel f iltration column chromatography. It is worth noting also that the meas urements were considerably faster (a few minutes as opposed to about 1 6 h). In addition, no radiolabelling was necessary as required for det ection on the P-4 columns. Mixtures of oligosaccharides from several g lycoproteins (ribonuclease B, human immunoglobulin G (IgG) transferrin , bovine fetuin and chicken ovalbumin) were examined and the patterns of the identified oligosaccharides were found to agree closely with th e known compositions of the sugar mixtures. The mass spectrometric res olution on the magnetic sector instrument was very much better (up to 3000, FWHM) than could be obtained with the linear TOF systems (200-40 0). The technique was used as a detection system for the products of e xoglycosidase digestion in experiments to determine the detailed struc ture of the oligosaccharide chains from human IgG.