Fingerprinting of large oligosaccharides linked to ceramide by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: highly heterogeneous polyglycosylceramides of human erythrocytes with receptor activity for Helicobacter pylori.

Highly microheterogeneous polyglycosylceramides (PGCs) of human erythrocytes with an average composition of about 25 monosaccharides linked to ceramide were analyzed by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS). The human gastric pathogen Helicobacter pylori was earlier shown to bind this glycosphingolipid mixture by thin-layer chromatogram binding assay. The receptor activity was present along the whole nonresolved chromatographic interval. Mass spectra of intact PGCs were compared with corresponding spectra of oligosaccharides enzymatically released from the ceramides. Two subfractions of PGCs containing less than one and more than one sialic acid residue per molecule were used. MALDI-MS spectra were recorded in both linear and reflectron mode with the accuracies of </=0.08% and </=0.02%, respectively, which allowed determination of the constituent parts of the detected ions in form of ceramide and number of hexoses, N-acetylhexosamines, fucoses and sialic acids. Molecular species were found based on ceramide with mainly sphingosine and fatty acids 24:0 and 24:1 (with less amounts of 22:0), and with a total number of monosaccharides ranging from 11 (neutral, m/z = 2605 for [M+Na](+)) to 41 (one sialic acid, m/z = 8057 for [M-H](-)). The saccharide composition obtained supported a successively extended and branched N -acetyllactosamine core with substitutions of fucoses (0 up to 8) and sialic acid (0 to 1). The reliable molecular analysis of large oligosaccharides linked to ceramide using this approach will be of great help for the further structure analysis in order to define the epitope for the sialic acid-dependent binding by the bacterium.