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 erythrocyte s with an average composition of about 25 monosaccharides linked to ceramid e were analyzed by matrix-assisted laser desorption-ionization time-of-flig ht mass spectrometry (MALDI-TOF MS). The human gastric pathogen Helicobacte r pylori was earlier shown to bind this glycosphingolipid mixture by thin-l ayer chromatogram binding assay. The receptor activity was present along th e whole nonresolved chromatographic interval, Mass spectra of intact PGCs w ere compared with corresponding spectra of oligosaccharides enzymatically r eleased from the ceramides, Two subfractions of PGCs containing less than o ne and more than one sialic acid residue per molecule were used. MALDI-MS s pectra were recorded in both linear and reflectron mode with the accuracies of less than or equal to 0.08% and less than or equal to 0.02%, respective ly, which allowed determination of the constituent parts of the detected io ns in form of ceramide and number of hexoses, N-acetylhexosamines, fucoses and sialic acids. Molecular species were found based on ceramide with mainl y sphingosine and fatty acids 24:0 and 24:1 (with less amounts of 22:0), an d with a total number of monosaccharides ranging from 11 (neutral, m/z = 26 05 for [M+Na](+)) to 41 tone sialic acid, m/z = 8057 for [M-H](-)). The sac charide composition obtained supported a successively extended and branched N-acetyllactosamine core with substitutions of fucoses (0 up to 8) and sia lic acid (0 to 1), The reliable molecular analysis of large oligosaccharide s linked to ceramide using this approach will be of great help for the furt her structure analysis in order to define the epitope for the sialic acid-d ependent binding by the bacterium.