Neutral O-linked oligosaccharides released from the salivary mucin MUC5B were separated and detected by negative ion LC-MS and LC-MS2. The resolution of the chromatography and the information obtained from collision induced dissociation of detected [M - H]- ions were usually sufficient to identify the sequence of individual oligosaccharides, illustrated by the fact that 50 different oligosaccharides ranging from disaccharides to nonasaccharides could be assigned from the sample. Fragmentation was shown to yield mostly reducing end sequence fragments (Zi and Yi), enabling primary sequence assignment. Specific fragmentation pathways or patterns were also detected giving specific linkage information. The reducing end core (Gal/GlcNAcβ1-3GalNAcol or Gal/GlcNAcβ1-3(GlcNAcβ1-6) GalNAcol) could be deduced from the pronounced glycosidic C-3 cleavage and Ai type cleavages of the reducing end GalNAcol, together with the non reducing end fragment from the loss of a single substituted GalNAcol. Substitution patterns on GlcNAc residues were also found, indicative for C-4 substitution (0,2Ai - H2O cleavage) and disubstitution of C-3 and C-4 (Zi/Zi cleavages). This kind of fragmentation can be used for assigning the mode of chain elongation (Galβ1-3/4GlcNAcβ1-) and identification of Lewis type antigens like Lewis a/x and Lewis b/y on O-linked oligosaccharides. In essence, negative ion LC-MS2 was able to generate extensive data for understanding the overall glycosylation pattern of a sample, especially when only a limited amount of material is available.
|Number of pages||14|
|Journal||Journal of the American Society for Mass Spectrometry|
|Publication status||Published - May 2004|