title: Multistep Fractionation and Mass Spectrometry Reveal Zwitterionic and Anionic Modifications of the N- and O-glycans of a Marine Snail.
abstract: Various studies in the past have revealed that molluscs can produce a wide range of rather complex N-glycan structures, which vary from those occurring in other invertebrate animals; particularly methylated glycans have been found in gastropods, and there are some reports of anionic glycans in bivalves. Due to the high variability in terms of previously described structures and methodologies, it is a major challenge to establish glycomic workflows that yield the maximum amount of detailed structural information from relatively low quantities of sample. In this study, we apply differential release with peptide:N-glycosidases F and A followed by solid-phase extraction on graphitized carbon and reversed-phase materials to examine the glycome of Volvarina rubella (C. B. Adams, 1845), a margin snail of the clade Neogastropoda. The resulting four pools of N-glycans were fractionated on a fused core RP-HPLC column and subject to MALDI-TOF MS and MS/MS in conjunction with chemical and enzymatic treatments. In addition, selected N-glycan fractions, as well as O-glycans released by β-elimination, were analyzed by porous graphitized carbon-LC-MS and MS(n). This comprehensive approach enabled us to determine a number of novel modifications of protein-linked glycans, including N-methyl-2-aminoethylphosphonate on mannose and N-acetylhexosamine residues, core β1,3-linked mannose, zwitterionic moieties on core Galβ1,4Fuc motifs, additional mannose residues on oligomannosidic glycans, and bisubstituted antennal fucose; furthermore, typical invertebrate N-glycans with sulfate and core fucose residues are present in this gastropod.
authors.affiliation: From the ‡Department für Chemie, Universität für Bodenkultur Wien, 1190 Wien, Austria;
authors.forename: Barbara
authors.identifier: http://orcid.org/0000-0003-0818-867X
authors.initials: B
authors.lastname: Eckmair
authors.affiliation: §Institutionen för Biomedicin, Göteborgs universitet, 405 30 Göteborg, Sweden;
authors.forename: Chunsheng
authors.initials: C
authors.lastname: Jin
authors.affiliation: ¶Haus des Meeres-Aqua Terra Zoo, 1060 Wien, Austria.
authors.forename: Daniel
authors.initials: D
authors.lastname: Abed-Navandi
authors.affiliation: From the ‡Department für Chemie, Universität für Bodenkultur Wien, 1190 Wien, Austria; katharina.paschinger@boku.ac.at.
authors.forename: Katharina
authors.identifier: http://orcid.org/0000-0002-3594-7136
authors.initials: K
authors.lastname: Paschinger
mesh_terms: D026921:Amino Acid Transport Systems, Neutral / Q000235:genetics*; D000818:Animals; D000838:Anions; D002851:Chromatography, High Pressure Liquid; D005643:Fucose / Q000378:metabolism; D054794:Glycomics; D008358:Mannose / Q000378:metabolism; D013058:Mass Spectrometry; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000235:genetics / Q000378:metabolism; D011134:Polysaccharides / Q000096:biosynthesis* / Q000235:genetics; D012908:Snails / Q000235:genetics* / Q000378:metabolism
chemical_list: D026921:Amino Acid Transport Systems, Neutral; D000838:Anions; D011134:Polysaccharides; D005643:Fucose; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D008358:Mannose