title: Discrimination of epimeric glycans and glycopeptides using IM-MS and its potential for carbohydrate sequencing.
abstract: Mass spectrometry is the primary analytical technique used to characterize the complex oligosaccharides that decorate cell surfaces. Monosaccharide building blocks are often simple epimers, which when combined produce diastereomeric glycoconjugates indistinguishable by mass spectrometry. Structure elucidation frequently relies on assumptions that biosynthetic pathways are highly conserved. Here, we show that biosynthetic enzymes can display unexpected promiscuity, with human glycosyltransferase pp-α-GanT2 able to utilize both uridine diphosphate N-acetylglucosamine and uridine diphosphate N-acetylgalactosamine, leading to the synthesis of epimeric glycopeptides in vitro. Ion-mobility mass spectrometry (IM-MS) was used to separate these structures and, significantly, enabled characterization of the attached glycan based on the drift times of the monosaccharide product ions generated following collision-induced dissociation. Finally, ion-mobility mass spectrometry following fragmentation was used to determine the nature of both the reducing and non-reducing glycans of a series of epimeric disaccharides and the branched pentasaccharide Man3 glycan, demonstrating that this technique may prove useful for the sequencing of complex oligosaccharides.
authors.affiliation: 1] School of Chemistry & Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, UK [2].
authors.forename: P
authors.initials: P
authors.lastname: Both
authors.affiliation: 1] School of Chemistry & Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, UK [2].
authors.forename: A P
authors.initials: AP
authors.lastname: Green
authors.affiliation: 1] School of Chemistry & Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, UK [2].
authors.forename: C J
authors.initials: CJ
authors.lastname: Gray
authors.affiliation: School of Chemistry & Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.
authors.forename: R
authors.initials: R
authors.lastname: Sardzík
authors.affiliation: College of Food Science and Technology, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, China.
authors.forename: J
authors.initials: J
authors.lastname: Voglmeir
authors.affiliation: Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden.
authors.forename: C
authors.initials: C
authors.lastname: Fontana
authors.affiliation: School of Chemistry & Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.
authors.forename: M
authors.initials: M
authors.lastname: Austeri
authors.affiliation: Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.
authors.forename: M
authors.initials: M
authors.lastname: Rejzek
authors.affiliation: School of Chemistry & Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.
authors.forename: D
authors.initials: D
authors.lastname: Richardson
authors.affiliation: Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.
authors.forename: R A
authors.initials: RA
authors.lastname: Field
authors.affiliation: Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden.
authors.forename: G
authors.initials: G
authors.lastname: Widmalm
authors.affiliation: 1] School of Chemistry & Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, UK [2].
authors.forename: S L
authors.identifier: 0000000232235926
authors.initials: SL
authors.lastname: Flitsch
authors.affiliation: 1] School of Chemistry & Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, UK [2].
authors.forename: C E
authors.initials: CE
authors.lastname: Eyers
mesh_terms: D000595:Amino Acid Sequence; D002241:Carbohydrates / Q000737:chemistry*; D006020:Glycopeptides / Q000737:chemistry*; D016695:Glycosyltransferases / Q000737:chemistry; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry*
chemical_list: D002241:Carbohydrates; D006020:Glycopeptides; D011134:Polysaccharides; D016695:Glycosyltransferases