title: Travelling-wave ion mobility and negative ion fragmentation of high-mannose N-glycans.
abstract: The isomeric structure of high-mannose N-glycans can significantly impact biological recognition events. Here, the utility of travelling-wave ion mobility mass spectrometry for isomer separation of high-mannose N-glycans is investigated. Negative ion fragmentation using collision-induced dissociation gave more informative spectra than positive ion spectra with mass-different fragment ions characterizing many of the isomers. Isomer separation by ion mobility in both ionization modes was generally limited, with the arrival time distributions (ATD) often showing little sign of isomers. However, isomers could be partially resolved by plotting extracted fragment ATDs of the diagnostic fragment ions from the negative ion spectra, and the fragmentation spectra of the isomers could be extracted by using ions from limited areas of the ATD peak. In some cases, asymmetric ATDs were observed, but no isomers could be detected by fragmentation. In these cases, it was assumed that conformers or anomers were being separated. Collision cross sections of the isomers in positive and negative fragmentation mode were estimated from travelling-wave ion mobility mass spectrometry data using dextran glycans as calibrant. More complete collision cross section data were achieved in negative ion mode by utilizing the diagnostic fragment ions. Examples of isomer separations are shown for N-glycans released from the well-characterized glycoproteins chicken ovalbumin, porcine thyroglobulin and gp120 from the human immunodeficiency virus. In addition to the cross-sectional data, details of the negative ion collision-induced dissociation spectra of all resolved isomers are discussed.
authors.affiliation: Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.|Department of Biological Sciences, University of Warwick, Coventry, CV47AL, UK.
authors.forename: David J
authors.initials: DJ
authors.lastname: Harvey
authors.affiliation: Department of Biological Sciences, University of Warwick, Coventry, CV47AL, UK.|Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, UK.
authors.forename: Charlotte A
authors.initials: CA
authors.lastname: Scarff
authors.affiliation: Department of Biological Sciences, University of Warwick, Coventry, CV47AL, UK.
authors.forename: Matthew
authors.initials: M
authors.lastname: Edgeworth
authors.affiliation: Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.|Chemistry Research Laboratory, 12 Mansfield Road, Oxford, OX1 3TA, UK.
authors.forename: Weston B
authors.initials: WB
authors.lastname: Struwe
authors.affiliation: Institute of Chemistry and Biochemistry, Freie Universität Berlin, Takustrasse. 3, 14159, Berlin, Germany.|Fritz Haber Institute of the Max Planck Society, Faradayweg 4-6, 14195, Berlin, Germany.
authors.forename: Kevin
authors.initials: K
authors.lastname: Pagel
authors.affiliation: Institute of Structural and Molecular Biology, Division of Biosciences, University College London, London, WC1E 6BT, UK.|Institute of Structural and Molecular Biology, Department of Biological Sciences, Birkbeck College, University of London, London, UK.
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Thalassinos
authors.affiliation: Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.
authors.forename: Max
authors.initials: M
authors.lastname: Crispin
authors.affiliation: Department of Biological Sciences, University of Warwick, Coventry, CV47AL, UK.
authors.forename: Jim
authors.initials: J
authors.lastname: Scrivens
mesh_terms: D000818:Animals; D002240:Carbohydrate Sequence; D002645:Chickens; D006023:Glycoproteins / Q000737:chemistry*; D007477:Ions / Q000032:analysis / Q000737:chemistry; D008358:Mannose / Q000032:analysis* / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D011134:Polysaccharides / Q000032:analysis / Q000737:chemistry*; D013552:Swine
chemical_list: D006023:Glycoproteins; D007477:Ions; D011134:Polysaccharides; D008358:Mannose
keywords: N-linked glycosylation; T-wave ion mobility; fragmentation; high mannose; isomers