title: Differentiation of isomeric N-glycan structures by normal-phase liquid chromatography-MALDI-TOF/TOF tandem mass spectrometry.
abstract: The detailed characterization of protein N-glycosylation is very demanding given the many different glycoforms and structural isomers that can exist on glycoproteins. Here we report a fast and sensitive method for the extensive structure elucidation of reducing-end labeled N-glycan mixtures using a combination of capillary normal-phase HPLC coupled off-line to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and TOF/TOF-MS/MS. Using this method, isobaric N-glycans released from honey bee phospholipase A2 and Arabidopsis thaliana glycoproteins were separated by normal-phase chromatography and subsequently identified by key fragment ions in the MALDI-TOF/TOF tandem mass spectra. In addition, linkage and branching information were provided by abundant cross-ring and "elimination" fragment ions in the MALDI-CID spectra that gave extensive structural information. Furthermore, the fragmentation characteristics of N-glycans reductively aminated with 2-aminobenzoic acid and 2-aminobenzamide were compared. The identification of N-glycans containing 3-linked core fucose was facilitated by distinctive ions present only in the MALDI-CID spectra of 2-aminobenzoic acid-labeled oligosaccharides. To our knowledge, this is the first MS/MS-based technique that allows confident identification of N-glycans containing 3-linked core fucose, which is a major allergenic determinant on insect and plant glycoproteins.
authors.affiliation: Chemistry Department, University of Cambridge, Lensfield Road, Cambridge, UK CB2 1EW.
authors.forename: Sarah
authors.initials: S
authors.lastname: Maslen
authors.forename: Pawel
authors.initials: P
authors.lastname: Sadowski
authors.forename: Alex
authors.initials: A
authors.lastname: Adam
authors.forename: Kathryn
authors.initials: K
authors.lastname: Lilley
authors.forename: Elaine
authors.initials: E
authors.lastname: Stephens
mesh_terms: D000586:Amination; D000818:Animals; D017360:Arabidopsis / Q000737:chemistry*; D001516:Bees / Q000737:chemistry*; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006023:Glycoproteins / Q000032:analysis / Q000737:chemistry; D007536:Isomerism; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000737:chemistry / Q000378:metabolism; D054467:Phospholipases A2; D011134:Polysaccharides / Q000032:analysis / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D062367:ortho-Aminobenzoates / Q000737:chemistry
chemical_list: D006023:Glycoproteins; D011134:Polysaccharides; D062367:ortho-Aminobenzoates; C031385:anthranilic acid; D010741:Phospholipases A; D054467:Phospholipases A2; C000219:anthranilamide