title: Mass spectrometric analysis of O-linked oligosaccharides from various recombinant expression systems.
abstract: Analysis of O-linked glycosylation is one of the main challenges during structural validation of recombinant glycoproteins. With methods available for N-linked glycosylation in regard to oligosaccharide analysis as well as glycopeptide mapping, there are still challenges for O-linked glycan analysis. Here, we present mass spectrometric methodology for O-linked oligosaccharides released by reductive β-elimination. Using LC-MS and LC-MS(2) with graphitized carbon columns, oligosaccharides are analyzed without derivatization. This approach provides a high-throughput method for screening during clonal selection, as well as product structure verification, without impairing sequencing ability. The protocols are exemplified by analysis of glycoproteins from mammalian cell cultures (CHO cells) as well as insect cells and yeast. The data shows that the method can be successfully applied to both neutral and acidic O-linked oligosaccharides, where sialic acid, hexuronic acid, and sulfate are common substituents. Further characterization of O-glycans can be achieved using permethylation. Permethylation of O-linked oligosaccharides followed by direct infusion into the mass spectrometer provide information about oligosaccharide composition, and subsequent MS (n) experiments can be carried out to elucidate oligosaccharide structure including linkage information and sequence.
authors.affiliation: School of Chemistry and Medical Biochemistry, National University Ireland Galway, Galway, Ireland.
authors.forename: Diarmuid T
authors.initials: DT
authors.lastname: Kenny
authors.forename: Stefan
authors.initials: S
authors.lastname: Gaunitz
authors.forename: Catherine A
authors.initials: CA
authors.lastname: Hayes
authors.forename: Anki
authors.initials: A
authors.lastname: Gustafsson
authors.forename: Magnus
authors.initials: M
authors.lastname: Sjöblom
authors.forename: Jan
authors.initials: J
authors.lastname: Holgersson
authors.forename: Niclas G
authors.initials: NG
authors.lastname: Karlsson
mesh_terms: D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification; D006031:Glycosylation; D006801:Humans; D015151:Immunoblotting; D007074:Immunoglobulin G / Q000737:chemistry* / Q000302:isolation & purification; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification; D008745:Methylation; D051379:Mice; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry* / Q000302:isolation & purification; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000302:isolation & purification; D012015:Reference Standards; D021241:Spectrometry, Mass, Electrospray Ionization / Q000592:standards
chemical_list: D006023:Glycoproteins; D007074:Immunoglobulin G; D008562:Membrane Glycoproteins; D009844:Oligosaccharides; D011993:Recombinant Fusion Proteins