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Mass spectrometric analysis of O-linked oligosaccharides from various recombinant expression systems

Journal article
Authors Diarmuid T. Kenny
S. Gaunitz
Catherine A Hayes
A. Gustafsson
M. Sjöblom
Jan Holgersson
Niclas G. Karlsson
Published in Methods in Molecular Biology
Volume 988
Pages 145-167
ISSN 1064-3745
Publication year 2013
Published at Institute of Biomedicine, Department of Clinical Chemistry and Transfusion Medicine
Institute of Biomedicine
Institute of Biomedicine, Department of Medical Biochemistry and Cell Biology
Pages 145-167
Language en
Keywords β-Elimination, Electrospray-MS, Glycomics, Graphitized carbon LC-MS, O-Linked oligosaccharides, Permethylation, carbon, glycoprotein, hexuronic acid, oligosaccharide, P selectin glycoprotein ligand 1, sialic acid, sulfate, animal cell, article, cation exchange, derivatization, female, gas flow, gel filtration, gene expression system, insect cell, liquid chromatography, mammal cell, mass spectrometry, methylation, nonhuman, phase separation, priority journal, temperature, yeast cell
Subject categories Pharmacy


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-MS2 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 exempli fied 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.

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