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-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 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 MSn experiments can be carried out to elucidate oligosaccharide structure including linkage information and sequence.
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Acknowledgements
This work was supported by the Swedish Research Council (621-2010-5322 to N.K. and K2011-65X-3031-01-6 to J.H.), the Swedish Foundation for International Research and Higher Education (to N. K.), the EU Marie Curie Program (PIRG-GA-2007-205302 to N.K.), the County Council of Västra Götaland (ALF to J.H.), and Recopharma AB. The mass spectrometers were obtained by grants from the Swedish Research Council (342-2004-4434).
Conflicts of interest. Jan Holgersson is founder, board member, and part-time CSO of Recopharma AB.
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Kenny, D.T. et al. (2013). Mass Spectrometric Analysis of O-Linked Oligosaccharides from Various Recombinant Expression Systems. In: Beck, A. (eds) Glycosylation Engineering of Biopharmaceuticals. Methods in Molecular Biology, vol 988. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-327-5_9
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DOI: https://doi.org/10.1007/978-1-62703-327-5_9
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