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Göteborgs universitets publikationer

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

Författare och institution:
Diarmuid T. Kenny (Institutionen för biomedicin); S. Gaunitz (-); Catherine A Hayes (Institutionen för biomedicin, avdelningen för medicinsk kemi och cellbiologi); A. Gustafsson (-); M. Sjöblom (-); Jan Holgersson (Institutionen för biomedicin, avdelningen för klinisk kemi och transfusionsmedicin); Niclas G. Karlsson (Institutionen för biomedicin, avdelningen för medicinsk kemi och cellbiologi)
Publicerad i:
Methods in Molecular Biology, 988 s. 145-167
ISSN:
1064-3745
E-ISSN:
1940-6029
Publikationstyp:
Artikel, refereegranskad vetenskaplig
Publiceringsår:
2013
Språk:
engelska
Fulltextlänk:
Sammanfattning (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 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.
Ämne (baseras på Högskoleverkets indelning av forskningsämnen):
MEDICIN OCH HÄLSOVETENSKAP ->
Medicinska grundvetenskaper ->
Farmaceutisk vetenskap ->
Biofarmaci
Nyckelord:
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
Postens nummer:
235864
Posten skapad:
2016-05-03 16:56
Posten ändrad:
2016-06-02 09:23

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