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

• 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.

Subject headings

MEDICIN OCH HÄLSOVETENSKAP  -- Medicinska och farmaceutiska grundvetenskaper -- Farmaceutiska vetenskaper (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Basic Medicine -- Pharmaceutical Sciences (hsv//eng)

TEKNIK OCH TEKNOLOGIER  -- Industriell bioteknik -- Bioprocessteknik (hsv//swe)

ENGINEERING AND TECHNOLOGY  -- Industrial Biotechnology -- Bioprocess Technology (hsv//eng)

Keyword

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

Biokemisk processteknik

Publication and Content Type

ref (subject category)

art (subject category)