Identification of N‐glycans from Ebola virus glycoproteins by matrix‐assisted laser desorption/ionisation time‐of‐flight and negative ion electrospray tandem mass spectrometry |
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| Authors: | Gayle Ritchie David J Harvey Ute Stroeher Friederike Feldmann Heinz Feldmann Victoria Wahl‐Jensen Louise Royle Raymond A Dwek Pauline M Rudd |
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| Institution: | 1. Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford UK;2. Special Pathogens Program, National Microbiology Laboratory, Public Health Agency of Canada, 1015 Arlington Street, Winnipeg, Manitoba, Canada, R3E 3R2;3. Department of Medical Microbiology, University of Manitoba, 543‐730 William Avenue, Winnipeg, Manitoba, Canada, R3E 0W3;4. Viral Therapeutics Branch, Virology Division, US Army Medical Research Institute for Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702, USA;5. National Institute for Bioprocessing Research and Training, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland |
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| Abstract: | The larger fragment of the transmembrane glycoprotein (GP1) and the soluble glycoprotein (sGP) of Ebola virus were expressed in human embryonic kidney cells and the secreted products were purified from the supernatant for carbohydrate analysis. The N‐glycans were released with PNGase F from within sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS‐PAGE) gels. Identification of the glycans was made with normal‐phase high‐performance liquid chromatography (HPLC), matrix‐assisted laser desorption/ionisation mass spectrometry, negative ion electrospray ionisation fragmentation mass spectrometry and exoglycosidase digestion. Most glycans were complex bi‐, tri‐ and tetra‐antennary compounds with reduced amounts of galactose. No bisected compounds were detected. Triantennary glycans were branched on the 6‐antenna; fucose was attached to the core GlcNAc residue. Sialylated glycans were present on sGP but were largely absent from GP1, the larger fragment of the transmembrane glycoprotein. Consistent with this was the generally higher level of processing of carbohydrates found on sGP as evidenced by a higher percentage of galactose and lower levels of high‐mannose glycans than were found on GP1. These results confirm and expand previous findings on partial characterisation of the Ebola virus transmembrane glycoprotein. They represent the first detailed data on carbohydrate structures of the Ebola virus sGP. Copyright © 2010 John Wiley & Sons, Ltd. |
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