Abstract
Three different analytical techniques (planar SDS-PAGE, CGE-on-a-chip and MALDI-TOF-MS) applied for determination of the molecular weight of intact and partly and completely de-N-glycosylated human serum glycoproteins (antithrombin III and coagulation factor IX) have been compared. N-Glycans were removed from the protein backbone of both complex glycoproteins using PNGase F, which cleaves all types of asparagine-attached N-glycan provided the oligosaccharide has at least the length of a chitobiose core unit. Two of the applied techniques were based on gel electrophoretic separation in the liquid phase while the third technique was the gas-phase technique mass spectrometry. It was demonstrated that the enzymatic de-N-glycosylation generally worked well (completely or partially) with both glycoproteins (one containing only N-glycans and the second N- and O-glycans). All three methods were suitable for monitoring the de-N-glycosylation progress. While the molecular weights determined with MALDI-TOF-MS were most accurate, both gel electrophoretic methods provided molecular weights that were too high because of the attached glycan structures.
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Acknowledgements
We want to thank F. Weigang (Agilent Technologies, Vienna, Austria) for early support with the Agilent 2100 Bioanalyzer. Further we thank K. Pock and A. Buchacher (Octapharma, Vienna, Austria) for samples of plasma-derived AT III and FIX.
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Müller, R., Marchetti, M., Kratzmeier, M. et al. Comparison of planar SDS-PAGE, CGE-on-a-chip, and MALDI-TOF mass spectrometry for analysis of the enzymatic de-N-glycosylation of antithrombin III and coagulation factor IX with PNGase F. Anal Bioanal Chem 389, 1859–1868 (2007). https://doi.org/10.1007/s00216-007-1586-3
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DOI: https://doi.org/10.1007/s00216-007-1586-3