Summary
Electrospray ionization-mass spectrometry (ESI-MS) is a powerful tool for the characterization of intact proteins. However, complex samples require separation in order to obtain clear mass spectra and avoid matrix interaction; capillary electrophoresis (CE) has been shown to be a powerful separation technique for intact proteins. Herein, we present a method based on capillary zone electrophoretic (CZE) separation coupled online with mass spectrometric protein detection. While this approach is suitable for the separation and characterization of various intact proteins, the emphasis is placed on the separation of glycoforms of various and rather complex glycoproteins. The method has been successfully applied to the analysis of glycoproteins, e.g., erythropoietin, fetuin, and alpha-acid glycoprotein.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Hutterer, K., and Dolník, V. (2004) Capillary electrophoresis of proteins 2001–2003, Elec-trophoresis, 22, 3998–4012.
Patrick, J. S., and Lagu, A. L. (2001) Review applications of capillary electrophoresis to the analysis of biotechnology-derived therapeutic proteins, Electrophoresis, 22, 4179–96.
Kamoda, S., and Kakehi, K. (2006) Capillary electrophoresis for the analysis of glycoprotein pharmaceuticals, Electrophoresis, 27, 2495–504.
Hernández-Borges, J., Neusüβ, C., Cifuentes, A., and Pelzing, M. (2004) On-line capillary elec-trophoresis-mass spectrometry for the analysis of biomolecules, Electrophoresis, 25, 2257–81.
Simpson, D. C., and Smith, R. D. (2005) Combining capillary electrophoresis with mass spectrometry for applications in proteomics, Electrophoresis, 26, 1291–305.
Bateman, K. P., Kelly, J. F., Thibault, P., Rama-ley, L., and White, R. L. (2003) Glycoprotein analysis by capillary zone electrophoresis-elec-trospray mass spectrometry, in Capillary Elec-trophoresis of Carbohydrates (ed. P. Thibault and S. Honda), Methods in Molecular Biology, Vol. 213, Humana, Totowa, NJ, pp. 219–39.
Neusüβ, C., Demelbauer, U., and Pelzing, M. (2005) Glycoform characterization of intact erythropoietin by capillary electrophoresis-electrospray-time of flight-mass spectrometry, Electrophoresis, 26, 1442–50.
Balaguer, E., and Neusüβ, C. (2006) Glyco-protein characterization combining intact protein and glycan analysis by capillary elec-trophoresis-electrospray ionization-mass spec-trometry, Analytical Chemistry, 78, 5384–93.
Balaguer, E., Demelbauer, U., Pelzing, M., Sanz-Nebot, V., Barbosa, J., and Neusüβ, C. (2006) Glycoform characterization of erythropoietin combining glycan and intact protein analysis by capillary electrophoresis – electrospray – time-of-flight mass spectrom-etry, Electrophoresis, 27, 2638–50.
Ohnesorge, J., Neusüβ, C., and Wätzig, H. (2005) Quantitation in capillary electro-phoresis – mass spectrometry, Electrophoresis, 26, 3973–87.
Acknowledgments
We thank Jonathan Moss for proof reading of the manuscript.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Neusüβ, C., Pelzing, M. (2009). Capillary Zone Electrophoresis-Mass Spectrometry for the Characterization of Isoforms of Intact Glycoproteins. In: Lipton, M.S., Paša-Tolic, L. (eds) Mass Spectrometry of Proteins and Peptides. Methods In Molecular Biology, vol 492. Humana Press. https://doi.org/10.1007/978-1-59745-493-3_11
Download citation
DOI: https://doi.org/10.1007/978-1-59745-493-3_11
Publisher Name: Humana Press
Print ISBN: 978-1-934115-48-0
Online ISBN: 978-1-59745-493-3
eBook Packages: Springer Protocols