Abstract
Separation and determination of proteins by capillary isoelectric focusing (CIEF) and mass spectrometry (MS) are essential and complementary techniques in the field of bioanalysis. The hyphenation of these two techniques is challenging due to the nonvolatile substances required for the CIEF separation. An additional separation step prior to MS enables the removal of the nonvolatile substances. However, it is complicated due to the small transfer volume and the required high voltages in the CIEF process. In order to remove nonvolatile substances and transfer the analytes toward the mass spectrometer, we applied a four-port valve to couple CIEF online to capillary electrophoresis-mass spectrometry. To demonstrate the power of this concept, hemoglobin and glycated hemoglobin with an isoelectric point difference of 0.037 were separated via isoelectric focusing and characterized by MS. In general, this setup guaranties interference-free mass spectra and will provide an information-rich and sensitive top down protein characterization.
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Acknowledgments
We gratefully thank Sabine Neuberger and Kevin Jooß from the University of Aalen for helpful suggestions during the work on this paper and Neil R Pattinson (Canterbury Scientific Ltd) for the kind gift of glycated hemoglobin. With regard to the polishing process of the valve, we gratefully thank Elvira Reiter, Department of Material Research, University of Aalen, Germany. We thank an anonymous reviewer for numerous valuable suggestions.
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Hühner, J., Neusüß, C. CIEF-CZE-MS applying a mechanical valve. Anal Bioanal Chem 408, 4055–4061 (2016). https://doi.org/10.1007/s00216-016-9498-8
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DOI: https://doi.org/10.1007/s00216-016-9498-8