title: CIEF-CZE-MS applying a mechanical valve.
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. Graphical abstract Interference free coupling of capillary isoelectric focusing to mass spectrometry by applying a mechanical valve. The focused proteins were tranferred from the isoelectric focusing to capillary electrophoresis by a mechanical valve. Afterwards, the transferred protein was sepearated from ionization interfering substances in the capillary electrophoresis prior to the mass spectrometry detection.
authors.affiliation: Faculty of Chemistry, Aalen University, Beethovenstr. 1, 73430, Aalen, Germany.|Institute of Pharmaceutical Science, University of Tübingen, Auf der Morgenstelle 8, 72076, Tübingen, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Hühner
authors.affiliation: Faculty of Chemistry, Aalen University, Beethovenstr. 1, 73430, Aalen, Germany. christian.neusuess@hs-aalen.de.
authors.forename: Christian
authors.initials: C
authors.lastname: Neusüß
mesh_terms: D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D006454:Hemoglobins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000295:instrumentation / Q000379:methods*; D007526:Isoelectric Point; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D006454:Hemoglobins
keywords: Capillary isoelectric focusing; Mass spectrometry; Online hyphenation; Protein; Two-dimensional separation