title: High-resolution separations of protein isoforms with liquid chromatography time-of-flight mass spectrometry using polymer monolithic capillary columns.
abstract: The separation of intact proteins, including protein isoforms arising from various amino-acid modifications, employing a poly(styrene-co-divinylbenzene) monolithic capillary column in high-performance liquid chromatography coupled on-line to a time-of-flight mass spectrometer (MS) is described. Using a 250 mm × 0.2 mm monolithic capillary column high-sensitivity separations yielding peak capacities of >600 were achieved with a 2h linear gradient and formic acid added in the mobile phase as ion-pairing agent. The combination of high-resolution chromatography with high-accuracy MS allowed to distinguish protein isoforms that differ only in their oxidation and biotinylation state allowing the separation between structural isoforms. Finally, the potential to separate proteins isoforms due to glycosylation is discussed.
authors: Vrije Universiteit Brussel, Department of Chemical Engineering, Pleinlaan 2, B-1050 Brussels, Belgium. seeltink@vub.ac.be
authors: Sebastiaan
authors: S
authors: Eeltink
authors: Bert
authors: B
authors: Wouters
authors: Gert
authors: G
authors: Desmet
authors: Mario
authors: M
authors: Ursem
authors: David
authors: D
authors: Blinco
authors: Glenwyn D
authors: GD
authors: Kemp
authors: Achim
authors: A
authors: Treumann
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D020033:Protein Isoforms / Q000032:analysis / Q000302:isolation & purification*; D012680:Sensitivity and Specificity
chemical_list: D020033:Protein Isoforms