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The application of 2DE to membrane proteins is hampered by the limited protein solubility. In addition, the gel matrix is a limiting factor in combination with downstream analytical techniques such as mass spectrometry. Free-Flow Electrophoresis (FFE) in the isoelectric focusing (IEF) mode provides a carrier-free alternative to 2DE. IEF-FFE has proven useful for soluble proteins since the methodology is not limited in pH or size range. Various detergents have been applied to handle more hydrophobic proteins under denaturing conditions. However, a prerequisite for IEF-FFE is the maintained solubility of the proteins at their pI. This is not the case for many membrane proteins which have high tendency to precipitate at their respective pI.
Here we present Interval Zone FFE (IZ-FFE) as a novel mode of FFE which facilitates separation of charged molecular species including proteins and protein complexes. In contrast to IEF, the separation is carried out at a constant pH relying on the net charges of the proteins. The applied pH may be significantly different from the pI of the proteins to be separated thus maintaining proteins in solution that would otherwise precipitate. Resolution and solubility may be increased further under denaturing conditions using urea and/or thiourea and by including non-ionic or zwitterionic detergents.
IZ-FFE was applied to a total protein extract from HeLa cells and compared to IEF-FFE. A significant increase in protein solubility was observed in the IZ-FFE mode. Separations were performed using detergent-free media at high loading rates without observing precipitation. This was in contrast to IEF-FFE where precipitation was observed even when detergents were included in the media. The detergent-free separation media facilitated the coupling to RPLC-MS/MS since no detergent removal was required and the proteins were maintained in solution.