Prefractionation of complex protein samples prior to mass spectrometry provides a method for the isolation of low-abundance proteins into specific fractions thereby enabling their identification. Free-flow electrophoresis in the isoelectric focusing mode (IEF-FFE) presents a complementary approach to established prefractionation methodologies. Proteins are separated in solution according to their isoelectric point (pI) with a high throughput of sample volume. The separation may be performed under denaturing or nondenaturing conditions and detergents may be added to promote protein solubilization. A protocol covering the pH range from pH 3 to 9 under denaturing conditions was used to illustrate the method of IEF-FFE including sample preparation prior to reversed-phase liquid chromatography and tandem mass spectrometry. The IEF-FFE separation was applied to a sample of human urine.
