Comprehensive analysis of complex proteomes using microscale solution isoelectrofocusing prior to narrow pH range two-dimensional electrophoresis.

Abstract

Comprehensive analysis of complex proteomes requires prefractionation of samples prior to two-dimensional gel electrophoresis (2-DE). This study demonstrates the utility of using a high resolution sample prefractionation method and slightly overlapping narrow pH range two-dimensional gel electrophoresis to enhance quantitative comparisons of complex proteomes. A key feature of this strategy is to prefractionate samples into a few well-defined pools using microscale solution isoelectric focusing (nusol-IEF) prior to 2-DE protein analysis. Sample prefractionation is achieved using a series of tandem small volume chambers (500 nuL) separated by thin membranes containing immobilines at specific pH's. The resulting well-resolved fractionated samples are optimally separated on a series of slightly overlapping narrow pH range immobilized pH gradient (IPG) gels, which are approximately 0.1 pH units wider than the nusol-IEF fractionated pools. When nusol-IEF prefractionation was applied to proteome analyses of mouse serum, it resulted in the capacity to separate much higher protein loads on narrow pH range IPG gels while retaining good resolution and spot recovery. More importantly, the prefractionation of serum greatly enhanced the ability to detect low abundance proteins, because major interfering proteins were removed from most fractions. At least 6- to 30-fold higher protein loads were possible for nonalbumin fractions on narrow pH range IPG gels. The dynamic range of protein detection is substantially increased since higher protein loads can be applied to narrow pH range 2-DE gels, and duplicate gels can be stained with colloidal Coomassie and silver stains for quantitation of abundant and minor proteins, respectively. Finally, the ability to effectively fractionate complex proteomes into very narrow ranges (< 0.5 pH units) strongly suggests that nusol-IEF could be used to prefractionate complex samples for subsequent direct analysis by liquid chromatography-tandem mass spectrometry methods as an alternative to using overlapping narrow pH range 2-DE gels.