A proteome strategy for fractionating proteins and peptides using continuous free-flow electrophoresis coupled off-line to reversed-phase high-performance liquid chromatography

Abstract

Extensive prefractionation is now considered to be a necessary prerequisite for the comprehensive analysis of complex proteomes where the dynamic range of protein abundances can vary from ∼106 for cells to ∼10 10 for tissues such as blood. Here, we describe a high-resolution 2D protein separation system that uses a continuous free-flow electrophoresis (FFE) device to fractionate complex protein mixtures by solution-phase isoelectric focusing (IEF) into 96 well-defined pools, each separated by ∼0.02-0.10 pH unit depending on the gradient created, followed by rapid (∼6 min per analysis) reversed-phase high-performance liquid chromatography (RP-HPLC) of each FFE pool. Fractionated proteins are ..