Electrophoretic prefractionation for comprehensive analysis of proteomes

Abstract

Two-dimensional polyacrylamide gel electrophoresis (2DE) has dominated protein profile analysis for more than 25 years, and it is still the method of choice in many laboratories for quantitatively comparing changes of proteins for proteome analysis experiments. Due to the limited capacities of both gel based and nongel based protein profiling methods, it has become apparent that more powerful and reliable methods are needed for prefractionation of complex proteomes prior to 2D gels and alternative LC-MS analysis. Although preparative IEF prefractionation methods are not orthogonal to 2D gels, they show the most promise due to the very high resolution that can be obtained. Alternative lower resolution prefractionation methods severely compromise the ability to perform comprehensive quantitative comparisons due to variable cross-contamination between adjacent fractions and greater fraction complexity. A number of preparative solution-based IEF methods have been productively integrated into quantitative protein profiling strategies including Rotofor, FFE, IsoPrime, the MCE and µsol-IEF. However, some of these methods require large sample amounts and result in large dilute fractions that are not compatible with direct analysis using downstream protein profiling methods.