Effects of post-electrophoretic analysis on variance in gel-based proteomics.

2D electrophoresis (2DE) is a prominent separation method for complex proteomes. Although recent advances have increased the utility of this method in quantitative proteomics studies, many sources of variance still exist. This review discusses the post-electrophoretic sources of variance in current 2DE analysis. The essential improvements in protein visualization and software algorithms that have made 2DE a leading quantitative proteomics method are briefly reviewed. A number of shortcomings in the post-electrophoretic analysis of 2DE data that require further attention are highlighted. Topics discussed include protein visualization and image acquisition, internal standards and normalization methods, background subtraction algorithms, normality of distribution, and the need for standardized tests for the evaluation of 2DE analysis software packages.