Efficient and reproducible sample preparation methods are key to successful separation in proteome. Meanwhile, efficient sample preparation also enriches target sample to some extent and plays a pre-separation role. In this study, for the first time we put forward using acidic and basic extraction buffer as complementary methods of standard ways to extract complex proteins in rat liver tissue. We also compared the effect of three extraction methods on sample pre-separation and enrichment. The acidic or basic condition was obtained by adding 0.1% trifluoroacetic acid or 40 mM Tris, respectively. The rat liver samples were divided into three equal groups and different extraction buffers were used for each group. The concentration of protein from different methods was measured and the corresponding sample was separated using two-dimensional get electrophoresis, followed by detection of MALDI-TOF-TOF mass spectrometry. Three extraction lysates were also separated by reversed-phase high performance liquid chromatography. The images in gel and mass spectrum indicated that the different methods can pre-separate and enrich some special proteins. Experimental results also showed that the three methods had good reproducibility and could be applied in proteomic analysis. (c) 2006 Elsevier B.V. All rights reserved.