Chromatographic prefractionation prior to two-dimensional electrophoresis and mass spectrometry identifies: Application to the complex proteome analysis in rat liver

In this paper, we describe an approach for fractionating complex protein samples from rat liver prior to two-dimensional gel electrophoresis (2-DE) using reversed-phase high-performance liquid chromatography (RP-HPLC). The whole lysate of liver tissue was prefractionated by RP-HPLC with an optimal multi-stage linear gradient elution. Successive fractions were analyzed using 2-DE and selected spots were identified by MALDI-TOF-TOF mass spectrometry. The reproducibility of this prefractionation technology allows pooling of several consecutive runs of the same sample, resulting in a highly enrichment of low abundance proteins. Computer-assisted calculation showed that the total spot number of samples prefractionated by RP-HPLC was nearly five times as many as that of unfractionated sample. The choice of Coomassie Blue staining rather than silver staining indicated that RP-HPLC prefractionation can provide strong enrichment effect which enabled us to visualize additional and less abundance proteins. Chromatographic enrichment was also demonstrated by the peptide mass fingerprint data, which gave mass spectra with increased number of peptide detected and improved signal intensity.