A multi-laboratory study assessing reproducibility of a 2D-DIGE differential proteomic experiment.

Abstract

Although two-dimensional gel electrophoresis (2DE) has long been used to study differential proteomics, its reproducibility has always been a major concern. In recent years, different methodological improvements have contributed to more robust 2DE workflows: use of immobilized isoelectric focusing (IEF) strips, fluorescence-based difference gel electrophoresis (DIGE), new software tools, etc. To assess the reproducibility of 2DE experiments across laboratories, we set up a multi-laboratory study, performed at 12 laboratories of the ProteoRed network (Spanish network of proteomics facilities). All participating laboratories received two protein extracts, prepared from cultured human adenocarcinoma MDA-MB-468 cells, treated or not with 50 ng/ml epidermal growth factor (EGF) for 24 h. Differential analysis was performed by a four-gel 2D-DIGE experiment, using four technical replicates of each sample, with Cy dye-swapping. Strictly defined 2DE conditions were followed by all laboratories. Each laboratory selected the 30 spots presenting the highest fold variations (with P<0.05) and attempted mass spectrometry (MS) protein identification.