In most proteomics analyses and in particular for the "off-gel" approaches, based essentially on chromatography, the complexity of the proteome should be reduced; otherwise identifications can be hindered, especially if the mass spectrometry analysis is not conducted using state-of-the-art instrumentation. Even if the method used is a bottom-up proteomics, it appears mandatory to pre-fractionate the proteins in order to reduce the complexity. We report here the development and validation of a

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... e-fractionation based on the differential solubilisation of proteins using increasing concentrations of acetonitrile (ACN). This "ACN fractionation" was applied to the study of the Triton X-100 soluble sub-proteome of brain capillary endothelial cells (BCEC) with re-induced blood-brain barrier (BBB) functions.