Extraction of Escherichia coli proteins with organic solvents prior to two-dimensional electrophoresis

Compared to soluble proteins, hydrophobic proteins, in particular membrane proteins, are an underrepresented protein species on two-dimensional (2-D) gels. One possibility is that many hydrophobic proteins are simply not extr acted from the sample prior to 2-D gel separation. We attempted to isolate hydrophobic proteins from Escherichia coli by extracting with organic solve nts, then reconstituting the extracted proteins in highly solubilising samp le solution amenable to 2-D electrophoresis using immobilized pH gradients (IPGs). This was conducted by an extraction with a mixture of chloroform an d methanol, followed by solubilisation using a combination of urea, thioure a, sulfobetaine detergents and tributyl phosphine. Peptide mass fingerprint ing assisted in the identification of 13 proteins, 8 of which have not prev iously been reported on 2-D gels. Five of these new proteins possess a posi tive hydropathy plot. These results suggest that organic solvent extraction s may be useful for selectively isolating some proteins that have previousl y been missing from proteome maps.