title: Extraction of Escherichia coli proteins with organic solvents prior to two-dimensional electrophoresis.
abstract: 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 extracted from the sample prior to 2-D gel separation. We attempted to isolate hydrophobic proteins from Escherichia coli by extracting with organic solvents, then reconstituting the extracted proteins in highly solubilising sample solution amenable to 2-D electrophoresis using immobilized pH gradients (IPGs). This was conducted by an extraction with a mixture of chloroform and methanol, followed by solubilisation using a combination of urea, thiourea, sulfobetaine detergents and tributyl phosphine. Peptide mass fingerprinting assisted in the identification of 13 proteins, 8 of which have not previously been reported on 2-D gels. Five of these new proteins possess a positive hydropathy plot. These results suggest that organic solvent extractions may be useful for selectively isolating some proteins that have previously been missing from proteome maps.
authors.affiliation: Australian Proteome Analysis Facility, School of Biological Sciences, Macquarie University, Sydney.
authors.forename: M P
authors.initials: MP
authors.lastname: Molloy
authors.forename: B R
authors.initials: BR
authors.lastname: Herbert
authors.forename: K L
authors.initials: KL
authors.lastname: Williams
authors.forename: A A
authors.initials: AA
authors.lastname: Gooley
mesh_terms: D001426:Bacterial Proteins / Q000302:isolation & purification*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry*; D010449:Peptide Mapping; D012997:Solvents
chemical_list: D001426:Bacterial Proteins; D012997:Solvents