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 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.
Molloy
M P
MP
Australian Proteome Analysis Facility, School of Biological Sciences, Macquarie University, Sydney.
Herbert
B R
BR
Williams
K L
KL
Gooley
A A
AA
D001426:Bacterial Proteins / Q000302:isolation & purification*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry*; D010449:Peptide Mapping; D012997:Solvents
D001426:Bacterial Proteins; D012997:Solvents