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

作者：Mark P. Molloy, Ben Herbert, Keith L. Williams, Andrew A. Gooley · 发表于：Electrophoresis · 年份：1999 · DOI：10.1002/(sici)1522-2683(19990101)20:4/5<701::aid-elps701>3.0.co;2-5 · 被引用次数：117 · 研究领域：Advanced Proteomics Techniques and Applications、Mass Spectrometry Techniques and Applications、Microfluidic and Capillary Electrophoresis Applications

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.