Isolation of Escherichia coli inner membranes by metal affinity two-phase partitioning.

摘要: As reduction of sample complexity is a central issue in membrane proteomic research, the need for new pre-fractionation methods significant. Here we present method fast and efficient enrichment Escherichia coli inner membranes expressing His-tagged integral L-fucose-proton symporter (FucP). An enriched fraction was obtained from crude mixture using affinity two-phase partitioning combination with nickel-nitrilotriacetic acid (Ni-NTA) immobilized on agarose beads. Due to interaction between beads FucP, were selectively partitioned bottom phase polymer/polymer aqueous system consisting poly(ethylene glycol) (PEG) dextran. The monitored by assaying activity an marker protein measuring total content both phases. proteins dextran also investigated methodology, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), trypsin digestion liquid chromatography tandem mass spectrometry (LC-MS/MS). Using high level significance (99.95%) subsequent database search, 36 assigned identified phase, compared 29 when standard sucrose gradient centrifugation isolation. Furthermore, metal up 10 times faster than centrifugation. separation conditions these model experiments provide basis selective isolation E. can therefore facilitate research such proteomes.