Proteomic analysis of the Escherichia coli outer membrane

Outer membrane proteins (OMPs) of Gram-negative bacteria are key molecules that interface the cell with the environment. Traditional biochemical and g enetic approaches have yielded a wealth of knowledge relating to the functi on of OMPs. Nonetheless, with the completion of the Escherichia coli genome sequencing project there is the opportunity to further expand our understa nding of the organization, expression and function of the OMPs in this Gram -negative bacterium. In this report we describe a proteomic approach which provides a platform for parallel analysis of OMPs. We propose a rapid metho d for isolation of bacterial OMPs using carbonate incubation, purification and protein array by two-dimensional electrophoresis, followed by protein i dentification using mass spectrometry. Applying this method to examine E. c oli K-12 cells grown in minimal media we identified 21 out of 26 (80%) of t he predicted integral OMPs that are annotated in SWISS-PROT release 37 and predicted to separate within the range of pH 4-7 and molecular mass 10-80 k Da. Five outer membrane lipoproteins were also identified and only minor co ntamination by nonmembrane proteins was observed. Importantly, this researc h readily demonstrates that integral OMPs, commonly missing from 2D gel map s, are amenable to separation by two-dimensional electrophoresis. Two of th e identified OMPs (YbiL, YeaF) were previously known only from their ORFs, and their identification confirms the cognate genes are transcribed and tra nslated. Furthermore, we show that like the E. coli iron receptors FhuE and FhuA, the expression of YbiL is markedly increased by iron limitation, sug gesting a putative role for this protein in iron transport. In an additiona l demonstration we show the value of parallel protein analysis to document changes in E. coli OMP expression as influenced by culture temperature.