Two-dimensional electrophoresis and peptide mass fingerprinting of bacterial outer membrane proteins

Mark P. Molloy, Nikhil D. Phadke, Janine R. Maddock, Philip C. Andrews

Research output: Contribution to journalArticle

90 Citations (Scopus)

Abstract

Many bacterial outer membrane proteins (OMPs) are missing from two-dimensional (2-D) gel proteome maps. Recently, we developed a technique for 2-D electrophoresis (2-DE) of Escherichia coli OMPs using alkaline pH incubation for isolation of OMPs, followed by improved solubilization conditions for array by 2-DE using immobilized pH gradients. In this report, we expanded our study, examining protein components from the outer membranes of two enteric bacteria, Salmonella typhimurium and Klebsiella pneumoniae (also known as Klebsiella aerogenes), as well as the unrelated, free-living α-proteobacteria Caulobacter crescentus. Patterns of OMPs expression appeared remarkably conserved between members of the Enterobacteriaceae, while C. crescentus was unique, displaying a greater number of clusters of higher-molecularweight proteins (>80 kDa). Peptide mass fingerprinting (PMF) was used for protein identification, and despite matching across-species boundaries, proved useful for first-pass protein assignment of enteric OMPs. In contrast, identification of C. crescentus OMPs was successful only when searching against its recently completed genome. For all three microorganisms examined, the majority of proteins identified on the 2-D gel appear localized to the outer membrane, a result consistent with our previous finding in Escherichia coli. In addition, we discuss some of the benefits and limitations of PMF in cross-species searching.

Original languageEnglish
Pages (from-to)1686-1696
Number of pages11
JournalElectrophoresis
Volume22
Issue number9
DOIs
Publication statusPublished - 2001
Externally publishedYes

Keywords

  • Bacteria
  • Mass spectrometry
  • Membrane proteins
  • Peptide mass fingerprinting
  • Two-dimensional gel electrophoresis

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