title: Rapid label-free quantitative analysis of the E. coli BL21(DE3) inner membrane proteome.
abstract: Biological membranes define cells and cellular compartments and are essential in regulating bidirectional flow of chemicals and signals. Characterizing their protein content therefore is required to determine their function, nevertheless, the comprehensive determination of membrane-embedded sub-proteomes remains challenging. Here, we experimentally characterized the inner membrane proteome (IMP) of the model organism E. coli BL21(DE3). We took advantage of the recent extensive re-annotation of the theoretical E. coli IMP regarding the sub-cellular localization of all its proteins. Using surface proteolysis of IMVs with variable chemical treatments followed by nanoLC-MS/MS analysis, we experimentally identified ∼45% of the expressed IMP in wild type E. coli BL21(DE3) with 242 proteins reported here for the first time. Using modified label-free approaches we quantified 220 IM proteins. Finally, we compared protein levels between wild type cells and those over-synthesizing the membrane-embedded translocation channel SecYEG proteins. We propose that this proteomics pipeline will be generally applicable to the determination of IMP from other bacteria.
authors.affiliation: Institute of Molecular Biology and Biotechnology, Foundation for Research & Technology, Iraklio, Greece.|Department Pathology & Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, USA.
authors.forename: Malvina
authors.initials: M
authors.lastname: Papanastasiou
authors.affiliation: Institute of Molecular Biology and Biotechnology, Foundation for Research & Technology, Iraklio, Greece.|Department of Biology, University of Crete, Iraklio, Greece.
authors.forename: Georgia
authors.initials: G
authors.lastname: Orfanoudaki
authors.affiliation: Institute of Molecular Biology and Biotechnology, Foundation for Research & Technology, Iraklio, Greece.
authors.forename: Nikos
authors.initials: N
authors.lastname: Kountourakis
authors.affiliation: Institute of Molecular Biology and Biotechnology, Foundation for Research & Technology, Iraklio, Greece.
authors.forename: Marina
authors.initials: M
authors.lastname: Koukaki
authors.affiliation: Institute of Molecular Biology and Biotechnology, Foundation for Research & Technology, Iraklio, Greece.|Laboratory of Molecular Bacteriology, Rega Institute, Department of Microbiology and Immunology, Katholieke Universiteit Leuven, Leuven, Belgium.
authors.forename: Marios Frantzeskos
authors.initials: MF
authors.lastname: Sardis
authors.affiliation: Institute of Molecular Biology and Biotechnology, Foundation for Research & Technology, Iraklio, Greece.
authors.forename: Michalis
authors.initials: M
authors.lastname: Aivaliotis
authors.affiliation: Institute of Molecular Biology and Biotechnology, Foundation for Research & Technology, Iraklio, Greece.|Department of Biology, University of Crete, Iraklio, Greece.|Laboratory of Molecular Bacteriology, Rega Institute, Department of Microbiology and Immunology, Katholieke Universiteit Leuven, Leuven, Belgium.
authors.forename: Konstantinos C
authors.initials: KC
authors.lastname: Tsolis
authors.affiliation: Institute of Molecular Biology and Biotechnology, Foundation for Research & Technology, Iraklio, Greece.|Laboratory of Molecular Bacteriology, Rega Institute, Department of Microbiology and Immunology, Katholieke Universiteit Leuven, Leuven, Belgium.
authors.forename: Spyridoula
authors.initials: S
authors.lastname: Karamanou
authors.affiliation: Institute of Molecular Biology and Biotechnology, Foundation for Research & Technology, Iraklio, Greece.|Department of Biology, University of Crete, Iraklio, Greece.|Laboratory of Molecular Bacteriology, Rega Institute, Department of Microbiology and Immunology, Katholieke Universiteit Leuven, Leuven, Belgium.
authors.forename: Anastassios
authors.initials: A
authors.lastname: Economou
mesh_terms: D002853:Chromatography, Liquid; D004926:Escherichia coli / Q000737:chemistry* / Q000166:cytology; D029968:Escherichia coli Proteins / Q000032:analysis*; D059748:Proteolysis; D020543:Proteome / Q000032:analysis*; D040901:Proteomics; D053719:Tandem Mass Spectrometry
chemical_list: D029968:Escherichia coli Proteins; D020543:Proteome
keywords: Inner membrane proteome; LC-MS/MS; Label-free quantitation; Microbiology; SecYEG over-expressed; Surface proteolysis