Characterization of the Salmonella typhimurium proteome by semi-automated two dimensional HPLC-mass spectrometry: detection of proteins implicated in multiple antibiotic resistance.
The proteome of Salmonella enterica serovar Typhimurium was characterized by 2-dimensional HPLC mass spectrometry to provide a platform for subsequent proteomic investigations of low level multiple antibiotic resistance (MAR). Bacteria (2.15 +/- 0.23 x 10(10) cfu; mean +/- s.d.) were harvested from liquid culture and proteins differentially fractionated, on the basis of solubility, into preparations representative of the cytosol, cell envelope and outer membrane proteins (OMPs). These preparations were digested by treatment with trypsin and peptides separated into fractions (n = 20) by strong cation exchange chromatography (SCX). Tryptic peptides in each SCX fraction were further separated by reversed-phase chromatography and detected by mass spectrometry. Peptides were assigned to proteins and consensus rank listings compiled using SEQUEST. A total of 816 +/- 11 individual proteins were identified which included 371 +/- 33, 565 +/- 15 and 262 +/- 5 from the cytosolic, cell envelope and OMP preparations, respectively. A significant correlation was observed (r2 = 0.62 +/- 0.10; P < 0.0001) between consensus rank position for duplicate cell preparations and an average of 74 +/- 5% of proteins were common to both replicates. A total of 34 outer membrane proteins were detected, 20 of these from the OMP preparation. A range of proteins (n = 20) previously associated with the mar locus in E. coli were also found including the key MAR effectors AcrA, TolC and OmpF.
Coldham
Nick G
NG
Department of Food and Environmental Safety, Veterinary Laboratories Agency, Addlestone, Surrey KT15 3NB, United Kingdom. n.g.coldham@vla.maff.gsi.gov.uk
Woodward
Martin J
MJ
D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000378:metabolism*; D004351:Drug Resistance / Q000502:physiology*; D013058:Mass Spectrometry; D010455:Peptides / Q000378:metabolism*; D020543:Proteome; D012486:Salmonella typhimurium / Q000378:metabolism*; D014357:Trypsin / Q000737:chemistry
D010455:Peptides; D020543:Proteome; D014357:Trypsin