Comparison of proteins expressed by Pseudomonas aeruginosa strains representing initial and chronic isolates from a cystic fibrosis patient: an analysis by 2-D gel electrophoresis and capillary column liquid chromatography-tandem mass spectrometry.
Isolates of Pseudomonas aeruginosa from chronic lung infections in cystic fibrosis (CF) patients have phenotypes distinct from those initially infecting CF patients, as well as from other clinical or environmental isolates. To gain a better understanding of the differences in these isolates, protein expression was followed using two-dimensional (2-D) gel electrophoresis and protein identification by peptide sequencing using micro-capillary column liquid chromatography-tandem mass spectrometry (microLC/MS/MS). The isolates selected for this analysis were from the sputum of a CF patient: strain 383 had a nonmucoid phenotype typical of isolates from the environment, and strain 2192, obtained from the same patient, had a mucoid phenotype typical of isolates from chronic CF lung infections. Strains 383 and 2192 were confirmed to be genetically identical by restriction endonuclease analysis, random amplified polymorphic DNA-PCR, and pulsed-field gel electrophoresis. Conditions of protein extraction were optimized for consistent high-resolution separation of several hundred proteins from these clinical isolates as detected by Coomassie staining of 2-D gels. Fourteen proteins were selected for analysis; this group included those whose expression was common between both strains as well as unique for each strain. The proteins were identified by microLC/MS/MS of the peptides produced by an in-gel tryptic digestion and compared to translated data from the Pseudomonas Genome Project; optimization of this technique has allowed for the comparison of proteins expressed by strains 383 and 2192.
Hanna
Sheri L
SL
Department of Microbiology1 and the W. M. Keck Biomedical Mass Spectrometry Laboratory, Biomolecular Research Facility2, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA.
Sherman
Nicholas E
NE
Department of Microbiology1 and the W. M. Keck Biomedical Mass Spectrometry Laboratory, Biomolecular Research Facility2, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA.
Kinter
Michael T
MT
Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195, USA3.|Department of Microbiology1 and the W. M. Keck Biomedical Mass Spectrometry Laboratory, Biomolecular Research Facility2, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA.
Goldberg
Joanna B
JB
Department of Microbiology1 and the W. M. Keck Biomedical Mass Spectrometry Laboratory, Biomolecular Research Facility2, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA.
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002908:Chronic Disease; D003550:Cystic Fibrosis / Q000382:microbiology*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D010641:Phenotype; D011552:Pseudomonas Infections / Q000382:microbiology; D011550:Pseudomonas aeruginosa / Q000145:classification / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020539:Sequence Analysis, Protein / Q000379:methods
D001426:Bacterial Proteins