Proteomic analysis of Pseudomonas aeruginosa grown under magnesium limitation.
In this study, large-scale qualitative and quantitative proteomic technology was applied to the analysis of the opportunistic bacterial pathogen Pseudomonas aeruginosa grown under magnesium limitation, an environmental condition previously shown to induce expression of various virulence factors. For quantitative analysis, whole cell and membrane proteins were differentially labeled with isotope-coded affinity tag (ICAT) reagents and ICAT reagent-labeled peptides were separated by two-dimensional chromatography prior to analysis by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) in an ion trap mass spectrometer (ITMS). To increase the number of protein identifications, gas-phase fractionation (GPF) in the m/z dimension was employed for analysis of ICAT peptides derived from whole cell extracts. The experiments confirmed expression of 1331 P. aeruginosa proteins of which 145 were differentially expressed upon limitation of magnesium. A number of conserved Gram-negative magnesium stress-response proteins involved in bacterial virulence were among the most abundant proteins induced in low magnesium. Comparative ICAT analysis of membrane versus whole cell protein indicated that growth of P. aeruginosa in low magnesium resulted in altered subcellular compartmentalization of large enzyme complexes such as ribosomes. This result was confirmed by 2-D PAGE analysis of P. aeruginosa outer membrane proteins. This study shows that large-scale quantitative proteomic technology can be successfully applied to the analysis of whole bacteria and to the discovery of functionally relevant biologic phenotypes.
Guina
Tina
T
Department of Pediatrics, Division of Infectious Diseases, University of Washington, Seattle, Washington 98195, USA. tguina@u.washington.edu
Wu
Manhong
M
Miller
Samuel I
SI
Purvine
Samuel O
SO
Yi
Eugene C
EC
Eng
Jimmy
J
Goodlett
David R
DR
Aebersold
Ruedi
R
Ernst
Robert K
RK
Lee
Kimberly A
KA
D001425:Bacterial Outer Membrane Proteins / Q000032:analysis / Q000235:genetics; D001426:Bacterial Proteins / Q000032:analysis* / Q000235:genetics; D002455:Cell Division; D015180:Electrophoresis, Gel, Two-Dimensional; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D008274:Magnesium / Q000378:metabolism* / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects; D020543:Proteome / Q000032:analysis* / Q000235:genetics; D040901:Proteomics; D011550:Pseudomonas aeruginosa / Q000166:cytology* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D014774:Virulence
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D020543:Proteome; D008274:Magnesium