Journal of the American Society for Mass Spectrometry

, Volume 14, Issue 7, pp 742–751

Proteomic analysis of Pseudomonas aeruginosa grown under magnesium limitation

Authors

    • Department of Pediatrics, Division of Infectious DiseasesUniversity of Washington
  • Manhong Wu
    • Departments of Microbiology, Medicine, and Genome SciencesUniversity of Washington
  • Samuel I. Miller
    • Departments of Microbiology, Medicine, and Genome SciencesUniversity of Washington
  • Samuel O. Purvine
    • Institute for Systems Biology
  • Eugene C. Yi
    • Institute for Systems Biology
  • Jimmy Eng
    • Institute for Systems Biology
  • David R. Goodlett
    • Institute for Systems Biology
  • Ruedi Aebersold
    • Institute for Systems Biology
  • Robert K. Ernst
    • Department of Medicine, Division of Infectious DiseasesUniversity of Washington
  • Kimberly A. Lee
    • Department of BiochemistryUniversity of Washington
Focus: Proteomics

DOI: 10.1016/S1044-0305(03)00133-8

Cite this article as:
Guina, T., Wu, M., Miller, S.I. et al. J Am Soc Mass Spectrom (2003) 14: 742. doi:10.1016/S1044-0305(03)00133-8

Abstract

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.

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© American Society for Mass Spectrometry 2003