FOCUS: PROTEOMICS 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 magne-sium limitation, an environmental condition previously shown to induce expression of various virulence factors. For quantitative analysis, whole cell and membrane proteins were differen-tially labeled with isotope-coded affinity tag (ICAT) reagents and ICAT reagent-labeled peptides were separated by two-dimensional chromatography prior to analysis by electros-pray 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. Thi