We set up the highly sensitive and accurate Selective Reaction Monitoring technique by using heavy isotope labeled peptides in order to obtain in vivo absolute quantification of the outer membrane proteome of Salmonella i.e. the copy number per cell. This targeted proteomics technique enables the envelope characterization of different subsets of Salmonella mutants in various environments and will be used as a guide to optimize in vitro conditions that mimic the in vivo Salmonella envelope properties.
The bacterial envelope is an interface to the host that is crucial for virulence and a potential target structure for vaccination. The outer membrane of Salmonella is a dynamic compartment that modulates its composition according to various stresses. However, our knowledge of in vivo quantitative envelope composition remains incomplete as most of the outer membrane proteins are expressed below the quantification threshold of standard shotgun proteomics approaches. To obtain absolute quantification of the outer membrane proteome of Salmonella in vivo, we set up the highly sensitive and accurate Selective Reaction Monitoring proteomics technique. As an example, we can identify a peptide from OmpF with high sensitivity and quantify it accurately using a mixed-in heavy isotope labeled synthetic peptide with the same sequence (Fig. 1A). This targeted approach enabled the characterization of 68 outer membrane proteins of different Salmonella strains and compare it to WT outer membrane proteins under various in vitro and in vivo conditions. Fig. 1B shows a comparison of WT Salmonella with an rpoE mutant that has a defect in responses to envelope stress. Most proteins are similarly abundant but the simple porin OmpD and the gated porin FhuE are more abundant in the mutant, while the efflux-associated outer membrane channel YohG is less abundant. We currently obtain large-scale data sets for Salmonella subpopulations with different growth rates and stress level from infected mouse tissues.
Proteome analysis of Salmonella outer membrane proteins. A) Identification and quantification of OmpF using an internal heavy isotope-labeled reference peptide. B) Comparison of outer membrane protein abundance in WT and rpoE Salmonella.
Outlook for last year
We will optimize the proteomics approach to cover all 85 relevant outer membrane proteins. We will enlarge our in vivo data set by including Salmonella samples from antibiotics-treated mice. Finally, the accumulating in vivo envelope composition data will be used as a guide to optimize in vitro conditions that mimic the in vivo Salmonella envelope properties.