The outer membrane protein contents of Salmonella enterica serovar Typhimurium strains with PhoP/PhoQ regulon mutations were compared by two-dimensional gel electrophoresis. At least 26 species of outer membrane proteins (OMPs) were identified as being regulated by PhoP/PhoQ activation. One PhoP/PhoQ-activated OMP was identified by semiautomated tandem mass spectrometry coupled with electronic database searching as PgtE, a member of the Escherichia coli OmpT and Yersinia pestis Pla family of outer membrane proteases. Salmonella PgtE expression promoted resistance to alpha-helical cationic antimicrobial peptides (␣-CAMPs). Strains expressing PgtE cleaved C18G, an 18-residue ␣-CAMP present in culture medium, indicating that protease activity is likely to be the mechanism of OmpT-mediated resistance to ␣-CAMPs. PhoP/PhoQ did not regulate the transcription or export of PgtE, indicating that another PhoP/PhoQ-dependent mechanism is required for PgtE outer membrane localization. PgtE is a posttranscriptionally regulated component of the PhoP/PhoQ regulon that contributes to Salmonella resistance to innate immunity.

An interface was developed for mass spectrometry based on dc induction. Ions were produced as liquid flowed through a concentric cylindrical capacitor at room temperature. No externally applied high voltages affecting the spray tip were used, thus eliminating corona discharge problems in negative ion mode. The voltages applied to the capacitor were lower than those used for electrospray on the same instrument. Stable signals were observed for positive ions infused at flow rates from 50 nL/min to 200 µL/min, anions in the range 50-500 nL/min, without addition of sheath liquid or gas. The minimum concentration required to generate an interpretable product ion spectrum from the [M -2H] 2precursor, for infused Salmonella typhimurium diphosphorylated lipid A, was improved by a factor of ∼500. Positive ion capillary LC/MS results for a horse apomyoglobin tryptic digest were similar to those observed for an optimized electrospray interface. Factor XIII b subunit, an 83-kDa protein, yielded an interpretable positive ion multiple charge envelope, under buffer conditions that did not allow collection of useful data with our low-flow electrospray source. Mass spectra were also generated for a single-stranded DNA 15-mer and 2′,4′-dideuteriotrifluoropropranolol, a small molecule that had been difficult to ionize by either electrospray or APCI.

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