No abstract

Fused-silica capillary LC columns (25-microm i.d.) with 3-microm-i.d. integrated electrospray emitters interfaced to a quadrupole ion trap mass spectrometer were evaluated for high-sensitivity LC-MS2. Column preparation involved constructing frits by in situ photopolymerization of glycidyl methacrylate and trimethylolpropane trimethacrylate, preparing the electrospray emitter by pulling the column outlet to a fine tip with a CO2 laser puller, and slurry-packing the column with 5-microm reversed-phase particles. Large-volume injections were facilitated by an automated two-pump system that allowed high-flow rates for sample loading and low-flow rates for elution. Small electrospray emitters, low elution flow rates, and optimization of gradient steepness allowed a detection limit of 4 amol, corresponding to 2 pM for 1.8 microL injected on-column, for a mixture of peptides dissolved in artificial cerebral spinal fluid. The system was coupled on-line to microdialysis sampling and was used to monitor and discover endogenous neuropeptides from the globus pallidus of anesthetized male Sprague-Dawley rats. Time-segmented MS2 scans enabled simultaneous monitoring of Met-enkephalin, Leu-enkephalin, and unknown peptides. Basal dialysate levels of Met-enkephalin and Leu-enkephalin were 60 +/- 30 and 70 +/- 20 pM while K+-stimulated levels were 1,900 +/- 500 and 1,300 +/- 300 pM, respectively (n = 7). Data-dependent and time-segmented MS2 scans revealed several unknown peptides that were present in dialysate. One of the unknowns was identified as peptide I(1-10) (SPQLEDEAKE), a novel product of preproenkephalin A processing, using MS2, MS3, and database searching.

Bacterial infections can be aggravated by antibiotic treatment that induces SOS response and vesiculation. This leads to a hypothesis concerning association of SOS with vesiculation. To test it, we conducted multiple analyses of outer membrane vesicles (OMVs) produced from thePseudomonas aeruginosawild type in which SOS is induced by ciprofloxacin and from the LexA noncleavable (lexAN) strain in which SOS is repressed. The levels of OMV proteins, lipids, and cytotoxicity increased for both the treated strains, demonstrating vesiculation stimulation by the antibiotic treatment. However, the further increase was suppressed in thelexANstrains, suggesting the SOS involvement. Obviously, the stimulated vesiculation is attributed by both SOS-related and unrelated factors. OMV subproteomic analysis was performed to examine these factors, which reflected the OMV-mediated cytotoxicity and the physiology of the vesiculating cells under treatment and SOS. Thus, SOS plays a role in the vesiculation stimulation that contributes to cytotoxicity.

No abstract