This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The long term goal of this collaboration is to determine the structures of F. tularensis surface molecules useful as protective antigens for vaccine production. Towards these ends we are characterizing the O-antigen portioni of the F. tularensis lipopolysaccharide. The structure of this O-antigen is known from nuclear spectroscopic evidence based on the bulk properties of the polysaccharide. In addition, the O-antigen composition consistent with this structure was reported based on matrix assisted laser desorption/ionization of the permethylated molecule. In initial studies, we are producing detailed mass profiles on O-antigen isolated from virulent and avirulent F. tularensis strains. We will characterize these structures in detail using tandem mass spectrometric methods developed at the Mass Spectrometry Resource. We have used a combination of chromatographic purification, immune assays and mass spectrometry to analyze O-antigen from two F. tularensis variants (LVS and SchuS4). Initial results for native O-antigen oligosaccharides identified components not previously observed from literature reports. These oligosaccharides had the same nominal mass values as expected O-antigen components. Fourier transform MS of the oligosaccharides assigned exact mass values to these previously unidentified components. It was observed that MALDI-TOF MS analysis of permethylated oligosaccharides failed to ionize these components. A series of high resolution tandem MS experiments demonstrated the presence of capusular oligosaccharides that lack the core hexasaccharide of O-antigen. We expect to publish these results by mid-2011.
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