| Two research projects will be presented with the underlining theme of using tandem mass spectrometry to achieve fundamental and structural information about relevant biomolecules. The first project involves the use of ECD, IRMPD, and CID techniques on modified and un-modified synthetic peptides for further understanding of the mechanistic details involved in electron-based dissociation methods. Here, both experimental and computational methods are utilized to provide an enhanced understanding of peptide cation-radical dissociations produced by electron capture or transfer. We report the internal energy gained by electron capture of the modified synthetic peptides resulted in redistribution of the internal energy over the entire peptide moiety, and when combined with additional IR excitation, induced proton-driven ion dissociations which occurred at sites that were remote from the site of electron capture. This example of spin-remote fragmentation provided the first clearcut experimental example of an ergodic dissociation upon electron capture dissociation.;The second project concerns the use of tandem mass spectrometry for complete structural characterization of lipid A extracted from Yersinia pestis . Structural elucidation of lipid A from Yersinia pestis involved determining the degree and extent of acylation followed by detection of an unique phosphorylation pattern referred to as pyrophosphoryl lipid A. Pyrophosphoryl lipid A was not only determined to be found in Yersinia pestis but also widespread among a host of common Gram-negative pathogens. Further confirmation and precise location of pyrophosphate in lipid A from Yersinia pestis was established via incorporation of specific lipid A phosphatase enzymes. Finally, we assessed the link between Yersinia pestis lipid A phosphorylation and its biological function by measuring its pro-inflammatory response in murine and human simulated cells. We report the removal of one phosphate group from diphosphoryl tetra-acylated lipid A had an immunomodulatory effect on murine type cells and immunostimulatory effect on human type cells. This investigation into Yersinia pestis lipid A provided valuable biochemical insight into the interaction of the pathogen:host relationship. |
