Top Down Tandem Mass Spectrometric Analysis of a Chemically Modified Rough-Type Lipopolysaccharide Vaccine Candidate.
Recent advances in lipopolysaccharide (LPS) biology have led to its use in drug discovery pipelines, including vaccine and vaccine adjuvant discovery. Desirable characteristics for LPS vaccine candidates include both the ability to produce a specific antibody titer in patients and a minimal host inflammatory response directed by the innate immune system. However, in-depth chemical characterization of most LPS extracts has not been performed; hence, biological activities of these extracts are unpredictable. Additionally, the most widely adopted workflow for LPS structure elucidation includes nonspecific chemical decomposition steps before analyses, making structures inferred and not necessarily biologically relevant. In this work, several different mass spectrometry workflows that have not been previously explored were employed to show proof-of-principle for top down LPS primary structure elucidation, specifically for a rough-type mutant (J5) E. coli-derived LPS component of a vaccine candidate. First, ion mobility filtered precursor ions were subjected to collision induced dissociation (CID) to define differences in native J5 LPS v. chemically detoxified J5 LPS (dLPS). Next, ultra-high mass resolving power, accurate mass spectrometry was employed for unequivocal precursor and product ion empirical formulae generation. Finally, MS3 analyses in an ion trap instrument showed that previous knowledge about dissociation of LPS components can be used to reconstruct and sequence LPS in a top down fashion. A structural rationale is also explained for differential inflammatory dose-response curves, in vitro, when HEK-Blue hTLR4 cells were administered increasing concentrations of native J5 LPS v. dLPS, which will be useful in future drug discovery efforts. Graphical Abstract ᅟ.
['Oyler|Benjamin L|BL|', 'Khan|Mohd M|MM|', 'Smith|Donald F|DF|', 'Harberts|Erin M|EM|', 'Kilgour|David P A|DPA|', 'Ernst|Robert K|RK|', 'Cross|Alan S|AS|', 'Goodlett|David R|DR|']
D002460:Cell Line D004926:Escherichia coli D004927:Escherichia coli Infections D022361:Escherichia coli Vaccines D006801:Humans D008070:Lipopolysaccharides D053719:Tandem Mass Spectrometry
Top down Lipopolysaccharide Vaccine Collision induced dissociation FT-ICR Lipid A Oligosaccharide