Characterization of Lipid A Acylation Patterns in Francisella tularensis F. novicida and F. philomiragia using Multiple-Stage Mass Spectrometry (MSn) on a vMALDI Linear Ion Trap

摘要

Lipopolysaccharide (LPS) is a major component of the outer membrane of gram-negative bacteria. The lipid A region of LPS stimulates the immune system in a structure dependent manner. We have previously identified the two major lipid A species from Francisella tularensis as asymmetric tetraacylated structures containing four long acyl chains (16 and 18 carbons) and a single phosphate group that is partially modified by galactosamine (Phillips, N.J.; Schilling B.; McLendon, M.K.; Apicella, M.A.; Gibson, B.W. Infect. Immun. >2004, 72, 5340–5348). In this current study, we used matrix-assisted laser desorption / ionization on a vacuum source (vMALDI) coupled to a linear ion trap (LIT) mass spectrometer in multiple-stage mass fragmentation mode (MSⁿ) to determine the structures of several minor and low abundant lipid A species present in F. tularensis, F. novicida, and F. philomiragia LPS that have not been previously characterized. Comprehensive vMALDI-MSⁿ fragmentation studies allowed us to deduce the composition and the position of the fatty acid substituents within the lipid A moieties. Unexpectedly, most of these minor lipid A species consisted of multiple isobaric species with acyl chains of various lengths. Moreover, we found that a small portion of these lipid A’s may be modified by the addition of a hexose or hexosamine sugar, in addition to the galactosamine that was previously identified. Overall, we found that MSⁿ analysis on the vMALDI-LIT MS platform was highly efficient and sensitive, allowing for thorough analysis of very minor lipid A species.