Characterization of intact lipopolysaccharides from the Haemophilus influenzae strain RM 118 using electrophoresis-assisted open-tubular liquid chromatography-mass spectrometry.

We have applied an electrophoresis-assisted open-tubular LC-MS method for analyzing intact lipopolysaccharides (LPSs) from Haemophilus influenzae strain RM118 (Rd). We were able to obtain structural information on both core oligosaccharides (OSs) and the lipid A moiety including the sialylation, glycylation, and the distribution of fatty acid residues on the disaccharide backbone of lipid A. The fragmentation patterns of sodiated and protonated LPS molecules were investigated for determining the location of sialic acid. It was found that the tandem mass spectra of sodiated ions provided unambiguous evidence of both sialylated lactose and sialylated lacto-N-neotetraose. In contrast, the fragment ions of protonated ions only offered the evidence for the existence of sialylated lacto-N-neotetraose. The lipid A of Gram-negative bacteria, as the principal endotoxic component of LPS, plays a major role in the pathogenesis of bacterial infections. We have previously characterized lipid A species after mild acid hydrolysis of LPS during which lipid A precipitates. In this study, intact LPS was directly introduced to a tandem mass spectrometer. In-source dissociation strategy was employed, followed by multiple-stage MS/MS on the ions originating from the lipid part to obtain structural information. This is the first time that the structure of lipid A of H. influenzae was characterized by MS/MS on intact LPS molecules without any prior chemical modifications. In the same way information on the OS can be obtained by MS/MS by focusing on ions originating from core OS.