Separation and characterization of O-deacylated lipooligosaccharides and glycans derived from Moraxella catarrhalis using capillary electrophoresis-electrospray mass spectrometry and tandem mass spectrometry.

Electrophoretic methods have been developed for the analysis of complex carbohydrates derived from lipooligosaccharides (LOS) of Moraxella catarrhalis using capillary electrophoresis coupled to electrospray mass spectrometry (CE-ESMS). Separation of lipooligosaccharides (LOS) arising from mild hydrazinolysis of the intact lipopolysaccharides (LPS) was achieved using aqueous ammonium formate, and enabled identification of sites of heterogeneity (phosphates, phosphoethanolamine, and pendant acyl groups) on either the lipid A or the core oligosaccharide. More complex mixtures of carbohydrates obtained from the complete deacylation and dephosphorylation of LOS were amendable to electrophoretic conditions using both anionic and cationic separation. In particular, electrophoretic conditions were developed which permitted resolution of closely related oligosaccharides according to the number of carbohydrate residues appended to the core structure. Structural characterization of carbohydrates and LOS released from the hydrazinolysis and acid hydrolysis treatment of the intact LPS was achieved using tandem mass spectrometry (MS-MS) for samples introduced by direct flow injection. Taken together, the combination of CE-ESI-MS and MS-MS analyses provided valuable information on the heterogeneity of the LOS population in which a significant level of variability was found mostly in the lipid A portion.