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Mass spectrometric analysis of Klebsiella pneumoniae ssp. pneumoniae rough strain R20 (O1-: K20-) lipopolysaccharide preparations: identification of novel core oligosaccharide components and three 3-deoxy-D-manno-oct-2-ulopyranosonic artifacts.

In an attempt to find the best approach for the mass spectrometric analysis of the whole range of lipopolysaccharide (LPS) structures from Klebsiella pneumoniae ssp. pneumoniae rough strain R20 (O1-:K20-), various methods of LPS preparation were applied and the products were analyzed using a range of mass spectrometric techniques. The most productive approach proved to be the removal of lipid A by mild acid hydrolysis and the study of the core oligosaccharide structures using nanoelectrospray time-of-flight mass spectrometry (TOF-MS) in combination with collision-induced dissociation tandem mass spectrometry. This procedure is very sensitive, but results in the generation of a reducing 3-deoxy-D-manno-oct-2-ulopyranosonic acid residue (Kdo) that is susceptible to the formation of artifacts, which give rise to pseudomolecular ions 18, 46, and 88 Da below the pseudomolecular ion for the unmodified species. Alternatively, matrix-assisted laser desorption/ionization TOF-MS combined with post-source decay can be used to study the de-O-acylated LPS preparation and especially to identify those residues bearing phosphate groups and the residues involved in the linkage between the core and lipid A. In addition to the five LPS core structures defined using NMR spectroscopy by Süsskind et al., several extra related LPS structure were identified. Larger LPS species were observed, which surprisingly do not represent species containing longer versions of the novel Klebsiella heptoglycan, but instead are species having the defined core and heptoglycan extended with up to three extra hexuronic acid and one or two extra hexose residues.