Characterization of the O-4 phosphorylated and O-5 substituted Kdo reducing end group and sequencing of the core oligosaccharide of Aeromonas salmonicida ssp salmonicida lipopolysaccharide using tandem mass spectrometry

The molecular structure of the wild strain of the lipopolysaccharide (LPS) core of Aeromonas salmonicida, ssp salmonicida has been sequenced using tandem mass spectrometry. The core oligosaccharide was determined to contain an O-4 phosphorylated and O-5 substituted Kdo reducing group and its structure is proposed as follows: L-alpha-D-Hepp 1 PO4 down arrow down arrow 6 4 R-L-alpha-D-Hepp-(1 --> 2)-L-alpha-D-Hepp-(1 --> 3)-L-alpha-D-Hepp-(1 --> 5)-alpha-Kdop-(2 --> lipid A) 4 up arrow 1 alpha-D-GlcpN-(1 --> 7)-L-beta-D-Hepp R = alpha-D-Galp-(1 --> 4)-alpha-D-Galp NAc-(1 --> 6)-alpha-D-Glcp-(1 --> 4) After the core oligosaccharide of LPS was released from the lipid A portion by conventional treatment with 1% acetic acid, we demonstrated the existence of a homogeneous mixture composed mainly of the native core oligosaccharide containing the Kdo with its O-4 phosphate group intact and a degraded core oligosaccharide mixture which eliminated the O-4 phosphate group with extreme ease. The precise molecular structure and glycone sequence of the homogeneous mixture of phosphorylated and dephosphorylated core oligosaccharides was determined by electrospray ionization mass spectrometry and tandem mass spectrometric (MS/MS) analysis. Collision-induced dissociation MS/MS of the homogeneous mixture of permethylated core oligosaccharides afforded a series of diagnostic product ions which confirmed the established sequence of the glycones to be determined. Matrix-assisted laser desorption/ionization MS/MS reconfirmed the molecular structure of the dephosphorylated homogeneous permethylated mixture of the core oligosaccharides containing the diastereomeric 4,8- and 4,7-anhydro-alpha-keto acids.