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

Eur. J. Mass Spectrom. 10, 715–730 (2004)
DOI: 10.1255/ejms.673

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

Joseph Banoub,^a,b* Anas El Aneed,^b Alejandro Cohen,^c and Patrick Martin^b
^aDepartment of Fisheries and Oceans, Science Branch, Special Projects, PO Box 5667, St. John’s, NL A1C 5X1, Canada. E-mail: banoubjo@dfo-mpo.gc.ca
^bMemorial University of Newfoundland, Biochemistry Department, St. John’s, NL A1C 5S7, Canada
^cIUT Bethune, Departement de Chimie, Université d’Artois, Bethune, France

ABSTRACT:

The molecular structure of the wild strain of the lipopolysaccharide 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 the follows:
R-L-α-D-Hepp- (1→2)-L-α-D-Hepp-(1→3)-L-α-D-Hepp-(1→5)-α-Kdop-(2→lipid A)
α-D-GlcpN-(1→7)-L-β– D-Hepp
R= α-D-Galp-(1→4)-α-D-Galp NAc-(1→6)-α-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 facility. The precise molecular structure and glycone sequence of the homogeneous mixture of phosphorylated and dephosphorylated core oligosaccharides was determined by electrospray ionization (ESI) mass spectrometry and tandem mass spectrometric analysis. CID-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 (MALDI) tandem mass spectrometry reconfirmed the molecular structure of the dephosphorylated homogeneous permethylated mixture of the core oligosaccharides containing the diastereomeric 4,8- and 4,7-anhydro-α-keto acids.

Keywords: structural investigation, Aeromonas. Salmonicida ssp salmonicida, lipopolysaccharide, native phosphorylated core oligosaccharide, degraded dephosphorylated core oligosaccharide, electrospray and matrix-assisted laser desorption/ionization, mass spectrometry and tandem mass spectrometry

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