Determination of distinctive carbohydrate signatures obtained from the Aeromonas hydrophila (chemotype II) core oligosaccharide pinpointing the presence of the 4‐O‐phosphorylated 5‐O‐linked Kdo reducing end group using electrospray ionization quadrupole orthogonal time‐of‐flight mass spectrometry and tandem mass spectrometry

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The electrospray quadrupole orthogonal time-of-flight mass spectrometric (ESI-QqTOF-MS) structural elucidation of the core oligosaccharide of Aeromonas hydrophila (chemotype II) lipopolysaccharide has been investigated and it was demonstrated that it contained an 4-O-phosphorylated Kdo reducing end group, which was glycosylated by the remaining outer core oligosaccharide through its O-5 position. After releasing the core oligosaccharide from the native LPS with acid, the phosphorylated Kdo residue eliminated phosphoric acid, to produce a core oligosaccharide containing a mixture of diastereomeric 4,8- and 4,7-anhydro-alpha-keto acids and an open-chain olefinic Kdo residue. The characteristic glycone sequence was elucidated by collision-induced dissociation tandem mass spectrometry (CID-MS/MS) of the protonated molecule of the native core oligosaccharide. In addition, the analysis of the Hakomori permethylated core oligosaccharide was carried out by electrospray ionization quadrupole orthogonal time-of-flight mass spectrometry (ESI-QqTOF-MS) and matrix-assisted laser desorption/ionization (MALDI)-QqTOF-MS analyses. The presence of more than nine isobaric isomers of this core was detected. The CID-MS/MS analysis of the various protonated permethylated core oligosaccharide molecules showed a similar and diagnostic fragmentation pattern. The over-methylation of the permethylated core oligosaccharide containing either the 4,7- or the 4,8-anhydro-alpha-keto acid unit and the open-chain olefinic Kdo unit was reported. It was realized that the extra minor satellite signals obtained in the ESI-QqTOF-MS and MALDI-TOF-MS analyses were dimethyl sulfoxide (DMSO) stable covalent addition products, which have occurred by a Michael addition on the 4,8-Kdo exocyclic double bond. The occurrence of this series of covalent addition products during the MS analysis of a permethylated core oligosaccharide should be considered as 'carbohydrate-distinctive signatures' establishing and confirming the presence of a 4-O-phosphorylated-5-O-linked Kdo reducing end group.