title: 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.
abstract: 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.
authors.affiliation: Department of Chemistry, Memorial University of Newfoundland, St. John's, Newfoundland, A1B 3V6, Canada.
authors.forename: Salim
authors.initials: S
authors.lastname: Sioud
authors.forename: Farid
authors.initials: F
authors.lastname: Jahouh
authors.forename: Mina
authors.initials: M
authors.lastname: Nashed
authors.forename: Nicolas
authors.initials: N
authors.lastname: Joly
authors.forename: Joseph H
authors.initials: JH
authors.lastname: Banoub
mesh_terms: D016980:Aeromonas hydrophila / Q000737:chemistry*; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D007536:Isomerism; D007651:Keto Acids / Q000737:chemistry; D008070:Lipopolysaccharides / Q000737:chemistry* / Q000302:isolation & purification; D009844:Oligosaccharides / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013400:Sugar Acids / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D007651:Keto Acids; D008070:Lipopolysaccharides; D009844:Oligosaccharides; D013400:Sugar Acids; C002532:2-keto-3-deoxyoctonate; D004121:Dimethyl Sulfoxide