Analysis of the Core Oligosaccharide of Aeromonas hydrophila (Chemotype III) Lipopolysaccharide Using Fast Atom Bombardment, Electrospray and Low Energy Tandem Mass Spectrometry

Abstract

Fast-atom bombardment mass spectrometry (FAB-MS) was employed for the structural analysis of the core oligosaccharide ofAeromonas hydrophila(Chemotype III) lipopolysaccharide. Positive ion FAB-MS of the underivatized core oligosaccharide gave the protonated molecular ion, confrrming the correct composition in terms of hexoses, heptoses and Kdo which was present as a bicyclic furanosidic lactone. Negative ion FAB-MS gave the deprotonated molecular ion and fragment ions which were derived from more than two cleavage events with charge retention at the reducing and non-reducing terminals. Positive ion F AB-MS of the permethylated core oligosaccharide afforded fragment ions consistent with the defined sequence and branching patterns of the sugar constituents. The electrospray mass spectrum (ESMS) in the positive ion mode of the underivatized core oligosaccharide afforded the protonated molecular ion in the singly and doubly charged forms. Low energy collision-activated dissociation tandem mass spectrometry (CAD MS/MS) analysis of the protonated molecular ion [M+2H]+2provided additional structural data. ESMS of the permethylated and N-acetylated permethylated core oligosaccharides provided useful structural indices and afforded a characteristic pattern for fragmentions resulting from the opening of the methylated bicyclic Kdo furanosidic 1,7- lactone, which was similar to that obtained in the corresponding FAB-MS.

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

“…We have previously established the chemical structure of the core oligosaccharide of Aeromonas hydrophila (chemotype II) SJ-26R using NMR, methylation analysis, partial hydrolysis with acid, periodate oxidation, Smith degradation, nitrous acid deamination, and oxidation with chromium trioxide. [18][19][20] After releasing the core oligosaccharide from the lipid A by hydrolysis of the rough LPS with 1% acetic acid, we have recently realized that the single reducing Kdo unit, which contained an O-4 phosphate group, eliminated phosphoric acid and produced a homogenous mixture of the nonphosphorylated core oligosaccharide with an olefinic chain on the Kdo residue. This open-chain olefinic Kdo cyclizes to produce a mixture of diastereomers of the 4,8-and the 4,7anhydro-a-keto acid derivatives,.…”

“…21,22 These over-methylation products were also noticed during the ESI-QqTOF-MS analyses of the deep mutant and smooth A. salmonicida and with the FAB-MS analysis of the Aeromonas hydrophila chemotype III. 20 The Hakomori methylation method has been maligned for a very long time and has been suspected to form side products, which are caused by the endothermic reactions of the dimsyl anion, the starting material and the methyl iodide. 16 Certainly, we know well that all methylations of carbohydrates occur through successive, base (B)-catalyzed ionization of the hydroxyl groups, followed by reaction with the methylating agent (MeI).…”