The chemical structure of lipid A, isolated by mild acid hydrolysis from a rough mutant and a wild strain of Aeromonas salmonicida lipopolysaccharide, was investigated using electrospray ionization quadrupole time-of-flight (QqToF) hybrid tandem mass spectrometry and showed a great degree of microheterogeneity. The chemical structure of the main constituent of this heterogeneous mixture was identified as a β-D-(1→6) linked D-glucosamine disaccharide substituted by two phosphate groups, one being bound to the non-reducing end at position O-4' and the other to the position O-1 of the reducing end of the D-glucosamine disaccharide. The location of the fatty acids linked to the disaccharide backbone was established by identifying diagnostic ions in the conventional QqToF-MS scan. Low-energy collision tandem mass spectrometry analysis of the selected precursor diagnostic ions confirmed, unambiguously, their proposed molecular structures. We have established that myristyloxylauric (C14:0(3-O(12:0))) acid residues were both N-2' and O-3' linked to the non-reducing end of the D-GlcN residue, and that two 3-hydroxymyristic (C14:0(3-OH)) acid chains acylated the remaining positions of the reducing end. The MS and MS/MS data obtained allowed us to determine the complex molecular structure of lipid A. The QqToF-MS/MS instrument has shown excellent superiority over a conventional quadrupole-hexapole-quadrupole tandem instrument which failed to fragment the selected precursor ion.
Lipopolysaccharide, lipid A, Aeromonas salmonicida, tandem mass spectrometry
NCBI PubMed ID: 15912470Publication DOI: 10.1002/rcm.1971Journal NLM ID: 8802365Publisher: John Wiley And Sons Ltd
Correspondence: banoubjo@dfo-mpo.gc.ca
Institutions: Memorial University of Newfoundland, Biochemistry Department, St. John's, NL, A1B 3X9, Canada, Department of Fisheries and Oceans, Science Branch, Special Projects, P.O. Box 5667, St. John’s, NL, A1B 3X9, Canada
Methods: GC-MS, ESI-MS, GC, ESI-QTOF-MS/MS
Found