title: Tandem mass spectrometry determination of the putative structure of a heterogeneous mixture of Lipid As isolated from the lipopolysaccharide of the Gram-negative bacteria Aeromonas liquefaciens SJ-19a.
abstract: BACKGROUND
  We report herein the electrospray ionization mass spectrometry (ESI-MS) and low-energy collision-induced dissociation tandem mass spectrometry analysis (CID-MS/MS) of a mixture of lipid As isolated from the rough lipopolysaccharide (LPS) of the mutant wild strain of the Gram-negative bacteria Aeromonas liquefaciens (SJ-19a, resistant) grown in the presence of phages. The interaction between the phages and the Gram-negative bacteria regulates host specificity and the heterogeneity of the lipid A component of the LPS.
  
  
  METHODS
  The heterogeneous mixture of lipid As was isolated by the aqueous phenol method from the LPS of the rough wild strain of Gram-negative bacteria Aeromonas liquefaciens (SJ-19a). Hydrolysis of the LPS was with 1% acetic acid, and purification was by chromatography using Sephadex G-50 and Sephadex G-15. ESI-MS and low-energy CID-MS/MS analyses were performed with a triple-quadrupole (QqQ) and a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer.
  
  
  RESULTS
  Preliminary analysis of the lipid As mixture was conducted by ESI-MS in the negative ion mode and the spectrum obtained suggested that the lipid A SJ-19a was composed of a heterogeneous mixture of different lipid A molecules. CID-MS/MS experiments confirmed the identities of the various mono-phosphorylated β-D-GlcpN-(1→6)-α-D-GlcpN disaccharide entities. This lipid As mixture was asymmetrically substituted with fatty acids such as ((R)-14:0(3-OH)), (14:0(3-(R)-(O-12:0)) and (14:0(3-(R)-O-(14:0)) located on the O-3, O-3', N-2 and N-2' positions, respectively.
  
  
  CONCLUSIONS
  Low-energy collision-induced dissociation tandem mass spectrometry in-space (QqQ-MS/MS) and in-time (FTICR-MS/MS) allowed the exact determination of the fatty acid acylation positions on the H2 PO3 →4-O'-β-D-GlcpN-(1→6)-α-D-GlcpN disaccharide backbones of this heterogeneous mixture of lipid As , composed inter alia of seven different substituted lipid As , formed from the incomplete biosynthesis of their respective LPS.
authors.affiliation: Chemistry Department, Memorial University of Newfoundland, St. John's, Newfoundland, Canada.
authors.forename: Mervt
authors.initials: M
authors.lastname: Almostafa
authors.affiliation: Chemistry Department, Memorial University of Newfoundland, St. John's, Newfoundland, Canada.
authors.forename: Bashaeer
authors.initials: B
authors.lastname: Allehyane
authors.affiliation: Chemistry Department, Memorial University of Newfoundland, St. John's, Newfoundland, Canada.
authors.forename: Stefana
authors.initials: S
authors.lastname: Egli
authors.affiliation: Chemistry Department, Memorial University of Newfoundland, St. John's, Newfoundland, Canada.
authors.forename: Christina
authors.initials: C
authors.lastname: Bottaro
authors.affiliation: Chemistry Department, Memorial University of Newfoundland, St. John's, Newfoundland, Canada.
authors.forename: Travis D
authors.initials: TD
authors.lastname: Fridgen
authors.affiliation: Chemistry Department, Memorial University of Newfoundland, St. John's, Newfoundland, Canada.|Special Projects, Science Branch, Department of Fisheries and Oceans Canada, St. John's, Newfoundland, Canada.
authors.forename: Joseph
authors.initials: J
authors.lastname: Banoub
mesh_terms: D000333:Aeromonas / Q000737:chemistry*; D008050:Lipid A / Q000032:analysis* / Q000737:chemistry*; D008070:Lipopolysaccharides / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D008050:Lipid A; D008070:Lipopolysaccharides