title: Application of capillary electrophoresis- electrospray-mass spectrometry to the separation and characterization of isomeric lipopolysaccharides of Neisseria meningitidis.
abstract: A capillary electrophoresis-electrospray-mass spectrometry technique for the characterization of lipopolysaccharides (LPSs) was developed, permitting the separation of trace-level O-deacylated LPS isoforms for subsequent structural characterization using tandem mass spectrometry (MS/MS). The separation buffer and electrospray interface were optimized first using O-deacylated LPS samples from large-scale preparations. It was found that with microelectrospray or sheath-solution interface, we could separate LPS in anionic forms and detect them using either negative or positive ion mode MS. For negative ion detection mode MS, 30 mM morpholine with addition of 5% v/v methanol was employed as separation buffer. When positive ion detection mode MS was required, 10 mM ammonium acetate with addition of 5% methanol was used as separation buffer. The structural assignments obtained from MS/MS and capillary zone electrophoresis-electrospray-MS (CZE-ESMS) analyses enabled the identification of isomeric glycoforms. Application of this technique to the analysis of LPS from the galE mutants of Neisseria meningitidis strain BZ157 B5+ revealed the presence of isomeric glycoforms, in which the location of a functional group phosphoethanolamine was found to be in either inner core or lipid A-OH regions. The described technique was also applied to the analysis of LPS samples from the galE mutant of N. meningitidis strains F1576 A4+ and A4-. The occurrence of isomeric LPS glycoforms differing by the location or presence of neutral sugar residues, such as hexoses, can also be characterized using MS/MS.
authors.affiliation: Institute for Biological Sciences, National Research Council, Ottawa, ON, Canada.
authors.forename: Jianjun
authors.initials: J
authors.lastname: Li
authors.affiliation: Institute for Biological Sciences, National Research Council, Ottawa, ON, Canada.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Cox
authors.affiliation: Institute for Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK.
authors.forename: Derek
authors.initials: D
authors.lastname: Hood
authors.affiliation: Institute for Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK.
authors.forename: E Richard
authors.initials: ER
authors.lastname: Moxon
authors.affiliation: Institute for Biological Sciences, National Research Council, Ottawa, ON, Canada.
authors.forename: James C
authors.initials: JC
authors.lastname: Richards
mesh_terms: D000107:Acetylation; D002021:Buffers; D019075:Electrophoresis, Capillary / Q000379:methods*; D006601:Hexoses / Q000032:analysis / Q000737:chemistry; D007536:Isomerism; D008070:Lipopolysaccharides / Q000737:chemistry / Q000302:isolation & purification*; D013058:Mass Spectrometry / Q000379:methods*; D008832:Microchemistry; D009154:Mutation; D009345:Neisseria meningitidis / Q000737:chemistry* / Q000235:genetics
chemical_list: D002021:Buffers; D006601:Hexoses; D008070:Lipopolysaccharides