title: Characterization of complex, heterogeneous lipid A samples using HPLC-MS/MS technique I. Overall analysis with respect to acylation, phosphorylation and isobaric distribution.
abstract: We established a new reversed phase-high performance liquid chromatography method combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry for the simultaneous determination and structural characterization of different lipid A types in bacteria (Escherichia coli O111, Salmonella adelaide O35 and Proteus morganii O34) showing serological cross-reactivity. The complex lipid A mixtures (obtained by simple extraction and acid hydrolysis of the outer membrane lipopolysaccharides) were separated and detected without phosphate derivatization. Several previously unidentified ions were detected, which differed in the number and type of acyl chains and number of phosphate groups. In several cases, we observed the different retention of isobaric lipid A species, which had different secondary fatty acyl distribution at the C2' or the C3' sites. The fragmentation of the various, C4' monophosphorylated lipid A species in deprotonated forms provided structural assignment for each component. Fragmentation pathways of the tri-acylated, tetra-acylated, penta-acylated, hexa-acylated and hepta-acylated lipid A components and of the lipid A partial structures are suggested. As standards, the hexa-acylated ion at m/z 1716 with the E. coli-type acyl distribution and the hepta-acylated ion at m/z 1954 with the Salmonella-type acyl distribution were used. The results confirmed the presence of multiple forms of lipid A in all strains analyzed. In addition, the negative-ion mode MS permitted efficient detection for non-phosphorylated lipid A components, too. Copyright © 2016 John Wiley & Sons, Ltd.
authors: Institute of Bioanalysis and Szentágothai Research Centre, University of Pécs, Szigeti út 12, 7624, Pécs, Hungary. sandor.viktor@pte.hu.
authors: Viktor
authors: V
authors: Sándor
authors: Department of Analytical and Environmental Chemistry, Faculty of Sciences, University of Pécs, Ifjúság útja 6, 7624, Pécs, Hungary.
authors: Ágnes
authors: http://orcid.org/0000-0002-6552-8522
authors: Á
authors: Dörnyei
authors: Institute of Bioanalysis and Szentágothai Research Centre, University of Pécs, Szigeti út 12, 7624, Pécs, Hungary.
authors: Lilla
authors: L
authors: Makszin
authors: Institute of Bioanalysis and Szentágothai Research Centre, University of Pécs, Szigeti út 12, 7624, Pécs, Hungary.|Department of Analytical and Environmental Chemistry, Faculty of Sciences, University of Pécs, Ifjúság útja 6, 7624, Pécs, Hungary.
authors: Ferenc
authors: F
authors: Kilár
authors: Department of Medical Microbiology and Immunology, Faculty of Medicine, University of Pécs, Szigeti út 12, 7624, Pécs, Hungary.
authors: Zoltán
authors: Z
authors: Péterfi
authors: Department of Medical Microbiology and Immunology, Faculty of Medicine, University of Pécs, Szigeti út 12, 7624, Pécs, Hungary.
authors: Béla
authors: B
authors: Kocsis
authors: MTA-PTE Molecular Interactions in Separation Science Research Group, Ifjúság útja 6, 7624, Pécs, Hungary.
authors: Anikó
authors: A
authors: Kilár
mesh_terms: D000215:Acylation; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004926:Escherichia coli / Q000737:chemistry; D006868:Hydrolysis; D008050:Lipid A / Q000737:chemistry*; D010766:Phosphorylation; D059748:Proteolysis; D011511:Proteus / Q000737:chemistry; D012475:Salmonella / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D008050:Lipid A
keywords: Escherichia coli; HPLC-MS/MS; Proteus morganii; Salmonella adelaide; fragmentation pathway; lipid A