title: Comprehensive structure characterization of lipid A extracted from Yersinia pestis for determination of its phosphorylation configuration.
abstract: We report on comprehensive structure characterization of lipid A extracted from Yersinia pestis (Yp) for determination of its phosphorylation configuration that was achieved by combining the methods of molecular biology with high-resolution tandem mass spectrometry. The phosphorylation pattern of diphosphorylated lipid A extracted from Yp has recently been found to be a heterogeneous mixture of C-1 and C-4' bisphosphate, C-1 pyrophosphate, and C-4' pyrophosphate (Proc. Natl. Acad. Sci. 2008, 105, 12742). To reduce the inherent phosphate heterogeneity of diphosphorylated lipid A extracted from Yp, we incorporated specific C-1 and C-4' position phosphatases into wild type KIM6+ Yp grown at 37 degrees C. Comprehensive high-resolution tandem mass spectrometric analyses of lipid A extracted from Yp modified with either the C-1 or C-4' phosphatase allowed for unambiguous structure assignment of monophosphorylated and diphosphorylated lipid A and distinction of isomeric bisphosphate and pyrophosphate forms. The prevalent aminoarabinose modification was determined to be exclusively attached to the lipid A disaccharide via a phospho-diester linkage at either or both the C-1 and C-4' positions.
authors.affiliation: Department of Medicinal Chemistry, University of Washington, Seattle, Washington, USA.
authors.forename: Jace W
authors.initials: JW
authors.lastname: Jones
authors.forename: Ilana E
authors.initials: IE
authors.lastname: Cohen
authors.forename: Frantisek
authors.initials: F
authors.lastname: Tureĉek
authors.forename: David R
authors.initials: DR
authors.lastname: Goodlett
authors.forename: Robert K
authors.initials: RK
authors.lastname: Ernst
mesh_terms: D011756:Diphosphates / Q000737:chemistry; D008050:Lipid A / Q000737:chemistry* / Q000302:isolation & purification; D008958:Models, Molecular; D008968:Molecular Conformation; D010710:Phosphates / Q000737:chemistry*; D010766:Phosphorylation; D053719:Tandem Mass Spectrometry / Q000379:methods*; D015010:Yersinia pestis / Q000737:chemistry*
chemical_list: D011756:Diphosphates; D008050:Lipid A; D010710:Phosphates