Comprehensive structure characterization of lipid A extracted from Yersinia pestis for determination of its phosphorylation configuration.
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.
Jones
Jace W
JW
Department of Medicinal Chemistry, University of Washington, Seattle, Washington, USA.
Cohen
Ilana E
IE
Tureĉek
Frantisek
F
Goodlett
David R
DR
Ernst
Robert K
RK
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*
D011756:Diphosphates; D008050:Lipid A; D010710:Phosphates