Open AccessComprehensive structure characterization of lipid a extracted fromYersinia pestisfor determination of its phosphorylation configuration
Author(s) -
Jace W. Jones,
Ilana E. Cohen,
František Tureček,
David R. Goodlett,
Robert K. Ernst
Publication year - 2010
Publication title -
journal of the american society for mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.961
H-Index - 127
eISSN - 1879-1123
pISSN - 1044-0305
DOI - 10.1016/j.jasms.2010.01.008
Subject(s) - chemistry , yersinia pestis , characterization (materials science) , phosphorylation , computational biology , biochemistry , chromatography , stereochemistry , nanotechnology , virulence , biology , materials science , gene
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.