Comprehensive Structure Characterization of Lipid A Extracted from Yersinia pestis for Determination of its Phosphorylation Configuration

Jace W. Jones, Ilana E. Cohen, Frantiek Tureek, David R. Goodlett, Robert K. Ernst

Research output: Contribution to journalArticlepeer-review

21 Scopus citations

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. 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.

Original languageEnglish (US)
Pages (from-to)785-799
Number of pages15
JournalJournal of the American Society for Mass Spectrometry
Volume21
Issue number5
DOIs
StatePublished - May 2010

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