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

Bibliographical note

Funding Information:
The authors thank the NIAID ( 1U54 AI57141 ) and NIEHS ( 5P30ES007033-10 ) for funding and support. Additional thanks are due to the Mass Spectrometry Facilities, located at the University of Washington School of Pharmacy, and the Proteomics Resource of the University of Washington School of Medicine (UWPR95794).

Copyright:
Copyright 2010 Elsevier B.V., All rights reserved.

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