Microwave-assisted sample preparation for rapid and sensitive analysis of H. pylori lipid A applicable to a single colony

Ping Zhou,Vandana Chandan,Xin Liu,Kenneth Chan,Eleonora Altman,Jianjun Li

doi:10.1194/jlr.d900013-jlr200

Abstract

The lipid A of Gram-negative bacteria plays a major role in the pathogenesis of bacterial infections. Lipid A diversity is observed both in the number and length of fatty-acid side chains and in the presence of terminal phosphate residues and associated modifications. In this report, we describe a new sample preparation method based on microwave-assisted enzymatic digestion and detergent-free mild hydrolysis, in conjunction with a MALDI-time-of-flight (TOF)/TOF analysis, to determine the structures of lipid A from Helicobacter pylori. The total time for sample preparation and mass spectrometric analysis is within 2 h and applicable to profiling the lipid A structures from dried bacterial cells on as little as 1 microg. The reliability of the technique was further demonstrated through the analysis of the lipid A from bacterial cells of different H. pylori strains. The phosphorylation and acylation patterns of lipid A could be elucidated using material from a single colony. Furthermore, we found unusual heptaacyl lipid A species present in H. pylori mutant that have not been previously reported, although the abundance was relatively low. The present study provides the first characterization of the lipid A component from a single bacterial colony sample by mass spectrometry.

Highlights

The lipid A of Gram-negative bacteria plays a major role in the pathogenesis of bacterial infections
We first prepared the H. pylori lipid A using this method and the MALDITOF mass spectra obtained from 100 ␮g and 10 ␮g of dried cells are presented in Fig. 1A and C
The unusual structure of H. pylori lipid A is believed to be responsible for its low endotoxicity as compared with lipid A of Salmonella and Escherichia coli [12]

Summary

Introduction

The lipid A of Gram-negative bacteria plays a major role in the pathogenesis of bacterial infections. In an effort to study the relationship between the lipid A structure and the role of H. pylori LPS in pathogenesis, we developed a fast and sensitive analytical method to analyze lipid A based on microwave-assisted enzymatic digestion and mild acid hydrolysis. The isolation of lipid A directly from bacterial cells has been developed by using an isobutyric acid-ammonium hydroxide hydrolysis procedure [17, 19].

Results

Conclusion