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Papers In Press, published online ahead of print September 1, 2009
J. Lipid Res., doi:10.1194/jlr.D900013-JLR200

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Journal of Lipid Research, Vol. 50, 1936-1944, September 2009
Copyright © 2009 by American Society for Biochemistry and Molecular Biology

Methods

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

Ping Zhou^*,, Vandana Chandan^*, Xin Liu^*, Kenneth Chan^*, Eleonora Altman^* and Jianjun Li^1,*

^* Institute for Biological Sciences, National Research Council Canada, Ottawa, Ontario, Canada
College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, Peoples' Republic of China

¹ To whom correspondence should be addressed. e-mail: Jianjun.Li{at}nrc-cnrc.gc.ca

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 µg. 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.

Supplementary key words Helicobacter pylori • acylation • microwave-assisted digestion • mass spectrometry • MALDI-TOF/TOF

Abbreviations: CMBT, 5-chloro-2-mercaptobenzothiazole; LPS, lipopolysaccharide; PEtn, phosphoethanolamine; TOF, time-of-flight

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