Micromethods for Lipid A Isolation and Structural Characterization
Lipopolysaccharides (LPSs) are major components of the external membrane of Gram-negative bacteria, and act as an effective permeability barrier. They are essentially composed of a hydrophilic polysaccharide region linked to an hydrophobic one, termed lipid A. Depending on their individual variable fine structures, they may be potent immunomodulators. Because of the structural importance and role of lipid A in bacterial pathogenesis, herein we describe two rapid practical micromethods for structural analysis. The first method allows the direct isolation of lipid A from whole bacteria cell mass; the second describes conditions for the sequential release of fatty acids, enabling the determination of their substitution position in the lipid A structure to be determined by matrix-assisted laser desorption/ionization mass spectrometry. Examples are given with reference to two major pathogens: Bordetella pertussis and Pseudomonas aeruginosa.
Key wordsGas chromatography 3-Deoxy-d-manno-oct-2-ulosonic acid Lipooligosaccharide Lipopolysaccharide Matrix-assisted laser desorption/ionization mass spectrometry Tumor necrosis factor Sodium dodecyl sulfate
Alexey Novikov is a recipient of a young researcher fellowship from INSERM (France). Part of this work was supported by the CNRS Group of Research GDR3048. This chapter is dedicated to Dr. Malcolm B. Perry (Ottawa, Canada) on his 80th birthday.
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