Analysis of Fatty Acid Binding Positions Lipid A Molecules by Periodate Oxidation and Mass Spectrometry
Keywords:
Lipid A, Fatty acid binding positions, Mass spectrometry, Periodate oxidation, Escherichia coliAbstract
The immunological activity of LPS is closely related to the localization of fatty acids and the acyloxyacyl structures formed by them in lipid A molecules. MALDI-TOF mass spectrometry (MS) is commonly used to determine fatty acid localization by detecting oxonium ions derived from non-reducing end glucosamine by the split of glucosamine disaccharide of lipid A. However, an alternative method is required because the sensitivity in MS is reduced when the purity of lipid A preparation is not enough. To solve this problem, we developed the method of periodate oxidation. The glucosamine disaccharide of lipid A was split by sodium periodate after reduction with NaBH4 and the hemiacetal ring of reducing end glucosamine was opened. Through this oxidation, non-reducing end glucosamine with fatty acids bound to it remained and could be detected by MALDI-TOF MS. This method was applied to the wild-type lipid A, and the modified lipid A of Escherichia coli strains KGU0485 and KGU0496 constructed in the previous study by the introduction of Klebsiella acyltransferase gene. The results indicated that the method was useful for the determination of fatty acid localization in lipid A molecules.
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