biostudies-literatureOkahashi NJapan Agency for Medical Research and DevelopmentJapan Science and Technology AgencyJapan Society for the Promotion of Science197https://www.ebi.ac.uk/biostudies/studies/S-EPMC8065654biostudies-literatureUnknown11(4)Lipid A is a characteristic molecule of Gram-negative bacteria that elicits an immune response in mammalian cells. The presence of structurally diverse lipid A types in the human gut bacteria has been suggested before, and this appears associated with the immune response. However, lipid A structures and their quantitative heterogeneity have not been well characterized. In this study, a method of analysis for lipid A using liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF/MS) was developed and applied to the analyses of <i>Escherichia coli</i> and Bacteroidetes strains. In general, phosphate compounds adsorb on stainless-steel piping and cause peak tailing, but the use of an ammonia-containing alkaline solvent produced sharp lipid A peaks with high sensitivity. The method was applied to <i>E. coli</i> strains, and revealed the accumulation of lipid A with abnormal acyl side chains in knockout strains as well as known diphosphoryl hexa-acylated lipid A in a wild-type strain. The analysis of nine representative strains of Bacteroidetes showed the presence of monophosphoryl penta-acylated lipid A characterized by a highly heterogeneous main acyl chain length. Comparison of the structures and amounts of lipid A among the strains suggested a relationship between lipid A profiles and the phylogenetic classification of the strains.MetabolitesAnalyses of Lipid A Diversity in Gram-Negative Intestinal Bacteria Using Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry.PMC8065654JP18gm0010003JPMJAX20B215H05897, 15H05898Ueda MMatsuda FArita MOkahashi NfalseAnalyses of Lipid A Diversity in Gram-Negative Intestinal Bacteria Using Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry.Lipid A is a characteristic molecule of Gram-negative bacteria that elicits an immune response in mammalian cells. The presence of structurally diverse lipid A types in the human gut bacteria has been suggested before, and this appears associated with the immune response. However, lipid A structures and their quantitative heterogeneity have not been well characterized. In this study, a method of analysis for lipid A using liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF/MS) was developed and applied to the analyses of <i>Escherichia coli</i> and Bacteroidetes strains. In general, phosphate compounds adsorb on stainless-steel piping and cause peak tailing, but the use of an ammonia-containing alkaline solvent produced sharp lipid A peaks with high sensitivity. The method was applied to <i>E. coli</i> strains, and revealed the accumulation of lipid A with abnormal acyl side chains in knockout strains as well as known diphosphoryl hexa-acylated lipid A in a wild-type strain. The analysis of nine representative strains of Bacteroidetes showed the presence of monophosphoryl penta-acylated lipid A characterized by a highly heterogeneous main acyl chain length. Comparison of the structures and amounts of lipid A among the strains suggested a relationship between lipid A profiles and the phylogenetic classification of the strains.2021-01-01T00:00:00Z2021 Mar2026-05-09T09:48:06.692Z2025-04-03T21:31:27.555ZS-EPMC80656543381039210.3390/metabo11040197