Metabolic Quenching and Growth Medium Shape Strain-Level Interpretation in Untargeted Escherichia coli Lipidomics

Abstract Untargeted bacterial lipidomics is increasingly used to investigate membrane adaptation, host–pathogen interactions, antimicrobial stress responses, and microbial phenotypic variation, but the measured lipidome depends strongly on experimental design. Preanalytical variables can alter lipid recovery, introduce handling-derived artifacts, and change biological interpretation. Here, we evaluated how methanol-based quenching, growth medium, and strain background affect LC-MS/MS untargeted Escherichia coli lipidomics. Three strains, ATCC 25922, O127:H6, and O157:H7, were grown in chemically defined M9 medium or tryptic soy broth to late stationary phase. Lipids were extracted from biomass-normalized bacterial pellets using dichloromethane and analyzed by reversed-phase LC–MS/MS. Methanol-based quenching did not improve reproducibility and altered the abundance of metabolically active lipid species, such as lysophospholipids and quinones. Growth medium had greater effects than strain identity, altering phospholipid balance and the detectability of strain-specific lipid phenotypes. Under controlled handling and culture conditions, pathogenic strains exhibited distinct lipidomic profiles, including increased cardiolipins and phosphatidic acids, elevated lysophospholipid ratios, and reduced diacylglycerol levels. These results define practical constraints for bacterial lipidomics workflows and show that strain-level lipid phenotypes require control of quenching strategy and growth medium.