Targeted lipopolysaccharide biosynthetic intermediate analysis with normal-phase liquid chromatography mass spectrometry

• DOI: 10.1371/journal.pone.0211803
• Publication Date: 2019-02-08T18:31:23Z
• Authors: William S. Sawyer; Lisha Wang; Tsuyoshi Uehara; Pramila Tamrakar; Ramadevi Prathapam; Mina Mostafavi; Louis E. Metzger; Brian Feng; Christopher M. Ba...
• Keywords: Lipopolysaccharides; 0301 basic medicine; Science; Q; R; 3. Good health; 03 medical and health sciences; Periplasm; Escherichia coli; Medicine; Acyltransferases; Research Article

Lipopolysacharride (LPS) forms the outer leaflet of membrane in Gram-negative bacteria and contributes to permeability barrier immune response. In this study, we established a method for monitoring LPS biosynthetic intermediates Raetz pathway (lpxA-lpxK) Escherichia coli. Metabolites from compound-treated cells genetically-perturbed were extracted whole concentrated by mixed-mode weak anion exchange (WAX) solid-phase extraction (SPE) prior analysis normal phase (NP)LC-MS/MS. Data was normalized cell density an internal standard comparison against untreated order determine fold accumulation depletion affected metabolites. Using LC-MS/MS method, able reliably monitor changes levels response compound-treatment genetic modification. addition, found that deletion periplasmic CDP-diacylglycerol pyrophosphatase dramatically increased UDP-containing intermediates, suggesting enzymatic breakdown during sample preparation. This assay allows probing key essential effort discover antibacterial agents inhibit enzymes pathway.