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

• Published in: PloS one Vol. 14; no. 2; p. e0211803
• Main Authors: Sawyer, William S, Wang, Lisha, Uehara, Tsuyoshi, Tamrakar, Pramila, Prathapam, Ramadevi, Mostafavi, Mina, Metzger, 4th, Louis E, Feng, Brian, Baxter Rath, Christopher M
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 08.02.2019; Public Library of Science (PLoS)
• Subjects: Acyltransferases - genetics; Anion exchange; Anion exchanging; Antibacterial agents; Antibiotics; Bacteria; Biology and Life Sciences; Biomaterials; Biomedical research; Biosynthesis; CDP-diacylglycerol; Cell density; Chemistry; Chromatography; Clonal deletion; Depletion; Diglycerides; Drug resistance; E coli; Enzymes; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Genetic modification; Genetically modified organisms; Gram-negative bacteria; Immune response; Immune system; Infectious diseases; Intermediates; Laboratories; Lipids; Lipopolysaccharides; Lipopolysaccharides - biosynthesis; Lipopolysaccharides - genetics; Liquid chromatography; Mass spectrometry; Mass spectroscopy; Medicine and Health Sciences; Membrane permeability; Metabolites; Mitogens; Periplasm - genetics; Periplasm - metabolism; Permeability; Physiological aspects; Pyrophosphatase; Research and Analysis Methods; Sample preparation; Scientific imaging; Solid phases; Spectroscopy; United States > US
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0211803

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