Open AccessTargeted lipopolysaccharide biosynthetic intermediate analysis with normal-phase liquid chromatography mass spectrometry
Author(s) -
William S. Sawyer,
Lisha Wang,
Tsuyoshi Uehara,
Pramila Tamrakar,
Ramadevi Prathapam,
Mina Mostafavi,
Louis E. Metzger,
Brian Y. Feng,
Christopher M. Rath
Publication year - 2019
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0211803
Subject(s) - lipopolysaccharide , escherichia coli , chemistry , periplasmic space , bacterial outer membrane , biochemistry , diacylglycerol kinase , bacteria , chromatography , enzyme , lysis , biology , gene , genetics , protein kinase c , endocrinology
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.