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

Sawyer, William and Wang, Lisha and Uehara, Tsuyoshi and Tamrakar, Pramila and Prathapam, Ramadevi and Mostafavi, Mina and Richie, Daryl and Metzger, Louis and Feng, Brian and Rath, Christopher (2018) Targeted lipopolysaccharide biosynthetic intermediate analysis with normal-phase liquid chromatography mass spectrometry. PLoS ONE.

Abstract

In this study, we established a method for monitoring the lipopolysacharride (LPS) biosynthetic intermediates of the Raetz pathway (lpxA-lpxK) in Gram-negative bacteria. LPS forms the outer leaflet of the outer membrane in Gram-negative bacteria and contributes to the permeability barrier and immune response. 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) LCMS/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 method, we were able to reliably monitor changes to levels of intermediates in this pathway in response to compound-treatment and genetic modification. This capability allows for probing a key essential pathway in Gram-negative bacteria in an effort to better understand its role as a permeability barrier to antibiotics.

Item Type: Article
Date Deposited: 12 Sep 2018 00:45
Last Modified: 12 Sep 2018 00:45
URI: https://oak.novartis.com/id/eprint/36126

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