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Quantitative lc‐ms/ms analysis of cytoplasmic bacterial cell wall biosynthesis intermediates: application to vancomycin resistance VRE (982.2)
Author(s) -
Vemula Harika,
Putty Sandeep,
Bobba Sudheer,
Gutheil William
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.982.2
Subject(s) - derivatization , chemistry , peptidoglycan , biosynthesis , glycopeptide , vancomycin , metabolite , biochemistry , chromatography , high performance liquid chromatography , bacteria , cell wall , antibiotics , biology , gene , genetics , staphylococcus aureus
Vancomycin resistance in vancomycin‐resistant enterococci (VRE) is due to an alternative cell wall biosynthesis pathway in which D‐Ala‐D‐Ala is replaced, most commonly by D‐Ala‐D‐Lac. Methods for quantitative analysis of these intermediates has been lacking, which has impeded progress in characterizing the details of this resistance, and in developing new agent to counter this resistance. Methods: LC‐MS/MS assays for both the alanine branch and the UDP‐linked cytoplasmic peptidoglycan intermediates have been developed. Alanine branch intermediates used Marfey’s reagent derivatization to enable analysis, and analysis of the UDP‐linked intermediates used an ion‐pairing approach. For the development of the UDP‐linked intermediate assays, these intermediate were purified from VRE using preparative ion‐ pairing HPLC. To demonstrate this assay, VRE was grown to log phase and treated with increasing concentrations of vancomycin. Results: Analysis of alanine branch and UDP‐linked intermediates demonstrates the utility of LC‐MS/MS‐based methods for the study of the of the metabolite level changes in VRE in response to vancomycin.