title: Ion-pairing liquid chromatography-tandem mass spectrometry-based quantification of uridine diphosphate-linked intermediates in the Staphylococcus aureus cell wall biosynthesis pathway.
abstract: Bacterial cell wall biosynthesis is the target of several antibiotics and is of interest as a target for new inhibitor development. The cytoplasmic steps of this pathway involve a series of uridine diphosphate (UDP)-linked peptidoglycan intermediates. Quantification of these intermediates is essential for studies of current agents targeting this pathway and for the development of new agents targeting this pathway. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for quantification of these intermediates in Staphylococcus aureus. To address the problem of poor retention of UDP-linked intermediates on reverse phase media, an ion-pairing (IP) approach using N,N-dimethylhexylamine was developed. MS/MS detection in negative mode was optimized for UDP-GlcNAc, UDP-MurNAc, UDP-MurNAc-L-Ala, UDP-MurNAc-L-Ala-D-Glu, UDP-MurNAc-L-Ala-D-Glu-L-Lys, and UDP-MurNAc-L-Ala-D-Glu-L-Lys-D-Ala-D-Ala. The lower limits of quantification (LLOQs) for these analytes were 1.8, 1.0, 0.8, 2.2, 0.6, and 0.5 pmol, respectively, which correspond to LLOQs of 6, 3, 3, 7, 2, and 2 nmol/g bacteria, respectively. This method was demonstrated for quantification of in vivo levels of these intermediates from S. aureus (0.3mg dry weight analyzed) treated with fosfomycin, D-boroAla, D-cycloserine, and vancomycin. Metabolite accumulation is consistent with the known targets of these antibiotics and indicates potential regulatory loops within this pathway.
authors.affiliation: Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, Kansas City, MO 64108, USA.
authors.forename: Harika
authors.initials: H
authors.lastname: Vemula
authors.affiliation: Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, Kansas City, MO 64108, USA.
authors.forename: Sudheer
authors.initials: S
authors.lastname: Bobba
authors.affiliation: Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, Kansas City, MO 64108, USA.
authors.forename: Sandeep
authors.initials: S
authors.lastname: Putty
authors.affiliation: XenoTech, Lenexa, KS 66219, USA.
authors.forename: Joanna E
authors.initials: JE
authors.lastname: Barbara
authors.affiliation: Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, Kansas City, MO 64108, USA. Electronic address: gutheilw@umkc.edu.
authors.forename: William G
authors.initials: WG
authors.lastname: Gutheil
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002473:Cell Wall / Q000737:chemistry / Q000378:metabolism*; D002853:Chromatography, Liquid; D013058:Mass Spectrometry; D013211:Staphylococcus aureus / Q000737:chemistry / Q000378:metabolism*; D014539:Uridine Diphosphate Sugars / Q000032:analysis / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D014539:Uridine Diphosphate Sugars
keywords: Antibacterial antibiotic; Cell wall; Ion-pairing chromatography; Peptidoglycan; Staphylococcus aureus; Uridine diphosphate-linked intermediates