Original Paper

Analytical and Bioanalytical Chemistry

, Volume 387, Issue 2, pp 497-511

First online:

Comprehensive profiling of N-acylhomoserine lactones produced by Yersinia pseudotuberculosis using liquid chromatography coupled to hybrid quadrupole–linear ion trap mass spectrometry

  • Catharine A. OrtoriAffiliated withCentre for Analytical Bioscience, School of Pharmacy, University of Nottingham
  • , Steve AtkinsonAffiliated withInstitute of Infection, Immunity and Inflammation, Centre for Biomolecular Sciences, University of Nottingham
  • , Siri Ram ChhabraAffiliated withInstitute of Infection, Immunity and Inflammation, Centre for Biomolecular Sciences, University of Nottingham
  • , Miguel CámaraAffiliated withInstitute of Infection, Immunity and Inflammation, Centre for Biomolecular Sciences, University of Nottingham
  • , Paul WilliamsAffiliated withInstitute of Infection, Immunity and Inflammation, Centre for Biomolecular Sciences, University of Nottingham
  • , David A. BarrettAffiliated withCentre for Analytical Bioscience, School of Pharmacy, University of Nottingham Email author 

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Abstract

A method for the comprehensive profiling of the N-acylhomoserine lactone (AHL) family of bacterial quorum-sensing molecules is presented using liquid chromatography (LC) coupled to hybrid quadrupole–linear ion trap (QqQLIT) mass spectrometry. Information-dependent acquisition (IDA), using triggered combinations of triple-quadrupole and linear ion trap modes in the same LC-MS/MS run, was used to simultaneously screen, quantify and identify multiple AHLs in a single sample. This MS method uses common AHL fragment ions attributed to the homoserine moiety and the 3-oxo-, 3-hydroxy- or unsubstituted acyl side chains, to identify unknown AHLs in cell-free culture supernatants in an unbiased manner. This LC-MS technique was applied to determine the relative molar ratios of AHLs produced by Yersinia pseudotuberculosis and the consequences of inactivating by mutation either or both of the AHL synthase genes (ypsI and ytbI) on AHL profile and concentration. The Y. pseudotuberculosis wild type but not the ypsI ytbI double mutant produced at least 24 different AHLs with acyl chains ranging from C4 to C15 with or without 3-oxo or 3-hydroxy substituents. YtbI, in contrast to YpsI, could direct the synthesis of all of the AHLs identified. The most abundant and hence most biologically relevant Y. pseudotuberculosis AHLs were found to be the 3-oxo-substituted C6, C7 and C8 AHLs and the unsubstituted C6 and C8 compounds. The LC-QqQLIT methodology is broadly applicable to quorum-sensing signal molecule analysis and can provide comprehensive AHL profiles and concentrations from a single sample and simultaneously collect confirmatory spectra for each AHL identified.

Keywords

Yersinia Quorum sensing N-Acylhomoserine lactones LC-MS Quadrupole linear ion trap