Detection and Quantitation of Bacterial Acylhomoserine Lactone Quorum Sensing Molecules via Liquid Chromatography–Isotope Dilution Tandem Mass Spectrometry

Abstract

A range of acylhomoserine lactones (AHLs) are used as intraspecies quorum sensing signals by Gram-negative bacteria, and the detection and quantitation of these molecules is of interest. This manuscript reports a liquid chromatographic-isotope dilution tandem mass spectrometric method for the quantitation of these molecules. A divergent solid-phase synthesis of stable-isotope-labeled AHLs suitable for use as an internal standard is reported. This route relies on the biomimetic conversion of a dideuterated methionine equivalent, N-Fmoc-(4,4-(2)H(2))methionine, to the desired labeled AHL, and a representative series of eight of these molecules was produced in >95% purity and yields up to ~50%. The representative AHL internal standards were then used to develop an optimized liquid chromatography-tandem mass spectrometric (LC-MS/MS) separation and detection protocol for these molecules, which relies on a high-efficiency C18 core-shell column to minimize the time necessary for separation. The addition of internal standards at different steps during sampling was also found to affect the analysis for hydrophobic AHLs with addition prior to cell removal giving the most accurate results. Taken together, the use of the internal standards and separation method reported herein provides a rapid and quantitative method for the study of AHL production in bacteria.

Phaeobacter sp. Strain Y4I Utilizes Two Separate Cell-to-Cell Communication Systems To Regulate Production of the Antimicrobial Indigoidine

“…AHL identification was accomplished using a previously developed high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method (40). Briefly, 10 l of the concentrated extract was injected onto a reverse-phase C 18 core-shell column (Phenomenex Kinetex, Torrance, CA) (2.6-m pore size, 100-Å particle size, 100 by 2.1 mm) by the use of a Surveyor Autosampler Plus autosampler (Thermo Electron, Waltham, MA) at 25°C.…”

“…The (40,41). Following accurate identification of the AHLs in question, subsequent experiments were performed with a TSQ Quantum Discovery Max triple-quadrupole MS as it provided more-accurate quantitation.…”

“…AHL extraction from the agar was performed by addition of 100 ml of EtOAc followed by stirring for 3 h. Afterward, the EtOAc was decanted and concentrated in vacuo to give an off-white solid, which was reconstituted with 300 l EtOAc and transferred to an autosampler vial for analysis via isotope dilution high-performance liquid chromatography-tandem mass spectrometry (ID-HPLC-MS/MS). AHL concentrations in the plate were calculated using the previously described ID-HPLC-MS/MS methodology (40). Briefly, the concentrations of the endogenous AHLs were determined by integrating the area under the peak and comparing it to the stable isotope-labeled internal standards using the following formula: [area of endogenous AHL/area of (D 2 )AHL] ϫ [final concentration of (D 2 )AHL in the plate].…”

Synthesis and analysis of stable isotope-labelled N-acyl homoserine lactones

“…When the isotope label is included in the lactone ring (analogues 3 and 4), all classes of AHLs can easily be accessed. 27,28 However, studies to investigate the metabolization of the fatty acid side chain are impossible with these analogues. To include the label in the side chain (analogues 2, 5 and 10), most synthetic routes rely on the use of rather expensive deuterated fatty acids.…”

“…Noteworthy, dideuterated AHLanalogues 4a-h were applied as internal standards in an isotope dilution tandem mass spectrometric method. 28 Therefore, a more general route was developed to gain access to the dideuterated analogues of all different types of AHLs. 49 This mild catalysis only allows selective deuterium introduction at the acidic -position.…”