Profiling of N‐acyl‐homoserine lactones by liquid chromatography coupled with electrospray ionization and a hybrid quadrupole linear ion‐trap and Fourier‐transform ion‐cyclotron‐resonance mass spectrometry (LC‐ESI‐LTQ‐FTICR‐MS)

Abstract

A method for the comprehensive profiling of the N-acyl-homoserine lactone (AHL) family of bacterial quorum-sensing molecules is presented using liquid chromatography (LC) coupled to a hybrid quadrupole linear ion trap (LTQ) and Fourier-transform ion-cyclotron-resonance mass spectrometer (FTICR). We demonstrate an increase in signal intensity in MS with electrospray ionization (ESI) of the protonated molecules, [M + H](+), by using acetonitrile (ACN) instead of methanol (MeOH) as the organic solvent under the conditions in which the samples were supplied to the probe by direct infusion at constant flow rates. The presence of ACN prevents the formation of methanol adducts such as [M + MeOH + H](+) and [M + MeOH + Na](+), while also lowering the signal intensity of sodiated [M + Na](+) ions. Sensitivity of these signaling molecules in terms of signal-to-noise ratio (S/N) using low-resolution LTQ-MS and high-resolution FTICR-MS were compared under reversed-phase (RP) LC separations with ESI interface. Special emphasis was paid to the choice of the separation column, its elution conditions and detection of the major AHL compounds produced by the Serratia liquefaciens strain ATCC 27592. The most promising results were obtained using a RP C16-amide column eluted with a linear mobile phase gradient ACN/H(2)O containing 0.1% formic acid. The whole set of AHL homologs in bacterial extracts was detected in the extracted-ion chromatographic (XIC) mode, and the calculations of molecular formulae were performed by including the isotopic pattern. This mode of displaying data, with a very narrow mass-to-charge ratio window (i.e. +/- 0.0010 as m/z unit) around each selected ion, has allowed the identification of all the eight known homoserine lactones, viz. C(4)-HSL, 3-oxo-C(6)-HSL, C(6)-HSL, 3-oxo-C(8)-HSL, C(8)-HSL, C(10)-HSL, C(12)-HSL and C(14)-HSL. In addition, at least four uncommon signaling mediators previously unreported, namely, 3-oxo-C(10:1)-HSL, 3-oxo-C(11:2)-HSL, 3-oxo-C(13:2)-HSL and 3-OH-C(16)-HSL, were identified and characterized; their roles in cell-to-cell communication has to be elucidated.

A high-throughput screen for quorum-sensing inhibitors that target acyl-homoserine lactone synthases

“…There has been little published on inhibitors of acyl-HSL synthases (4,10,12,13), at least in part, because of the fact that inhibition is difficult to measure, particularly in cell-based assays. The unique product of acyl-HSL synthase activity is the acyl-HSL itself, which can be measured by using a bioassay (27,28), by mass-spectrometric techniques (27,29,30), or by measuring incorporation of radiolabeled SAM into the product (24,25). The previously described DCPIP assay, which measures the reactive thiol of the ACP product of the reaction, is not amenable to high-throughput screening because many compounds will affect absorbance and the assay lacks sensitivity (20).…”

The Legionella Autoinducer Synthase LqsA Produces an α-Hydroxyketone Signaling Molecule

“…Reporter strain assays are highly sensitive and widely used tools for the detection of autoinducer molecules. However, due to their intrinsic specificity, these bioassays are often very selective and can neither provide a comprehensive profile nor an accurate quantification of signaling molecules (48,49). To trigger signaling of the CAI-1 reporter strain, both backbone length and stereochemistry of ␣-hydroxyketones were found to be important, yet in addition to CAI-1 (C 13 ) smaller homologues (C 12 and C 11 ), activated the V. cholerae quorum sensing circuit as well (35).…”

Acyl homoserine lactone-based quorum sensing in a methanogenic archaeon

“…Using ESI (positive ion mode) coupled with Fourier transform ion cyclotron resonance MS APEX IV (ESI-FT-ICR; Bruker Daltonics, Billerica, MA, USA), high-resolution data of the compounds in methanol were determined. The Bruker Compass Data Analysis software (version 4.0) was used for data acquisition and processing (Cataldi et al, 2008).…”