Identification of unsaturated N-acylhomoserine lactones in bacterial isolates of Rhodobacter sphaeroides by liquid chromatography coupled to electrospray ionization-hybrid linear ion trap-Fourier transform ion cyclotron resonance mass spectrometry

The identification of two unsaturated N-acylhomoserine lactones (AHLs) produced by Rhodobacter sphaeroides bacteria, based on liquid chromatography (LC) coupled to a hybrid quadrupole linear ion trap (LTQ)-Fourier transform ion cyclotron resonance (FTICR) mass spectrometer upon electrospray ionization (ESI), is presented. Along the confirmation of the signaling molecule already described in the literature, i.e. (Z)-N-tetradec-7-enoyl-homoserine lactone (C14:1-HSL), we have discovered the occurrence, at low yet significant levels, of another monounsaturated compound, C12:1-HSL, which may extend the number of small diffusible chemical signals known for R. sphaeroides. Both unsaturated AHLs were identified by high resolution FTICR mass spectrometry in extracts of bacterial culture media and the occurrence of a C=C bond was assessed upon their conversion to bromohydrins. Collision induced dissociation (CID) spectra were then collected on the LTQ mass analyser. A careful comparison of tandem MS spectra of monounsaturated (i.e., C12:1-HSL and C14:1-HSL) and saturated AHLs (i.e. C12-HSL and C14-HSL) led to emphasise two series of product ions, exhibiting 14 Da-spaced m/z ratios. Both series were referred to progressive fragmentations at the aliphatic end of the AHL acyl chains, followed by neutral losses of terminal alkenes (i.e., CH2=CH(CH2)nH). In particular, the series located at the higher end of the explored m/z range (> 200 Da), observed only for monounsaturated species, enabled the location of the C=C bond between carbons 7 and 8 of the acyl chain.