Identification of Unanticipated and Novel N-Acyl L-Homoserine Lactones (AHLs) Using a Sensitive Non-Targeted LC-MS/MS Method

N -acyl L-homoserine lactones (AHLs) constitute a predominant class of quorum-sensing signaling molecules used by Gram-negative bacteria. Here, we report a sensitive and non-targeted HPLC-MS/MS method based on parallel reaction monitoring (PRM) to identify and quantitate known, unanticipated, and novel AHLs in microbial samples. Using a hybrid quadrupole-high resolution mass analyzer, this method integrates MS scans and all-ion fragmentation MS/MS scans to allow simultaneous detection of AHL parent-ion masses and generation of full mass spectra at high resolution and high mass accuracy in a single chromatographic run. We applied this method to screen for AHL production in a variety of Gram-negative bacteria (i.e. B . cepacia , E . tarda , E . carotovora , E . herbicola , P . stewartii , P . aeruginosa , P . aureofaciens , and R . sphaeroides ) and discovered that nearly all of them produce a larger set of AHLs than previously reported. Furthermore, we identified production of an uncommon AHL (i.e. 3-oxo-C7-HL) in E . carotovora and P . stewartii , whose production has only been previously observed within the genera Serratia and Yersinia . Finally, we used our method to quantitate AHL degradation in B . cepacia , E . carotovora , E . herbicola , P . stewartii , P . aeruginosa , P . aureofaciens , the non-AHL producer E . coli , and the Gram-positive bacterium B . subtilis . We found that AHL degradation ability varies widely across these microbes, of which B . subtilis and E . carotovora are the best degraders, and observed that there is a general trend for AHLs containing long acyl chains (≥10 carbons) to be degraded at faster rates than AHLs with short acyl chains (≤6 carbons).