MALDI-MS-Based Quantitative Analysis for Ketone Containing Homoserine Lactones in <i>Pseudomonas aeruginosa</i>

<i>N</i>-Acyl homoserine lactones (AHLs), quorum sensing molecules produced by Gram-negative bacteria, are used as important secondary metabolites for antibacterial drug development and cell-to-cell communication. Although various analytical techniques have been developed for detection and quantitation of AHLs from more complex bacterial culture media, only a few methods have been applied to AHL identification in physiological samples. Here, we developed a highly sensitive and reliable MALDI-based 3-oxo AHL quantitation method by employing Girard’s reagent T (GT) to produce a permanent cationic charge state [M]<sup>+</sup> at the ketone group of AHLs. After extracting AHLs from the supernatant of bacterial cultures using ethyl acetate, the extracts were subsequently derivatized with GT without any additional purification or desalting steps. The chemical derivatization of 3-oxo AHLs dramatically enhanced sensitivity (up to 60 000 times) by lowering the limit of detection (LOD, ∼0.5 fmol)/limit of quantitation (LOQ, ∼2.5 fmol). Additionally, the GT-derivatized 3-oxo AHLs allowed more accurate quantitative analysis from the <i>Pseudomonas aeruginosa</i> PAO1 culture supernatants. This method may be applied for developing high-throughput and sensitive detection methods of quorum sensing signal molecules in biofilm-related clinical applications such as virulence factor characterization and antibacterial drug development.