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MALDI-MS-Based Quantitative Analysis for Ketone Containing Homoserine Lactones in Pseudomonas aeruginosa
journal contribution
posted on 2015-01-20, 00:00 authored by Yoon-Woo Kim, Changmin Sung, Seulee Lee, Kyoung-Jin Kim, Yung-Hun Yang, Byung-Gee Kim, Yoo Kyung Lee, Hee Wook Ryu, Yun-Gon KimN-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]+ 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 Pseudomonas aeruginosa 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.