Identification and Quantification of N-Acyl Homoserine Lactones Involved in Bacterial Communication by Small-Scale Synthesis of Internal Standards and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

J Am Soc Mass Spectrom. 2017 Dec;28(12):2538-2547. doi: 10.1007/s13361-017-1777-x. Epub 2017 Aug 30.


N-acyl homoserine lactones (AHL) are small signal molecules involved in the quorum sensing of many gram-negative bacteria, and play an important role in biofilm formation and pathogenesis. Present analytical methods for identification and quantification of AHL require time-consuming sample preparation steps and are hampered by the lack of appropriate standards. By aiming at a fast and straightforward method for AHL analytics, we investigated the applicability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Suitable MALDI matrices, including crystalline and ionic liquid matrices, were tested and the fragmentation of different AHL in collision-induced dissociation MS/MS was studied, providing information about characteristic marker fragments ions. Employing small-scale synthesis protocols, we established a versatile and cost-efficient procedure for fast generation of isotope-labeled AHL standards, which can be used without extensive purification and yielded accurate standard curves. Quantitative analysis was possible in the low pico-molar range, with lower limits of quantification reaching from 1 to 5 pmol for different AHL. The developed methodology was successfully applied in a quantitative MALDI MS analysis of low-volume culture supernatants of Pseudomonas aeruginosa. Graphical abstract ᅟ.

Keywords: Internal standard; Ionic liquid matrices; Isotope labeling; Low molecular weight; Quantification; Quantitative MALDI; Quorum sensing.

MeSH terms

  • Acyl-Butyrolactones / analysis*
  • Acyl-Butyrolactones / chemical synthesis
  • Acyl-Butyrolactones / metabolism
  • Chemistry Techniques, Synthetic
  • Humans
  • Ionic Liquids / chemistry
  • Pseudomonas Infections / microbiology
  • Pseudomonas aeruginosa / chemistry*
  • Pseudomonas aeruginosa / physiology
  • Quorum Sensing
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / methods*
  • Tandem Mass Spectrometry / methods


  • Acyl-Butyrolactones
  • Ionic Liquids