Systematic Proteome Research & Bioanalytics, Institute for Experimental Medicine, University of Kiel, Niemannsweg 11, 24105, Kiel, Germany.
Department of Internal Medicine, University Hospital Schleswig-Holstein, Campus Kiel, Arnold-Heller-Straße 3, 24105, Kiel, Germany.
Anal Bioanal Chem. 2018 Jul;410(19):4737-4748. doi: 10.1007/s00216-018-0937-6. Epub 2018 Feb 22.
The identification and quantification of molecules involved in bacterial communication are major prerequisites for the understanding of interspecies interactions at the molecular level. We developed a procedure allowing the determination of 2-heptyl-4(1H)-quinolone (HHQ) and 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) and the virulence factor pyocyanin (PYO) formed by the Gram-negative bacterium Pseudomonas aeruginosa. The method is based on dispersive liquid-liquid microextraction from small supernatant volumes (below 10 μL) followed by quantitative matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). The use of ionic liquid matrix led to a lowered limit of detection for pyocyanin and, due to suppression of matrix background signals, easy to interpret mass spectra compared to crystalline matrices. Using an isotope-labeled pyocyanin standard synthesized in small-scale synthesis, quantitative analysis spanning approximately one order of magnitude (0.5 to 250 fmol) was feasible. The method was successfully applied to the detection of the signaling molecules PQS and HHQ in cultures of P. aeruginosa strains isolated from sputum of cystic fibrosis patients and allowed a highly sensitive quantification of PYO from these cultures. Hence, the developed method bears the potential to be used for screening purposes in clinical settings and will help to decipher the molecular basis of bacterial communication. Graphical abstract Ionic liquid matrices for the detection and quantification of the toxin pyocyanin and other signaling molecules from P. aeruginosa by MALDI MS.
鉴定和量化参与细菌通讯的分子是理解种间相互作用的分子水平的主要前提。我们开发了一种允许确定革兰氏阴性菌铜绿假单胞菌形成的 2-庚基-4(1H)-喹啉（HHQ）和 2-庚基-3-羟基-4(1H)-喹啉（PQS）以及毒力因子绿脓菌素（PYO）的方法。该方法基于从小上清液体积（低于 10μL）进行分散液-液微萃取，然后进行定量基质辅助激光解吸/电离（MALDI）质谱（MS）分析。离子液体基质的使用降低了绿脓菌素的检测下限，并且由于抑制了基质背景信号，与晶体基质相比，很容易解释质谱。使用从小规模合成合成的同位素标记的绿脓菌素标准品，可实现约一个数量级（0.5 到 250 fmol）的定量分析。该方法成功地应用于从囊性纤维化患者痰液中分离的铜绿假单胞菌菌株培养物中信号分子 PQS 和 HHQ 的检测，并允许从这些培养物中高度敏感地定量 PYO。因此，所开发的方法具有在临床环境中用于筛选目的的潜力，并将有助于破译细菌通讯的分子基础。
