Lc ms grade

Capillary liquid chromatography of tryptic peptides was performed with a Waters NanoAcquity Ultra-Performance Liquid Chromatography system equipped with a 200 μm x 5 mm PepSwift Monolithic Column (PS-DVB) thermostated at 55°C and a 2.6 μL PEEKSIL-sample loop (SGE, Darmstadt, Germany). The aqueous mobile phase (mobile phase A) was H₂O (LC-MS grade, Roth, Freiburg, Germany) with 0.1% formic acid. The organic mobile phase (mobile phase B) was 0.1% formic acid in acetonitrile (LC-MS grade, Roth). Sample (1 μL injection) were loaded onto the column in direct injection mode with 1% mobile phase B for 4 min at 800 nL/min. Peptides were eluted from the column with a gradient from 1–50% mobile phase B over 40 min at 800 nL/min followed by a 7 min rinse of 99% mobile phase B. The column was immediately re-equilibrated at initial conditions (1% mobile phase B) for 8 min. [Glu¹]fibrinopeptide was used as lockmass at 100 fmol/μL. Lockmass solution was delivered from the auxiliary pump of the NanoAcquity system at 500 nL/min to the reference sprayer of the NanoLockSpray source.

For a quick estimation of phenazine production, the concentration of oxidized pyocyanin was determined spectrophotometrically at 691 nm after vigorously vortexing the culture supernatant for 30 s. The concentrations of pyocyanin were calculated via Lambert–Beer's law using an extinction coefficient of 4.31 mM⁻¹cm⁻¹ for pyocyanin (Filloux and Ramos, 2014 ).
Quantitative phenazine analysis was performed via reversed phase high performance liquid chromatography RP-HPLC (Prominence UFLC, Shimadzu) using a C18 column (C18 Nucleodur ec, Macherey Nagel, Germany), equipped with a photo diode array UV/VIS detector (SPD-M20A, Shimadzu), which enabled detection and quantification of the phenazines at the following wavelengths: pyocyanin-319 nm, phenazine-1-carboxylic acid-366 nm, 1-hydroxyphenazine-257 nm (Mavrodi et al., 2001 (link)). Separation was achieved with a gradient of 25 mM ammonium acetate (eluent A) and acetonitrile (eluent B, LC-MS grade, Carl Roth GmbH, Germany) as eluents (with eluent A at 20% for 5 min, 80% for 10 min, and again 20% for 5 min) at a flow rate of 0.35 ml/min and a column temperature of 20°C. Phenazine peak areas were correlated to standard curves obtained by measuring pure commercial phenazines: PYO (Sigma Aldrich), PCA (Princeton BioMolecular Research Inc., New Jersey, USA), 1-OHPHZ (TCI Europe N.V., Belgium).

AHLs were extracted from sterile culture supernatants by liquid-liquid extraction (LLE) in pro-analysis-grade ethyl acetate (Carl Roth, Karlsruhe, Germany) containing 1% (v/v) analytical grade anhydrous acetic acid (Merck, Darmstadt, Germany). For this, acidified ethyl acetate was added to collected supernatant (1:3 v/v) and vortexed for 1 min. Afterwards, the organic phase was collected, and the remaining aqueous phase was extracted again as described. In total, five extraction cycles were performed. Pooled organic phases were concentrated by evaporation before drying using a SpeedVac vacuum concentrator (Eppendorf, Hamburg, Germany) for 1 h at RT. For LC-MS/MS analysis, the residue was reconstituted in 200 µL acetonitrile (LC-MS-grade, Carl Roth, Karlsruhe, Germany). To determine the performance and recovery rates achieved using the LLE protocol, we spiked the culture supernatants with 100 ng of OHHL and HHL as additional samples.