1290 infinity 2 uhplc system

Standards and samples were acquired on an 1290 Infinity II UHPLC system (Agilent) coupled to a ZenoTOF 7600 mass spectrometer with a DuoSpray TurboV source (SCIEX). Prior to MS analysis, samples were chromatographically separated on an Agilent InfinityLab Poroshell 120 EC-C18 1.9 µm, 2.1 mm × 50 mm column heated to 30°C. A 5 min 0.8 ml/min flow rate of linear-gradient ramping from 3% ACN/0.1% formic acid to 40% ACN/0.1% formic acid was applied (Messner et al., 2021 (link)). 1 µl of each dilution series sample was loaded prior to samples entering MS. A SWATH acquisition scheme with 60 variable-size windows and 11 ms accumulation time was used (Supplementary file 2). The Zeno SWATH MS has the same 60 windows as the SWATH MS and 13 ms accumulation time. For both acquisition methods, ion source gas 1 (nebuliser gas), ion source gas 2 (heater gas), and curtain gas were set to 60, 65, and 55 psi, respectively; CAD gas was set to 7, source temperature to 700°C, and spray voltage to 3500 V.

For detection of coumarins in root exudates, lyophilized samples were analyzed on a 1290 Infinity II UHPLC system equipped with an InfinityLab Poroshell 120 PFP, 2.1 × 100 (P/N: 695675-408) in combination with a Poroshell 120 PFP, with a 2.1 mm HPLC guard column and a 1.9 µm LC column (P/N: 821725-942; Agilent Technologies) coupled to an Agilent Technologies 6470A Tandem Quadrupole Mass Spectrometer (TQ-MS–MS). Coumarins were subsequently analyzed using MS with an ESI source in positive ion mode. Further details are described in Methods S1.

LC-MS analysis was performed by an Agilent 1290 Infinity II UHPLC system (Santa Clara, CA, USA) coupled to an Agilent 6545 UHD and an Accurate-Mass Q-TOF/MS. A 2 μL aliquot of the filtrate was injected into a Waters XSelect HSS T3 column (100 × 2.1 mm, 2.5 µm, Waters, Manchester, UK) held at 25 °C. The column was eluted with gradient elution using mobile phases A and B. Mobile phase A is an aqueous solution containing 0.1% formic acid (Sigma-Aldrich). Mobile phase B is an acetonitrile solution (HPLC grade, Merck, Germany) containing 0.1% formic acid. The optimized gradient elution condition was: 0–2 min, 5% B; 2–10 min, 5–95% B; 10–15min, 95% B; 15–18 min, 95–5% B. The post time was set as 3 min for system balance.
Mass spectrometry was operated in both positive and negative ionization modes. The parameters optimized were as follows: capillary voltage, 4 kV in positive mode and 3.5 kV in negative mode; drying gas flow, 10 L/min; gas temperature, 325 °C; nebulizer pressure, 20 psig; fragmentor voltage, 120 V; skimmer voltage, 45 V. The mass data were collected at the range of m/z 100–1700.

M382 biofilms were cultured in 2216E and 1/10 2216E media in 6-well plates at 25 °C for 24 h. The cells were harvested and stored using the same procedures as those used in the preparation for transcriptomic sequencing. UHPLC-MS/MS analysis was performed on an Agilent 1290 Infinity II UHPLC system linked to a 6470A Triple Quadrupole mass spectrometry (Santa Clara, CA, USA) at the Profleader Biotech Company (Shanghai, China). The weighted samples were spiked with 10 mL of 6M HCl and 0.5% β-mercaptoethanol, sealed under nitrogen, and hydrolyzed at 110 °C for 22 h. After hydrolysis, 100 μL of the hydrolysate was dried under nitrogen and HCl traces were removed by deionized water. The samples were then dried and dissolved in 50 μL of sodium carbonate (100 mM) and derivatized by the addition of 50 μL of 2% benzoyl chloride. The derivatized samples were isometrically mixed with internal standard solutions before UHPLC-MS/MS analysis. The raw data were processed by MassHunter Workstation Software (version B.08.00, Agilent, Santa Clara, CA, USA) with default parameters. Calibration curves of ten points were constructed by plotting the peak area ratio of each compound to the internal standard against the concentration of each compound.

Samples were analysed using a 1290 Infinity II UHPLC system coupled to a 6550 iFunnel Q-TOF mass spectrometer with an electrospray ionization source (Agilent, Waldbronn, Germany). Aliquots of the sample (0.75 µg) were injected onto a reversed phase C4 column (Aquity UPLC Protein BEH C4 Column, 300 Å, 1.7 µm, 2.1 mm, 100 mm, Waters, Manchester, UK) and the column temperature was maintained at 80 °C. Mobile phase A was 0.1% (v/v) formic acid in water, and mobile phase B was 0.1% (v/v) formic acid in ACN. The flow rate was 0.5 mL/min. The complete run consisted of 1 min isocratic elution (20% solvent B), followed by a 40 min gradient (20% to 32% solvent B), a subsequent purge step (quick rise to isocratic elution at 95% solvent B), and a final 5 min reequilibration step (quick decrease to isocratic elution at 20% solvent B).
Mass spectrometer settings included a mass range of m/z 600 to m/z 3,200; nebulizer: 2.8 bar, drying gas flow: 14 L/min; drying gas temperature: 290 °C; sheath gas flow: 12 L/min; sheath gas temperature: 375 °C; fragmentor voltage: 400 V. Spectra were acquired at 2 spectra/s. Internal mass calibration was achieved by continuously spraying of internal reference compounds through the reference nebulizer.
Data acquisition was controlled with the MassHunter software (Agilent, Santa Clara, CA, USA).