Accella1250 hplc system

HPLC-MS analysis was performed using an Accella1250 HPLC system coupled with the benchtop ESI-MS Orbitrap Exactive (Thermo Fisher Scientific, San Jose, CA, USA) in negative and positive ion mode. Samples were analyzed on a C18 column (Shim Pack Shimadzu XR-ODS 3 × 75 mm) using a gradient of water/acetonitrile with 0.1% formic acid (0–5 min, 98–90% H₂O; 5–10 min, 90–5% H₂O; 10–13 min, 5% H₂O; 13–14 min, 98% H₂O). Data analysis was performed using Qual Browser Thermo Xcalibur software (version 2.2 SP1.48).
HPLC-MS analysis of alumina extraction samples was performed using a Waters Acquity Class-I UPLC (Waters Chromatography B.V, Etten-Leur, The Netherlands) system coupled to a MaXis Plus Q-TOF (Bruker, Billerica, MA, USA) on negative ion mode with post-column addition of 3 μL/min ESI Tune Mix (G1969-85000; Agilent Technologies, Middelburg, The Netherlands) for mass calibration. Samples were analyzed on a C18 column (Shim Pack Shimadzu XR-ODS 3 × 75 mm) using a gradient of water/acetonitrile with 0.1% formic acid (0–5 min, 98–90% H₂O; 5–10 min, 90–5% H₂O; 10–13 min, 5% H₂O; 13–15 min, 2% H₂O; 15–17 min, 98% H₂O). Data analysis was performed using Bruker Compass Data Analysis (version 4.2 SR1).

For identification of R. jostii RHA1 γ-butyrolactone-like molecules, HPLC-MS analysis was performed using an Accella1250™ HPLC system coupled with the benchtop ESI-MS Orbitrap Exactive™ (Thermo Fisher Scientific, San Jose, CA). A Reversed Phase C18 (Shim Pack Shimadzu XR-ODS 3 × 75 mm) column was used and a gradient from 2% to 95% of acetonitrile:water (0.1% Formic Acid) as follows: 2 min 2% acetonitrile, 2–10 min gradient to 95% acetonitrile, 1 min 95% acetonitrile. To separate further the peaks from A-factor and 6-dehydro SCB2, a gradient from 2% to 80% acetonitrile was applied to the separation: 2 min 2% acetonitrile, 2–25 min in 2–80% acetonitrile, 1 min 80% acetonitrile. Data was analysed using Xcalibur software from Thermo Scientific. LC-MS analysis was performed with 2–4 biological replicates per strain.

LC-MS analysis was performed using an Accella1250 HPLC system coupled with the benchtop ESI-MS Orbitrap Exactive (Thermo Fisher Scientific, Waltham, Massachusetts, USA) on positive ion mode. Samples were analyzed on a C18 column (Shim Pack Shimadzu XR-ODS, 2.2 μ 3 × 75 mm, Shimadzu Corporation, Kyoto, Japan) or on XB-C18 column (Kinetex 2.6 μm, XB-C18, 100 Å, 150 x 1.0 mm, Phenomenex, Utrecht, the Netherlands) using a gradient of water/acetonitrile with 0.1% formic acid (0 to 5 min, 98% to 90% H₂O; 5 to 10 min, 90 to 5% H₂O; 10 to 13 min, 5% H₂O; 13 to 14 min, 98% H₂O). Data analysis was performed using Qual Browser Thermo Xcalibur software (version 2.2 SP1.48).