Vanquish binary pump h system

UHPLC-LTQ-Orbitrap-ESI-MS/MS analysis was performed using a UHPLC system equipped with a Vanquish binary pump H system (Thermo Fisher Scientific, Waltham, MA, USA) coupled with auto-sampler and column compartment. A Phenomenex KINETEX^® C18 column (100 mm × 2.1 mm, 1.7 µm particle size; Torrance, CA, USA) was used to separate a sample analyte. The analytical parameters were adopted from a study reported by Kwon et al. [17 (link)].

For UHPLC-LTQ-Orbitrap-MS/MS analysis, each dried sample (10 mg/mL) was dissolved in 100% methanol and used. UHPLC-LTQ-Orbitrap-MS/MS analysis was performed using a UHPLC system equipped with the Vanquish binary pump H system (Thermo Fisher Scientific, Waltham, MA, USA) coupled with an auto-sampler and column compartment. The chromatographic separation was performed on the Phenomenex KINETEX^® C18 Column (100 × 2.1 mm, 1.7 μm; Torrance, CA, USA) and the operational parameters were adapted from a study reported by Lee et al. [48 (link)]. The chromatograms of samples are shown in Figure S2.

UHPLC-LTQ-Orbitrap-MS/MS analysis was performed using an UHPLC system equipped with a Vanquish binary pump H system (Thermo Fisher Scientific, Waltham, MA, USA) coupled with an auto-sampler and column compartment. Chromatographic separation was performed on a Phenomenex KINETEX^® C18 column (100 mm × 2.1 mm, 1.7 um particle size; Torrance, CA, USA) and the injection volume was 5 μL. The column temperature was set to 40 °C, and the flow rate was 0.3 mL/min. The mobile phase consisted of 0.1% v/v formic acid in water (A) and 0.1% v/v formic acid in acetonitrile (B). The gradient parameters were set as follows: 5% B was maintained initially for 1 min, followed by a linear increase to 100% B over 9 min, then sustained at 100% B for 1 min, with a gradual decrease to 5% B over 3 min. The total run time was 14 min. The MS data were collected in the range of 100–2000 m/z using an ion trap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The probe heater and capillary temperature were set to 300 °C and 350 °C, respectively. The capillary voltage was set to 2.5 kV in negative mode (positive mode, 3.7 kV).

Metabolite profiling of LpCC and NCC juice extracts (10 mg/mL) was done by using UHPLC-LTQ-Orbitrap-MS/MS. Chloramphenicol (2.5 mg/mL) was used as an internal standard (IS). The sample injection volume of 5 μL was injected at the flow rate of 0.3 mL/min into a UHPLC system equipped with a Vanquish binary pump H system (Thermo Fisher Scientific, Waltham, MA, USA), an auto-sampler, and the column compartment. The chromatographic separation was achieved on a Phenomenex KINETEX^® C18 column (100 mm × 2.1 mm, 1.7 μm particle size; Torrance, CA, USA) maintained at the column temperature of 40 °C. The mobile phase consisted of 0.1% formic acid in water (v/v, solvent A) and 0.1% formic acid in acetonitrile (v/v, solvent B). The mobile phase solvent gradient was programmed as follows: 5% solvent B for 1 min, gradually increased to 100% solvent B over 9 min, and maintained as such for next 1 min followed by a decrease to 5% solvent B over 3 min. The MS data were collected by using an Orbitrap Velos ProTM system equipped with an ion-trap mass spectrometer and HESI-II probe. The mass spectra were acquired over the range of 100–2000 m/z. The probe heater and capillary temperatures were set to 300 °C and 350 °C, respectively. The capillary voltage was set to 2.5 KV and 3.7 KV in negative and positive ion modes, respectively.

For ultra-high-performance liquid chromatography quadrupole orbitrap ion trap tandem mass spectrometry (UHPLC-Q-orbitrap-MS/MS) analysis, each dried sample (20 mg/ml) was dissolved in aqueous methanol (80%) and used. UHPLC-Orbitrap-MS/MS analysis was performed using a UHPLC system equipped with a Vanquish Binary Pump H System (Thermo Fisher Scientific, USA) coupled with auto-sampler and column compartment. The chromatographic separation was performed on a Phenomenex KINETEX C18 column (100 mm × 2.1 mm, 1.7 μm; USA) and the operational parameters were adapted from a study by Lee et al. [14 (link)].

A UHPLC system equipped with a Vanquish binary pump H system (Thermo Fisher Scientific, Waltham, MA, USA) was united with an auto-sampler and column compartment. A Phenomenex KINETEX^® C18 column (100 mm × 2.1 mm, 1.7 µm particle size; Torrance, CA, USA) was used to chromatographic separation. The injection volume was 5 µL. The column temperature was set to 40 °C, and the flow rate was 0.3 mL/min. The mobile phase consisted of 0.1% v/v formic acid in water (A) and acetonitrile (B). The gradient parameters were configured as follows: Five percent solvent B was maintained initially for 1 min, followed by a linear increase to 100% solvent B over 9 min and then sustained at 100% solvent B for 1 min, with a gradual decrease to 5% solvent B over 3 min. The total run time was 14 min. The MS data were collected in a range of 100–1500 m/z (in negative- and positive-ion mode) using an Orbitrap Velos Pro system (Thermo Fisher Scientific, Waltham, MA, USA) coupled with an ion-trap mass spectrometer coupled with a heated electrospray ionization (HESI-II) probe. The probe heater and capillary temperatures were set to 300 °C and 350 °C, respectively. The capillary voltage was set to 2.5 kV in negative mode (positive mode 3.7 kV).

Analytical samples for metabolite profiling were prepared by diluting culture supernatant broth extracts (0.2 ml) with HPLC-grade water (0.2 ml). The UHPLC-LTQ-Orbitrap-MS/MS system used was equipped with a Vanquish binary pump H system (Thermo Fisher Scientific, USA) coupled with an autosampler and a column compartment. Chromatographic separation was performed on a Phenomenex KINETEX C18 column (100 mm × 2.1 mm, 1.7μm; Torrance, USA) with an injection volume of 5 μl. The column temperature was set to 40°C, and the flow rate was 0.3 ml/min. The mobile phases consisted of 0.1% v/v formic acid in water (A) and 0.1% v/v formic acid in acetonitrile (B). The gradient parameters were set as described by Kwon et al. [19 (link)]. The MS data were collected in the range of 100–1500 m/z (under negative- and positive-ion mode) using an Orbitrap Velos ProTM system, which is combined with an Ion-Trap Mass Spectrometer (Thermo Fisher Scientific) coupled with a HESI-II probe. The probe heater and capillary temperatures were set to 300°C and 350°C, respectively. The capillary voltage was set to 2.5 kV in negative mode (positive mode, 3.7 kV).