Xevo g2 xs qtof mass spectrometer

The enzymatic activity assay was carried out in a 500 µl reaction system comprising 100 µg purified proteins, 1 mM caffeoyl-CoA or p-coumaroyl-CoA as the acyl donors, 1 mM pHPL or DHPL as the acyl receptors. The reactions were incubated at 25 °C for 60 min and terminated by adding 10 µl of 10 M acetic acid. Controls were carried out using total proteins from E. coli transformed with the empty pGEX-4T-1 vector. Reaction products were collected and analyzed using ACQUITY UPLC system (Waters, Milford, MA, USA). MS/MS data were recorded on a Xevo G2-XS Q-ToF Mass Spectrometer (Waters, Milford, MA, USA) coupled to a Waters Acquity I-Class UPLC system (Waters, Milford, MA, USA). MS/MS analyses were conducted in negative-ion mode. The samples were separated on an ACQUITY UPLC BEH C18 column (1.7 μm, 100×2.1 mm) at 25°C. The mobile phase A was 0.1% (v/v) formic acid-acetonitrile. The mobile phase B was 0.1% (v/v) formic acid in water. The flow rate was 0.3 mL min⁻¹. The mobile phases changed with the following gradient: 0–6 min, 5% A and 95% B; 6–8 min, 20% A and 80% B; 8–14 min, 21% A and 79% B; 14–18 min, 95% A and 5% B. MS was analyzed using electrospray ionization (ESI) at negative ion mode. MS-MS data were analyzed using the MssLynx V4.1 software (Waters) as described previously (Pan et al., 2023 (link)).

Melting points were obtained on a SGW X-4 micro melting point apparatus (INESA Co., Shanghai, China). Optical rotations were measured on a SGW-533 automatic polarimeter (INESA Co., Shanghai, China). The 1D and 2D NMR spectra were measured with a Bruker DRX-600 instrument (Bruker BioS-pin GmbH Company, Rheinstetten, Germany) with TMS as the internal standard. ESIMS data were recorded on an API QSTAR mass spectrometer (Applied Biosystem/MSD Sciex, Concord, Ontario, Canada); HRESIMS were recorded on a Waters Xevo G2-XS QTof mass spectrometer (USA). ECD spectra were recorded on a JASCO J-810 circular dichroism (CD) spectropolarimeter (JASCO Co., Tokyo, Japan). Column chromatography was performed on silica gel 60 (100–200 mesh, Qingdao Marine Chemical Ltd., Qingdao, China), sephadex LH-20 (GE Healthcare, Uppsala, Sweden), and Develosil ODS (50 μm, Nomura Chemical Co. Ltd., Osaka, Japan). The preparation of TLC was performed on HSGF254 plates (Jiangyou silicone Development Co., Ltd., Yantai, China).

Specific rotations were measured using a DIP-360 digital polarimeter (JASCO, Easton, PA, USA). Nuclear magnetic resonance (NMR) spectra were recorded on a JEOL ECX 400 FT-NMR spectrometer (JEOL, Tokyo, Japan) at room temperature. Electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) experiments were performed using a Waters Xevo G2-XS Q-TOF mass spectrometer (Waters, Milford, MA, USA). Column chromatography was performed on Silica Gel 60 (Nacalai Tesque, Kyoto, Japan, 230–400 mesh) and YMC ODS-A gel (YMC Co. Ltd., Kyoto, Japan, 50 µm). Thin-layer chromatography (TLC) was performed on TLC Silica Gel 60F254 (Merck, Damstadt, Germany) and TLC Silica Gel 60 RP-18 F254S (Merck, Damstadt, Germany) plates. The spots were visualized by spraying with 10% aq. sulfuric acid followed by heating. High-performance liquid chromatography (HPLC) was performed using a UV-8020 UV-VIS detector (Tosoh Corp., Tokyo, Japan), DP-8020 pump (Tosoh Corp., Tokyo, Japan), and DP-8020 degasser (Tosoh Corp., Tokyo, Japan). An XBridge BEH C18 Column (Waters, Milford, MA, USA, 130 Å, 3.5 µm, 10 mm × 250 mm) was used for preparative purposes.

An ACQUITY UPLC connected to a XEVO-G2XS QTOF mass spectrometer (Waters, Manchester, UK) equipped with an electrospray ion source was used. Liquid chromatography was performed using a Titan™ C18 UHPLC column (2.1 x 100 mm, 1.9 μm, Supelco). The column temperature was maintained at 45°C. The separation was performed at a flow rate of 0.4 mL min^-1 under a gradient program in which the mobile phase consisted of (A) 0.1% formic acid (v/v) and (B) pure methanol. The gradient program was applied as follows (in % B): (t) = 0 min, 1%; t = 2.0 min, 1%; t = 8.0 min, 38%; t = 20 min, 99.5%; t = 25 min, 99.5%; t = 25.1 min, 1%; and t = 28 min, 1%, for a total analysis time of 28 minutes. The injection volume was 0.2 μL. Positive ion mode was recorded, and the instrument was operated in data-independent acquisition mode (MS^E). The m/z range was 100–1700, with an acquisition rate of 0.5 sec per scan. The following instrumental parameters were used: capillary: 3.0 kV; cone: 40,000 V; desolvation temperature: 550°C; cone gas flow: 10 L h^-1; desolvation gas flow: 900 L h^-1. The collision energy was 20 to 60 eV for fragmentation. Leucine encephalin (molecular weight = 555.62; 200 pg μL^-1 in 1:1 acetonitrile:water) was used as the lock mass for accurate mass measurements, and a 0.5 mM sodium formate solution was used for calibration. Samples were randomly analyzed.

Chemical diversity of the selected bioactive crude extracts was explored via an untargeted UPLC-QToF-MS/MS-based metabolomics approach. ULC-MS grade solvents were purchased from Biosolve Chimie or from LGC Standards (Wesel, Germany). LC-MS/MS analyses were performed on an Acquity UPLC I-Class system coupled to a Xevo G2-XS QToF mass spectrometer (Waters, Milford, MA, USA), equipped with an Acquity UPLC HSS T3 column (High Strength Silica C18, 1.8 μm, 2.1 × 100 mm, Waters) operating at 40 °C. Crude extracts were dissolved in MeOH at a concentration of 1.0 mg/mL and the injection volume was 0.3 µL. A binary mobile phase system (A: 0.1% formic acid in ultra-purified water, B: 0.1% formic acid in acetonitrile) was pumped at a flow rate of 0.6 mL/min by applying a linear gradient (% of A given): initial, 99%; 11.5 min, 1%; 14.5 min, 1%; washing and reconditioning of the column until 16 min. Acquisition of MS and MS/MS spectra was performed as previously described [47 (link)], despite the following modifications: spectra were recorded in positive mode and the acquisition range was set to m/z 50–1200. The capillary voltage was kept at 3 kV. Solvent (MeOH) and media controls (CAG, GYM, MB, PDA) were analyzed using the same conditions.

UPLC-ESI-Q-TOF-MS analysis was performed on a Waters Xevo G2-XS QTOF mass spectrometer (Milford, MA, USA) equipped with a UPLC system and an electrospray ionization source (ESI). The column was Waters ACQUITY UPLC BEH C₁₈ (100 × 2.1 mm, 1.7 μm) (Milford, MA, USA), and the temperature of the column was set at 30 °C. The mobile phases consisted of eluent A (0.1% formic acid acetonitrile solution, v/v) and eluent B (0.1% formic acid aqueous solution, v/v), and the flow rate was 0.4 mL/min. The gradient elution conditions were as follows: 0–2 min, linear gradient 86–93% A; 93% A in 2–8 min. The mass spectrometry analysis was performed using an ESI, and the accurate molecular mass was corrected by leucine enkephalin solution. The negative ion mass spectrometry scanning mode was used for detection. The scanning m/z range was 100–1200 Da, the scanning time was 0.2 s, the sprayer flow rate was 800 L/h, and the cone voltage was set to 40 V. The sample of RCOO was dissolved in methanol (through 0.22 μm microporous membrane) and analyzed by mass spectrometry. The mass spectrometry data were collected and recorded by Masslynx workstation (version 4.1, waters, Manchester, UK).