Compounds POM2-C-HMBP (6 ), and POM2-C-HMHP (11 ) or control (T cell media) were added separately to PBMCs (1,000,000 cells/mL) and at a final concentration of 10 μM in 100 μL cells suspended in media in a 96-well plate. Aliquots were incubated at 37 °C with time points of 0, 6, 24, and 48 hours. At each time point, 100 μL of sample was collected, spun (600 rcf, 3 minutes), and 80 μL of the supernatant was collected. Acetonitrile (320 μL) was added to the supernatant and the samples were vortexed and spun (10,000 rcf, 2 minutes); 300 μL of the supernatant was collected and stored at −20 °C until analysis. Analysis was completed using UPLC-MS (Waters Xevo G2-XS QTOF-MS) using a C18 column. The gradient ranged from 25% acetonitrile to 80% acetonitrile over 8 minutes and was held for 1 minute before re-equilibration. Integrated value of the molecular ion is reported relative to control (t = 0).
G2 xs qtof ms
Manufactured by Waters Corporation
Sourced in United States
The G2-XS QTOF-MS is a high-resolution mass spectrometry system designed for advanced analytical applications. It features a quadrupole time-of-flight (QTOF) mass analyzer that provides accurate mass measurements and high-resolution capabilities. The core function of this instrument is to perform sensitive and precise analysis of a wide range of sample types.
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4 protocols using g2 xs qtof ms
1
Kinetics of POM-C-HMBP and POM-C-HMHP in PBMCs
2
LC-QTOF Sample Preparation for Okara Analysis
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Sample preparation for LC-QTOF analysis was performed according to the method of Chan et al. (2021) (link) with slight modifications. Ctrl, LAC, and PCC samples (24 h) were centrifuged at 4 °C, 9,956 x g for 10 min, and the supernatant was collected and frozen at −80 °C before freeze-drying (Buchi Standalone Freeze Dryer, Essen, Germany). The freeze-dried sample (0.1 g) was dissolved in 1 mL of LC-MS grade water to obtain a concentration of 100 mg/mL. An aliquot of 350 μL of dissolved sample was mixed with 1.4 mL of cooled acetonitrile to obtain a concentration of 20 mg/mL. Samples were kept at −20 °C for 1 h to precipitate the proteins, before centrifuging at 4 °C, 10,000 x g for 5 min. The supernatant was filtered through a 0.22-μm hydrophilic PTFE syringe filter and 1.0 mL was added into a HPLC vial for analysis on a LC-QTOF MS apparatus (Waters Xevo G2-XS QTOF MS). Technical duplicates were prepared for each sample. A standard was prepared using paracetamol (a small molecule that is not found in okara and would not be generated during fermentation) and added to each vial to obtain a concentration of 10.0 μg/mL. Quality control (QC) samples were prepared by mixing 200 µL of solution from each vial.
3
Mass Fragmentation of Isolated Compounds
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The mass fragmentation of isolated compounds (1 –9 ) was performed on a Waters UPLC system (Waters, Milford, MA, USA) coupled with Waters column and a Xevo G2-XS Q-TOF MS with electrospray ionization source (Waters, Milford, MA, USA). Triple TOF MS equipped with a DuosprayTM ion source was used to complete the high-resolution experiment. For mass detection, the instrument was operated in the negative ion electrospray mode, and the conditions of the MS/MS detector were as follows: the de-solvation gas was 800 L/h at 400 °C, the cone gas was 50 L/h, the source offset voltage was 80 eV, and the source temperature was 120 °C. A full scan was run in the negative mode with a mass range from m/z 50 to 1250 amu. The capillary voltage was 3.0 kV, and the sampling cone voltage was 40 eV. Sodium formate was used for mass spectrometer instrument calibration in the resolution mode. Leucine encephalin, which generated the reference ion (m/z 554.2615 [M-H]-), was used to ensure accuracy throughout the mass spectrometry analysis. All data acquisition and processes were performed for qualitative analysis by using MassLynx V4.1 and UNIFI V1.9 (Waters, Milford, MA, USA). The gradient elution of the mobile phase was conducted following the above established gradient elution.
4
Quantification and Identification of Polyphenolic Compounds
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For the quantification and identification of polyphenolic compounds, LC analysis was conducted using Acquity Ultra Performance Liquid Chromatography system (Waters, Milford, MA, USA), equipped with a C18 column (2.1 × 100 mm, 1.7 μm, Waters, Milford, MA, USA). The column, constant at 40 °C, was eluted with a linear gradient mobile phase at 0–28 min: 2–50% B, 28–28.5 min: 50–100% B, 28.5–30.5 min: 100% B, 30.5–32 min: 100–2% B, 32–34 min: 2% B, where A = water with 0.1% acetic acid and B = acetonitrile. The flow rate was 0.3 mL/min, and the injected volume was 1 μL.
The mass spectrometric data of the full scan mode was collected using a G2-XS QT of MS (Waters, Milford, MA, USA). The scan range was from m/z 100 to 2000 with a scan time of 0.3 s. The source temperature was set at 120 °C with a cone gas flow of 50 L/h. The gas flow was set to 800 L/h at a temperature of 400 °C. The capillary was set at 1 kV for ESI− mode with the cone voltage at 40 V. The MSn analysis was carried out on a Waters-Micro mass Quattro Premier triple quadrupole mass spectrometer. The collision energy was optimized according to the specific precursor ions.
The mass spectrometric data of the full scan mode was collected using a G2-XS QT of MS (Waters, Milford, MA, USA). The scan range was from m/z 100 to 2000 with a scan time of 0.3 s. The source temperature was set at 120 °C with a cone gas flow of 50 L/h. The gas flow was set to 800 L/h at a temperature of 400 °C. The capillary was set at 1 kV for ESI− mode with the cone voltage at 40 V. The MSn analysis was carried out on a Waters-Micro mass Quattro Premier triple quadrupole mass spectrometer. The collision energy was optimized according to the specific precursor ions.
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