The serum samples were collected and stored at −80 °C until analyzed. The samples were extracted and subjected to the ultra-high performance liquid chromatography – high resolution mass spectrometry UHPLC-HRMS procedure as outlined by Oliver Fiehn's group35 (link) and (http://www.metabolomicsworkbench.org/protocols/getprotocol.php?file_id=163 ). The analysis was conducted on a Waters Acquity UPLC coupled to a Waters Xevo G2-qTOF (quadrupole time-of-flight) mass spectrometer. The samples were analyzed in positive electrospray mode, and the data were acquired using the MSe acquisition format with a scan rate of 2 Hz. High-energy MSe scans were ramped from 15 eV to 30 eV. A leucine enkaphlin (2 μg/ml) solution was used as lockspray solution. Data processing, including chromatographic alignment and peak picking occurred in Progenesis QI 2.0. Potential markers were identified by matching the molecular ion (5 p.p.m. error) and fragment ions (50 p.p.m. error) to the lipidblast database using the database search feature within progenesis QI. Further processing occurred by exporting the data to EZinfo 3.0.
Xevo g2 qtof
Manufactured by Waters Corporation
Sourced in United States, United Kingdom
The Xevo G2 QTof is a high-resolution quadrupole time-of-flight mass spectrometer designed for advanced analytical applications. It provides accurate and precise mass measurements for a wide range of molecules.
Automatically generated - may contain errors
Lab products found in correlation
Acquity uplc, by Waters Corporation (11 mentions)
Acquity uplc system, by Waters Corporation (9 mentions)
Acquity uplc beh c18 column, by Waters Corporation (7 mentions)
Nanoacquity uplc system, by Waters Corporation (5 mentions)
Beh c18 column, by Waters Corporation (5 mentions)
Protein lynx global server version 2, by Waters Corporation (4 mentions)
Uplc system, by Waters Corporation (3 mentions)
Acquity uplc 1 class, by Waters Corporation (3 mentions)
Scaffold v4, by Proteome Software (3 mentions)
C18 waters trizaic nanotile, by Waters Corporation (3 mentions)
T3 c18 uplc column, by Waters Corporation (2 mentions)
J 715 spectropolarimeter, by Jasco (2 mentions)
400 mhz spectrometer, by Bruker (2 mentions)
Beh glycan column, by Waters Corporation (2 mentions)
Quanlynx software, by Waters Corporation (2 mentions)
Amino acid standard, by Agilent Technologies (2 mentions)
Masslynx 4, by Waters Corporation (2 mentions)
300 mhz nmr spectrometer, by Bruker (2 mentions)
Acquity uhplc system, by Waters Corporation (2 mentions)
Acquity uplc 1 class system, by Waters Corporation (2 mentions)
89 protocols using xevo g2 qtof
1
Metabolic Profiling of Serum Samples via UHPLC-HRMS
2
Coumarin Metabolic Stability Screening
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Coumarin metabolic stability screening in expressed human CYP and UGT isoforms (CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4 and UGT1A1, UGT1A3, UGT1A6, UGT1A9, UGT2B7) identified CYP2A6 as the only enzyme metabolizing coumarin (Supplementary material ). Pooled human cryopreserved primary hepatocytes (human tissues are only obtained from donors with valid, written consent following full ethics approval prior to tissue collection; 50 multidonors, BioIVT, lot no. YQV) in suspension at a cell density of 0.5 million cells per ml, were incubated at 37°C up to 90 min with 10 µM coumarin (final concentration of 0.25% DMSO) without and with tranylcypromine (0.5, 2 µM) to inhibit the CYP2A6-specific reaction. A second experiment was conducted at a higher concentration of coumarin (1 mM) without the inhibitor, to saturate the 7-hydroxycoumarin pathway; all experiments were performed in duplicate. Full scan liquid chromatography-mass spectrometry data were acquired using multienergy time-of-flight acquisition (Waters Xevo G2 Q-ToF in MSE mode). The data were interrogated for the masses associated with the metabolites predicted by Meteor Nexus (Supplementary material ). All possible metabolites were reported based on the Meteor Nexus output and observed fragments. Metabolites were reported as “% total peak area” and “% parent peak area at T = 0.”
3
Metabolic Profiling of COVID-19 Plasma
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A total of 123 blood samples were included in our final analysis, covering both the viral replication (72 samples) and convalescent (41 samples) phases, and 10 plasma samples were collected from healthy participants to serve as controls. The collected samples were centrifuged at 3,000 rpm for 5 min and the supernatant was stored at −80°C before analysis. Metabolites were extracted from plasma, and UPLC-MS/MS analysis was performed using a Waters Acquity UPLC coupled with a Xevo G2-Q-Tof (Waters, Milford, MA, USA) in both positive and negative modes. The obtained raw data were pre-processed using Progenesis QI ver. 2.2 (Nonlinear Dynamic). Metabolites were identified by searching the HMDB library (https://hmdb.ca/spectra/ms/search ). Pathway analysis was performed using the MetaboAnalyst 4.0 online tool (http://www.metaboanalyst.ca/ ). Detailed information is outlined in supporting documents.
4
Spectroscopic Analysis of Natural Products
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Optical rotation was measured with a Jasco P2000 polarimeter (Jasco Corporation, Tokyo, Japan), and FT-IR spectra using a Jasco FT/IR-4200 (Jasco corporation, Japan). ECD and UV spectra were recorded with an Applied Photophysics Chirascan-plus CD spectrometer. 1H, 13C and 2D NMR spectra were obtained on a Varian 400 (Varian, Palo Alto, CA, USA)-400MHz. Waters Xevo G2 Q-TOF, (Waters, Milford, MA, USA) spectra were measured on a Q-TOF mass spectrometer. Semi-preparative high-performance liquid chromatography (HPLC) was performed on a Gilson 321 pump, Gilson 172 Diode Array Detector (Gilson, Middleton, WI, USA). YMC-pack Ph, 250 × 20 mm (YMC, Tokyo, Japan) and Luna 5u C18 column 250 × 10 nm (Phenomenex) as HPLC columns were used. MPLC was run on Isolera One (Biotage, Cardiff, UK). Solvents for HPLC were acetonitrile (MeCN) (HPLC grade) and methanol (HPLC grade), purchased from SK Chemical (Seoul, Korea). Water was purified using a MIlli-Q system (Millipore, Bedford, MA, USA). Column chromatography was performed on C-18 RP silica gel (Cosmosil, Kyoto, Japan) and Sephadex LH-20 (GE Healthcare, Stockholm, Sweden). TLC analysis was run on silica gel 60 F254 plates (Marck, Darmstadt, Germany). The spots were visualized by spraying with 10% aqueous H2SO4.
5
Electrolysis Technique Characterization
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Unless otherwise noted, materials were obtained from commercial suppliers and used without further purification. The instrument for electrolysis is a dual display potentiostat (DJS-292B) (made in China). The anodic electrode was a BDD (20 mm by 30 mm by 0.625 mm) or platinum plate electrode (20 mm by 20 mm by 0.2 mm), and the cathodic electrode was a platinum plate (20 mm by 20 mm by 0.2 mm). Thin-layer chromatography was performed using glass 0.25-mm silica gel plates. Flash column chromatography was carried out using 300- to 400-mesh silica gel at medium pressure. Cyclic voltammograms were recorded on a CHI 760E potentiostat. 1H nuclear magnetic resonance (NMR) and 13C NMR spectra were recorded at 25°C on Bruker Advance 400 M NMR spectrometers and Bruker Advance 500 M NMR or 600 M spectrometers. High-resolution mass spectral analysis was performed on a Waters XEVO G2 Q-TOF. Optical rotations were determined at 589 nm (sodium D line) by using a Perkin-Elmer-343 polarimeter. The measurement of enantiomeric excesses was performed on a Waters-Alliance (2998, Photodiode Array Detector).
6
Purification and NMR Characterization of Compounds
7
UPLC-MS Protocol for Compound Analysis
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Ultra-performance liquid chromatography (UPLC) determinations were performed using an AQUITYTM UPLC system (Waters Corp., Milford, MA, USA) equipped with a binary gradient system, an auto-injector, and a UV-Visible detector. Samples (2.0 μL) were separated on a BEH C18 column (2.1 × 100 mm, 1.7 μm) at a flow rate of 0.4 mL/min and eluted using a linear gradient of two mobile phases containing 0.1% formic acid (A: water; B: acetonitrile). A chromatographic gradient was optimized as follows: 0 min, 10% B; 0–8 min, 10–25% B; 8–11 min, 25–90% B; 11–12 min, 90–100% B; 12–13.3 min, 100% B; and 13.4 min, back to 100–10% B. Mass spectrometry was performed using a quadrupole time-of-flight mass spectrometer (Xevo G2 QT of, Waters Corp.) equipped with an electrospray ionization (ESI) interface in the negative ion mode. It was operated using the following parameters: cone voltage 40 V, capillary voltage 2500 V, source temperature 110 °C, and desolvation temperature 350 °C. A sprayer with a reference solution of leucine-enkephalin ([M − H]−m/z 554.2615) was used as the lock mass. All the extraction and chromatographic solvents were LC/GC-MS grade for analysis (J. T. Baker, Phillipsburg, NJ, USA).
8
Platinum-Quadruplex DNA Interactions
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Negative electrospray ionization mass spectrometry (ESI-MS) was performed on the Xevo G2 QTOF (Waters) or solariX ESI (Bruker) mass spectrometer. G-quadruplex DNA samples were in 10 μM concentration with 100 mM ammonium acetate buffer (pH 7.0). Different equivalents of the Pt-tripod were added and incubated for 2 h before measurement.
9
Quantitative Proteomics Analysis by UPLC-MS/MS
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Quantitative analysis will be performed essentially on a Waters Xevo G2 qTof mass spectrometer (Waters). In brief, the tryptic peptide sample will be chromatographically separated on M-class UPLC separations module (Waters) incorporating 50 femtomole tryptic digested BSA as the internally spiked protein quantification standard. Peptide elution will be executed through a 75 μm × 25 cm BEH C-18 column (Waters) under gradient conditions at a flow rate of 300 nL/min over 70 min at 40 °C. The mobile phase will be composed of acetonitrile as the organic modifier and formic acid (0.1% v/v) for molecule protonation. Mass spectrometry was performed on Xevo G2 qTof (waters) instrument equipped with a nanoflow electrospray ionization (ESI) interface and operated in the data-independent collection mode (MSE). Parallel ion fragmentation will be programmed to switch between low (4 eV) and high (15–45 eV) energies in the collision cell, and data will be collected from 300 to 2000 m/z utilizing glu-fibrinopeptide B (Sigma-Aldrich, m/z 785.8426) as the separate data channel lock mass calibrant. Data will be processed with ProteinLynx GlobalServer v3.0 (waters) for qualification and Progenesis QI for proteomics (Waters) for relative quantification, respectively. Deisotoped results will be searched for protein association and modification from the Uniprot (www.uniprot.org ) human protein database.
10
Ara-Man-mannitol LC-MS/MS Analysis
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The above LC-purified, prereduced, per-O-methylated Araf-(1→6)-Manp-(1→6)-mannitol sample was separated using a Waters UPLC system with a Waters T3 C18 UPLC column (1 × 100 mm, 1.8 µM). Mobile phase A consisted of water + 0.1% formic acid, and mobile phase B consisted of acetonitrile + 0.1% formic acid. Flow rate was held constant at 0.2 mL/min, and the column temperature was set to 50 °C. The gradient consisted of 99.9% A for 1 min, a linear ramp to 99.9% B over 12 min, a 3-min hold at 99.9% B, and a return to starting conditions over 0.05 min with 3.95 min of equilibration. For MS/MS analysis, the UPLC eluent was directed to a Waters Xevo G2 Q-TOF running in positive-ionization mode. Mass was calibrated using sodium formate with less than 1-ppm mass error. LockMass reference of leucine enkaphalin was used to ensure mass accuracy through the run. The electrospray source conditions included capillary voltage of 2.2 kV, cone voltage = 30 V, extraction cone = 4 V, temperature = 150 °C, desolvation temperature of 350 °C, and desolvation gas flow of 800 L/h. Data were acquired in centroid mode from m/z 50–1200 using 0.2-s scan times. Collision energy 45 V was identified optimal to induce MS/MS fragmentation. Data analysis were performed using MassLynx V4. 1 SCN803 (Waters) software.
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