U2OS and hepatocyte cell pellets were thawed on ice and metabolites were extracted using a modified Bligh-Dyer method (Bligh and Dyer, 1959 (link); Tambellini et al., 2013 (link)). Briefly, a methanol:chloroform (2:1) mixture (300μl) was added to the cell pellets, then vortexed and sonicated for 15 min. Chloroform and water (100μl each) were then added and samples were vortexed. Organic and aqueous layers were separated by centrifugation at 13,300rpm, for 7mins at 4°C. Aqueous layer was dried in a speed vacuum for 4 h until dry and re-dissolved in a mixture of 50:50 acetonitrile:water. The samples were centrifuged at 13300rpm for 5min to remove fine particulates. The supernatant was transferred to a LC-MS certified sample vial for Ultraperformance liquid chromatography with 95:5 water:acetonitrile 20mM ammonium acetate, pH 9 (solvent A) and 100% acetonitrile (Solvent B), on a Waters Acquity UPLC coupled to a Waters TQD mass spectrometer (Waters Corporation, USA). The same procedure was used for liver tissue analysis with 50 mg of liver tissue extracted per animal.
Waters tqd mass spectrometer
Manufactured by Waters Corporation
Sourced in United States
The Waters TQD mass spectrometer is a high-performance analytical instrument designed for precise and sensitive measurement of chemical compounds. It utilizes tandem quadrupole technology to provide accurate mass analysis and quantification of a wide range of analytes in complex samples.
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3 protocols using waters tqd mass spectrometer
1
Metabolite Extraction from Cell Pellets
2
Oxidation of α-CTx TxID Peptide
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Met is easily modified to methionine sulfoxide under oxidative conditions. α-CTx TxID (0.2 mg) firstly dissolved in 100 μL of 60% acetonitrile in ddH2O, which was incubated with 5 mL of 10% hydrogen peroxide (H2O2) in 20 min at room temperature. Then reaction products were separated using semi-preparative RP-HPLC with a linear gradient as above conditions. The purity of oxidative products was identified by Waters ACQUITY UPLC™ System with BEH300 C18 column (50 × 2.1 mm, 1.7 µm) (Waters Corporation, Milford, MA, USA). The molecular mass was measured on a Waters TQD mass spectrometer equipped with an electrospray ionization source (Waters Corporation, Milford, MA, USA).
3
Comprehensive Characterization of Compounds
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All commercially available chemicals and reagents were used without any further purification. Melting points of compounds were measured on Büchi SMP-560 apparatus (Büchi Labortechnik AG, Flawil, Switzerland) and were given as uncorrected. NMR spectra were recorded on a JEOL ECZ-500R spectrometer equipped with a JEOL Royal Probe HFX (JEOL USA Inc., Peabody, MA, USA) at 500 MHz for 1H and at 126 MHz for 13C; with solvent as the internal standard. The UPLC–MS system equipped with a Waters ACQUITY® UPLC® (Waters Corporation, Milford, MA, USA) and a Waters TQD mass spectrometer (electrospray ionization (ESI) mode tandem quadrupole) was used for the analysis. The investigated compounds were analyzed on a Acquity UPLC BEH (bridged ethyl hybrid) C18 column (2.1 × 100 mm, 1.7 µm), comprising an Acquity UPLC BEH C18 VanGuard precolumn (2.1 × 5 mm, 1.7 µm). The purity of compounds was determined by UPLC-MS and was above 98%.
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