https://doi.org/10.1038/s41598-023-27538-y
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Ultra-high-performance liquid chromatography tandem mass spectrometry
Although radiometric assays are highly sensitive in their ability to detect extremely low levels of enzyme activity, they do not provide information on identities of the reaction products. Thin-layer chromatography is useful for visualization of a range of reaction products, but the technique often lacks resolution. uHPLC-MS/MS, however, provides both a high level of sensitivity and resolution in analyte identification and represents a gold standard for analyte separation, detection, and quantification. Additionally, our uHPLC-MS/MS method utilizes multiple techniques to definitively confirm analyte identification, including retention time via uHPLC, triple quadrupole multiple reaction monitoring of both precursor and product ions, and spiked deuterated internal reference standards for NMT and DMT.
To determine concentrations of NMT and DMT, 10 µL of the 30% methanol resuspension (see ‘Enzyme assays’ section) was spiked with 10 µL of a mixture containing 100 nM d3-NMT, and d6-DMT (Toronto Research Chemicals) as deuterated internal standards to normalize for extraction efficiency and mass spectrometry ionization efficiency. Calibration solutions of NMT and DMT were prepared in 30% methanol, to create a calibration range of 0.625–125 nM for NMT, and 0.125–25 nM for DMT. Calibration standards were spiked with the internal standard solution as described above and injected prior to each day of analysis. 6-point calibration curves were determined based on the peak area ratio of the calibration standard to the internal standard by linear regression. All samples and standards were analyzed using a Phenomenex Kinetex C18 chromatography column (100 × 2.1 mm, 1.7 μm, 100 Å) on a Vanquish uHPLC (Thermo Fisher Scientific, Gemering, Germany) interfaced to a TSQ Quantum Ultra triple quadrupole mass spectrometer (Thermo Fisher Scientific, San Jose, CA). Mobile phase A was 10 mM ammonium formate with 0.15% (v/v) formic acid in water. Mobile phase B was acetonitrile. The gradient used was as follows: initial, 5% B; 0.01 min, 19% B; 0.68 min, 26% B, 1.05 min, 75% B; 1.8 min, 100% B; 2.2 min, 100% B; 2.3 min, 5% B; 3.0 min, 5% B at 600 μL/min. The sample injection volume was 7.5 µL, the autosampler was kept at ambient temperature, and the column was held at 30 °C in still air mode. NMT eluted at 0.92 min, and DMT at 0.96 min. Electrospray ionization was used in positive mode at 4 kV. The capillary temperature was 325 °C, the vaporizer temperature was 300 °C, the sheath gas pressure was 50, and the auxiliary gas pressure was 10. Ions were detected in tandem mass spectrometry mode. The scanning parameters for all 4 analytes were as follows. NMT: precursor ion m/z = 175, product ion m/z = 143.8, collision energy = 14, tube lens = 53. d3-NMT: precursor ion m/z = 178, product ion m/z = 143.8, collision energy = 14, tube lens = 65. DMT: precursor ion m/z = 189.1, product ion m/z = 144.1, collision energy = 19, tube lens = 49. d6-DMT: precursor ion m/z = 195.1, product ion m/z = 144.1, collision energy = 19, tube lens = 49. Automated peak integration was performed using XCalibur 3.0 MS software, and all peaks were visually inspected to ensure proper integration.
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