Thermo vanquish uplc system

Samples were analysed by LC-MS/MS using Thermo TSQ Quantiva with Thermo Vanquish UPLC system (Thermo Fisher Scientific, Hemel Hempstead, UK) with a calibration range of 2.5–1000 nM for DMT. Separation of analytes was performed using Luna Omega 2.1  ×  50 mm, 2.6 µm column at 65 °C with a 1 µl/sample. The mobile phase consisted of solvent A [0.1% (v/v) formic acid in water] and solvent B [0.1% (v/v) formic acid in acetonitrile] using a gradient of 5–99% of B (0–1.35 min), 5% (1.36–11.8) and flow rate of 0.8 ml/min. MS was performed with an electrospray ionisation (ESI) source in the positive-ionization mode with multiple reaction monitoring and a sheath gas of 54 Arb and aux gas of 17 Arb. The ion spray voltage was set at 4000 V and the source temperature was 450 °C. The mass-to-charge transition (m/z) of precursor and product ions for DMT was identified as: (m/z) 189.13 → 58.15, 144.04.

A Thermo Vanquish UPLC system (ThermoFisher, Waltham, MA, USA) coupled with a Hybrid Quadrupole Orbitrap Q Exactive™ mass spectrometer (ThermoFisher) was used for small molecule analysis of study samples. Bourbon molecules were separated using an Xbridge BEH Amide (2.5 μm, 2.1 × 150 mm, Waters, Milford, MA, USA) column. The mobile phases consisted of solvents A and B, where A contained 5 mM ammonium acetate, 0.1% acetic acid in 90% H2O/10% acetonitrile (ACN), and B contained 5 mM ammonium acetate, 0.1% acetic acid in 10% H2O/90% ACN. A gradient of mobile phase injection was used for 20 min at a flow rate of 0.3 ml/min, starting with 30% of mobile phase A and increasing to 70% at 5 min. The proportion of mobile phase A was maintained at 70% until 9 min and then started to decrease to 30% until 11 min. The percentage of solvent A was then kept at 70% until 20 min, after which it gradually returned to 30% to prepare for the next injection. To observe and prove the consistency of the instrument during testing and to enable data normalization, a pooled QC was added to the sample tray after every 10 sample injections in both sequences. A blank sample was added after each QC sample.

Reagents used for molecular
biology experiments were purchased from New England Biolabs (NEB,
Ipswich, MA). Other chemicals were purchased from Sigma-Aldrich (St.
Louis, MO) or Thermo Fisher Scientific (Waltham, MA), unless stated
otherwise. Escherichia coli DH5α,
BL21(DE3), and Shuffle T7 Express (Δgor/ΔtrxB/DsbC*) strains were purchased from NEB, and Origami2
strain (Δgor/ΔtrxB)
was purchased from Novagen/EMD-Millipore. Plasmid vectors were obtained
from Novagen/EMD-Millipore for pET expression vectors. Media was purchased
from Teknova (Hollister, Ca). LC–MS analyses were performed
using a TripleTOF 6600 Quadrupole Time-Of-Flight (SCIEX) equipped
with an ACQUITY ultraperformance liquid chromatography (UPLC) system
(Waters). Plasma stability assay and in vivo PK assay were performed
on a Thermo Q Exactive HF-X mass spectrometer coupled to a Thermo
Vanquish UPLC system.