Rxi 1ms column

Accurate mass measurements were performed at the University of Hull using a Bruker Maxis Impact QqTOF MSMS (Coventry, UK). Before mass measurement the instrument was calibrated against sodium formate over the range 90 to 1550 Da. Resolution used was typically 45000. Samples (as solutions in methanol, 10–5 M) were injected into a solvent stream from a syringe pump at 3 µL min⁻¹ via a 5 µL loop injector. The data was then internally mass measured against an internal calibrant peak from hexakis(1H,1H,4H-hexafluorobutyloxy)phosphazine (CAS No. 186406-47-2) C₂₄H₁₈O₆N₃P₃F₃₆m/z 1220.99064. An average result from 3–5 separate injections is quoted. The mass was measured and calculated using Bruker DataAnalysis 4.2 software (Coventry, UK).
GCMS. A Perkin-Elmer Autosystem XL GC coupled with a Perkin-Elmer Turbomass (Seer Green, UK) quadrupole MS was used, equipped with a Restek Rxi-1MS column 30 m (l) × 0.25 mm (i.d.) 0.25 µm film thickness. He was used as the carrier gas (mobile phase) at 1 mL·min⁻¹. A 1.0 µL sample injection volume with 20:1 split was employed. The scan range spanned m/z 600 to m/z 30 in 1 sec in conjunction with positive mode electron ionization at 70 eV.

A published method was adapted for the MaNifB-dependent production of methanethiol^{13 (link),51 (link)}. The procedure involved removal of excess DT from MaNifB via gel filtration with Sephadex G-25 fine resin that was equilibrated with a buffer containing 25 mM Tris–HCl (pH 8). Subsequently, 40 nmol of MaNifB was mixed with 400 nmol SAM in a total volume of 100 μL in a sealed 300-μL glass vial, incubated for 30 min at 25 °C, and quenched by 25 μL of 1 M HCl. The acid-quenched sample was then incubated at 60 °C for 15 min to release the volatile methanethiol into the headspace, followed by equilibration of the sample to room temperature for 10 min, and injection of the entire headspace by a gas-tight syringe onto a Restek Rxi-1ms column (30 m, 0.32 mm ID, 4 μm df) for analysis by GC–MS (Thermo-Fisher Scientific Trace 1300 GC connected to a Thermo-Fisher Scientific ISQ QD single quadrupole mass spectrometry). During each GC–MS run, the GC inlet and oven temperatures were kept at 30 °C, and the mass spectrometry transfer line and ion source were kept at 250 °C. Using SIM conditions in electron ionization mode, methanethiol was detected at an m/z ratio of 47.