Methanethiol Production from MaNifB and SAM

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.