Ultimate 3000 rslc binary pump

LC-MS analysis was performed on an Acquity CSH C18 column held at 50 °C (100 mm × 2.1 mm × 1.7 μL particle size; Waters) using an Ultimate 3000 RSLC Binary Pump (400 μL/min flow rate; Thermo Scientific). Mobile phase A consisted of 10 mM ammonium acetate in ACN/H₂O (70:30, v/v) containing 250 μL/L acetic acid. Mobile phase B consisted of 10 mM ammonium acetate in IPA/ACN (90:10, v/v) with the same additives. Mobile phase B was held at 50% for 1.5 min and then increased to 95% over 6.5 min where it was held for 2 min. The column was then reequilibrated for 3.5 min before the next injection. 10 μL of sample were injected by an Ultimate 3000 RSLC autosampler (Thermo Scientific). The LC system was coupled to a Q Exactive mass spectrometer by a HESI II heated ESI source kept at 300 °C (Thermo Scientific). The inlet capillary was kept at 300 °C, sheath gas was set to 25 units, auxiliary gas to 10 units, and the spray voltage was set to 4,000 V and 5,000 V for positive and negative mode respectively. CoQ and its intermediates were targeted for quantification. The MS was operated in positive or negative mode depending on the intermediate being targeted. Metabolite signals were integrated and normalized to a CoQ₆ internal standard. Error bars represent s.d. of biological triplicate measurements. See Table S1 for targeted CoQ species.

Sample analysis by LC–MS/MS was performed in randomized order on an Acquity CSH C18 column held at 50 °C (2.1 mm × 100 mm × 1.7 μm particle diameter; Waters) using an Ultimate 3000 RSLC binary pump (400 μl min⁻¹ flow rate; Thermo Fisher) or a Vanquish binary pump. The same mobile phase and gradient as for the DO samples were used.
For the validation experiments, 10 μl of caecal or culture extract were injected by a Vanquish Split Sampler HT autosampler (Thermo Fisher) coupled to a Q Exactive HF mass spectrometer by a HESI II heated ESI source. Both source and inlet capillary were kept at 350 °C (Thermo Fisher). Sheath gas was set to 25 units, auxiliary gas to 15 units and spare gas to five units, while the spray voltage was set to 3,500 V and the S-lens RF level to 90. The MS was operated in polarity switching dd-MS2 mode (Top2), acquiring positive and negative mode MS1 and MS2 spectra during the same separation. MS acquisition parameters were 30,000 resolution, 1 × 10⁶ AGC target for MS1 and 5 × 10⁵ AGC target for MS2 scans, 100 ms MS1 and 50 ms MS2 ion accumulation time, 200 to 2,000 Th MS1 scan range, 1.0 Th isolation width for fragmentation and stepped HCD collision energy (20, 30, 40 units).