Mass spectrometry-based cellular thiamine-uptake assays

The uptake of deuterated thiamine (thiamine-d3) was measured in hSLC19A3-wt and mock transfected cells in using LC-MS/MS. For this assay, Expi293F^TM cells were transfected as described above and incubated for 48 h at 37 °C, 8% (v/v) CO₂, and 270 rpm prior to the experiment. The cells were then pelleted for 5 min at 250×g and resuspended in 1×PBS (pH 7.4) at a cell density of 2 million cells per mL. The cell suspension was subsequently transferred to a 96-deep well plate in three technical replicates per condition (n = 3, 1 mL per well, corresponding to 2 million cells per condition). 200 μM of the selected compounds were added to the respective wells and the cells were incubated for 1 min on a shaking platform. Subsequently, 2 μM of thiamine-d3 were added and the plate was incubated for another 5 min on a shaking platform. Afterwards, the cells were pelleted for 5 min at 500×g and washed 3× with 1 mL 1×PBS (pH7.4), before transferring them to a fresh 96-deep well plate. The cell pellets were stored at –70 °C. For the LC-MS/MS analysis, the samples were extracted by adding 500 µL of a mixture of H₂O:MeOH:ACN (1:1:1, v/v), containing 5% (v/v) formic acid. After vortexing and ultrasonication in a water bath for 5 min at 4 °C, samples were incubated for 20 min at –20 °C. Ultimately, samples were centrifuged at 15,000×g and 4 °C for 10 min using a 5415 R microcentrifuge (Eppendorf, Hamburg, Germany). The supernatants were then transferred for LC-MS analysis, which was initiated within one hour after sample preparation. LC-MS/MS analysis was performed on a Vanquish UHPLC system coupled to an Orbitrap Exploris 240 high-resolution mass spectrometer (Thermo Fisher Scientific, MA, USA) in positive ESI (electrospray ionization) mode. Chromatographic separation was carried out on an Atlantis Premier BEH Z-HILIC column (Waters, MA, USA; 2.1 mm × 100 mm, 1.7 µm) at a flow rate of 0.25 mL/min. The mobile phase consisted of water:acetonitrile (9:1, v/v; mobile phase phase A) and acetonitrile:water (9:1, v/v; mobile phase B), which were modified with a total buffer concentration of 10 mM ammonium formate. The aqueous portion of each mobile phase was adjusted to pH 3.0 via addition of formic acid. The following gradient (8 min total run time including re-equilibration) was applied (time [min]/%B): 0/90, 3/85, 3.5/60, 4/60, 5/90, 8/90. Column temperature was maintained at 40 °C, the autosampler was set to 4 °C and sample injection volume was set to 3 µL. Analytes were recorded via a full scan with a mass resolving power of 120,000 over a mass range from 60 to 900 m/z (scan time: 100 ms, RF lens: 70%). MS/MS fragment spectra were recorded via targeted product ion scans for Thiamine ([M]⁺, m/z = 265.1118) and Thiamine-d₃ ([M]⁺, m/z = 268.1306) at a resolving power of 15,000, stepped collision energies [%]: 20/35/50, and total cycle time of 3 s. Ion source parameters were set to the following values: spray voltage: 3500 V, sheath gas: 30 psi, auxiliary gas: 5 psi, sweep gas: 0 psi, ion transfer tube temperature: 350 °C, vaporizer temperature: 300 °C.

All experimental samples were measured in a randomized manner. Pooled quality control (QC) samples were prepared by mixing equal aliquots from each processed sample. Multiple QCs were injected at the beginning of the analysis in order to equilibrate the analytical system. A QC sample was analyzed after every 6^th experimental sample to monitor instrument performance throughout the sequence. For determination of background signals and subsequent background subtraction, an additional processed blank sample was recorded. Data was processed using TraceFinder 5.1 and raw peak area data was exported for relative metabolite quantification.