Quantifying Cysteine and Glutathione Pools

For determination of the relative Cys and GSH pool sizes across compartments, previously established LC-MS conditions were employed^{5 (link),24} For chromatographic metabolite separation, a Vanquish UPLC system was coupled to a Q Exactive HF (QE-HF) mass spectrometer equipped with HESI (Thermo Fisher Scientific, Waltham, MA, USA). Samples were run on an Atlantis Premier BEH Z-HILIC VanGuard FIT column, 2.5 µm, 2.1 mm × 150 mm (Waters, Milford, MA, USA). The mobile phase A was 10 mM (NH₄)₂CO₃ and 0.05% NH₄OH in H₂O, while mobile phase B was 100% ACN. The column chamber temperature was set to 30 °C. The mobile phase condition was set according to the following gradient: 0-13 min: 80% to 20% of mobile phase B, 13–15 min: 20% of mobile phase B. The ESI ionization mode was negative, and the MS scan range (m/z) was set to 65-975. The mass resolution was 120,000 and the AGC target was 3 × 10⁶. The capillary voltage and capillary temperature were set to 3.5 kV and 320 °C, respectively. 5 μL of sample was loaded. For each experimental run, 5 μL of extraction solvent were run in triplicate as blank controls; these were disbursed throughout the run to signal changes in the integrity of metabolite detection. The LC-MS metabolite peaks were manually identified and integrated by El-Maven (Version 0.10.0) by matching with a previously established in-house library^{5 (link)}.