Sample preparation (A)

Flash frozen liver sections (~25 mg) from WT or Gclc^−/− mice (3 biological replicates each per TMT sample) were thawed on ice and resuspended in DPBS, supplemented with protease (cOmplete, EDTA-free protease inhibitor cocktail, Roche, #11873580001) and phosphatase (PhosSTOP, Roche, #4906845001) inhibitor tablets. Tissue was homogenized by probe sonication (2 × 10 pulses, 40% power output), and particulate matter was removed by passing samples through 0.4 μm syringe filters. The proteome concentration of the tissue lysates was determined using the DC protein assay (Bio-Rad), normalized to 2 mg/mL, and then 100 μL of each sample was transferred to a LoBind Eppendorf tube containing 48 mg of urea. Samples were reduced with DTT (5 μL of 200 mM stock in H₂O, 10 mM final concentration) and incubated at 65 °C for 15 min, then alkylated with iodoacetamide (5 μL of 400 mM stock in H₂O, 20 mM final concentration) and shaken at 37 °C for 30 min in the dark. Ice-cold MeOH (600 μL), CHCl₃ (200 μL), and H₂O (500 μL) samples were vortexed and then centrifuged (10,000 g, 10 min, 4 °C) to precipitate proteins. The upper layer of supernatant was removed, and ice-cold MeOH (600 μL) was added to wash the protein disc. Samples were vortexed again, centrifuged (16,000 g, 10 min, 4 °C) and then all supernatant was removed to leave a protein pellet. Samples were resuspended in 160 μL EPPS buffer (200 mM, pH 8.0) using a probe sonicator (1 × 10–15 pulses, power output 20%) and then digested with LysC (4 μL of 0.5 μg/μL per sample, resuspended in HPLC grade water, Wako-chemicals, Fujifilm #125-05061) for 2 h at 37 °C in a shaker incubator. Samples were then digested with trypsin (11 μL of 0.5 μg/μL per sample, resuspended in trypsin resuspension buffer containing 20 mM CaCl₂; Promega, #V542A) overnight at 37 °C in a shaker incubator. After incubation with trypsin, the peptide concentration in samples was estimated using a Micro BCA Protein Assay (Thermo Scientific, #23235), and a volume corresponding to 25 μg peptides was transferred to a new low-bind Eppendorf tube per sample. Sample volumes were normalized to 35 μL with EPPS buffer (200 mM, pH 8), diluted with HPLC grade CH₃CN (9 μL), and then labeled (5 μL of 20 μg/μL per sample) with the corresponding TMTsixplex Isobaric Mass Tag (Themo Scientific, #90064B). Samples were incubated at room temperature for 1 h, vortexing intermittently, and then quenched by the addition of hydroxylamine (5 μL of 5% w/v in HPLC water per sample) and incubated for 15 min at room temperature. Samples were then acidified with formic acid (2.5 μL), and 2 µL of each sample was combined in a LoBind Eppendorf and dried using a Speedvac to perform a ratio check. Remaining samples were stored at −80 °C until after experimentally determining TMT channel intensities.