Mitochondrial Proteome Isolation and Digestion

Isolated mitochondria were lysed in urea lysis buffer (8 M urea in 40 mM Tris, 30 mM NaCl, 1 mM CaCl₂, 1× cOmplete ULTRA mini EDTA-free protease inhibitor tablet; pH = 8.0), as described previously.^{16 (link),20 (link)} The samples were subjected to three freeze-thaw cycles and sonicated with a probe sonicator in three 5s bursts (Q Sonica #CL-188; the amplitude of 30). Samples were centrifuged at 10 000×g for 10 min at 4°C. Protein concentration was determined by BCA. Equal amounts of protein were reduced with 5 mM DTT at 37°C for 30 min and then alkylated with 15 mM iodoacetamide for 30 min in the dark at room temperature. Unreacted iodoacetamide was quenched with DTT (15 mm). Initial digestion was performed with Lys C (ThermoFisher Cat# 90307; 1:100 w:w) for 4 h at 37°C. Following dilution to 1.5 M urea with 40 mM Tris (pH = 8.0), 30 mM NaCl, 1 mM CaCl₂, samples were digested overnight with trypsin (Promega; Cat# V5113; 50:1 w/w) at 37°C. Samples were acidified to 0.5% TFA and then centrifuged at 4000×g for 10 min at 4°C. The supernatant containing soluble peptides was desalted, as described previously^{20 (link)} and then eluate was frozen and subjected to speedvac vacuum concentration.