ABSTRACT: OMA1 KO MEFs (10 cm dish) and its rescued cells stably expressing WT or EQ mutant OMA1-Myc were incubated with 0.2% (w/v) formaldehyde for 15 min at 37° C, followed by quenching with 100 mM glycine-NaOH (pH 7.5) for 10 min at room temperature. After once wash with 1× PBS (pH 7.4), the cells were scraped and mitochondria were isolated from the cells as described above. The mitochondrial fractions were then lysed in 1 mL of lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10% (w/v) glycerol, 1 mM EDTA, 0.5% (w/v) digitonin, and protease inhibitor cocktail. After centrifugation, the clarified supernatants were incubated with an antibody against Myc (My3; MBL Life Science, Tokyo, Japan) for 2 h at 4° C. The reactants were then incubated with magnetic SureBeads Protein G (Bio-Rad) overnight at 4° C, and the next day the beads were washed three times with 1× PBS (pH 7.4) and twice with 50 mM ammonium bicarbonate. Proteins on the beads were digested by adding 200 ng trypsin/Lys-C mix (Promega) for 16 h at 37° C. The digests were reduced, alkylated, acidified, and desalted using GL-Tip SDB, and the eluates were evaporated and dissolved in 0.1% trifluoroacetic acid (TFA) and 3% acetonitrile.
LC-MS/MS analysis of the resulting peptides was performed on an EASY-nLC 1200 UHPLC connected to an Orbitrap Fusion mass spectrometer through a nanoelectrospray ion source. Peptides were separated on a 75 µm inner diameter × 150 mm C18 reverse phase column (Nikkyo Technos, Tokyo, Japan) with a linear gradient of 4%-32% acetonitrile for 0-100 min followed by an increase to 80% acetonitrile for 100-110 min. The mass spectrometer was operated in a data-dependent acquisition mode with a maximum duty cycle of 3 s. MS1 spectra were measured with a resolution of 120,000, an automatic gain control (AGC) target of 4e5, and a mass range of 375 to 1,500 m/z. HCD MS/MS spectra were acquired in the linear ion trap with an AGC target of 1e4, an isolation window of 1.6 m/z, a maximum injection time of 35 ms, and a normalized collision energy of 30. Dynamic exclusion was set to 20 s. Raw data were analyzed directly against the Swiss-Prot database restricted to Mus musculus using Proteome Discoverer version 2.4 (Thermo Fisher Scientific) for identification and label-free precursor ion quantification. Search parameters were as follows: (a) trypsin as an enzyme with up to two missed cleavages; (b) precursor mass tolerance of 10 ppm; (c) fragment mass tolerance of 0.6 Da; and (d) cysteine carbamidomethylation as a fixed modification; and (e) protein N-terminal acetylation and methionine oxidation as variable modifications. Peptides were filtered with a false discovery rate of 1% using the percolator node. Normalization was performed so that the total sum of abundance values for each sample was equal across all peptides.
