Shotgun proteomics (mouse)

Per sample 10μg of isolated mitochondria were used. SDS was added to a final concentration of 2% for efficient solubilization, prior to tryptic protein digest using a modified FASP protocol (Wiśniewski et al., 2009). Proteomic measurements were performed on a Q-Exactive HF mass spectrometer (Thermo Scientific) online coupled to an Ultimate 3000 nano-RSLC (Dionex). Peptides were separated on a C18 nanoEase MZ HSS T3 column (100Å, 1.8 µm, 75 µm x 250 mm; Waters) in a 95 min non-linear acetonitrile gradient. Precursor (scan range 300 – 1500 m/z) and TOP10 fragment spectra of charges 2-7 were acquired in the orbitrap mass detector of the mass spectrometer, at resolutions of 60000 and 15000 respectively with each a maximum injection time of 50 ms and a dynamic exclusion of 30 s.