Shotgun proteomics

Samples for mass spectrometry were obtained from 8- to 9-week-old, male mice. Mitochondria were immunopurified from cerebellum according to the described protocol with the alteration that the final mitochondrial pellet was washed twice in IB without EDTA and BSA. Sample were lysed in 100 µL SDT lysis buffer (4% w:v SDS, 100 mM DTT in 100 mM Tris-HCl pH 7.6) by heating for 5 min at 95°C and ultrasonication (Vialtweeter: 6 times for 30 s, 100% Amplitude, 50% cycle, max power; Hielscher Ultrasonics). Debris and non-dissolved material was removed by centrifugation for 10 min at 20,000×g. Protein concentrations were estimated using the Pierce 660 nm assay supplemented with the ionic detergent compatibility reagent (Thermo Fisher Scientific) with a dilution series of bovine albumin in SDT buffer for calibration. An amount of 15 µg was subjected to protein digestion using the filter-aided sample preparation (FASP) with small modifications. Briefly, 30 kDa Vivacon filters (Sartorius) were used. The alkylation step was followed by incubation with 50 units of benzonase (Sigma-Aldrich) in 50 mM Tris-HCl, pH 8 and 1 mM MgCl2. Afterwards, three washing steps with 100 µl of 8M urea in 100 mM Tris-HCl pH 8 were implemented. Proteins were digested for 16 h with 0.3 µg of LysC (Promega) followed by 4 h incubation with 0.15 µg of trypsin (Promega). Peptides were eluted into collection tubes and desalted using C18 stop-and-go extraction. Samples were dried after elution by vacuum centrifugation. Samples were analyzed on an Easy nLC-1000 nano UHPLC (Thermo Fisher Scientific) coupled online via a Nanospray Flex electrospray ion source (Thermo Fisher Scientific) equipped with a column oven (Sonation) to either a Q-Exactive or a Q-Exactive HF mass spectrometer (Thermo Fisher Scientific). An amount of 1.3 µg of peptides were separated on self-packed C18 columns (300 mm × 75 µm, ReproSil-Pur 120 C18-AQ, 1.9 µm; Dr. Maisch) using a binary gradient of water (A) and acetonitrile (B) supplemented with 0.1% FA (0 min, 2% B; 5 min, 5% B; 185 min, 25% B; 230 min, 35% B; 250 min, 60% B). The dataset from Purkinje cells was analyzed on a Q-Exactive MS. Full MS spectra were acquired at a resolution of 70,000 (AGC target: 3E+6). The 10 most intense peptide ions were chosen for fragmentation by higher-energy collisional dissociation (resolution: 17.5 k, isolation width: 2 m:z, AGC target: 1E+5, NCE: 25%). A dynamic exclusion of 120 s was applied for fragment ion spectra acquisition.