Shotgun proteomics

Post-nuclear supernatants were prepared from testis of wild type and SPPL2c-/- mice (n=5 per genotype). After removal of the tunica albuginea, testicles from two wild type mice were minced with an ultraturrax in 250 mM sucrose, 10 mM HEPES-NaOH, pH 7.4 and 1 mM EDTA (HB, homogenisation buffer) and then further homogenised by eight strokes of a Potter homogeniser. For sedimentation of nuclei, the tissue homogenate was centrifuged at 750 x g for 10 min. The supernatants were collected as post-nuclear supernatants. Total membrane fractions were obtained by ultracentrifugation at 146,944 x gmax for 1 h at 4°C. The supernatant was recovered as the cytosolic fraction for further analysis. The sedimented membranes were resuspended in 0.1 M Na2CO3, pH 11.5, incubated on ice for 1 h and centrifuged again as described above. Membranes were washed once in 10 mM HEPES-NaOH, pH 7.4, and resuspended in the same buffer after a final sedimentation. Aliquots of both the cytosolic and the carbonate-washed membrane fractions were subjected to proteomic analysis. A protein amount of 15 µg per sample was subjected to proteolytic digestion with 0.3 µg LysC (Promega) and 0.15 µg trypsin (Promega) using the filter assisted sample preparation (FASP) protocol with 30 kDa Vivacon spin filters (Sartorius). Proteolytic peptides were desalted by stop and go extraction with C18 tips. The purified peptides were dried by vacuum centrifugation. Samples were dissolved in 20 µL 0.1% formic acid. Peptides were analysed on an Easy nLC 1000 nanoHPLC (Thermo Scientific) which was coupled online via a Nanospray Flex Ion Source (Thermo Sientific) equipped with a PRSO-V1 column oven (Sonation) to a Q-Exactive HF mass spectrometer (Thermo Scientific). An amount of 1.3 µg of peptides was separated on an in-house packed C18 column (30 cm x 75 µm ID, ReproSil-Pur 120 C18-AQ, 1.9 µm, Dr. Maisch GmbH) using a binary gradient of water (A) and acetonitrile (B) supplemented with 0.1% formic acid (0 min., 2% B; 3:30 min., 5% B; 137:30 min., 25% B; 168:30 min., 35% B; 182:30 min., 60% B) at 50°C column temperature. A data-dependent acquisition method was used. Full MS scans were acquired at a resolution of 120,000 (m/z range: 300-1400, AGC target: 3E+6). The 15 most intense peptide ions per full MS scan were selected for peptide fragmentation (resolution: 15,000, isolation width: 1.6 m/z, AGC target: 1E+5, NCE: 26%). A dynamic exclusion of 120 s was used for peptide fragmentation.