Shotgun proteomics

Cells were harvested and centrifuged 400g for 10 min at 4°C. Cell pellets were resuspended in STE-Buffer (250mM sucrose, 5mM Tris pH 7, 1mM EGTA, PI mix 1:500) and lysed with a 27-gauge needle. Samples were centrifuged 10 min at 800g to remove nuclei, then 10 min at 15.000g to remove mitochondria and finally 1 hour 100.000g. The resulting pellets were washed twice with 100mM Na2CO3 and centrifuged 30 min at 100000g after each wash. Pellets from membrane preparations were dissolved in lysis buffer (150 mM NaCl, 50 mM TrisHCl pH 7.5, 2 mM EDTA, 1% Triton X-100). The protein concentration was estimated using the Pierce 660nm protein assay (Thermo Fisher Scientific, US). A protein amount of 15 µg was subjected to proteolytic digestion with trypsin and LysC (Promega, Germany) using the filter-aided sample preparation (FASP) with Vivacon centrifugal concentrators (30 kDa cut-off, Sartorius, Germany) according to a standard protocol. Peptides were enriched and desalted using stop and go extraction with self-packed C18 Tips (3M Empore, US). Eluted peptides were dried by vacuum centrifugation and dissolved in 20 µL 0.1% formic acid. Peptides were analyzed on an Easy nLC 1000 nanoHPLC (Thermo Sientific, US) which was coupled online via a Nanospray Flex Ion Source (Thermo Sientific, US) equipped with a PRSO-V1 column oven (Sonation, Germany) to a Q-Exactive mass spectrometer (Thermo Sientific, US). 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, Germany) 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 mass spectrometric MS scans were acquired at a resolution of 70,000 (m/z range: 300-1400, AGC target: 3E+6). The ten most intense peptide ions per full MS scan were chosen for peptide fragmentation (resolution: 17,500, isolation width: 2.0 m/z, AGC target: 1E+5, NCE: 25%). A dynamic exclusion of 120 s was used for peptide fragmentation.