Shotgun proteomics

LUHMES cell culture LUHMES cells were cultured and differentiated as described previously (Sholz et al., 2011). 0.5 Million undifferentiated LUHMES (unLUHMES) cells were seeded into a Poly-D-Lysine coated 6-well containing growth media (DMEM F12, 1% N2 Supplement, 0.04 µg/mL bFGF) and harvested at day three in vitro. The live cell count was around 1 Million cells using Trypan blue and an automated cell counter (Biorad). For differentiation, 1 Million unLUHMES were seeded into a Poly-D-Lysine coated 6 well plate containing differentiation media (DMEM F12, 1% N2 Supplement, 1 µg/mL Tetracycline, 2 ng/mL GDNF, 500 µg/mL Dibutyril cAMP). At day 6 in vitro the morphology of difLUHMES were validated under the microscope and the cells were harvested. Primary murine midbrain neuron culture The midbrains were isolated at embryonic day 16.5 from C57BL/6 mice and cultured as described by Kuhn et al., 2012. In brief, midbrains were isolated, after removal of the meninges the tissue was digested with papain, dissociated and 1 Million living cells were seeded onto a Poly-D-Lysine coated 6-well dish using Neurobasal Medium containing B27. The live cell count was determined using Trypan blue and an automated cell counter (Biorad). After 4h and at day 4 in culture, the medium was exchanged with fresh cultivation media (Neurobasal + B27 + 0.5mM glutamine + 1% P/S). The neurons were lysed at day 6 in vitro. Sample preparation Cells were washed three times with 1 mL 1x PBS before being lysed in 250 µL STET buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 50mM Tris pH 7.5). Lysate was incubated for 20 min on ice and cleared from cellular debris by a centrifugation step at maximum speed for 10 min at 4°C. Whole protein concentration was determined with a BCA protein assay kit according to manufacturer´s instructions (Interchim, UP40840A). 40 µg protein extract was digested of each biological replicate using the Filter Aided Sample Preparation (FASP) protocol (Wisniewski et al., 2009). 30 kDa Vivacon filters were used. Desalting was performed using self-made C18 columns for stop-and-go extraction (Rappsilber et al., 2003). Afterwards, peptides were dried by vacuum centrifugation and resolved in 0.5% formic acid. Mass spectrometry analysis Proteome analysis was performed on a LC-MS/MS set up by coupling an EASY-nLC 1200 UHPLC system (Thermo Fischer Scientific) with a Q-ExactiveTM Hybrid Quadrupole-OrbitrapTM mass spectrometer (Thermo Fischer Scientific). 1.5 µg were injected of each biological replicate. A C18 column (30cm length, 75 µm ID, self-made) packed with ReproSil-Pur 120 C18-AQ resin (Dr. Maisch GmbH, 1.9 µm) was utilized for peptide separation. A binary gradient of mass spectrometry grade H2O and 80% acetonitrile (B) was chosen with a gradient time of 120 min, 50°C column temperature, flow rate of 250 nL/min: (3% B 0 min, 6% B 2 min, 30% B 92 min, 44%B 112 min, 75% B 121 min). All samples were analyzed wit data dependent acquisition by choosing the top 15 most abundant peptides for collision-induced dissociation (CID) fragmentation. A scan range of 300 to 1400 m/z, full scan at 120,000 resolution, maximum injection time (IT) of 50 ms and automatic gain control (AGC) of 3x106 were used for MS1. The following settings were chosen for MS2: Resolution of 15,000; maximum IT of 100 ms; AGC of 1x105; Isolation window of 1.6 m/z; dynamic exclusion of 120 s.