Shotgun proteomics (mouse)

Mice were sacrificed by cervical dislocation (n=4) and brains were subsequently extracted and the injury-site and corresponding area on the contralateral side was removed using a 2.5 mm biopsy punch and the white matter was removed. Samples were homogenized using a (100 μl) dounce (Wheaton #357844) in 100 ul PBS (with protease inhibitor cocktail and Ethylenediaminetetraacetic acid (EDTA)) and directly frozen in liquid nitrogen and stored in -80 °C until tissue protein fractionation. Following centrifugation, we collected the supernatant (protein fraction 0) and then sequentially extracted proteins using the MS analysis adapted decellularization protocol of Schiller et al. (Schiller et al. 2015). In short, we resuspended the pellet in three buffers, each followed by centrifugation for 20 min at 16,000 g. The samples were incubated in buffer 1 (150 mM NaCl, 50 mM Tris–HCl (pH 7.5), 5% glycerol, 1% IGEPAL, 1 mM MgCl2, protease inhibitors (+EDTA), 1% benzonase, 1× phosphatase inhibitors) and buffer 2 (150 mM NaCl, 50 mM Tris–HCl (pH 7.5), 5% glycerol, 1.0% IGEPAL, 0.5% sodium deoxycholate, 0.1% SDS, 1× protease inhibitors (+EDTA), and 1% benzonase) for 20 min on ice, and in buffer 3 (500 mM NaCl, 50 mM Tris–HCl (pH 7.5), 5% glycerol, 1.0% IGEPAL, 2% sodium deoxycholate, 1% SDS, 1× protease inhibitors (+EDTA), and 1% benzonase) for 20 min at RT. Each of the supernatant from the buffer treatment resulted in fraction 1, 2, and 3, with the residual insoluble material resulting in our fraction 4. Fraction 0 and 1 were combined to generate our 1st fraction. All four fractions were precipitated in 80% acetone and sonicated for 5x30 sec (Bioruptor, model UCD-200, Diagenode). Samples then were incubated at -20 °C for a minimum of 1h and were then centrifuged. The precipitation was repeated once in order to remove any residual detergent. Alkylation/reduction buffer (100 mM Tris-HCl (pH 8.5), 6M GDmCl, 10 mM TCEP, and 50 mM 2-chloroacetamide) was added to the samples and then boiled at 99 °C for 15 min, followed by sonication for 10 x 30 sec. Protein concentration was determined using the BCA method (Micro BCA protein assay kit, ThermoFisher Scientific) according to manufacturer instructions. Enzymatic digestion was done in two steps. First, samples were incubated at 37 °C for 2h with LysC (1/50) and then with LysC (1/50) and Trypsin (1/25) overnight. Both digestions were aided by 10 x 30 sec sonification. Samples were then acidified by adding 1% TFA followed by desalting using the StageTip method (Kulak et al. 2014) with SDB-RPS filters. Each protein lysate was eluted into three peptide fractions using three buffers (buffer 1: 150 mM NH4HCO2, 40% acetonitrile, 0.5% Formic acid (FA); Buffer 2: 150 mM NH4HCO2, 60% acetonitrile, 0.5% FA and buffer 3: 5% ammonia (from 25% stock solution) and 80% acetonitrile) resulting in a total of 12 fractions per sample. Mass spectromtery Samples we loaded at approximately 2 μg of peptides in buffer A (0.1% (v/v) formic acid). We separated peptides by a 2h gradient in a 50 cm long C18 column (75 μm inner diameter filled in house with ReproSil-Pur C18-AQ 1.9-lm resin (Dr. Maish GmbH)). Samples were eluted in 5–60% buffer B (0.1% (v/v) formic acid, 80% (v/v) acetonitrile) at a flow rate of 250 nl/ min using a nanoflow UHPLC (Easy nLC, Thermo Fisher Scientific) online coupled to the mass spectrometer (Q Exactive HF Orbitrap, Thermo Fisher Scientific). Each gradient was followed by a wash with buffer B and recalibration with buffer A. Survey scans had a resolution of 70,000 at m/z 400 with a maximum injection time of 20 ms. Target value for the full scan MS spectra was 3 × 106 and isolation window of 1.6 m/z with 10 most abundant precursor ions chosen for fragmentation. MS/MS scans had a resolution of 17,500 at m/z 400 with a maximum injection time of 120 ms. Ion target value for the MS/MS scan was 1 × 105.