Shotgun proteomics (mouse)

Library-Matched Single Shot (LMSS) method Mice were sacrificed by cervical dislocation and brains were subsequently extracted and put into cold phosphate buffered saline (PBS). The ventricular walls were laid bare by removing the dorsal ventricular wall and all tissue above it, as well as the choroid plexus. Brains were then snap-frozen on dry ice and cut into 100 μm sections on a cryostat (Leica CM1000S). The medial (MEZ) and lateral ventricular (SEZ) walls were then manually dissected under a light microscope (Leica MZ6). 8-10 sections from each animal were collected per sample (n=8) and kept on dry ice until tissue lysis. Somatosensory cortex (Cx) samples were dissected by removing corpus callossum and top layer of cortex (including meninges). Olfactory bulb (OB) was dissected by cutting out the core of the OB approximately along the external plexiform layer. Libray proteome was prepared and single shot sampes were processed as in Kulak et al. 2017. Quantitative Detergent Solubility Profile (QDSP) method Mice were sacrificed by cervical dislocation and brains were subsequently extracted and put into cold PBS. The olfactory bulb (OB) was removed by dissection at the base of the OB. Somatosensory cortex (Cx) was removed using a 2.5 mm biopsy punch and the white matter was removed. Both subependymal zones (SEZs) were dissected as previously described (Ortega et al. 2011). Samples then went through step-wise detergent lysis and processed as samples in Schiller et al. 2015. For both the LMSS (including each library sample) and QDSP samples we loaded 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.