Shotgun proteomics (mouse)

3-4 months old male and female C57BL/6J mice (n=10 intact controls, n=10 SW-injured, n=5 LPS injected) were sacrificed through cervical dislocation, brains were removed and placed into cold PBS. Biopsy punches (2.5 mm diameter) of the visual cortex of both hemispheres were dissected whereas meninges and white matter were carefully taken off. The contralateral (uninjured) cortices of the SW-injured brains were not considered for proteome analysis (intact: n=20, SW: n=10, LPS: n=10). Samples were placed into low-protein binding Eppendorf tubes, frozen on dry ice, and stored at - 80 °C until further processing. Tissue samples were lysed in NP40 buffer (1% NP40 in 10 mM Tris, pH 7.4, 150 mM NaCl) in a Precellys homogenizator (VWR) and 10 ?g total protein per sample were proteolyzed with Lys-C and trypsin using a modified FASP procedure (Grosche et al., 2016). LC-MS/MS analysis was performed on a Q Exactive HF mass spectrometer (Thermo Fisher Scientific) online coupled to a nano-RSLC (Ultimate 3000 RSLC; Dionex). Tryptic peptides were accumulated on a nano trap column (Acclaim PepMap 100 C18, 5 ?m, 100 Å, 300 ?m inner diameter (i.d.) × 5 mm; Thermo Fisher Scientific) at a flow rate of 30 ?l/min followed by separation by reversed phase chromatography (?PAC™ column, 200 cm length, with pillar array backbone at interpillar distance of 2.5 ?m, PharmaFluidics) using a non-linear gradient for 240 minutes from 3 to 42% buffer B (acetonitrile [v/v]/0.1% formic acid [v/v] in HPLC-grade water) in buffer A (2% acetonitrile [v/v]/0.1% formic acid [v/v] in HPLC-grade water) at a flow rate of 300 nl/min. MS spectra were recorded at a resolution of 60,000 with an AGC target of 3 x 106 and a maximum injection time of 50 ms, at a range of 300 to 1500 m/z. From the MS scan, the 10 most abundant ions were selected for HCD fragmentation with a normalized collision energy of 27, an isolation window of 1.6 m/z, and a dynamic exclusion of 30 s. MS/MS spectra were recorded at a resolution of 15,000 with an AGC target of 105 and a maximum injection time of 50 ms.