Shotgun proteomics (mouse)

8 months old control C57BL/6J (n=10), 17 months aged C57BL/6J (n=4), and 8 months old APP/PS1 mice (n=5) were sacrificed through cervical dislocation, brains were removed and placed into cold 1x PBS. Biopsy punches of the visual cortex of both hemispheres were taken with a tissue puncher (2.5 mm diameter) and meninges and white matter were carefully removed using forceps to have a tissue sample consisting of grey matter only. Each sample was put into a low-protein binding Eppendorf tube, frozen on dry ice, and stored at - 80°C until further processing. Tissue samples were lysed in NP40 buffer (1% NP40 in 10mM Tris, pH 7.4, 150mM NaCl) in a Precellys homogenizator (VWR) and 10µg total protein per sample were proteolyzed with Lys-C and trypsin using a modified FASP procedure (Grosche et al., 2016). LC-MSMS 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 µm, 100 Å, 300 µm inner diameter (i.d.) × 5mm; Thermo Fisher Scientific) at a flow rate of 30µl/min and then separated by reversed phase chromatography (µPAC™ column, 200cm length, with pillar array backbone at interpillar distance of 2.5µm, PharmaFluidics, Zwijnaarde, Belgium) 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 300nl/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 50ms 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 50ms.