Shotgun proteomics (mouse)

Dissection of ventral pial cerebral vessels: Mice were anesthetized with Ketamine (100 mg/kg) and Xylazine (10 mg/kg) and transcardially perfused with 20 ml PBS, followed by 2 ml of 2% Evan’s blue (w/v) prepared in PBS. After brain harvest, pial vessels were collected from the ventral face of the brain with micro forceps under a M205 A dissection microscope (Leica) and snap frozen on dry ice. Lysis of cerebral vessels. Vessels were mixed with 4% (w/v) SDS, 100 mM dithiothreitol, 100 mM Tris–HCl, pH 7.6 and homogenized in a TissueLyser LT bead mill (Qiagen, 2 x 3 min at 50 Hz). After heating for 3 min at 95°C, samples were sonicated 5 times for 30 s at 4°C in a VialTweeter sonicator (Hielscher, amplitude 100%, duty cycle 50%), then centrifuged at 16,000 g for 15 min. The supernatant was stored at -80°C before further processing. For MS analysis, proteins were precipitated overnight at -80°C after adding NaCl and ethanol to a final concentration of 50 mM and 90% (v/v), respectively. Precipitated proteins were washed once with 90% EtOH and the precipitate was dissolved in 50 mM Tris pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100. LC–MS/MS: An amount of 25 units Benzonase (Sigma-Aldrich) was added to the entire sample volume and samples were incubated for 30 min at 37°C at 1,400 rpm in a Thermomixer (Eppendorf, Germany) to remove remaining DNA. Afterwards, samples were digested with LysC and trypsin, using single-pot solid-phase-enhanced sample preparation (SP3)42. Proteolytic peptides were dried by vacuum centrifugation and dissolved in 20 µl 0.1% (v/v) formic acid. 1.2 µg of peptide mixture was separated on a nanoLC system (EASY-nLC 1200, Thermo Fisher Scientific) using an in-house packed C18 column (30 cm × 75 µm ID, ReproSil-Pur 120 C18-AQ, 1.9 µm, Dr. Maisch GmbH) with a binary gradient of water and 80% acetonitrile (ACN) containing 0.1% formic acid (0 min, 3% ACN; 3.5 min, 6% ACN; 137.5 min, 30% ACN; 168.5 min, 44% ACN; 182.5 min, 75% ACN; 185 min, 99% ACN; 200 min, 99% ACN) at 50 °C column temperature. The nanoLC was coupled online via a nanospray flex ion source equipped with a column oven (Sonation) to a Q-Exactive HF mass spectrometer (Thermo Fisher Scientific). Full MS spectra were acquired at a resolution of 120,000 and a m/z range from 300 to 1,400. The top 15 peptide ions were chosen for collision induced dissociation (resolution: 15,000, isolation width 1.6 m/z, AGC target: 1E+5, NCE: 26%). A dynamic exclusion of 120 s was used for peptide fragmentation.