Shotgun proteomics (rat)

The neurons were lysed direct on the plate in RIPA lysis buffer using a cell scraper. The lysate was transferred into a fresh Eppendorf tube and undissolved material was removed by centrifugation for 5 min at 20,000 g and 4°C. A protein assay was performed and 15 µg of protein were subjected to proteolytic digestion with the SP3 protocol. Proteins were reduced by addition of 9 µL of 200 mM dithiothreitol (Biozol, Germany) in 50 mM ammonium bicarbonate and incubation for 30 min at 37°C. Cysteine residues were alkylate by addition of 18 µL 400 mM iodoacetamide (Sigma Aldrich, US) and incubation for 30 min at room temperature in the dark. Afterwards, the reaction was quenched by adding another 9 µL of 200 mM dithiothreitol. Proteolytic digestion was performed using a modified protocol for single-pot solid-phase enhanced sample preparation (SP3) (Sielaff et al., 2017). Briefly, after binding of proteins to 40 µg of a 1:1 mixture of hydrophilic and hydrophobic magnetic Sera-Mag SpeedBeads (GE Healthcare, US) with a final concentration of 70% acetonitrile for 30 min at room temperature, beads were washed twice with 200 µL 70% ethanol and twice with 180 µL acetonitrile. For proteolytic digestion, 125 ng LysC and 125 ng trypsin (Pomega, Germany) were added in 20 µL 50 mM ammonium bicarbonate followed by an incubation for 16 h at room temperature. The supernatants were transferred to fresh 0.5 mL protein lobind tubes (Eppendorf, Germany). For improved peptide recovery, 20 µL 0.1% formic acid were added to the magnetic beads followed by sonication for 30 s in a sonication bath (Hielscher Ultrasonics GmbH, Germany). The supernatants were combined and dried by vacuum centrifugation. Peptides were redissolved in 20 µL 0.1% formic. The peptide concentration was estimated using Nanodrop at 280 nm (Thermofisher, US). Peptides from neuronal lysate digestion were analysed on an Easy nLC 1200 nanoHPLC (Thermo Scientific) which was coupled online via a Nanospray Flex Ion Source (Thermo Sientific, US) equipped with a PRSO-V1 column oven (Sonation, Germany) to a Q-Exactive HF mass spectrometer (Thermo Scientific, US). An amount of 1 µg of peptides per sample was separated on an in-house packed C18 column (30 cm x 75 µm ID, ReproSil-Pur 120 C18-AQ, 1.9 µm, Dr. Maisch GmbH,Germany) using a binary gradient of water (A) and acetonitrile (B) supplemented with 0.1% formic acid min (gradient: 0 min., 2.4% B; 2 min., 4.8% B; 92 min., 24% B; 112 min., 35.2% B; 121 min., 60% B). Full MS spectra were acquired at a resolution of 120,000. The top 15 peptide ions were chosen for Higher-energy C-trap Dissociation (HCD) with a normalized collision energy of 26%. Fragment ion spectra were acquired at a resolution of 15,000. A dynamic exclusion of 120 s was used for peptide fragmentation.

The exosome pellets were lysed in 80 µL of a modified RIPA lysis buffer (50 mM TrisHCl pH 8, 150 mM NaCl, 5 mM EDTA, 1% (v/v) Triton X-100, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v)) with protease inhibitors (Sigma Aldrich, US) on ice with intermediate vortexing. 20 µL H2O, 10 µL 100 mM MgCl2, and 25 units Benzonase (Sigma Aldrich, US) were added followed by an incubation for 30 min at 37°C at 1400 rpm in a Thermomixer (Eppendorf, Germany). Undissolved material was removed by centrifugation for 5 min at 20,000 g and 4°C. The supernatants were transferred to 1.5 mL protein Lobind Tubes. Proteins were reduced by addition of 9 µL of 200 mM dithiothreitol (Biozol, Germany) in 50 mM ammonium bicarbonate and incubation for 30 min at 37°C. Cysteine residues were alkylate by addition of 18 µL 400 mM iodoacetamide (Sigma Aldrich, US) and incubation for 30 min at room temperature in the dark. Afterwards, the reaction was quenched by adding another 9 µL of 200 mM dithiothreitol. Proteolytic digestion was performed using a modified protocol for single-pot solid-phase enhanced sample preparation (SP3) (Sielaff et al., 2017). Briefly, after binding of proteins to 40 µg of a 1:1 mixture of hydrophilic and hydrophobic magnetic Sera-Mag SpeedBeads (GE Healthcare, US) with a final concentration of 70% acetonitrile for 30 min at room temperature, beads were washed twice with 200 µL 70% ethanol and twice with 180 µL acetonitrile. For proteolytic digestion, 125 ng LysC and 125 ng trypsin (Promega, Germany) were added in 20 µL 50 mM ammonium bicarbonate followed by an incubation for 16 h at room temperature. The supernatants were transferred to fresh 0.5 mL protein lobind tubes (Eppendorf, Germany). For improved peptide recovery, 20 µL 0.1% formic acid were added to the magnetic beads followed by sonication for 30 s in a sonication bath (Hielscher Ultrasonics GmbH, Germany). The supernatants were combined and dried by vacuum centrifugation. Peptides were redissolved in 20 µL 0.1% formic. The peptide concentration was estimated using Nanodrop at 280 nm (Thermofisher, US). Peptides were analysed on an Easy nLC 1000 nanoHPLC (Thermo Scientific) which was coupled online via a Nanospray Flex Ion Source (Thermo Sientific, US) equipped with a PRSO-V1 column oven (Sonation, Germany) to a Q-Exactive mass spectrometer (Thermo Scientific, US). An amount of 1 µg of peptides per sample was separated on an in-house packed C18 column (30 cm x 75 µm ID, ReproSil-Pur 120 C18-AQ, 1.9 µm, Dr. Maisch GmbH,Germany) using a binary gradient of water (A) and acetonitrile (B) supplemented with 0.1% formic acid (0 min., 2% B; 3:30 min., 5% B; 137:30 min., 25% B; 168:30 min., 35% B; 182:30 min., 60% B) at 50°C column temperature. A data-dependent acquisition method was used. Full MS scans were acquired at a resolution of 70,000 (m/z range: 300-1400, AGC target: 3E+6). The 10 most intense peptide ions per full MS scan were selected for peptide fragmentation (resolution: 17,000, isolation width: 2 m/z, AGC target: 1E+5, NCE: 25%). A dynamic exclusion of 120 s was used for peptide fragmentation.