Shotgun proteomics

2 x 108 wild-type or Tspan15-knockout HEK-293T cells were lysed in 1% digitonin lysis buffer containing protease inhibitor cocktail (Sigma-Aldrich). Lysates were pre-cleared with protein G sepharose prior to immunoprecipitation with Tspan15 mAb 1C12 chemically cross-linked to protein G sepharose with dimethyl pimelimidate (Thermo Fisher Scientific). Five independent immunoprecipitations were carried out for each cell type. Immunoprecipitation samples in non-reducing Laemmli buffer were subjected to a modified single-pot solid-phase-enhanced sample preparation (SP3) protocol (Hughes et al. 2019). Briefly, 10 µl of a 4 µg/µl bead slurry of Sera-Mag SpeedBeads A and B (GE Healthcare, Chicago, IL) were added to the samples. Protein binding to the magnetic beads was achieved by adding acetonitrile to a final volume of 70% (v/v) and mixing at 1200 rpm at 24 °C for 30 minutes in a Thermomixer (Eppendorf, Hamburg, Germany). Magnetic beads were retained in a DynaMag-2 magnetic rack (Thermo Fisher Scientific) and the supernatant was discarded. Disulfide bridges were reduced by adding 20 µl of 30 mM dithiothreitol (Biozol, Eching, Germany) and incubating at 1200 rpm at 37 °C for 30 minutes. Cysteines were alkylated by adding 25 µl of 80 mM iodoactemamide (Sigma-Aldrich) and incubating at 1200 rpm at 24 °C for 30 minutes in the dark in a Thermomixer. The reaction was quenched by adding 3 µl of 200 mM dithiothreitol. Protein binding to the beads was repeated in 70% (v/v) acetonitrile for 30 minutes. After removing the solvent, beads were washed twice in 200 µl 70% (v/v) ethanol and twice in 180 µl of 100% (v/v) acetonitrile. Next, 250 ng of LysC and 250 ng of trypsin (Promega, Mannheim, Germany) were added in 20 µl of 50 mM ammonium bicarbonate (Sigma Aldrich). The protein digestion was performed for 16 hours at room temperature. Samples were acidified with formic acid to a final concentration of 1% (v/v) and placed in a magnetic rack. The supernatants were transferred into fresh 0.5 ml protein LoBind tubes (Eppendorf). A volume of 20 µl of 2% (v/v) dimethyl sulfoxide was added to the beads and subjected to sonication for 30 seconds in a water bath. Tubes were placed in the magnetic rack and the supernatants were transferred. The samples were dried in a vacuum centrifuge and dissolved in 20 µl 0.1% formic acid. Samples were analyzed by LC-MS/MS for relative label free protein quantification. A volume of 10 µl per sample was separated on a nanoLC system (EASY-nLC 1200, Thermo Fisher Scientific) using an in-house packed C18 column (30 cm x 75 µm ID, ReproSil-Pur 120 C18-AQ, 1.9 µm; Dr. Maisch GmbH, Ammerbuch, Germany) with a binary gradient of water (A) and acetonitrile (B) containing 0.1% formic acid at 50 °C column temperature and a flow rate of 250 nl/minute (gradient: 0 minutes 2.4% B; 2 minutes, 4.8% B; 92 minutes, 24% B; 112 minutes, 35.2% B; 121 minutes, 60% B). The nanoLC was coupled online via a nanospray flex ion source (Proxeon, Thermo Fisher Scientific) equipped with a PRSO-V2 column oven (Sonation, Biberach an der Riss, Germany) to a Q-Exactive HF mass spectrometer (Thermo Fisher Scientific). 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) (normalized collision energy of 26%, automatic gain control 1E+5 ions, intensity threshold 5E+3 ions, maximum ion trapping time 100 ms). Fragment ion spectra were acquired at a resolution of 15,000. A dynamic exclusion of 120 s was used for peptide fragmentation.