For proteomics, 10 µg of proteins were reduced using TCEP for each sample of cells and EVs at morphine concentrations of 0, 1, 10, 25, 50 and 100 μM (final concentration 5 mM, 30 min, 37 °C) (Sigma-Aldrich, St. Louis, MO, USA), alkylated using iodoacetamide (final concentration 15 mM, 60 min, RT, in dark conditions) (Sigma-Aldrich, St. Louis, MO, USA) and digested by an overnight tryptic digestion (w/w ratio 1:50) (Promega, Madison, WI, USA). The RapiGest surfactant was cleaved by incubating samples with 0.5% trifluoacetic acid (Sigma-Aldrich, St. Louis, MO, USA) for 45 min at 37 °C. Samples were then desalted on a C18 reverse phase column (Harvard Apparatus, Holliston, MA, USA), peptides were dried under vacuum and subsequently resuspended in 5% ACN 0.1% FA (peptides final concentration of 0.5 µg/µL and spiked with iRT peptide (Biognosys, Schlieren, Switzerland) (1:20)).
For metabolomics, cell samples (morphine exposure: 0, 5 and 50 μM) were prepared based on Meister et al. [15 (link)] work, as described inSupplementary File S1 .
For metabolomics, cell samples (morphine exposure: 0, 5 and 50 μM) were prepared based on Meister et al. [15 (link)] work, as described in
Free full text: Click here
