Rapigest

The biotinylated ODN-bound proteins were precipitated with streptavidin beads from mitochondria lysates and resolved by PAGE (Bio-rad). The PAGE gels were stained using a silver staining kit (Pierce). Silver-stained gel pieces were excised, washed, destained and digested in-gel with 200 ng modified trypsin (sequencing grade, Promega) and Rapigest (TM, Waters Corp.) for 18 h at 37° C. In-solution samples were precipitated with 5:1 v/v of cold acetone at −20° C for 18 h, then centrifuged and the acetone was removed prior to treatment with Rapigest (100 °C for 10 min), followed by addition of trypsin. The resulting peptides were extracted and analyzed by high-sensitivity LC-MS/MS on an Orbitrap Fusion mass spectrometer (Thermo Scientific, Waltham MA). Proteins were identified by database searching of the fragment spectra against the SwissProt (EBI) protein database using Mascot (v 2.6, Matrix Science, London, UK) and Proteome Discoverer (v 2.2, Thermo Scientific). Typical search settings were: mass tolerances, 10 ppm precursor, 0.8d fragments; variable modifications, methionine sulfoxide, pyro-glutamate formation; enzyme, trypsin, up to 2 missed cleavages. Peptides were subject to 1% FDR using reverse-database searching.

The total protein concentration of serum from each patient was determined by a BCA assay (Thermo Fisher Scientific, Waltham, MA). Serum protein (100 µg) was used for trypsin digestion. The serum proteins were denatured using 0.1% RapiGest (Waters, Milford, MA) and 50 mM ammonium bicarbonate, reduced with 5 mM dithiothreitol for 30 min at 60 °C, and then alkylated with 15 mM iodoacetamide for 30 min at room temperature in the dark. Subsequently, trypsin (Promega, Madison, WI) was added at an enzyme/protein ratio of 1:50, followed by incubation for 18 h at 37 °C. The digestion was terminated by adding trifluoroacetic acid (Sigma, St. Louis, MO) to a final concentration of 0.5% (v/v) followed by incubation for 30 min at 37 °C. RapiGest was removed by centrifugation at 13,000 rpm for 10 min. After RapiGest removal, the digested serum was desalted and concentrated using C18 Sep-Pak cartridges containing 1 mg sorbent (Waters) according to the manufacturer’s instructions. The eluted peptides were frozen for 30 min at − 80 °C and then dried by vacuum centrifugation. The peptides were resuspended in 5% acetonitrile and 0.1% formic acid, and the peptide concentration was measured using a NanoDrop (Thermo Fisher Scientific).

EV pellets were resuspended in 100 ml (50 ml for normal cell controls) of 50 mM ABC containing 0.5% Rapigest (Waters Corp.), sonicated 2 × 10 s, and incubated with shaking for 1 h at RT, digested with trypsin with a final concentration of 0.1% Rapigest. Sequencing grade trypsin (Promega) was added with a 1:100 (enzyme: substrate) ratio and digested O/N at 37 °C. Peptide concentration was determined by nanodrop (A280nm). Capillary-liquid chromatography-nanospray tandem mass spectrometry (Capillary-LC/MS/MS) of protein identification was carried out on a Thermo Scientific orbitrap fusion mass spectrometer. This spectrometer was equipped with an EASY-Spray™ Sources which operated in positive ion mode. In this experiment two mobile phase were used. Mobile phase A constituted of 0.1% Formic Acid in water. The mobile phase B comprised of acetonitrile (with 0.1% formic acid). Raw files were converted into a merged file (.mgf) using MS convert (ProteoWizard) in order to process the sequence information. The resulting mgf files were searched using Mascot Daemon by Matrix Science version 2.3.2 (Boston, MA). Proteomics data were summarized in scaffold and spectral counting was used for protein quantitation.