Proteomic Analysis of Exosome Fractions

Proteomic experiments were performed in biological duplicate (n = 2) as previously described, with MIAPE-compliance171 (link)174 (link). Briefly, exosomes from each cell line (10 μg) were lysed in SDS sample buffer (4% (w/v) SDS, 20% (v/v) glycerol, 0.01% (v/v) bromophenol blue, 0.125 M Tris-HCl, pH 6.8), and proteins separated by short-range SDS-PAGE (10 × 6 mm), and visualized by Imperial Protein Stain (Invitrogen). Individual samples were excised into equal fractions (n = 2), destained (50 mM ammonium bicarbonate/acetonitrile), reduced (10 mM DTT (Calbiochem) for 30 min), alkylated (50 mM iodoacetic acid (Fluka) for 30 min) and trypsinized (0.2 μg trypsin (Promega Sequencing Grade) for 16 hr at 37 °C). A nanoflow UPLC instrument (Ultimate 3000 RSLCnano, Thermo Fisher Scientific) was coupled on-line to an LTQ Orbitrap Elite mass spectrometer (Thermo Fisher Scientific) with a nanoelectrospray ion source (Thermo Fisher Scientific). Peptides were loaded (Acclaim PepMap100, 5 mm × 300 μm i.d., μ-Precolumn packed with 5 μm C18 beads, Thermo Fisher Scientific) and separated (Acquity UPLC M-Class Peptide BEH130, C18, 1.7 μm, 75 μm × 250 mm, Waters). Data was acquired using Xcalibur software v2.1 (Thermo Fisher Scientific). Details of the operation of the mass spectrometer are described previously174 (link).