μ precolumn

The dried fractionated peptides were re-dissolved in buffer C (2% ACN, 0.1% formic acid (FA)) and centrifuged at 20,000× g for 10 min to collect supernatants. Peptides were separated by Shimadzu LC-20AD, on which a 300 μm × 4 mm (μ-Precolumn, Thermo Scientific, Waltham, MA, USA) for peptide enrichment and a 75 μm × 15 cm in-house column for peptide separation were equipped. After enrichment and desalting in a trap column, each fraction was separated in in-house column by a 65-min gradient at 300 nl/min: 5% buffer D (98% ACN, 0.1% FA) for 8 min, 8–35% buffer D for 35 min, 35–60% buffer D for 5 min, 60–80% buffer D for 2 min, 80% buffer D for 5 min, and 5% buffer D for 10 min. Peptides were detected by the Q-Exactive tandem mass spectrometer (ThermoFisher Scientific, San Jose, CA, USA) that coupled to the Shimadzu LC-20AD at a data-dependent acquisition (DDA) mode. MS parameters were set as follows: spray voltage 1.6kv; scan range 350–1600 m/z; MS resolution 70,000; MS/MS resolution 17,500; dynamic exclusion duration 15s.

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).

Another LC-MS/MS was performed using a Q Exactive HF coupled to an UltiMate 3000 UHPLC system (Thermo Scientific). The peptides of each fraction from the Hp-RP were separated by a 75 μm × 25 cm in-house analytical column that packed with Ultimate LP-C18 particles (3 μm, 120 Å; Materials Inc.) at a flow rate of 300 nL/min. Each fraction was loaded on a trap column (30 μm × 5 mm, μ-Precolumn; Thermo Fisher Scientific) with buffer E (2% ACN, 0.1% FA) in 5 min, followed by a linear 40-min gradient of 5%–35% buffer F (98% ACN, 0.1% FA), and then increased to 80% in 5 min. Mass spectrometry data were acquired with a top30 data-dependent mode scan method. The electrospray voltage was set to1.6 kV and full scan range was set to 350–1600 m/z. We used a resolution of 60,000 at m/z 200 for survey scans. Precursor ions were fragmented by high-energy collisional dissociation (NCE 27%), and fragment ions were detected in the Orbitrap (R = 15,000 at m/z 200). Dynamic exclusion duration was set to 30 s.