Iodixanol

Plasma EVs were isolated and purified as previously described by Onódi et al.^{25 (link)}. In brief, extracellular vesicle isolation was performed by iodixanol (60 w/V% iodixanol in ultrapure water; Axis-Shield, Oslo, Norway) density gradient ultracentrifugation (24 h, 120.000 × g, 4 °C). The EV-rich DGUC fractions (Supplementary Figure S1) were loaded into a HiScreen Capto Core 700 column (GE Healthcare Life Sciences) and size exclusion chromatography-based purification was performed. Vezics system (vezics.com) was used to implement the isolation.

Primary MEFs were grown in DMEM lacking Lysine, Arginine and Leucine (Sigma) with 10% dialyzed FBS, and normal isotopic Lys, Arg, Leu (Sigma) for the light sample, or Leu, ¹³C₆¹⁵N₂ Lys, ¹³C₆¹⁵N₄ Arg (Sigma) for the heavy sample. Cells were grown until > 99% incorporation of isotopic amino acids, which was confirmed by LC-MS/MS. Cells were trypsinised and pooled together in 0.25 M sucrose, 1 mM EDTA, 20 mM Hepes buffer before being broken by pulling through a 21G needle. The plasma membrane fraction was isolated by flotation through an iodixanol (Axis-Shield) discontinuous density gradient and the samples were collected from the 17.5%-25% interphase. Proteins were methanol precipitated and samples were prepared for mass spectrometric analysis using a filter-aided sample preparation method, essentially as described previously [48 (link)], using Lys C and Trypsin digest. The resulting peptides were fractionated by liquid isoelectric focusing using Agilent 3100 OFFGEL fractionator (Agilent Technologies), and analysed by LC-MS/MS and quadrupole ion trap mass spectrometer (Orbitrap LTQ XL, ThermoScientific). Data analysis was performed using MaxQuant software [49 (link)], which utilizes Mascot search engine programme (Matrix Science) for peptide identification.

Cell surface biotinylation of CHO cells was performed as described^{18 (link)}. Briefly, cells were serum deprived at a confluence of 80% for 16 h, washed thrice with HBSS supplemented with 0.5 mM CaCl₂ and 2 mM MgCl₂ (HBSS-2+), and incubated for 1 h at 4 °C with 0.5 mg/ml of sulfo-NHS-LC-biotin (Pierce) in HBSS-2+ . After washing twice with 100 mM glycine, cells were washed with HBSS-2+ and incubated without or with chicken NCAM antibody for 30 min.
The Subcellular Protein Fractionation Kit for Cultured Cells (ThermoFisher Scientific) was used for the isolation of membrane, cytoplasmic and nuclear fractions, and the ER isolation kit (Sigma-Aldrich) was used for the isolation of ER-enriched fractions. The isolation of endosomal fractions was performed as described^{18 (link)}. Briefly, cells were homogenized and centrifuged at 1,000 × g for 10 min followed by centrifugation of the resulting supernatant at 17,000 × g for 15 min and centrifugation of the 17,000 × g supernatant at 100,000 × g for 1 h. The 100,000 × g pellet was layered on a step gradient of 10, 15, 20 and 30% iodixanol (Axis-Shield, Oslo, Norway) and centrifuged at 100,000 × g for 3 h and the material from the interphases was collected. Isolation of biotinylated proteins using streptavidin-conjugated magnetic beads (Invitrogen) has been described^{18 (link)}.