*2.9. Mass Spectrometry (MS)*

EV protein identification was performed using nano-liquid chromatography-nanospray tandem mass spectrometry (LC/MS/MS) on a Thermo Scientific Q Exactive mass spectrometer equipped with an EASY-Spray™ Sources operated in positive ion mode. Each sample was injected into a μ-Precolumn Cartridge (Thermo Scientific) and desalted with 0.1% formic acid in water for 5 min. Samples were then separated on an easy spray nano column (PepmapTM RSLC, C18 3<sup>μ</sup> 100 A, 75 <sup>μ</sup>m <sup>×</sup> 150 mm, Thermo Scientific) using a two-dimensional (2D) RSLC high-performance liquid chromatography system (Thermo Scientific). Mobile phase A was 0.1% formic acid in water and mobile phase B was acetonitrile/0.1% formic acid. Peptide elution was achieved with a multi-linear gradient comprising 2–35% B over 225 min, 35–55% B over 35 min, 55–90% B over 10 min and then 90% B isocratic for 5 min. After each run, the column was equilibrated in 2% B for 20 min before the next sample injection.

The MS/MS method was a Top10 method: the analysis was programmed for a full scan recorded between *m*/*z* 400–1600 and an MS/MS scan to generate product ion spectra to determine amino acid sequence in consecutive scans starting from the most abundant peaks in the spectrum, and then selecting the next nine most abundant peaks. To achieve high mass accuracy MS determination, the full scan was performed at a resolution setting of 70,000. The AGC target ion number for full scan was set at 3 <sup>×</sup> 106 ions, maximum ion injection time was set at 100 ms. MS/MS was performed using a stepped normalized CE of 25, 30, and 35, acquired at a resolution of 17,500 with an AGC target of <sup>1</sup> <sup>×</sup> 105 ions and a maximum injection time of 50 ms. Dynamic exclusion was enabled with a repeat count of 1 within 30 s.

Sequence information from the MS/MS data was processed by converting the raw files into a merged file (.mgf) using MS convert (ProteoWizard). The resulting mgf files were searched using Mascot Daemon by Matrix Science version 2.6.0 (Boston, MA, USA) and the database searched against Uniprot Mouse database. The mass accuracy of the precursor ions was set to 10 ppm, and accidental inclusion of 1 13C peaks was also included into the search. The fragment mass tolerance was set to 0.05 Da. Four missed cleavages for the enzyme were permitted. A decoy database was also searched to determine the false discovery rate (FDR) and peptides were filtered according to the FDR. Proteins with less than 1% FDR as well as a minimal of two significant peptides detected were considered as valid proteins. Proteomics data was summarized in scaffold to allow for spectral counting analysis. Complete MS datasets are available in the Supplemental Data section as Tables S2–S4.
