EVs isolated from bile were lysed in 40 µL of 2% RapiGest SF (Waters, MA, USA) in 50 mM ammonium bicarbonate (Honeywell Fluka, Thermo Fisher Scientific), supplemented with 50 mM DTT (FUJIFILM Wako Pure Chemical Corporation) and incubated at 60 °C for 30 min. The samples were then cooled to room temperature; 4 µL of 150 mM 2-iodoacetamide (Nacalai Tesque, Kyoto, Japan) was added to each sample after which the samples were incubated at room temperature for 30 min in the dark. The lysates were incubated with 1 µg/mL of Mass Spec Grade Trypsin/Lys-C Mix (Promega, WI, USA) at 37 °C overnight. Next a 4 µL of 10% TFA was added to the digested mixture, which was incubated at 37 °C for 30 min. After centrifugation at 13,000×g for 10 min at room temperature, the supernatant was lyophilized in a miVac system (Genevac Ltd, Ipswich, UK) and desalted using Pierce C-18 Spin Columns (Thermo Fisher Scientific) according to the manufacturer’s instructions. The resultant peptide was eluted in 70% acetonitrile and was then lyophilized using a miVac system and stored at − 80 °C.

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