Sensitive Sp3 Protein Cleanup and Digestion

The lysates of the equivalent to 4 × 10⁸ purified sEV were processed according to the sensitive Sp3 protocol [19 (link)]. The cysteine residues were reduced in 100 mM DTT and alkylated in 100 mM iodoacetamide (Acros Organics). 20 ug of beads (1:1 mixture of hydrophilic and hydrophobic SeraMag carboxylate-modified beads, GE Life Sciences) were added to each sample in 50% ethanol. Protein clean-up was performed on a magnetic rack. The beads were washed two times with 80% ethanol and once with 100% acetonitrile (Fisher Chemical). The captured beads proteins were digested overnight at 37 °C under vigorous shaking (1200 rpm, Eppendorf Thermomixer) with 0.5 ug Trypsin/LysC (MS grade, Promega) prepared in 25 mM Ammonium bicarbonate. Next day, the supernatants were collected and the peptides were purified using a modified Sp3 clean-up protocol and finally solubilized in the mobile phase A (0.1% Formic acid in water), sonicated, and the peptide concentration was determined through absorbance at 280 nm measurement using a nanodrop instrument.