Pierce c18 spin column

Following the lysis of the exosome samples, the FASP method was used to perform tryptic digestion. The lysed sample was reduced with 100 mM dithiothreitol for 10 min at 70°C. The solution was allowed to cool down and then was mixed with 200 µL of 8 M urea in 100 mM TrisHCl (pH 8.5), transferred to a centrifugal spin filter with a MW cutoff of 30 kDa (YM-30, Millipore), and centrifuged for 15 min at 14,000×g. The same centrifugation conditions were used for the following steps: The sample was washed again with 200 µL of the urea buffer. Alkylation was performed by adding 100 µL of 50 mM iodoacetamide (IAA) in the urea buffer, followed by vortexing for 1 min and incubation for 20 min in the dark at room temperature. To remove the remaining IAA, the protein mixture was centrifuged and washed twice with the urea buffer. The sample was washed three times with 100 µL of 50 mM ammonium bicarbonate. Then, tryptic digestion was performed overnight at 37 °C by adding trypsin (Sequencing grade modified, Promega) in a 1:20 ratio (w/w). Digested peptides were collected by centrifugation with 40 µL of 50 mM ammonium bicarbonate three times. After tryptic digestion, the samples were desalted using Thermo Scientific Pierce C18 Spin Columns before LC-MS/MS analysis.