Quantitative Proteomics Analysis of Exosome Proteins

About 1% protease inhibitor was added to the sample and ultrasonic lysis was used to extract protein. Trypsin was added at 1:50 trypsin-to-protein mass ratio for the first digestion overnight and 1:100 trypsin-to-protein mass ratio for a second 4 h-digestion. The peptide was desalted by Strata X C18 SPE column (Phenomenex) and vacuum dried. The peptide was reconstituted in 0.5 M TEAB and processed according to the protocol of the manufacturer for the TMT kit/iTRAQ kit. The peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo) coupled online to the UPLC. The m/z scan range was 350–1,800 for a full scan, and intact peptides were detected in the Orbitrap at a resolution of 70,000. The resulting MS/MS data were processed using the Maxquant search engine (v.1.5.2.8). Quantitative proteomics analysis was repeated three times.