Quantitative Proteomic Analysis of Exosomes

For western blot analysis, total protein was extracted from exosomes, CRC cells, or mouse liver metastasis tissue using RIPA lysis buffer and analyzed as previously described (Fang et al., 2020 (link)). The exosomes were labeled with iTRAQ reagents using the iTRAQ multiplex kit (AB Sciex, USA) and followed analyzed according to previous publication (Kawakami et al., 2017 (link)). Labeled samples were separated and automatically spotted onto a MALDI plate, and mass spectra were acquired using the AB Sciex TOF/TOF 5800 system. All MS/MS data were analyzed via MASCOT and the Protein Pilot software (version 4.5; AB Sciex) to identify and quantify corresponding proteins in different groups (Supplementary Table S2). Protein identification was considered correct, based on the selection criteria (Kawakami et al., 2017 (link)).