Itraq multiplex kit

To prepare cell membrane proteins, vascular endothelial cells were collected via centrifugation at 500 × g for 5 min, followed by washing with PBS. The cell pellet was then lysed using the Mem-PER Plus Membrane Protein Extraction Kit, according to the manufacturer’s instructions (Thermo Fisher Scientific). The extracts were stored at -80℃ until use.
For western blot analysis, total protein was extracted from the exosomes and HUVECs using RIPA lysis buffer and assessed as previously described [18 (link)]. For quantitative protein analysis, exosomes were labeled with iTRAQ reagents using an iTRAQ multiplex kit (AB Sciex, USA) and analyzed as previously described [19 (link)]. Labeled samples were separated and automatically spotted onto a MALDI plate; mass spectra were then acquired using an AB Sciex TOF/TOF 5800 system. All tandem mass spectrometry data were analyzed using MASCOT and Protein Pilot software (version 4.5; AB Sciex) to identify and quantify the corresponding proteins in different groups (Table S2). Protein identification was considered correct based on the selection criteria [19 (link)].

Proteomic analysis was performed as previously described [16 (link)]. In brief, exosomes were labeled with iTRAQ reagents using the iTRAQ multiplex kit (AB Sciex, Foster City, CA, USA). Labeled samples were separated and automatically spotted onto a MALDI plate using the direct nanoLC and MALDI fraction system DiNa-MaP (KYA Technologies, Tokyo Japan). Mass spectra were acquired using the AB Sciex TOF/TOF 5800 system operated on the TOF/TOF Series Explorer software version 4.1 (AB Sciex). All MS/MS data were submitted to the ProteinPilot software version 4.5 (AB Sciex). Protein identification was considered to be correct based on the following selection criteria: protein having at least 2 peptides with an ion score above 95% confidence; and protein with protein score (ProtScore) > 1.3 (unused, p < 0.05, 95% confidence).

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)).