Proteome discoverer analytical software

Exosomes were lysed with RIPA lysis buffer and Halt Protease/Phosphatase inhibitor (ThermoFisher Scientific, Rockford, IL). Next, exosomal lysate (50 μg) was electrophoresed on a 10% 1D SDS-PAGE and the gel was stained with Coomassie brilliant blue and cut into 5 slices. Gel slices were digested using a protocol modified from Lee et al.^{30 (link)} Proteins were processed by LC/MS/MS at Proteomics and Metabolomics Shared Resource at Wake Forest Baptist Medical Center. The annotated human protein database from UniProtKB (20,161 entries, date; 10–1-2014) was used within Proteome Discoverer analytical software (v1.4, Thermo Scientific, Rockford, IL, USA) applying the generic MASCOT search algorithm. The protein subcellular localizations and functions were determined from the Ingenuity Systems software (Redwood City, CA, USA; http://www.ingenuity.com/index.html). Pathway analysis and network constructions were assembled using the Ingenuity software.

Exosomes were isolated from PLSCs conditioned media by Exoquick™ reagent (System Bioscience, (Palo Alto, CA) following Vendor’s instructions. Exosomes were lysed with RIPA lysis buffer and Halt Protease/Phosphatase inhibitor (ThermoFisher Scientific, Rockford, IL). Next, exosomal lysate (50 μg) was electrophoresed on a 10% 1D SDS-PAGE and the gel was stained with Coomassie brilliant blue and cut into 5 slices. Gel slices were digested using a protocol modified from Lee et al [13 (link)]. Proteins were processed by LC/MS/MS at Proteomics and Metabolomics Shared Resource at Wake Forest Baptist Medical Center. The annotated human protein database from UniProtKB (20,161 entries, date; 10-1-2014) was used within Proteome Discoverer analytical software (v1.4, Thermo Scientific, Rockford, IL, USA) applying the generic MASCOT search algorithm. There were at least 2 peptides per hit, and the protein subcellular localizations and functions were determined from the Ingenuity Systems software (Redwood City, CA, USA; http://www.ingenuity.com/index.html). Pathway analysis and network constructions were assembled using the Ingenuity software.