Exosome Harvesting and Proteomic Analysis

Laboratory investigators (NKG, LMW, JSS, and KMD) were blinded to patient characteristics until completion of all assays and data generation. Exosomes were harvested from human serum via Exoquick (Systems Biosciences, Inc., Mountain View, CA) after filtration through a 0.2 micron filter. Protein content of the purified exosomes was quantitated by micro bicinchoninic acid assay (Thermo Fisher Scientific, Inc. Rockford, IL). Twenty µg of protein of each sample was resolved by SDS-PAGE and processed via digestion. Samples subjected to full proteomic characterization by LC-MS/MS were divided into ten gel fractions prior to digestion [12] (link). Exosome samples for MRM-MS were left unfractionated. All samples were digested with trypsin at a 20:1 ratio (sample:protease), as previously published [12] (link).