Phosphoproteome Characterization Of Human Colorectal Cancer Cell-derived Exosomes And New Phosphosite Discovery

Identification of all post translational modification(PTM)forms of known proteins is a major goal of the Chromosome-Centric Human Proteome Project(C-HPP).Recent studies have found that certain phosphoproteins can be encapsulated in exosomes and function as key regulators in tumor microenvironment.Due to major technological hindrances,such as large-amount of exosome isolation and microscale enrichment of phosphopeptides,there is no phosphoproteome of human exosomes has been reported to date.As such,exosomes may be source of new human protein phosphosites.Here,we realized high purity SW620 exosome isolation and performed MS analyses on the exosomal phosphoproteome.We compiled three phosphosite databases,dbPTM,PhosphoSitePlus? and SubPhosDB to generate the multiple phosphoprotein database(MPD),which contained 42,143 protein/isoform entries and 184,804 phosphosite entries.With stringent data quality control,313 phosphoproteins with 924 phosphosites were confidently identified from the SW620 exosome,from which we detected 202 new phosphosites,which were not recorded in MPD.Exosomal phosphoproteins were significantly enriched in chromosome 11,showing a significant difference to cellular phosphoprotein chromosomal distribution.In addition,exosomes derived from colorectal cancer cells,at least SW620 and HCT116 cells,had a remarkably high level of tyrosine(Y)-phosphosites(~6%)compared to cells.Bioinformatic analysis revealed that exosomal phosphoproteins,especially Y-phosphoproteins were not only critical for exosome life cycle,but also targeted ephrin signaling-directed cytoskeleton remodeling and tight junction.In conclusion,we here report the first high-coverage phosphoproteome of human cell-secreted exosomes,which provide high-confident new phosphosites and new phosphorylated forms of protein compliant with the C-HPP criteria.Our study demonstrate that exosomes are critical phosphorylation signaling transporter and contain human new phosphoproteins.