Identification of all phosphorylation 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, but no deep coverage phosphoproteome of human exosomes has been reported to date, which makes the exosome a potential source for the new phosphosite discovery. In this study, we performed highly optimized MS analyses on the exosomal and cellular proteins isolated from human colorectal cancer SW620 cells. With stringent data quality control, 313 phosphoproteins with 1091 phosphosites were confidently identified from the SW620 exosome, from which 202 new phosphosites were detected. Exosomal phosphoproteins were significantly enriched in the 11q12.1-13.5 region of chromosome 11 and had a remarkably high level of tyrosine-phosphorylated proteins (6.4%), which were functionally relevant to ephrin signaling pathway-directed cytoskeleton remodeling. In conclusion, we here report the first high-coverage phosphoproteome of human cell-secreted exosomes, which leads to the identification of new phosphosites for C-HPP. Our findings provide insights into the exosomal phosphoprotein systems that help to understand the signaling language being delivered by exosomes in cell-cell communications. The mass spectrometry proteomics data have been deposited to the ProteomeXchange consortium with the data set identifier PXD004079, and iProX database (accession number: IPX00076800).
Keywords: C-HPP; exosome; new phosphosites; phosphoproteome; signaling pathway.