Phosphoproteome Characterization of Human Colorectal Cancer SW620 Cell-Derived Exosomes and New Phosphosite Discovery for C‑HPP

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).