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

Abstract

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).

SWATH-based proteomics identified carbonic anhydrase 2 as a potential diagnosis biomarker for nasopharyngeal carcinoma

“…We performed core analysis of IPA for fold changes of DEPs in each MS method as we describe previously2850. The Upstream Analysis was used to find out upstream regulators of DEPs with activation/inhibition status predictions51.…”

Cytoskeleton-centric protein transportation by exosomes transforms tumor-favorable macrophages

“…The detailed instrumental setting could be found in our previously publications [34, 46, 47]. All of the MS raw data were deposited in the iProX database (http://www.iprox.org, accession number: IPX00029500).…”

Deep Coverage Tissue and Cellular Proteomics Revealed IL-1β Can Independently Induce the Secretion of TNF-Associated Proteins from Human Synoviocytes

“…We next performed in-solution protein digestion following our published procedure (11,12). The phosphopeptide enrichment was conducted as we previously reported (12).…”

“…We next performed in-solution protein digestion following our published procedure (11,12). The phosphopeptide enrichment was conducted as we previously reported (12). Briefly, Titansphere TiO particles (GL Sciences, Tokyo, Japan) were used to trap phosphopeptides, which were eluted by 5% NH 3 •H 2 O. Phosphopeptides were then fractionated by gradient elutions with 0, 2, 5, 8, 10, and 40% acetonitrile in 5% NH 3 •H 2 O. Mono Tip C18 Pipette Tips (GL Sciences) were employed for desalting.…”