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Proteomics of Extracellular Vesicles Harvested with ExtraPEG
Polyethylene glycol (PEG) is commonly used to isolate virus particles from culture medium. This project adapted and optimized the virus procedure for harvesting extracellular vesicles (of similar biophysical properties to viruses) from media. An 8% PEG solution harvested the purest vesicles. Followed by a wash step (ultracentrifugation of concentrated vesicles in saline), this method produced preparations comparable or superior to commonly used methods in terms of purity, abundance, and cost. It is also highly adaptable, allowing the procedure to be used for enriching vesicles from a variety of biological fluids. As proof of principle, extracellular vesicle protein was successfully harvested from HeLa cell medium using the described PEG-based harvest method and analyzed by mass spectrophotometry.
Sample Processing Protocol
HeLa cell extracellular vesicle proteins were harvested using the ExtraPEG protocol (8% Polyethylene Glycol precipitation of vesicles, followed by an ultracentrifugation wash of the re-suspended precipitates in saline; described above). Vesicles were lysed and proteins were harvested, quantified, and gel-purified. The pure proteins were trypsin digested in the gel. Eluted peptides were lyophilized prior to mass spectrophotometry analysis. The biosample was run with three technical replicates.
Data Processing Protocol
All .raw files were uploaded into proteome discoverer (Thermo Scientific, version 126.96.36.1998) and searched with Mascot and Sequest HT (version 2.4.0). Trypsin was selected as the enzyme and a maximum of 2 miss cleavages were permitted. Dynamic modifications were carbamiodomethyl (C residues), Oxidation (M residues), and Phosphorylation (S, T, and Y residues). The resulting .msf files (technical replicate) were then uploaded into the Scaffold software (Proteome Software, version 188.8.131.52). Technical replicates were uploaded as an individual biosample, and data were searched with X!Tandem. All database searches used the swisprot human database with appended reverse sequences for target-decoy filtering, as well as a list of common contaminants of proteomics experimts (Contaminant sequences in the common Repository of Adventitious Proteins [cRAP] were downloaded from the Global Proteome Machine website). In Scaffold, the protein FDR was set to 1.0%, the peptide FDR was set to 1.0%, and the minimum number of peptides was set to 2.
Rider MA, Hurwitz SN, Meckes DG Jr. ExtraPEG: A Polyethylene Glycol-Based Method for Enrichment of Extracellular Vesicles. Sci Rep. 2016 Apr 12;6:23978 PubMed: 27068479
|#||Accession||Title||Proteins||Peptides||Unique Peptides||Spectra||Identified Spectra||View in Reactome|
|1||62178||no assay title provided (mzIdentML)||6520||13527||10146||11774||11773||
|2||62176||no assay title provided (mzIdentML)||6738||13758||10234||11877||11875||
|3||62177||no assay title provided (mzIdentML)||6956||13870||10257||11909||11906||