Journal
JOURNAL OF PROTEOME RESEARCH
Volume 17, Issue 9, Pages 3308-3316Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.jproteome.8b00459
Keywords
extracellular vesicles; EVs; exosomes; urine; phosphoproteomics; EVTRAP
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Funding
- NIH [1R01GM111788, 1R41CA210772]
- National Science Foundation [1506752]
- Purdue University Center for Cancer Research (NIH Grant) [P30 CA023168]
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Analysis of protein phosphorylation in extracellular vesicles (EVs) offers an unprecedented potential for understanding cancer signaling and early stage disease diagnosis. However, prior to the phosphoproteome analysis step, the isolation of EVs from biofluids remains a challenging issue to overcome due to the low yield and impurity from current isolation methods. Here, we carry out an extensive assessment of several EV isolation methods including a novel rapid isolation method EVTRAP for highly efficient capture of extracellular vesicles from human urine sample. We demonstrate that over 95% recovery yield can be consistently achieved by EVTRAP, a significant improvement over current standard techniques. We then applied EVTRAP to identify over 16 000 unique peptides representing 2000 unique EV proteins from 200 mu L urine sample, including all known EV markers with substantially increased recovery levels over ultracentrifugation. Most importantly, close to 2000 unique phosphopeptides were identified from more than 860 unique phosphoproteins using 10 mL of urine. The data demonstrated that EVTRAP is a highly effective and potentially widely implementable clinical isolation method for analysis of EV protein phosphorylation.
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