Highly Efficient Phosphoproteome Capture and Analysis from Urinary Extracellular Vesicles

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 μL
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.