Plasma-Derived Extracellular Vesicle Phosphoproteomics through Chemical Affinity Purification

The invasive nature
and the pain caused to patients inhibit the
routine use of tissue biopsy-based procedures for cancer diagnosis
and surveillance. The analysis of extracellular vesicles (EVs) from
biofluids has recently gained significant traction in the liquid biopsy
field. EVs offer an essential “snapshot” of their precursor
cells in real time and contain an information-rich collection of nucleic
acids, proteins, lipids, and so on. The analysis of protein phosphorylation,
as a direct marker of cellular signaling and disease progression could
be an important stepping stone to successful liquid biopsy applications.
Here we introduce a rapid EV isolation method based on chemical affinity
called EVtrap (extracellular vesicle total recovery and purification)
for the EV phosphoproteomics analysis of human plasma. By incorporating
EVtrap with high-performance mass spectrometry (MS), we were able
to identify over 16 000 unique peptides representing 2238 unique
EV proteins from just 5 μL of plasma sample, including most
known EV markers, with substantially higher recovery levels compared
with ultracentrifugation. Most importantly, more than 5500 unique
phosphopeptides representing almost 1600 phosphoproteins in EVs were
identified using only 1 mL of plasma. Finally, we carried out a quantitative
EV phosphoproteomics analysis of plasma samples from patients diagnosed
with chronic kidney disease or kidney cancer, identifying dozens of
phosphoproteins capable of distinguishing disease states from healthy
controls. The study demonstrates the potential feasibility of our
robust analytical pipeline for cancer signaling monitoring by tracking
plasma EV phosphorylation.