Glass Fiber-Supported Hybrid Monolithic Spin Tip for Enrichment of Phosphopeptides from Urinary Extracellular Vesicles

Extracellular vesicles (EVs) are
attracting increasing interest
with their intriguing role in intercellular communications. Protein
phosphorylation in EVs is of great importance for understanding intercellular
signaling processes. However, the study of EV phosphoproteomics is
impeded by their relatively low amount in limited clinical sample
volumes, and it is necessary to have a sensitive and efficient enrichment
method for EV phosphopeptides. Herein, a novel Ti­(IV)-functionalized
and glass fiber-supported hybrid monolithic spin tip, termed PhosTip,
was prepared for enriching phosphopeptides from urinary EVs. Glass
fiber as the stationary phase positions the hybrid monolith in a standard
pipet tip and prevents the monolith from distortion during experiments.
The preparation procedure for the new PhosTip is simple and time-saving.
The hybrid monolithic PhosTip provides excellent enrichment efficiency
of low-abundance phosphopeptides from cell digests and urinary EVs
with minimum contamination and sample loss. Using the PhosTip, we
demonstrate that 5373 and 336 unique phosphopeptides were identified
from 100 and 1 μg of cell lysates, while 3919 and 217 unique
phosphopeptides were successfully identified from 10 and 1 mL of urinary
samples, respectively. The PhosTip was finally applied to enrich phosphopeptides
in urine EVs from prostate cancer patients and healthy controls and
quantify 118 up-regulated proteins with phosphosites in prostate cancer
samples. These results demonstrated that the PhosTip could be a simple
and convenient tool for enriching phosphopeptides from clinical samples
and for broader applications in biomarker discovery.