Profiling Phosphoproteome Landscape in Circulating Extracellular Vesicles from Microliters of Biofluids through...

Area of Science:

• Biochemistry
• Proteomics
• Biomaterials

Background:

• Protein phosphorylation regulates critical biological processes.
• Monitoring phosphorylation in biofluids is challenging but clinically relevant.
• Extracellular vesicles (EVs) are promising biomarkers.

Purpose of the Study:

• To develop a streamlined method for EV isolation, protein extraction, and phosphopeptide enrichment.
• To enable sensitive phosphoproteomic analysis from small biofluid volumes.
• To demonstrate clinical utility in monitoring disease treatment.

Main Methods:

• Introduced EVTOP: a one-pot strategy using functionalized magnetic beads for EV isolation, lysis, and protein digestion.
• Utilized Ti(IV) ions and octa-arginine (R8+) for efficient EV capture and protein retention.
• On-bead digestion and phosphopeptide enrichment for subsequent analysis.

Main Results:

• Quantified over 500 unique EV phosphopeptides from a few μL of plasma.
• Identified over 1200 phosphopeptides from 100 μL of cerebrospinal fluid (CSF).
• Successfully monitored chemotherapy outcomes in primary central nervous system lymphoma (PCNSL) patients using CSF.

Conclusions:

• EVTOP is an ultra-sensitive platform for phosphoproteomics.
• The method significantly simplifies analysis from minimal biofluid samples.
• Presents a powerful tool for broad clinical applications, including disease monitoring.