Profiling Phosphoproteome Landscape in Circulating Extracellular Vesicles from Microliters of Biofluids through Functionally Tunable Paramagnetic Separation

Many biological processes are regulated through dynamic protein phosphorylation. Monitoring disease-relevant phosphorylation events in circulating biofluids is highly appealing but also technically challenging. We introduce here a functionally tunable material and a strategy, extracellular vesicles to phosphoproteins (EVTOP), which achieves one-pot extracellular vesicles (EVs) isolation, extraction, and digestion of EV proteins, and enrichment of phosphopeptides, with only a trace amount of starting biofluids. EVs are efficiently isolated by magnetic beads functionalized with Ti-IV ions and a membrane-penetrating peptide, octa-arginine R-8(+), which also provides the hydrophilic surface to retain EV proteins during lysis. Subsequent on-bead digestion concurrently converts EVTOP to Ti-IV ion-only surface for efficient enrichment of phosphopeptides for phosphoproteomic analyses. The streamlined, ultra-sensitive platform enabled us to quantify 500 unique EV phosphopeptides with only a few mu L of plasma and over 1200 phosphopeptides with 100 mu L of cerebrospinal fluid (CSF). We explored its clinical application of monitoring the outcome of chemotherapy of primary central nervous system lymphoma (PCNSL) patients with a small volume of CSF, presenting a powerful tool for broad clinical applications.

Automated summary

Researchers introduced a functionally tunable material and strategy, extracellular vesicles to phosphoproteins (EVTOP), for monitoring disease-relevant phosphorylation events in circulating biofluids. EVs were efficiently isolated by magnetic beads and enriched for phosphopeptides for phosphoproteomic analyses. This platform enables the quantitative detection of a large number of EV phosphopeptides in a small volume of biofluids, presenting broad clinical applications.