Synergistically Bifunctional Paramagnetic Separation Enables Efficient Isolation of Urine Extracellular Vesicles and Downstream Phosphoproteomic Analysis

Extracellular vesicles (EVs) have emerged as important carriers for intercellular communication and biological sources for diagnosis and therapeutics. Low efficiency in EV isolation from biofluids, however, severely restricts their downstream characterization and analysis. Here, we introduced a novel strategy for EV isolation from urine for prostate cancer diagnosis using bifunctionalized magnetic beads through high affinity Ti­(IV) ions and the insertion of a phospholipid derivative, 1,2-distearoyl-<i>sn</i>-glycero-3-phosphoethanolamine, into the EV membrane synergistically. We demonstrated its efficient isolation of EVs from urine samples with low contamination, high recovery (>80%), and short separation time (within 1 h), resulting in the identification of 36,262 unique EV peptides corresponding to 3302 unique proteins and 3233 unique phosphopeptides representing 1098 unique phosphoproteins using only 100 μL and 5 mL urine samples, respectively. Coupled with trapped ion mobility spectrometry and parallel accumulation–serial fragmentation for phosphosite-specific resolution, quantitative phosphoproteomics of urine samples from prostate cancer patients and healthy individuals revealed 121 upregulated phosphoproteins in cancer patients in contrast to the healthy group. These particular advantages indicate that the novel bifunctional material enables sensitive EV phosphoproteomic analysis for noninvasive biomarker screening and early cancer diagnosis.