Increasing efforts have been made to develop proteins in circulating extracellular vesicles (EVs) as potential disease markers. It is in particular intriguing to measure post-translational modifications (PTMs) such as phosphorylation, preserved and sta-ble in EVs. To facilitate the quantitative measurement of EV protein phosphorylation for potential clinical use, a label-free (LF) multiple reaction monitoring (MRM) strategy is introduced by utilizing a synthetic phosphopeptide set (phos-iRT) as inter-nal standards and a local normalization method. The quantitation method was investigated in terms of its linear dynamic range, sensitivity, accuracy, precision, and matrix effect, with a dynamic range spanning from 10 to 1000 ng/mL and accura-cy ranging from 82.4% to 116.8% for EV samples. Then, the LF-MRM-based local normalization method was utilized to evaluate and optimize our recently developed EVTOP method for the enrichment of phosphopeptides from EVs. Finally, we applied the optimized EV enrichment approach and the LF-MRM-based local normalization method to quantify phospho-peptides in urine EVs from patients with prostate cancer (PCa) and healthy individuals, showcasing the strategy's superiori-ty in quantifying phosphopeptides without isotopic internal standards and validating the method is generally applicable in MRM-based EVs phosphopeptide quantification.

The frozen urine samples were thawed in a 37 °C water bath. Subsequently, the thawed urine samples were spiked with one-tenth volume of 0.1% NP40/Triton X-100 buffer, and an appropriate amount of EVTOP was added to the sample and mixed thoroughly. The sample was incubated at room temperature for 60 min, to allow for EVTOP-assisted capture of EVs from the urine samples. EVs were collected by discarding the supernatant using a magnetic stand. Afterwards, these EV-bound magnetic beads were washed with 0.01% NP40/Triton X-100 buffer once and PBS twice, along with the magnet separation and superna-tant removal. Finally, the EVs from urine samples were captured on the surface of EVTOP. EV Phosphopeptide Preparation for LC-MRM-MS Anal-ysis Prior to LC-MRM-MS measurement, urinary EVs collected by EVTOP were subjected to protein extraction using a phase-transfer-surfactant (PTS) lysis solution at 95 ℃ for 10 min. This solution contained 12 mM sodium lauroyl sarcosinate, 12 mM sodium deoxycholate, 50 mM Tris·HCl with pH 8.5, 40 mM 2-Chloroacetamide (CAA), 10 mM Tris (2-carboxyethyl) phosphine (TCEP), and phosphatase in-hibitor cocktail 2 (Sigma-Aldrich), which facilitated protein denaturation, reduction, and alkylation. The samples were diluted 5-fold with 50 mM triethylammonium bicarbonate (TEAB), and subsequently digested by Lys-C (Wako) in a 1:100 (w/w) enzyme-to-substrate ratio at 37 °C for 3 h under shaking at 1200 rpm. After digestion, trypsin was spiked in a 1:50 (w/w) enzyme-to-substrate ratio for a 12 h digestion at 37 °C. The digestion process was quenched by adding a trifluoroacetic acid (TFA) aqueous solution to 1% final concentration. Afterwards, ethyl acetate was added in a 2:1 ratio to the mixture, which was vortexed for 3 min and cen-trifuged at 20,000 g for 3 min to remove the organic phases (top layer) and collect the aqueous phase (bottom layer) prior to the phosphopeptide enrichment process. LC-MRM-MS analysis and data analysis are shown in detail in Sup-porting Information.