Label-free Multiple Reaction Monitoring-Mass Spectrometry for Quantifying Phosphopeptides from Extracellular Vesicles

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.