Label-Free Multiple Reaction Monitoring–Mass\nSpectrometry for Quantifying Phosphopeptides from Extracellular Vesicles

Increasing\nefforts have been made to develop proteins in circulating\nextracellular vesicles (EVs) as potential disease markers. It is in\nparticular intriguing to measure post-translational modifications\n(PTMs) such as phosphorylation, preserved and stable in EVs. To facilitate\nthe quantitative measurement of EV protein phosphorylation for potential\nclinical use, a label-free (LF) multiple reaction monitoring (MRM)\nstrategy is introduced by utilizing a synthetic phosphopeptide set\n(phos-iRT) as the internal standards and a local normalization method.\nThe quantitation method was investigated in terms of its linear dynamic\nrange, sensitivity, accuracy, precision, and matrix effect, with a\ndynamic range spanning from 10 to 1000 ng/mL and an accuracy ranging\nfrom 82.4 to 116.8% for EV samples. Then, the LF-MRM-based local normalization\nmethod was utilized to evaluate and optimize our recently developed\nEVTOP method for the enrichment of phosphopeptides from EVs. Finally,\nwe applied the optimized EV enrichment approach and the LF-MRM-based\nlocal normalization method to quantify phosphopeptides in urine EVs\nfrom patients with prostate cancer (PCa) and healthy individuals,\nshowcasing the strategy’s superiority in quantifying phosphopeptides\nwithout isotopic internal standards and validating that the method\nis generally applicable in MRM-based EV phosphopeptide quantification.