Paper Title
A NOVEL STRATEGY FOR BIOMARKER DISCOVERY: NEUTRAL LOSS-DEPENDENT ACQUISITIONOF CIRCULATING EXTRACELLULAR VESICLES FOR THE DETECTION OF PHOSPHOPEPTIDES

Abstract
Circulating extracellular vesicles (EVs) play a crucial role in intercellular communication and carry diverse biological signaling molecules. Emerging research increasingly indicates that phosphoproteins, prevalent in circulating EVs, are promising candidates as biomarkers for diagnosing diseases and assessing prognosis, particularly in cancer. In this study, we employed EVtrap (extracellular vesicle total recovery and purification) to isolateurinary EVs couple with an integrated liquid chromatography tandem mass spectrometry (LC−MS/MS) strategy, to carry out the neutral loss-information-dependent acquisition-enhanced product ion (NL-IDA-EPI) analysis, which was specifically designed to quantify endogenous phosphopeptides from urinary EVs. A notable advantage of this methodologyis its ability to directly analyze phosphopeptides without necessitating a prior enrichment step for phosphopeptides, streamlining the process and potentially reducing sample bias. Based on the fragmentation patterns observed in representative EV phosphoserine- or phosphothreonine-containing peptides, a distinct mass loss of 49 Da was identified as a marker for the neutral loss phenomenon in MS2 spectra, acquired via EPI scan mode. This marker is pivotal for the non-targeted screening of phosphopeptide. Upon comparative analysis of samples from high- and low-risk prostate cancer (PCa) patients, along with a healthy control group, six differentially expressed phosphopeptides emerged as a promising panel of potential biomarkers. This result highlights the feasibility of our robust analytical pipeline for monitoring cancer signaling by tracking phosphorylation events inner EVs using NL-IDA-EPI. Keywords - Extracellular Vesicles; Neutral Loss; Phosphopeptide; NL-IDA-EPI; prostate cancer.