Proteomic and Phosphoproteomic Landscape of Salivary Extracellular Vesicles to Assess OSCC Therapeutical Outcomes.

Circulating extracellular vesicles (EVs) have emerged as an appealing source for surrogates to evaluate the disease status. Herein, we present a novel proteomic strategy to identify proteins and phosphoproteins from salivary EVs to distinguish oral squamous cell carcinoma (OSCC) patients from healthy individuals and explore the feasibility to evaluate therapeutical outcomes. Bi-functionalized magnetic beads (BiMBs) with Ti (IV) ions and a lipid analog, 1,2-Distearoyl-3-sn-glycerophosphoethanolamine (DSPE) are developed to efficiently isolate EVs from small volume of saliva. In the discovery stage, label-free proteomics and phosphoproteomics quantification showed 315 upregulated proteins and 132 upregulated phosphoproteins in OSCC patients among more than 2,500 EV proteins and 1,000 EV phosphoproteins, respectively. We further applied targeted proteomics by coupling parallel reaction monitoring with parallel accumulation-serial fragmentation (prm-PASEF) to measure panels of proteins and phosphoproteins from salivary EVs collected before and after surgical resection. A panel of 3 total proteins and 3 phosphoproteins, most of which have previously been associated with OSCC and other cancer types, show sensitive response to the therapy in individual patients. Our study presents a novel strategy to the discovery of effective biomarkers for non-invasive assessment of OSCC surgical outcomes with small amount of saliva. Significance Statement We reported here the first time to large-scale profile proteins and phosphoproteins in saliva extracellular vesicles (EVs) and demonstrate the feasibility of monitoring EV phosphoproteins to assess therapeutic outcomes with small amount of saliva. Salivary EVs were isolated by a novel bi-functionalized magnetic beads (BiMBs) with high efficiency and specificity, facilitating downstream proteomic and phosphoproteomic analyses and longitudinal measurement of individual patients before and after oral surgery. This article is protected by copyright. All rights reserved.