Extracellular vesicles (EVs) have emerged as important carriers for intercellular communication and biological sources for diagnosis and therapeutics. Low efficiency in EV isolation from biofluids, however, severely restricts their downstream characterization and analysis. Here, we introduced a novel strategy for EV isolation from urine for prostate cancer diagnosis using bifunctionalized magnetic beads through high affinity Ti­(IV) ions and the insertion of a phospholipid derivative, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, into the EV membrane synergistically. We demonstrated its efficient isolation of EVs from urine samples with low contamination, high recovery (>80%), and short separation time (within 1 h), resulting in the identification of 36,262 unique EV peptides corresponding to 3302 unique proteins and 3233 unique phosphopeptides representing 1098 unique phosphoproteins using only 100 μL and 5 mL urine samples, respectively. Coupled with trapped ion mobility spectrometry and parallel accumulation–serial fragmentation for phosphosite-specific resolution, quantitative phosphoproteomics of urine samples from prostate cancer patients and healthy individuals revealed 121 upregulated phosphoproteins in cancer patients in contrast to the healthy group. These particular advantages indicate that the novel bifunctional material enables sensitive EV phosphoproteomic analysis for noninvasive biomarker screening and early cancer diagnosis.

Neoantigen-based immunotherapy has yielded promising results in clinical trials. However, it is limited to tumor-specific mutations, and is often tailored to individual patients. Identifying suitable tumor-specific antigens is still a major challenge. Previous proteogenomics studies have identified peptides encoded by predicted non-coding sequences in human genome. To investigate whether tumors express specific peptides encoded by non-coding genes, we analyzed published proteomics data from five cancer types including 933 tumor samples and 275 matched normal samples and compared these to data from 31 different healthy human tissues. Our results reveal that many predicted non-coding genes such as DGCR9 and RHOXF1P3 encode peptides that are overexpressed in tumors compared to normal controls. Furthermore, from the non-coding genes-encoded peptides specifically detected in cancers, we predict a large number of “dark antigens” (neoantigens from non-coding genomic regions), which may provide an alternative source of neoantigens beyond standard tumor specific mutations.

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 2500 EV proteins and 1000 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 three total proteins and three 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.

Extracellular vesicles (EVs) are attracting increasing interest with their intriguing role in intercellular communications. Protein phosphorylation in EVs is of great importance for understanding intercellular signaling processes. However, the study of EV phosphoproteomics is impeded by their relatively low amount in limited clinical sample volumes, and it is necessary to