Titanium(IV) immobilized affinity chromatography facilitated phosphoproteomics analysis of salivary extracellular vesicles for lung cancer.

Extracellular vesicles (EVs) play critical roles in intercellular communications, which contain valuable biomarkers for the detection of cancers. Phosphoproteomics analysis of human saliva EVs (sEVs) can help to discover lung cancer-related candidates. Due to the low abundance of phosphoproteins in sEVs, an efficient, reproducible, and cost-effective strategy is required for their enrichment. Here, we compared the latest phosphopeptide techniques, including TiO2, ZrO2, CaTiO3, and Ti4+-IMAC (immobilized metal affinity chromatography) methods, for phosphopeptide isolation. Our data demonstrated that Ti4+-IMAC was the superior one. By using the optimized Ti4+-IMAC approach, we identified more than 500 sEV phosphopeptides. Quantitative proteomics was employed to comprehensively decipher the sEV phosphoproteome of the normal group (n = 6) and lung cancer group (n = 6). Accordingly, 524 and 333 phosphopeptides were enriched, respectively, which corresponded to 439 and 282 phosphoproteins. In total, 857 unique sEV phosphopeptides corresponding to 721 phosphoproteins were revealed. Among 493 identified phosphosites, 37 were upregulated (> 1.5) and 217 were downregulated (< 0.66) in the cancer group. Our data collectively demonstrated that Ti4+-IMAC is an efficient and reproducible technology for comprehensive analysis of sEV phosphoproteome. Differentially expressed sEV phosphoproteins and phosphosites might be used for the detection of lung cancer non-invasively.