Affinity chromatography assisted comprehensive phosphoproteomics analysis of human saliva for lung cancer.

Affinity chromatography is a powerful technology for phosphopeptide enrichment from body fluids. Saliva is a non-invasive body fluid for disease diagnosis, while few studies applied affinity enrichment for saliva phosphoproteome. In this study, we tested two kinds of affinity chromatography materials, Ti4+-IMAC (immobilized metal affinity chromatography) and CaTiO3, for the enrichment of phosphopeptides. Through comparison, Ti4+-IMAC method was demonstrated as the superior one, which was utilized for the comprehensive analysis of salivary phosphoproteome. More than 360 phosphoproteins were specifically extracted and identified from human saliva. Ti4+-IMAC method was further applied to compare the phosphoprotein profiling in the saliva of lung cancer group and normal control group through label-free quantification. Accordingly, 477 and 699 phosphopeptides were enriched, respectively, which corresponded to 339 and 466 proteins. In total, 796 unique phosphopeptides were revealed for 517 saliva phosphoproteins. In particular, 709 phosphorylation sites were identified, among which 26 were up-regulated (>1.5) and 149 were down-regulated (<0.66) in lung cancer. Their corresponding proteins were mainly associated with cancer promotion, system disorder, and organismal injury. Our data collectively demonstrated that salivary phosphopeptides can be comprehensively characterized through Ti4+-IMAC method. These discovered phosphoprotein candidates might be used for lung cancer detection through salivary diagnostics.