Exploring the Effects of Pepsin on Salivary Peptidome: Insights from a Proof-of-Concept Proteomic Profiling Study Using MALDI-ToF Mass Spectrometry

Background: Pepsin, a component of gastric refluxate, has been investigated as a potential salivary biomarker for gastro-esophageal reflux disease (GERD), although its diagnostic accuracy remains uncertain. This proof-of-concept study aimed to characterize the effects of pepsin on the human salivary peptidome using matrix-assisted laser desorption/ionization–time of flight (MALDI-ToF) mass spectrometry. Methods: Whole saliva samples were collected from ten healthy adult volunteers under fasting conditions and divided into untreated controls and aliquots digested with pepsin at acidic pH. MALDI-ToF MS was used to profile digestion-induced changes in peptide mass patterns. Spectral data were analyzed using multivariate statistical approaches, including principal component analysis (PCA), linear discriminant analysis (LDA), and hierarchical clustering. Results: Pepsin digestion increased peptide signal intensity and spectral complexity compared with controls. PCA demonstrated clear separation between native and digested samples along the first principal component. Ten peptide m/z features showed the strongest association with pepsin exposure based on PCA loadings. LDA and hierarchical clustering further supported this distinction, with the top 15 discriminative m/z features showing consistent enrichment in digested samples despite inter-individual variability. Conclusions: Pepsin exposure induces reproducible remodeling of the salivary peptidome detectable by MALDI‑ToF MS. Although this peptide‑level approach cannot resolve the full diversity of salivary proteoforms, the resulting signatures support the feasibility of identifying markers of reflux‑associated enzymatic activity and provide a basis for future validation in clinical GERD cohorts.