Impact of Tobacco Consumption on Salivary Protein Profiles: A MALDI-TOF Mass Spectrometry Study.

Objective: To investigate the impact of tobacco consumption on salivary protein profiles and identify proteomic alterations associated with tobacco use, using advanced mass spectrometry techniques.

Methods: This cross-sectional study involved 100 adults aged 18-60, divided into two groups: 50 tobacco users (smokers and smokeless users) and 50 non-users. Participants were selected based on strict inclusion criteria, excluding individuals with systemic diseases, ongoing medications, or cancer history to minimize confounding factors. Unstimulated saliva samples were collected under standardized conditions, centrifuged, and stored at -80°C. Proteins were extracted using trichloroacetic acid (TCA) precipitation, quantified via Bradford assay, and analyzed with Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). Bioinformatics tools, including Cytoscape and heatmap analysis, were utilized to identify protein interaction networks and significant proteomic alterations. Statistical significance was set at p < 0.05.

Results: Tobacco users exhibited significant proteomic alterations compared to non-users, with marked upregulation of inflammatory proteins such as TRML1, MAFB (p < 0.05), and NINJ1, indicating chronic inflammation. Oxidative stress markers, including mitochondrial proteins NU4M and GSAS1, were significantly elevated (p < 0.01), reflecting tobacco-induced cellular stress. DNA repair proteins like O6C70 and PP4RL showed increased expression (p < 0.05), suggesting an adaptive response to smoking-induced DNA damage. Cancer-related proteins TWST1 and CD82 were upregulated (p < 0.05), highlighting processes linked to carcinogenesis, such as epithelial-mesenchymal transition (EMT). Cytoscape analysis revealed highly interconnected protein networks in smokers, with central nodes involving JUNB, EGR1, and FOSB, indicating systemic biological disruptions. In contrast, non-smokers displayed modular and compartmentalized networks reflecting stable cellular processes. Heatmap analysis further emphasized significant differences in protein expression patterns, with smokers showing elevated levels of stress-related proteins while non-smokers exhibited a balanced proteomic profile associated with immune homeostasis and metabolic regulation.

Conclusion: Tobacco consumption significantly alters salivary protein profiles, promoting chronic inflammation, oxidative stress, and DNA repair dysregulation, all of which may contribute to oral carcinogenesis. These findings emphasize the utility of MALDI-TOF MS in detecting salivary biomarkers for early diagnosis of tobacco-related oral diseases and provide insights into the molecular pathways disrupted by tobacco use.