Optimization of protein extraction and digestion workflows for proteomic analysis of saliva, salivary stones and kidney stones

Background Salivary and kidney stone diseases are associated with pathological calcification, yet their molecular composition remains incompletely characterized. Proteomic analysis of saliva and stone material may provide valuable insights into these processes; however, such analyses are technically challenging and strongly dependent on efficient and reproducible sample preparation workflows. Methods In this study, we systematically evaluated and optimised protein extraction and digestion conditions for proteomic analysis of saliva, salivary stones and kidney stones. Different lysis buffers, sample amounts, sonication parameters and digestion strategies were tested. Additional biological materials, including salivary gland tissue and Raoultella ornithinolytica , were analysed to assess the broader applicability of the developed workflows. Results Optimised workflows significantly improved peptide and protein identification, reduced the proportion of missed cleavages and enhanced sequence coverage across multiple biological matrices. Sonication-assisted protocols proved particularly effective for stone-derived materials, although optimal conditions varied depending on the sample type. Conclusion This pilot study demonstrates that careful optimisation of protein extraction and digestion is essential for reliable proteomic analysis of challenging biomaterials such as saliva and pathological calcifications. The workflows established here provide a robust methodological foundation for future large-scale and biomarker-oriented proteomic studies of stone disease and related calcification disorders.