ORIGINAL RESEARCH article
Front. Mol. Biosci.
Sec. Protein Biochemistry for Basic and Applied Sciences
Optimization of protein extraction and digestion workflows for proteomic analysis of saliva, salivary stones and kidney stones
Provisionally accepted
Paulina Czaplewska1*
Natalia Musiał1*
Martyna Iwaniec1Inez Mruk1Michał Puchalski1Dmitry Tretiakow2Andrzej Skorek2Konrad Szydłowski2Jan Szlęzak2- 1University of Gdansk, Gdansk, Poland
- 2Clinical Department of Otolaryngology, Academy of Applied Medical and Social Sciences, The Nicolaus Copernicus Hospital in Gdansk, Copernicus Healthcare Entity,, Gdańsk, Poland
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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. Conclusions: 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.
Keywords: Biocalcification, biomarkers, Kidney Stones, Proteomics, Saliva, Salivary stones, sample preparation, Sonication
Received: 17 Dec 2025; Accepted: 16 Feb 2026.
Copyright: © 2026 Czaplewska, Musiał, Iwaniec, Mruk, Puchalski, Tretiakow, Skorek, Szydłowski and Szlęzak. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence:
Paulina Czaplewska
Natalia Musiał
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