{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,16]],"date-time":"2026-03-16T06:19:36Z","timestamp":1773641976899,"version":"3.50.1"},"reference-count":76,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,3,16]],"date-time":"2026-03-16T00:00:00Z","timestamp":1773619200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Mol. Biosci."],"abstract":"<jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>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.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      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\n                      <jats:italic>Raoultella ornithinolytica<\/jats:italic>\n                      , were analysed to assess the broader applicability of the developed workflows.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>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.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>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.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.3389\/fmolb.2026.1769952","type":"journal-article","created":{"date-parts":[[2026,3,16]],"date-time":"2026-03-16T05:22:28Z","timestamp":1773638548000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Optimization of protein extraction and digestion workflows for proteomic analysis of saliva, salivary stones and kidney stones"],"prefix":"10.3389","volume":"13","author":[{"given":"Natalia","family":"Musia\u0142","sequence":"first","affiliation":[{"name":"Laboratory of Mass Spectrometry, Intercollegiate Faculty of Biotechnology, University of Gdansk","place":["Gdansk, Poland"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Martyna","family":"Iwaniec","sequence":"additional","affiliation":[{"name":"Laboratory of Mass Spectrometry, Intercollegiate Faculty of Biotechnology, University of Gdansk","place":["Gdansk, Poland"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Inez","family":"Mruk","sequence":"additional","affiliation":[{"name":"Laboratory of Mass Spectrometry, Intercollegiate Faculty of Biotechnology, University of Gdansk","place":["Gdansk, Poland"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Micha\u0142","family":"Puchalski","sequence":"additional","affiliation":[{"name":"Laboratory of Biopolymers Structure, Intercollegiate Faculty of Biotechnology, University of Gdansk","place":["Gdansk, Poland"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dmitry","family":"Tretiakow","sequence":"additional","affiliation":[{"name":"Clinical Department of Otolaryngology, Academy of Applied Medical and Social Sciences, The Nicolaus Copernicus Hospital in Gdansk, Copernicus Healthcare Entity","place":["Gdansk, Poland"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Andrzej","family":"Skorek","sequence":"additional","affiliation":[{"name":"Clinical Department of Otolaryngology, Academy of Applied Medical and Social Sciences, The Nicolaus Copernicus Hospital in Gdansk, Copernicus Healthcare Entity","place":["Gdansk, Poland"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Konrad","family":"Szyd\u0142owski","sequence":"additional","affiliation":[{"name":"Clinical Department of Otolaryngology, Academy of Applied Medical and Social Sciences, The Nicolaus Copernicus Hospital in Gdansk, Copernicus Healthcare Entity","place":["Gdansk, Poland"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jan","family":"Szl\u0119zak","sequence":"additional","affiliation":[{"name":"Clinical Department of Otolaryngology, Academy of Applied Medical and Social Sciences, The Nicolaus Copernicus Hospital in Gdansk, Copernicus Healthcare Entity","place":["Gdansk, Poland"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Paulina","family":"Czaplewska","sequence":"additional","affiliation":[{"name":"Laboratory of Mass Spectrometry, Intercollegiate Faculty of Biotechnology, University of Gdansk","place":["Gdansk, Poland"]}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2026,3,16]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"997","DOI":"10.1099\/00221287-136-6-997","article-title":"Listericidal activity of human neutrophil cathepsin G","volume":"136","author":"Alford","year":"1990","journal-title":"J. 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