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However, a critical step for the success of these analyses is the protein extraction from the tissue of interest, with well-described and reproducible protocols still scarce in the current literature, especially for bone tissues. Therefore, the main objective of this study was to present a reliable and standardized methodology for protein extraction from hard tissue samples. The methodology employed was based on an extraction protocol using demineralized bone tissue, with more detailed experimental parameters such as incubation times, temperatures and sample storage conditions. As a result, 1429 proteins previously described for the species Rattus norvegicus were identified, along with 138 uncharacterized proteins and the detection of signalling pathways strongly associated with bone metabolism. The proposed protocol has the potential for adaptation to different experimental contexts, being adjustable for various species and types of bone, considering variations in density, degree of mineralization and structural characteristics of the analysed tissue.<\/jats:p>","DOI":"10.1098\/rsos.261692","type":"journal-article","created":{"date-parts":[[2026,10,14]],"date-time":"2026-10-14T00:16:44Z","timestamp":1791937004000},"source":"Crossref","is-referenced-by-count":0,"title":["Recent advances in protein extraction methods from bone tissue for proteomics analysis"],"prefix":"10.1098","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4478-4615","authenticated-orcid":false,"given":"Karoline B. do.","family":"Nascimento","sequence":"first","affiliation":[{"name":"Universidade Estadual Paulista Julio de Mesquita Filho 1 , Araraquara, S\u00e3o Paulo, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0064-2527","authenticated-orcid":false,"given":"Bruno L. Graciliano","family":"Silva","sequence":"additional","affiliation":[{"name":"Universidade Estadual Paulista Julio de Mesquita Filho 1 , Araraquara, S\u00e3o Paulo, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Felipe E.","family":"Pinotti","sequence":"additional","affiliation":[{"name":"Pontifica Universidade Cat\u00f3lica de Campinas 2 , Campinas, S\u00e3o Paulo, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3384-8863","authenticated-orcid":false,"given":"Julio Cesar","family":"S\u00e1nchez-Puetate","sequence":"additional","affiliation":[{"name":"Universidad San Francisco de Quito 3 , Quito, Pichincha, Ecuador"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Morgana R.","family":"Guimar\u00e3es-Stabili","sequence":"additional","affiliation":[{"name":"Universidade Estadual Paulista Julio de Mesquita Filho 1 , Araraquara, S\u00e3o Paulo, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Camila C.","family":"Marcantonio","sequence":"additional","affiliation":[{"name":"Universidade Estadual Paulista Julio de Mesquita Filho 1 , Araraquara, S\u00e3o Paulo, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rosemary Adriana C.","family":"Marcantonio","sequence":"additional","affiliation":[{"name":"Universidade Estadual Paulista Julio de Mesquita Filho 1 , Araraquara, S\u00e3o Paulo, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"175","published-online":{"date-parts":[[2026,10,14]]},"reference":[{"key":"2026102010504592200_RSOS261692C1","doi-asserted-by":"crossref","first-page":"36","DOI":"10.1016\/j.jprot.2018.12.012","article-title":"Proteomics turns functional","volume":"198","author":"Monti","year":"2019","journal-title":"J. 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