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The prevalence of fungal biofilm formation during infections, with its heightened resistance to antifungal drugs, highlights the urgency for the discovery and development of new antifungals with antibiofilm activity. Current advances in mass spectrometry-based proteomics and computational platforms provide a powerful toolkit to accelerate drug discovery from target identification to optimization of a lead molecule. In this review, we highlight fungal biofilms of four critical priority fungal pathogens (as deemed by the World Health Organization) and define important technological considerations for proteomics and computational methodologies. Additionally, we explore recent proteomics and computational applications within fungal biofilms for the identification and elucidation of biological mechanisms underscoring biofilm formation as well as the discovery of novel putative antibiofilm targets.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/mbio.02303-24","type":"journal-article","created":{"date-parts":[[2025,7,22]],"date-time":"2025-07-22T13:01:44Z","timestamp":1753189304000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":5,"title":["Breaking down biofilms across critical priority fungal pathogens: proteomics and computational innovation for mechanistic insights and new target discovery"],"prefix":"10.1128","volume":"16","author":[{"given":"Oscar","family":"Romero","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01r7awg59","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Molecular and Cellular Biology, University of Guelph","place":["Guelph, Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4257-2096","authenticated-orcid":true,"given":"Jennifer","family":"Geddes-McAlister","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01r7awg59","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Molecular and Cellular Biology, University of Guelph","place":["Guelph, Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_2_2_2","doi-asserted-by":"publisher","DOI":"10.1016\/S1473-3099(23)00692-8"},{"key":"e_1_3_2_3_2","unstructured":"World Health Organization. 2024. 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