{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,7]],"date-time":"2026-08-07T15:23:08Z","timestamp":1786116188668,"version":"build-2736575974"},"reference-count":20,"publisher":"Wiley","license":[{"start":{"date-parts":[[2026,8,6]],"date-time":"2026-08-06T00:00:00Z","timestamp":1785974400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"},{"start":{"date-parts":[[2026,8,6]],"date-time":"2026-08-06T00:00:00Z","timestamp":1785974400000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/100009619","name":"Japan Agency for Medical Research and Development","doi-asserted-by":"publisher","award":["25ama121018j"],"award-info":[{"award-number":["25ama121018j"]}],"id":[{"id":"10.13039\/100009619","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["analyticalsciencejournals.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Proteomics"],"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:label\/>\n                    <jats:p>Formalin\u2010fixed paraffin\u2010embedded (FFPE) tissues represent a vast archive for translational research that offers access to specimens with clinical outcomes. However, formalin\u2010induced crosslinking hinders efficient protein extraction and limits proteomic applications. We systematically compared three protein extraction workflows, adaptive focused acoustics (AFA) with Covaris buffer (AFA\/Covaris\u2010b), AFA with SDS buffer (AFA\/SDS\u2010b), and SDS\u2010based lysis followed by SP3 clean\u2010up and digestion (SP3\/SDS\u2010b), using hepatocellular carcinoma (HCC) tissues archived for over 30 years. Benchmarking was performed on two HCC cases using three serial tumor sections each, followed by protein quantification using data\u2010independent acquisition mass spectrometry on a ZenoTOF 7600+ instrument. All workflows identified 5206 proteins, with similar mean identifications (AFA\/Covaris\u2010b: 4068; AFA\/SDS\u2010b: 4134; SP3\/SDS\u2010b: 4048), but distinct reproducibility (CVs: 11.9%, 12.2%, and 20.8%, respectively), leading to the exclusion of SP3. Method selection across eight HCC cases, including paired tumor and adjacent non\u2010tumorous tissues, revealed a total of 6018 proteins, with mean identifications of 3876 (AFA\/Covaris\u2010b) and 3852 (AFA\/SDS\u2010b). Both AFA workflows showed high reproducibility, with slightly lower variability for AFA\/Covaris\u2010b (CVs: HCC: 13.5%, non\u2010tumorous liver tissue (NTL): 11.0%) compared with AFA\/SDS\u2010b (CVs: HCC: 14.8%, NTL: 11.3%). While upregulated pathways were not consistently detected across workflows, downregulated pathways showed greater concordance between methods. These findings should be interpreted cautiously given the lack of appropriate temporal or fresh\u2010frozen controls. Collectively, AFA\u2010based workflows enable reproducible proteomic profiling of long\u2010archived FFPE tissues and provide a practical approach for methodological evaluation in retrospective proteomics studies.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Significance of the study<\/jats:title>\n                    <jats:p>\n                      <jats:list list-type=\"bullet\">\n                        <jats:list-item>\n                          <jats:p>FFPE tissues are one of the most valuable clinically annotated biospecimen resources worldwide. However, their routine use in proteomics is hindered by extensive protein cross\u2010linking, particularly in long\u2010term archived blocks. Our study addresses this challenge by systematically evaluating and validating protein extraction strategies for FFPE hepatocellular carcinoma tissues stored for more than 30 years. By directly benchmarking AFA\u2010based and SP3 workflows, we show that AFA protocols, particularly those with the Covaris buffer, provide superior reproducibility while maintaining broad proteome coverage. Importantly, we showed that archival samples yielded more than 6,000 quantifiable proteins, with reproducible pathway\u2010level patterns observed across methods, although interpretation of biological signals remains limited by the absence of appropriate controls and potential extraction\u2010related biases. These findings suggest that long\u2010preserved FFPE specimens can serve as useful resources for modern data\u2010independent acquisition mass spectrometry. The ability to extract reproducible proteomic information from decades\u2010old FFPE blocks has important implications; it enables retrospective biomarker discovery, integration with genomic data, and validation of therapeutic targets in richly annotated patient cohorts. By providing a reproducible workflow, this study supports the expanded use of archived FFPE tissues and facilitates future efforts for large\u2010scale, longitudinal cancer proteomics.<\/jats:p>\n                        <\/jats:list-item>\n                      <\/jats:list>\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1002\/pmic.70169","type":"journal-article","created":{"date-parts":[[2026,8,6]],"date-time":"2026-08-06T07:45:41Z","timestamp":1786002341000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Comparative Evaluation of Adaptive Focused Acoustics\u2010 and SP3\u2010Based Workflows for Deep Proteomic Profiling of 30\u2010Year\u2010Old Formalin\u2010Fixed Paraffin\u2010Embedded Tissues"],"prefix":"10.1002","author":[{"given":"Rei","family":"Noguchi","sequence":"first","affiliation":[{"name":"Division of Rare Cancer Research National Cancer Center Research Institute  Chuo\u2010ku Tokyo Japan"},{"name":"Department of Pharmacology Tokyo Women's Medical University  Shinjuku\u2010ku Tokyo Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Masatsugu","family":"Ishii","sequence":"additional","affiliation":[{"name":"Department of Surgery Keio University School of Medicine  Shinjuku\u2010ku Tokyo Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuki","family":"Adachi","sequence":"additional","affiliation":[{"name":"Division of Rare Cancer Research National Cancer Center Research Institute  Chuo\u2010ku Tokyo Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuki","family":"Yoshimatsu","sequence":"additional","affiliation":[{"name":"Department of Patient\u2010Derived Cancer Model Tochigi Cancer Center Research Institute  Utsunomiya Tochigi Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kazuki","family":"Sasaki","sequence":"additional","affiliation":[{"name":"Department of Oncopeptidomics Tochigi Cancer Center  Tochigi Japan"},{"name":"Department of Peptidomics Sasaki Institute  Chuo\u2010ku Tokyo Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nobuyoshi","family":"Hiraoka","sequence":"additional","affiliation":[{"name":"Department of Diagnostic Pathology National Cancer Center Hospital  Chuo\u2010ku Tokyo Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sumio","family":"Ohtsuki","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Microbiology Graduate School of Pharmaceutical Sciences Kumamoto University  Chuo\u2010ku Kumamoto Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tadashi","family":"Kondo","sequence":"additional","affiliation":[{"name":"Division of Rare Cancer Research National Cancer Center Research Institute  Chuo\u2010ku Tokyo Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,8,6]]},"reference":[{"key":"e_1_2_14_2_1","doi-asserted-by":"publisher","DOI":"10.1089\/bio.2012.0052"},{"key":"e_1_2_14_3_1","doi-asserted-by":"publisher","DOI":"10.1039\/B800098K"},{"key":"e_1_2_14_4_1","doi-asserted-by":"publisher","DOI":"10.1021\/pr1004168"},{"key":"e_1_2_14_5_1","doi-asserted-by":"publisher","DOI":"10.1038\/labinvest.3700343"},{"key":"e_1_2_14_6_1","doi-asserted-by":"publisher","DOI":"10.1586\/epr.09.106"},{"key":"e_1_2_14_7_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467\u2010021\u201023855\u2010w"},{"key":"e_1_2_14_8_1","doi-asserted-by":"publisher","DOI":"10.1002\/pmic.201100550"},{"key":"e_1_2_14_9_1","doi-asserted-by":"publisher","DOI":"10.15252\/msb.20145625"},{"key":"e_1_2_14_10_1","unstructured":"L.Schweizer F.Coscia J.M\u00fcller S.Doll M.Wierer andM.Mann \u201cAFA\u2010sonication Followed by Modified Protein Aggregation Capture (APAC) Enables Direct Reproducible and Non\u2010Toxic Sample Preparation of FFPE Tissue for Mass Spectrometry\u2010based Proteomics \u201d Covaris Inc. (2020) https:\/\/www.covaris.com\/wp\/wp\u2010content\/uploads\/2020\/06\/M020141.pdf."},{"key":"e_1_2_14_11_1","doi-asserted-by":"publisher","DOI":"10.1021\/jasms.3c00099"},{"key":"e_1_2_14_12_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jproteome.9b00686"},{"key":"e_1_2_14_13_1","doi-asserted-by":"publisher","DOI":"10.1186\/s12014\u2010018\u20109202\u20104"},{"key":"e_1_2_14_14_1","doi-asserted-by":"publisher","DOI":"10.1002\/path.5420"},{"key":"e_1_2_14_15_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.analchem.8b01838"},{"key":"e_1_2_14_16_1","doi-asserted-by":"publisher","DOI":"10.1369\/jhc.2009.953497"},{"key":"e_1_2_14_17_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0016353"},{"key":"e_1_2_14_18_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jproteome.4c00149"},{"key":"e_1_2_14_19_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.mcpro.2025.101044"},{"key":"e_1_2_14_20_1","doi-asserted-by":"crossref","first-page":"951","DOI":"10.1021\/acs.jproteome.2c00758","article-title":"Proteome Coverage After Simultaneous Proteo\u2010Metabolome Liquid\u2010Liquid Extraction","volume":"22","author":"Pijkeren A.","year":"2023","journal-title":"Journal of Proteome Research"},{"key":"e_1_2_14_21_1","doi-asserted-by":"publisher","DOI":"10.1002\/pmic.70095"}],"container-title":["PROTEOMICS"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/analyticalsciencejournals.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/pmic.70169","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/analyticalsciencejournals.onlinelibrary.wiley.com\/doi\/full-xml\/10.1002\/pmic.70169","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/analyticalsciencejournals.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/pmic.70169","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,6]],"date-time":"2026-08-06T07:45:43Z","timestamp":1786002343000},"score":1,"resource":{"primary":{"URL":"https:\/\/analyticalsciencejournals.onlinelibrary.wiley.com\/doi\/10.1002\/pmic.70169"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,6]]},"references-count":20,"alternative-id":["10.1002\/pmic.70169"],"URL":"https:\/\/doi.org\/10.1002\/pmic.70169","archive":["Portico"],"relation":{},"ISSN":["1615-9853","1615-9861"],"issn-type":[{"value":"1615-9853","type":"print"},{"value":"1615-9861","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,6]]},"assertion":[{"value":"2025-12-13","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-07-21","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-08-06","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e70169"}}