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In recent years, proteomics based on molecular sexual dimorphism has emerged as a preferred method, particularly because of its minimally invasive approach for extracting amelogenin X and Y proteins from tooth enamel. However, there is an increasing demand to accelerate this process and facilitate the analysis of large archeological assemblages. This study presents a novel high-throughput targeted paleoproteomics method for biological sex estimation using MALDI-CASI-FTICR mass spectrometry. This approach combines the strengths of existing methods, including ultrahigh resolution, significantly reduced processing times, targeted analysis, and scalability to large archeological sample sets. The method was initially validated on modern individuals with known sex and subsequently applied to 130 adult and juvenile individuals from medieval Great Moravia (present-day Czech Republic). Biological sex was successfully estimated for all but one of the individuals. The results not only provide a more efficient biological sex estimation but also help to resolve a few errors in sex assessment previously encountered with osteomorphological and tooth morphometric techniques. The implementation of this method significantly improves the accuracy and efficiency of biological sex estimation, offering a powerful tool for anthropological research.<\/jats:p>","DOI":"10.1021\/acs.jproteome.6c00163","type":"journal-article","created":{"date-parts":[[2026,9,2]],"date-time":"2026-09-02T07:47:15Z","timestamp":1788335235000},"page":"5173-5188","source":"Crossref","is-referenced-by-count":0,"title":["High-Throughput\nTargeted Paleoproteomics Sex Estimation\non Medieval Great Moravian Individuals Using MALDI-CASI-FTICR Mass\nSpectrometry"],"prefix":"10.1021","volume":"25","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4723-8206","authenticated-orcid":true,"given":"Fabrice","family":"Bray","sequence":"first","affiliation":[{"name":"Univ. Lille , , ,","place":["Lille, France, F-59000"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anez\u030cka","family":"Pilmann Kote\u030crova\u0301","sequence":"additional","affiliation":[{"name":"Charles University , , Vinic\u030cna\u0301 7 , ,","place":["Prague 2, Czech Republic, 128 00"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lisa","family":"Garbe\u0301","sequence":"additional","affiliation":[{"name":"Univ. Lille , , ,","place":["Lille, France, F-59000"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3115-7978","authenticated-orcid":true,"given":"Marc","family":"Haegelin","sequence":"additional","affiliation":[{"name":"Univ. Lille , , ,","place":["Lille, France, F-59000"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Benoi\u0302t","family":"Bertrand","sequence":"additional","affiliation":[{"name":"Univ. 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Lille , , ,","place":["Lille, France, F-59000"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Petr","family":"Velemi\u0301nsky\u0301","sequence":"additional","affiliation":[{"name":"National Museum , , Va\u0301clavske\u0301 na\u0301me\u030csti\u0301 1700\/68 , ,","place":["Prague 1, Czech Republic, 110 00"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jaroslav","family":"Bru\u030az\u030cek","sequence":"additional","affiliation":[{"name":"Charles University , , Vinic\u030cna\u0301 7 , ,","place":["Prague 2, Czech Republic, 128 00"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7904-7108","authenticated-orcid":true,"given":"Marine","family":"Morvan","sequence":"additional","affiliation":[{"name":"Vilnius University , , Universiteto g. 7 , ,","place":["Vilnius, Lithuania, LT-01513"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,28]]},"reference":[{"key":"2026100208413978700_cit1","doi-asserted-by":"publisher","first-page":"110854","DOI":"10.1016\/j.forsciint.2021.110854","article-title":"How Low Can\nWe Go? A Skeletal Maturity Threshold for Probabilistic Visual Sex\nEstimation from Immature Human Os Coxae","volume":"325","author":"Corron","year":"2021","journal-title":"Forensic\nSci. Int."},{"issue":"15","key":"2026100208413978700_cit2","doi-asserted-by":"publisher","first-page":"2300183","DOI":"10.1002\/jssc.202300183","article-title":"Peptide Analysis of\nTooth Enamel \u2013 A Sex Estimation Tool for Archaeological, Anthropological,\nor Forensic Research","volume":"46","author":"Miks\u030ci\u0301k","year":"2023","journal-title":"J. Sep. Sci."},{"key":"2026100208413978700_cit3","doi-asserted-by":"publisher","first-page":"204","DOI":"10.1016\/j.culher.2023.11.021","article-title":"Undertaking\nthe Biological\nSex Assessment of Human Remains: The Applicability of Minimally-Invasive\nMethods for Proteomic Sex Estimation from Enamel Peptides","volume":"66","author":"Bru\u030az\u030cek","year":"2024","journal-title":"J. Cult. Herit."},{"key":"2026100208413978700_cit4","doi-asserted-by":"publisher","DOI":"10.1017\/CBO9780511542473","volume-title":"The Bioarchaeology\nof Children: perspectives from Biological and Forensic Anthropology","author":"Lewis","year":"2006","edition":"1st"},{"issue":"66","key":"2026100208413978700_cit5","doi-asserted-by":"publisher","first-page":"61673","DOI":"10.1039\/C6RA05120K","article-title":"The Identification of Peptides by nanoLC-MS\/MS from\nHuman Surface Tooth Enamel Following a Simple Acid Etch Extraction","volume":"6","author":"Stewart","year":"2016","journal-title":"RSC Adv."},{"issue":"1","key":"2026100208413978700_cit6","doi-asserted-by":"publisher","first-page":"13130","DOI":"10.1038\/s41598-019-49562-7","article-title":"Enamel Peptides\nReveal the Sex of the Late Antique \u2018Lovers of Modena","volume":"9","author":"Lugli","year":"2019","journal-title":"Sci. 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Practical adjustments from Van Vark\u2019s\nprocedure","volume":"9","author":"Murail","year":"1999","journal-title":"Int. J. Osteoarchaeol."},{"key":"2026100208413978700_cit23","volume-title":"Velkomoravske\u0301\npohr\u030cebis\u030cte\u030c v Rajhrade\u030c","author":"Hana\u0301kova\u0301","year":"1986","edition":"1st"},{"key":"2026100208413978700_cit24","unstructured":"Pospi\u0301s\u030cilova\u0301, K.\n          \n          Odhad ve\u030cku\npuberty v bioarcheologii: aplikace v populaci Velke\u0301 Moravy\n(9.\u201311. stoleti\u0301). Master thesis, Katedra antropologie\na genetiky c\u030clove\u030cka, Praha; Univerzita Karlova, Pr\u030ci\u0301rodove\u030cdecka\u0301 fakulta, 2017."},{"issue":"1","key":"2026100208413978700_cit25","doi-asserted-by":"publisher","first-page":"158","DOI":"10.1016\/j.jas.2007.02.013","article-title":"Sample-Specific\n(Universal) Metric Approaches for Determining the Sex of Immature\nHuman Skeletal Remains Using Permanent Tooth Dimensions","volume":"35","author":"Cardoso","year":"2008","journal-title":"J. Archaeol. Sci."},{"issue":"25","key":"2026100208413978700_cit26","doi-asserted-by":"publisher","first-page":"7369","DOI":"10.1002\/anie.201713020","article-title":"Palaeoproteomic\nProfiling of Conservation Layers on a 14th Century Italian Wall Painting","volume":"57","author":"Mackie","year":"2018","journal-title":"Angew. Chem., Int. Ed."},{"issue":"52","key":"2026100208413978700_cit27","doi-asserted-by":"publisher","first-page":"13649","DOI":"10.1073\/pnas.1714926115","article-title":"Sex Determination\nof Human Remains from Peptides in Tooth Enamel","volume":"114","author":"Stewart","year":"2017","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2026100208413978700_cit28","doi-asserted-by":"publisher","first-page":"169","DOI":"10.1016\/j.jas.2018.08.011","article-title":"Sex Estimation Using\nSexually Dimorphic Amelogenin Protein Fragments in Human Enamel","volume":"101","author":"Parker","year":"2019","journal-title":"J. Archaeol. Sci."},{"key":"2026100208413978700_cit29","doi-asserted-by":"publisher","first-page":"104430","DOI":"10.1016\/j.jasrep.2024.104430","article-title":"Advancing\nSex Estimation\nfrom Amelogenin: Applications to Archaeological, Deciduous, and Fragmentary\nDental Enamel","volume":"54","author":"Gamble","year":"2024","journal-title":"J. Archaeol. Sci. 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Sci."},{"key":"2026100208413978700_cit43","doi-asserted-by":"publisher","first-page":"105354","DOI":"10.1016\/j.jas.2021.105354","article-title":"AMELY Deletion Is Not Detected in\nSystematically Sampled Reference Populations: A Reply to S\u030ctamfelj","volume":"130","author":"Parker","year":"2021","journal-title":"J. Archaeol. Sci."},{"issue":"1","key":"2026100208413978700_cit44","doi-asserted-by":"publisher","first-page":"4431399","DOI":"10.1155\/tswj\/4431399","article-title":"Comparative Evaluation\nof Deciduous and Permanent Coronal Caries Using Polarizing Light Microscopy\nand Scanning Electron Microscopy","volume":"2025","author":"Thomas","year":"2025","journal-title":"Sci. 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