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We present SurfDiff, a structure- and sequence-informed framework for protein surface comparison. SurfDiff supports one-to-one and one-to-many comparisons by combining local structural alignments with physicochemically and spatially aware neighbourhood analysis. It assigns residue-level uniqueness and similarity scores, which can be aggregated into surface-level similarity, selectivity, and a discriminability score that captures conservation across desired targets while penalising similarity to undesired off-targets. These scores, calculated solely from the targets without any knowledge of the binders, reliably predict known experimental selectivity for diverse binders including small molecules, peptides, antibodies and antibody mimetics across viral antigens, cytokine isoforms, GPCR subtypes and serum albumin. They also correlate strongly with binding and neutralisation data across pathogen variants, and substantially outperform commonly used bioinformatic metrics such as sequence substitution matrices and structural similarity measures. SurfDiff provides a generalisable and interpretable approach to protein surface profiling, enabling selective or cross-reactive binder design, cross-species prioritisation, and facilitating immunogen selection. We make SurfDiff available as downloadable open-source software: (\n                  <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" ext-link-type=\"uri\" xlink:href=\"https:\/\/gitlab.developers.cam.ac.uk\/ch\/sormanni\/surfdiff\">https:\/\/gitlab.developers.cam.ac.uk\/ch\/sormanni\/surfdiff<\/jats:ext-link>\n                  ) and as a webserver:\n                  <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" ext-link-type=\"uri\" xlink:href=\"https:\/\/www-cohsoftware.ch.cam.ac.uk\/index.php\/surfdiff\">www-cohsoftware.ch.cam.ac.uk\/index.php\/surfdiff<\/jats:ext-link>\n                  .\n                <\/jats:p>","DOI":"10.64898\/2025.12.23.696170","type":"posted-content","created":{"date-parts":[[2025,12,24]],"date-time":"2025-12-24T03:50:16Z","timestamp":1766548216000},"source":"Crossref","is-referenced-by-count":0,"title":["SurfDiff: protein surface profiling for selective or broadly reactive epitope prioritisation in binder and immunogen design"],"prefix":"10.64898","author":[{"given":"Benedikt","family":"Jedlitzke","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Oliver","family":"Wissett","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tung H.","family":"Nguyen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1515-725X","authenticated-orcid":false,"given":"Elodie","family":"Ghedin","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6228-2221","authenticated-orcid":false,"given":"Pietro","family":"Sormanni","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2025122903300751000_2025.12.23.696170v1.1","doi-asserted-by":"crossref","first-page":"101","DOI":"10.1293\/tox.2024-0033","article-title":"Therapeutic antibodies: technical points to consider in tissue cross-reactivity studies","volume":"37","year":"2024","journal-title":"J. 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