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We describe a rapid method for epitope mapping, employing barcoded charged scanning mutagenesis libraries displayed on the yeast surface, and screening using flow cytometry coupled with deep sequencing. Prior scanning mutagenesis data suggest that mutations to a charged residue, such as Aspartic acid or Arginine, will be well tolerated at exposed positions of an antigen and minimally affect protein stability and expression. Yet such substitutions at epitope residues strongly perturb binding to a cognate partner. We constructed an Aspartate scanning library of SARS\u2010CoV\u20102 receptor binding domain and linked every mutation in the library to a defined unique barcode. The approach was used to map epitopes targeted in polyclonal sera of mice immunized with different SARS\u2010CoV\u20102 immunogens. In contrast to complete mutational scans, charged scanning mutagenesis with the introduced barcoding strategy employs libraries with &gt;50\u2010fold lower diversity, facilitating library construction, screening, and downstream analysis, and also allowing for further multiplexing of samples, thus accelerating interaction site identification, as well as vaccine and inhibitor development.<\/jats:p>","DOI":"10.1002\/pro.70433","type":"journal-article","created":{"date-parts":[[2025,12,22]],"date-time":"2025-12-22T13:51:20Z","timestamp":1766411480000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Charged scanning mutagenesis as a high\u2010throughput approach for epitope mapping"],"prefix":"10.1002","volume":"35","author":[{"given":"Kawkab","family":"Kanjo","sequence":"first","affiliation":[{"name":"Molecular Biophysics Unit (MBU) Indian Institute of Science  Bengaluru India"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Munmun","family":"Bhasin","sequence":"additional","affiliation":[{"name":"Molecular Biophysics Unit (MBU) Indian Institute of Science  Bengaluru India"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chandrani","family":"Dey","sequence":"additional","affiliation":[{"name":"Molecular Biophysics Unit (MBU) Indian Institute of Science  Bengaluru India"},{"name":"Department of Biotechnology and Dr. B. 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