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Here, we developed a DMS by phage display protocol to define the specificity determinants of short linear motifs (SLiMs) binding to peptide\u2010binding domains. We first designed a benchmarking DMS library to evaluate the performance of the approach on well\u2010known ligands for 11 different peptide\u2010binding domains, including the talin\u20101 PTB domain, the G3BP1 NTF2 domain, and the MDM2 SWIB domain. Comparison with a set of reference motifs from the eukaryotic linear motif (ELM) database confirmed that the DMS by phage display analysis correctly identifies known motif binding determinants and provides novel insights into specificity determinants, including defining a non\u2010canonical talin\u20101 PTB binding motif with a putative extended conformation. A second DMS library was designed, aiming to provide information on the binding determinants for 19 SLiM\u2010based interactions between human and SARS\u2010CoV\u20102 proteins. The analysis confirmed the affinity determining residues of viral peptides binding to host proteins and refined the consensus motifs in human peptides binding to five domains from SARS\u2010CoV\u20102 proteins, including the non\u2010structural protein (NSP) 9. The DMS analysis further pinpointed mutations that increased the affinity of ligands for NSP3 and NSP9. An affinity\u2010improved cell\u2010permeable NSP9\u2010binding peptide was found to exert stronger antiviral effects than the wild\u2010type peptide. Our study demonstrates that DMS by phage display can efficiently be multiplexed and applied to refine binding determinants and shows how the results can guide peptide\u2010engineering efforts.<\/jats:p>","DOI":"10.1002\/pro.70174","type":"journal-article","created":{"date-parts":[[2025,5,24]],"date-time":"2025-05-24T11:34:49Z","timestamp":1748086489000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Defining short linear motif binding determinants by phage display\u2010based deep mutational scanning"],"prefix":"10.1002","volume":"34","author":[{"given":"Caroline","family":"Benz","sequence":"first","affiliation":[{"name":"Department of Chemistry \u2013 BMC Uppsala University  Uppsala Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lars","family":"Maassen","sequence":"additional","affiliation":[{"name":"Department of Chemistry \u2013 BMC Uppsala University  Uppsala 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