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Here, we develop qPISA (quantitative Protease specificity Inference from Substrate Analysis) to study Dipeptidyl Peptidase Four (DPP4), a key regulator of blood glucose levels. We use mass spectrometry to quantify &gt;40,000 peptides from a complex, commercially available peptide mixture. By analyzing changes in substrate levels quantitatively instead of focusing on qualitative product identification through a binary classifier, we can reveal cooperative interactions within DPP4\u2019s active pocket and derive a sequence motif that predicts activity quantitatively. qPISA distinguishes DPP4 from the related\n                    <jats:italic>C. elegans<\/jats:italic>\n                    DPF-3 (a DPP8\/9-orthologue), and we relate the differences to the structural features of the two enzymes. We demonstrate that qPISA can direct protein engineering efforts like the stabilization of GLP-1, a key DPP4 substrate used in the treatment of diabetes and obesity. Thus, qPISA offers a versatile approach for profiling protease and especially exopeptidase specificity, facilitating insight into enzyme mechanisms and biotechnological and clinical applications.\n                  <\/jats:p>","DOI":"10.1038\/s44320-024-00071-4","type":"journal-article","created":{"date-parts":[[2024,10,28]],"date-time":"2024-10-28T11:03:07Z","timestamp":1730113387000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Deep quantification of substrate turnover defines protease subsite cooperativity"],"prefix":"10.1038","volume":"20","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7364-9279","authenticated-orcid":false,"given":"Rajani Kanth","family":"Gudipati","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dimos","family":"Gaidatzis","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7858-2720","authenticated-orcid":false,"given":"Jan","family":"Seebacher","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sandra","family":"Muehlhaeusser","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0228-6730","authenticated-orcid":false,"given":"Georg","family":"Kempf","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Simone","family":"Cavadini","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1642-5404","authenticated-orcid":false,"given":"Daniel","family":"Hess","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3833-2169","authenticated-orcid":false,"given":"Charlotte","family":"Soneson","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8169-6905","authenticated-orcid":false,"given":"Helge","family":"Gro\u00dfhans","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2024,10,28]]},"reference":[{"key":"71_CR1","doi-asserted-by":"publisher","first-page":"412","DOI":"10.1110\/ps.03460604","volume":"13","author":"K Aertgeerts","year":"2004","unstructured":"Aertgeerts K, Ye S, Tennant MG, Kraus ML, Rogers J, Sang BC, Skene RJ, Webb DR, Prasad GS (2004) Crystal structure of human dipeptidyl peptidase IV in complex with a decapeptide reveals details on substrate specificity and tetrahedral intermediate formation. 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