{"doi":"10.1101/2023.11.17.567387","title":"Massively parallel quantification of substrate turnover defines protease subsite cooperativity","abstract":"Abstract Substrate specificity determines protease functions in physiology and in clinical and biotechnological application, yet quantitative cleavage information is often unavailable, biased, or limited to a small number of events. Here, we develop qPISA (quantitative Protease specificity Inference from Substrate Analysis) to study D i p eptidyl P eptidase Four (DPP4), a key regulator of blood glucose levels. We use mass spectrometry to quantify its effects on >40,000 peptides from a complex, commercially available peptide mixture. By determining substrate turnover instead of focusing on product identification, we can reveal cooperative interactions within DPP4’s active pocket and derive a sequence motif that predicts activity quantitatively. qPISA distinguishes DPP4 from the related C. elegans DPF-3 (a DPP8/9 orthologue), and we relate the differences to structural features of the two enzymes. We demonstrate that qPISA can direct protein engineering efforts like stabilization of GLP-1, a key DPP4 substrate used in 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.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2023,"id":401612,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9433,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2023-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":339437,"name":"Dimos Gaidatzis","orcid":"0000-0003-4294-4504","position":1,"is_corresponding":false},{"id":867821,"name":"Jan Seebacher","orcid":"0000-0002-7858-2720","position":2,"is_corresponding":false},{"id":1179044,"name":"Sandra Muehlhaeusser","orcid":null,"position":3,"is_corresponding":false},{"id":1019609,"name":"Georg Kempf","orcid":"0000-0002-0228-6730","position":4,"is_corresponding":false},{"id":1019610,"name":"Simone Cavadini","orcid":"0000-0003-2777-7584","position":5,"is_corresponding":false},{"id":81034,"name":"Daniel Heß","orcid":"0000-0002-1642-5404","position":6,"is_corresponding":false},{"id":230638,"name":"Charlotte Soneson","orcid":"0000-0003-3833-2169","position":7,"is_corresponding":false},{"id":372024,"name":"Helge Großhans","orcid":"0000-0002-8169-6905","position":8,"is_corresponding":false},{"id":568754,"name":"Rajani Kanth Gudipati","orcid":"0000-0002-7364-9279","position":0,"is_corresponding":true}],"reference_count":74,"raw_metadata":null,"created_at":"2026-07-19T01:20:12.316631Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}