{"doi":"10.1101/2023.02.12.528234","title":"An expanded 2-pyridinecarboxaldehyde (2PCA)-based chemoproteomics toolbox for probing protease specificity","abstract":"Summary Proteomic profiling of protease-generated N termini, or N terminomics, provides key insights into protease function and specificity. However, current N terminomics technologies have sequence limitations or require specialized synthetic reagents for N-terminal peptide isolation. Here, we introduce an expanded N terminomics toolbox that is based on 2-pyridinecarboxaldehyde (2PCA) reagents. These tools enable efficient enrichment of protein N termini by combining selective N-terminal biotinylation using 2PCA reagents with chemically cleavable linkers for N-terminal peptide recovery. By incorporating a commercially available alkyne-modified 2PCA in combination with Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), our strategy eliminates the need for chemical synthesis of N-terminal probes. Using these reagents, we developed PICS2 ( P roteomic Identification of C leavage S ites with 2 PCA reagents) to profile the specificity of subtilisin/kexin-type proprotein convertases (PCSKs). We also implemented CHOPPER ( Ch emical enrichment O f P rotease substrates with P urchasable, E lutable R eagents) for global sequencing of apoptotic proteolytic cleavage sites. Based on their broad applicability and ease of implementation, PICS2 and CHOPPER are useful tools that will advance our understanding of protease biology.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2023,"id":394150,"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":3,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9481,"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":1046710,"name":"Clara L. Frazier","orcid":"0000-0003-0765-4827","position":1,"is_corresponding":false},{"id":566381,"name":"Amy M. Weeks","orcid":"0000-0003-4700-8256","position":2,"is_corresponding":false},{"id":1047086,"name":"Haley N. Bridge","orcid":null,"position":0,"is_corresponding":true}],"reference_count":61,"raw_metadata":null,"created_at":"2026-07-19T01:19:10.334330Z","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":[]}