{"doi":"10.1039/d2mo00244b","title":"Deciphering <i>O</i> -glycoprotease substrate preferences with O-Pair Search","abstract":"Abstract O-Glycoproteases are an emerging class of enzymes that selectively digest glycoproteins at positions decorated with specific O-linked glycans. O-Glycoprotease substrates range from any O-glycoprotein (albeit with specific O-glycan modifications) to only glycoproteins harboring specific O-glycosylated sequence motifs, such as those found in mucin domains. Their utility for multiple glycoproteomic applications is driving the search to both discover new O-glycoproteases and to understand how structural features of characterized O-glycoproteases influence their substrate specificities. One challenge of defining O-glycoprotease specificity restraints is the need to characterize O-glycopeptides with site-specific analysis of O-glycosites. Here, we demonstrate how O-Pair Search, a recently developed O-glycopeptide-centric identification platform that enables rapid searches and confident O-glycosite localization, can be used to determine substrate specificities of various O-glycoproteases de novo from LC-MS/MS data of O-glycopeptides. Using secreted protease of C1 esterase inhibitor (StcE) from enterohemorrhagic Escherichia coli and O-endoprotease OgpA from Akkermansia mucinophila, we explore numerous settings that effect O-glycopeptide identification and show how non-specific and semi-tryptic searches of O-glycopeptide data can produce candidate cleavage motifs. These putative motifs can be further used to define new protease cleavage settings that lower search times and improve O-glycopeptide identifications. We use this platform to generate a consensus motif for the recently characterized immunomodulating metalloprotease (IMPa) from Pseudomonas aeruginosa and show that IMPa is a favorable O-glycoprotease for characterizing densely O-glycosylated mucin-domain glycoproteins.","journal":"Molecular Omics","year":2022,"id":247568,"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":33,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9519,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2022-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":218284,"name":"Carolyn R. Bertozzi","orcid":"0000-0003-4482-2754","position":1,"is_corresponding":false},{"id":218283,"name":"Nicholas M. Riley","orcid":"0000-0002-1536-2966","position":0,"is_corresponding":true}],"reference_count":73,"raw_metadata":null,"created_at":"2026-07-19T00:23:57.824223Z","pmid":"36373229","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":[]}