{"doi":"10.1016/j.crmeth.2024.100744","title":"Global O-glycoproteome enrichment and analysis enabled by a combinatorial enzymatic workflow","abstract":"A comprehensive analysis of site-specific protein O-glycosylation is hindered by the absence of a consensus O-glycosylation motif, the diversity of O-glycan structures, and the lack of a universal enzyme that cleaves attached O-glycans. Here, we report the development of a robust O-glycoproteomic workflow for analyzing complex biological samples by combining four different strategies: removal of N-glycans, complementary digestion using O-glycoprotease (IMPa) with/without another protease, glycopeptide enrichment, and mass spectrometry with fragmentation of glycopeptides using stepped collision energy. Using this workflow, we cataloged 474 O-glycopeptides on 189 O-glycosites derived from 79 O-glycoproteins from human plasma. These data revealed O-glycosylation of several abundant proteins that have not been previously reported. Because many of the proteins that contained unannotated O-glycosylation sites have been extensively studied, we wished to confirm glycosylation at these sites in a targeted fashion. Thus, we analyzed selected purified proteins (kininogen-1, fetuin-A, fibrinogen, apolipoprotein E, and plasminogen) in independent experiments and validated the previously unknown O-glycosites.","journal":"Cell Reports Methods","year":2024,"id":440068,"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":9,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9592,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1216170,"name":"Rohit Budhraja","orcid":"0000-0002-4209-2180","position":1,"is_corresponding":false},{"id":1247992,"name":"Jin Yong Kim","orcid":"0000-0003-0115-4215","position":2,"is_corresponding":false},{"id":1220594,"name":"Neha Joshi","orcid":"0000-0001-9232-1589","position":3,"is_corresponding":false},{"id":1117240,"name":"Kishore Garapati","orcid":"0000-0002-2759-609X","position":4,"is_corresponding":false},{"id":60408,"name":"Akhilesh Pandey","orcid":"0000-0001-9943-6127","position":5,"is_corresponding":false},{"id":485313,"name":"Taewook Kang","orcid":"0000-0003-3959-4777","position":0,"is_corresponding":true}],"reference_count":28,"raw_metadata":null,"created_at":"2026-07-19T02:00:57.233748Z","pmid":"38582075","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":[]}