biostudies-literatureKang TNational Cancer InstituteNCI NIH HHSNIH100744https://www.ebi.ac.uk/biostudies/studies/S-EPMC11046030biostudies-literatureUnknown4(4)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 tCell reports methodsGlobal O-glycoproteome enrichment and analysis enabled by a combinatorial enzymatic workflow.PMC11046030U01 CA271410P30CA15083U01CA271410Garapati KJoshi NKang TBudhraja RKim JPandey AfalseGlobal O-glycoproteome enrichment and analysis enabled by a combinatorial enzymatic workflow.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 t2024-01-01T00:00:00Z2024 Apr2026-06-01T19:57:45.149Z2026-05-20T03:07:43.565ZS-EPMC110460303858207510.1016/j.crmeth.2024.100744