Enrichment strategies for mucin-domain O-glycoproteins

Mucin-domain glycoproteins are densely O-glycosylated proteins that comprise a major component of the glycocalyx and play central roles in immune regulation, host-pathogen interactions, cancer biology, and cell-cell communication. Despite their biological importance, the structural and functional characterization of mucin-type O-glycosylation remains analytically challenging due to extensive microheterogeneity, resistance to conventional proteases, and the combinatorial complexity of glycoforms. This chapter outlines practical, mass spectrometry-compatible enrichment and analysis strategies designed to overcome these barriers and enable robust O-glycoproteomic investigations. Two complementary enrichment approaches are described. The first leverages an inactive point mutant of the mucinase StcE (StcE^E447D) conjugated to a solid support to selectively isolate mucin-domain glycoproteins from complex biological samples, functioning as a targeted mucinome probe. The second, termed GlycoFASP, employs molecular weight cut-off filters in combination with O-glycoproteases or mucinases to enrich O-glycopeptides more broadly, making it well suited for biofluids and diverse O-glycoproteomes. Both workflows are coupled to enzymatic digestion strategies that rely on glycoproteases with dual recognition motifs dependent on peptide sequence and glycosylation status, followed by desalting and LC-MS analysis with electron-based fragmentation to enable site-specific glycan localization. Detailed protocols, rationale, optimization strategies, and troubleshooting guidance are provided to facilitate implementation across a range of sample types. Together, these approaches offer accessible and adaptable solutions for mapping mucin-domain O-glycoproteins with molecular precision, advancing the study of glycoprotein structure and function in health and disease.