Characterization of O-glycoproteases for applications in mass spectrometry

This chapter describes practical workflows for the characterization and application of O-glycoproteases and mucinases in mass spectrometry-based analysis of mucin-domain glycoproteins. Dense O-glycosylation limits the effectiveness of conventional proteases, making specialized enzymes essential for generating informative peptides. We outline a stepwise strategy that begins with rapid gel-based assays to determine enzyme activity, glycan dependence, and sensitivity to sialylation. These qualitative experiments identify suitable substrates and guide optimization of enzyme to substrate ratios and digestion conditions. We then detail preparation of mucin samples for LC-MS/MS, including optional glycosidase treatment, controlled proteolysis, cleanup, and acquisition parameters that enable confident identification of glycopeptides. Particular emphasis is placed on interpreting fragmentation data and combining collisional and electron-based dissociation to determine peptide sequence and glycosite localization. Because automated searches remain error prone for O-glycopeptides, we provide guidelines for manual validation and for constructing cleavage motifs that incorporate both amino acid and glycan preferences. Together, these approaches establish a general framework for defining enzyme specificity and selecting appropriate tools for complex biological samples. Comprehensive characterization of mucin-degrading enzymes expands the analytical toolbox for studying mucin biology and improves the ability to map site-specific O-glycosylation with molecular precision.