Deciphering O-glycoprotease substrate preferences with O-Pair Search

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 that can be 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.