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. These putative motifs can be further 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.