Deciphering O-glycoprotease substrate preferences with O-Pair Search

Area of Science:

• Biochemistry and enzymology
• Glycoproteomics
• Mass spectrometry

Background:

• O-Glycoproteases are enzymes that cleave O-linked glycoproteins at specific glycan sites.
• Understanding O-glycoprotease substrate specificity is crucial for glycoproteomic applications.
• Characterizing O-glycoprotease specificity requires site-specific analysis of O-glycopeptides.

Purpose of the Study:

• To demonstrate the utility of the O-Pair Search platform for determining O-glycoprotease substrate specificities.
• To identify O-glycoprotease cleavage motifs and characterize enzyme specificities using LC-MS/MS data.
• To explore O-glycopeptide identification settings and improve O-glycopeptide discovery.

Main Methods:

• Utilized the O-Pair Search platform for O-glycopeptide-centric identification and O-glycosite localization.
• Analyzed LC-MS/MS data from O-glycopeptides using secreted protease of C1 esterase inhibitor (StcE) and O-endoprotease OgpA.
• Performed non-specific and semi-tryptic searches to generate candidate cleavage motifs.

Main Results:

• The O-Pair Search platform enables rapid and confident O-glycosite localization and de novo determination of O-glycoprotease specificities.
• Non-specific and semi-tryptic searches identified putative cleavage motifs for StcE and OgpA.
• A consensus motif for immunomodulating metalloprotease (IMPa) was generated, highlighting its utility for mucin-domain glycoproteins.

Conclusions:

• The O-Pair Search platform is effective for characterizing O-glycoprotease specificities and identifying cleavage motifs.
• Optimized search settings can improve O-glycopeptide identification efficiency.
• IMPa is a promising O-glycoprotease for studying densely O-glycosylated mucin-domain glycoproteins.