Electron-Based Dissociation Is Needed for O-Glycopeptides Derived from OpeRATOR Proteolysis

Area of Science:

• Biochemistry
• Proteomics
• Glycobiology

Background:

• O-glycoproteomics is crucial for understanding biological processes.
• Characterizing O-glycopeptides is challenging due to complex glycosylation patterns.
• The bacterial O-glycoprotease OpeRATOR offers orthogonal cleavage for O-glycopeptide analysis.

Purpose of the Study:

• To investigate the utility of OpeRATOR for O-glycopeptide characterization.
• To determine the number of O-glycosites in OpeRATOR-digested O-glycopeptides.
• To assess the necessity of different fragmentation methods for O-glycopeptide analysis.

Main Methods:

• O-glycoprotein digestion using OpeRATOR and trypsin.
• Combined collision- and electron-based fragmentation techniques.
• Mass spectrometry/tandem mass spectrometry (MS/MS) analysis.

Main Results:

• OpeRATOR cleaves N-terminally to O-glycans on serine and threonine residues.
• Over 50% of O-glycopeptides from combined OpeRATOR and trypsin digestion contained multiple O-glycosites.
• Collision-based fragmentation alone was insufficient for characterizing all O-glycopeptides.

Conclusions:

• OpeRATOR is a valuable tool for O-glycoproteomics, providing orthogonal cleavage.
• Multiple O-glycosites are common in OpeRATOR-digested O-glycopeptides.
• Electron-based dissociation methods are essential for comprehensive O-glycopeptide diversity analysis.