Characterization of Site-Specific N- and O-Glycopeptides from Recombinant Spike and ACE2 Glycoproteins Using LC-MS/MS...

Area of Science:

• Biochemistry and Molecular Biology
• Virology
• Immunology

Background:

• The COVID-19 pandemic, caused by SARS-CoV-2, necessitates understanding viral protein structure and function.
• Spike (S) protein and ACE2 receptor glycosylation critically influence SARS-CoV-2 entry and immunogenicity.
• Detailed site-specific glycoproteomic analysis is crucial for therapeutic and vaccine development.

Purpose of the Study:

• To characterize the N- and O-glycosylation profiles of recombinant SARS-CoV-2 spike protein domains (RBD, S2) and ACE2.
• To identify and quantify site-specific glycosylation patterns and associated post-translational modifications (PTMs).
• To provide insights into how glycosylation affects protein function and immunogenicity for improved COVID-19 countermeasures.

Main Methods:

• Recombinant receptor-binding domain (RBD) and S2 subunit of spike protein, and ACE2 were expressed in Expi293F and plant cells.
• High-resolution mass spectrometry (Orbitrap Eclipse Tribrid) coupled with the Integrated GlycoProteome Analyzer (I-GPA) was employed.
• Site-specific N- and O-glycopeptides were characterized, and glycan types and PTMs were analyzed.

Main Results:

• Characterized 148 N- and 28 O-glycopeptides from RBD, 71 N-glycopeptides from S2, and 139 N-glycopeptides from ACE2.
• Identified novel glycan PTMs, including mannose-6-phosphate (M6P) and O-acetylation, on RBD and ACE2.
• Major N-glycosylation sites identified at Asn331 (RBD), Asn1098 (S2), and Asn103 (ACE2), with complex glycans on RBD/ACE2 and high-mannose on S2.

Conclusions:

• Detailed glycoproteomic maps of key SARS-CoV-2 proteins were established.
• Site-specific glycosylation patterns significantly influence protein structure and function.
• Findings provide a foundation for designing more effective COVID-19 vaccines and antibody-based therapies.