N-Glycan Analysis of ACE2 as SARS-CoV-2 Receptor

Importance of the Topic

N-glycan profiling and characterization of sialic acid linkage isomers are critical for understanding glycoprotein function, viral infectivity, and receptor binding. In particular, the linkage type of sialic acids (α2,3 vs. α2,6) on host cell receptors influences viral tropism and pathogenicity. A robust, simple mass spectrometric method to discriminate linkage isomers can accelerate research in viral glycomics, therapeutic development, and quality control of biopharmaceuticals.

Objectives and Study Overview

This study demonstrates an approach to release, derivatize, label, and analyze N-glycans from human ACE2—the SARS-CoV-2 host receptor—using the SialoCapper™-ID Kit. The main goals are to stabilize sialic acid residues, distinguish α2,3- and α2,6-linked isomers in a single MS1 measurement, and quantify glycan populations without liquid chromatography or enzymatic link-specific cleavage.

Methodology and Instrumentation

The workflow comprises the following steps:

• Denaturation and reduction of recombinant human ACE2 using SDS and DTT, followed by nonionic surfactant addition and overnight PNGase F digestion at 37 °C to release N-glycans.
• In-solution, linkage-specific derivatization of sialic acids via the patented SALSA method with the SialoCapper-ID Kit, converting α2,6-linked residues to isopropylamide and α2,3-linked residues to methylamide.
• Removal of excess derivatization reagents using HILIC-based GL-Tip Amide purification.
• Labeling of glycan reducing ends with 2-aminobenzamide (AB) and subsequent clean-up by GL-Tip Amide.
• Deposition of sample and matrix onto a MALDI target, followed by MALDI-TOF MS analysis on the MALDI-8020 instrument using Super-DHB matrix.
• Data processing with the Supporting Tool for SialoCapper-ID Kit software to assign monosaccharide compositions and calculate relative abundances.

Used Instrumentation

• SialoCapper™-ID Kit for sialic acid stabilization and linkage-specific derivatization.
• MALDI-8020 MALDI-TOF mass spectrometer.
• GL-Tip Amide (GL Science) for HILIC purification.
• Supporting Tool for SialoCapper-ID Kit software for composition analysis.
• Standard reagents: PNGase F, SDS, DTT, NP-40, 2-aminobenzamide, Super-DHB matrix.

Results and Discussion

MALDI-8020 MS1 spectra revealed a range of complex-type N-glycans, both neutral and sialylated. Software-assisted composition assignments matched known biosynthetic pathways. The SialoCapper-ID Kit neutralization equalized ionization across glycan types, enabling direct comparison of peak intensities. Quantitative analysis indicated that recombinant ACE2 carries a higher proportion of α2,3-linked sialic acids compared to α2,6, highlighting potential implications for SARS-CoV-2 binding affinity and tropism.

Benefits and Practical Applications

• Simultaneous detection and relative quantification of neutral and sialylated N-glycans in a single run.
• Discrimination of sialic acid linkage isomers by MS1, eliminating the need for LC separation or sialidase digestion.
• Streamlined sample preparation suitable for high-throughput glycomics in virology, biopharma QA/QC, and basic research.

Future Trends and Applications

Advances may include integration of linkage-specific derivatization with imaging mass spectrometry, automation of sample prep, and coupling with machine-learning for glycan pattern recognition. Broad adoption could facilitate large-scale glyco-profiling of viral receptors, biomarker discovery in infectious diseases, and improved characterization of therapeutic glycoproteins.

Conclusion

The SialoCapper™-ID Kit combined with MALDI-TOF MS provides a rapid, robust platform for sialic acid linkage discrimination and glycan profiling of ACE2. This workflow supports detailed glycomic analysis without complex instrumentation or enzymatic treatments, offering valuable insights into virus–host interactions.

References

1. Shajahan A, et al. Comprehensive Characterization of N- and O-Glycosylation of SARS-CoV-2 Human Receptor Angiotensin Converting Enzyme 2. Glycobiology. (In press).
2. Nishikaze T, et al. Differentiation of Sialyl Linkage Isomers by One-Pot Sialic Acid Derivatization for Mass Spectrometry-Based Glycan Profiling. Analytical Chemistry. 2017;89:2353–2360.
3. Hanamatsu H, et al. Sialic Acid Linkage Specific Derivatization of Glycosphingolipid Glycans by Ring-Opening Aminolysis of Lactones. Analytical Chemistry. 2018;90:13193–13199.