Decipher intricate glycoproteins using data-independent acquisition-proton transfer charge reduction and native top-down mass spectrometry

Significance of Glycoprotein Characterization

Viral glycoproteins play a pivotal role in host cell recognition, immune evasion and vaccine efficacy. Detailed mapping of glycan heterogeneity and proteoforms is crucial for understanding infection mechanisms and for guiding therapeutic and vaccine design.

Objectives and Study Overview

This study implements a combined data-independent acquisition–proton transfer charge reduction (DIA-PTCR) and native top-down mass spectrometry workflow to achieve comprehensive characterization of glycosylation and proteoforms in key glycoproteins: human fetuin, SARS-CoV-2 nucleocapsid, receptor-binding domain (RBD) and full-length spike protein.

Methodology and Instrumentation

Samples were buffer-exchanged into 200 mM ammonium acetate and analyzed by static nanospray on Thermo Scientific Orbitrap Tribrid platforms. Key instrumental configurations included:

• Orbitrap Ascend Structural Biology Tribrid: native MS, quadrupole isolation up to m/z 8000, DIA-PTCR with 5–20 Th windows, PTCR reaction times 7–15 ms
• Orbitrap Eclipse Tribrid: native top-down ETD/EThcD and HCD fragmentation for sequence and PTM site mapping
• BioPharma Finder 5.0 for deconvolution, proteoform assignment and glycoform annotation

Main Results and Discussion

• Human Fetuin: Two N-linked, seven O-linked and multiple phosphorylation sites generate ~37 kDa heterogeneity. Quadrupole isolation plus PTCR outperformed ion trap isolation in signal-to-noise and resolution, assigning >300 glycoforms versus 29 from full MS. Native top-down ETD/EThcD revealed disulfide connectivity linking the B-chain N-terminus to the A-chain C-terminus.
• SARS-CoV-2 Nucleocapsid: Full MS showed a 49 kDa species with overlapping charge states. DIA-PTCR resolved covalent modifications and noncovalent adducts. Top-down ETD de novo sequencing identified a 3686.68 Da N-terminal tag. ETD and HCD localized an exposed N-terminus, a C-terminal dimerization domain, and Na+ binding sites.
• Spike RBD: Native MS indicated a ~31.8 kDa distribution from combined N- and O-glycans. PNGase F treatment removed Hex(9)HexNAc(10)dHex(3) (∆4 216 Da), simplifying the profile to ~27.6 kDa and enabling assignment of major O-glycoforms and sequence variants (R37T, K21I).
• Full-Length Spike Protein: MS1 spectra were unresolved above m/z 4000. DIA-PTCR with 5 Th isolation and 5 ms PTCR separated monomeric (160–200 kDa glycosylated), trimeric and impurity species, providing rapid mass profiling of heavy glycoproteins.

Benefits and Practical Applications

• Extended quadrupole isolation up to m/z 8000 enhances native omics throughput and sensitivity.
• DIA-PTCR streamlines intact mass measurement of heterogeneous glycoproteins.
• Native top-down MS delivers sequence, structural and PTM localization in a single experiment.

Future Trends and Potential Applications

• Automation of DIA-PTCR workflows for high-throughput vaccine antigen screening.
• Integration of AI-driven annotation tools for rapid proteoform and glycoform identification.
• Extension to other viral and therapeutic glycoproteins to accelerate biopharmaceutical development.
• Combined structural approaches, coupling native MS data with cryo-EM for holistic glycoprotein models.

Conclusion

The integration of DIA-PTCR and native top-down MS on advanced Tribrid platforms provides an unmatched toolkit for dissecting glycoprotein complexity. This approach enhances detection of low-abundance proteoforms, refines glycan composition analysis and informs vaccine and therapeutic design.

References

1. Huguet R., et al. Proton transfer charge reduction enables high-throughput top-down analysis of large proteoforms. Analytical Chemistry, 2019, 91(24):15732–15739.
2. Schachner L.F., et al. Exposing the molecular heterogeneity of glycosylated biotherapeutics. Nature Communications, 2024, 15(1):3259.