An urgent need for expanded virus research

Importance of the topic

Emerging viral pathogens such as SARS-CoV-2 highlight the urgent need for rapid, detailed characterization of virus structure, function and host interactions. Mass spectrometry (MS)–based approaches enable researchers to probe intact viral particles, glycoprotein modifications, protein dynamics, and virus–host complexes with high specificity and sensitivity. These insights underpin improved detection methods, antiviral drug discovery and vaccine design.

Objectives and study overview

This white paper reviews state-of-the-art MS techniques for:

• Structural analysis of viral proteins and capsids
• Site-specific glycosylation profiling
• Conformational dynamics by hydrogen-deuterium exchange
• Virus–host protein interactions via crosslinking and affinity purification
• Characterization of host antigen presentation (immunopeptidomics)

It aims to inform virology researchers on methods routinely used in specialized laboratories and to encourage broader adoption and method development.

Methodology and instrumentation

Key MS approaches discussed include:

• Native mass spectrometry for intact particle stoichiometry, subunit connectivity and proteoform distributions
• Top-down fragmentation of isolated capsids to map protein composition and posttranslational modifications
• Bottom-up glycoproteomics and glycomics (LC-MS/MS) for site-specific N- and O-glycan profiling
• Hydrogen-deuterium exchange MS (HDX-MS) to monitor conformational changes and assembly dynamics
• Crosslinking MS (XL-MS) to define distance constraints in virus–host complexes and low-resolution topology
• Affinity purification MS (AP-MS) with quantitative labels (TMT, SILAC, LFQ) to map dynamic interactomes
• Immunopeptidomics workflows using monoclonal capture of MHC complexes and LC-MS/MS for viral epitope identification

Instrumentation used

• High-resolution Orbitrap and time-of-flight mass spectrometers
• Ion mobility devices for native MS assemblies
• Reversed-phase nano-LC systems with nanoelectrospray sources
• Cryogenic electron microscopy for integrative structural biology
• Automated HDX-MS platform with online digestion and temperature control
• MS-cleavable crosslinkers and dedicated XL-MS analysis software
• Tandem mass tag (TMT) multiplexing kits for quantitative AP-MS and temporal proteomics

Main results and discussion

Native MS studies resolved distinct hepatitis B virus capsid populations (3–4 MDa) and ejected individual subunits through tandem MS. Glycoproteomics of SARS-CoV-2 spike protein revealed site-specific glycan heterogeneity similar to SARS-CoV-1 but distinct from HIV-1, guiding vaccine antigen design. HDX-MS mapped conformational changes in HIV-1, Rous sarcoma virus and HBV capsids, elucidating assembly pathways. XL-MS identified novel human receptor factors (AXL, M6PR) for vaccinia virus and interaction topologies for plant and human viruses. Quantitative AP-MS with TMT tracked >1,100 plasma membrane protein changes during HCMV infection. Immunopeptidomics uncovered viral peptides presented by MHC class I, informing epitope selection for vaccines and therapies.

Benefits and practical applications

• Rapid structural characterization accelerates antiviral drug screening and inhibitor design
• Detailed glycan mapping informs immunogen design for broadly protective vaccines
• Dynamics and topology data support rational engineering of viral antigens and nanoparticles
• Quantitative interactomes reveal host factors essential for viral entry, replication and egress
• Immunopeptidomics defines naturally presented epitopes, aiding T-cell vaccine development and immunotherapy

Future trends and potential uses

• Integration of MS with cryo-EM and X-ray crystallography for high-resolution, dynamic models
• Deep-learning spectral prediction (e.g., PROSIT) to expand confident epitope discovery
• Advanced glycoproteomics workflows for microheterogeneity and site occupancy under immune pressure
• Single-cell and spatial proteomics to map infection microenvironments
• Expanded use of quantitative proteomics and multiplexed labeling to monitor viral evolution and vaccine responses in real time

Conclusion

Mass spectrometry has evolved into a versatile toolkit for virus research, offering unparalleled insights into virion architecture, glycosylation, protein dynamics, host interactions and immune presentation. Its integration with other structural and computational methods will continue to drive rapid advances in antiviral strategies and vaccine design.

Reference

• Vankadari N, Wilce JA. Emerging WuHan (COVID-19) coronavirus: Glycan shield and structure prediction of spike glycoprotein and its interaction with human CD26. Emerg Microbes Infect. 2020;9(1):601–604.
• Thermo Fisher Scientific. Native MS for structural biology research. White Paper 73306.
• Snijder J, Rose RJ, Veesler D, et al. Studying 18 MDa virus assemblies with native mass spectrometry. Angew Chem Int Ed Engl. 2013;52(14):4020–4023.
• Uetrecht C, Versluis C, Watts NR, et al. High-resolution mass spectrometry of viral assemblies: Molecular composition and stability of dimorphic hepatitis B virus capsids. Proc Natl Acad Sci U S A. 2008;105:9216–9220.
• Shoemaker GK, Van Duijn E, Crawford SE, et al. Norwalk virus assembly and stability monitored by mass spectrometry. Mol Cell Proteomics. 2010;9:1742–1751.
• Uetrecht C, Barbu IM, Shoemaker GK, et al. Interrogating viral capsid assembly with ion mobility–mass spectrometry. Nat Chem. 2011;3:126–132.
• Watanabe Y, Allen JD, Wrapp D, McLellan JS, Crispin M. Site-specific glycan analysis of the SARS-CoV-2 spike. Science. 2020;May 4:eabb9983.