It’s all in the technique: how MS provides information on virus structure and function

Andreas Huhmer, Senior Director, Proteomics and Metabolomics, Thermo Fisher Scientific, examines the mass spectrometry techniques providing detailed insight into virus structure and function

Our ability to quickly and accurately understand new viruses has been significantly tested in 2020, with the emergence of SARS-CoV-2, the coronavirus causing the COVID-19 pandemic. Since the first reports of an outbreak of novel pneumonia cases in Wuhan, China in December 2019, SARS-CoV-2 has spread around the world, resulting in more than one million deaths globally (1) and spurring an international race to develop effective detection, vaccine and therapeutic strategies.

To rapidly respond to harmful new viruses, as well as develop new therapeutics for known ones, scientists need access to detailed and reliable insight into virus structure and function. Such knowledge is fundamental for the creation of virus detection strategies and to inform drug and vaccine development programs, as learning what makes a particular virus tick enables specific structural and mechanistic vulnerabilities to be used against it. Mass spectrometry (MS) has established itself as one of the most versatile tools for virus characterisation, capable of providing researchers with information on viral architecture, cell-surface interactions and routes of infection. Here, we consider some of the most effective MS strategies being used to advance virus research and make progress in the fight against SARS-CoV-2.

Virus structure and function

Animal viruses come in a variety of shapes and sizes, and may adopt a range of genetic strategies for replication (2). However, most share a number of common structural features. Viruses typically consist of a payload of genetic material (which can be RNA or DNA), surrounded by a protective shell of proteins and glycoproteins (known as a capsid). Some viruses are also further enclosed by a membrane envelope composed of lipids and proteins, the surface of which may include glycoproteins involved in binding to and facilitating viral entry into host cells.

The large transmembrane glycoprotein trimers present on the surface of coronaviruses such as SARS-CoV-2 (Figure 1), for example, play a key role in targeting and entry into host cells. These so-called spike proteins are a major focus of current research, as they may be used as vaccine immunogens to generate an immune response, or serve as promising targets for small molecule antiviral drugs (3). Other important areas of virus research include the virus capsid and virus-host cell protein interactions, which may also be exploited for vaccine development or targeted for therapeutics.

Accurate structural insight is critical to inform effective drug and vaccine development programmes, which demand reliable techniques for protein characterisation. Given their elaborate three-dimensional complexity, elucidating the structure and function of viruses often requires integrating several complementary characterisation strategies, including techniques such as protein X-ray crystallography, cryogenic electron microscopy (cryo-EM), as well as an expanding range of versatile MS approaches (Figure 2).

Characterising viral glycosylation using native MS and glycoproteomics

Viral envelope proteins are often modified by the attachment of glycans, which can make up as much as half of the molecular weight of these glycoproteins (4). Coronavirus spike proteins, for instance, are extensively glycosylated, encoding between 66 and 87 N-linked glycosylation sites per trimeric spike (5). Many viruses, including the HIV-1 and influenza viruses, use the glycosylation of surface antigens to evade recognition by the immune system – essentially using the host’s own cellular components to mask the virus’s protein surface (4). Viral glycans also play an important role in virus-host receptor binding and conformational dynamics, influencing binding efficacy. Determining the extent of glycoprotein glycosylation is, therefore, critical for the generation of effective vaccine candidates that mimic circulating viruses, and for the development of therapeutics designed to prevent viral infection by blocking receptor attachment.

One of the most useful tools for characterising glycosylation on viral glycoproteins is native MS, a powerful technique that allows researchers to study proteins in their intact or ‘native’ state (6). The approach, based on electrospray ionisation, involves introducing biological analytes into the mass spectrometer using a non-denaturing solvent that preserves the non-covalent interactions holding a protein’s three-dimensional structure together (7).

Unlike other biological MS approaches that typically utilise denaturing conditions, the relatively mild experimental conditions employed in native MS serve to maintain proteins in a similar structural configuration to the one they exist in under biological conditions. As such, native MS can reveal a great deal of useful structural information on proteins and protein complexes, including the mass and stoichiometry of intact macromolecular complexes and interactions between their components. Native MS can even be used to identify stable subcomplexes and assign the relative position of subunits (6).

Native MS is particularly useful for studying virus structure as it permits different proteoforms, such as differential glycosylations, to be detected simultaneously, a task difficult to do using other structural biology techniques. As such, native MS allows the number and type of glycosylations to be determined, and other post-translational modifications such as phosphorylation to be studied. Furthermore, the technique can also be used to determine the mass of the intact protein, offering critical information to distinguish proteins of interest from other analytes.

When it comes to building a more complete picture of viral glycosylation, bottom-up glycoproteomics strategies, involving the enzymatic digestion, enrichment and analysis of viral surface glycoproteins, can be highly effective at supplementing the insight obtained through native MS. Bottom-up glycoproteomics strategies can identify considerably more site-specific glycans than is possible with native MS, providing information on glycosylation sites and glycan compositions, as well as revealing minor glycoforms and O-linked glycosylation that might be missed or suppressed in native MS approaches.

Bottom-up glycoproteomics strategies are already helping researchers better understand the glycosylation of the SARS-CoV-2 spike protein (8), with site-specific MS approaches being used to successfully identify glycan structures on a recombinant SARS-CoV-2 spike immunogen, generating a map of the glycan-processing states across the spike protein. With global research teams pursuing a growing number of glycoprotein-based vaccine candidates, this detailed analysis of site-specific glycan signatures provides a route to measure antigen quality as vaccines and antibody tests are developed.

Studying capsid assembly using hydrogen-deuterium exchange MS

Another important focus of structural virology centres on understanding the construction of the capsid surrounding the virus’ genetic material. The protein capsid serves three key functions: to shield the genetic material against digestive enzymes, to aid attachment of the virus to the host cell membrane, and to enable delivery of the genetic information into the cell. Understanding the structure and conformational dynamics of capsids is vital for elucidating the specific role these structures play in the lifecycle of the virus, and can potentially reveal insights that may be used in the fight against viral infection.

One MS approach that has been used to determine local conformational dynamics and gain insight into the mechanisms of capsid assembly and maturation is hydrogen-deuterium exchange MS (HDX-MS). HDX-MS is a powerful integrated structural biology technique that exploits the labile nature of the protons found on protein backbone amides. In deuterated buffer, certain protons present on the protein will freely exchange with deuterons from the solvent. However, the rate of exchange at individual sites will depend on the specific structural characteristics at that position, as only protons located on backbone amides will exchange on a rate measurable by HDX-MS experiments. Protons positioned on the functional groups of amino acid side chains will exchange at a rate too fast to measure, while those attached to carbon atoms will exchange too slowly. By measuring the rate of hydrogen-deuterium exchange at specific sites of interest, HDX-MS experiments can therefore be used to infer information on protein structure, including protein-protein and protein-ligand interaction sites, allosteric effects, intrinsic disorder and conformational changes induced by post-translational modifications.

An important advantage of HDX-MS compared with other integrated structural biology approaches is that it is not limited by the size of proteins or protein complexes, making it a highly versatile tool for examining virus structure. A highly sensitive technique, HDX-MS can be used to detect co-existing protein conformations and probe membrane proteins that are challenging to study using more traditional approaches. HDX-MS has been successfully used to study capsid assembly and maturation of the HIV-1 virus (10), the capsid protein dynamics of the hepatitis B virus (11), and the higher-order structure of the Rous sarcoma virus spacer peptide assembly domain (12).

Understanding virus-host interactions with crosslinking MS

Examining how viruses interact with host cells is critical for understanding processes such as viral entry and genome replication, potentially yielding valuable information to support the development of vaccines and antiviral therapeutics. Like many other viruses, SARS-CoV-2 binds and enters host cells using the spike glycoprotein trimers present on the surface of the virus (Figure 3). Given their important role in host cell targeting and entry, spike proteins are frequently used as immunogens for vaccines to generate neutralising antibodies, and targeted for inhibition by small molecules that might block host receptor binding or membrane fusion (3). However, mapping the topology of the proteins involved in virus-host binding and understanding how these structures interact can be exceptionally challenging, not least because viruses are conformationally dynamic, and these interactions are often transient.

Crosslinking mass spectrometry (XL-MS) is widely seen as a powerful technique for studying transiently interacting protein complexes. XL-MS strategies involve the use of chemical crosslinking reagents of fixed length that covalently bind to specific protein sites in close proximity to each other. Crosslinked proteins are subsequently digested using an appropriate enzyme and the resulting peptides are then enriched, separated and analysed using liquid chromatography-mass spectrometry (LC-MS). Using the distance constraints provided by the known length of the crosslinker, XL-MS workflows allow scientists to reconstruct three-dimensional maps of viral proteins or visualise interacting regions between viruses and host protein receptors. Furthermore, because crosslinking strategies permit the examination of protein topology under near-physiological conditions, they can provide more biologically-relevant structural information that can help to better inform vaccine and antiviral drug research.

XL-MS is often used in combination with high-resolution structural biology techniques such as cryo-EM and protein X-ray crystallography to obtain highly detailed structural information on protein complexes, subunits and stoichiometry. However, unlike cryo-EM and protein X-ray crystallography, XL-MS does not require the use of highly pure protein samples, making it amenable to a much broader range of samples.

Examining virus-host interaction dynamics with affinity purification MS

Other MS techniques are helping researchers probe protein-protein interactions within protein complexes and study these structures at the interactome level. Qualitative and quantitative MS workflows can be coupled to enrichment strategies, such as affinity purification (AP-MS), to study specific proteins or protein complexes within analytically challenging samples. AP-MS takes advantage of specific binding interactions between molecules to isolate proteins or protein complexes of interest. Samples are typically passed through a column containing an active ligand chemically bound to a solid support. By utilising ligands that interact more strongly with the proteins of interest than other sample components, the analytes can be concentrated on the column and later washed off, effectively enriching the proteins of interest for subsequent MS analysis.

Affinity purification is a highly versatile technique that may be used in combination with a variety of quantitative methods, including label-free quantitation (LFQ), stable isotope labelling by amino acids in cell culture (SILAC) and isobaric labelling approaches. AP-MS supports a wide range of purification techniques, such as antigen purification, immunoprecipitation, and co-immunoprecipitation approaches, as well as pull-down assays, fusion tag protein purification and avidin-biotin systems. Furthermore, because quantitative AP-MS workflows are well suited to the study of proteins under different conditions, they can offer a very dynamic perspective on protein-protein interactions and the role post-translational modifications play in facilitating these interactions.

Building a comprehensive picture of virus structure and function with MS

Knowledge of a virus’s structure and how it interacts with host cells potentially enables researchers to develop vaccines and treatments. MS has established itself as a powerful tool for elucidating virus structure and function, complementing structural biology tools such as cryo-EM and protein X-ray crystallography. The integration of MS techniques such as native MS, HDX-MS, AP-MS and XL-MS, together with ongoing improvements in quantitative MS, are helping researchers obtain more useful information more quickly, accelerating the development of vaccines and therapeutics.

Figure 1. The structure of SARS-CoV-2. Created with



Figure 3. SARS-CoV-2 binding to host cell receptor, and entry by membrane fusion and endocytosis. Created with


Volume 22, Issue 1 – Winter 2020/21


  1. Center for Systems Science and Engineering at Johns Hopkins University. COVID-19 Dashboard. Accessed October 2020
  2. Lodish H, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000. Section 6.3, Viruses: Structure, Function, and Uses
  3. Zhao P, et al. Virus-Receptor Interactions of Glycosylated SARS-CoV-2 Spike and Human ACE2 Receptor. Cell Host & Microbe. 2020. DOI: 10.1016/j.chom.2020.08.004
  4. Grant OC, et al. Analysis of the SARS-CoV-2 spike protein glycan shield reveals implications for immune recognition. Scientific Reports. 2020. DOI: 10.1038/s41598-020-71748-7
  5. Watanabe Y, et al. Vulnerabilities in coronavirus glycan shields despite extensive glycosylation. Nature Communications. 2020. DOI: 10.1038/s41467-020-16567-0
  6. Erba EB, et al. Exploring the structure and dynamics of macromolecular complexes by native mass spectrometry. Journal of Proteomics. 2020. DOI: 10.1016/j.jprot.2020.103799
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  8. Wanatabe Y, et al. Site-specific glycan analysis of the SARS-CoV-2 spike. Science. 2020. DOI: 10.1126/science.abb9983
  9. Huang RY-C and Chen G. Higher order structure characterization of protein therapeutics by hydrogen/deuterium exchange mass spectrometry. Analytical and Bioanalytical Chemistry. 2014. DOI: 10.1007/s00216-014-7924-3
  10. Monroe EB, et al. Hydrogen/Deuterium Exchange Analysis of HIV-1 Capsid Assembly and Maturation. Structure. 2010. DOI: 10.1016/j.str.2010.08.016
  11. Bereszczak JZ, et al. Assessment of differences in the conformational flexibility of hepatitis B virus core-antigen and e-antigen by hydrogen deuterium exchange-mass spectrometry. Protein Science. 2014. DOI: 10.1002/pro.2470
  12. Bush DL, et al. Higher-order structure of the Rous sarcoma virus SP assembly domain. American Society for Microbiology. 2014. DOI: 10.1128/JVI.02659-13




Figure 1. The structure of SARS-CoV-2. Created with


Figure 2. Integrative structural biology draws upon a broad range of tools and techniques

Figure 3. SARS-CoV-2 binding to host cell receptor, and entry by membrane fusion and endocytosis. Created with






Figure 1. The structure of SARS-CoV-2. Created created with


Figure 2. Integrative structural biology draws upon a broad range of tools and techniques.

Figure 3. SARS-CoV-2 binding to host cell receptor, and entry by membrane fusion and endocytosis.

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