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COVID-19 Puts Mass Spectrometry in the Spotlight

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The COVID-19 pandemic has brought the fields of virology and vaccine development firmly into the limelight. As the global scientific community has raced to find drugs and vaccine candidates against the SARS-CoV-2 virus, it has relied on tools that can quickly solve the structure of the virus and how it interacts with human host cell proteins. These technologies have become an important part of our arsenal against current and future infectious diseases.

A key addition to the virologist’s structural biology toolkit is the use of advanced mass spectrometry (MS), which has been an invaluable asset alongside cryo-electron microscopy (cryo-EM) for studying SARS-CoV-2. MS approaches can be used to study proteins at the individual level and as larger complexes. At an individual protein level, MS can reveal protein structure and ligand interactions and characterize post-translational modifications (PTMs). For large protein complexes, MS can reveal insights into protein stoichiometry and determine the interfaces involved in protein-protein interactions (PPIs).

Studying viruses requires integrating several complementary MS techniques. In this article, we review a range of approaches that can address questions about virus structure and function.

Revealing viral protein structure and function

Advances in MS means there is now a broader range of methods available for studying virus structure. For example, bottom-up proteomics (which involves proteolytic digestion of proteins before MS analysis) combined with peptide sequencing provides information about primary protein structure, and can determine the identity of single proteins or several proteins within large complexes. Alternatively, limited proteolysis-coupled to MS on a single protein (which involves digesting proteins with low amounts of proteases before MS analysis) can provide insights on the higher order structure of single proteins and their folded states. Both methods are powerful tools for identifying viral proteins within host cells and understanding their different structures and conformations as viruses move through their life cycle.

Another popular MS tool for understanding protein structure and function is hydrogen deuterium exchange mass spectrometry (HDX-MS). This measures the rate of isotope exchange between hydrogens in the protein backbone and surrounding solvent, providing information on structure and conformational changes of single proteins or PPIs, as well as insights into protein-ligand interaction sites.

HDX-MS was recently used to evaluate the binding of prospective therapeutic antibody pairs that could simultaneously neutralize the receptor-binding domain (RBD) of SARS-CoV-2 spike protein.1 The technique successfully enabled researchers to group antibodies based on their RBD binding pattern. This was important as the goal was to find antibodies that would bind at separate sites and be ideal partners for a therapeutic antibody cocktail.

These MS methods all provide basic structural information about viral proteins. In the next sections, we look at how MS has been used to reveal further insights about these proteins to understand their potential as therapeutic targets.

Figure 1: Image of the SARS-CoV-2 structure showing key proteins discussed. Credit: Thermo Fisher Scientific.

Examining glycoprotein spikes

Glycosylation is one of the most-studied PTM of eukaryotic cell proteins because of the vast array of physiological functions glycans are involved in. Glycoproteins are also one of the major components of human pathogenic viruses, playing a key role in the interactions between virus and host cells. Their function in host-cell entry makes them an ideal target of anti-viral drugs and vaccines. Understanding the site-specific glycosylation pattern on viral proteins is, therefore, essential. MS can be used to study glycosylation sites at a large-scale proteomic level and examine unique glycoproteins on individual viruses.

Like other coronaviruses, SARS-CoV-2 has a viral envelope of three proteins: the membrane, envelope and spike proteins. The spike protein is highly glycosylated and plays a crucial role in penetrating host cells and initiating infection, making it a prime target for vaccines. MS approaches were used in some of the studies that revealed essential information about SARS-CoV-2 glycoproteins, which underpins the COVID-19 vaccines we have today.

In one such study,2 researchers used nano-liquid chromatography coupled to mass spectrometry (nano-LC-MS) to resolve the site-specific glycosylation of a purified recombinant SARS-CoV-2 spike protein. Glycopeptides were analyzed by nano-LC-MS and the glycan compositions determined for all 22 N-linked glycan sites of the spike protein. Researchers combined this with the cryo-EM structure to reveal how the N-linked glycans shield certain regions across the surface of the SARS-CoV-2 spike. This shielding is characteristic of viral glycoproteins, including those of HIV and influenza, and locating which regions are protected helps to highlight the most conserved and potentially vulnerable areas of the virus’ structure.

In a separate study,3 researchers used MS methods to characterize site-specific differences in glycans on the SARS-CoV-2 spike protein and determine how these influence interactions between the spike and human ACE2. A combination of different proteolysis strategies with advanced MS technologies revealed microheterogeneity between glycans at the 22 known glycosylation sites. This information could then be mapped onto 3D structural models of the SARS-CoV-2 spike protein in complex with its target ACE2 protein to help predict epitope accessibility and immunogenicity of both current SARS-CoV-2 virus proteins and those of emerging new variants. 

These studies illustrate the power of combining detailed chemical information from MS analysis with complementary 3D images from cryo-EM, to provide in-depth characterization of viral glycoprotein structure and function.

Characterizing virus’ molecular machinery

In addition to glycoprotein spikes that help viruses gain entry to human cells, viruses also encode key cellular machinery once inside host cells to enable them to replicate, assemble new virus particles and infect further cells. The study of these proteins is important both for understanding the virus lifecycle and evaluating their potential as targets for antiviral therapies and vaccines.

Traditionally, studying these proteins in their physiological conformation and in complex with binding partners has been challenging. But, advances in MS technology have led to the adoption of native MS for investigating intact protein complexes in their natural state, providing a more accurate understanding of structure and function under physiological conditions.4 This approach has been used to characterize two important components of the SARS-CoV-2 molecular machinery – the nucleocapsid protein5 and main protease.6

The SARS-CoV-2 nucleocapsid protein is the most immunogenic of the virus’ structural proteins and is involved in several stages of the virus life cycle – from binding and protecting the viral genome to enhancing its transcription. Using native MS, researchers have been able to characterize the RNA binding properties of the full-length nucleocapsid protein in intricate detail, including which forms of the nucleocapsid protein generated the highest antibody response using convalescent plasma from recovered COVID-19 patients.5 By using native MS, they could determine the protein’s assembly state as dimers or monomers when it binds RNA under different conditions.

In SARS-CoV-2, the main protease (Mpro) plays a critical role in processing viral proteins translated from RNA. Native MS was used by one team to characterize the functional unit of the main protease and determine the balance of dimer to monomer forms, revealing a strong preference towards the dimer and providing insights on binding affinity.6 This data could then be used for subsequent screening of inhibitors against Mpro, providing a starting point for drug development.

These proteins are highly desirable targets in the design of antiviral agents, and it is important to gain an understanding of their function in a form that mirrors their physiological context as closely as possible. These examples show the power of native MS for rapidly providing detailed insights into protein higher structures, and natural dynamics and behaviours.

Figure 2: Integrative structural biology schematic. Credit: Thermo Fisher Scientific.

Revealing interactions between viruses and host proteins

Viruses rely on their ability to interact with host cell proteins. Usefully, one of main strengths of MS technology is the range of methods that can characterize PPIs.

For example, limited proteolysis coupled to MS is a simple and fast way to determine the exact protein residues that interact with each other: if one protein is bound to another it will make protease sites inaccessible, providing information about the location of the interface.

Another method is affinity purification MS (AP-MS), which uses the specific binding interactions between molecules to isolate proteins of interest in an affinity-enrichment step. By coupling affinity purification to quantitative MS, PPIs can be studied under different conditions. This can also be expanded to study how PTMs facilitate PPIs. AP-MS was used to great effect in a drug repurposing study,7 where researchers expressed 26 tagged SARS-CoV-2 proteins in human cells and then identified the proteins that physically associated with them. Through this approach they identified 332 PPIs, of which 66 were druggable, and 29 were the targets of approved medicines. This rapidly enabled teams to highlight many potential drug candidates against SARS-CoV-2.

An alternative approach to studying interactions between virus and human proteins is crosslinking MS (XL-MS). In this method, soluble crosslinkers or photoactivatable amino acids are used to crosslink residues in proximity.8 These crosslinked proteins are then digested into peptides and analyzed by tandem MS; the data can be combined with other available structural information to model the shape of protein complexes.

A UV-based XL-MS approach was used by one group to map interactions between SARS-CoV-2 RNA and human host cell proteins.9 They used RNA antisense purification coupled with MS, which allows rapid identification of protein interaction partners of a specific RNA molecule. This enabled characterization of the entire SARS-CoV-2-human cell interactome in a single experiment and identified 104 proteins of potential therapeutic importance.


Understanding the structural, temporal and spatial complexity of virus-host PPIs is essential for developing new treatments and vaccines against existing viral pathogens, and to protect us against emerging viruses that could cause future pandemics. Today, a range of structural biology tools exist to enable greater understanding of virus protein structure, function and dynamics. Central to this toolkit are innovations in MS technologies that provide a range of complementary methods to the conventional structural biology techniques of cryo-EM, X-ray crystallography and mutagenesis. Pioneering research during the COVID-19 pandemic illustrates the powerful role MS can play as part of an integrated structural biology toolkit for virology and vaccinology.


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