SARS-CoV-2 studies highlight mass photometry’s ability to characterise complex heterogeneous systems
The SARS-CoV-2 pandemic has altered our lives and shaped the course of scientific research, bringing an explosion in studies on the virus. Mass photometry has proved valuable in coronavirus research, especially in characterising complex and heterogeneous proteins, and in analysing protein interactions and stoichiometry. In this blog post, we highlight ways that mass photometry has helped investigate previously intractable questions and advance our understanding of SARS-CoV-2.
Figure 1. The principle of mass photometry, as applied to SARS-CoV-2 spike protein measurements. Light scattered by spike trimers at the measurement surface interferes with light reflected at that surface to produce the mass photometry signal, which is proportional to protein mass. Read our blog post on how mass photometry works for more details. Characterisation of the SARS-CoV-2 spike protein mass and oligomerisation in solution was among a multitude of applications of mass photometry in SARS-CoV-2 research.
Overcoming glycosylation and heterogeneity for insights into structure and binding
Mass photometry can, unlike other techniques, operate despite heavy glycosylation and the heterogeneity it brings. The SARS-CoV-2 spike (S) protein – infamous for its role in viral fusion to and entry into host cells – is heavily glycosylated. The S protein’s dense glycan shield, which helps it evade immune detection, also makes it difficult to study with, for example, native mass spectrometry and x-ray crystallography. As these techniques are often used to gain insights into protein structure and interactions, it has been challenging to study the spike protein and its interactions with other proteins, such as the human ACE2 receptor – the virus’s entry point into human cells.
Mass photometry has helped researchers characterise S trimers (Fig. 1), and interactions between the S protein and ACE2 receptor. One study using mass photometry to measure the mass of the S protein identified trimers of mass 474 ± 44 kDa, a small number of monomers and no higher-order species . The measurement, which was consistent with results from charge detection mass spectrometry (CD-MS), indicates that the S protein’s glycan shield accounts for an impressive 30% of its mass. According to lead author Victor Yin, who presented the study and its other findings in a recent webinar, mass photometry enables the team to measure complex, heterogeneous systems that have been beyond the reach of other mass analysis techniques.
Mass photometry also helped researchers from AstraZeneca and Karlstad University to analyse the kinetics of the multivalent interaction between the S protein and the ACE2 receptor . Using mass photometry, they found that the ectodomain of ACE2 forms multivalent complexes with that of the S protein, with 0, 1, 2 or 3 ACE2 molecules for every S trimer. This heterogeneity poses challenges for kinetic binding studies, which the authors overcame by combining mass photometry with size exclusion chromatography (SEC) and the protein switchavidin to separate the species. The resulting fractions were amenable to kinetic analysis using surface plasmon resonance and modelling, enabling the team to explore the question of whether a high affinity for ACE2 may make SARS-CoV-2 highly infective.
Directly reporting stoichiometry in solution
Numerous studies have capitalised on the power of mass photometry to characterise the stoichiometry of molecular assemblies quickly and easily, and that capability has also proved useful in SARS-CoV-2 studies. With its ability to rapidly analyse samples directly in solution, mass photometry provides a straightforward way to identify the makeup of molecular complexes and quantify their distribution.
The finding that the ACE2 receptor ectodomain binds the S trimer multivalently was confirmed in multiple mass photometry-based studies –. The clear evidence from mass photometry that S trimers bind up to three ACE2 receptors in solution (Fig. 2) aids the interpretation of cryogenic electron microscopy (cryo-EM) findings. Cryo-EM studies had shown mainly structures with 1:1 binding alongside some structures with other stoichiometries, leaving unresolved the question of which stoichiometries occur in solution.
Figure 2. Mass photometry characterisation of the SARS-CoV-2 spike ectodomain and its interaction with ACE2.The upper mass histogram shows recombinant S protein ectodomain appearing mainly as a trimer. The lower mass histogram shows the ACE2 receptor and its binding with the S trimer (inset). Data from collaboration with A. Gunnarsson and S. Geschwindner of AstraZeneca, Gothenburg, Sweden .
Meanwhile, interactions between the S protein and antibodies are also an important avenue for exploration in SARS-CoV-2 research. The flexibility of full-length antibodies makes them difficult to study with conventional structural methods (e.g. EM), necessitating the use of antigen-binding fragments (Fabs) whose behaviour may not match that of their full-length counterparts. Studies of the S protein are often applied to truncated version of the receptor binding domain for similar reasons.
For Yin and colleagues at the University of Utrecht, mass photometry offered a way to explore interactions between the S protein ectodomain and full-length IgG antibodies – and gain a more representative picture . Mass photometry enabled them to rapidly assess S protein binding stoichiometry across a panel of 12 monoclonal anti-S-trimer IgGs. They found, surprisingly, that none of the antibodies bound the S trimers in a full (3:1) stoichiometry, meaning that at least one S monomer always remained unbound. A second surprise was that this partial antibody binding (2:1) was enough to prevent the S protein from binding ACE2 (Fig. 3).
These findings offer valuable insights for understanding how antibodies function in SARS-CoV-2. Prior to this work, structural studies on protein fragments had revealed which regions interacted but not the overall assembly. This study showed that steric hindrance or allosteric effects – occurring outside the regions captured in earlier structural studies – are likely an important part of the puzzle. The study also demonstrated the need to go beyond protein fragments to discover how proteins interact and assemble, and mass photometry is ideal for that type of analysis.
Figure 3. Mass photometry revealed that partial antibody binding was sufficient to prevent the SARS-CoV-2 spike protein from binding to the ACE2 receptor. Mass histograms show that, in the presence of antibodies COVA2-15 or COVA1-18, the spike protein did not bind to the ACE2 receptor, which was instead bound primarily to only 1-2 antibodies. Figure adapted from .
The research response to the Covid pandemic demonstrated how quickly science can move and the importance of having access to the right tools. While mass photometry is just one of many valuable bioanalytical tools, it has a unique combination of strengths. As the studies above have demonstrated, mass photometry is quick and simple to use, and it can provide crucial information on complex systems involving extensive glycosylation or the formation of heterogeneous complexes directly from in-solution measurements. It also provides valuable data on its own and can complement other techniques. Mass photometry is truly a versatile, universal solution for biomolecular characterisation.
To learn more about the use of mass photometry in SARS-CoV-2 research, we recommend:
Victor Yin, a postdoctoral researcher of biomolecular mass spectrometry and proteomics at the University of Utrecht, discusses how mass photometry and charge detection mass spectrometry enable the study of several challenging protein systems, including the interaction of full antibodies with SARS-CoV-2.
This brief application note shows how mass photometry can be used to study the mechanism by which the SARS-CoV-2 spike protein binds to the ACE2 receptor.
Read our technical blog explaining the principle behind mass photometry and why mass photometry is useful.
 V. Yin et al., ‘Probing Affinity, Avidity, Anticooperativity, and Competition in Antibody and Receptor Binding to the SARS-CoV-2 Spike by Single Particle Mass Analyses’, ACS Central Science, vol. 7, no. 11, pp. 1863–1873, Nov. 2021, https://doi.org/10.1021/acscentsci.1c00804
 A. Gutgsell, A. Gunnarsson, P. Forssen, E. Gordon, T. Fornstedt, and S. Geschwindner, ‘Biosensor-Enabled Deconvolution of the Avidity-Induced Affinity Enhancement for the SARS-CoV-2 Spike Protein and ACE2 Interaction’, Analytical Chemistry, https://doi.org/10.1021/acs.analchem.1c04372
 Y. Higuchi et al., ‘Engineered ACE2 receptor therapy overcomes mutational escape of SARS-CoV-2’, Nature Communications, vol. 12, no. 1, p. 3802, Jun. 2021, https://doi.org/10.1038/s41467-021-24013-y
 A. Gunnarsson, S. Geschwindner, J. Andrecka, K. Haeussermann, and J. Wilkinson, ‘Mass photometry on SARS-CoV-2, Refeyn Application Note’. Refeyn Ltd. [Online]. Available: https://www.refeyn.com/applications