How does mass photometry work?

Updated: Sep 17


Mass photometry is a novel bioanalytical technology that measures molecular mass by quantifying light scattering from individual biomolecules in solution.



What is the principle behind mass photometry?

The principle behind mass photometry is simple: a single molecule in contact with a measurement surface (e.g. a coverslip) exposed to a beam of light produces a small but measurable light scattering signal, which is directly proportional to the molecule’s mass. The greater the mass of the molecule, the more intense the signal (Fig. 1).


Simple explanation of the mass photometry principle.

Figure 1 Mass photometry concept. A biomolecule (light blue) that is exposed to light (yellow) and placed in a mass photometer generates a light-scattering signal (grey circles beneath the biomolecules). The signal’s intensity is correlated with the molecule’s mass.




In a mass photometry measurement, a sample of proteins or other biomolecules is illuminated with a beam of light. Some of that light is reflected by the measurement surface and some is scattered by molecules in contact with the measurement surface (Fig. 2).


Mass photometry measures the interference between the light scattered by the molecule and the light reflected by the measurement surface. The signal measured is called the mass photometry contrast (or interferometric contrast) and is directly correlated with molecular mass (Young, et al. 2018).




The principle of mass photometry. The light scattered by a molecule in contact with a measurement surface interferes with light reflected by that surface. The interference signal scales linearly with the molecule’s mass.

Figure 2 The principle of mass photometry. The light scattered by a molecule in contact with a measurement surface interferes with light reflected by that surface. The interference signal scales linearly with the molecule’s mass.




Why mass photometry is useful

Mass photometry has seven valuable benefits that distinguish it from other bioanalytical techniques:


1. It measures true molecular mass. Unlike other techniques, mass photometry does not infer the mass indirectly from a different physical parameter, such as the hydrodynamic radius. Instead, the mass photometry signal measured is directly correlated with the true molecular mass, enabling measurement of the mass of molecules in the range 30 kDa to 5 MDa.


2. It shows molecular heterogeneity. Mass photometry measures the mass of each molecule that is in contact with the measurement surface. Single molecule measurements make it possible to detect subpopulations of protein species or detail the molecular heterogeneity – aspects of a sample that are invisible to methods that use bulk measurement.


3. It works in solution. Mass photometry measurements are performed in solution, and mass photometry is compatible with water as well as a wide range of buffers. Measuring biomolecules in an environment that mimics the intracellular aqueous environment allows their true native behaviour to be studied.


4. It uses minimal amounts of sample. Mass photometry requires very little sample. Volumes as little as 10 µL can be enough for a single set of measurements, and the recommended concentration range is 100 pM – 100 nM. The low concentration range means that biomolecules can be studied at concentrations that are physiologically relevant – again helping to mimic the intracellular environment and observe native behaviour.


5. It requires no modifications of the sample. Mass photometry does not require any labelling of the molecules under investigation. This simplifies sample preparation and eliminates the risk of labels interfering with the native behaviour of a molecule being analysed.


6. It provides results rapidly. Mass photometry workflows are very quick, with both sample preparation and measurement together often taking just minutes.


7. It enables dynamic measurements. Mass photometry can gather data over time, capturing the dynamic behaviour of biomolecules. These dynamic data can be used to monitor shifts in chemical equilibria or the assembly/disassembly of macromolecular complexes, or reveal the existence of transient intermediates or steps in the formation of protein oligomers – all phenomena that could be missed when relying on static snapshots (and/or population-averaged data).




What types of molecules can you measure with mass photometry?

Mass photometry is a bioanalytical method suitable for measuring the mass of water-soluble molecules in the 30 kDa – 5 MDa mass range.


Most published studies have applied mass photometry to proteins – to study protein-protein interactions (Higuchi, et al. 2021), protein oligomerisation mechanisms (Naftaly, et al. 2021) and characterise protein samples in terms of stability (Nuber, et al. 2021) or another variable.


Mass photometry has also been used successfully to study heteromolecular interactions such as DNA-protein interactions (Hickman, et al. 2020).


Mass photometry can be applied to nucleic acids, enabling the detection and quantification of nucleic acids at sub-picomolar concentrations (Li, et al. 2020).


Mass photometry has also been applied to vesicles and micelles (Lebedeva, et al. 2020), and polysarcosine star polymers for drug delivery (England, et al. 2020).


The correlation between the mass photometry signal and molecular mass applies to a variety of biomolecules (Young, et al. 2018) – making mass photometry a universal bioanalytical tool for biomolecules in solution. The precise linear relationship between the mass photometry signal and molecular mass will be different for each class of molecule, requiring calibration with an appropriate standard (i.e. a protein calibrant for mass photometry measurements of proteins, a DNA calibrant for measurements of DNA, etc).




How is mass photometry used?

With mass photometry, you can measure the molecular mass of single biomolecules, oligomers, polymers, macromolecular assemblies and nanostructures (Chen, et al. 2021, England, et al. 2020, Bertosin, et al. 2021, Naftaly, et al. 2021).


You can also quantify the oligomerisation and aggregation of biomolecules (Naftaly, et al. 2021), characterise sample heterogeneity (Olerinyova, et al. 2021, Sonn Segev, et al. 2020), monitor the stability of sample components (Nuber, et al. 2021), and study the effects of molecular or experimental modifications on sample integrity (Bertosin, et al. 2021).


Mass photometry is especially valuable for studying biomolecular interactions – including protein-protein interactions (Higuchi, et al. 2021; Soltermann, et al. 2020) and protein-DNA interactions (Hickman, et al. 2020; Acharya, et al. 2021). Mass photometry makes it possible to determine stoichiometries in biochemical reactions (Chen, et al 2021), and quantify affinities and rate constants in molecular interactions (Wu and Piszczek 2020; Soltermann, et al. 2020).


Mass photometry applications have been rapidly growing as an increasing number of users discover the technology and its versatility. Mass photometry has been used recently to characterise AAV gene therapy vectors (Wu, et al. 2021). Mass photometry has also been used in SARS-CoV-2 research – to study oligomerisation of the SARS-CoV-2 spike protein, and the stoichiometry of its interactions with antibodies and with the ACE2 receptor (Brun, et al. 2021; Yin, et al. 2021; Higuchi, et al. 2021). With applications in areas ranging from fundamental bioscience to the development and manufacture of therapeutics, and beyond, the impact of mass photometry can only grow.




Further resources

If you would like to learn more about the biophysics behind mass photometry, we recommend the following resources:



Webinar: Measuring molecules with light by Prof. Philipp Kukura, University of Oxford

Prof. Kukura, who led the development of mass photometry, explains the principle of mass photometry and talks you through the steps that he and his team of scientists took while developing mass photometry as an analytical tool for biomolecules. You can also hear about examples of mass photometry applications.




Webinar: Applications of mass photometry by Prof. Justin Benesch, University of Oxford

Prof. Benesch, who co-developed mass photometry, presents the data firmly establishing mass photometry as a reliable method for molecular mass measurement. He also discusses experimental data on mass photometry applications and how mass photometry compares to alternative analytical methods.





References

Acharya, A. et al., 2021. Distinct RPA domains promote recruitment and the helicase-nuclease activities of Dna2. Research Square, pp. DOI: 10.21203/rs.3.rs-358230/v1.


Bertosin, E. et al., 2021. Cryo-Electron Microscopy and Mass Analysis of Oligolysine-Coated DNA Nanostructures. ACS Nano, p. 15(6):9391–9403.


Brun, J. et al., 2021. Assessing Antigen Structural Integrity through Glycosylation Analysis of the SARS-CoV-2 Viral Spike. ACS Central Science, p. 7(4):586–593.


Chen, H., et al, 2021. Single-molecule microscopy for in-cell quantification of protein oligomeric stoichiometry. Current Opinion in Structural Biology, pp. 66:112-118.


Cole, D. et al., 2017. Label-Free Single-Molecule Imaging with Numerical-Aperture-Shaped Interferometric Scattering Microscopy. ACS Photonics, p. 4(2): 211–216.


England, R. M. et al., 2020. Synthesis and Characterization of Dendrimer-Based Polysarcosine Star Polymers: Well-Defined, Versatile Platforms Designed for Drug-Delivery Applications. Biomacromolecules, p. 21(8):3332–3341.


Hickman, A. B. et al., 2020. Casposase structure and the mechanistic link between DNA transposition and spacer acquisition by CRISPR-Cas. eLife, p. 9:e50004.


Higuchi, Y. et al., 2021. Engineered ACE2 receptor therapy overcomes mutational escape of SARS-CoV-2. Nature Communications, p. 12:3802.


Lebedeva, M. A., et al, 2020. Emergence and Rearrangement of Dynamic Supramolecular Aggregates Visualized by Interferometric Scattering Microscopy. ACS Nano, pp. 14(9):11160-11168.


Li, Y., et al, 2020. Single-molecule mass photometry of nucleic acids. Nucleic Acids Research, p. e97.


Melo, L. et al., 2021. Size distributions of colloidal gold nanoparticles measured in solution by single-particle mass photometry. arXiv, p. 2107.06247.


Naftaly, A., et al, 2021. Revealing Advanced Glycation End Products Associated Structural Changes in Serum Albumin. ACS Biomaterials Science & Engineering, p. 7(7):3179–3189.


Nuber, F. et al., 2021. Biochemical consequences of two clinically relevant ND-gene mutations in Escherichia coli respiratory complex I. Scientific Reports volume, p. 11:12641.


Olerinyova, A. et al. 2021. Mass Photometry of Membrane Proteins. Chem 7, 224–236.


Ortega-Arroyo, J. & Kukura, P., 2012. Interferometric scattering microscopy (iSCAT): new frontiers in ultrafast and ultrasensitive optical microscopy. Physical Chemistry Chemical Physics, pp. 14:15625-15636.


Soltermann, F. et al., 2020. Quantifying Protein-Protein Interactions by Molecular Counting with Mass Photometry. Angewandte Chemie International Edition in English, pp. 59(27):10774-10779.


Verschueren, H., 1985. Interference reflection microscopy in cell biology: methodology and applications. Journal of Cell Science, pp. 75(1):279-301.


Wu, D. et al., 2021. Rapid Characterization of AAV gene therapy vectors by Mass Photometry. bioRxiv, p. doi: https://doi.org/10.1101/2021.02.18.431916.


Wu, D. & Piszczek, G., 2020. Measuring the affinity of protein-protein interactions on a single-molecule. Analytical Biochemistry, p. 592:113575.