Optimized membrane protein analytics with mass photometry

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Enabling rapid structural studies

Membrane proteins make up one quarter of the proteome and >60% of drug targets, but are difficult to purify and study. Membrane mimetics – such as detergents, nanodiscs, and SMALPs – help stabilize and purify fully functional membrane proteins, but optimizing membrane extraction is often a painstaking process.


Mass photometry is compatible with most membrane mimetics and offers a non-destructive method for optimizing extraction and purification conditions – enabling structural studies of transmembrane proteins by cryo-EM, X-ray crystallography and other methods.

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Mass photometry supports membrane protein analysis

Mass photometry is a powerful analytical technique that can help overcome the bottlenecks associated with membrane protein characterization. It quantifies samples at the single-molecule level by measuring the light scattered by individual biomolecules in solution (Figure 1). Its versatility extends to a wide array of applications, from assessing assembled/disassembled complexes to identifying impurities and protein aggregates. 

Mass photometry is uniquely equipped to deliver fast, precise results using minimal sample input, and is compatible with most membrane mimetics. As this process is non-destructive and does not require labels, researchers can identify optimal conditions for their structural studies without wasting valuable resources. Furthermore, its user-friendly nature and small instrument footprint make mass photometry highly accessible and adaptable for routine use in laboratory settings. 

Figure 1. The principle of mass photometry. The light scattered by a molecule that has landed on a measurement surface interferes with light reflected by that surface. The interference signal scales linearly with mass. 

Discover the basic principles of how mass photometry works
A revolutionary analytical method for biomolecules

Compatibility with detergents

Detergents are a popular method for membrane protein purification because of their comparative ease of use. However, detergents may destabilize and denature proteins, in addition to increasing sample heterogeneity. Given the huge variety of detergents currently available, identifying a detergent that fulfils specific protein requirements, in terms of stability and functionality, can be a painstaking trial-and-error process.

Compatibility with detergents
 
Figure 2. Mass photometry measurements of U. maydis virulence-associated membrane proteins in detergent. The left plot shows the mass distribution for 25 nM Vmp1 samples solubilized in the detergent LDAO. A measured mass of close to 42 kDa suggests Vmp1 exists as monomers. The right plot shows the mass distribution of 100 nM Vmp2 samples solubilized in the detergent LDAO. A measured mass of 81 kDa indicates Vmp2 exists mainly as dimers. Adapted from Figures 5 and 6 in https://doi.org/10.3389/fpls.2021.669835 
 

Mass photometry not only eliminates the necessity for complete detergent removal but also enables quick assessment of solubility conditions in a wide range of buffers (Figure 2). As measurements take less than five minutes, researchers can rapidly analyze different detergent concentrations and buffer conditions to pinpoint the optimal parameters for membrane protein stability and functionality.

Discover our 3-step fast dilution procedure for analyzing detergent samples

Compatibility with other membrane mimetics

Compatibility with other membrane mimetics

Mass photometry is readily compatible with a variety of membrane mimetics beyond detergents, including nanodiscs, SMALPs, and peptidiscs (Young et al., 2024). The high resolution of mass photometry enables the analysis of complex samples, including nanodiscs of varying compositions or sizes. Operating under non-disruptive conditions, mass photometry enables measurements of samples embedded within nanodiscs, facilitating the assessment of results from antibody assays or functional assays (Figure 3), as showcased in studies like Olerinyova et al. (2021). This capability provides valuable insights into the structure and function of membrane proteins. 

Figure 3. Antibody binding assay confirms the presence of AQP4 in a purified sample. Blue histogram: Antibody binding assay using an anti-AQP4 antibody. The peak at 449 kDa corresponds to AQP4 singlets bound to anti-AQP4 antibody. Grey histogram: Negative control with an anti-GFP antibody. The peak at 145 kDa corresponds to free anti-GFP antibody. Source: Mass photometry in membrane protein purification | Refeyn 
Find out how mass photometry is used to characterize nanodisc-embedded membrane protein samples

Mass photometry solutions for protein interaction analytics

  • TwoMP

  • For molecular mass measurements with unmatched sensitivity, speed and simplicity of use, a TwoMP mass photometer offers a wide mass range and single-molecule resolution. 

    • High-fidelity measurements of molecular mass 

    • Little sample required 

    • ​Intuitive acquisition and data analysis software  

    • ​Easy setup – a compact, benchtop instrument with minimal installation requirements 

    Learn more

  • TwoMP Auto

  • The TwoMP automated mass photometer frees up operator time and offers enhanced precision, enabling rapid measurement of multiple samples with low sample consumption.  

    • Rapid, automated measurement of multiple samples 

    • Ideal for screening and titration assays 

    • Highly reproducible data with user-friendly software 

    Learn more

  • Refeyn consumables
  • By using Refeyn consumables, you can spend less time preparing for measurements and gain greater confidence in your data. The consumables range includes calibrants, sample carrier slides and more.  
     

    Learn more

For molecular mass measurements with unmatched sensitivity, speed and simplicity of use, a TwoMP mass photometer offers a wide mass range and single-molecule resolution. 

  • High-fidelity measurements of molecular mass 

  • Little sample required 

  • ​Intuitive acquisition and data analysis software  

  • ​Easy setup – a compact, benchtop instrument with minimal installation requirements 

Learn more

The TwoMP automated mass photometer frees up operator time and offers enhanced precision, enabling rapid measurement of multiple samples with low sample consumption.  

  • Rapid, automated measurement of multiple samples 

  • Ideal for screening and titration assays 

  • Highly reproducible data with user-friendly software 

Learn more

By using Refeyn consumables, you can spend less time preparing for measurements and gain greater confidence in your data. The consumables range includes calibrants, sample carrier slides and more.  
 

Learn more

What mass photometry users are saying

“A major advantage of MP measurement is the small amount and the low concentration of the protein sample employed (1 μL at 200 nM concentration). This is extremely relevant for eukaryotic membrane proteins as they are difficult to purify in large amounts. In the case of HCN proteins, such limitation has long restricted the biochemical studies of HCN-TRIP8b interaction to their isolated soluble domains and thus prevented the study of the complex in full-length proteins”

Saponaro et al. (2022) Front. Physiol.,

“The underlying physics behind how it works are very complicated, but using the instrument is very much plug-and-play […] One of the routine things that we are doing now is after solubilizing the protein and purifying it, we put it into the mass photometer to check if there is a single peak of the right size before we do the expensive cryoEM experiments.”

Philip Kitchen

School of Biosciences, Aston University

View further resources related to membrane protein analytics

  • Niebling, S., Veith, K., Vollmer, B. et al. 2022. Biophysical screening pipeline for Cryo-EM grid preparation of membrane proteins. Frontiers in Molecular Biosciences, 9, p.882288. [Link]

  • Young, J.W., Pfitzner, E., van Wee, R., et al. 2024. Characterization of membrane protein interactions by peptidisc-mediated mass photometry. Iscience, 27(2). [Link]​

  • Olerinyova, A., Sonn-Segev, A., Gault, J., Eichmann, C., et al. 2021. Mass photometry of membrane proteins. Chem, 7(1), pp.224-236. [Link]

  • Weiland, P. and Altegoer, F., 2021. Identification and characterization of two transmembrane proteins required for virulence of Ustilago maydis. Frontiers in Plant Science, 12, p.669835. [Link] 

  • Webby, M.N., Oluwole, A.O., Pedebos, C., Inns, P.G., Olerinyova, A., Prakaash, D., Housden, N.G., Benn, G., Sun, D., Hoogenboom, B.W. and Kukura, P., (2022). Lipids mediate supramolecular outer membrane protein assembly in bacteria. Science Advances, 8(44), p.eadc9566. [Link] 

View resources

Learn more about how mass photometry can optimize your membrane protein purification

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