Sponsored Content by RefeynReviewed by Olivia FrostJul 19 2024
Automated mass photometry combines the unparalleled simplicity and sensitivity of Refeyn’s molecular mass measurement technology with the efficiency and ease of automation.
This approach offers enhanced precision while freeing up valuable operator time, facilitating the rapid measurement of multiple samples with minimal sample requirements.
Image Credit: vchal
About mass photometry
Mass photometry is a powerful analytical tool that investigates biomolecules at the single-molecule level, without the need for labels, and in the biomolecules’ native state.
This technique is used to measure the interference between light reflected by a glass surface and light scattered by individual molecules adhering to the glass, utilizing this signal to measure the molecules’ mass by counting them (Figure 1).
Mass photometry is employed in a wide range of application areas, including the study of oligomerization, molecular interactions, and macromolecular assembly. It also facilitates easy sample quality and stability assessments as a core element of rapid and responsive process optimization cycles.
Figure 1. The principle of mass photometry. The light scattered by a molecule interacting with the measurement interface interferes with light reflected at that interface. The signal intensity scales linearly with mass. Image Credit: Refeyn Ltd.
Automated mass photometry
The automated mass photometry solution from Refeyn is comprised of a robotically-controlled mass photometer able to autonomously measure 24 samples, including calibrants, in just 90 minutes.
This technology is ideally suited to a wide range of applications that require precise and efficient molecular mass characterization on a repeated basis, including titration assays and screening.
Automated mass photometry is ideal for any task that requires precise and efficient repeated molecular mass characterization. The robotics unit can be retrofitted to both the OneMP and TwoMP mass photometers produced by Refeyn. It is also available together with the TwoMP mass photometer as the TwoMP Auto.
The TwoMP Auto from Refeyn is easy to use thanks to an intuitive single software interface that allows the operator to directly control the whole data acquisition process.
The operator defines a sample mixing and measurement protocol before loading the sample and any required buffers, calibrants and solutions onto a 96-well plate. It is also possible to automatically dilute samples immediately before measurement, minimizing the possibility of well adsorption.
Once it has been defined, the robot follows this protocol to transfer components from the 96-well plate to a cassette on the mass photometer. This multi-sample well cassette is able to accommodate 24 samples (Figure 2), with its first and final wells typically used for calibrants, ensuring the presence of an internal validation control.
The robot will autonomously run the mass measurements, returning relevant data to the operator for further analysis.
All the benefits of mass photometry are afforded by automated mass photometry; for example, each experiment takes just minutes, uses a minimal amount of sample, and the results can be intuitively interpreted, all with the more consistent and convenient sample manipulation inherent to automated processes.
Figure 2. A close-up of the robotic pipetting arm used in automated mass photometry. The liquid-handling robot uses the pipette tips (1) to transfer sample from the 96-well plate (2) to the multi-sample well cassette (3), in preparation for a measurement. Image Credit: Refeyn Ltd.
Figure 3. Studying protein oligomerization with automated mass photometry. Mass distributions of citrate synthase samples in a pH titration series performed using the TwoMP Auto. The inset plots show detailed molecular mass distribution histograms for the highest (8.5) and lowest (6.5) pH values measured. Data courtesy of Stefano Lometto and Dr. Georg Hochberg of the Max Planck Institute for Terrestrial Microbiology, Marburg. Image Credit: Refeyn Ltd.
Oligomerization studies
Oligomerization is a key process in the function of a significant number of proteins, but effectively capturing and characterizing protein oligomerization can be challenging. This is especially the case where oligomeric species are only present at very low concentrations in the sample.
Mass photometry is a powerful tool that provides high-resolution distributions of molecular mass directly in a solution while maintaining single-molecule sensitivity. These qualities make it ideally suited to detecting a protein’s different oligomeric states, including rare species representing less than 1 % of a sample population.
The TwoMP Auto builds on the strengths of mass photometry, allowing users to rapidly screen a protein’s oligomerization states with ease. This is possible under a range of different experimental conditions, including variations in buffer pH (Figure 3).
In the example presented here, the TwoMP Auto was employed in the measurement of mass distributions in citrate synthase samples in a pH titration series. At certain pH values, citrate synthase will oligomerize into inactive filaments that register in a mass photometry histogram as peaks at higher molecular masses, therefore representing higher-order oligomers.
Straightforward titration
Running titration assays like the one displayed in Figure 4 is simple and straightforward using automated mass photometry. In the example shown here, mass photometry analysis revealed that the protein inosine-5′-monophosphate dehydrogenase (IMPDH) exists in two different oligomeric states: a tetramer (known to be catalytically active) and an octamer (known to be inactive).1
Protein concentration was held constant throughout this experiment, while the ligand diadenosine tetraphosphate (Ap4A) concentration was varied (this ligand is known to promote octamer formation).
It was observed that octamer concentration rose sharply before becoming saturated at higher Ap4A concentrations. Mass photometry measurements exhibited high consistency in this experiment, with close values confirmed between repeated measurements.
Figure 4. A ligand titration series. Mass photometry analysis shows two oligomeric states of IMPDH: a tetramer (light blue peaks) and an octamer (dark blue peaks). Inset: The proportion of octamers to tetramers increased as the concentration of the ligand Ap4A was increased. Each measurement was performed twice, as shown in the inset, and one set is shown in the main plot. Data courtesy of Pietro Giammarinaro, Prof. Dr. Gert Bange and Dr. Georg Hochberg, Max Planck Institute for Terrestrial Microbiology, Marburg. Image Credit: Refeyn Ltd.
TwoMP Auto: The automated mass photometer
Combining the efficiency and ease of automation with the simplicity and sensitivity of mass photometry, the TwoMP Auto enables rapid measurement of multiple samples while maintaining consistently high reproducibility.
Image Credit: Refeyn Ltd.
Reproducibility
A combination of automated liquid handling and the capacity to create and use standardized protocols is key to the simplification of mass photometry experiments. These characteristics also ensure improved reproducibility of results versus manual operation (Figure 5).
There was clear consistency across measurements between mass measurements (147 ± 1.0 kDa for monomers, 304 ± 2.4 kDa for dimers) and the proportions of monomers and dimers (90 % monomers ± 0.7 %), indicating the reproducibility and reliability of mass photometry. Mass measurements were found to match the reported mass of both IgG monomers (150 kDa) and dimers (300 kDa).
Figure 5. Automated mass photometry measurements are highly consistent. Autonomously run mass photometry measurements of IgG (10 nM) illustrate the reproducibility of the system. A: The mass distributions (inset: mass histograms for each of 10 wells, shaded light blue) form two peaks – monomers and dimers. The measured mass, calculated from the location of the maximum of the lower-mass peak in each measurement (B), counts associated with the lower-mass peak (C), and relative proportions of monomers and dimers (D) are shown across the 10 wells. Image Credit: Refeyn Ltd.
Accessories and consumables
Refeyn offers a number of accessories and consumables that are ideal for mass photometry measurements.
These products afford users confidence in their data by helping maintain consistent measurement conditions while simultaneously reducing the number of steps required to obtain each measurement.
Image Credit: Refeyn Ltd.
Refeyn’s range of accessories and consumables includes:
- Ready-to-use sample carrier slides
- Multi-sample well cassettes
- Magnetic slide holders
- An alignment tool and tweezers
References and further reading
- Giammarinaro, Pietro I., et al. Nat Microbiol, 2022
Acknowledgments
Produced from materials originally authored by Refeyn Ltd.
About Refeyn Ltd.
Refeyn are the innovators behind mass photometry, a novel biotechnology that allows users to characterise the composition, structure and dynamics of single molecules in their native environment. We are producing a disruptive generation of analytical instruments that open up new possibilities for research into biomolecular functions.
Spun out of Oxford University in 2018 by an experienced team of scientific professionals, Refeyn aims to transform bioanalytics for scientists, academic researchers, and biopharma companies around the world.
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