The analysis of biomolecular interactions is simple and straightforward using mass photometry. This popular and widely used technology allows the quantitative analysis of macromolecular binding, oligomerization, and assembly, making it possible to do so both in solution and without the need for labels.
Photo credit: Shutterstock/Siwakorn TH
THE MassFluidix® HC microfluidic system Refeyn offers users a significantly expanded range of sample concentrations suitable for mass photometric study, enabling applications such as the characterization of low-affinity interactions.
Expanding the scope of analysis of mass photometry
This bioanalytical technology allows rapid measurement of the mass distribution of biomolecules in a sample, in just one minute, in solution and without the need to use labels.
This technology offers significant value when used in applications such as bond assessment, sample purity and oligomerization.
The concentration of the measured sample should ideally be below 100 nM to ensure optimal mass photometry measurements. This concentration is perfectly viable for a large number of measurement contexts, but it may be necessary to analyze the behavior of the sample at a higher concentration in some cases.
Complexes formed via low-affinity interactions dissociate at low concentrations, meaning they can usually only be detected at higher concentrations.
Refeyn MassFluidix Microfluidic System® HC successfully addresses this problem, significantly expanding the scope of samples suitable for measurement by mass photometry.
MassFluidix® HC increases the upper limit of sample concentration from the nanomolar to the micromolar range. This article explores how the system works and describes an example application in which it is used to measure interactions between an immunoglobulin G (IgG) antibody and the neonatal IgG Fc receptor (FcRn).1
Characterization of low affinity interactions
Using MassFluidix® With the HC system, it is possible to perform measurements at an optimal concentration for mass photometry while simultaneously capturing the state of biomolecular interactions occurring at micromolar concentrations.
The system achieves this by rapidly diluting the sample and circulating it across the measurement surface, before the equilibrium of the biomolecular interaction is disturbed by the dilution.
This microfluidic system is capable of diluting a sample up to 10,000 times, by passing it directly over the measuring surface. In less than 50 milliseconds after the start of the dilution, the sample has reached the observation window and is ready for analysis.
Up to 98% of the complex will remain intact when the sample is measured under these conditions, even considering low affinity interactions (µM KD) with dissociation rates as fast as one second.-1.2.3
Samples rapidly diluted via MassFluidix® HCs retain their pre-dilution properties, meaning that users can use mass photometry to characterize biomolecular activity that only occurs at higher concentrations, for example, low-affinity interactions.
The MassFluidix® HC system
The computer-controlled MassFluidix® The HC system includes both fluid controls and a fast dilution chip. Dilution occurs on the MassFluidix® HC chip, with sample and buffer combined in an inverted Tesla valve mixer before the diluted sample flows through the observation area. The sample then leaves the chip through an outlet. (Figure 1A).
The larger system includes a central unit, computer, pump, flow monitors, and pressure regulators that work together to ensure that sample and buffer enter the chip at the correct flow rates (Figure 1B). This simplified setup ensures rapid sample dilution that is suitable for mass photometric measurement.
Figure 1. The MassFluidix HC system is designed for rapid dilution. A: Illustration of the MassFluidix HC chip. Undiluted sample (orange) and buffer (grey) enter through inlets on opposite sides of the chip. Sample is diluted where the channels meet and into the mixer, reaching the target concentration (dark blue) near the observation area. The colour of the fluid indicates the concentration (see colour bar). B: The computer (top left) is connected to the MassFluidix HC CPU (shown next to the buffer and sample tubes) and the mass photometer (bottom right). Flow through the sample line and buffer line is monitored by the small flow monitor (FRMS) and large flow monitor (FRML), respectively. The sample and buffer line tubing connects to a channel on the MassFluidix HC chip, which is placed inside the mass photometer. Image credit: Refeyn Ltd.
A key difference between the MassFluidix® HC The special feature of traditional mass photometry is that the molecules under study are subjected to a flow. Refeyn has verified – both theoretically and empirically – that the flow itself has only a negligible effect on the measurement.
From a theoretical point of view, the presence of no-slip boundary conditions means that the fluid velocity at the measuring surface is zero. It therefore remains negligible in the space where biomolecules are typically measured: up to about 20 nm from the surface.
Importantly, there is also no concentration variation across the channel as the sample is homogeneously mixed when it reaches the measuring surface.
Empirically, measurements of a range of proteins in the system (performed by Refeyn) confirmed that counts accumulate as smoothly as they do when using conventional measurements.
MassFluidix® HC reveals low affinity complexes
In the example shown here, the MassFluidix® The HC system was used in a study of the binding of the IgG monoclonal antibody trastuzumab to the soluble domain of FcRn.1
A regular mass photometry measurement was performed on a sample containing the two proteins in a 1:10 ratio (IgG at 2 µM and FcRn at 20 µM). This sample had been manually diluted to 10 nM.
Two main peaks were observed in this case, which is consistent with previously published data,1 one of which corresponded to FcRn monomers and the other to unbound IgG (Figure 2). There was no evidence of the presence of IgG-FcRn complexes.
However, it was evident that FcRn-IgG complexes were present when MassFluidix® The HC system was used to measure the same proteins. The sample had an initial concentration of 22 µM in this measurement and was rapidly diluted 2000-fold to 11 nM in the system.
Figure 2. Mass histograms reveal low-affinity complexes only after rapid dilution of the sample. Mass histograms and corresponding best-fit Gaussian distributions are shown for measurements of samples containing IgG and FcRn (mixed in a ratio of 1:10) after manual dilution (orange) or rapid dilution via the Microfluidix HC system (blue). After manual dilution, peaks corresponding to FcRn monomers and IgG monomers were observed. After rapid dilution, in addition to FcRn and IgG monomers, peaks corresponding to FcRn dimers and IgG-FcRn complexes with stoichiometry of 1:1 and 1:2 were also clearly observed. Image credit: Refeyn Ltd.
Peaks corresponding to 1:1 FcRn-IgG and 2:1 FcRn-IgG complexes were also clearly visible in this measurement, as well as the aforementioned peaks corresponding to FcRn monomers and unbound IgG (Figure 2).
This example shows how mass photometry measurements performed using the MassFluidix® HCs were able to detect low-affinity complexes that had not been observed before.
Low-affinity complexes can only form if the interacting biomolecules are present at higher concentrations. In this case, the results therefore suggest that FcRn-IgG complexes possess relatively low binding affinities.
The differences in complex formation at 10 nM versus 22 µM clearly highlight the value of studying biomolecular interactions at higher concentrations in order to achieve a more complete understanding of the dynamics of biomolecular systems.
The MassFluidix® The HC system allows users to perform these measurements using mass photometry.
Conclusion
THE MassFluidix® HC system uses rapid dilution to expand the range of sample concentrations that can be efficiently analyzed using mass photometers, e.g., Refeyn’s OneDeputy And twoDeputy mass photometers.
Using mass photometry, measuring the mass distribution of biomolecular samples in solution is quick and easy, without labels and using minimal sample amounts.
This technology and combination of equipment also allows the study of biomolecular assembly, oligomerization and interactions, as well as the evaluation of sample purity.® HC also allows for the efficient characterization of low-affinity interactions, further expanding the applicability of mass photometry.
Experimental details
- The lamella was coated with a cationic coating
- Samples were at pH 5.0 and diluted in PBS, pH 5.0
- Flow rates were 0.5 µL/minute (sample) and 1 mL/minute (buffer)
- The measurement time was 60 seconds
- One. TwoDeputy A mass photometer was used for all data collections
References and further reading
- Soltermann et al., Angew. Chemistry. Int. Ed., 2020
- Pollard TD, MBoC, 2017
- Jarmoskaite et al., eLife, 2020
Thanks
Produced from materials originally written by Refeyn Ltd.
About Refeyn Ltd.
Refeyn is the originator of mass photometry, a new biotechnology that allows users to characterize the composition, structure and dynamics of individual molecules in their native environment. We are producing a generation of revolutionary analytical instruments that open up new possibilities for research into biomolecular functions.
Founded at the University of Oxford in 2018 by an experienced team of scientific professionals, Refeyn aims to transform bioanalysis for scientists, academic researchers and biopharmaceutical companies around the world.
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