Mass photometry techniques with detergents

Detergents are commonly used throughout the biochemistry industry, but these tend to display complex behavior in aqueous solutions. Mass photometry is tool well suited to the study of biomolecules in solutions that contain detergent, as well as the evaluation of micelle formation and molecular aggregation.

Image Credit: Shutterstock/Mongkolchon Akesin

This article explores how detergents impact mass photometry measurements, and summarizes several key recommendations around the optimization of conditions for mass photometry experiments with detergents.

Detergents have a wide range of uses throughout the industry, including the extraction and solubilization of membrane proteins, the prevention of nonspecific binding, and the control of conditions affecting protein crystallization.

Detergents exhibit remarkable chemical properties and complex behavior in aqueous solutions; however, the presence of detergents imposes considerable downstream limitations on many widely utilized analytical technologies.

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Mass photometry measures the mass of individual biomolecules in solution, making it an ideal solution to this particular challenge. It is also compatible with a diverse array of buffers, effectively eliminating the need for complete detergent removal.

Properly used, mass photometry provides a rapid, streamlined means of determining the impact of detergents on sample solubility and the ways in which detergent behavior varies in different buffers and at different concentrations.

The effect of detergents on mass photometry

Mass photometry is unable to detect individual detergent molecules as the amount of light they scatter is typically below the technique’s detection threshold. Detergents may also produce noise (signal fluctuations) across ratiometric mass photometry images.

This can be the result of water molecules forming large solvation shells around detergent molecules, detergent molecules generating dynamic structures on the glass surface, or a range of other factors that may impact the refractive index at the glass-water interface.

Detergents can also impact mass photometry measurements via the formation of micelles when the detergent concentration in an aqueous solution is greater than the critical micelle concentration (CMC).

Smaller micelles (those below the detection limit) behave in a similar fashion to individual detergent molecules in that these generate noise in a mass photometry measurement.

It is possible to visualize larger micelles directly¹ in mass photometry in the same way as biomolecules,² but many standard protocols necessitate the use of high concentrations of detergent, leading to micelle concentrations that are too high to permit individual micelle masses to be quantified by mass photometry.

Many overlapping events will be observed in this scenario as the micelles meet the glass-water interface. These overlapping events produce a pattern of noise that is similar to the pattern generated by individual molecules and smaller micelles but with a stronger signal.

The overall result is a random noise pattern in the ratiometric mass photometry image (Figure 1, upper panel), which prevents the detection of macromolecules with a signal in the same range or lower, effectively raising the lower limit for mass detection (Table 1).

It is also important to note that these noise patterns can result in incorrect or misleading mass photometry signals, much like the signals generated by macromolecules.

When this occurs, conventional mass photometry image analysis will incorrectly read these patterns as a macromolecule landing on the surface. Should the patterns occur more than once, they will result in a peak in a histogram with a certain apparent mass but no biological significance.

The lower panel of Figure 1 highlights the presence of a mirror-image peak with an equivalent, ‘negative’ apparent mass and the same height. It is possible to use this signature mirror-imaging to differentiate peaks arising from noise to those representing biomolecules that land on the measurement surface because negative mass results from particles moving away from the glass surface, not particles landing on it.

Figure 1. Typical detergent noise signature. Top: PBS buffer alone and with detergent Tween^®20 at two concentrations. Bottom: Superposition of histograms of PBS (grey) with PBS supplemented with Tween^®20 at concentrations below (0.003 mM, mid blue) and above (0.3 mM, dark blue) the CMC. Apparent mass and sigma values of Gaussian fits are indicated. Values measured on the One^MP. Image Credit: Refeyn Ltd.

It is important to note that mass photometry measurements of proteins and similar biomolecules generally yield no, or very small, negative peaks as the biomolecules will usually interact with the glass surface rather than moving away from this.

Measuring samples containing detergents

Mass photometry will only image biomolecules with mass significantly greater than the apparent mass corresponding to the detergent noise peak. Therefore, a lower detergent concentration will usually result in improved resolution, superior accuracy and a lower mass detection limit (Figure 2).

Mass photometry measurements should be conducted at the lowest possible detergent concentration. In certain cases, however, this may still correspond to a mass detection limit that is too large for meaningful measurement via mass photometry.

If the mass detection limit is too large for meaningful measurement but the detergent and protein are sufficiently bonded, it is possible to employ an in-drop, fast dilution procedure to facilitate mass photometry measurements of proteins at detergent concentrations below that which is otherwise considered the minimum for protein stability.⁵

This straightforward procedure can only be used if the detergent-protein interaction remains stable for the entire 1-minute duration of the measurement and the protein does not aggregate. The procedure is completed using the following steps:

1. Load buffer with no detergent onto the coverslip
2. Find the focus
3. Add protein with detergent to the original buffer. This should then be mixed by using the pipette to gently aspirate in and out.
4. Conduct the mass photometry measurement

To assess the apparent mass of the detergent noise peak, this procedure should be completed following an appropriate control measurement of the buffer at an identical detergent concentration.

Table 1 provides approximations of the effective detection limits for different detergents after dilution in PBS. This should be regarded as a general guide, since actual detection limits may vary based on the buffer’s characteristics; for example, ionic strength or pH.

It is, therefore, prudent to assess the mass detection limit of detergent-containing solutions on a case-by-case basis. This is best achieved by conducting control measurements of the solution without the biomolecules of interest present.

Certain proteins are only soluble above a specific detergent concentration, and these proteins will form large aggregates when below this concentration. Optimal conditions for each individual protein/detergent combination are difficult to anticipate due to their dependence on a number of different factors.

Figure 2. Mass photometry measurement of a protein in detergent. Histograms represent measurements of buffer with 0.0025% LMNG alone, and 10 nM protein in buffer with 0.0025% and 0.00025% LMNG. Excessive dilution of detergent may result in protein aggregation, as illustrated in ratiometric frames showing soluble protein (light blue) and aggregated protein (mid blue). Data courtesy of Blanca López Méndez and Vadym Tkach, University of Copenhagen. Values measured on the One^MP.

Table 1. Effective lower detection limits corresponding to relative concentrations of detergents. Estimates of lowest detectable protein mass in kDa for One^MP (light blue) and Two^MP (mid blue) are based on the noise peak detected at the respective detergent concentration (mM, grey gradient). Detergents were diluted in PBS. CMC is indicated in grey. N/A: Detection limit of the instrument applies. Source: Refeyn Ltd.

These should also be assessed experimentally on a case-by-case basis, ideally using mass photometry. This technique represents an ideal choice for the screening of solubility conditions because it requires very little sample and can be performed quickly. Aggregates are also easily identifiable when viewed in a ratiometric mass photometry movie (Figure 2).

Using mass photometry to assess the CMC

Detergents commonly generate a low mass photometry background below the CMC, with the mass photometry background increasing abruptly above the CMC.

Figure 3. Detergent behavior above the CMC varies by detergent. Mass photometry measurements of two different detergents, DDM (dark blue) and OG (orange), show sigmoidal (DDM) vs linear (OG) increases in background as detergent concentration is increased above the CMC. The approximate CMC (in PBS) is indicated in grey. The background was quantified as the standard deviation of contrast for each ratiometric image, averaged over 3000 frames. Values measured on the One^MP. Image Credit: Refeyn Ltd.

Background intensity can be seen to plateau for detergents that form micelles of a single size, for example, DDM (n-dodecyl-ß-D-maltoside). This background may also continue to increase with some detergents, should the micelle size increase with concentration; for example, in the case of OG (octyl glucoside) (Figure 3).

The CMC in these cases in largely dependent on factors such as the nature of the biomolecules or the ionic strength and pH of the buffer. These factors can lead to a detergent’s observed CMC to vary significantly from the CMC reported for that detergent in water, even when investigated under experimental conditions.

This variability is especially apparent in ionic detergents like SDS (sodium dodecyl sulfate)³ (Figure 4, top).

Detergents are also prone to displaying complex micelle formation behavior. For example, CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate) has been found to possess two distinct CMCs (around 7 and 32 mM) and has been seen to form micelles 1.8 times larger above the second CMC⁴ (Figure 4, bottom).

The capacity to monitor micelle formation under a range of experimental conditions is valuable because this allows users to optimize detergent concentration, using only the necessary amount of detergent in each instance.

There are a number of challenges associated with measuring the CMC in practice; however, experiments are generally performed with detergent concentrations far above the required concentrations.

Mass photometry offers a potential solution to this issue. It offers a rapid, convenient means of evaluating detergent behavior under a range of experimental conditions, making it easy to establish the optimal parameters for any given experiment.

Figure 4. Detergent micelle formation may be sensitive to buffer composition or display complex behavior. Top: The background measured using mass photometry for increasing concentrations of SDS in water (blue), 0.1x PBS (grey) and 1x PBS (orange). Bottom: Similar measurements for increasing concentrations of CHAPS in PBS. CMCs reported in the literature³ are indicated as grey areas. The background was quantified as in Fig. 3. Values measured on the One^MP. Image Credit: Refeyn Ltd.

References and further reading

1. Lebedeva et al., ACS Nano 2020
2. Young et al., Science 2018
3. Danov et al., Adv Colloid Interface Sci 2014
4. Qin et al. J Phys Chem B 2010
5. Olerinyova et al., Chem 2021

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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