Improving the ability to accurately measure protein concentration via mass photometry

The current gold standard methods of measuring protein concentration include spectrophotometric or nanodrop methods such as UV-vis spectroscopy, probing the absorbance of a solution of protein at 280nm.

Conversely, the concentration may be determined by reacting the protein with inorganic metal ions or organic dyes in a colorimetric assay. UV-vis spectroscopy has been the preferred method due to its simplicity and fast measurement.

However, the measurement is susceptible to inaccuracies such as impurities and buffer components that may also absorb 280nm light.

Furthermore, there is an assumption that the optical properties of aromatic residues remain constant across the many complex layers of possible protein folding and oligomerisation.

Each protein contains a varying number of tyrosine and tryptophan residues, and therefore the extinction coefficient of a given protein may be an estimation, resulting in an inaccurate approach for concentration determination.

Concentration is a critical parameter for understanding key biophysical interactions, such as enzyme kinetics or protein binding constants, important in biochemical processes such as amyloid aggregation.

The potential impact and utility of a technique that can provide greater accuracy in concentration determination will therefore provide the foundation for a better quantitative understanding of protein dynamics.

Mass photometry (MP) uses the principle of interferometric light scattering (iSCAT) to detect the landing events of single protein molecules to a glass substrate. No prior assumption of the extinction of the protein of interest is required to determine concentration.

MP also provides a label free detection method of mass, as the measured scattering signal upon binding is proportional to the molecule's mass.

By improving the resolution and dynamic range of this technique, it may be possible to provide a fast, reproducible, and more accurate method of concentration determination relative to currently utilised techniques. Establish the current extent to which MP can provides an accurate determination of protein concentration.

This will require the accumulation of highly precise measurements from a wide variety of proteins to determine the full dynamic range of the technique.

Develop novel optical hardware to improve the resolution and dynamic range of MP, thus further enhancing the suitability of this measurement.

Create a user-friendly analysis package coupled to the new instrument, enabling the rapid and accurate determination of protein concentration with MP.

If successful, MP will provide a level of accuracy in protein concentration measurement that has not been achieved to date, yet still maintains the key benefits of UV-vis spectroscopy; namely fast, reproducible measurement with a non-reliance on additional reagents.

This project will require the development of novel optical microscopy hardware, overall resulting in high resolution and accurate visualisation and quantification of individual protein molecules in solution.

By combining real time imaging with the single molecule counting capability of MP, parameters such as diffusion coefficients can also be measured, enabling a multi-faceted novel analytic approach. This project falls within the EPSRC Physical Sciences research area.

Understanding the physics of life is a key EPSRC research area, and if successful, this project provides a clear platform for furthering our quantitative understanding of protein dynamics.