Extending the Functionality of Single Molecule Mass Photometry with Tandem Ultraviolet Illumination

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Mass photometry is a relatively new interferometric scattering microscopy technique capable of single biomolecule detection which has seen rapid uptake in recent years. In a simple mass photometry measurement, the species of interest binds to a glass coverslip where its landing may be detected via ratiometric imaging of the interferometric contrast between light reflected by the glass coverslip and light scattered by the incoming species. The signal generated scales linearly with the mass of the binding species, therefore revealing the mass of the complex if correctly calibrated. This technique is most commonly applied to proteins, however, may also be used for species such as nucleic acids and vesicles. This project aims to bring further functionality to interferometric scattering microscopy through the addition of a high-intensity ultraviolet (UV) laser to a mass photometry setup and demonstrate its relevance to the study of biological molecules, most notably proteins. The UV laser will most likely be used after an initial mass photometry measurement, giving the technique a working title of tandemMP. The first objective will be to build and refine the mass photometry setup with the additional UV laser. The second objective is to prove its applicability to single biomolecule studies through proof-of-concept experiments. Two potential objectives to demonstrate the technique are described, however, further concepts may be developed and explored throughout the project. Mass photometry can provide a mass distribution of proteins within a sample. However, without a high level of knowledge of the system, the oligomeric composition of proteins within the system remains unknown, especially for protein mixtures. Illuminating the bound oligomers with a high-intensity UV laser may decompose the structures into smaller component pieces which can unbind. Through mass photometry these unbinding events can be temporally resolved and through sequential unbinding of smaller subunits, each with a detected mass, it may be possible to build a clearer image of the composition of an oligomeric protein. This decomposition data may also elucidate the relative binding strengths between subunits, providing further structural information. In a second application of such a device, a low-power UV laser may also be utilised within mass photometry for specific protein mass measurement via photocleavable linkers. Currently, simple mass photometry measurements result in non-specific protein detection. It is possible that by specifically binding proteins to photocleavable antibodies or DNA aptamers on a coverslip surface, unbinding events may be observed under moderate UV illumination. Proteins bound non-specifically to the glass surface should be unaffected. Specific unbinding may allow multiplexed measurement of specific proteins within complex biofluid samples. The novelty of the research methodology is highlighted in the aims above. Firstly, mass photometry will be combined in tandem with a UV laser for the first time. Hence, the possibilities offered by such a device have not been explored before and there is much scope for further development. If successful, tandemMP may provide a new method to extract further information from protein mass measurements, including a simple pathway for structural analysis of oligomeric proteins as well as an approach towards specific protein detection. This project falls within the EPSRC chemical biology and biological chemistry research area. New methods to probe single biomolecules forms the heart of this project and as such is most likely to impact this area. However, this project will also fall into the broader physical science theme, where it is applicable to the physical and mathematical sciences strategic priority.