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

质量光度法是一种相对较新的干涉散射显微镜技术，能够进行单生物分子检测，近年来得到了迅速的应用。在简单的质量光度测量中，感兴趣的物质结合到玻璃盖片，其中可以经由由玻璃盖片反射的光与由入射物质散射的光之间的干涉对比度的比率成像来检测其着陆。产生的信号与结合物质的质量成线性比例，因此如果正确校准，则揭示复合物的质量。该技术最常应用于蛋白质，然而，也可用于核酸和囊泡等物质。该项目旨在通过将高强度紫外（UV）激光添加到质量光度测定设置中，为干涉散射显微镜带来更多功能，并展示其与生物分子研究的相关性，特别是蛋白质。紫外激光器将最有可能在最初的质量光度测量后使用，使该技术的工作名称为tandemMP。第一个目标将是建立和完善质量测光设置与额外的紫外激光器。第二个目标是通过概念验证实验证明其对单生物分子研究的适用性。两个潜在的目标，以证明该技术的描述，但是，进一步的概念可能会开发和探索整个项目。质量光度法可以提供样品内蛋白质的质量分布。然而，没有高水平的知识的系统，系统内的蛋白质的寡聚体组成仍然是未知的，特别是对于蛋白质的混合物。用高强度UV激光照射结合的低聚物可以将结构分解成可以解结合的更小的组成部分。通过质量光度测定，这些解结合事件可以在时间上分辨，并且通过较小亚基的顺序解结合，每个亚基具有检测到的质量，可以建立寡聚蛋白质的组成的更清晰的图像。这种分解数据也可以阐明亚基之间的相对结合强度，提供进一步的结构信息。在这种装置的第二种应用中，低功率UV激光器也可以在质量光度法中用于经由光可裂解连接体的特定蛋白质质量测量。目前，简单的质量光度测量导致非特异性蛋白质检测。通过将蛋白质特异性结合到盖玻片表面上的光可裂解抗体或DNA适体，可以在中等UV照射下观察到解结合事件。与玻璃表面非特异性结合的蛋白质应不受影响。特异性解结合可以允许复杂生物流体样品内的特异性蛋白质的多重测量。上述目标突出了研究方法的新奇。首先，质量测光将首次与紫外激光器相结合。因此，这种装置所提供的可能性以前没有被探索过，有很大的进一步发展的余地。如果成功的话，tandemMP可能提供一种新的方法来从蛋白质质量测量中提取更多的信息，包括一种简单的寡聚蛋白质结构分析途径以及一种特异性蛋白质检测方法。该项目属于EPSRC化学生物学和生物化学研究领域的福尔斯。探测单个生物分子的新方法构成了该项目的核心，因此最有可能影响这一领域。然而，该项目也将属于更广泛的物理科学主题，适用于物理和数学科学战略优先事项。

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.