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能够检测单个生物分子的干涉散射显微技术，近年来得到了迅速的应用。在一种简单的质量光度测量中，感兴趣的物种结合到玻璃盖板上，通过对玻璃盖板反射的光和进入的物种散射的光之间的干涉对比进行比率成像，可以检测到它的着陆。产生的信号与结合物种的质量呈线性关系，因此如果正确校准，就能揭示络合物的质量。这项技术最常应用于蛋白质，但也可能用于核酸和囊泡等物种。该项目旨在通过将高强度紫外线(UV)激光添加到质量光度测量装置中来为干涉散射显微镜带来进一步的功能，并展示其与生物分子(尤其是蛋白质)研究的相关性。紫外激光很可能会在最初的质量光度测量之后使用，这使得这项技术有了一个暂定的标题：TandemMP。第一个目标将是建立和改进使用额外的UV激光的质量光度测量系统。第二个目标是通过概念验证实验证明其在单生物分子研究中的适用性。描述了演示该技术的两个潜在目标，然而，在整个项目中可能会开发和探索更多的概念。质量光度法可以提供样品中蛋白质的质量分布。然而，如果没有对该系统的高水平的了解，该系统内蛋白质的寡聚组成仍然未知，特别是对于蛋白质混合物。用高强度的紫外线激光照射结合的低聚物可能会将结构分解成更小的组成部分，从而可以解开。通过质量光度法，这些解离事件可以在时间上被分解，并且通过顺序地解离较小的亚基，每个亚基具有检测到的质量，有可能建立低聚蛋白质组成的更清晰的图像。这种分解数据还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。在这种装置的第二个应用中，低功率的UV激光也可以在质量光度法中用于通过可光裂解连接物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的检测。有可能通过将蛋白质与盖玻片表面的可光切割抗体或DNA适配子特异性结合，在适度的紫外光照射下可以观察到解离事件。与玻璃表面非特异性结合的蛋白质不应受到影响。特异解结合可能允许对复杂生物体液样本中的特定蛋白质进行多路测量。上述目标突出了研究方法的新颖性。首先，将首次将质量光度学与紫外光激光串联起来。因此，这种设备提供的可能性以前从未被探索过，还有很大的发展空间。如果成功，tandemMP可能会提供一种从蛋白质质量测量中提取进一步信息的新方法，包括一种分析寡聚蛋白质结构的简单途径，以及一种检测特定蛋白质的方法。该项目属于EPSRC化学生物学和生物化学研究领域。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，在那里它适用于物理和数学科学的战略优先事项。

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.