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

质谱法是一种相对较新的干涉散射显微镜技术，能够检测单个生物分子，近年来发展迅速。在简单的质谱测量中，感兴趣的物质与玻璃盖盖结合在一起，通过玻璃盖盖反射的光与入射物质散射的光之间的干涉对比的比例成像，可以检测到它的着陆。产生的信号与结合物质的质量成线性关系，因此，如果校准正确，就能揭示复合物的质量。这项技术最常应用于蛋白质，然而，也可用于诸如核酸和囊泡之类的物种。该项目旨在通过在质谱装置中添加高强度紫外线（UV）激光，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。紫外激光将最有可能在最初的质量光度测量之后使用，给这项技术一个临时的标题——串联分光光度测量。第一个目标将是建立和完善质量测光装置与额外的紫外激光。第二个目标是通过概念验证实验证明其对单个生物分子研究的适用性。描述了演示该技术的两个潜在目标，但是，在整个项目中可能会开发和探索进一步的概念。质谱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，系统内蛋白质的低聚组成仍然是未知的，特别是蛋白质混合物。用高强度紫外激光照射结合的低聚物可以将结构分解成更小的组分，这些组分可以解除结合。通过质谱法，这些解结合事件可以暂时解决，并且通过较小亚基的顺序解结合，每个亚基都具有检测到的质量，可以建立更清晰的低聚蛋白组成图像。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，低功率紫外激光器也可以在质谱测定中用于通过光可切割连接剂进行特定蛋白质质量测量。目前，简单的质谱测定法只能检测非特异性蛋白质。这是可能的，通过特异性结合蛋白到光切割抗体或DNA适体在盖盖表面上，解结合事件可以在中等紫外线照射下观察到。非特异性结合到玻璃表面的蛋白质应该不受影响。特异性解结合可以允许在复杂的生物流体样品中对特定蛋白质进行多重测量。研究方法的新颖性在上述目标中得到突出体现。首先，质量光度法将首次与紫外激光串联使用。因此，这种装置所提供的可能性以前没有被探索过，还有很大的发展空间。如果成功，tandemMP可能提供一种从蛋白质质量测量中提取进一步信息的新方法，包括低聚蛋白质结构分析的简单途径以及特定蛋白质检测的方法。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这个项目的核心，因此最有可能影响这个领域。然而，这个项目也将属于更广泛的物理科学主题，它适用于物理和数学科学的战略重点。

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.