Characterisation of amyloid assembly using mass spectrometry

蛋白质由共价结合的氨基酸单位链组成，不同的氨基酸形成链的顺序是该蛋白质独一无二的。在活细胞中，蛋白质链折叠成独特的三维结构。通常，它们与其他蛋白质以及细胞内的小分子和离子形成非共价相互作用，形成大分子生物复合体。细胞中的大部分工作是由这种复合体完成的，而不是由单独工作的单个蛋白质完成的。重要的是，我们要试图了解蛋白质是如何形成这种复合体的，这样我们才能了解蛋白质的功能。如果一种蛋白质从其三维结构中展开，然后不能正确地折叠回来，那么该蛋白质可能会出现功能障碍。一些蛋白质错误折叠，然后聚合，形成被称为淀粉样纤维的大而有序的聚合物。这些纤维与几种备受瞩目的疾病有关，包括阿尔茨海默病、II型糖尿病、血液透析相关的淀粉样变性，以及包括牛海绵状脑病(疯牛病)和克雅氏病在内的普恩疾病。尽管淀粉样纤维对人类健康具有重要意义，以及以有益的方式利用蛋白质纤维的其他令人兴奋的潜力，但目前我们对正常情况下可溶的蛋白质如何组装成淀粉样纤维知之甚少。在这项提案中，我们将使用质谱学来阐明新的信息和蛋白质自组装机制。这种方法包括将溶液中的蛋白质注入质谱仪，在质谱仪上使用一种称为电喷雾电离的技术将蛋白质分子电离，然后根据其质量电荷比进行分离。这些数据被记录在光谱上，根据光谱可以确定蛋白质的分子质量。然而，除了分子质量信息，电喷雾电离质谱仪还可以揭示蛋白质的额外细节，例如蛋白质是否正确折叠，以及它是单体还是寡聚形式。我们一直在研究蛋白质β-2-微球蛋白。在健康人体中，β-2-微球蛋白是从肾脏排泄出来的。如果一个人患有肾衰竭，正在接受透析治疗，β-2-微球蛋白不会被排泄出来，并形成不可溶的纤维，在脚踝、膝盖、髋关节、肘关节和肩关节堆积。这种疾病被称为血液透析相关的淀粉样变性，会引起很大的疼痛，最终是致命的。我们已经开发了定量折叠、部分折叠和未折叠的β-2微球蛋白分子混合物的方法；我们还监测了蛋白质单体的聚集，并看到二聚体、三聚体、四聚体等在纤维形成过程中出现和消失。此外，我们还开发了一些方法来观察纤维的结构，方法是使用酶从纤维中去除可访问的蛋白质片段，然后通过质谱仪对这些片段进行表征。在这里，我们建议检测β-2-微球蛋白在其自然环境中，即被生物物种包围和在生理pH下，以发现蛋白质如何在体内自组装。我们还将开发新的质谱学方法，以更准确地破译形成的纤维的聚集反应机理和结构。

Proteins consist of a chain of covalently bound amino acid units, and the order in which the different amino acids form the chain is unique to that protein. In the living cell, protein chains fold into a unique 3-dimensional configuration. Often they form non-covalent interactions with other proteins, as well as small molecules and ions within the cell, to make macromolecular biological complexes. Most of the work in cells is performed by such complexes, rather than by individual proteins working alone. It is important that we try to understand how proteins form such complexes, so that we can understand how proteins function. If a protein unfolds from its 3-dimensional structure and then does not fold back again correctly, the protein can mis-function. Some proteins misfold and then polymerise to form large, well-ordered polymers known as amyloid fibrils. These fibrils are associated with several high-profile diseases including Alzheimer's disease, Type II diabetes, haemodialysis-related amyloidosis and the prion diseases including bovine spongiform encephalopathy ('mad cow' disease) and Creutzfeldt-Jakob disease. Despite the significance of amyloid fibrils in human health, and other exciting potentials for utilising protein fibrils in a beneficial way, we currently know little about how normally soluble proteins assemble into amyloid fibrils. In this proposal, we will use mass spectrometry to elucidate new information and protein self-assembly mechanisms. This method involves injecting a protein in solution into a mass spectrometer whereupon protein molecules are ionised, using a technique known as electrospray ionisation, and then separated according to their mass-to-charge ratio. These data are recorded onto a spectrum from which the molecular mass of the protein can be determined. As well as molecular mass information however, electrospray ionisation mass spectrometry can also reveal additional details about a protein, such as whether the protein is correctly folded and whether it is in monomeric or oligomeric form. We have been studying the protein beta-2-microglobulin. In healthy humans, beta-2-microglobulin is excreted from the kidney. If a person is suffering from kidney failure and undergoing dialysis treatment, beta-2-microglobulin is not excreted, and forms insoluble fibrils that build up in the ankle, knee, hip, elbow and shoulder joints. This disease, known as haemodialysis-related amyloidosis, causes much pain and eventually is fatal. We have already developed methods to quantify a mixture of folded, partially folded and unfolded beta-2-microglobulin molecules; we have also monitored the aggregation of the protein monomer and seen the dimer, trimer, tetramer, etc appear and disappear during fibril formation. Also we have developed methods to look at the structure of the fibrils by using enzymes to remove accessible protein fragments from the fibrils, and then characterising these fragments by mass spectrometry. Here we propose to examine beta-2-microglobulin in its natural environment, i.e. surrounded by biological species and at physiological pH, to discover how the protein might self-assemble in vivo. We will also develop new mass spectrometry methods to decipher more precisely the aggregation reaction mechanism and structure of the fibrils formed.