Characterisation of amyloid assembly using mass spectrometry

蛋白质由共价结合的氨基酸单元链组成，不同氨基酸形成链的顺序对该蛋白质来说是独一无二的。在活细胞中，蛋白质链折叠成独特的三维结构。它们通常与细胞内的其他蛋白质以及小分子和离子形成非共价相互作用，形成大分子生物复合物。细胞中的大部分工作都是由这些复合物完成的，而不是由单个蛋白质单独完成的。重要的是，我们试图了解蛋白质是如何形成这种复合物的，这样我们就可以了解蛋白质是如何起作用的。如果一种蛋白质从它的三维结构中展开，然后不能正确地折叠回来，这种蛋白质就会出现功能错误。一些蛋白质错误折叠，然后聚合形成大而有序的聚合物，即淀粉样原纤维。这些原纤维与几种引人注目的疾病有关，包括阿尔茨海默病、II型糖尿病、血液透析相关淀粉样变性和朊病毒疾病，包括牛海绵状脑病（“疯牛病”）和克雅氏病。尽管淀粉样原纤维对人类健康具有重要意义，并且在有益利用蛋白原纤维方面具有其他令人兴奋的潜力，但我们目前对通常可溶蛋白如何组装成淀粉样原纤维知之甚少。在这个提议中，我们将使用质谱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射到质谱仪中，然后使用一种称为电喷雾电离的技术将蛋白质分子电离，然后根据它们的质量电荷比分离。这些数据被记录在光谱上，由此可以确定蛋白质的分子质量。然而，除了分子质量信息，电喷雾电离质谱法还可以揭示蛋白质的其他细节，例如蛋白质是否正确折叠，以及它是单体形式还是寡聚形式。我们一直在研究-2-微球蛋白。在健康人体内，β -2微球蛋白从肾脏排出。如果一个人患有肾衰竭并正在接受透析治疗，β -2微球蛋白不会排出体外，而是形成不溶性原纤维，在脚踝、膝盖、臀部、肘关节和肩部积聚。这种疾病被称为血液透析相关淀粉样变，会引起疼痛，最终是致命的。我们已经开发出方法来量化折叠、部分折叠和未折叠的β -2微球蛋白分子的混合物；我们还监测了蛋白质单体的聚集，并观察到在纤维形成过程中二聚体、三聚体、四聚体等的出现和消失。我们还开发了一些方法来观察原纤维的结构，通过使用酶从原纤维中去除可接近的蛋白质片段，然后用质谱法对这些片段进行表征。在这里，我们建议检查β -2微球蛋白在其自然环境中，即被生物物种包围和生理pH值，以发现蛋白质如何在体内自组装。我们还将开发新的质谱方法，以更精确地破译形成的原纤维的聚集反应机制和结构。

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.