Characterisation of amyloid assembly using mass spectrometry

使用质谱法表征淀粉样蛋白组装

• 批准号: BB/D010284/1
• 负责人: Alison Ashcroft
• 金额: $ 31.1万
• 依托单位: University of Leeds
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2007
• 资助国家: 英国
• 起止时间: 2007 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FD010284%2F1
• 关键词: Characterisation; amyloid; assembly; using; mass

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.

蛋白质由共价结合的氨基酸单元链组成，并且不同氨基酸形成链的顺序对于该蛋白质是独特的。在活细胞中，蛋白质链折叠成独特的三维构型。它们通常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，以形成大分子生物复合物。细胞中的大部分工作都是由这种复合物完成的，而不是由单独的蛋白质完成的。重要的是，我们试图了解蛋白质如何形成这种复合物，这样我们就可以了解蛋白质如何发挥作用。如果一个蛋白质从它的三维结构展开，然后没有正确地折叠回来，那么这个蛋白质可能会功能失调。一些蛋白质错误折叠，然后聚合形成大的，有序的聚合物，称为淀粉样纤维。这些原纤维与几种备受关注的疾病相关，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。尽管淀粉样纤维在人类健康中的重要性，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，但我们目前对正常可溶性蛋白质如何组装成淀粉样纤维知之甚少。在这个计划中，我们将使用质谱来阐明新的信息和蛋白质自组装机制。该方法包括将蛋白质溶液注入质谱仪，使用称为电喷雾电离的技术使蛋白质分子电离，然后根据其质荷比分离。这些数据被记录在光谱上，从中可以确定蛋白质的分子量。然而，除了分子质量信息之外，电喷雾电离质谱还可以揭示关于蛋白质的其他细节，例如蛋白质是否正确折叠以及它是否处于单体或寡聚体形式。我们一直在研究β-2-微球蛋白。在健康人中，β-2-微球蛋白从肾脏排出。如果一个人患有肾衰竭并接受透析治疗，β-2-微球蛋白不会被排出，并形成不溶性纤维，在脚踝，膝盖，臀部，肘部和肩关节中积聚。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β-2-微球蛋白分子混合物的方法;我们还监测了蛋白单体的聚集，并观察到二聚体、三聚体、四聚体等在原纤维形成过程中出现和消失。此外，我们已经开发了通过使用酶从原纤维中去除可接近的蛋白质片段，然后通过质谱法表征这些片段来观察原纤维结构的方法。在这里，我们建议在其自然环境中检查β-2-微球蛋白，即在生理pH值下被生物物种包围，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。

项目成果

Monitoring copopulated conformational states during protein folding events using electrospray ionization-ion mobility spectrometry-mass spectrometry.

• DOI: 10.1016/j.jasms.2007.09.017
• 发表时间: 2007-12
• 期刊: Journal of the American Society for Mass Spectrometry
• 影响因子: 3.2
• 作者: Smith DP;Giles K;Bateman RH;Radford SE;Ashcroft AE
• 通讯作者: Ashcroft AE

Protein misfolding diseases : current and emerging principles and therapies

蛋白质错误折叠疾病：当前和新兴的原理和疗法

• DOI: 10.1002/9780470572702
• 发表时间: 2010
• 期刊: Nature Communications
• 影响因子: 16.6
• 作者: M. Ramirez;J. Kelly;C. Dobson
• 通讯作者: C. Dobson

Elongated oligomers in beta2-microglobulin amyloid assembly revealed by ion mobility spectrometry-mass spectrometry.

通过离子迁移谱-质谱法揭示 β2-微球蛋白淀粉样蛋白组装中的延长寡聚体。

• DOI: 10.1073/pnas.0913046107
• 发表时间: 2010
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Smith DP