Probing biomolecular interactions by combining ETD-tandem mass spectrometry with chemical footprinting methodologies.

在生命中，大多数蛋白质是由蛋白质、核酸、小分子和金属离子组成的生物分子复合体的组成部分，所有这些都在特定的3D结构中结合在一起。功能正常的蛋白质在植物和动物中维持健康，后者包括人类。然而，蛋白质功能失调可能导致疾病：例如，不必要的蛋白质自我聚集可能会产生不可溶的淀粉样斑块，与阿尔茨海默氏症、帕金森氏症和II型糖尿病等众所周知的疾病有关。我们建议购买一台最先进的质谱仪，它将使我们能够定位蛋白质-蛋白质和蛋白质-配体结合位点。我们的策略包括对天然(3D)构象的蛋白质或蛋白质复合体进行化学修饰(或“足迹”)技术。将被化学修饰的蛋白质区域是暴露在其结构外部的那些区域，而位于结构内部的区域是不可访问的，并且保持不变。为了确定化学修饰的位置，我们需要使用先进的质谱学技术，它不仅可以告诉我们蛋白质的分子质量，而且还可以告诉我们它的氨基酸序列。在蛋白质测序过程中，由于化学修饰导致残基质量的变化，哪些氨基酸发生了明显的修饰。例如，如果氨基酸被氧化，它将获得一个氧原子，并相应增加16Da。我们提出的先进的质谱学与化学修饰方法的结合将提供有关以下方面的新信息：a.通过揭示结构表面的哪些区域和结构内部的哪些区域来揭示蛋白质的3D结构；b.蛋白质-蛋白质和蛋白质-配体结合位置，通过比较蛋白质单独的“足迹”与结合到小分子或另一种蛋白质上的蛋白质的足迹，我们可以辨别配体的结合位置；c.蛋白质的功能。我们可以随着时间的推移监测蛋白质的化学足迹，以绘制发生的任何结构变化，例如展开、结合、自我聚集。这将为蛋白质的功能和错误功能提供关键的见解；d.潜在疗法的设计。为了防止蛋白质功能失常，通常可以添加小分子药物来防止不想要的事件，例如，为了防止蛋白质在自我聚集后展开，可以添加一个小分子，它将成功竞争蛋白质-蛋白质结合位点。我们的方法将定位结合部位。这些拟议研究的受益者将是生物科学家，他们将对蛋白质和其他生物分子的功能和错误功能的方式有显著的了解。这将导致制药和生物制药公司改进药物设计，进而为我们所有人带来更健康的生活。

In life, most proteins function as an integral part of a biomolecular complex consisting of proteins, nucleic acids, small molecules and metal ions, all held together in a specific 3D architecture. Correctly functioning proteins maintain health in plants and animals, the latter including humans. However, protein mis-function can lead to disease: for example, unwanted protein self-aggregation can produce insoluble amyloid plaques which are associated with well-known diseases such as Alzheimer's, Parkinson's and Type II diabetes. We propose to purchase a state-of-the-art mass spectrometer which will enable us to pin-point protein-protein and protein-ligand binding sites. Our strategy involves subjecting a protein, or protein complex, which is in its native (3D) conformation, to chemical modification (or "footprinting") techniques. The regions of the protein that will be modified chemically are those which are exposed on the exterior of its structure, whilst the regions lying in the interior of the structure are inaccessible and remain unmodified.In order to identify the sites of chemical modification, we need to use advanced mass spectrometric techniques that will tell us not only the molecular mass of the protein, but also its amino acid sequence. During protein sequencing, it is apparent which amino acids have been modified due to the change in the mass of the residue due to the chemical modification. For example, if the amino acid is oxidised, it will gain an oxygen atom and a corresponding increase of 16 Da. Our proposed combination of advanced mass spectrometry with chemical modification methods will provide new information concerning: a. the 3D structure of the protein by disclosing which regions are on the surface and which are in the interior of the structure; b. protein-protein and protein-ligand binding sites, by comparing the "footprint" of the protein alone with that of the protein bound to a small molecule or another protein, we can discern where the ligand is bound; c. protein function. We can monitor the protein's chemical footprint over time to map any structural changes occurring e.g. unfolding, binding, self-aggregation. This will provide key insights into how proteins function and mis-function; d. the design of potential therapeutics. To prevent a protein from mis-functioning, it is often possible to add a small molecule drug to prevent an unwanted event e.g. to prevent a protein from unfolding followed by self-aggregation, it is possible to add a small molecule which will compete successfully for the protein-protein binding site. Our methods will locate the binding site.The beneficiaries of these proposed studies will be the bio-scientists who will gain a significantly improved understanding of the ways in which proteins and other biomolecules function and mis-function. This will lead to improved drug design by pharmaceutical and biopharma companies, which in turn will lead to a healthier life for all of us.