New Detection Methods for Biomarkers of Protein Oxidation

该项目旨在开发可用于生物样品的检测蛋白质损伤的改进方法。蛋白质对身体的正常运作是必不可少的。它们会被活性化学物质破坏，如自由基或氧的衍生物。这些导致蛋白质的化学氧化，破坏其结构，从而破坏其功能。这被认为是各种疾病的重要事件，特别是神经退行性疾病和其他与衰老有关的疾病。在使用蛋白质和酶的工业过程中，它也可能是一个问题。因此，能够准确测量蛋白质氧化，并确定已被损坏的精确蛋白质（体内有数千种不同的蛋白质，甚至在单细胞中）以及已发生的损伤的确切性质是很重要的。后者很重要，因为不同的破坏性化学物质对蛋白质有不同的影响，因此蛋白质可以告知破坏性化合物的存在，这可能对理解疾病至关重要。检测蛋白质氧化的最常见方法是测量所有氧化蛋白质的一般特征的非特异性方法。最近，这些方法已经与识别单个蛋白质的方法（类似于指纹）相结合，因此只有被氧化损伤的蛋白质才能被检测和识别。通常，这仍然不能提供关于蛋白质如何被破坏的确切信息。或者，蛋白质（或生物样品中的蛋白质混合物）可以被分解成它们的单个组成部分（氨基酸），这些可以被测试以查看哪些被损坏。这种方法通常用于寻找血清或尿液中蛋白质损伤的证据。这允许观察到某些破坏性化学物质的特定类型的损伤特征，但是如果使用蛋白质的混合物，则无法精确定位遭受损伤的蛋白质。该项目将开发涉及质谱法（可以测量蛋白质或氨基酸的重量）的方法，以确定已识别蛋白质中的特定损伤。这将涉及结合蛋白质的“指纹”，已经是一种常规方法，将蛋白质分解为氨基酸并检测受损的氨基酸。在某些情况下，为了提供特定类型损伤的特异性标记，可能需要进一步分解氨基酸。为此，将设计新的机器程序和协议。首先，它们将在单个氨基酸和蛋白质上进行测试，然后在复杂的混合物上进行测试，以表明这些方法将足以检测临床或生物样品中的特定损伤。质谱仪将被设置为寻找氧化损伤的“标记”，如果发现任何标记，将进行测试以识别它们来自的蛋白质。其目的是设计一种方法，可以一次查找许多不同类型的损坏，从而在一个单一的测试方法中提供更多的信息。本提案中描述的工作非常重要，因为它将提供更有效和信息量更大的蛋白质损伤测量。最初，这将对生物科学研究人员有很大的帮助，他们试图了解蛋白质何时受损，以及这是如何在身体或工业过程中引起问题的。随后，经过更多的测试，当更好地了解蛋白质氧化在疾病中的作用时，它可能会作为相关疾病的筛查方法。

This project aims to develop improved methods for detecting protein damage that could be used in biological samples. Proteins are essential for the correct functioning of the body. They can be damaged by reactive chemicals, like free radicals or derivatives of oxygen. These cause a chemical oxidation of the protein, and disrupt its structure, and consequently its function. This is thought to be an important event in various diseases, especially neurodegenerative diseases and other diseases linked to ageing. It can also be a problem in industrial processes where proteins and enzymes are used. It is therefore important to be able to measure protein oxidation accurately, and identify both the precise protein that has be damaged (there are many thousands of different ones in the body, and even in single cells), and the exact nature of the damage that has occurred. The latter is important because different damaging chemicals have different effects on the proteins, so the protein can inform about the presence of damaging compounds, which may be critical in understanding a disease. The most common ways of detecting protein oxidation are non-specific methods that measure generic characteristics of all oxidized proteins. More recently, these approaches have been combined with methods of identifying individual proteins (analogous to fingerprinting), so that only proteins that have been damaged by oxidation are detected and identified. Usually this still doesn't give information about exactly how the protein has been damaged. Alternatively, proteins (or mixtures of proteins in biological samples) can be broken down into their individual component parts (amino acids) and these can be tested to see which are damaged. This method is often used to look for evidence of protein damage in serum or urine. This allows particular types of damage characteristic for certain damaging chemicals to be observed, but the protein that suffered the damage cannot be pinpointed if a mixture of proteins is used. This project would develop methods involving mass spectrometry (which can measure the weight of proteins or amino acids) to identify specific damage in identified proteins. This will involve the combination of 'fingerprinting' of proteins, already a routine method, with breakdown of the proteins to amino acids and detection of damaged amino acids. In some cases, to provide a specific marker for a particular type of damage, it might be necessary to breakdown the amino acids further. To do this, new machine procedures and protocols will be designed. To start with, they will be tested on individual amino acids and proteins, and then on complex mixtures, to show that the methods will be powerful enough to detect specific damage in clinical or biological samples. The mass spectrometer will be set up to look for 'markers' of damage by oxidation, and if it finds any, to run a test to identify the protein they came from. The aim is to design methods that can look for a number of different types of damage all at once, thus providing much more information in a single test method. The work described in this proposal is very important because it will provide much more efficient and informative measurements of protein damage. Initially this will be a great help to researchers in biological sciences who are trying to understand when proteins become damaged and how this causes problems in the body or in industrial processes. Subsequently, after more testing and when a better understanding of the role of protein oxidation in disease is reached, it could be useful as a screening method for relevant diseases.