Single-molecule proteomics: next-generation analysis of proteins in individual cells

单分子蛋白质组学：单个细胞中蛋白质的下一代分析

• 批准号: BB/W00349X/1
• 负责人: Justin Benesch
• 金额: $ 700.78万
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FW00349X%2F1
• 关键词: Single; molecule; proteomics; next; generation

Proteins, the molecules that work together to enable life, are formed from strings of amino acids and encoded by genes. Although we have about 20,000 genes, there are many more than 20,000 "proteoforms"-altered forms of a protein that can function very differently despite sharing the same amino acid sequence. Post-translational modifications (PTMs) are an important source of these alterations and one of the most common PTMs is phosphorylation-the naturally occurring addition of a phosphoryl group to an amino acid on a protein. There are hundreds of different types of PTMs, and they often co-occur on the same protein; other PTMs involve the addition of sugars (glycosylation), lipids (lipidation) and acetyl groups (acetylation). Due in large part to the complexity arising from PTMs, the field of proteomics - which focuses on identifying and quantifying proteins - has so far struggled in its efforts to fully describe how proteins in a given cell function and work together. Rather, two fundamental limitations to current proteomics strategies have emerged. The first is a reliance on costly and complex technology. The second is the insensitivity of the technology to the complexity and combinatorics of many PTMs, including some forms of phosphorylation. Still, the ability to distinguish different proteoforms and understand the effects of PTMs remains essential. Even if difficult to detect, PTMs affect nearly all proteins, and proteomics is incomplete without them.We propose to transform the capabilities of proteomics by developing a next-generation approach that overcomes the above limitations. Instead of relying on mass spectrometry, the dominant proteomics technology, we will bring together three complementary new technologies. The first, nanopore technology, can be used to infer a protein's amino acid sequence. The second, electrometry, measures electrical charge. The third, mass photometry, measures mass. We will combine these measurements with microfluidics so that we can analyse the protein content of single cells. Our hypothesis is that bringing together the three types of measurements of a given protein (along with existing data about the proteins in the types of cells under study and applying machine learning) will enable identification of individual proteins and detection of their PTMs. We have three main objectives. The first two centre on developing, validating and refining our platform. The third is to apply our approach in bacteria, where the most common forms of phosphorylation tend to be more unstable and difficult to detect using existing proteomics methods. There are critical gaps in our knowledge of phosphorylation in the complex protein networks fundamental for bacterial life. We will study ubiquitous regulatory systems (known as two-component systems) in the opportunistic, disease-causing and increasingly multi-drug resistant bacterial pathogen P. aeruginosa. Our experienced and accomplished team includes the inventors of the three nanometric technologies, who are all based at the University of Oxford's Department of Chemistry. Other team members, based in Oxford, at the University of Liverpool and at the Wellcome Sanger Institute in Cambridge, bring expertise in microfluidics, machine learning, bioinformatics, biochemistry and microbiology, while the input from two supportive companies aligned with our vision will also be welcome. Our platform will make it possible to capture the PTMs that enable the rich complexity of protein function but are currently effectively invisible. This ambitious work will give rise to numerous valuable insights-both during development and once the platform is established. It will transform proteomics research across the life and environmental sciences, may bring economic impacts through commercialisation of the technology, and enhance our knowledge of PTMs' roles in disease, and bacterial virulence and drug resistance.

蛋白质是由一串氨基酸组成并由基因编码的分子，它们共同作用使生命得以存在。虽然我们有大约20，000个基因，但有超过20，000个"蛋白质形式"-蛋白质的改变形式，尽管具有相同的氨基酸序列，但功能却非常不同。翻译后修饰（PTMs）是这些改变的重要来源，并且最常见的PTMs之一是磷酸化-在蛋白质上的氨基酸上天然发生的磷酰基基团的添加。有数百种不同类型的PTM，它们通常共同出现在同一蛋白质上;其他PTM涉及添加糖（糖基化），脂质（脂化）和乙酰基（乙酰化）。在很大程度上，由于PTM带来的复杂性，蛋白质组学领域-专注于识别和定量蛋白质-迄今为止一直在努力充分描述给定细胞中的蛋白质如何发挥功能并共同工作。相反，目前蛋白质组学策略的两个基本限制已经出现。第一个是对昂贵而复杂的技术的依赖。第二是该技术对许多PTM的复杂性和组合不敏感，包括某些形式的磷酸化。尽管如此，区分不同蛋白形式和理解PTM作用的能力仍然至关重要。即使很难检测到，PTM影响几乎所有的蛋白质，蛋白质组学是不完整的，没有他们。我们建议通过开发下一代的方法，克服上述限制，改变蛋白质组学的能力。我们将不依赖于质谱分析，这是占主导地位的蛋白质组学技术，而是将三种互补的新技术结合在一起。第一种是纳米孔技术，可以用来推断蛋白质的氨基酸序列。第二种是电测法，测量电荷。第三种是质量测光，测量质量。我们将联合收割机将这些测量与微流体技术结合起来，这样我们就可以分析单细胞的蛋白质含量。我们的假设是，将给定蛋白质的三种类型的测量（沿着关于正在研究的细胞类型中的蛋白质的现有数据并应用机器学习）结合在一起将能够识别单个蛋白质并检测它们的PTM。我们有三个主要目标。前两个中心是开发、验证和完善我们的平台。第三是将我们的方法应用于细菌，其中最常见的磷酸化形式往往更不稳定，难以使用现有的蛋白质组学方法检测。在我们对细菌生命所必需的复杂蛋白质网络中磷酸化的知识中存在着关键的空白。我们将研究无处不在的监管系统（称为双组分系统）的机会，致病和日益多药耐药的细菌病原体铜绿假单胞菌。我们的团队经验丰富，成就卓著，包括三项纳米技术的发明者，他们都来自牛津大学化学系。其他团队成员来自牛津大学、利物浦大学和剑桥的Wellcome桑格研究所，他们带来了微流体、机器学习、生物信息学、生物化学和微生物学方面的专业知识，同时也欢迎两家支持公司与我们的愿景保持一致。我们的平台将使捕获PTM成为可能，这些PTM能够实现蛋白质功能的丰富复杂性，但目前有效地不可见。这项雄心勃勃的工作将带来许多有价值的见解-无论是在开发期间还是在平台建立之后。它将改变整个生命和环境科学的蛋白质组学研究，可能通过技术的商业化带来经济影响，并提高我们对PTM在疾病，细菌毒力和耐药性中的作用的认识。