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

蛋白质是由一串串氨基酸组成，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，但还有远远超过20,000个“蛋白质形式”--一种蛋白质的不同形式，尽管它们拥有相同的氨基酸序列，但它们的功能却截然不同。翻译后修饰(PTM)是这些改变的一个重要来源，最常见的PTM之一是磷酸化--蛋白质上的氨基酸自然地增加了一个磷酸基。有数百种不同类型的PTM，它们经常在同一蛋白质上共存；其他PTM涉及添加糖(糖基化)、脂类(脂基化)和乙酰基(乙酰化)。在很大程度上，由于PTMS引起的复杂性，蛋白质组学领域--侧重于识别和量化蛋白质--迄今在努力充分描述特定细胞中的蛋白质如何发挥功能和相互作用方面遇到了困难。相反，目前的蛋白质组学策略出现了两个根本性的限制。首先是对昂贵而复杂的技术的依赖。第二个问题是该技术对许多PTM的复杂性和组合性不敏感，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PTMS的影响的能力仍然是必不可少的。即使很难检测，PTMS几乎影响所有的蛋白质，没有它们蛋白质组学是不完整的。我们建议通过开发一种克服上述限制的下一代方法来改变蛋白质组学的能力。我们将把三项互补的新技术结合在一起，而不是依赖于占主导地位的蛋白质组学技术--质谱学。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，用来测量电荷。第三种是质量光度法，用来测量质量。我们将把这些测量与微流控技术结合起来，这样我们就可以分析单个细胞的蛋白质含量。我们的假设是，将给定蛋白质的三种类型的测量结果(以及关于所研究的细胞类型中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其PTM。我们有三个主要目标。前两项工作的重点是开发、验证和完善我们的平台。第三是将我们的方法应用于细菌，在细菌中，最常见的磷酸化形式往往更不稳定，使用现有的蛋白质组学方法很难检测到。在对细菌生命至关重要的复杂蛋白质网络中，我们对磷酸化的了解存在着严重的空白。我们将研究在机会主义、致病和日益耐药的铜绿假单胞菌中普遍存在的调控系统(称为双组分系统)。我们经验丰富的团队包括三项纳米技术的发明者，他们都在牛津大学的化学系工作。其他团队成员在牛津、利物浦大学和剑桥的惠康·桑格研究所工作，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，同时也欢迎两家与我们的愿景保持一致的支持公司的投入。我们的平台将使捕获PTM成为可能，这些PTMS使蛋白质功能的丰富复杂性成为可能，但目前实际上是不可见的。这项雄心勃勃的工作将产生许多有价值的见解--无论是在开发过程中，还是在平台建立之后。它将改变生命和环境科学中的蛋白质组学研究，可能通过技术商业化带来经济影响，并增强我们对PTMS在疾病中的作用以及细菌毒力和耐药性的知识。

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.