ALACATS: bespoke solutions for absolute protein quantification

著名的生物化学家亚瑟·科恩伯格（Arthur Kornberg）因其在DNA从一个细胞复制到另一个细胞、一代又一代的机制方面的研究而获得诺贝尔奖。但是，他敏锐地意识到，虽然DNA（以及相关的RNA）是蓝图，但生命的真正使命在于核酸蓝图中所描述的机器的行为，即蛋白质。为了真正理解生命过程，我们需要对蛋白质世界有详细的定量了解。正如基因组学领域改变了我们对DNA的认识一样，"蛋白质组学"领域也极大地促进了我们对蛋白质世界的理解。蛋白质组学的核心技术是基于精密的质谱仪，能够精确地分析百万分之一克的肽（我们使用肽作为其母体蛋白质的代理分子）。但是，尽管质谱仪很复杂，但它们都有一个固有的局限性-它们对来自同一蛋白质的不同肽给出不同的信号强度，即使它们的量相同。然而，为了理解细胞是如何表现其所包含的蛋白质的，我们需要能够准确测量任何一种蛋白质有多少拷贝。为了克服这一限制，我们使用准确已知的标准品，这些标准品由质谱仪共同分析，其优点是标准品和真正的细胞蛋白质可以分别测量，因为我们将标准品设计成"更重"，从而在质谱仪中可辨别。因此，如果我们加入1000个标准品分子，而细胞成分给我们的信号是两倍大，我们可以自信地断言样品中含有2,000个该蛋白质的拷贝。大约12年前，我们发明了一种新方法来产生大量的定量蛋白质组学标准品。我们创造了新的“设计师蛋白质”，这在地球上是前所未有的，可以通过简单的细菌生产来制造。这些人工蛋白质中的每一个都包含了多达50种蛋白质的肽标准品，因为这些蛋白质是根据其编码的蛋白质预先设计的，这意味着它们并不总是完美地适应单个科学家的需求。我们需要的是“构建自己的”设计蛋白质的能力。在这个建议中，我们设计了一种方法来做到这一点。在未来，无论感兴趣的系统是什么，科学家都能够“拨号”他们感兴趣的蛋白质，我们将能够组装，“点菜”蛋白质标准。我们将创建一个构建模块库（我们称之为“Qbricks”，是“量化生物砖”的缩写），并使用先进的合成生物学DNA操作方法，我们将能够在两天内创建完美的标准蛋白质。我们称之为"ALACAT"是因为"点菜"的设计理念。这是绝对定量蛋白质组学的革命性方法，对增强我们对蛋白质世界的理解具有巨大潜力。该项目将建立核心技术和方法，并建立一套Qbricks，用于为蛋白质组学社区创建标准和研究工具。我们将展示ALACAT理念如何作为一种技术资源开发，易于被许多研究小组借鉴，从而以可持续的方式实现广泛的一系列研究计划。

A famous biochemist, Arthur Kornberg, won the Nobel Prize for his work on the mechanisms by which DNA copies itself from cell to cell, generation to generation. But, he was acutely aware that whilst DNA (and by association, RNA) are the blueprint, the true vocation of life lies in the actions of the machines that are described in the nucleic acid blueprint, the proteins. To truly understand living processes, we need to gain a detailed quantitative understanding of the protein world. And, just as the field of genomics has transformed our knowledge of DNA, so an equivalent field of 'proteomics' has hugely advanced our understanding of the protein world.The core technology in proteomics is based on sophisticated mass spectrometers, capable of analysing one million millionths of a gram of peptide in exquisite detail (we use peptides as the proxy molecule for their parent proteins). But sophisticated as they are, mass spectrometers all have one intrinsic limitation - they give different signal intensities for different peptides from the same protein, even though they are in the same amount. Yet, to understand how a cell is the manifestation of the proteins it contains, we need to be able to measure exactly how many copies of any one protein there are. To overcome this limitation, we use accurately known standards that are co-analysed by the mass spectrometer, with the advantage that the standard and true cellular protein can be separately measured because we engineer the standard to be 'heavier' and thus discernible in the mass spectrometer. Thus, if we add 1000 molecules of a standard, and the cell component gives us a signal that is twice as large, we can confidently assert that the sample contains 2,000 copies of that protein.About 12 years ago, we invented a new method to generate large numbers of standards for quantitative proteomics. We created new 'designer proteins', never seen before on the planet, that could be made, in heavy form, by simple production in bacteria. These artificial proteins each contained peptide standards for up to 50 proteins.Because these proteins were pre-designed in terms of the proteins that were encoded within it, it meant that they were not always perfectly tuned to the needs of individual scientists. What was needed was the ability to 'build your own' designer protein. In this proposal, we have devised a way to do exactly this. In future, no matter what the system of interest, scientists will be able to 'dial up' their interesting proteins, and we will be able to assemble, 'a la carte' a protein standard. We will create a library of building blocks (we call them 'Qbricks', short for 'Quantification biobricks') and using advanced synthetic biology methods of DNA manipulation we will be able to create, in two days, the perfect standard protein for their research. We call these 'ALACATs' because of the 'à la carte' design philosophy.This is a revolutionary approach to absolute quantitative proteomics, and has huge potential to enhance our understanding of the protein world. This project will establish the core technology and methodologies, and build a set of Qbricks that will be used to create standards and research tools for the proteomics community. We will show how the ALACAT philosophy can be developed as a technical resource, readily drawn upon by many research groups, and thus, enabling a broad series of research programmes in a sustainable fashion.