PFI-TT: A highly multiplexed readout system for single-molecule analysis

这个创新-技术转化伙伴关系(PFI-TT)项目的更广泛的影响/商业潜力是开发能够对细胞蛋白质进行深入分析的仪器。这些方法将在基础生物学研究中得到广泛应用。该项目的中心目标是开发和验证研究蛋白质组学的仪器--蛋白质组是构成所有生命形式背后的分子机制的蛋白质集合。基因是细胞器的分子前体，是编码蛋白质在细胞内如何构建的模板。基因组学革命最近使人类基因组的破译成为可能。蛋白质组学技术没有出现过类似的进步，因为基因组研究依赖于基因放大来制作数十亿个相同的拷贝，从而能够进行集体分析。蛋白质不存在类似的分子放大过程，因此单分子分析是关键。这项拟议的研究依赖于来自学术界和工业界的跨物理、工程学、化学、生物学和数学领域的研究人员的合作努力。将寻求可行的、可商业化的仪器的组件技术。该项目的高度协作性和丰富的研究环境为研究生提供了一个机会，他们将参与到这一努力中来获得重要的职业技能。拟议中的项目将开发仪器，以揭开蛋白质生物学惊人的复杂性。单个哺乳动物细胞大约由3B蛋白质组成，涵盖约7000种独特类型，浓度跨度跨度为8个数量级--从仅有几个拷贝的细胞蛋白质，到表达水平为100M量级的蛋白质。在人类血清中，这一范围是1000倍，跨越11个订单。解析蛋白质组成的全谱对于生物学和医学中的基本问题是至关重要的。详细的分析--在种群水平上，但只有单分子分辨率--将对现有的共识类型蛋白质组学方法中丢失的精细细节进行分层，这些方法“平均”于蛋白质种群，从而只提供关于最流行物种的信息。健康和疾病中的关键过程通常只由细胞内蛋白质分子的几个拷贝决定。因此，在大量最普遍的物种存在的情况下对蛋白质和蛋白质复合体进行单分子解析是至关重要的。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

The broader impact/commercial potential of this Partnerships for Innovation - Technology Translation (PFI-TT) project is to develop instrumentation that will enable deep profiling of cellular proteins. The methods will be made broadly applicable in fundamental biological research. The project’s central goal is to develop and validate instrumentation for studying proteomics – the collection of proteins constituting the molecular machinery underlying all life forms. Genes, the molecular precursors of cellular machinery, are templates encoding how proteins are constructed within cells. The genomics revolution has recently enabled decoding of the human genome. Similar advances in proteomics technology has not occurred since genomic studies rely upon gene amplification to making billions of identical copies to enable analysis en masse. No similar molecular amplification process exists for proteins, hence single-molecule analyses are key. The proposed research relies upon the collaborative efforts of researchers spanning physics, engineering, chemistry, biology, and mathematics – from both academia and industry. Component technologies for a feasible, commercializable instrument will be pursued. This project’s highly collaborative and rich research environment provides an opportunity for the graduate student who will be involved in this effort to gain important career skills. The proposed project will develop instrumentation to unravel the staggering complexity of protein biology. An individual mammalian cell comprises roughly 3B proteins, spanning about 7,000 unique types, with concentrations spanning 8 orders of magnitude – ranging from cellular proteins present with just a few copies, to those with expression levels of order 100M. In human blood serum this range is a 1000X greater, spanning 11 orders. Resolving the full spectrum of protein constituents is essential to fundamental questions in biology and medicine. Detailed analyses – at the population level, yet with single-molecule resolution – will stratify fine details lost in existing consensus-type approaches to proteomics that “average over” the protein population, thus providing information only about the most prevalent species. Critical processes in health and disease are often determined by only a few copies of a protein molecule within a cell. Accordingly, single-molecule resolution of proteins and protein complexes in the presence of an immense overabundance of the most prevalent species is essential.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.