BRIGE: Microfractionation in Droplets (µFD) - Linking Proteomic Separations to High Throughput Functional Screening

1032603基本智能优点：在蛋白质组学领域，LC/MS等分析分离技术可以分离、定量和鉴定数以千计的蛋白质；但它们不能执行功能分析来确定蛋白质的催化或结合活性。另一方面，制备技术可以将蛋白质分离成组分，同时保留它们的生物结构，以便它们可以用于功能筛选分析。然而，由于馏分收集技术的限制，制备技术只能分离相对较少的馏分。现有的组份收集器需要机械臂和专门的容器来存放每个组份，无法容纳典型蛋白质组中的数千种蛋白质。只有开发出可扩展的分级分离技术，才能实现“全蛋白质组”功能分析的愿景。这对许多领域(包括制药研究、系统生物学和工业生物技术)构成了障碍，在这些领域，蛋白质的功能和反应性比其结构或序列更重要。这项建议的目的是开发一种高度可扩展的分离技术，所需的全蛋白质组功能筛选。我们的方法是液滴中的微量分馏(？FD)，这是一种“无容器”分馏技术，可以优雅地将数千个分离的馏分收集并分离成微尺度的液滴。？FD是一种高通量技术，每秒可产生数千个分数，体积小至50pl。另一个重要的好处是能够将液滴组分直接耦合到下游筛选分析。除了我们支持性的初步数据外，我们还组建了一个跨学科团队，其中包括1)A.Basu(Pi)，他发表了？FD技术，并在液滴微流体领域拥有8年的经验；2)B.Shay，在分离科学方面拥有15年的工业和学术经验；以及3)R.Kilkuskie，密歇根高通量筛选中心主任。这项提议将开发一种概念证明，表明？fd可以将蛋白质分离成液滴，并将它们耦合到功能蛋白质分析上。这3个具体目标包括：1)建立一个将微分离系统与尺寸排除层析(SEC-FD)相结合的系统；2)使用SEC-FD形成蛋白质文库；以及3)将该蛋白质文库用于液滴形式的模型酶筛选实验。这个跨学科的项目创造性地将微流控技术与分离科学结合在一起，可以解决蛋白质组学中的一个限制性问题。这项研究的最终愿景是一个能够对蛋白质组中的所有蛋白质进行功能分析的系统，从而实现上述愿景。广泛影响：发现蛋白质的功能已经并将继续对许多国家优先考虑的领域产生重大影响，包括生物研究、制药、工业/农业生物技术和替代能源。这项研究还将通过提供一种低成本、高性能的微孔板替代品，在高通量筛选方面产生革命性的影响。作为Brige计划的一部分，PI制定了一个多方面的教育推广计划，向多个人口群体推广STEM，包括K-12学生、代表不足的少数族裔、本科生、女性和20-30岁的“GenXers”。计划包括底特律科学中心为中小学生提供的“Nanoday”计划，通过韦恩州立大学的SURA和REU计划为少数族裔和本科生提供的研究体验，底特律科学中心工程项目的女性，以及底特律大都会地区面向20-30岁学生和年轻专业人员的非正式科学咖啡馆。

1032603BasuIntellectual Merit: In the field of proteomics, analytic separation techniques like LC/MS can separate, quantify, and identify thousands of proteins; however, they cannot perform functional assays for determining the catalytic or binding activity of the protein. Preparative techniques, on the other hand, can isolate proteins into fractions while preserving their biological structure so that they can be used in functional screening assays. However, preparative techniques can only isolate relatively small number of fractions due to limitations in fraction collection technology. Existing fraction collectors, which require robotic arms and dedicated containers for each fraction, cannot accommodate the thousands of proteins in a typical proteome. The vision of "whole-proteome" functional assays cannot be realized until a scalable fractionation technology is developed. This represents a barrier to many fields (including pharmaceutical research, systems biology, and industrial biotechnology) where the function and reactivity of a protein is more important than its structure or sequence. The objective of this proposal is to develop a highly scalable fractionation technology needed for whole-proteome functional screening. Our approach is micro-fractionation in droplets (ìFD), a "containerless" fractionation technique which can elegantly collect and isolate thousands of separated fractions into microscale droplets. ìFD is a high throughput technology which can generate thousands of fractions per second with volumes as small as 50 pL. Another important benefit is the ability to couple the droplet fractions directly to a downstream screening assay. In addition to our supportive preliminary data, we have assembled an interdisciplinary team, including 1) A. Basu (PI), who has published the ìFD technique and has 8 years experience in droplet-based microfluidics; 2) B. Shay, with 15 years industrial and academic experience in separation science; and 3) R. Kilkuskie, director of the Michigan High Throughput Screening Center. This proposal will develop a proof of concept showing that ìFD can fractionate proteins into droplets and couple them to a functional protein assay. The 3 specific aims include 1) Build a system for integrating the microfractionation system with size exclusion chromatography (SEC- ìFD); 2) Form a protein library using SEC-ìFD; and 3) Utilize the protein library in a model enzyme screening assay in droplet format. This interdisciplinary project creatively couples microfluidic technology with separation science in a way that can address a limiting problem in proteomics. The ultimate vision for this research is a system which can perform functional assays on all the proteins in a proteome, thereby realizing the vision described above.Broader Impact: Discovering the function of proteins has had and will continue to have substantial impact on many areas of national priority, including biological research, pharmaceuticals, industrial/agricultural biotechnology, and alternative energy. This research will also have transformative impact in high throughput screening by providing a low cost, high performance alternative to microplates. As part of the BRIGE program, the PI has developed a multi-faceted educational outreach plan which promotes STEM to multiple demographics, including K-12 students, underrepresented minorities, undergraduates, women, and 20-30 year old "GenXers". Programs include the "Nanodays" program at the Detroit Science Center for elementary and middle school students, research experiences for minorities and undergraduates via Wayne State's SURA and REU programs, women in engineering programs at the Detroit Science Center, and informal Science Cafes targeted towards 20-30 year old students and young professionals in the metro Detroit area.