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

1032603 Basu智力优点：在蛋白质组学领域，分析分离技术如LC/MS可以分离，定量和鉴定数千种蛋白质;然而，它们不能进行功能测定以确定蛋白质的催化或结合活性。另一方面，分离技术可以将蛋白质分离成级分，同时保留其生物结构，以便它们可以用于功能筛选测定。然而，由于馏分收集技术的限制，制备技术只能分离相对少量的馏分。现有的组分收集器需要机械臂和用于每个组分的专用容器，无法容纳典型蛋白质组中的数千种蛋白质。“全蛋白质组”功能分析的愿景在可扩展的分离技术开发之前无法实现。这代表了许多领域（包括药物研究，系统生物学和工业生物技术）的障碍，其中蛋白质的功能和反应性比其结构或序列更重要。本提案的目的是开发一个高度可扩展的分馏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分馏（micro-fractionation in droplets，简称CFDFD），这是一种“无容器”分馏技术，可以优雅地收集和分离数千个分离的馏分到微尺度液滴中。CFDFD是一种高通量技术，每秒可产生数千个体积小至50 pL的馏分。另一个重要的益处是将液滴级分直接偶联到下游筛选测定的能力。除了我们的初步数据支持，我们已经组建了一个跨学科的团队，包括1）A。Basu（PI），他发表了微滴FD技术，并在基于液滴的微流体方面有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; Kilkuskie是密歇根州高血压筛查中心的主任。该提案将开发概念证明，表明CRFD可以将蛋白质破碎成液滴，并将其与功能性蛋白质测定偶联。3个具体目标包括1）构建用于整合微分级系统与尺寸排阻色谱（SEC-SDFD）的系统; 2）使用SEC-SDFD形成蛋白质文库;和3）在液滴形式的模型酶筛选测定中利用蛋白质文库。这个跨学科的项目创造性地将微流体技术与分离科学结合起来，以解决蛋白质组学中的一个限制性问题。该研究的最终目标是建立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，从而实现上述目标。更广泛的影响：发现蛋白质的功能已经并将继续对许多国家优先领域产生重大影响，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，对高通量筛选产生变革性影响。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，将STEM推广到多种人口，包括K-12学生，代表性不足的少数民族，本科生，女性和20-30岁的“GenXers”。方案包括在底特律科学中心的“纳米日”方案的小学和中学生，通过韦恩国家的SURA和REU方案的少数民族和本科生的研究经验，妇女在底特律科学中心的工程方案，和非正式的科学咖啡馆针对20-30岁的学生和年轻专业人士在底特律大都会地区。

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.