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

Wayne State University, Detroit MI

Investigators

Abstract

1032603 Basu Intellectual 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.