IDBR: TYPE A: Automated protein analysis: Gel-to-Mass Spectrometer Coupling Device

Carnegie Mellon University, Pittsburgh PA

Investigators

Abstract

An award is made to Carnegie Mellon University to advance the field of protein discovery for basic biological and medical research. Cells contain thousands of different types of proteins. The goal of comparative proteomics is to discover important proteins that differ between cells, tissues and organisms grown under different conditions, with different genetic backgrounds, or at different stages of development or disease. These "difference-proteins" represent what makes these cells behave differently under different circumstances. Because cells contain thousands of proteins, individual proteins are routinely separated from one another in thin "Jello"-like slabs by a process called gel electrophoresis, which allows us discover the difference-proteins. To figure out what genes encode the proteins-of-interest, the proteins need to be removed from the gel and injected into a mass spectrometer, a machine that very accurately determines the identity of the protein. There is a severe bottleneck in moving proteins from the gel to the mass spectrometer. This project will build a device to greatly increases the speed and efficiency of this transfer process. A key element of this project is to introduce undergraduate students to these complex, highly technical experiments. The goal is to build the students' identity as professional scientists. The unique feature of this program is that the students work as an integrated team to work on collaborative projects. They progress from trainees to trainers as they move through their academic careers. They learn valuable team management and communication skills. All of the students graduating from this group to date have gone onto STEM careers. The goal of this project is to develop a device whose purpose is to couple the transfer of proteins out of electrophoresis gels, to enzymatically digest the isolated proteins into peptides, and to the transfer the resultant peptides into a mass spectrometer (MS) for protein identification. We will develop an automated device for enhanced protein identification that will enable the identification of proteins isolated from electrophoresis gels down to 0.1 fmol (10-16 moles), representing a 100-fold improvement over existing methods. In addition, this automated system will accelerate the entire process from 24-48 hours per sample to about 1 hour. Thus enabling researchers to perform their proteomics experiments with far greater sensitivity and speed. This will be accomplished by developing a disposal tip for electrophoresing proteins-of-interest out the electrophoresis gel. The isolated proteins will be digested into peptides within the tip. The peptides will then be introduced into the MS through a microfluidic device. This device will benefit any biologist doing MS identification of proteins isolated from electrophoresis gels.