A grant has been awarded to Drs. Tolley and McCarroll at Southern Illinois University to develop an instrument that can systematically identify the proteins in a cell that interact with a specific molecule. These proteins are generally referred to as targets. There is currently no genomic or proteomic approach for performing this type of analysis. Identifying the targets is necessary because there are many molecules that have significant effects on cells, and yet their mode of action and target is unknown. The identification of the target proteins of a molecule is critical to evaluate and understand biological systems. The method to do this was invented by the PIs and is called DIABLA (dynamic isoelectric binding ligand assay). A prototype instrument has been constructed and has demonstrated DIABLA?s great potential and many advantages, but it is labor intensive and not suitable for everyday use. In order to unlock DIABLA?s potential, additional instrumental development must be performed.
Two different instrumental configurations will be constructed as part of this grant award, each having capabilities that the other lacks. The first will be referred to as a discovery system. In this configuration, DIABLA will be able to incrementally search the biological sample for targets and then isolate each target with a very high precision. The targets will then be identified using mass spectrometry. This will not be a rapid analysis, but it will effectively discover and identify new protein targets. The second configuration is called screening mode. Each sample is rapidly separated and analyzed to determine its binding profile. Though each target will not be identified, the overall pattern of targets will be visualized, permitting rapid comparisons between different samples. This binding profile will be able to provide information critical to understanding the biological pathways that the molecule influences. The instrument will have the capability of analyzing eight different samples simultaneously. This could be used to measure the binding of eight different chemicals to a single sample, or to screen eight different tissue types against a single molecule to determine organ-specific effects, for instance. Each analysis would take less than thirty minutes, and future embodiments of the instrument could easily accommodate additional samples.
There are significant broader impacts that the proposed research will have on many research areas. It will be important because it can provide data and perform analyses that are not possible using other methods, enabling future research in understanding RNA splicing, environmental pollutants and other areas where protein interactions need to be identified and studied. The screening capabilities of DIABLA will enable the protein binding patterns from different samples to be rapidly compared. The samples could be from different tissue types, organisms or sub-cellular protein fractions. The binding patterns can then be compared to understand the observed biological effects and help to integrate primary and applied protein research. A derivative of a plasticizer could be tested, for instance, to determine if it has the same binding pattern as known endocrine disruptors. This capability will accelerate development of new products and increase consumer and environmental safety.
