Caldera Pharmaceutical's aim is to develop a broadly applicable, quantitative research tool which addresses core technical challenges in the field of proteomics, including protein-drug interactions, protein-protein interactions, and post-translational modifications. We propose to create a new proteomics technology involving a novel X- ray translucent flow cell design. If our aims are achieved, more accurate, sensitive, and affordable, high-throughput proteomics methods will be available. Our goals are: 1. recognition of protein interactions, and 2. characterization of post- translational modifications. Our long-term objective is to solve key and fundamental problems with proteomic technologies, as described by the world's leading scientists. There is a desperate public health need for proteomics, quantitative chemistry, pharmacogenetics, and others to contribute to reducing the incidence of adverse treatment reactions, discovering diseases earlier, and optimizing and individualizing treatments. We intend to focus on the development of technologies that will permit observations of unaltered small molecules which are quantitative and made in real time. Real time measurements can be made using Caldera's technology because reactions are performed in solution. Caldera Pharmaceuticals, in collaboration with Los Alamos National Laboratory (LANL), developed Reagentless Pharmacoproteomic Measurement (RPM), a patented technology which uses micro-X-ray fluorescence to quantify the binding of drugs to proteins. It is a unique label-free system. Currently, the binding kinetics of only a few proteins and their inhibitors can be measured accurately. Most proteins do not have a readily measurable reaction product (as is the case for protein kinases), so these proteins remain very difficult to test. Thus, even though tethering a protein [as with surface plasmon resonance (SPR) systems] can lead to incorrect binding constants by orders-of-magnitude, most pharmaceutical companies have purchased SPR instruments. With Caldera's proposed innovation, researchers will have a major technological advantage, for the first time; truly label-free binding measurements available for all proteins and most drugs. Furthermore, our technology has the potential to provide benefit to all biomedical and pharmaceutical research which involves drug to protein binding. The proposed project is a method and apparatus for rapidly characterizing proteins for their binding properties and post-translational modifications. This characterization will be achieved using X-ray fluorescence measurements of proteins, their post-translational modifications, and any binding partners (drugs, proteins, co-factors). These measurements use native, unmodified proteins and reagents without the need either to label the drug or to tether the protein. This measurement technique eliminates the experimental biases introduced by existing technologies. The primary experimental biases to be removed are labeling and albumin elimination. The significance of Caldera's innovative tool is that determining protein characterization currently requires significant sample preparations. For example, measuring drug-protein binding kinetics has previously required labeling of the drug and/or tethering of the protein to a surface. Both of these procedures render kinetic data unreliable or incorrect (slower rates and other constants off by as much as four orders of magnitude). Funding will allow Caldera to optimize a tool that has the potential to address proteomics research for many public health problems. Our long-term objective is to solve key and fundamental problems with proteomic technologies, as described by the world's leading scientists. After the completion of the human genome project, it became apparent that secrets to better healthcare lie in the field of proteomics. The phrase, """"""""one gene: one protein"""""""" appears to be simplistic; sequencing of the human genome has led to estimates of approximately """"""""30,000 genes, yielding more than 100,000 transcripts, resulting in more than 1,000,000 different protein molecular species."""""""" There is a desperate public health need for proteomics, quantitative chemistry, pharmacogenetics, and others to contribute to reducing the incidence of adverse treatment reactions, discovering diseases earlier, and optimizing and individualizing treatments. With Caldera's proposed innovation, researchers will have a major technological advantage, for the first time; truly label-free binding measurements available for all proteins and most drugs! Furthermore, our technology has the potential to provide benefit to all biomedical and pharmaceutical research which involves drug to protein binding. ? ? ?
