Novel ultra-stable enzymes and methods for proteomics

PROJECT SUMMARY/ABSTRACTThe highly technical and rapidly advancing field of modern proteomics relies heavily on enzyme technologies that were established over one hundred and fifty years ago, namely trypsin. We propose to apply our entirely novel ultra-stable Archaeal proteases to make superior front-end digestion products for proteomics. Our patented technologies allow efficient production of ultra-stable enzymes with optima near the boiling point of water at highly acidic pH with half-lives on the order of weeks, not hours. The hot-acid conditions of CinderBio enzymatic digest reactions denatures target proteins and allows faster and more effective target digestion without added chaotropes like urea. Our hyperthermoacidic enzymes generate protein coverage and protein identification metrics that are competitive with overnight trypsin/urea digests with less sample, fewer steps, and five-minute reaction times. Here we propose to build on over a decade of our work with these enzyme systems to commercialize proteomic digestion products with novel capabilities and cleavage specificities to empower proteomic research. We have established academic and corporate partnerships to facilitate this work to expedite product development and deployment of our novel capabilities into the proteomics markets within the next two years. With the astonishing rate of technical advances in proteomics it is remarkable that front-end enzymatic digests have changed very little since the beginning of the field. Here we propose to bring to market novel enzyme products and capabilities to advance proteomic capabilities, reproducibility, and throughput with our efficient proteomic digestion products.PROJECT NARRATIVE ! Proteomics is critical for health research as it enables analysis of proteins involved in a broad range of biological processes. Although proteomics has laid the foundation for many advances in disease diagnosis, treatment, and prevention, improvements in sample preparation have lagged far behind impressive developments in mass spectrometry technologies. Here we propose to bring an entirely new class of ultrastable enzymes and their capabilities to the forefront of proteomics research. We think these advances will not only address known protease limitations, but also expand the scope and effectiveness of proteomic analyses for biological and medical research.