In the past few years, Next generation sequencing (NGS) has made rapid advances, enabling unprecedented experimental potential in cancer research. With substantial reduction in cost and significant increase in throughput and accuracy, many researchers and clinics are utilizing this technology to find answers to critical challenges in cancer biology, diagnosis and therapeutics. On the other hand, the field of proteomics plays a vital role in disease etiology and treatment. One of the major challenges in biology is elucidation of physiological function of all proteins. In the field of functional proteomics, new high-throughput (HT) methods to study protein function will enable to discover novel interacting partners, biochemical pathways and undiscovered therapeutic hits, a critical need in cancer research. Integrating the potential of NGS with high-throughput functional proteomics could revolutionize cancer research by converting information about protein interactions, protein-drug profiling and early cancer screening into genetic information that can be measured in a massively parallel manner. In order to exploit this unprecedented experimental potential we propose to develop Multiplexed In Solution Protein Array (MISPA) with broad utility in cancer biology and diagnostics. The crux of this method includes barcoding individual proteins, which can then interact in solution with a query (i.e., another protein, biological fluids, drug molecules etc) and assessed quantitatively by NGS. One of the important pre-requisite for this approach is the need for large collections of full length cDNAs of human genes (and other organisms) in a format compatible to HT protein expression. We have ~ 13,000 full length human genes along with large collections for other organisms including many pathogenic viruses and bacteria in our DNASU plasmid repository that can be easily transferred into any protein expression system through HT Gateway cloning, giving us the benefit of exploring many different combinations of proteins in MISPA platform. The proposed platform has many advantages: proteins in our cocktail mix will be produced in a human milieu, preserving the functionality; fresh proteins will be produced within two hours in equimolar quantities; solution phase kinetics will facilitate efficient binding of targets to the query molecule with high sensitivity to detect strong and weak interactions; expanded dynamic range and massively parallel quantitative profiling; multiplexed readouts and easy implementation into clinical diagnostics. We will demonstrate the effectiveness of this approach by 1). investigating protein-protein interactions in the B-cell receptor (BCR) pathway, a highly targeted pathway in cancer and 2). implementing a quantitative, multiplexed, robust, clinical assay for early detection of oropharyngeal carcinomas.
|Effective start/end date||8/15/15 → 7/31/18|
- HHS-NIH: National Cancer Institute (NCI): $672,075.00