(including UNDERLYING PREMISE and STRATEGY to ENSURE ROBUSTNESS): Our premise is that developing a better method for analyzing proteomes is required to take full advantage of the technological breakthroughs that have occurred in the last 15 years in DNA sequencing. We believe that the difficulties associated with previous proteome analysis methods can be overcome by using an entirely different approach. We propose to test this premise by obtaining affinity reagents to N-terminal amino acids that can be used in a new, massively-parallel method for protein sequencing. Our strategy to ensure a robust, unbiased and reduced risk approach is to develop in parallel the directed evolution of two protein scaffolds and compare them to commercially available and naturally-occurring IgG affinity reagents. We have chosen methods that are extremely well- established in our lab: phage display screening in both bulk and emulsions, phage display library construction, and affinity maturation. The two scaffolds for display include lipocalins and scFv Abs. While not discussed in the proposal, if there is time remaining or if a specific need arises, we may try other scaffolds.
(and OVERALL IMPACT): The overall impact of RNA sequencing and digital counting is significant in biological research. But RNA molecule counting can only be used as a surrogate for protein abundance. That results in a couple of outstanding problems. In many cases there appears to be a limited correlation for RNA levels and protein abundance. And RNA abundance gives absolutely no information with regard to post-translational modification of proteins that is so important to cell signaling and other biological processes. We believe the proposed development of a ProCode system will significantly impact many areas of science, just as NextGen sequencing has done for genome analysis. There are several components to the proposed system: (1) development of the assay, including proprietary reagents (the focus of this proposal), (2) instrumentation, and (3) software, which includes instrumentation control and bioinformatics. This proposal will focus on component (1), the development of the binding reagents needed for the ProCode assay. Specifically, we will describe the experiments needed to generate the N-terminal amino acid (NTAA)-recognizing affinity reagents for use in the ProCode assay.