Characterizing complex protein states with next generation HX-MS concepts

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This proposal describes new concepts in hydrogen/deuterium exchange mass spectrometry (HX-MS) for characterizing highly-complex protein systems relevant to drug development activities in pharmaceutical research programs. HX-MS provides conformational data that link protein structure to protein function. Conceptually, HX processes offer an excellent opportunity to generate maps of structure-activity relationships and support the selection of leads in early stages of drug research. However, the complexity of available HX-MS technology has restricted its use in this context. Conventional solutions have low spectral capacity and under-developed data analysis routines, limiting HX-MS to simpler protein systems and low throughput. The research proposed here will overcome these barriers by developing a new, scalable nanoHX-MS^2 capability, supported by informatics solutions that evaluate the high volume of data for underlying structure-activity relationships. We will build on recent HX innovations from my laboratory, combined with new QTOF-based mass spectrometry technology from Sciex, a leading technology provider in the analytical instruments market. The major challenge for the research program will be to profile ultracomplex protein complexes or networks without loss of sequence coverage, and remove the need for manual intervention in both data collection and analysis. Through effective use of the MS^2 dimension, and the development of new processing reagents and informatics strategies, we anticipate a platform that can generate complete HX maps of protein states at least 10-fold more complex than current best capabilities. The platform will be developed with the active participation of pharmaceutical partners and benefit their drug development efforts. Pfizer will provide test scenarios in the form of complex protein-ligand systems, and Genentech will provide therapeutic proteins to support the development of a derivative mode of HX-MS^2 operation targeting the biologics market. Together, we anticipate that our research will address the key barriers to implementing HX-MS in a wider pharmaceutical R&D market, and shorten the path to commercializing systems, software and reagents.