Mass spectrometry (MS) based protein footprinting is a new addition to structural biology tool box. The sensitivity and speed of MS provide a bridge between low resolution methods, like circular dichroism, and high resolution methods, like NMR and X-ray crystallography. Footprinting modifies proteins in biologically relevant environments. The modified peptides can be qualitatively and quantitatively analyzed by MS to generate maps of the solvent accessibility of target proteins. This view of the protein structure at the peptide level is sufficient to determine protein conformational changes introduced by the functional switch of proteins and can be investigated without limitations of purity and size. Two approaches are deuterium (hydrogen deuterium exchange, or HDX) and hydroxyl radical labeling (fast photochemical oxidation of proteins, or FPOP). Both of them can label more than half of the amino acids in biologically relevant environments. FPOP can monitor very fast events, down to a microsecond. Phenomena such as fast protein folding, conformational changes, and aggregation can now be probed. In addition, other radicals that react with specific residues will be developed. Combining HDX with MS allows much deeper investigation. Titration-based HDX will be exploited to provide protein-ligand binding affinities. Pulsed HDX will be developed to look at challenging protein aggregates such as amyloid beta (implicated in Alzheimer's Disease). Both technologies will be pushed to determine if they can provide information at the amino acid residue level.
-Public Health Relevance. The Washington University Biomedical Mass Spectrometry Resource has a longstanding history as an active and productive citizen in the NIH Biotechnology Research Resources community. We propose to extend our mission by advancing mass spectrometry technology, development, and research, applying these discoveries to answer critical biomedical research questions, and training the next generation of researchers, towards the ultimate goal of improving public health.
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