Protein footprinting by mass spectrometry: H/D exchange, specific amino acid labeling, and fast photochemical oxidation of proteins

An emerging approach for analyzing protein structure is mass spectrometry-based footprinting. Here, a footprint is installed within a protein by executing a chemical reaction. Often this is accomplished in a differential manner where one compares the footprint of one state (wild-type, unbound) with another (mutant, bound). The approach is also applicable to one state where the extent of footprinting is a measure of order or disorder, solvent accessibility, or buried structure. One example of footprinting is a reversible reaction utilized by hydrogen/deuterium exchange (HDX), now productively and extensively coupled with mass spectrometry. Irreversible (or nearly irreversible) footprinting is achieved by specific amino acid footprinting whereby one reactive, usually nucleophilic amino acid side chain is specifically labeled. Another approach is reactive species footprinting, where several amino acid side chains are modified in time frames as fast as microseconds, affording broader coverage than obtainable by specific amino acid footprinting but not as extensive as provided by HDX. In this chapter, we describe these footprinting approaches and provide examples from our work to illustrate their capabilities.