Fast Radical Footprinting for Protein Ligand Interaction Analysis

Another approach, fast radical footprinting, uses reactions of a radical (e.g. -OH) with amino acid side-chains. It is complementary to PLIMSTEX because it exploits side-chain reactivity instead of exchange of backbone amides. It can be readily expanded to locating the amino acid residue that has reacted, because it introduces an irreversible modification (stable covalent bond) to the protein and can utilize any protease to locate reaction sites, taking advantage of in silico prediction of cleavage sites. Whereas PLIMSTEX must use pepsin to cleave the ana-lyte under quenching conditions and necessitates rapid LC runs to minimize back exchange, any proteomic method can be used when probing protein interfaces that are determined by chemical reactions that make an irreversible change to the protein. A chemical reaction method such as hydroxyl radical footprinting probes specific functions (e.g. hydrophobic and sulfur-containing amino acid side-chains) whereas H/D exchange probes the exchange rate of every backbone amide hydrogen in the protein.

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