Investigating Protein-Nucleic\nAcid Binding Interactions\nwith Diethylpyrocarbonate Covalent Labeling–Mass Spectrometry

Nucleic acids are\nimportant biomolecules that facilitate numerous\ncellular functions and have in recent years become promising candidates\nfor treating disease. Consequently, there is a need for methods to\ncharacterize protein interactions with these molecules. Here, we demonstrate\nthat diethylpyrocarbonate (DEPC) covalent labeling–mass spectrometry\n(CL-MS) can provide structural information for protein-nucleic acid\nbinding by characterizing the binding sites of two DNA aptamers specific\nto thrombin. Reductions in thrombin labeling are observed at the pair’s\nbinding interfaces. Furthermore, we find that binding of the aptamers\ncauses changes in labeling at residues in the thrombin active site\nand known exosites for each aptamer, showcasing the sensitivity of\nDEPC CL-MS to significant allosteric changes.