Protein structural investigation using high spatial resolution hydroxyl radical protein footprinting coupled with liquid chromatography and tandem mass spectrometry
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Hydroxyl radical protein footprinting (HRPF) coupled with liquid chromatography and tandem mass spectrometry (LC-MS/MS) has become a very promising technique to study intact protein, protein conformational change and protein – ligand interactions in solution. HRPF oxidizes amino acid side chains at a rate based on a combination of the inherent reactivity and the accessibility to the hydroxyl radical diffusing in solution, giving its advantages of fast time frame for protein modification, high labeling efficiency and stability of labeled analyte. By monitoring the change in the amount of oxidation under different experimental conditions, changes in protein structure can be determined by how the structural changes affect the solvent accessibility of the different regions of the protein. The work presented describes a newly developed method using high spatial resolution HRPF coupled with electron-transfer dissociation (ETD)-based LC-MS/MS to investigate complex molecular interaction interfaces at residue level. Protein-GAG interaction and chemokine association binding interfaces were successfully determined by this method. An improvement of the method by adding a supercharging reagent m-NBA to increase sequence coverage and spatial resolution for protein quantitation is also presented.
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