Multiplex labeling chemistry methods for protein footprinting

Abstract Protein footprinting (PF) is a powerful medium resolution structural biology technique for assessing protein structure and dynamics that relies on “bottom-up” mass spectrometry (MS) to detect, identify, and quantitatively analyze the small (5-15 residue) peptides that are generated in protease-based workflows. Early on hydrogen deuterium exchange (HDX) led the way, later

advances in irreversible reagent development, such as hydroxyl radical footprinting (HRF) mediated by radiolysis; or methods utilizing photolysis of peroxide, and recently plasma, have been introduced and refined. As a result, PF is routinely applied in understanding the effects of protein-ligand binding on higher order interactions in solution, even for large macromolecules like

antibodies, large molecular complexes, and membrane proteins; all of which are important drug targets and biological machines. However, state of the art HRF-based PF studies typically report data from only