Application of Amide Proton Exchange Mass Spectrometry for the Study of Protein‐Protein Interactions

Abstract

This protocol describes amide proton exchange experiments that probe for changes in solvent accessibility at protein-protein interfaces. The simplest version of the protocol, termed the "on-exchange" experiment, detects protein-protein interfaces by taking advantage of the fact that solvent deuterium oxide (D2O) molecules are excluded from the surface of a protein to which another protein is bound. A more complete version of the experiment can also be performed in which the rate of surface deuteration is initially measured separately for each of the proteins involved in the interaction, after which the deuterated proteins are allowed to complex and the rate of "off-exchange" (i.e., replacement of surface deuterons by protons from solvent H2O molecules) at the resulting protein-protein interface is measured. This version of the experiment yields additional kinetic information that can help to define the solvent-inaccessible "core" of the interface.