Measuring Protein-Protein Interactions and Quantifying Their Dissociation Constants with Mass Photometry

Protein-protein interactions underlie most biological processes, and determining the affinity and abundance of binding partners for each interaction is often a challenging task because these interactions often involve multiple ligands and binding sites. Standard methods for determining the affinity of protein interactions often require a large amount of starting material in addition to potentially disruptive labeling or immobilization of the binding partners. Mass photometry is a bioanalytical technique that measures the mass of single biomolecules in solution, quickly and with minimal sample requirements. This article describes how mass photometry can be used to determine the mass distribution of binding partners, the complexes they form, the relative abundance of each species, and, accordingly, the dissociation constant (K_D ) of their interactions. © 2024 Refeyn Ltd. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Using mass photometry to measure protein-protein binding and quantify the K_D of this interaction.