Quantifying Protein-Protein Interactions by Molecular Counting with Mass Photometry

Abstract

Interactions between biomolecules control the processes of life in health and their malfunction in disease, making their characterization and quantification essential. Immobilization- and label-free analytical techniques are desirable because of their simplicity and minimal invasiveness, but they struggle with quantifying tight interactions. Here, we show that mass photometry can accurately count, distinguish by molecular mass, and thereby reveal the relative abundances of different unlabelled biomolecules and their complexes in mixtures at the single-molecule level. These measurements determine binding affinities over four orders of magnitude at equilibrium for both simple and complex stoichiometries within minutes, as well as the associated kinetics. These results introduce mass photometry as a rapid, simple and label-free method for studying sub-micromolar binding affinities, with potential for extension towards a universal approach for characterizing complex biomolecular interactions.

Keywords: antibodies; mass photometry; protein-protein interactions; receptors; single-molecule studies.

Figure 1

Principle of single‐molecule counting by mass photometry. a) Label‐free single‐molecule detection by imaging the interference of scattered and reflected light arising from individual protein landing events at a glass‐water interface over time. b) Scatter plot of single‐molecule contrasts and resulting mass distribution for a 1:1 monomer/dimer 2G12 mixture. c) Mass distributions for varying 2G12 monomer/dimer ratios. d) Comparison of monomer/dimer ratios measured by MP compared to expectations based on UV‐VIS absorption characterization.

Figure 2

Single‐shot K _d and kinetics measurements of IgG‐FcγRIa interactions. a) MP mass distributions of IgG (red), FcγRIa (purple) and a 1:1 mixture of IgG‐FcγRIa (blue). b) MP distributions of deglycosylated IgG (red), FcγRIa (black) and 1:1 mixture of IgG‐FcγRIa (blue). c) Mass distributions for a 1:1 mixture of deglycosylated IgG‐FcγRIa at total IgG concentrations ranging from 300 pm to 5 nm and respective K _d calculated from a single‐shot measurement. d) Mass distributions for a 1:1 mixture of deglycosylated IgG‐FcγRIa at 1.5 nm total IgG concentration, ranging from 0 to 30 minutes after dilution from 2.9 μm. e,f) Mole fraction of assembled IgG‐FcγRIa and deglycosylated IgG‐FcγRIa complexes as a function of time after dilution from 2.7 μm to 0.3 nm total IgG concentration, and 2.6 μm to 5 nm total deglycosylated IgG concentration and corresponding single exponential fits. g,h) Corresponding SPR analysis of IgG‐FcγRIa and deglycosylated IgG‐FcγRIa (h).

Figure 3

Binding stoichiometry and affinity of the IgG‐FcRn interaction as a function of pH. a) Self‐assembly of FcRn dimers at pH 5 (red) and 5.5 (dotted red) and equivalent pH measurements of IgG at pH 5 (grey) and 5.5 (dotted grey). b) IgG‐FcRn complexes (1:1 mixture) at pH 5, 5.5 and 6. c) Associated pH dependent binding affinities of interaction revealing cooperativity in FcRn binding (PDB: 4N0U, 3FRU).