Measuring the affinity of protein-protein interactions on a single-molecule level by mass photometry

• Published in: Analytical biochemistry Vol. 592; p. 113575
• Main Authors: Wu, Di, Piszczek, Grzegorz
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-03-2020
• Subjects: Animals; Antibodies, Monoclonal, Murine-Derived - metabolism; Antibody; Association equilibrium constant; Binding cooperativity; Humans; iSCAMS; Label free; Nanotechnology - methods; Photometry - methods; Protein Binding; Protein Interaction Mapping; Protein-protein interaction; Recombinant Proteins - metabolism; Single Molecule Imaging - methods; Thrombin - metabolism; Association equilibrium constant; MP; iSCAMS; Protein-protein interaction; Binding cooperativity; Antibody; Label free; HT; AHT; BLI
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2020.113575

Measurements of biomolecular interactions are crucial to understand the mechanisms of the biological processes they facilitate. Bulk-based methods such as ITC and SPR provide important information on binding affinities, stoichiometry, and kinetics of interactions. However, the ensemble averaging approaches are not able to probe the intrinsic heterogeneity often displayed by biological systems. Interactions that involve cooperativity or result in the formation of multicomponent complexes pose additional experimental challenges. Single-molecule techniques have previously been applied to solve these problems. However, single-molecule experiments are often technically demanding and require labeling or immobilization of the molecules under study. A recently developed single-molecule method, mass photometry (MP), overcomes these limitations. Here we applied MP to measure the affinities of biomolecular interactions. We have demonstrated how MP allows the user to study multivalent complexes and quantify the affinities of different binding sites in a single measurement. Results obtained from this single-molecule technique have been validated by ITC and BLI. The quality and information content of the MP data, combined with simple and fast measurements and low sample consumption makes MP a new preferred method for measuring strong protein-protein interactions.
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•MP measures protein-protein interaction affinities on a single-molecule level without sample labeling or modification.•MP can easily estimate the cooperativity of multivalent interactions.•MP requires much less material and time to measure affinities than traditional methods.