Quantifying Protein–Protein Interactions by Molecular Counting with Mass Photometry
| Autor: | Fabian Soltermann, Justin L. P. Benesch, Martin R. Galpin, Eric D. B. Foley, Veronica Pagnoni, Weston B. Struwe, Philipp Kukura |
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| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
receptors
protein–protein interactions single-molecule studies 010402 general chemistry 01 natural sciences Catalysis Protein–protein interaction Photometry (optics) 03 medical and health sciences mass photometry antibodies Equilibrium constant 030304 developmental biology Binding affinities chemistry.chemical_classification 0303 health sciences Molecular mass 010405 organic chemistry Biomolecule Communication Single‐Molecule Studies | Hot Paper Proteins General Medicine General Chemistry Communications 3. Good health 0104 chemical sciences Kinetics chemistry Spectrophotometry Ultraviolet Biological system |
| Zdroj: | Angewandte Chemie (International Ed. in English) Angewandte Chemie International Edition |
| ISSN: | 1521-3773 1433-7851 |
| Popis: | 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. Keeping count: Reported is the quantification of protein–protein interactions with light using mass photometry, which detects, images, and quantifies light scattering of single proteins upon binding to an illuminated glass‐water interface. The resulting signals are proportional to the molecular mass of each species and overall abundance, providing a new means to measure the relative abundances of proteins and their complexes, thereby enabling measurement of binding affinities and kinetics by molecular counting. |
| Databáze: | OpenAIRE |
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