Quantifying Protein-Protein Interactions by Molecular Counting with Mass Photometry

Area of Science:

• Biochemistry and biophysics
• Analytical chemistry
• Molecular biology

Background:

• Biomolecular interactions are crucial for life processes and disease.
• Label-free analytical methods are desired for simplicity and minimal invasiveness.
• Quantifying tight biomolecular interactions remains a challenge for current techniques.

Purpose of the Study:

• To demonstrate mass photometry as a method for label-free quantification of biomolecules and their complexes.
• To assess the capability of mass photometry in determining binding affinities and kinetics.
• To explore the potential of mass photometry for analyzing complex biomolecular interactions.

Main Methods:

• Utilizing mass photometry for single-molecule level detection and quantification.
• Analyzing unlabeled biomolecules and their complexes in mixtures.
• Measuring binding affinities at equilibrium and associated kinetics.

Main Results:

• Mass photometry accurately counts and distinguishes biomolecules by mass in mixtures.
• The technique quantifies relative abundances of different species at the single-molecule level.
• Binding affinities were determined over four orders of magnitude within minutes for various stoichiometries.

Conclusions:

• Mass photometry is a rapid, simple, and label-free method for studying biomolecular interactions.
• The technique enables the characterization of sub-micromolar binding affinities.
• Mass photometry shows potential as a universal approach for complex biomolecular interaction analysis.