Interactions between biomolecules control the processes of life in health and their malfunction in disease, making their characterization and quantification essential. We first explore the capabilities of established methods for binding affinity measurements of protein-protein interactions and present mass photometry in this context. We explain that immobilization- and label-free analytical techniques are desirable because of their simplicity and minimal invasiveness, but that they struggle with quantifying tight interactions and resolving co-existing species. After a bench-marking process we show that mass photometry can accurately count different unlabelled biomolecules at the single-molecule level, distinguishing them by molecular mass, thereby revealing the relative abundances of different oligomeric states and stoichiometries of complexes in vitro. We develop and characterize a single-shot measurement method to determine binding affinities over four orders of magnitude at equilibrium for both simple and complex stoichiometries, as well as the associated kinetics in experiments with minimal sample consumption (fmol) and efficient experimental procedures (< few minutes per sample). We then present applications of this method for protein-protein interactions quantification of biotherapeutics and glycoproteins in the field of cancer, autoimmune disease, HIV and SARS-CoV-2 research. These results introduce mass photometry as a rapid, simple and label-free method for studying sub-micro molar binding affinities, with potential to become a universal approach for characterizing wide range of biomolecular interactions.
