Abstract
AbstractInteractions 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.
Highlights
We have recently developed mass photometry (MP), originally introduced as interferometric scattering mass spectrometry, as a means for detecting and measuring the mass of single proteins and the complexes they form in solution.[26]
To explore this capability in the context of biomolecules, we chose monomers and domain exchanged dimers of the HIV-1 neutralizing antibody 2G12, which produced mass distributions with the expected major bands at 147 kDa and 291 kDa (Figure 1 b). Repeating these experiments for monomer/dimer ratios ranging from 0.15 to 8.1 (Figure 1 c) revealed close agreement with UVVIS-based characterization within the experimental error (4.6 % RMS), except for noticeable deviations ( % 20 %) for the lowest ratios (Figure 1 d). We found that such deviations could almost exclusively be attributed to sample preparation, such as an additional dilution step required to reach subnanomolar concentrations, leading to variations in counts arising from nonspecific protein adsorption to the sample tube
Equipped with these benchmarking results, we set out to investigate the suitability of MP to characterize interactions of varying affinities, using the immunoglobulin G (IgG) monoclonal antibody trastuzumab (Herceptin) binding to soluble domains of IgG Fc receptors or ErbB2 (HER2) antigens
Summary
IgG N-glycan removal (see Figure S8) weakened FcR binding[29] resulting in a 1:1 mixture of FcgRIa and deglycosyated IgG exhibiting considerably less bound antibody At concentrations above the Kd value we found mostly bound complexes, with free species dominating below the Kd value, but all measurements yielded similar binding affinities (Kd = 1.0 Æ 0.1, 0.6 Æ 0.1 and 0.7 Æ 0.3 nm), suggesting that they were performed at or close to equilibrium (see Figure S11).
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