Mass photometry is a revolutionary new way to analyse molecules. It enables the accurate mass measurement of single molecules in solution, in their native state and without the need for labels. The ability of mass photometry to yield rapid and intuitive feedback on a variety of applications opens up new possibilities for bioanalytics and research into the functions of biomolecules.

How does it work?

Mass photometry builds on the principles of interference reflection microscopy and interferometric scattering microscopy. Through carefully controlled illumination, a novel spatial-filtering strategy in the detection beam path and careful image analysis, the group of Prof. Kukura at Oxford University has recently demonstrated that the minute amount of light scattered by single molecules can be reliably detected and, more importantly, that it is directly correlated with molecular mass (Fig 1) .

Fig 1: The principle of mass photometry. The light scattered by a molecule attached to the measurement interface interferes with light reflected at that interface. The interference contrast scales linearly with mass.

The light scattered by a particle scales linearly with particle volume and refractive index. As the optical properties and density of proteins vary only by a few percent, their scattering signal is directly proportional to their sequence mass – making it possible to weigh single molecules with light, with high accuracy and over a large mass range (Fig 2). The correlation of scattering signal with mass holds true for a variety of biomolecules (glycoproteins, nucleic acids or lipids), making mass photometry a universal analysis tool for biomolecules in solution.

Fig 2: Mass photometry measures the molecular mass of proteins and protein assemblies in solution. Via calibration, mass photometry enables the mass measurement of unknowns with high accuracy (2% average).

Benefits of mass photometry

Accurate measurement of true native behaviour

  • In solution, in a variety of buffers and compatible with membrane proteins
  • Label-free, without the need to modify samples

Information on all sub-populations in samples 

  • Single molecule counting 
  • Wide mass range and high dynamic range

One assay format delivering multiple results

  • Homogeneity, structural integrity and activity
  • Quick, simple, minimal sample amounts 

Applications

Sample purity and composition

Mass photometry measures the molecular mass of proteins and protein assemblies in solution with high accuracy. The mass photometry signal scales linearly with the known sequence mass of proteins (as well as other biomolecules) and is independent of their shape. As a result, via simple calibration, mass photometry enables the mass measurement of unknowns with high accuracy (<5 kDa, 1.9% average).

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Protein complexes – Assembly and stoichiometry

The self- and co-assembly of proteins underpins their biological function, while protein aggregation can lead to ineffective function, or even toxicity. Mass photometry provides a unique means to measure the mass distribution of proteins, detecting quantitatively even very rare stoichiometries, in solution. This provides an unparalleled view of the composition of a protein solution, and an avenue to test and optimise solution conditions that may influence oligomerization or aggregation. 

Mass photometry allows the determination of stoichiometry distributions of proteins. During a mass photometry experiment, the signal corresponding to individual protein landing events are detected independently from those of others. Single particle detection delivers a high dynamic range (3 orders of magnitude in this spectrum) and the ability to detect low abundance species, such as bovine serum albumin (BSA) tetramers (0.25%). The polydispersity of BSA (67 kDa monomer) is baseline-resolved and the relative amounts of monomer, dimer, trimer and tetramer can be quantified directly from the number of landing events.

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Mass measurement of complex biomolecules

Mass photometry is universal in its applicability, as all molecules scatter light, irrespective of whether they are based on amino-acids, lipids, nucleic-acids, carbohydrates, or other building blocks. This means that not just molecules of simple composition, but also those comprising multiple biomolecular classes (e.g. glycoproteins, membrane proteins solubilised by detergent, protein-DNA complexes) are amenable to accurate mass measurement using mass photometry.

Differences in the molecular mass of heterogeneous biomolecules, such as lipid nanodiscs, are distinguishable and highly reproducible. Measurements using alternative techniques – size-exclusion chromatography, nuclear magnetic resonance spectroscopy, dynamic light scattering, and native mass spectrometry – return masses differing from each other by ~30% (orange lines). Mass photometry can assign accurate mass values to nanodiscs with variable protein belt components and lipid compositions. Analysis of lipid bilayer mimetics, such as nanodiscs, is a key capability for structural and functional studies of membrane proteins.

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Protein-protein interactions

The function of proteins, and their malfunction in disease, is governed by their interactions with each themselves and with other biomolecules. Mass photometry provides a new and uniquely powerful means to quantify these interactions in terms of physically meaningful quantities, such as dissociation constants and free energies. This opens the door for measuring the manipulation of these interactions in drug development.

Complex protein-protein interactions can be quantified by mass photometry. The Env glycoprotein of the HIV virus is targeted by antiviral lectins which either bind to single Envs or cross-links Env units. At low concentration of lectin, the Env is primarily a single unit. At higher lectin concentration, higher order Env units are detected, and found to include bound lectins. From the titration curve and relative amounts of Env-lectin complexes, the associated binding of each interaction can be quantified providing key information for understanding anti-viral mechanisms.

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