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How pure is your sample?

16 Apr 2020
Proteasome - negative stain micrograph

Sample purity is central to in vitro studies of protein function and regulation, and to the efficiency and success of structural studies using techniques such as x-ray crystallography and cryo-electron microscopy. In a study published online by Nature Communications, structural biologists involving IMP-researchers show that mass photometry (MP) can accurately characterise the heterogeneity of a sample with high molecular resolution within minutes while requiring minimal sample volume.

The structural and biophysical characterisation of biomolecular complexes generally requires the isolation of molecular species from a heterogeneous background of cellular components. The molecules of interest themselves can range from monomeric species to large multimeric machines that undergo conformational rearrangements and form transient interactions with binding partners. As a result, sample heterogeneity remains a significant challenge to the routine and high-throughput structure determination of protein complexes. 

The methods for protein characterisation that researchers typically rely upon all come with their particular limitations. Those that are based on gel electrophoresis reveal molecular weights, but not stoichiometry or interactions. Size-exclusion chromatography on the other hand reports on stokes radii, but not the actual molecular weight, and dynamic light scattering has limited mass accuracy and resolution. To determine and optimise sample quality therefore requires the appropriate combination of these techniques – a time consuming procedure.

Weighing molecules with light

An interdisciplinary team from the labs of structural biologist David Haselbach (IMP Vienna), biochemist Nicholas Brown (University of North Carolina) and physicist Philipp Kukura (Oxford University) now present a technique that has unique advantages over current methods in terms of rapid sample characterisation, prioritisation and workflow optimisation. Supported by expertise from the MIT, the MPI of Biochemistry (Martinsried) and the University of Freiburg, the researchers tested and optimised a technology called mass photometry (MP) as an efficient screening tool that is applicable to a wide range of samples. Mass photometry uses the minute amount of light scattered by a biomolecule for accurate mass measurements of single molecules in solution, in their native state and without the need for labels - basically weighing molecules with light.

To benchmark their approach, the researchers used negative stain electron microscopy (nsEM), a popular method for EM sample screening. The results showed that mass photometry provides information on sample composition that closely correlates with those obtained by nsEM, but with a number of key advantages. Certain limitations of the method, such as a currently limited concentration range, are expected to be overcome with technical improvements in the near future. Overall, the authors are convinced that mass photometry will be of tremendous value to the cryo-EM and X-ray crystallography communities. More generally, the capabilities of mass photometry will likely impact the broader life science community to enable accurate sample characterisation for the majority of biochemical and biophysical in vitro studies.

The image shows a negative-stain micrograph of proteasome complexes generated by nsEM analysis (Katarina Belacic, IMP)

Original Publication:

Sonn-Segev, A., Belacic, K., Bodrug, T. et al. Quantifying the heterogeneity of macromolecular machines by mass photometry. Nat Commun 11, 1772 (2020).