Quantifying the heterogeneity of macromolecular machines by mass photometry

Abstract

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 (cryo-EM). Here, we show that mass photometry (MP) can accurately characterize the heterogeneity of a sample using minimal material with high resolution within a matter of minutes. To benchmark our approach, we use negative stain electron microscopy (nsEM), a popular method for EM sample screening. We include typical workflows developed for structure determination that involve multi-step purification of a multi-subunit ubiquitin ligase and chemical cross-linking steps. When assessing the integrity and stability of large molecular complexes such as the proteasome, we detect and quantify assemblies invisible to nsEM. Our results illustrate the unique advantages of MP over current methods for rapid sample characterization, prioritization and workflow optimization.

Mass Photometry of Membrane Proteins

“…MP reveals true equilibrium distributions, reports on populations rather than ensembles due to its single-molecule nature and requires minimal sample volumes (mL) and concentrations (<<mM). Moreover, MP naturally extends toward characterizing structural heterogeneity 9 and protein-protein interactions, 10 resulting in a universal platform for studying biomolecules.…”

The Human RNA Helicase DDX21 Presents a Dimerization Interface Necessary for Helicase Activity

“…We next investigated the oligomeric state of DDX21 in the presence of RNA. The protein-RNA complexes were unstable under the conditions suitable for SEC-MALS analysis; therefore we used mass photometry to estimate the molecular mass of the macromolecular assemblies in solution (Sonn-Segev et al, 2020). The basis of mass photometry is the optical detection of the scattering signal generated by a single particle at the glass-water interface.…”

The linear ubiquitin chain assembly complex LUBAC generates heterotypic ubiquitin chains

“…The precise stoichiometry and oligomerization state of the LUBAC complex have not been established although recent structural work has suggested a 1:1:1 stoichiometry between the three core components (Fujita et al, 2018). To determine the stoichiometry and oligomerization state of the LUBAC complex we used mass photometry (MP), a technique that enables accurate mass measurements based on the scattering of light by single macromolecules in solution (Sonn-Segev et al, 2019, Young et al, 2018 ( Figure 2D and Figure EV4). MP measurements showed that the majority of species present in the samples had average masses of 231 kDa (Peak II), and 454 kDa (Peak III) (EV Table 1).…”