Quantitative mass imaging of single molecules in solution

Abstract

Abstract:The cellular processes underpinning life are orchestrated by proteins and their interactions. Structural and dynamic heterogeneity, despite being key to protein and drug function, continues to pose a fundamental challenge to existing analytical and structural methodologies used to study these associations. Here, we use interferometric scattering microscopy to mass-image single biomolecules in solution with <2% mass error, up to 19-kDa resolution and 1-kDa precision. Thereby, we resolve oligomeric distributions at high dynamic range, detect small-molecule binding, and mass-image biomolecules composed not only of amino acids, but also heterogeneous species, such as lipo-and glycoproteins. These capabilities enable us to characterize the molecular mechanisms of processes as diverse as oligomeric selfassembly, glycoprotein cross-linking, amyloidogenic protein aggregation, and actin polymerization. Interferometric scattering mass spectrometry (iSCAMS) provides spatially resolved access to the dynamics of biomolecular interactions ranging from those involving small molecules to mesoscopic assemblies, one molecule at a time.

Myosin II filament dynamics in actin networks revealed with interferometric scattering microscopy

“…Regions exhibiting consecutive deposition of actin filaments within a diffraction-limited spot, displayed a step-wise increase in the interferometric signal (Sup Fig. 1D, E; Video 1), in line with the scaling of the interferometric signal with molecular mass (20). These results suggest, that the iSCAT signal grows proportionally with the amount of protein in the region of interest even for large structures such as myosin filaments and can be translated into the number of actin filaments stacked on top of each other.…”

Single Molecule Mass Photometry Reveals Dynamic Oligomerization of Plant and Human Peroxiredoxins for Functional Conservation and Diversification

“…To address these open questions, we applied single molecule mass photometry to reveal the oligomeric behavior of 2-CysPRXs for static and dynamic physicochemical environments and selected single point mutants from different organisms. [19] 2. Results…”

Identification and Quantification of Proteoforms by Mass Spectrometry

“…On a longer horizon, a variety of exciting new single‐molecule analysis platforms are actively under development around the world, including nanoscale cantilevers, nanopore strategies, interferometric light scattering, cryo‐electron microscopy, x‐ray scattering, and others. The yet‐to‐be‐determined and evolving strengths and limitations of such new strategies for proteoform identification will dictate how these approaches supplant or synergize with today's technologies in ways that we cannot presently foresee.…”