Quantitative mass imaging of single biological macromolecules

Abstract

The cellular processes underpinning life are orchestrated by proteins and their interactions. The associated structural and dynamic heterogeneity, despite being key to function, poses a fundamental challenge to existing analytical and structural methodologies. We used interferometric scattering microscopy to quantify the mass of single biomolecules in solution with 2% sequence mass accuracy, up to 19-kilodalton resolution, and 1-kilodalton precision. We resolved oligomeric distributions at high dynamic range, detected small-molecule binding, and mass-imaged proteins with associated lipids and sugars. These capabilities enabled us to characterize the molecular dynamics of processes as diverse as glycoprotein cross-linking, amyloidogenic protein aggregation, and actin polymerization. Interferometric scattering mass spectrometry allows spatiotemporally resolved measurement of a broad range of biomolecular interactions, one molecule at a time.

Single molecule mass photometry of nucleic acids

“…Microscope coverglass (24 x 50mm # 1, 5 SPEZIAL, Menzel-Glaser) and APTES-functionalised coverslips were prepared as described previously (16,20). Briefly, coverslips were cleaned by sequential sonication in 2% Hellmanex (Hellma Analytics), water and iso-propanol for 10 minutes before plasma cleaning with oxygen (Diener electronic Zepto) for 8 minutes.…”

“…To account for differences in binding rate and thus molecule counts caused by differences in diffusion coefficient, we applied a correction to the measured mass distributions (16). We assumed that the binding rate constant scales with the diffusion coefficient, which has been reported to be roughly proportional to (base pair) -0.72 for DNA (i.e., * = α * /0.23 , where ki is the binding rate constant for DNA component i and α is a scaling factor) (21).…”

Recent advancement of light‐based single‐molecule approaches for studying biomolecules

“…Strikingly, iSCAT demonstrates the capability of quantitative mass measurement (Young et al, ). This differs from other single‐molecule techniques in that the signal contrast is linearly related to the polarizability and thus mass of a protein.…”

Optical imaging of single protein size, charge, mobility, binding and conformational change

“…Several technologies have been demonstrated to detect single proteins without using fluorescent labels. [14][15][16] One is to detect refractive index changes of proteins resulted from local heating by light. 14 A more direct method is to measure protein binding to plasmonic hotspots on the nanorod surface from plasmonic absorption.…”

“…Recently, a light interference method has been developed to quantify the protein size based on optical scattering intensity. 16 These label-free methods are attractive for protein analysis because they measure the size, an intrinsic property of proteins. However, size alone provides only limited information.…”