Weighing Molecules With Light

Schematic view of a light-scattering approach for accurate mass determination of proteins as small as 20 Kilodaltons. (Source: G. Young et al. / AAAS)

Using a micro­scope that detects light scattering rather than fluores­cence, the researchers have demon­strated that single molecules can be observed, and their mass measured, in solution. The research has been carried out in colla­boration with insti­tutions in Germany, Sweden, Switzer­land and the US. Senior author Professor Philipp Kukura, from Oxford’s Depar­tment of Chemistry, said: “This research has emerged from a decade of work which involved making an ever more sensi­tive light micro­scope.”

“Single molecules have been observed in light micro­scopes since the late 1980s, but essen­tially all optical techniques rely on fluores­cence, which is the emission of light by a material after being excited by the absorp­tion of electro­magnetic radiation. As immensely powerful as that is, it is not universal”, Kukura said. The researchers first demons­trated the use of light scat­tering to visualise individual proteins in 2014. But it was not until last year that they were able to improve the image quality suffi­ciently to compete with fluores­cence.

Kukura said: “We then addressed the question of whether we could use our visua­lisation approach to quantify, rather than just detect, single molecules. We realised, given that the volume and optical proper­ties of biomo­lecules scale directly with mass, that our micro­scope should be mass sensitive. This turned out indeed to be the case, not only for proteins but also for molecules con­taining lipids and carbo­hydrates.” It is this generality that excites the authors. Justin Benesch of Oxford’s Department of Chemistry, an expert in mass measure­ment, said: “The beauty of mass is that it is both a universal property of matter and extremely diag­nostic of the molecule under inves­tigation. Our approach is therefore broadly appli­cable and, unlike tradi­tional single-molecule micro­scopy, does not rely on the addition of labels to make molecules visible.”

The researchers say the inter­ferometric scattering mass spectro­metry (iSCAMS) could have appli­cations ranging from studies of protein-protein inter­actions to drug disco­very and even point-of-care diag­nostics. Kukura said: “iSCAMS has lots of advan­tages. It measures mass with an accuracy close to that of state-of-the-art mass spectro­metry, which is expensive and operates in vacuum – not necessarily represen­tative of bio­logical systems – whereas iSCAMS does so with only a very small volume of sample and works in essen­tially any aqueous environ­ment.”

Benesch added: “This enables a lot of the things that researchers want to quantify: do certain molecules interact and, if yes, how tightly? What is the compo­sition of the protein in terms of how many pieces it contains, and how does it grow or fall apart?” Because essen­tially every physio­logical and patho­logical process is controlled by biomo­lecular interactions in solution, the researchers say this technology has consi­derable potential impact. Kukura said: “The universal applica­bility, combined with the fact that the instru­ments are close to shoebox size, can be operated easily, and allow the user to see the molecules in real time, is tremen­dously exciting.” The team is in the process of commercia­lising the tech­nology to provide access to other researchers who are not experts or may not even use optical micro­scopy. (Source: Oxford Univ.)

Reference: G. Young et al.: Quantitative mass imaging of single biological macromolecules, Science 360, 423 (2018); DOI: 10.1126/science.aar5839

Link: Physical and Theoretical Chemistry Laboratory, Dept. of Chemistry, University of Oxford, Oxford, UK

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