To date, we have made considerable progress on objective 1,3 and 5, with 2 and 4 being largely held back by lack of appropriate samples from collaborators strongly impacted by the Covid19 epidemic.
For objective 1, we performed a series of experiments starting from the basics of detecting and counting single biomolecules using mass photometry. We began by understanding and quantifying the basics of the technology: does it count and identify molecules correctly, and can one use the results to determine the interaction strengths and dynamics between molecules. We used commonly employed antibodies, given their therapeutic importance and method of action based on (un)binding to specific targets. We then continued by expanding our approach to more complex systems beyond 1:1 interactions, and showed that the range of species formed for some systems depends not only on environmental factors, but in fact controls protein function and varies substantially between different organisms.
Objective 3 benefitted considerably from the advances made in objective 1: we could now reveal how strongly proteins interact with each other at the single molecule level, as well as obtaining quantitative information on distributions ranging from single proteins to complexes containing a few hundred molecules. Equipped with this capability, we could directly observe how individual proteins dissolved in solution can turn into long filaments, a process that is central to a number of physiological and pathological processes. Interestingly, the mechanism we observed did not match that developed from ensemble observations made over the past 50 years.
Objective 5 has made considerable progress, most importantly through the development of a completely new approach to mass photometry. Until now, mass photometry was almost exclusively performed on glass surfaces, meaning that essentially everything present in solution produced a signal. This meant that we could only study systems consisting of highly purified proteins. Inspired by nature's use of lipid bilayer membranes, we used a related approach to passivate and at the same time selectively activate glass surfaces. In this way, we could remove the need for purification, making mass photometry applicable to a much broader range of systems.
