| Summary: | <p>Mass Photometry (MP), an emergent technology based on interferometric scattering principles, has revolutionised the field of biophysics by enabling label-free, solution-phase mass measurements at the single-molecule level. Traditionally, MP analysis and experiments focus on discrete events, characterising distributions of measurements from single molecules interacting with a glass surface. Our research challenges this convention, demonstrating that these events are not isolated but interconnected, encoding deeper insights of the underlying particle. </p>
<p>In this context, we introduce Tandem-MP, a novel approach that assigns binding and unbinding events to individual particles, thereby facilitating the measurement of dissociation at the single-molecule level. This method reveals intricate patterns that
illuminate the structural organisation of protein complexes in unprecedented detail. </p>
<p>Recognising that conventional analysis methods utilise only a small fraction of the information encoded within the data, we developed an innovative particle-centric analysis pipeline. This framework automatically tailors the analysis to each molecule, enabling
continuous tracking of both mass and position of particles without the need for experimental adjustments. This approach not only addresses the limitations inherent in standard MP methodologies, but also unveils a wealth
of quantitative, orthogonal information. </p>
<p>To exemplify the utility of our approach, we investigated two distinct systems:
citrate synthase and adeno-associated viruses. For citrate synthase, our method uncovered complex dissociation pathways at the level of individual molecules. In the study of adeno-associated viruses, we identified unique signatures of their surface interactions, a means to directly report capsid stability, and a way to differentiate between equal-mass serotypes. </p>
<p>This research demonstrates the true capabilities of MP by providing a proof-of-concept framework that leverages and exploits single-molecule measurement, paving the way for new discoveries in molecular biophysics.</p>
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