Time-Resolved Native Mass Spectrometry for Direct Measurement of Biomolecular Kinetics.

The functional outcomes of biomolecular interactions depend on the kinetics of association and dissociation between proteins and their binding partners, ranging from small molecules to other proteins, and are fundamental to understanding cooperativity, allostery, and drug action. However, existing kinetic methods, such as surface plasmon resonance and biolayer interferometry (BLI), require immobilization or labeling of one binding partner and are often indirect measurements. Here, we introduce a transformative time-resolved native mass spectrometry (MS) approach that enables direct, label-free, and immobilization-free quantification of biomolecular kinetics across diverse interactions within minutes using only picomolar sample amounts. We benchmarked the approach using well-characterized systems and obtained kinetic parameters that agreed with those measured by BLI. We further demonstrate the utility of time-resolved native MS in quantifying the kinetics of protein-small-molecule interactions, including those involving an irreversible inhibitor. By capturing the association and dissociation of biomolecular interactions in real time, time-resolved native MS overcomes longstanding limitations of conventional kinetic assays and transforms native MS from a static technique to a dynamic, quantitative tool for probing biomolecular kinetics and mechanisms that underpin therapeutic discovery.