QuantDSF: an Open-Source Platform for Comprehensive Nano Differential Scanning Fluorimetry Analysis

Nano differential scanning fluorimetry (nanoDSF) is widely used for label-free protein stability and ligand binding analysis, yet most workflows reduce each experiment to a single melting temperature (Tm), discarding the native-state fluorescence, concentration dependence, and temperature-resolved binding information present in the raw data. Here, we present QuantDSF, an open-source, fully transparent platform that converts standard concentration-series nanoDSF experiments into three layers of quantitative information typically obtained from separate assays: thermal stability, affinity, and binding thermodynamics. From a single experiment, QuantDSF reports Tm and ligand-induced shifts, legacy 4PL midpoints (Cmid) with method-aware quality control, and apparent ΔH and ΔS via isothermal slicing and Van’t Hoff regression. Tm is determined through three complementary algorithms, letting users match the method to their data. Native-state static fluorescence quenching and Gaussian deconvolution of overlapping transitions further capture binding events that Tm-only analyses miss. We validate QuantDSF on small molecules, nucleic acids, metal ions, and lipids, with orthogonal validation against isothermal titration calorimetry and surface plasmon resonance. In addition, QuantDSF enables label-free characterization of proteolysis-targeting chimera (PROTAC)−induced ternary complex formation across both VHL and CRBN E3 platforms. We further show that QuantDSF resolves the BCL-xL versus BCL-2 selectivity of DT2216, a BCL-xL−selective PROTAC, that proximity-based assays miss because it reports the thermodynamic stability of the ternary complex rather than spatial proximity alone. By exposing the full analytical content of nanoDSF data through reproducible open-source workflows, QuantDSF extends nanoDSF from endpoint stability screening into a quantitative platform for protein−ligand binding, thermodynamic analysis, and ternary complex characterization.