Quantifying Protein Homodimer Affinities and the Effect of Molecular Glues and Interface Residues Using Native Mass Spectrometry

Biological processes rely on finely tuned homo- and heteromeric interactions between (biomacro)molecules. The strength of an interaction, typically given by the dissociation constant (KD), plays a crucial role in basic research and must be monitored throughout the development of drugs and agrochemicals. An ideal method for KD determination is applicable to various analytes with a large range of affinities, tolerates complex matrix compositions, does not require labeling, and simultaneously provides information on the structural integrity of the binding partners. Native mass spectrometry meets these criteria but typically struggles with homooligomeric complexes due to overlapping mass signals. To overcome this, we resolve monomer/dimer contributions to overlapping MS-peaks by separately analyzing the charge state distribution of each oligomeric species via sample dilution and covalent cross-linking. Following this approach, we show that quantitative laser-induced liquid bead ion desorption mass spectrometry (qLILBID-MS) accurately captures the affinities of Bovine Serum Albumin (BSA) and chemically induced dimers of Tryparedoxin (Tpx), an oxidoreductase from human pathogenic Trypanosoma brucei parasites, with various molecular glues and homodimer affinities. Conveniently, qLILBID-MS requires a fraction of sample used by other methods such as isothermal titration calorimetry (ITC) and yields previously inaccessible protein homodimer KDs in the high micromolar range, which allowed us to monitor the gradual decrease in homodimer affinity via mutation of crucial dimer interface contacts. Overall, qLILBID-MS is a sensitive, robust, fast, scalable, and cost-effective alternative to quantify protein/protein interactions, that can accelerate contemporary drug discovery workflows, e.g. the efficient screening for proximity inducing molecules like proteolysis targeting chimera (PROTACs) and molecular glues.