Charge Detection Native Mass Spectrometry for Quantitative Insights into Heterogeneous Protein Interactions

Abstract Native mass spectrometry (nMS) is a powerful label-free method for detecting biomolecular complexes, resolving stoichiometry, and quantifying affinity (Kd). However, signal overlap in heterogeneous systems often limits its accuracy. Charge detection (CD)-nMS, which independently measures the mass-to-charge ratio and charge of individual ions, overcomes this challenge, enabling quantitative analysis of polydisperse and high molecular weight complexes with unresolved charge states. Here, we systematically validate CD-nMS for precise Kd determination using soluble protein–ligand complexes of known Kd and extend its application to quantify glycan ligand binding by a highly glycosylated immune lectin. We then demonstrate the implementation of slow mixing mode (SLOMO), a time-resolved mixing method that corrects for nonuniform response factors of interacting species, using CD-nMS to enable robust quantification of protein–protein interactions. Finally, we apply SLOMO-CD-nMS to directly detect and quantify bacterial toxin binding to glycolipids embedded in membrane-like assemblies, a capability not accessible with conventional nMS. These measurements uncovered previously unrecognized assembly pathways and demonstrate, for the first time, that SLOMO-CD-nMS can resolve and quantify multivalent lectin engagement with glycolipids in a native-like membrane context. Collectively, these results establish CD-nMS, alone or in combination with SLOMO, as a broadly applicable assay for quantitative characterization of complex biomolecular interactions across soluble, glycosylated, and membrane-associated systems.