Quantifying Biomolecular Interactions Using Slow Mixing Mode (SLOMO) Nanoflow ESI-MS

[Image: see text] Electrospray ionization mass spectrometry (ESI-MS) is a powerful label-free assay for detecting noncovalent biomolecular complexes in vitro and is increasingly used to quantify binding thermochemistry. A common assumption made in ESI-MS affinity measurements is that the relative ion signals of free and bound species quantitatively reflect their relative concentrations in solution. However, this is valid only when the interacting species and their complexes have similar ESI-MS response factors (RFs). For many biomolecular complexes, such as protein–protein interactions, this condition is not satisfied. Existing strategies to correct for nonuniform RFs are generally incompatible with static nanoflow ESI (nanoESI) sources, which are typically used for biomolecular interaction studies, thereby significantly limiting the utility of ESI-MS. Here, we introduce slow mixing mode (SLOMO) nanoESI-MS, a direct technique that allows both the RF and affinity (K(d)) for a biomolecular interaction to be determined from a single measurement using static nanoESI. The approach relies on the continuous monitoring of interacting species and their complexes under nonhomogeneous solution conditions. Changes in ion signals of free and bound species as the system approaches or moves away from a steady-state condition allow the relative RFs of the free and bound species to be determined. Combining the relative RF and the relative abundances measured under equilibrium conditions enables the K(d) to be calculated. The reliability of SLOMO and its ease of use is demonstrated through affinity measurements performed on peptide–antibiotic, protease–protein inhibitor, and protein oligomerization systems. Finally, affinities measured for the binding of human and bacterial lectins to a nanobody, a viral glycoprotein, and glycolipids displayed within a model membrane highlight the tremendous power and versatility of SLOMO for accurately quantifying a wide range of biomolecular interactions important to human health and disease.