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

Abstract

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.

Structure of the AlgKX modification and secretion complex required for alginate production and biofilm attachment in Pseudomonas aeruginosa

“…The K a,kx for the interaction of AlgK Pp and AlgX Pp with the formation of AlgKX Pp complex was obtained using the Slow Mixing Mode (SLOMO) ESI-MS assay 71 . Briefly, ~2 µL of a solution of AlgX Pp (5 µM) and AlgK Pp (3–10 µM) was introduced into the nanoESI tip, followed by injection of 10 µL of solution of AlgX Pp (5 µM) and AlgK Pp (45 µM).…”

How Choice of Model Membrane Affects Protein–Glycosphingolipid Interactions: Insights from Native Mass Spectrometry

“…This observation likely reflects the low ESI-MS response factors of the CTB 5 −GM1 complex ions, which are released from the model membrane during ESI or in the gas phase, relative to those of free CTB 5 ions, which originate directly from solution. 32,35 At higher concentrations, ligand binding to CTB 5 is observed, and binding site occupancy increases with GM1 concentration, reaching a maximum value of ∼90% (Figure S9). As discussed elsewhere, this observation likely reflects the incomplete extraction of GM1 (bound to CTB 5 ) from the membrane in the gas phase.…”

Determination of dissociation constants via quantitative mass spectrometry

“…During these experiments, the two binding partners are incubated with a varying concentration of one of them (Jecklin et al, 2009;Báez Bolivar et al, 2021) (Figures 1A-D). This can be done either by preparation of a titration series or with a continuous ESI source, which allows the gradual combination of the two components (Fryčák and Schug, 2007;Bui et al, 2022). The latter is for example used in the "gap sampler" by the Zenobi group for bioaffinity screening with high-throughput and low sample consumption (Neu et al, 2013;Kaeslin et al, 2021).…”