Sliding Window Adduct Removal Method (SWARM) for Enhanced Electrospray Ionization Mass Spectrometry Binding Data

Abstract

Electrospray ionization mass spectrometry (ESI-MS) screening of compound libraries against target proteins enables the rapid identification of ligands and measurement of the stoichiometry and affinity of the interactions. However, non-specific association of buffer or salts (added or present as impurities) to the protein ions during gas-phase ion formation can complicate the analysis of ESI-MS data acquired for mixtures of compounds with similar molecular weights. Spectral overlap of ions corresponding to free protein and protein-ligand complexes and their corresponding adducts can hinder the identification of ligands and introduce errors in the measured affinities. Here, we present a straightforward approach, called the sliding window adduct removal method (SWARM), to quantitatively correct ESI mass spectra of low-to-moderate resolution for signal overlap associated with adducts. The method relies on the statistical nature of adduct formation in ESI and the assumption that the distributions of adducts associated with a given protein (free protein and ligand-bound forms) are identical at a given charge state. Analysis of ESI mass spectra measured for protein-oligosaccharide interactions using solutions that produced either low- or high-abundance adducts provides support for this assumption. Implementation of SWARM involves the stepwise subtraction of the adduct signal associated with the detected protein-ligand complexes from the mass spectrum. This is accomplished using the adduct distribution measured for an appropriate reference species (usually free protein). To demonstrate the utility of the method, we applied SWARM to ESI-MS screening data acquired for libraries of oligosaccharides and bifunctional ligands consisting of a sulfonamide moiety linked to human glycan structures. Graphical Abstract.

Keywords: Adducts; Affinity; Electrospray ionization mass spectrometry; Library screening.