Optimization of Electrospray Ionization by Statistical Design of Experiments and Response Surface Methodology: Protein-Ligand Equilibrium Dissociation Constant Determinations.

Liliana Pedro,Ronald J Quinn,Wesley C Van Voorhis

doi:10.1007/s13361-016-1417-x

Abstract

Electrospray ionization mass spectrometry (ESI-MS) binding studies between proteins and ligands under native conditions require that instrumental ESI source conditions are optimized if relative solution-phase equilibrium concentrations between the protein–ligand complex and free protein are to be retained. Instrumental ESI source conditions that simultaneously maximize the relative ionization efficiency of the protein–ligand complex over free protein and minimize the protein–ligand complex dissociation during the ESI process and the transfer from atmospheric pressure to vacuum are generally specific for each protein–ligand system and should be established when an accurate equilibrium dissociation constant (KD) is to be determined via titration. In this paper, a straightforward and systematic approach for ESI source optimization is presented. The method uses statistical design of experiments (DOE) in conjunction with response surface methodology (RSM) and is demonstrated for the complexes between Plasmodium vivax guanylate kinase (PvGK) and two ligands: 5′-guanosine monophosphate (GMP) and 5′-guanosine diphosphate (GDP). It was verified that even though the ligands are structurally similar, the most appropriate ESI conditions for KD determination by titration are different for each.Graphical ᅟElectronic supplementary materialThe online version of this article (doi:10.1007/s13361-016-1417-x) contains supplementary material, which is available to authorized users.

Highlights

Binding interactions between ligands and proteins are crucial for a wide variety of biological processes and for the effectiveness of many therapeutic compounds
To determine Plasmodium vivax guanylate kinase (PvGK)–guanosine diphosphate (GDP) KD by competition, the ion abundance of the reference protein –ligand complex with a known binding constant (PvGK–guanosine monophosphate (GMP), with KD determined by titration) was monitored in the presence and in the absence of the competitor ligand (GDP) and correlated to the corresponding solution equilibrium concentrations
Summary of ESI source parameters studied by statistical design of experiments (DOE) and response surface methodology (RSM) and their influence on relative ion abundances for (a) PvGK-GMP system and (b) PvGK-GDP system: parameters inside white boxes had no significant impact on relative ion abundances; parameters inside colored boxes had significant main effect on relative ion abundances; connecting lines denote significant interactions or quadratic effects flow rate 38 L/min, drying gas temperature 101 °C, and nebulizer gas pressure 72 psi

Summary

Introduction

Binding interactions between ligands and proteins are crucial for a wide variety of biological processes and for the effectiveness of many therapeutic compounds. Before establishing any correlation between the relative abundance of the gas-phase ions in the mass spectra and relative concentrations in solution, differences in ionization and detection efficiencies (response factors) between the free protein and protein–ligand complex, as well as processes such as nonspecific ligand binding and protein–ligand complex dissociation, need to be considered and, if possible, minimized.

Results

Conclusion