Some critical aspects in the determination of binding constants by electrospray ionisation mass spectrometry at the example of arsenic bindings to sulphur‐containing biomolecules

The influences of reactant concentrations, solvent type, acid strength, pH conditions and ionic strength on the determination of apparent gas‐phase equilibrium constants K using electrospray ionisation mass spectrometry (ESI‐MS) were elucidated. As example serves the interaction of the tripeptide glutathione (GSH) with phenylarsine oxide (PAO). It was shown that rising initial concentrations of both reactants were not adequately compensated by increasing signal intensities of the reaction products in the mass spectra. The equilibrium constant for the formation of the phenylarsenic‐substituted peptide species decreased from 1.42 × 10⁵ ± 1.81 × 10⁴ l µmol^?1 to 1.54 × 10⁴ ± 1.5 × 10³ l µmol^?1 with rising initial GSH concentrations from 1 to 10 µM at fixed PAO molarity of 50 µM . K values resulting from a series with a fixed GSH molarity of 5 µM and a PAO molarity varied from 10 to 100 µM remained in a narrower range between 4.59 × 10⁴ ± 2.15 × 10⁴ l µmol^?1 and 1.07 × 10⁴ ± 4.0 × 10³ l µmol^?1. In contrast, consumption numbers calculated from the ion intensity ratios of reaction products to the unreacted peptide were not influenced by the initial reactant concentrations. In a water–acetonitrile–acetic acid mixture (48:50:2, v:v), the consumption of 5 µ M GSH increased from 8.3 ± 1.4% to 39.6 ± 1.6% with increased molar excess of PAO from 2 to 20, respectively. The GSH consumption was considerably enhanced in a changed solvent system consisting of 25% acetonitrile and 75% 10 mM ammonium formate, pH 5.0 (v:v) up to 80% of the original peptide amount at an only threefold molar arsenic excess. Copyright © 2010 John Wiley & Sons, Ltd.