Influence of response factors on determining equilibrium association constants of non‐covalent complexes by electrospray ionization mass spectrometry

Abstract

A method for determining the equilibrium association constant of a complexation reaction A + B left harpoon over right harpoon AB by electrospray ionization mass spectrometry is described. The method consists in measuring the relative intensities of the peaks corresponding to A and to AB in equimolar A-B solutions at different concentrations C(0). The results are fitted by a non-linear least-squares procedure, with the two variable parameters being the equilibrium association constant K(a) and a factor R, defined by I(AB)/I(A) = R x [AB]/[A]. The factor R is the ratio between the response factors of AB and A, and corrects for the relative electrospray responses of the complex and the free substrate A, mass discrimination of instrumental origin and/or moderate in-source dissociation. The method is illustrated with the following two systems: complexes between a double-stranded 12-base pair oligonucleotide and minor groove binders, and cyclodextrin complexes with alpha,omega-dicarboxylic acids. For the oligonucleotide complexes, it is found that the response of the complex is not dramatically different to the response of the free oligonucleotide duplex, as the double helix conformation is disturbed by the drug only to a minor extent. In the case of cyclodextrin complexes, these complexes were found to have a much higher response than free cyclodextrin. This may be due to the fact that cyclodextrin is neutral in solution, whereas the complex is charged, but it can also stem from the fact that a significant proportion of the complex is in a non-inclusion geometry. The present method requires the exact determination of the concentrations of the reactants and is applicable to 1 : 1 complexes.

Determination of Noncovalent Binding Using a Continuous Stirred Tank Reactor as a Flow Injection Device Coupled to Electrospray Ionization Mass Spectrometry

“…K d values determined using the equimolar binding model developed by Gabelica et al [36], and combined with the CSTR dilution model, are presented in Table1.The results obtained for Ac-L-Lys(Ac)-D-Ala-D-Ala are similar to those reported in prior literature [1,6,7,13], where a variety of different analytical methods were used. However, the reported literature values do vary quite a bit, and this is most certainly due to slight differences in solution conditions and associated methodological accuracies.…”

“…Because this device allows an exponential dilution of the host-guest solution over time, a simple model could be developed to fit the obtained data. This approach was aided by the use of an equimolar titration model [36]. The technique was first simulated.…”

“…In the current embodiment, an equimolar solution of host and guest was pulse-injected into the device. By combining an exponential dilution model with a previously established equimolar binding model [36], binding constants could be determined for host-guest complexation reactions with a single injection. This work-flow was successfully modeled and then applied as a proof-of-principle for the determination of binding constants between vancomycin and Ac-Lys(Ac)-Ala-Ala tripeptide stereoisomers.…”

“…Schematic of the experimental manifold for noncovalent binding determinations using a continuous stirred tank reactor (CSTR) and ESI-MS equivalent concentration, the equimolar binding model developed by Gabelica et al [36] was used. An association constant is calculated by:…”

Noncanonical interactions between serum transferrin and transferrin receptor evaluated with electrospray ionization mass spectrometry

“…1D) acquired under the same conditions as Tf/TfR spectra (vide supra) shows unbound HSA and TfR as the principal species, while the contribution from the higher molecular weight species (putative nonspecific ðHSAÞ n :TfR complexes, m∕z > 6; 000) to the total ionic signal is negligible. estimate the magnitude of the binding constant (19); however, nonlinearity of ionic signal dependence on solution concentration often raises concerns over the reliability of such calculations (20). We approached assessment of aTf receptor affinity by monitoring its interaction with TfR in the presence of other Tf species.…”