Determination of Equilibrium Association Constants of Ligand–DNA Complexes by Electrospray Mass Spectrometry

International audienceElectrospray mass spectrometry can be used to detect ligand-DNA noncovalent complexesformed in solution. This chapter describes how to determine equilibrium associationconstants of the complexes. Particular attention is devoted to describing how to tune anelectrospray mass spectrometer using a 12-mer oligodeoxynucleotides duplex in order toperform these experiments. This protocol can then be applied to any nucleic acid structurethat can be ionized with electrospray mass spectrometry

A Simple Method to Determine Electrospray Response Factors of Noncovalent Complexes

The quantitative study of noncovalent complexes by electrospray mass spectrometry requires the determination of the relative response of each species. The method proposed here to determine the electrospray response factors is based on the use of (1) an internal standard and (2) the mass balance equation applied to one binding partner M, for which different complexes MxLy are detected in the electrospray mass spectra. A set of experiments providing various ratios between the complexes (e.g. different ligand concentrations in a titration experiment or different time points in a kinetics experiment) is used to generate a set of independent linear equations that can be solved using simple matrix algebra to find the response factors of each MxLy complex relative to that of the internal standard. The response factors can then be used to determine equilibrium dissociation constants or for the quantitative monitoring of reaction kinetics. The first is illustrated with a study of DNA-ligand complexes, where we show that neither minor groove binding nor intercalation dramatically affects the DNA response factor. The second is illustrated with a study of the association kinetics of the telomeric G-quadruplex dGGG(TTAGGG)3 with its complementary strand, where the response factors allow correcting for the relative response of the quadruplex and the long duplex and obtaining reproducible association rate constants independently of the source tuning potentials