Article ID Journal Published Year Pages File Type
1196165 Journal of the American Society for Mass Spectrometry 2006 10 Pages PDF
Abstract

We have applied laser spray mass spectrometry developed by Hiraoka et al. to investigate the binding affinity of protein–mutant DNA complexes. The results were compared with our previous data of collision-induced dissociation (CID) experiments using electrospray ionization mass spectrometry (ESI-MS). Systematic experiments were carried out on the complexes of the c-Myb DNA binding domain (c-Myb DBD) bound to eight kinds of 16- or 22-mer point mutant double-stranded DNA (dsDNA), whose solution Kd values are different in the range from 10−9 M to 10−7 M. The dissociation curve as a function of laser power was plotted for each complex, and the laser power where 50% of complex was dissociated (E50%) in population was obtained. The correlation coefficient between E50% and the relative binding free-energy change (ΔΔG) of each complex formation in solutions was 0.9808, which is much better than the coefficient obtained by the previous ESI-CID experiments that was 0.859. In addition, complexes of the c-Myb DBD with five other mutant dsDNA were also examined to confirm that laser spray can be used to estimate the Kd values of a DNA–protein complex in solutions if an appropriate calibration curve is available. In the process of laser spray, dissociations of these noncovalent complexes occur in solutions, but not in the gas phase. This differs greatly from ESI-CID. Laser spray mass spectrometry has been found to be better than ESI-CID in evaluating binding affinity of a protein to various mutant DNA.

Related Topics
Physical Sciences and Engineering Chemistry Analytical Chemistry
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