Evaluation of protein-DNA binding affinity by electrospray ionization mass spectrometry

Abstract

Binding affinity of complexes between a DNA-binding domain (DBD) of a transcription factor, c-Myb, and several double-stranded DNA (dsDNA) were evaluated by collision-induced dissociation (CID) of the multiply protonated molecules generated by electrospray ionization mass spectrometry (ESI-MS). Complexes of the c-Myb DBD and dsDNA were prepared in solution and analyzed by ESI-MS. Multiply protonated molecules of a high-affinity complex, the c-Myb DBD and dsDNA with a specific sequence, were clearly observed in ESI mass spectrum. Protonated molecules of the complex were quite stable in the gas-phase, and not easily dissociated even if high cone voltage was applied in the first vacuum chamber source when the sample was prepared in 10 mM ammonium acetate. As for the sample prepared in buffer with higher concentration of ammonium acetate, such as 500 mM ammonium acetate, protein-dsDNA complexes could easily be dissociated with an increase in the cone voltage, giving multiply protonated molecules of free c-Myb DBD and some DNA fragments. Systematic CID experiments were carried out on seven complexes between the c-Myb DBD and 22-mer dsDNA with different solution-Kd values in the range of 10 Ϫ9 M to 10 Ϫ7 M. For each complex dissociation curve as a function of cone voltage was plotted, and the cone voltage where 50% of the complex was dissociated (V 50% ) was calculated. Consequently, positive correlation was obtained between V 50% and relative binding free energy change (⌬⌬G) in complex formation in solution. This suggests that ESI-CID experiments can provide quantitative evaluation of the stability of protein-DNA complexes based on proper calibration. (J Am Soc Mass Spectrom 2005, 16, 116 -125)

Evaluation of binding affinity of protein-mutant dna complexes in solution by laser spray mass spectrometry

“…The binding affinity of the c-Myb DBD to dsDNA-m22 was confirmed by gel-mobility assay (data not shown) and ESI mass spectrometry, which was consistent with our previous report [9]. The ion source used in the present study is the same as the conventional ESI source except that the commercial desolvation chamber is removed for the consecutive laser spray experiments in the open air.…”

“…In addition, dissociation of the complex can also be achieved by changing the mass spectrometric parameters. We have previously applied ESI-MS to examine the binding affinity of a protein to various double-stranded DNA (dsDNA) oligomers [9] by using a minimal DNA-binding domain (DBD) of a transcription factor c-Myb [10].The c-Myb DBD, 13 kDa, specifically recognizes dsDNA with the consensus sequence of AACNG (N denotes A or T or G or C) [11][12][13]. We have carried out systematic investigation on the gas-phase stability of the complexes of the c-Myb DBD bound to seven 22-mer dsDNA oligomers.…”

“…However, some problems still existed that had to be resolved for rigorous quantification of the binding affinity of a protein to dsDNA. When the cone voltage was increased over 70 V in ESI-CID experiments, significant degradation of DNA was recognized [8,9] in addition to the dissocia- …”

“…We have carried out systematic investigation on the gas-phase stability of the complexes of the c-Myb DBD bound to seven 22-mer dsDNA oligomers. The dsDNA are single-point mutants and the complexes of the c-Myb DBD with dsDNA mutants have different solution K d values in a wide range of 6.3 ϫ 10 Ϫ7 M to 2.8 ϫ 10 Ϫ9 M. Multiply protonated molecules of the complexes were generated by ESI-MS, and successively subjected to collision induced dissociation (CID) in the first vacuum region [9]. A dissociation curve as a function of cone voltage was plotted, and the cone voltage where 50% of the population of the complex was dissociated (V 50% ) was calculated for each protein-DNA complex.…”

“…In addition, dissociation of the complex can also be achieved by changing the mass spectrometric parameters. We have previously applied ESI-MS to examine the binding affinity of a protein to various double-stranded DNA (dsDNA) oligomers [9] by using a minimal DNA-binding domain (DBD) of a transcription factor c-Myb [10].…”