The laser spray developed in our laboratory was applied to the analysis of bovine serum albumin (BSA), double-stranded DNA (dsDNA) and a protein-DNA complex. The tip of a stainless-steel capillary was irradiated with a 10.6 micro m infrared laser by increasing the laser power from 0 W (electrospray) to 1.4 W. The laser beam was focused to about 0.3 mm at the tip of the stainless-steel capillary. When BSA aqueous solution was irradiated by the laser, highly charged monomer ions were newly observed in addition to the multiply charged ions of non-denatured monomer, dimer and trimer moieties. This indicates that BSA suffers from denaturation on irradiation with an infrared laser in solution. A 1.4 W laser power is not sufficient to cause the complete denaturation of BSA under the present experimental conditions. Whereas dsDNA was found to dissociate almost completely to single-stranded DNA constituents on laser irradiation with a power of 1.2 W, no fragmentation of DNA molecules was observed. For a protein-DNA complex, i.e. a complex of c-Myb DNA binding domain and dsDNA, dissociation of the complex to the component moieties was observed. These findings indicate that the laser spray can selectively dissociate non-covalent complexes into subunits without causing dissociation of the covalent bonds of the subunits. The laser spray will be a versatile method for the investigation of the structures and stabilities of biomolecules including non-covalent complexes.
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)
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