Selective dissociation of non‐covalent bonds in biological molecules by laser spray

Abstract

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.

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

“…Other activation methods, prompt heating of the inlet of the sample solution, for example, might be effective for more quantitative analysis of the dissociation [21]. We are now trying to apply laser spray, developed by Hiraoka et al [22,23], for quantification of binding affinity of c-Myb DBD-dsDNA complexes [24]. This technique might be promising for the quantification of binding affinity of complexes stabilized mainly by hydrogen bonds and/or electrostatic interactions.…”

Development of New Ionization Methods for GC/MS and LC/MS Interfaces

“…In this section, an application of laser spray to the characterization of biomolecules, such as a protein, double-strand DNA (dsDNA), and a protein῍ DNA complex is described. 63) A protein c-Myb is a transcription factor, and its DNA binding domain (DBD), 13 kDa, specifically recognizes dsDNA with the consensus sequence of AACNG (N denotes A or T or G or C). Figure 16 shows electrospray (laser o#) and laser spray mass spectra of a complex of c-Myb DBD and 22mer dsDNA, whose solution K d had been determined to be 2.2ῌ10 ῌ9 M. 64) In electrospray mass spectrum the complex of c-Myb DBD and dsDNA gave three peaks, with 8, 9, and 10 positive charges, while peaks of free c-Myb DBD were observed with 7῍18 positive charges.…”

Application of Laser Spray for Liquid Chromatography/Mass Spectrometry Interface

“…7) In contrast, collision induced dissociation (CID) of non-covalent complex causes considerable cleavages of covalent bonds as well as the dissociation of non-covalent bonds. 8) Selective cleavage of the non-covalent bonds established by laser spray is highly preferable for the study of the stability and structure of non-covalent complexes.…”