Due to its lower critical consolute temperature, we can use a nanosecond laser T-jump to induce spinodal demixing in the triethylamine/water binary mixture. Using a time-resolved Raman probe, we obtained direct molecular level evidence for liquid restructuring in the early stage (<200 ns) of this spinodal decomposition. From these Raman data, we concluded that in this system the early and intermediate stage spinodal dynamics were apparently over within 1 µs. In addition to Raman spectroscopy, we developed a novel shadowgraphic microscopic time-resolved imaging system to get information about morphological changes during demixing, such as phase domain growth rate. In the microsecond time scale, the characteristic scale of length ( ) of phase domains increased with time following a simple power law ∼ t 0.76((0.04) , while the structure maintained its self-similarity. In this case, the onset of late stage spinodal phase change is several orders of magnitude faster than has been reported for other simple binary mixtures because of the depth of the jump into the two-phase region brought about by our heating pulse.
Laser spray, which is a newly developed ionization technique, can characterize the stability of noncovalent complexes in the solution phase. By using this advantage, laser spray has been applied to probe the intrinsic stability of double-stranded DNA (dsDNA) sequences and their binding affinities with various drugs in the solution phase. Systematic experiments were carried out using six 16-mer and three 22-mer dsDNA oligomers, together with the complexes of the 16-mer dsDNA with minor groove binders: berenil, Hoechst 33342, DAPI, and netropsin. Dissociation curves for each dsDNA or each complex were plotted as a function of laser power. The laser power (E50%), where 50% of each dsDNA or each complex was dissociated, was compared with its melting temperature (Tm) determined by UV spectroscopy. Linear correlations between E50% and Tm were obtained not only for the dsDNA oligomers (correlation factor r = 0.9835) but also for the 16-mer dsDNA complexes with minor groove binders (r = 0.9966). In addition, laser spray has successfully clarified the binding affinities of a 16-mer dsDNA with two intercalators: daunomycin and nogalamycin. In the case of the dsDNA-daunomycin complex, by changing the molar ratio of dsDNA : drug from 1 : 1 to 1 : 5, the concentration-dependent stability of the complex was confirmed by laser spray. The present results demonstrate that laser spray mass spectrometry can be a powerful and convenient method to investigate the relative binding affinities of dsDNA-ligand complexes in the solution phase, which could be applied to the early stage of high-throughput screening of drugs targeting for dsDNA.
In laser spray, the tip of an electrospray capillary is irradiated with a continuous CO(2) laser beam. Here, we report results from a modified laser spray method that employs a relatively low laser irradiance level. With a laser power of approximately 2 W and a focal spot size ( approximately 0.3 mm), which covered the entire front surface of the electrospray capillary, the irradiance was approximately 3 x 10(3) W/cm(2). This resulted in a quiescent and smooth vaporization of aqueous solutions. This "evaporation-mode" laser spray method yielded the best results so far obtained in our laboratory with laser-irradiated electrospray, producing higher and more stable signals. The method was applied to the analysis of aqueous solutions of lysozyme and myoglobin. Mass spectra were obtained as a function of laser power from 0 W (electrospray) to approximately 2 W. The spray generated at the tip of the stainless steel capillary was observed with a CCD camera. With increase of laser power, the droplets in the spray became finer and the Taylor cone became progressively smaller. The strongest ion signals were recorded when the sample solution protruded only slightly from the tip of the capillary. A broadening of the lysozyme charge-state distribution, attributable to protein unfolding, was observed with a laser power of 2 W. No denaturation of myoglobin took place up to a laser power of 1.6 W. However, a sudden onset of denaturation was observed at 1.8 W as a broadening of the myoglobin charge distribution and the appearance of apo-myoglobin peaks. These findings demonstrate that laser spray is capable of dissociating the noncovalent complexes selectively without breaking covalent bonds.
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 K d values are different in the range from 10 Ϫ9 M to 10 Ϫ7 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 (E 50% ) in population was obtained. The correlation coefficient between E 50% and the relative binding free-energy change (⌬⌬G) 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 K d 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. (J Am Soc Mass Spectrom 2006, 17, 611-620) © 2006 American Society for Mass Spectrometry E lectrospray ionization mass spectrometry (ESI-MS) has been successfully applied to detect the protonated molecules of noncovalent biomolecular complexes that are involved in various important biological events [1][2][3][4][5][6][7][8]. Although mass spectrometry cannot provide structural information of a biological complex at an atomic level, it can rapidly offer information on the binding stoichiometry, specificity, and stability of the complex with a small amount of the sample. 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. 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 ...
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.
The effect of the presence of foreign salts (NaCl, aerosol OT, tetra-n-hexylammonium bromide, and CH3COONH4) on the formation of gaseous ions for electrospray (ES) and laser spray (LS) was studied in the positive and negative modes of operations. The ion signals for amino acids show sudden decrease with the concentration of foreign salts greater than 10(-5) M for both ES and LS. When the surface-active counter ions were added, the signal intensities showed a marked decrease for both ES and LS. This may be due to the enrichment of the surface-active counter ions on the surface of the charged droplets. When CH3COONH4 was added to an aqueous solution of 10(-6) M lysozyme chloride, an increase of the signal intensities for (lysozyme+nH)n+ and a decrease in the values of n were observed. The decrease in n may be due to the salt formation of (lysozyme+nH)n+ with the negative acetate ion leading to the reduction of positive charges.
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