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

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(-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 (E(50%)) in population was obtained. The correlation coefficient between E(50%) and the relative binding free-energy change (DeltaDeltaG) 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.

Stability analysis for double-stranded DNA oligomers and their noncovalent complexes with drugs by laser spray.

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.

Experimentally determined growth exponents during the late stage of spinodal demixing in binary liquid mixtures.

Spinodal demixing was initiated in two systems, with critical and off-critical compositions, using nanosecond pulsed laser-induced temperature jumps (T-jumps) of various magnitude. In this way, deep quenches could be imposed on the systems. One system was the simple triethylamine (TEA)/water mixture and the other was the ionic mixture of 2-butoxyethanol (2BE)/water/KCl. The demixing process was followed using the technique of nanosecond time-resolved microscopic shadowgraphy. The growth of the evolving phase-separated domains followed a simple power law with respect to time in every case. For a given composition, the magnitude of the T-jump had little effect on the growth exponent, however the composition was found to influence the rate of domain growth. At off-critical mole fractions of 0.2 with respect to TEA, the domains grew according to the following expression: L(t)=t(0.70) (where L(t)= the domain size) whereas at the critical TEA mole fraction of 0.08 the domains grew as L(t)=t(0.52). 2BE/water/KCl mixtures quenched at the just off-critical composition of fraction with respect to 2BE evolved as L(t)=t(0.63). These results will be compared to theoretical models and simulations and discussed in terms of estimated Reynolds numbers as well as the consumption and conversion of the available surface energy that fuels the demixing process.

Denaturation of lysozyme and myoglobin in laser spray.

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.

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

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.

Ultrafast laser-induced molecular and morphological changes during spinodal demixing of water/2-butoxyethanol/KCl.

We initiated morphological and molecular level changes in the spinodal decomposition (SD) of H(2)O/2-butoxyethanol/KCl with a pulsed ir laser. Transient Raman spectra gave us a molecular level view of the early stage of this process that could be linked to later morphological events. Chemical changes during SD, such as reorganization of H bonds and forced hydrophobic interactions, ended after 1 micros; however, phase domains continued to grow with self-similarity after 30 micros. The growth of the phase domains satisfied the power law L(t) approximately t(0.55) and was consistent with the late stage of SD. The time scale for the onset of late stage SD is many orders of magnitude faster than previously reported in ionic and nonionic conditions.

Observation of dimethylaminoethyl methacrylate-myoglobin binding reaction using laser spray mass spectrometry.

Covalent bonds are often created by a reaction between chemicals and protein before causing various adverse effects in a cell. Dimethylaminoethyl methacrylate (DMAEMA), which has moderate toxicity, causes skin inflammation and throat irritation. For this study, we investigated a reaction mechanism between myoglobin and (DMAEMA) using a new analytical tool developed at our laboratory: laser spray mass spectrometry technique. It was found that initially DMAEMA was added to the amino group of protein by the Michael addition mechanism; the added DMAEMA was hydrolyzed to methacrylic acid using an autocatalytic system. The results of this study indicate the feasibility of the laser spray technique in analyses of reaction dynamics.

Thermal unfolding of proteins probed by laser spray mass spectrometry.

The stability and conformational changes of cytochrome c (cyt c) at different temperatures and pH have been well examined so far by using various analytical methods. We have found that laser spray mass spectrometry enables much faster and more convenient monitoring of those changes of cyt c compared with other methods. The results correlated well with circular dichroism (CD) experiments under relatively acidic conditions, which destabilize the protein. Laser spray mass spectra of cyt c at various pH were obtained at different levels of laser power. Bimodal charge-state distributions of the protein were observed in laser spray mass spectra, indicating the two-state model of structural change; the lower charges correspond to the folded state, the higher charges to the unfolded state. Based on this result, the presumed denaturation curve of the protein was plotted as a function of laser power, and laser power by which 50% of the protein was assumed to be denatured, E50%, as obtained at each pH. We also examined the melting temperatures, Tm, of cyt c at various values of pH by using CD spectroscopy. The correlation coefficient between E50% and Tm for cyt c was 0.999, demonstrating an excellent correlation. Furthermore, laser spray analysis of ubiquitin, which is found to be more thermally stable than cyt c, gave a higher E50% than cyt c. These results indicate that laser spray mass spectrometry can be an extremely convenient method for probing thermal stabilities and dynamic conformational changes of proteins with subtle structural differences caused by slight changes in pH.

Measurement of sugars using the laser spray technique with a gold capillary.

A gold (Au) capillary has higher thermal conductivity than a stainless steel capillary and can withstand capillary over-heating induced by high CO(2) laser irradiation (over 2.5 W) better than a stainless steel capillary. For this study, a laser spray using an Au capillary was applied for the detection of sugars. The signal of cationized compounds [M+Na](+) can be detected with higher sensitivity than with conventional laser sprays using high laser power (over 2.7 W). Using 3.5 W of laser power, the signal intensity is 15 times higher than the maximum value with stainless steel (2.3 W) in a 10(-5) M maltose aqueous solution. It is considered that almost all the water molecules evaporate by laser irradiation, which is impossible to achieve using a stainless steel capillary.

Denaturation of alpha-lactalbumin and ubiquitin studied by electrospray and laser spray.

Electrospray and laser spray mass spectra of human alpha-lactalbumin and bovine ubiquitin were studied, with an emphasis on the denaturation induced by laser spray. There were no remarkable differences in the electrospray and laser spray mass spectra for acidic and basic aqueous solutions of alpha-lactalbumin in positive and negative modes of operations. This originates from the fact that this protein is tightly folded with four disulfide bonds. For ubiquitin, however, denaturation was induced by laser spray for the positive mode of operation and the [M+nH](n+) with a maximum of n = 13 was observed, i.e., all the acidic amino acid residues are fully neutralized (protonated). In contrast, the laser-induced denaturation was not observed for the negative mode of operation, i.e., denaturation of ubiquitin is largely suppressed in the negatively charged liquid droplets. The marked difference observed in the positive and negative modes of operations for ubiquitin is ascribed to the difference in the susceptibility of side-chain/main-chain interactions in the positive-ion excess and in the negative-ion excess liquid droplets. That is, the interactions between the basic residues and main-chain amide carbonyl groups (-NH(3) (+)***O=C< or -NH(2)***O=C<) which play an important role in stabilizing the protein structures are not so affected in the negative mode of operation but are weakened in the positive mode of operation.

Atmospheric-pressure Penning ionization of aliphatic hydrocarbons.

A study has been made of the atmospheric-pressure Penning ionization (APPeI) of aliphatic hydrocarbons (pentane, hexane, heptane, and octane) with long-lived rare gas atoms (Rg*). The metastable rare gas atoms (He*, Ne*, Ar* and Kr*) were generated by the negative-mode corona discharge of atmospheric-pressure rare gases. In the Rg*APPeI mass spectra for aliphatic hyrocarbons, the relative abundances of fragment ions were found to increase in the order of He* --> Ne* --> Ar* --> Kr*. The order is in the opposite direction to the internal energies of the Rg*. The less fragmentation observed for He* may be because the nascent molecular ions [M(+.)]* formed by Penning ionization have lifetimes long enough for them to be collisionally deactivated in the atmospheric-pressure ion source. It was found that the relative abundances of fragment ions in Ar*APPeI increased when the sample pressure in the ion source was reduced. This is attributed to the collision of Ar* with molecular ions followed by fragmentation.

The effect of the presence of foreign salts on the formation of gaseous ions for electrospray and laser spray.

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.