Theory of DNA electrophoresis: a look at some current challenges

Although electrophoresis is one of the basic methods of the modern molecular biology laboratory, new ideas are being suggested at an accelerated rate, in large part because of the pressing demands of the biomedical community. Although we now have, at least for some methods, a fairly good theoretical understanding of the physical mechanisms that lead to the observed peak spacings, widths and shapes, this knowledge is often too qualitative to be used to guide further technical developments and improvements. In this article, we review some selected elements of the current state of our theoretical ignorance, focusing mostly on DNA electrophoresis, and we offer several suggestions for further theoretical investigations.     

Association of Cu2+ with uracil and its thio derivatives: a theoretical study.

The structures and relative stabilities of the complexes between Cu2+ and uracil, 2-thiouracil, 4-thiouracil, and 2,4-dithiouracil were investigated by B3LYP/6-311+G(2df,2p)//B3LYP/6-31G* DFT calculations. In those systems in which both types of basic centers, that is, a carbonyl and a thiocarbonyl group, are present, association of Cu2+ with the oxygen atom is systematically favored, in contrast to what was found for the corresponding Cu+ complexes. This can be understood by considering that association of Cu2+ is immediately followed by oxidation of the base, which accumulates the negative charge at the oxygen atoms. Similarly, for 2,4-dithiouracil the most basic site for Cu+ attachment is the sulfur atom at the 4-position, while for association of Cu2+ it is sulfur at the 2-position. In contrast, differences between uracil-Cu+ and uracil-Cu2+ complexes are very small, and in both cases the oxygen atom at the 4-position is the most basic. Cu2+ binding energies are about 4 and 1.2 times larger than Cu+ binding energies and proton affinities, respectively. Uracil- and thiouracil-Cu2+ complexes are thermodynamically unstable but kinetically stable with respect to their dissociation into uracil*+ + Cu+ or thiouracil*+ + Cu+. The Cu2+ binding energies vary with the difference between the second ionization potential of the metal and the first ionization potential of the base. regardless of the reference acid (H+, Cu+, Cu2+) the basicity trend is 2,4-dithiouracil > 4-thiouracil > 2-thiouracil > uracil.     

Desorption electrospray ionization ‐ orbitrap mass spectrometry of synthetic polymers and copolymers

Desorption ElectroSpray Ionization (DESI) ‐ Orbitrap Mass Spectrometry (MS) was evaluated as a new tool for the characterization of various industrial synthetic polymers (poly(ethylene glycol), poly(propylene glycol), poly(methylmethacrylate), poly(dimethylsiloxane)) and copolymers, with masses ranging from 500 g.mol⁻¹ up to more than 20 000 g.mol⁻¹. Satisfying results in terms of signal stability and sensitivity were obtained from hydrophobic surfaces (HTC Prosolia) with a mixture water/methanol (10/90) as spray solvent in the presence of sodium salt. Taking into account the formation of multiplied charged species by DESI‐MS, a strategy based on the use of a deconvolution software followed by the automatic assignment of the ions was described allowing the rapid determination of M_n, M_w and PDI values. DESI‐Orbitrap MS results were compared to those obtained from matrix‐assisted laser desorption/ionization‐ time‐of‐flight MS and gel permeation chromatography. An application of DESI‐Orbitrap MS for the detection and identification of polymers directly from cosmetics was described. Copyright © 2012 John Wiley & Sons, Ltd.     

Modelling peptide-metal dication interactions: formamide-Ca(2+) reactions in the gas phase

The collision induced dissociation of formamide-Ca(2+) complexes produced in the gas phase through nanoelectrospray ionization yields as main products ions [CaOH](+), [HCNH](+), [Ca(NH(2))](+), HCO(+) and [Ca(NH(3))](2+) and possibly [Ca(H(2)O)](2+) and [C,O,Ca](2+), the latter being rather minor. The mechanisms behind these fragmentation processes have been established by analyzing the topology of the potential energy surface by means of B3LYP calculations carried out with a core-correlated cc-pWCVTZ basis set. The Ca(2+) complexes formed by formamide itself and formimidic acid play a fundamental role. The former undergoes a charge separation reaction yielding [Ca(NH(2))](+) + HCO(+), and the latter undergoes the most favorable Coulomb explosion yielding [Ca-OH](+) + [HCNH](+) and is the origin of a multistep mechanism which accounts for the observed loss of water and HCN. Conversely, the other isomer of formamide, amino(hydroxyl)carbene, does not play any significant role in the unimolecular reactivity of the doubly charged molecular cation.     

Structural characterization of hexoses and pentoses using lead cationization. An electrospray ionization and tandem mass spectrometric study

The analytical potential of the complexation of isomeric underivatized hexoses (D-glucose, D-galactose, D-mannose, D-talose, D-fructose), methylglycosides (1-O-methyl-alpha-D-glucose and 1-O-methyl-beta-D-glucose) and pentoses (D-ribose, D-xylose, D-arabinose and D-lyxose) by Pb(2+) ions, was investigated by electrospray ionization and tandem mass spectrometry (MS/MS). Pb(2+) ions react mainly with monosaccharides by proton abstraction to generate [Pb(monosaccharide)(m) - H](+) ions (m = 1-3). At low cone voltage, a less abundant series of doubly charged ions of general formula [Pb(monosaccharide)(n)](2+) is also observed. The maximum number n of monosaccharides surrounding a single Pb(2+) ion depends on the metal : monosaccharide ratio. Our study shows that MS/MS experiments have to be performed to differentiate Pb(2+)-coordinated monosaccharides. Upon collision, [Pb(monosaccharide) - H](+) species mainly dissociate according to cross-ring cleavages, leading to the elimination of C(n)H(2n)O(n) neutrals. The various fragmentation processes observed allow the C(1), C(2) and C(4) stereocenters of aldohexoses to be characterized, and also a clear distinction aldoses and fructose. Furthermore, careful analysis of tandem mass spectra also leads to successful aldopentose distinction. Lead cationization combined with MS/MS therefore appears particularly useful to identify underivatized monosaccharides.     

Determination of citric acid in soft drinks using flow injection with potentiometric detection

A flow injection analysis (FIA) system with potentiometric detection has been developed for the determination of citric acid in commercial fruit juices using a copper-selective tubular electrode. It consists of the complexation of citrate ion with copper (II) ion, monitoring the free copper (II) concentration. Linear relationships between the potential and log concentrations of citric acid in the range of 1 × 10^–3 mol/L – 1 × 10^–2 mol/L were attained with samples injected in the system without prior treatment. Repeatability was determined by assessing the relative standard deviation for ten replicate injections of different juice samples which varied from 1.2% to 3.0%. The results provided by the automated system were compared with those of an enzymatic conventional method used for the analysis of citric acid in food and a relative deviation of less than 4.0% was found. Recovery trials resulted in values from 97.0% to 101.0%. The system allows a sampling frequency of 60 samples/h. Received: 22 October 1998 / Revised: 14 December 1998 / Accepted: 16 December 1998     

Perturbation of the intramolecular hydrogen bonds of glucose by Cu+ association

A density functional theory study of glucose and glucose–Cu⁺ complexes has been performed to investigate the changes undergone by the set of intramolecular hydrogen bonds of the neutral system upon Cu⁺ association. The geometries of the different species investigated were optimized at the B3LYP/6‐31G(d,p) level. The same level of theory was used to obtain the harmonic vibrational frequencies and to analyze the electron charge density by means of the atoms in molecules theory. We have shown that the interaction with Cu⁺ strongly perturbs the set of intramolecular hydrogen bonds of the neutral. Some of these changes are a direct consequence of the conformational changes induced by the metal, which result in the breaking of some of the existing bonds or in the formation of new ones. The most important point, however, is that the intramolecular hydrogen bonds that remain are perturbed to a different extent. In general, all hydrogen bonds in which the OH donor interacts directly with the metal cation are significantly stabilized while the remaining ones become weaker. These changes influence the relative stability of the complexes as well as its capacity to interact with other systems. © 2001 John Wiley & Sons, Inc. Int J Quantum Chem, 2001     

Analyses of biodynamic responses of seated occupants to uncorrelated fore-aft and vertical whole-body vibration

The apparent mass and seat-to-head-transmissibility response functions of the seated human body were investigated under exposures to fore-aft (x), vertical (z), and combined fore-aft and vertical (x and z) axis whole-body vibration. The coupling effects of dual-axis vibration were investigated using two different frequency response function estimators based upon the cross- and auto-spectral densities of the response and excitation signals, denoted as H₁ and H_v estimators, respectively. The experiments were performed to measure the biodynamic responses to single and uncorrelated dual-axis vibration, and to study the effects of hands support, back support and vibration magnitude on the body interactions with the seatpan and the backrest, characterized in terms of apparent masses and the vibration transmitted to the head. The data were acquired with 9 subjects exposed to two different magnitudes of vibration applied along the individual x- and z-axis (0.25 and 0.4 m/s² rms), and along both the axis (0.28 and 0.4 m/s² rms along each axis) in the 0.5-20 Hz frequency range. The two methods resulted in identical single-axis responses but considerably different dual-axis responses. The dual-axis responses derived from the H_v estimator revealed notable effects of dual-axis vibration, as they comprised both the direct and cross-axis responses observed under single axis vibration. Such effect, termed as the coupling effect, was not evident in the dual-axis responses derived using the commonly used H₁ estimator. The results also revealed significant effects of hands and back support conditions on the coupling effects and the measured responses. The back support constrained the upper body movements and thus showed relatively weaker coupling compared to that observed in the responses without the back support. The effect of hand support was also pronounced under the fore-aft vibration. The results suggest that a better understanding of the seated human body responses to uncorrelated multi-axis whole-body vibration could be developed using the power-spectral-density based H_v estimator.