Feasibility of nonvolatile buffers in capillary electrophoresis-electrospray ionization-mass spectrometry of proteins

The combination of capillary electrophoresis (CE) and electrospray ionization-mass spectrometry (ESI-MS) via a triaxial interface was studied as a potential means for the characterization of intact proteins. To evaluate the possibility to use a nonvolatile electrolyte for CE, the effect of sodium phosphate and ammonium borate on the MS signal of the proteins insulin, myoglobin, and bovine serum albumin (BSA) was investigated by employing infusion experiments, and compared to the effect of ammonium formate and formic acid. The study shows that with formic acid (50 mM, pH 2.4) the most intense protein signals were obtained, while the use of sodium phosphate buffer (5 and 10 mM, pH 7.5) almost completely diminished the MS response. Ammonium formate and ammonium borate (up to 100 mM, pH 8.5) also caused protein ion suppression, but especially with the borate buffer significant MS intensity remained. MS analysis of myoglobin revealed the loss of the heme group when an acidic CE electrolyte was used. Using a background electrolyte containing 25 mM ammonium borate (pH 8.5), it is demonstrated that a CE separation of a protein test mixture can be monitored with ESI-MS without degrading the MS performance allowing molecular weight determinations of the separated compounds. In the presence of borate, detection limits were estimated to be 5-10 microM (ca. 100 fmol injected). The usefulness of the CE-MS system employing a borate buffer is indicated by the analysis of a stored sample of BSA revealing several degradation products. A sample of placental alkaline phosphatase (PLAP), a potential therapeutic agent, was also analyzed by CE-MS indicating the presence of a protein impurity. Probably due to insufficient ionization of the PLAP (a complex glycoprotein), no MS signals of the intact protein were observed.     

Low-frequency mode activity of heme: femtosecond coherence spectroscopy of iron porphine halides and nitrophorin

The low-frequency mode activity of metalloporphyrins has been studied for iron porphine-halides (Fe(P)(X), X = Cl, Br) and nitrophorin 4 (NP4) using femtosecond coherence spectroscopy (FCS) in combination with polarized resonance Raman spectroscopy and density functional theory (DFT). It is confirmed that the mode symmetry selection rules for FCS are the same as for Raman scattering and that both Franck-Condon and Jahn-Teller mode activities are observed for Fe(P)(X) under Soret resonance conditions. The DFT-calculated low-frequency (20-400 cm (-1)) modes, and their frequency shifts upon halide substitution, are in good agreement with experimental Raman and coherence data, so that mode assignments can be made. The doming mode is located at approximately 80 cm (-1) for Fe(P)(Cl) and at approximately 60 cm (-1) for Fe(P)(Br). NP4 is also studied with coherence techniques, and the NO-bound species of ferric and ferrous NP4 display a mode at approximately 30-40 cm (-1) that is associated with transient heme doming motion following NO photolysis. The coherence spectra of three ferric derivatives of NP4 with different degrees of heme ruffling distortion are also investigated. We find a mode at approximately 60 cm (-1) whose relative intensity in the coherence spectra depends quadratically on the magnitude of the ruffling distortion. To quantitatively account for this correlation, a new "distortion-induced" Raman enhancement mechanism is presented. This mechanism is unique to low-frequency "soft modes" of the molecular framework that can be distorted by environmental forces. These results demonstrate the potential of FCS as a sensitive probe of dynamic and functionally important nonplanar heme vibrational excitations that are induced by the protein environmental forces or by the chemical reactions in the aqueous phase.     

Competing C-4 and C-5-Acyl Stabilization of Uronic Acid Glycosyl Cations

Uronic acids are carbohydrates carrying a terminal carboxylic acid and have a unique reactivity in stereoselective glycosylation reactions. Herein, the competing intramolecular stabilization of uronic acid cations by the C-5 carboxylic acid or the C-4 acetyl group was studied with infrared ion spectroscopy (IRIS). IRIS reveals that a mixture of bridged ions is formed, in which the mixture is driven towards the C-1,C-5 dioxolanium ion when the C-5,C-2-relationship is cis, and towards the formation of the C-1,C-4 dioxepanium ion when this relation is trans. Isomer-population analysis and interconversion barrier computations show that the two bridged structures are not in dynamic equilibrium and that their ratio parallels the density functional theory computed stability of the structures. These studies reveal how the intrinsic interplay of the different functional groups influences the formation of the different regioisomeric products.     

Minimal shape and intensity cost path segmentation

A new generic model-based segmentation algorithm is presented, which can be trained from examples akin to the active shape model (ASM) approach in order to acquire knowledge about the shape to be segmented and about the gray-level appearance of the object in the image. Whereas ASM alternates between shape and intensity information during search, the proposed approach optimizes for shape and intensity characteristics simultaneously. Local gray-level appearance information at the landmark points extracted from feature images is used to automatically detect a number of plausible candidate locations for each landmark. The shape information is described by multiple landmark-specific statistical models that capture local dependencies between adjacent landmarks on the shape. The shape and intensity models are combined in a single cost function that is optimized noniteratively using dynamic programming, without the need for initialization. The algorithm was validated for segmentation of anatomical structures in chest and hand radiographs. In each experiment, the presented method had a significant higher performance when compared to the ASM schemes. As the method is highly effective, optimally suited for pathological cases and easy to implement, it is highly useful for many medical image segmentation tasks.     

How accurately do mechanophores report on bond scission in soft polymer materials?

Mechanophores are molecules that are incorporated into a host material and react to the local mechanical condition—the state of stress or strain—of that host material. Among their many purposes is that of a reporter: Mechanophores whose optical activity changes in response to mechanical cues can reveal bulk material processes that are ordinarily hidden, such as fatigue and fracture. Moreover, they may do so well before a material is fully fractured. To extract quantitative information from the optical signals from embedded mechanophores it is important that the mechanophores, which are generally a minority component of the material, report proportionally and unambiguously on the mechanical condition of the bulk. This is particularly important for early reporting of damage and wear, for which the optical signal from the mechanophore should accurately reflect bulk bond scission. In this article, we develop and analyze a general theory for the quality of optical mechanoreporting by mechanophores in soft materials, based on the Bell-Evans theory of bond breaking. We find, that at the typical low fractions in which mechanophores are incorporated the overall change in strength is limited, but that the proportionality of the reporting can be off by significant amounts, particularly at short times after loading but, for non-scissile bonds, at long times as well.     

Solvent effects on IR and VCD spectra of natural products: an experimental and theoretical VCD study of pulegone

The VCD spectra of pulegone, dissolved in CDCl3, CD2Cl2 and CS2 have been recorded in the frequency range from 1000 to 3000 cm(-1). The assignment of the absolute configuration was performed by comparing the experimental data with theoretical spectra computed at the B3LYP/6-311+G(d,p) level. Analysis of the agreement in several spectral regions revealed significant shortcomings when comparing with vacuum calculations. It is shown that the agreement improves when the solvent effects are taken into account by a continuum model. For the measurements in CDCl3 and CD2Cl2 further improvement was found when considering explicitly 1 : 1 complexes between a pulegone and a CDCl3 or CD2Cl2 solvent molecule in vacuo, while the best agreement was obtained when embedding these in a continuum model. The presence of the chiral solute was found to induce a VCD active C-D stretch band which could be modeled also at ab initio level.     

Pattern Theorems,Ratio Limit Theorems and Gumbel Maximal Clusters for Random Fields

We study occurrences of patterns on clusters of size n in random fields on ℤ ^d . We prove that for a given pattern, there is a constant a>0 such that the probability that this pattern occurs at most na times on a cluster of size n is exponentially small. Moreover, for random fields obeying a certain Markov property, we show that the ratio between the numbers of occurrences of two distinct patterns on a cluster is concentrated around a constant value. This leads to an elegant and simple proof of the ratio limit theorem for these random fields, which states that the ratio of the probabilities that the cluster of the origin has sizes n+1 and n converges as n→∞. Implications for the maximal cluster in a finite box are discussed.     

A Combined Experimental/Quantum-Chemical Study of Tetrel,Pnictogen, and Chalcogen Bonds of Linear Triatomic Molecules

Linear triatomic molecules (CO₂, N₂O, and OCS) are scrutinized for their propensity to form perpendicular tetrel (CO₂ and OCS) or pnictogen (N₂O) bonds with Lewis bases (dimethyl ether and trimethyl amine) as compared with their tendency to form end-on chalcogen bonds. Comparison of the IR spectra of the complexes with the corresponding monomers in cryogenic solutions in liquid argon enables to determine the stoichiometry and the nature of the complexes. In the present cases, perpendicular tetrel and pnictogen 1:1 complexes are identified mainly on the basis of the lifting of the degenerate ν 2 bending mode with the appearance of both a blue and a red shift. Van ′t Hoff plots of equilibrium constants as a function of temperature lead to complexation enthalpies that, when converted to complexation energies, form the first series of experimental complexation energies on sp¹ tetrel bonds in the literature, directly comparable to quantum-chemically obtained values. Their order of magnitude corresponds with what can be expected on the basis of experimental work on halogen and chalcogen bonds and previous computational work on tetrel bonds. Both the order of magnitude and sequence are in fair agreement with both CCSD(T) and DFA calculations, certainly when taking into account the small differences in complexation energies of the different complexes (often not more than a few kJ mol⁻¹) and the experimental error. It should, however, be noted that the OCS chalcogen complexes are not identified experimentally, most probably owing to entropic effects. For a given Lewis base, the stability sequence of the complexes is first successfully interpreted via a classical electrostatic quadrupole–dipole moment model, highlighting the importance of the magnitude and sign of the quadrupole moment of the Lewis acid. This approach is validated by a subsequent analysis of the molecular electrostatic potential, scrutinizing the σ and π holes, as well as the evolution in preference for chalcogen versus tetrel bonds when passing to “higher” chalcogens in agreement with the evolution of the quadrupole moment. The energy decomposition analysis gives further support to the importance/dominance of electrostatic effects, as it turns out to be the largest attractive term in all cases considered, followed by the orbital interaction and the dispersion term. The natural orbitals for chemical valence highlight the sequence of charge transfer in the orbital interaction term, which is dominated by an electron-donating effect of the N or O lone-pair(s) of the base to the central atom of the triatomics, with its value being lower than in the case of comparable halogen bonding situations. The effect is appreciably larger for TMA, in line with its much higher basicity than DME, explaining the comparable complexation energies for DME and TMA despite the much larger dipole moment for DME.     

The Fluctuation Theorem as a Gibbs Property

Common ground to recent studies exploiting relations between dynamical systems and nonequilibrium statistical mechanics is, so we argue, the standard Gibbs formalism applied on the level of space-time histories. The assumptions (chaoticity principle) underlying the Gallavotti–Cohen fluctuation theorem make it possible, using symbolic dynamics, to employ the theory of one-dimensional lattice spin systems. The Kurchan and Lebowitz–Spohn analysis of this fluctuation theorem for stochastic dynamics can be restated on the level of the space-time measure which is a Gibbs measure for an interaction determined by the transition probabilities. In this note we understand the fluctuation theorem as a Gibbs property, as it follows from the very definition of Gibbs state. We give a local version of the fluctuation theorem in the Gibbsian context and we derive from this a version also for some class of spatially extended stochastic dynamics.     

Digital watermarking for DVD video copy protection

We illustrate the various issues that play a role in designing a copy-protection system for digital versatile disk (DVD) video as perceived by Millennium, one of the two contenders in the DVD-video copy-protection standardization activity. We present the Millennium watermark system, the systems proposed for DVD video copy protection by Philips, Macrovision, and Digimarc. We also address some specific system aspects, such as watermark detector location and copy generation control