Liquid chromatography-atmospheric pressure electron capture dissociation mass spectrometry for the structural analysis of peptides and proteins

Atmospheric pressure electron capture dissociation (AP-ECD) is an emerging technique with the potential to be a more accessible alternative to conventional ECD/electron transfer dissociation (ETD) methods because it can be implemented using a stand-alone ion source device suitable for use with any existing or future electrospray ionization mass spectrometer. With AP-ECD, no modification of the main instrument is required, so it may easily be retrofitted to instruments not originally equipped with ECD/ETD capabilities. Here, we present our first purpose-built AP-ECD source and demonstrate its use in conjunction with capillary LC for the analysis of substance P, a tryptic digest of bovine serum albumin, and a phosphopeptide mixture. Quality ECD spectra were obtained for all the samples at the low femtomole level, proving that LC-AP-ECD-MS is suitable for the structural analysis of peptides and protein digests, in this case using an unmodified quadrupole time-of-flight mass spectrometer built ca. 2002.     

Assessment of cellular oxygen gradients with a panel of phosphorescent oxygen-sensitive probes

The supply of oxygen (O(2)) to respiring tissue, cells, and mitochondria regulates metabolism, gene expression, and cell fate. Depending on the cell type and mitochondrial function, O(2) gradients between extra- and intracellular compartments may vary and play important physiological roles such as the regulation of activity of prolyl hydroxylases and adaptive responses to hypoxia. Here we present a new methodology for the analysis of localized O(2) gradients in cultures of adherent cells, using three phosphorescent Pt-porphyrin based probes with different localization. One new O(2) probe targeted to the cell membrane was developed and used together with existing MitoXpress and Nano2 probes to monitor mean pericellular (PC), extracellular (EC), and intracellular (IC) O(2) concentrations, respectively. Mouse fibroblasts and neuronal PC12 cells cultured in standard microplates were stained with probes and measured on a commercial time-resolved fluorescence reader in phosphorescence lifetime mode. Respiring cells exposed to various levels of atmospheric O(2) showed differences in oxygenation of their IC, PC, and EC compartments. Experiments with different cell numbers and modulation of respiration activity demonstrated that these gradients are dynamic and regulated by the O(2) diffusion and consumption rate. The new method facilitates the assessment of such gradients.     

High-precision isothermal titration calorimetry with automated peak-shape analysis

Isothermal titration calorimetry (ITC) is a powerful classical method that enables researchers in many fields to study the thermodynamics of molecular interactions. Primary ITC data comprise the temporal evolution of differential power reporting the heat of reaction during a series of injections of aliquots of a reactant into a sample cell. By integration of each injection peak, an isotherm can be constructed of total changes in enthalpy as a function of changes in solution composition, which is rich in thermodynamic information on the reaction. However, the signals from the injection peaks are superimposed by the stochastically varying time-course of the instrumental baseline power, limiting the precision of ITC isotherms. Here, we describe a method for automated peak assignment based on peak-shape analysis via singular value decomposition in combination with detailed least-squares modeling of local pre- and postinjection baselines. This approach can effectively filter out contributions of short-term noise and adventitious events in the power trace. This method also provides, for the first time, statistical error estimates for the individual isotherm data points. In turn, this results in improved detection limits for high-affinity or low-enthalpy binding reactions and significantly higher precision of the derived thermodynamic parameters.     

Vibrations of a vehicle excited by real road profiles

Based on experimental data obtained from the Lublin III delivery car, we have performed a vibration analysis of the vehicle suspension system. Vertical accelerations on the left and right sides of the suspension system were measured. Experiments were carried out on three types of road surfaces: (a) asphalt, (b) sett, and (c) railway cross. The acceleration signals were examined using Fourier transform, multiscale entropy analysis and continuous wavelet transform. These methods reveal the characteristics of the vibration patterns produced by the various road surface profiles. Our results can be used to assess the efficacy of a vehicle suspension system under different road conditions.     

Preparation and characterization of magnetic carbon nanomaterials bearing APTS–silica on their surface

The formation of metal-encapsulated carbon nanomaterials by using metallic catalysts (iron, cobalt, and nickel) has been studied. Moreover, these materials were coated with silica surface modified by (3-Aminopropyl)-trimethoxysilane (APTS). Each intermediate structure was confirmed by X-ray diffraction (XRD) and transmission electron microscopy (TEM). The surface morphology of silica-coated carbon nanomaterials was analyzed by scanning electron microscopy (SEM). The modified, APTS–silica surface was additionally characterized by Fourier transform infrared spectroscopy (FT-IR), elemental (EA), and thermogravimetric analysis (TGA).     

Shape design of internal cooling passages within a turbine blade

The article concerns the optimization of the shape and location of non-circular passages cooling the blade of a gas turbine. To model the shape, four Bezier curves which form a closed profile of the passage were used. In order to match the shape of the passage to the blade profile, a technique was put forward to copy and scale the profile fragments into the component, and build the outline of the passage on the basis of them. For so-defined cooling passages, optimization calculations were carried out with a view to finding their optimal shape and location in terms of the assumed objectives. The task was solved as a multi-objective problem with the use of the Pareto method, for a cooling system composed of four and five passages. The tool employed for the optimization was the evolutionary algorithm. The article presents the impact of the population on the task convergence, and discusses the impact of different optimization objectives on the Pareto optimal solutions obtained. Due to the problem of different impacts of individual objectives on the position of the solution front which was noticed during the calculations, a two-step optimization procedure was introduced. Also, comparative optimization calculations for the scalar objective function were carried out and set up against the non-dominated solutions obtained in the Pareto approach. The optimization process resulted in a configuration of the cooling system that allows a significant reduction in the temperature of the blade and its thermal stress.     

Modeling lubricant flow between thrust-bearing pads

Inlet temperature is one of the main inputs in all models for the analysis of fluid film bearing performance. At the same time, inlet temperature distribution and also oil velocity distribution at the inlet are the result of flow phenomena in the gap between the bearing pads. These phenomena are complex and in many cases further affected by any special arrangements of forced oil supply to the gap between pads. The reason for such arrangements is the more efficient introduction of externally cooled lubricant. Not many details are known about any flow phenomena in the gap between the pads and even less if the bearing is fitted with any kind of directed lubrication system. Contemporary tools of computational fluid dynamics (CFD) enable one to study the flow between bearing pads and even to build models of a whole hydrodynamic bearing. Preliminary results of modeling lubricant flow in the gap, in a bearing with a direct oil supply system will be presented in this paper. The effects of modified inlet oil temperature on some aspects of bearing performance are also presented.     

ABTS-modified multiwalled carbon nanotubes as an effective mediating system for bioelectrocatalytic reduction of oxygen

The ability of such a common redox mediator as 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) to undergo sorption on carbon surfaces is explored here to convert multiwalled carbon nanotubes (CNTs) into a stable colloidal solution of ABTS-modified carbon nanostructures, the diameters of which are approximately 10 nm (as determined by transmission electron microscopy). Subsequently, inks composed of fungal laccase (Cerrena unicolor) mixed with the dispersion of ABTS-modified CNTs and stabilized with Nafion, were deposited on glassy carbon and successfully employed to the reduction of oxygen in McIlvain buffer at pH 5.2. For comparison, the systems utilizing only ABTS-free CNTs and laccase as well as ABTS-modified CNTs did not show appreciable activity toward the oxygen reduction. The three-dimensionally distributed ABTS-modified CNTs are expected to improve the film's overall conductivity and to facilitate electrical connection between the electrode and the enzyme. The network film of ABTS-modified CNTs is rigid, and it is characterized by charge propagation capabilities comparable to the conventional redox polymers. The whole concept of utilization of CNTs modified with ultrathin films of redox mediators in the preparation of efficient bioelectrocatalytic films seems to be of general importance to electroanalytical chemistry and to the development of biosensors.     

Profile of fatty acids, muscle structure and shear force of musculus longissimus dorsi (MLD) in growing pigs as affected by energy and protein or protein restriction followed by realimentation

Forty-eight gilts were submitted to a 30% restriction of feed (groups F and F1) or protein intake (group P) from 90 to 118 days of age, followed by realimentation from 119 to 168 days of age. Control pigs (C) were fed during the whole experiment according to a semi ad libitum scale. During realimentation all animals were fed according to semi ad libitum scale except pigs F1 which were fed ad libitum. Six pigs from each group were slaughtered at the end of restriction and realimentation. Restriction decreased the weight of musculus longissimus dorsi (MLD) and increased shear force. Restriction of feed intake depressed MUFA concentration and increased n-6/n-3 ratio while protein restriction decreased n-6/n-3 and PUFA:SFA ratios. Structure of fibers was not affected. After realimentation MLD mass was still lower in all previously restricted pigs, shear force was the lowest in F1 pigs. Only percentage of fast twich oxidative fibers was significantly greater in F1 pigs than in others. Significant correlations between parameters investigated during the study were found.