A direct introduction of <Superscript>18</Superscript>O isotopes into peptides and proteins for quantitative mass spectroscopy analysis

A method for direct introduction of ¹⁸O isotopes into carboxyl groups of peptides and proteins via the exchange with H₂ ¹⁸O in the presence of TFA is described. The isotope label is sufficiently stable in a wide pH range. Since the compounds labeled by this method retain their physicochemical characteristics, they can be used as an internal standard in quantitative assay of authentic compounds in the analyzed objects by means of mass spectrometry. This method is applicable to quantitative analysis of peptides and proteins in biological environments, as well as for quantitative kinetic studies of metabolism and enzyme activity. The quantitative analysis of polypeptides and proteins is combined with trypsinolysis. When necessary, the isotope label can be simultaneously introduced into all peptides and proteins in a control biosample, making it applicable as a standard for comparative analysis of experimental biosamples.     

High-sensitive chemiluminescent assay for cholesterol

Chemiluminescent measurement of cholesterol can be performed in various biological tissues and fluids. The method described in this study has a sensitivity of 54 pmol. The tissue samples used for the determination of cholesterol can be reduced to as little as 1 mg and assay can be performed on diluted biological fluids, allowing sampling of plasma or serum as little as 5 μl. Cholesterol is solubilized in sodium cholate and aliquots are added to a reaction mixture containing cholesterol oxidase, luminol and peroxidase. Cholesterol oxidase, in the presence of cholesterol yields H₂O² which produces light in presence of luminol and peroxidase. Emitted light is quantified at a wavelength of 420 nm by means of a photomultiplier. Optimal conditions of the assay were determined and examples of cholesterol determinations, in blood plasma and nervous tissues, are presented.     

Specimen preparation for quantitative microradiography

Summary The great importance of the pretreatment of biological objects for quantitative microradiography is discussed. Freeze-drying was proved a suitable method in obtaining dehydrated sections without any measurable change in the dry substance. The different steps of the freeze-drying procedure are analyzed with special reference to their effects on mass determinations. The most important errors are the effects of shrinkage and incomplete drying, their magnitude being discussed. For microradiographic determination of dry mass per unit volume the thickness of the freeze-dried tissue sections must be measured. Different methods for thickness determination are discussed and for freeze-dried sections a procedure for focusing in a microscope proved appropriate, the error in a single determination being less than 1 m.This work was supported by grants from the Swedish Medical Research Council (12X 587), Stiftelsen Therese and Johan Anderssons Minne, and Karolinska Institutet (Reservationsanslaget).     

A sensitive atomic fluorescence system for metals in biological systems

An atomic fluorescence spectrometric system for trace elemental determinations in biological samples is described. A heated graphite atomization furnace is used, with continuous sample introduction. Carbonic anhydrase and DNA dependent RNA polymerase enzymes are employed as application models, and accurate Zn determinations at the 10^?7m level are made on enzyme samples of 0.1 mg and less with a precision of 1–2%. The instrumentation is relatively simple, the system is versatile and has excellent stability.     

Synthesis, characterization and selective fluorescent zinc(II) sensing property of three Schiff-base compounds

Three Schiff-base compounds, 4-methyl-2,6-bis(1-(2-piperidinoethyl)iminomethyl)-phenol (HL¹), 4-methyl-2,6-bis(1-(2-pyrrolidinoethyl)- iminomethyl)-phenol (HL²) and (4-methyl-2,6-bis(1-(2-morpholinoethyl)iminomethyl)-phenol) (HL³), have been synthesized and characterized by elemental analysis, FT-IR, ¹H NMR, UV-Vis, electrospray ionisation mass and fluorescence spectroscopy. The emission quantum yield of the compounds increases by ca. 10-17 times by the addition of Zn²⁺ ion. Introduction of other metal ions of biological and environmental relevance either keeps unaltered or quenches the emission intensity of the ligands. This happens because of large binding constant (∼10⁴ M⁻¹) of the ligand with Zn²⁺ ion in acetonitrile. Each of the three ligands forms 1: 2 (ligand:metal) complexes which are characterized by single crystal X-ray diffraction analyses. This imposes rigidity to the ligand due to the complexation and, as a result, the radiative decay constant increases and the corresponding nonradiative decay parameter decreases. All of the ligands react with zinc chloride in acetonitrile to form dinuclear complexes which have been characterized by the elemental analysis, FT-IR, UV-Vis, electrospray ionisation mass spectroscopies and single crystal X-ray structural determinations.     

The X-ray microanalysis of frozen-hydrated sections in scanning electron microscopy: An evaluation

The present status of the technique to measure concentrations of electrolyte elements and dry mass in 1 μm thick frozen-hydrated sections of soft biological tissues with electron probe X-ray microanalysis in a scanning electron microscope is critically reviewed. The technique is to quench-freeze fresh specimens to < − 180°C, cut 1 μm thick hydrated cryosections −70°C), transfer on to a cold stage (< −170°C) of a suitable microanalytical arrangement, obtain scanning transmission images to identify the cell and tissue compartments, locate an electron probe (several μm² to 100 nm) on the areas of interest and collect X-ray quanta. The X-ray quanta are collected with suitable spectrometers (WDS and EDS) and processed with a computer using a comprehensive programme based on continuum normalization procedures (‘Hall’ programme). The cryosections are analysed first in a hydrated state and second after dehydration within the microanalyser column to obtain directly elemental concentrations in mM kg⁻¹ wet wt and mM kg⁻¹ dry wt of the compartments identified under the beam. The local water-fractions are estimated and the elemental concentrations converted into mM 1⁻¹ water. In the past 7 years the technique has been applied to obtain fully quantitative information on Na, K, Cl, P, S, Ca and H₂O in more than ten types of tissue.     

Collision/Reaction Cell ICP-MS with Shielded Torch and Sector Field ICP-MS for the Simultaneous Determination of Selenium Isotopes in Biological Matrices

The determinations of selenium isotopes in biological samples were performed using both inductively coupled plasma collision/reaction cell quadruple mass spectrometer (CRC-ICP-QMS) and inductively coupled plasma sector field mass spectrometers (SF-ICP-MS). To significantly decrease the argon-based interferences at m/z 74 (³⁶Ar³⁸Ar), 76 (³⁸Ar³⁸Ar, ⁴⁰Ar³⁶Ar), 78 (³⁸Ar⁴⁰Ar), and 80 (⁴⁰Ar⁴⁰Ar), the gas-flow rates of a helium and hydrogen mixture used in the collision cell were optimized to 1.0 mL/min H₂ and 3.5 mL/min He. Under the optimized condition, the precisions for natural selenium isotope ratio measurements of both instruments were evaluated and compared using 100 ppb Se standard solution. A modified external calibration quantification method was applied for the simultaneous determination of clinically used enriched selinocompounds (⁷⁷Se-selenate, ⁸²Se-selenite, ⁷⁶Se-methylseleninic acid^IV, ⁷⁸Se-methylselenonic acid^VI) and to examine their fate in rat organs (liver, kidney, and lung).     

Quality-control method for the determination of biological activity of engineered calcineurin subunit B

The aim of this study was to establish a quality-control method for calcineurin subunit B(CNB) biological activity determinations. CNB enhances the p-nitrophenylphosphate(p NPP) dephosphorylating activity of calcineurin subunit A Δ316 mutant(CNAΔ316). A series of CNB concentrations were fitted to a four-parameter equation to calculate the corresponding p NPP maximum dephosphorylation rates. Values were calculated based on biological activity references using a parallel line method. The method was then validated for accuracy, precision, linearity, linear range, sensitivity, specificity, and robustness. The recovery results were greater than 98%. Intra-plate precision was 6.7%, with inter-plate precision of 10.8%. The coefficient of determination was greater than 0.98. The linear range was 0.05–50 μg m L?1, with sensitivity of 50 μg m L?1. Tested cytokines did not induce CNAΔ316 dephosphorylation of p NPP. The chosen CNAΔ316 concentration range did not affect activity determinations.     

Isotopic Analysis of some Romanian Wines by 2H NMR and IRMS

Isotopic analyses are now official or standard methods in Europe and North America for routine use in testing the authenticity of several food products. These methods are based on the measurement of stable isotope content (²H, ¹³C, ¹⁸O) of the product or of a specific component such as an ingredient or target molecule of the product. The determinations carried out using nuclear magnetic resonance (NMR), and Isotopic Ratio Mass Spectrometry (IRMS), provide information on the botanical and geographical origin of the food product. A deuterium natural abundance quantitative NMR method (SNIF-NMR: Site-specific Natural Isotope Fractionation) was developed as an efficient and powerful tool capable of characterizing the chemical origins of organic molecules and distinguishing their biological and geographical origin. The SNIF method is based on the measurement of deuterium / hydrogen (D/H) ratios at the specific sites of the ethanol. Using these methods, we present the obtained results for a series of Romanian wines. Our results may be used like reference data set for authenticity and origin control of wines.     

Determination of cobalt in urine by gas chromatography—mass spectrometry employing nickel as an internal standard

A gas chromatographic—mass spectrometric method for the determination of cobalt in biological materials employing stable enriched ⁶²Ni as an internal standard and using lithium bis(trifluoroethyl)dithiocarbamate as a chelating agent is described. The method involves the addition of a known amount (1 μg) of ⁶²Ni to the sample, the formation of the chelate and the determination by selected-ion monitoring of the m/z ratio, which corresponds to . No appreciable memory effect was observed, and an acceptable dynamic range of 100 was found. There was good agreement between the cobalt concentration values determined by gas chromatography—mass spectrometry and electrothermal atomic absorption spectrometry. The present method has high sensitivity and can be used for the quantitation of cobalt at concentrations as low as 1 μg/l. The use of enriched ⁶²Ni circumvents the problem caused by endogenous nickel and simultaneously provides data on the nickel concentration in the biological sample without any additional experimental effort.     

Small-angle X-ray scattering study of ribosomal proteins S3, S4 S7 and s20.

Ribosomal proteins S3, S4, S7, S20 from Escherichia coli have been studied by small-angle X-ray scattering techniques. The molecular weights found by X-ray scattering agree with other determinations. The large values of the radii of gyration indicate anisometric particles. A more detailed morphological analysis is hindered by low solubility. An interpretation of the experimental results is given in terms of compact objects of simple shape. Yet the possibility is envisaged that these proteins may be less rigid and compact than other proteins.     

Selenomethionine incorporation into a protein by cell-free synthesis

Multi-wavelength anomalous diffraction phasing is especially useful for high-throughput structure determinations. Selenomethionine substituted proteins are commonly used for this purpose. However, the cytotoxicity of selenomethionine drastically reduces the efficiency of its incorporation in in vivo expression systems. In the present study, an improved E. coli cell-free protein synthesis system was used to incorporate selenomethionine into a protein, so that highly efficient incorporation could be achieved. A milligram quantity of selenomethionine-containing Ras was obtained using the cell-free system with dialysis. The mass spectrometry analysis showed that more than 95% of the methionine residues were substituted with selenomethionine. The crystal of this protein grew under the same conditions and had the same unit cell constants as those of the native Ras protein. The three-dimensional structure of this protein, determined by multi-wavelength anomalous diffraction phasing, was almost the same as that of the Ras protein prepared by in vivo expression. Therefore, the cell-free synthesis system could become a powerful protein expression method for high-throughput structure determinations by X-ray crystallography.     

Use of a recycle-type SEC method as a powerful tool for purification of thiosialoside derivatives

An efficient separation between fully acetylated thiosialoside methyl esters and fully acetylated Neu5Ac2en methyl esters was accomplished by means of a size-exclusion chromatography (SEC) method. Purity determinations and structural elucidation of the isolated compounds were performed by a combination of elemental analyses and spectroscopic analyses, including IR, ¹H, and ¹³C NMR, and mass spectroscopic analyses.     

Development of a dispersive liquid-liquid microextraction method for spectrophotometric determination of barbituric acid in pharmaceutical formulation and biological samples

In this paper, a novel and simple method for the determination of trace amounts of barbituric acid in water and biological samples was developed by using dispersive liquid–liquid microextraction (DLLME) techniques combined with spectrophotometric analysis. The procedure is based on color reaction of barbituric acid with p-dimethylaminobenzaldehyde and extraction of the color product using the DLLME technique. Some important parameters such as reaction conditions and the type and volume of extraction and dispersive solvents as well as the extraction time were investigated and optimized in detail. Under the optimum conditions, the calibration graphs were linear over the range of 5.0 to 200 ng ml⁻¹ with limit of detection of 2.0 ng ml⁻¹. Relative standard deviation for five replicate determinations of barbituric acid at 50 ng ml⁻¹ concentration level was calculated to be 1.64%. Average recoveries for spiked samples were determined to be between 94% and 105%. The proposed method was applied for the determination of barbituric acid in pharmaceutical formulation and biological samples.     

Using Molecular Dynamics Simulations on Crambin to Evaluate the Suitability of Different Continuum Dielectric and Hydrogen Atom Models for Protein Simulations

Abstract

Molecular dynamics simulations of enzymes with enough explicit waters of solvation to realistically account for solute-solvent interactions can burden the computational resources required to perform the simulation by more than two orders of magnitude. Since enzyme simulations even with an implicit solvation model can be imposing for a supercomputer, it is important to assess the suitability of different continuum dielectric models for protein simulations. A series of 100-picosecond molecular dynamics simulations were performed on the X-ray crystal structure of the protein crambin to examine how well computed structures, obtained using seven continuum dielectric and two hydrogen atom models, agreed with the X-ray structure. The best level of agreement between computed and experimental structures was obtained using a constant dielectric of 2 and the all-hydrogen model. Continuum dielectric models of 1,1^*r, and 2^*r also led to computed structures in reasonably good agreement with the X-ray structure. In all cases, the all-hydrogen model gave better agreement than the united atom model, although, in one case, the difference was not significant. Dielectric models of 4, 80, and 4^*r with either hydrogen model yielded significantly poorer fits. It is especially noteworthy that the observed trends did not semiquantitatively converge until about 50 picoseconds into the simulations, suggesting that validation studies for protein calculations based on energy minimizations or short simulations should be viewed with caution.     

Cobalt determination in serum and urine by electrothermal atomic absorption spectrometry

Cobalt determinations in biological fluids are of great interest in biological or toxicological research programs. Cobalturia is often chosen as an indicator for a biological monitoring program in occupational exposure to cobalt dusts. The method described here derives from the IUPAC reference method for nickel determination. It enables cobaltemia and cobalturia to be measured in small samples (1 mL). The mean usual values for cobalt in biological fluids are very low (2.7 nmol L⁻¹ for serum and 6.7 nmol L⁻¹ for urine), and therefore, thus require an analytical procedure with preconcentration and extraction. The sample is mineralized by wet acid digestion. After digestion, inorganic cobalt is extracted in form of ammonium pyrrolidine dithiocarbamate complex into isobutyl methyl ketone and measured in the organic layer by electrothermal atomic absorption spectrometry. The analytical parameters are described in detail. The extraction output is about 99%. The detection limits are 1.93 and 1.89 nmol L⁻¹ for serum and urine, respectively. Sensitivity (expressed as the concentration that gives a 0.044 absorbance) is 3.4 nmol L⁻¹ for serum and 3.3 nmol L⁻¹ for urine. Within-run precision ranged between 3.9 and 2.5% (coefficients of variation) for serum and 4.2 and 1.1% for urine, at 87 and 136 nmol L⁻¹ levels, respectively. Between-run precision ranged between 4.3 and 3.3% (coefficients of variation) for serum and 4.2 and 2.3% for urine, at 87 and 136 nmol L⁻¹ levels, respectively. At very low concentration, 5.7 nmol L⁻¹ for serum and 2.5 nmol L⁻¹ for urine, the between-run precision is, respectively, 19.5 and 28%. Linearity is effective between 0 and 272 nmol L⁻¹. Interferences and matrix effects are negligible for urine, serum, or plasma samples without hemoglobin. The method is easily applicable for routine determinations.     

X-ray spectra from laser targets in experiments on the SOKOL-P facility

X-ray spectra from targets irradiated by picosecond laser pulses with intensities of 3 × 10¹⁷-10¹⁸ W/cm² have been studied experimentally on the SOKOL-P facility. Both massive metal targets and multilayer targets with a buried emitting layer have been examined. The measurement results are interpreted by using numerical simulations and theoretical analysis. Experimental data on the X-ray continuum in the photon energy range of 0.8–6 keV and the line spectra of hydrogen-and helium-like aluminum ions are found to agree satis-factorily with numerical results.     

Quantitative X-ray microanalysis of calcium with the Camebax-TEM system in frozen,freeze-substituted and resin-embedded tissue sections

Summary The relevance of the continuum method for a quantitative X-ray microanalysis of epon embedded tissue sections in the particular conditions offered by the Camebax-TEM system was tested and an improved model of specimen holder is proposed.The absolute calcium concentration [Ca] of membrane-bound intracellular glio-interstitial granules was determined by X-ray microanalysis in transmission electron microscopy of Mytilus retractor muscle. The Ca peak and background values were measured by the wavelength-dispersive spectrometer of the Camebax; the mass thickness of the section was recorded simultaneously with an added energy-dispersive detector. The tissue was frozen at 77 K in a mixture of liquid propane and butane, freeze-substituted in the presence of oxalic acid and embedded in epoxy resin. The calcium concentration of glio-intestitial granules can be as high as 180 mmol·kg^–1 of epoxy-embedded tissue, with an average of 40 mmol·kg^–1. The sampling of the data through repcated experiments is discussed and it is proposed that the cell would be the main level of variation. The Ca content of glio-interstitial granules is significantly lower in the tissbes of animals submitted to high-potassium artificial seawater for 10 min. This finding was predicted by the hypothesis that glio-interstitial tissue is a regulator of calcium concentration in extracellular spaces.     

Creating Objects and Object Categories for Studying Perception and Perceptual Learning

In order to quantitatively study object perception, be it perception by biological systems or by machines, one needs to create objects and object categories with precisely definable, preferably naturalistic, properties¹. Furthermore, for studies on perceptual learning, it is useful to create novel objects and object categories (or object classes) with such properties².Many innovative and useful methods currently exist for creating novel objects and object categories^3-6 (also see refs. 7,8). However, generally speaking, the existing methods have three broad types of shortcomings.First, shape variations are generally imposed by the experimenter^5,9,10, and may therefore be different from the variability in natural categories, and optimized for a particular recognition algorithm. It would be desirable to have the variations arise independently of the externally imposed constraints.Second, the existing methods have difficulty capturing the shape complexity of natural objects^11-13. If the goal is to study natural object perception, it is desirable for objects and object categories to be naturalistic, so as to avoid possible confounds and special cases.Third, it is generally hard to quantitatively measure the available information in the stimuli created by conventional methods. It would be desirable to create objects and object categories where the available information can be precisely measured and, where necessary, systematically manipulated (or ''tuned''). This allows one to formulate the underlying object recognition tasks in quantitative terms.Here we describe a set of algorithms, or methods, that meet all three of the above criteria. Virtual morphogenesis (VM) creates novel, naturalistic virtual 3-D objects called ''digital embryos'' by simulating the biological process of embryogenesis¹⁴. Virtual phylogenesis (VP) creates novel, naturalistic object categories by simulating the evolutionary process of natural selection^9,12,13. Objects and object categories created by these simulations can be further manipulated by various morphing methods to generate systematic variations of shape characteristics^15,16. The VP and morphing methods can also be applied, in principle, to novel virtual objects other than digital embryos, or to virtual versions of real-world objects^9,13. Virtual objects created in this fashion can be rendered as visual images using a conventional graphical toolkit, with desired manipulations of surface texture, illumination, size, viewpoint and background. The virtual objects can also be ''printed'' as haptic objects using a conventional 3-D prototyper.We also describe some implementations of these computational algorithms to help illustrate the potential utility of the algorithms. It is important to distinguish the algorithms from their implementations. The implementations are demonstrations offered solely as a ''proof of principle'' of the underlying algorithms. It is important to note that, in general, an implementation of a computational algorithm often has limitations that the algorithm itself does not have.Together, these methods represent a set of powerful and flexible tools for studying object recognition and perceptual learning by biological and computational systems alike. With appropriate extensions, these methods may also prove useful in the study of morphogenesis and phylogenesis.     

Quantitative Proteomic Analysis of Ribosome Assembly and Turnover In Vivo

Although high-resolution structures of the ribosome have been solved in a series of functional states, relatively little is known about how the ribosome assembles, particularly in vivo. Here, a general method is presented for studying the dynamics of ribosome assembly and ribosomal assembly intermediates. Since significant quantities of assembly intermediates are not present under normal growth conditions, the antibiotic neomycin is used to perturb wild-type Escherichia coli. Treatment of E. coli with the antibiotic neomycin results in the accumulation of a continuum of assembly intermediates for both the 30S and 50S subunits. The protein composition and the protein stoichiometry of these intermediates were determined by quantitative mass spectrometry using purified unlabeled and ¹⁵N-labeled wild-type ribosomes as external standards. The intermediates throughout the continuum are heterogeneous and are largely depleted of late-binding proteins. Pulse-labeling with ¹⁵N-labeled medium time-stamps the ribosomal proteins based on their time of synthesis. The assembly intermediates contain both newly synthesized proteins and proteins that originated in previously synthesized intact subunits. This observation requires either a significant amount of ribosome degradation or the exchange or reuse of ribosomal proteins. These specific methods can be applied to any system where ribosomal assembly intermediates accumulate, including strains with deletions or mutations of assembly factors. This general approach can be applied to study the dynamics of assembly and turnover of other macromolecular complexes that can be isolated from cells.