E. N. Nikolaev

Expression and characterization of a new esterase with GCSAG motif from a permafrost metagenomic library.

As a result of construction and screening of a metagenomic library prepared from a permafrost-derived microcosm, we have isolated a novel gene coding for a putative lipolytic enzyme that belongs to the hormone-sensitive lipase family. It encodes a polypeptide of 343 amino acid residues whose amino acid sequence displays maximum likelihood with uncharacterized proteins from Sphingomonas species. A putative catalytic serine residue of PMGL2 resides in a new variant of a recently discovered GTSAG sequence in which a Thr residue is replaced by a Cys residue (GCSAG). The recombinant PMGL2 was produced in Escherichia coli cells and purified by Ni-affinity chromatography. The resulting protein preferably utilizes short-chain p-nitrophenyl esters (C4 and C8) and therefore is an esterase. It possesses maximum activity at 45°C in slightly alkaline conditions and has limited thermostability at higher temperatures. Activity of PMGL2 is stimulated in the presence of 0.25-1.5 M NaCl indicating the good salt tolerance of the new enzyme. Mass spectrometric analysis demonstrated that N-terminal methionine in PMGL2 is processed and cysteine residues do not form a disulfide bond. The results of the study demonstrate the significance of the permafrost environment as a unique genetic reservoir and its potential for metagenomic exploration.

Ion motion stability diagram for distorted square waveform trapping voltage

Ion motion in a periodic radio frequency (RF) quadrupole electric field is analysed theoretically by the matrix method and direct trajectory calculation. General properties of the ion motion: stability condition, oscillation spectrum and secular frequency are derived analytically from the elements of the transformation matrix. Stability diagrams for ion motion in the Paul ion trap are presented for rectangular waveforms with different duty cycles (duration of pulse over period). Calculation of the secular frequencies of the ion motion in the ion trap is performed for the first time. The relation of radial and axial secular frequencies along the RF scan line was found to be practically identical in both the square waveform and harmonic voltage cases. Pulse shape distortions, due to resistive-inductive-capacitive filtering in real devices, are considered. Stability diagrams of ion motion in the ion trap with distorted voltage waveforms are calculated. Distortion of the waveform is shown to introduce minor changes in the diagram shape with respect to the diagram for an ideal square wave. Within the first stable region, distortion of the waveform does not lead to any auxiliary parametric resonances of the ion motion. Ion trapping with a pure random pulsed voltage is investigated by means of direct trajectory simulations. It is shown that, in this case, the ion motion can be conditionally stable for a considerable length of time.

Solid phase isotope exchange of deuterium and tritium for hydrogen in human recombinant insulin

Reaction of a high-temperature solid-phase catalytic isotope exchange in peptides and proteins under the action of the catalytically activated spillover hydrogen was studied. The reaction of human recombinant insulin with deuterium and tritium at 120–140°C resulted in an incorporation of 2–6 isotope hydrogen atoms per one insulin molecule. The distribution of the isotopic label by amino acid residues of the tritium-labeled insulin was determined by the oxidation of the protein S-S-bonds by performic acid, separation of polypeptide chains, their subsequent acidic hydrolysis, amino acid analysis, and liquid scintillation counts of tritium in the amino acids. The isotopic label was shown to be incorporated in all the amino acid residues of the protein, but the higher inclusion was observed for the FVNQHLCGSHLVE peptide fragment (B1–13) of the insulin B-chain, and the His5 and His10 residues of this fragment contained approximately 45% of the whole isotopic label of the protein. Reduction of the S-S-bonds by 2-mercaptoethanol, enzymatic hydrolysis by glutamyl endopeptidase from Bacillus intermedius, and HPLC fractionation of the obtained peptides were also used for the analysis of the distribution of the isotopic label in the peptide fragments of the labeled insulin. Peptide fragments which were formed after the hydrolysis of the Glu-Xaa bond of the B-chain were identified by mass spectrometry. The mass spectrometric analysis of the isotopomeric composition of the deuterium-labeled insulin demonstrated that all the protein molecules participated equally in the reaction of the solid-phase hydrogen isotope exchange. The tritium-labeled insulin preserved the complete physiological activity.

Exogenous proteins in exhaled human breath condensate

In the course of analysis of protein composition of exhaled breath to diagnose diseases of the respiratory system the problem is raised to distinguish between proteins, expressed in lung tissues and in respiratory tract (endogenous) and those that got into the respiratory system from the ambient air in the process of respiration (exogenous). In this work, an attempt is made to estimate the constitution of exogenous proteins in exhaled air with mass spectrometry and nanoflow high performance liquid chromatography (nano-HPLC). Six months’ indoors isolation of healthy donors with air being cleaned of dust leads to the removal from the spectrum of exhaled proteins of some keratins that are therefore considered to be exogenous. Nonkeratin proteins may also circulate between ambient air and human airways, but their concentration appears to be significantly lower than keratin concentrations (especially than the epidermis keratin). Among nonkeratins, dermicidin seems to be the most significant exogenous protein of the exhaled air. Conclusions concerning the diagnostic value of exhaled proteins can be made only after careful comparison of results of quantitative and qualitative analyses of their normal and pathological composition for a statistically significant sample of donors.

Determination of the non-constant component of the ion mobility using the spectrometer of ion mobility increment.

A new method for determination of the non-constant component, α(E), of an ion mobility, k(E), is suggested. The method uses the relationship U C (U S ) that can be experimentally obtained with a spectrometer of ion mobility increment with planar drift chamber. (U C is a compensating voltage, U S is separating voltage amplitude.) A general equation for α(E) has been derived. We have explored the possibility of determination of α(E) from the experimental data for different types of U S (t). In two specific cases, analytical solutions have been obtained.

Modification of the catalytic subunit of plasma fibrin-stabilizing factor under induced oxidation

For the first time, by using mass-spectrometry method, the oxidation-mediated modification of the catalytic FXIII-A subunit of plasma fibrin-stabilizing factor, pFXIII, has been studied. The oxidative sites were identified to belong to all structural elements of the catalytic subunit: the β-sandwich (Tyr104, Tyr117, and Cys153), the catalytic core domain (Met160, Trp165, Met266, Cys328, Asp352, Pro387, Arg409, Cys410, Tyr442, Met475, Met476, Tyr482, and Met500), the β-barrel 1 (Met596), and the β-barrel 2 (Met647, Pro676, Trp692, Cys696, and Met710), which correspond to 3.9%, 1.11%, 0.7%, and 3.2%, respectively, of oxidative modifications as compared to the detectable amounts of amino acid residues in each of the structural domains. Lack of information on some parts of the molecule may be associated with the spatial unavailability of residues, complicating analysis of the molecule. The absence of oxidative sites localized within crucial areas of the structural domains may be brought about by both the spatial inaccessibility of the oxidant to amino acid residues in the zymogen and the screening effect of the regulatory FXIII-B subunit.

Oxidation-induced modification of the fibrinogen polypeptide chains

By using the mass-spectrometry method, the oxidative modifications of the fibrinogen Aα, Bβ, and γ polypeptide chains induced by its oxidation have been studied. The αC-region has been proven to be the most vulnerable target for the oxidizer (ozone) as compared with the other structural elements of the Aα chain. The Bβ chain mapping shows that the oxidative sites are localized within all the structural elements of the chain in which the β-nodule exhibits high susceptibility to oxidation. The γ chains are the least vulnerable to the oxidizer action. The results obtained demonstrate convincingly that the self-assembly centers dealing with interactions of knob “A”: hole “a” are not involved in oxidative modification. It is concluded that the numerous oxidative sites revealed are mainly responsible for inhibiting lateral aggregation of protofibrils. The part of amino acid residues subjected to oxidation is supposed to carry out the antioxidant function.

A new method for normalization of the peptide retention times in chromatographic/mass-spectrometric experiments

Proteomic studies with the use of mass spectrometry required comparison of different experimental data (for example, control with a pathology or labeled and unlabeled samples). Identification of chromatographic peaks of the same substance in different chromatograms (time normalization of chromatograms) is complicated if peptides are identified in different experiments according only to their exactly evaluated masses and retention times on a chromatographic column. Retention times of the same peptides would vary from one experiment to another due to inevitable differences in experimental chromatographic conditions (replacement of chromatographic columns, slight changes in flow rates of a mobile phase or in a solvent concentration, or in other conditions). We proposed a reliable method for selection of peaks that corresponded to the same peptides from chromatography/mass spectra for the subsequent alignment of retention times (both a linear and some other monotone function).

Analysis of non-linear ion drift in spectrometers of ion mobility increment with cylindrical drift chamber

A model for the drift of ions under a non-uniform, high-frequency electric field in the drift chamber of a spectrometer of ion mobility increment is developed. For the general dependence of the ion mobility on the electric field strength and the general time-dependence of the separating voltage, we suggest a procedure for calculating of the ion peak form. The shape of the peak for the ion focusing and defocusing conditions has been obtained.

Hydrothermal treatment of organic waste

The yield and properties of solid and liquid products of hydrothermal treatment of organic waste were determined with cheese, meat, and apples as an example. The solid products of hydrothermal treatment of cheese and apples have higher carbon content, lower oxygen content, and, correspondingly, higher heat of combustion compared to the initial biomass, which allows these products to be considered as a promising solid biofuel. The oils obtained in experiments with cheese and meat also have higher carbon content and higher heat of combustion compared to the initial substances, which allows these products to be considered as a promising liquid biofuel.