[Show abstract][Hide abstract] ABSTRACT: We report the first direct electrochemistry of CYP109C1, CYP109C2 and CYP109D1 from the myxobacterium Sorangium cellulosum So ce56 immobilized on the screen-printed graphite electrode (SPE) modified by a didodecyldimethylammonium bromide (DDAB) film. Electrochemical response was investigated by cyclic voltammetry. Cyclic voltammograms in a deoxygenated argon saturated 0.1 M potassium phosphate buffer, pH 7.4, demonstrated a reversible redox process with E°‘ of −0.305 ± 0.005 V, −0.313 ± 0.005 V, −0.309 ± 0.005 V (vs. Ag/AgCl) for CYP109C1, CYP109C2 and СYP109D1, respectively. The apparent surface coverages were (2.5 ± 0.2) × 10−11, (2.5 ± 0.2) × 10−11, (3.1 ± 0.2) × 10−11 mol cm−2, which corresponded to 5-6% of the total amount of protein that was consumed by the immobilization process. The rate constant ks of heterogeneous electron transfer between the hemoproteins and the SPE modified by DDAB film (SPE/DDAB) were determined as 0.39 ± 0.01 s−1, 0.35 ± 0.01 s−1, and 0.43 ± 0.02 s−1 for CYP109C1, CYP109C2 and СYP109D1, respectively. The investigated CYPs (CYP109C1, CYP109C2 and CYP109D1) demonstrated electrocatalytic activity detected by an increase of the reduction current in the presence of dissolved oxygen. Upon addition of the substrates (myristic acid and the norisoprenoid α-ionone) in the air-saturated solution, the reduction peak current of dissolved oxygen increased, which manifested the catalytic behavior of CYP109C2 and CYP109D1 towards the substrates. CYP109D1-depended electrocatalytic hydroxylation of the myristic acid was analyzed by mass-spectrometry after electrolysis at controlled working electrode potential.
[Show abstract][Hide abstract] ABSTRACT: A method for detection and identification of the hepatitis C virus antigen (HCVcoreAg) in human serum with consideration for possible amino acid substitutions is proposed. The method is based on a combination of biospecific capturing and concentrating of the target protein on the surface of the chip for atomic force microscope (AFM chip) with subsequent protein identification by tandem mass spectrometric (MS/MS) analysis. Biospecific AFM-capturing of viral particles containing HCVcoreAg from serum samples was performed by use of AFM chips with monoclonal antibodies (anti-HCVcore) covalently immobilized on the surface. Biospecific complexes were registered and counted by AFM. Further MS/MS analysis allowed to reliably identify the HCVcoreAg in the complexes formed on the AFM chip surface. Analysis of MS/MS spectra, with the account taken of the possible polymorphisms in the amino acid sequence of the HCVcoreAg, enabled us to increase the number of identified peptides.
No preview · Article · Jan 2016 · Journal of virological methods
[Show abstract][Hide abstract] ABSTRACT: Microwave radiation at 3.4–4.2GHz frequency of the cytochrome P450 CYP102A1 (BM3) solution was registered during the lauric acid hydroxylation reaction. The microwave radiation generation was shown to occur following the addition of electron donor NADPH to a system containing an enzyme and a substrate. The radiation occurs for the enzyme solutions with enzyme concentrations of 10−8 and 10−9М. The microwave radiation effect elicited by the aqueous enzyme solution was observed for the first time. The results obtained can be used to elaborate a new approach to enzyme systems research, including studying of the mechanism of interaction of a functioning enzyme system with microenvironment.
[Show abstract][Hide abstract] ABSTRACT: In order to obtain more information about human proteome, especially about proteoforms (protein species) coded by 18th chromosome, proteins from human cancer cell line (HepG2) were separated by two-dimensional gel electrophoresis (2DE). Initially, proteins in major spots were identified. According to parameters (pI/Mw) of identified proteins the gel was calibrated. Using this calibrated gel, a virtual two-dimensional (2D) map of proteoforms coded by chromosome 18 was constructed. Next, the produced gel was divided into 96 sections with determined coordinates. Each section was cut, shredded, and treated by trypsin according to mass-spectrometry protocol. After protein identification by shotgun mass-spectrometry using LC ESI-MS/MS, a list of 22771 proteoforms (product of 3789 genes) was generated. Among them, 176 proteoforms are representing 39 genes of 18th chromosome. The 3D-graphs showing distribution of different proteoforms from the same gene in 2D map were generated. This is a first step in creation of 2DE-based knowledge database of proteins coded by 18th chromosome.
No preview · Article · Dec 2015 · Journal of Proteome Research
[Show abstract][Hide abstract] ABSTRACT: Hard conditions of long-term manned spaceflight can affect functions of many biological systems including a system of drug metabolism. The cytochrome P450 (CYP) superfamily plays a key role in the drug metabolism. In this study we examined the hepatic content of some P450 isoforms in mice exposed to 30 days of space flight and microgravity. The CYP content was established by the mass-spectrometric method of selected reaction monitoring (SRM). Significant changes in the CYP2C29, CYP2E1 and CYP1A2 contents were detected in mice of the flight group compared to the ground control group. Within seven days after landing and corresponding recovery period changes in the content of CYP2C29 and CYP1A2 returned to the control level, while the CYP2E1 level remained elevated. The induction of enzyme observed in the mice in the conditions of the spaceflight could lead to an accelerated biotransformation and change in efficiency of pharmacological agents, metabolizing by corresponding CYP isoforms. Such possibility of an individual pharmacological response to medication during long-term spaceflights and early period of postflight adaptation should be taken into account in space medicine.
[Show abstract][Hide abstract] ABSTRACT: Tyrosine based electrochemical analysis of various isoforms of the amyloid-β fragment 1-16 (Aβ16), representing the metal-binding domain of Alzheimer's human Aβ peptide with amino acid substitutions and post-translational modification (D7H, D7N, H6R, H6A-H13A, E11K, and pS8), was carried out by square wave voltammetry on carbon screen printed electrodes. Electrochemical analysis allowed for distinguishing: (i) some isoforms under study from the "normal variant" of the Aβ16; and (ii) the isoforms from one another. Effects of Zn(II) ions on Aβ16 isoforms' oxidation were studied within a wide range of Zn(II) ion concentrations in HEPES-buffers with the pH values of 5.5 to 9. Except for H6A-H13A-Aβ16, addition of Zn(II) ions significantly reduced the intensity of oxidation signals for Aβ16 and its isoforms and shifted the peaks to the more positive potentials. H6A-H13A-Aβ16 demonstrated distinctly different electrochemical behavior both in the absence and presence of Zn(II) ions. The observed effects were discussed in the light of known modes of the Zn(II) ions binding to Aβ16 and its isoforms. The proposed electrochemical assay based on the direct oxidation signal of a tyrosine residue emerges as a very promising tool for monitoring the conformational changes of Aβ peptides in vitro as well as for studying the effects of point mutations and amino acid modifications on the conformation of peptide-metal ion complexes.
No preview · Article · Nov 2015 · Electrochimica Acta
[Show abstract][Hide abstract] ABSTRACT: Virtual and experimental 2DE coupled with ESI LC-MS/MS was introduced to obtain better representation of the information about human proteome. The proteins from HEPG2 cells and human blood plasma were run by two-dimensional gel electrophoresis (2DE). After staining and protein spot identification by MALDI-TOF mass-spectrometry, the protein maps were generated. The experimental physicochemical parameters (pI/Mw) of the proteoforms further detected by ESI LC-MS/MS in these spots were obtained. Next, the theoretical pI and Mw of identified proteins were calculated using program Compute pI/Mw (http://web.expasy.org/compute_pi/pi_tool-doc.html). Accordingly, the relationship between theoretical and experimental parameters was analyzed, and the correlation plots were built. Additionally, virtual/experimental information about different protein species/proteoforms from the same genes was extracted. As it was revealed from the plots, the major proteoforms detected in HepG2 cell line have pI/Mw parameters similar to theoretical values. In opposite, the minor protein species have mainly very different from theoretical pI and Mw parameters. A similar situation was observed in plasma in much higher degree. It means that minor protein species are heavily modified in cell and even more in plasma proteome. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: New types of organic-inorganic hybrid nanocomposites based on nanosized Titanium (IV) oxide TiO2 (<100 nm particle size) and carbon nanotubes (CNT, outer diameter 10-15 nm, inner diamentre 2-6 nm, length 0.1-10 m) and phosphatidilcholine were elaborated for improvement of analytical characteristics of screen printed electrodes. These nanomaterials were employed as an interface for the immobilization of skeletal myoglobin. Electrochemical behavior of myoglobin on such interfaces was characterized with cyclic voltammetry (CV) and square wave voltammetry (SWV). Direct unmediated electron transfer between myoglobin and electrodes modified with organic-inorganic hybrid nanocomposites was registered. TiO2 film and CNT film are biocompartible nanomaterials for myoglobin as was demonstrated with UV-Vis spectra. The midpoint potential of Fe3+/Fe2+ pair of myoglobin corresponded to Е1/2 = -0,263 V for CNT film, and Е1/2 = -0,468 V for TiO2 nanocomposite (vs. Ag/AgCl reference electrode).
[Show abstract][Hide abstract] ABSTRACT: Molecularly imprinted poly-o-phenylenediamine with template myoglobin molecules (i.e., polymeric antibodies to myoglobin, molecularly imprinted polymer, MIP) was synthesized via electropolymerization. Electropolymerization, washing, and the interaction of the polymeric antibodies with myoglobin was examined by square wave voltammetry and microgravimetry. The analysis of myoglobin was carried out through direct electrochemical detection of the reduction peak of Fe3+ of the hemeprotein on screen-printed graphite electrodes modified by the MIP. According to the electrochemical analysis, MIP surfaces demonstrated remarkably higher ability to bind the protein compared to that of surfaces prepared by the same route under the same conditions but in the absence of myoglobin (surfaces of the non-imprinted polymer, NIP). The imprinting factor I
max(NIP) was found to be 2–4. The equilibrium dissociation constant K
d of the interaction of myoglobin with MIP electrodes was evaluated as (2.4 ± 0.5) × 10–8 M. The lower detection limit of myoglobin by a MIP sensor was determined as 0.5 × 10–9 M, the range of detectable concentrations being 10–9–10–5 M.
[Show abstract][Hide abstract] ABSTRACT: The electrochemical activity of cytochrome P450 (CYP) 3A4, 2C9 and 2D6 on a screen-printed graphite electrode nanostructured with gold nanoparticles and stabilized with didodecyldimethylammonium bromide was examined. The analysis of the CYP catalytic activity was carried out using a variety of electrochemical techniques, such as cyclic voltammetry, square wave voltammetry and amperometry. A sensitive electrochemical CYP-sensor system was proposed as a reliable candidate for investigating the influence of medicinal preparations - ethoxidol (2-ethyl-6-methyl-3-hydroxypyridine malate), mexidol (2-ethyl-6-methyl-3-hydroxypyridine succinate), cytochrome c and l-carnitine - on the CYP redox behavior and electrocatalytic activity. In the presence of antioxidants, the enhancement of the electrochemical signal of CYP was registered. It was shown that ethoxidol, in the concentration range of 90-600 μM, stimulates the electrochemical reduction of P450 cytochromes, with the maximum stimulating effects for CYP3A4, CYP2C9 and CYP2D6 being estimated as 181 ± 15%, 240 ± 7% and 110 ± 4% (at 540 μM ethoxidol), respectively.
[Show abstract][Hide abstract] ABSTRACT: The detection of cancer protein marker D-NFATc1 in the serum with the reusable nanowire (NW) chip based on silicon-on-insulator (SOI) structures, was demonstrated. The NW surface was modified with aptamers against D-NFATc1 to attain the biospecific detection of target protein. Two fabricated NW chip types with narrow NWs (w=90 nm) and wide NWs (w=3 μm) were compared with respect to their reuse, i.e. realizability of the repeated detection-regeneration cycles upon D-NFATc1 detection in the serum. The analysis of the serum has shown that the signal obtained with wide NWs was much more stable than that obtained with the narrow NWs. This makes SOI-NW biosensor with wide NWs much more suitable for protein analysis in biological fluids. The signal stability exhibited by the wide NWs allowed to perform repeated detection-regeneration cycles of this chip for multiple detection of D-NFATc1 protein in the serum with 10^(-14) M sensitivity. Although the narrow NW chip allows to attain higher sensitivity (with the concentration detection limit DL=10^(-15) M), it exhibits much lesser signal stability upon analysis of multicomponent biological fluid (serum).
No preview · Article · Jul 2015 · Analytical methods
[Show abstract][Hide abstract] ABSTRACT: Alzheimer’s disease is the most prevalent neurodegenerative pathology. According to the amyloid cascade hypothesis, transition of the amyloid-β peptide (Aβ) from the monomeric form to the aggregated state is a key event in pathogenesis of the Alzheimer’s disease. The mechanism of Aβ aggregation is intensively studied in vitro, by means of synthetic peptides and various physico-chemical methods allowing evaluation of size, molecular structure, and morphology of the formed aggregates. The review considers both the wellknown and recently introduced physico-chemical methods for analysis of Aβ aggregation, including microscopy, optical and fluorescent methods, electron paramagnetic resonance, electrochemical and electrophoretic methods, gel-filtration, and mass spectrometric methods. Advantages and disadvantages of these methods are considered. Special attention is paid to the unique possibility of simultaneous analysis of both Aβ monomers and its oligomers as well as large aggregates by means of atomic force microscopy or fluorescence correlation spectroscopy. The high detection sensitivity of the latter method provides opportunity for investigating the aggregation process in Aβ solutions of low peptide concentrations. Among mass spectrometric methods, the ion mobility mass spectrometry is considered as a method enabling to obtain information about both the spectrum of Aβ oligomers and their structure. Simultaneous employment of several methods providing complementary data about Aβ aggregates is the best experimental approach for studying the process of Aβ aggregation in vitro.
No preview · Article · Jul 2015 · Biochemistry (Moscow) Supplement Series B Biomedical Chemistry
[Show abstract][Hide abstract] ABSTRACT: Own experimental studies on the development of highly sensitive methods of electrochemical analysis applicable for biochemical research in the postgenomic era as well as electrochemical sensor systems for analysis of various biological objects have been summarized. Electroanalysis of catalytic activity of cytochrome P450 resulted in the development of a system for screening potential substrates and/or inhibitors of this class hemoproteins, as well as biologically active compounds modulating the catalytic function of this protein. The study of kinetics of bioaffinity troponin I/anti-troponin I (antibody to TnI) interactions in human plasma resulted in the development of a highly sensitive piezoelectric immunosensor, performing direct registration of biochemical interactions based on the difference of the kinetic parameters of specific and nonspecific bioaffinity interactions without additional administration of labels and without chemical modifications. The developed methods of direct registration of the electrochemical activity of bacterial cells Escherichia coli JM109 are applicable for real time evaluation of antibacterial activity of drug substances; this requires minimal volumes of cells (106 CFU/electrode). Special attention is paid to experimental data on preparation of polymers with molecular imprints (molecularly imprinted polymers, MIP) as analogues of antibodies and biorecognizing elements, carrying selective complementary analytes binding based on the “lock and key” principle.
Full-text · Article · Jul 2015 · Biochemistry (Moscow) Supplement Series B Biomedical Chemistry
[Show abstract][Hide abstract] ABSTRACT: The fundamental mission of Chromosome-centric Human Proteome Project (C-HPP) is the research of human proteome diversity including rare variants. Liver tissues, HepG2 cells and plasma were selected as one of the major objects for C-HPP studies. Proteogenomic approach, a recently introduced technique, is a powerful method to predict and validate proteoforms coming from alternative splicing, mutations and transcript editing. We developed PPLine, Python-based proteogenomic pipeline providing automated SAP/indels and alternative spliced variants discovery basing on raw transcriptome/exome sequence data, SNP annotation and filtration, prediction of proteotypic peptides (available at https://sourceforge.net/projects/ppline). In this work, we performed deep transcriptome sequencing of HepG2 cells and liver tissues using two platforms - Illumina HiSeq and Applied Biosystems SOLiD. Using PPLine, we revealed 7756 SAP and indels for HepG2 and liver (including 659 variants non-annotated in dbSNP). We found 17 indels in transcripts associated with translation of alternate reading frames (ARF) longer than 300 bp. ARF products of two genes, SLMO1 and TMEM8A, demonstrate signatures of caspase binding domain and Gcn5-related N-acetyltransferase. Alternative splicing analysis predicted novel proteoforms encoded by 203 (liver) and 475 (HepG2) genes according to both Illumina and SOLiD data. The results of present work represent a basis for subsequent proteomic studies of C-HPP consortium.
Full-text · Article · Jul 2015 · Journal of Proteome Research
[Show abstract][Hide abstract] ABSTRACT: This paper summarizes the recent activities of the Chromosome-Centric Human Proteome Project (C-HPP) consortium, which develops new technologies to identify yet-to-be annotated proteins (termed "missing proteins") in biological samples that lack sufficient experimental evidence at the protein level for confident protein identification. The C-HPP also aims to identify new protein forms that may be caused by genetic variability, post-translational modifications, and alternative splicing. Proteogenomic data integration forms the basis of the C-HPP's activities; therefore, we have summarized some of key approaches and their roles in the project. We present new analytical technologies that improve the chemical space and lower detection limits coupled with bioinformatics tools and some publicly available resources that can be used to improve data analysis or support the development of analytical assays. Most of this paper's contents have been compiled from posters, slides, and discussions presented in the series of C-HPP workshops held during 2014. All data (posters, presentations) used are available at the C-HPP Wiki (http://c-hpp.webhosting.rug.nl/) and in the supporting information.
Full-text · Article · Jun 2015 · Journal of Proteome Research
[Show abstract][Hide abstract] ABSTRACT: Nanocomposite materials were prepared by sequential drop casting of multi-walled carbon nanotube (MWCNT) suspensions and amphiphilic polybutadiene-block-poly(2-(N,N-dimethylamino)ethyl methacrylate) (PB290-b-PDMAEMA240) diblock copolymer micelles on screen-printed electrodes (SPEs). This nanocomposite material was found to be very favorable for integration of myoglobin (Mb) and facilitates a direct electron transfer from an electrode to heme proteins. In that respect, PB290-b-PDMAEMA240 was demonstrated to be a well-suited binding agent. In aqueous solutions, the diblock copolymer forms core-corona micelles (shown by cryogenic transmission electron microscopy, cryo-TEM, and nanoparticle tracking analysis, NTA), which at pH 7 in phosphate buffer exhibit good adhesion to carbon materials (shown by atomic force microscopy, AFM, scanning electron microscopy, SEM, and scanning transmission electron microscopy, STEM) and builds up uniform thin films on a hydrophobic graphite-based substrate. As demonstrated by a quartz crystal microbalance with dissipation monitoring (QCM-D), attractive interactions of Mb and PB290-b-PDMAEMA240 take place when both components are subsequently deposited onto a solid substrate. Spectroscopic studies confirmed that the absorption maximum of Mb remains unaltered, suggesting that at least some protein globules retain their tertiary structure. Cyclic voltammetry and square wave voltammetry show a remarkable (ca 180-fold) increase of the reductive current of Mb after its incorporation into the SPE/MWCNTs/PB290-b-PDMAEMA240 matrix. The herein developed analytical approach was used for the detection of cardiac myoglobin as a very early marker of acute myocardial infarction (AMI) both in plasma of healthy donors and patients with AMI.
[Show abstract][Hide abstract] ABSTRACT: The functional significance of cytochrome P450 (P450) enzymes includes their ability to catalyze the biotransformation of xenobiotics (foreign compounds) and endogenous compounds. P450 enzymes play an important role in the detoxification of exogenous bioactive compounds and hydrophobic xenobiotics (e.g. carcinogens, drugs, environment pollutants, food supplements, medicines, plant products) and in the biotransformation of endogenous bioactive compounds (e.g. amino acids, cholesterol, eicosanoids, saturated/unsaturated fatty acids, melatonin, steroid hormones). Electrode/P450 systems are analyzed in terms of the mechanisms underlying P450-catalyzed reactions. Bioelectrocatalysis-based screening of potential substrates or inhibitors of P450 enzymes, the stoichiometry of the electrocatalytic cycle, oxidation-reduction (redox) thermodynamics, and the peroxide shunt pathway are described. Electrochemical techniques are utilized for investigating the influence of (1) the vitamin B group, (2) vitamins (e.g. vitamins A and B) and antioxidants (e.g. taurine), and (3) drugs and antioxidants (e.g. mexidol, ethoxidol) on biocatalysis using P450 enzymes, and on the metabolism of drugs catalyzed by P450 3A4. The characteristics, performance and potential applications of P450 electrochemical systems are also discussed.
No preview · Article · May 2015 · Advances in Experimental Medicine and Biology
[Show abstract][Hide abstract] ABSTRACT: In the review the main approaches to creation of recognition materials capable of competing with biological specific receptors, (polymeric analogs of antibodies or molecularly imprinted polymers, MIP) for the electro analysis of functionally significant proteins such as a myoglobin, troponin T, albumin, human ferritin, calmodulin are considered. The main types of monomers for MIP fabrication, and methods for MIP/protein interactions, such as a surface plasmon resonance (SPR), nanogravimetry with use of the quartz crystal resonator (QCM), spectral and electrochemical methods are discussed. Experimental data on electrochemical registration of a myoglobin using MIP/electrode are presented. For a development of electrochemical sensor systems based on MIPs, o-phenylenediamine (1,2-diaminobenzene was used as a monomer. It was shown that the imprinting factor Imax(MIP)/Imax(NIP), calculated as a myoglobin signal ratio when embedding in MIP to a myoglobin signal when embedding in the polymer received without molecular template (NIP) corresponds 2-4.