[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: 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).
[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.
Journal of Proteome Research 07/2015; 14(9). DOI:10.1021/acs.jproteome.5b00490 · 4.25 Impact Factor
[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.
Journal of Proteome Research 06/2015; 14(9). DOI:10.1021/pr5013009 · 4.25 Impact Factor
[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.
Advances in Experimental Medicine and Biology 05/2015; 851:229-246. DOI:10.1007/978-3-319-16009-2_9 · 1.96 Impact Factor
[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.
[Show abstract][Hide abstract] ABSTRACT: For more than 40 years L-asparaginases are used in combined therapy of acute lymphoblastic leukemia in children and the range of tumors sensitive to these enzymes constantly extends. This review summarizes results of studies aimed at creation of new systems for heterological expression of bacterial L-asparaginases as Erwinia carotovora (EwA), Helicobacter pylori (HpA), Yersinia pseudotuberculosis (YpA) and Rhodospirillum rubrum (RrA); special attention is paid to isolation of purified enzymes and their crystallization, modification by chitosan/polyethylene, physicochemical, kinetic and structural properties characterization, and the study of the cytotoxic or anti-proliferative activity of new recombinant L-asparaginases on cell cultures in vitro. The resultant recombinant L-asparaginases (EwA, YpA, HpA и RrA) exhibit reasonable cytotoxic action on the human leukemia cells comparable to the pharmacologically available L-asparaginase EcA and represent practical interest in respect to creation, on their basis, new effective antineoplastic remedies. Further prospects of researches on bacterial L-asparaginases are associated with development of analogs of Rhodospirillum rubrum L-asparaginase (RrA) by means of directed changes of the protein structure using genetic engineering, development of chito-PEGylation for receiving L-asparaginase preparations with improved pharmacokinetic characteristics.
[Show abstract][Hide abstract] ABSTRACT: A method of atomic force microscopy-based fishing (AFM fishing) has been developed for protein detection in the analyte solution using a chip with an immobilized aptamer. This method is based on the biospecific fishing of a target protein from a bulk solution onto the small AFM chip area with the immobilized aptamer to this protein used as the molecular probe. Such aptamer-based approach allows to increase an AFM image contrast compared to the antibody-based approach. Mass spectrometry analysis used after the biospecific fishing to identify the target protein on the AFM chip has proved complex formation. Use of the AFM chip with the immobilized aptamer avoids interference of the antibody and target protein peaks in a mass spectrum.
[Show abstract][Hide abstract] ABSTRACT: Change in temperature is one of the factors affecting the activity of enzymes. In this work, the thermal denaturation and aggregation of cytochrome P450 BM3 were studied by atomic force microscopy. The specific temperature transitions were studied by fluorescence analysis. In the low melting temperature range (10-33°C), a decrease in the fluorescence intensity of the aromatic residues was observed simultaneously with an increase in the fluorescence intensity of the flavin groups. The protein melting in this range indicated three narrow S-shaped cooperative transitions at 16, 22, and 29°C. Atomic force microscopy analysis in this temperature range showed that the BM3 molecules retained a globular shape as compact objects (heights, h < 7 nm; lateral dimensions, d < 50 nm), but the protein oligomeric state changed. The first two transitions were accompanied by a decrease in the degree of oligomerization and the third one by its increase.
[Show abstract][Hide abstract] ABSTRACT: A combination of (atomic force microscopy)-based fishing (AFM-fishing) and mass spectrometry allows to capture protein molecules from solutions, concentrate and visualize them on an atomically flat surface of the AFM chip and identify by subsequent mass spectrometric analysis. In order to increase the AFM-fishing efficiency we have applied pulsed voltage with the rise time of the front of about 1 ns to the AFM chip. The AFM-chip was made using a conductive material, highly oriented pyrolytic graphite (HOPG). The increased efficiency of AFM-fishing has been demonstrated using detection of cytochrome b 5 protein. Selection of the stimulating pulse with a rise time of 1 ns, corresponding to the GHz frequency range, by the effect of intrinsic emission from water observed in this frequency range during water injection into the cell.
Biochemistry (Moscow) Supplement Series B Biomedical Chemistry 04/2015; 9(2):121-129. DOI:10.1134/S1990750815020080
[Show abstract][Hide abstract] ABSTRACT: A new method for the analysis of blood lipid based on direct mass spectrometry of lipophilic low molecular weight fraction of blood plasma has been considered. Such technique allows quantification of hundreds of various types of lipids and this changes existing concepts on diagnostics of lipid disorders and related diseases. The versatility and quickness of the method significantly simplify its wide use. This method is applicable for diagnostics of atherosclerosis, diabetes, cancer and other diseases. Detalization of plasma lipid composition at the molecular level by means of mass spectrometry allows to assess the effectiveness of therapy and to optimize the drug treatment of cardiovascular diseases by phospholipid preparations.
[Show abstract][Hide abstract] ABSTRACT: Achievement of the concentration detection limit for proteins at the level of the reverse Avogadro number determines the modern development of proteomics. In this review, the possibility of approximating the reverse Avogadro number by using nanotechnological methods (AFM-based fishing with mechanical and electrical stimulation, nanowire detectors, and other methods) are discussed. The ability of AFM to detect, count, visualize and characterize physico-chemical properties of proteins at concentrations up to 10-17-10-18 M is demonstrated. The combination of AFM-fishing with mass-spectrometry allows the identification of proteins not only in pure solutions, but also in multi-component biological fluids (serum). The possibilities to improve the biospecific fishing efficiency by use of SOMAmers in both AFM and nanowire systems are discussed. The paper also provides criteria for evaluation of the sensitivity of fishing-based det ection systems. The fishing efficiency depending on the detection system parameters is estimated. The practical implementation of protein fishing depending on the ratio of the sample solution volume and the surface of the detection system is discussed. The advantages and disadvantages of today's promising nanotechnological protein detection methods implemented on the basis of these schemes.
[Show abstract][Hide abstract] ABSTRACT: Alzheimer's disease is the most prevalent neurodegenerative pathology. According to the amyloid cascade hypothesis, a key event of the Alzheimer's disease pathogenesis is a transition of the b-amyloid peptide (Аb) from the monomeric form to the aggregated state. The mechanism of Аb 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 paper reviews both the well-known and recently introduced physico-chemical methods for analysis of Аb aggregation, including microscopу, optical and fluorescent methods, method of electron paramagnetic resonance, electrochemical and electrophoretic methods, gel-filtration, and mass spectrometric methods. Merits and drawbacks of the methods are discussed. The unique possibility to simultaneously observe Аb monomers as well oligomers and large aggregates by means of atomic force microscopy or fluorescence correlation spectroscopy is emphasized. The high detection sensitivity of the latter method, monitoring the aggregation process in Аb solutions at low peptide concentrations is underlined. Among mass spectrometric methods, the ion mobility mass spectrometry is marked out as a method enabling to obtain information about both the spectrum of Аb oligomers and their structure. It is pointed out that the use of several methods giving the complementary data about Аb aggregates is the best experimental approach to studying the process of b-amyloid peptide aggregation in vitro.
[Show abstract][Hide abstract] ABSTRACT: Huge range of concentrations of different protein and insufficient sensitivity of methods for detection of proteins at a single molecule level does not yet allow obtaining the whole image of human proteome. In our investigations, we tried to evaluate the size of different proteomes (cells and plasma). The approach used is based on detection of protein spots in 2-DE after staining by protein dyes with different sensitivities. The function representing the dependence of the number of protein spots on sensitivity of protein dyes was generated. Next, by extrapolation of this function curve to theoretical point of the maximum sensitivity (detection of a single smallest polypeptide) it was calculated that a single human cell (HepG2) may contain minimum 70000 proteoforms, and plasma - 1.5 mln. Utilization of this approach to other, smaller proteomes showed the competency of this extrapolation. For instance, the size of mycoplas ma (Acholeplasma laidlawii) was estimated in 1100 proteoforms, yeast (Saccharomyces cerevisiae) - 40000, E. coli - 6200, P. furiosus - 3400. In hepatocytes, the amount of proteoforms was the same as in HepG2 - 70000. Significance of obtained data is in possibilities to estimating the proteome organization and planning next steps in its study.
[Show abstract][Hide abstract] ABSTRACT: In the review, authors discussed recently published experimental data concerning highly sensitive electrochemical methods and technologies for biomedical investigations in the postgenomic era. Developments in electrochemical biosensors systems for the analysis of various bio objects are also considered: cytochrome P450s, cardiac markers, bacterial cells, the analysis of proteins based on electro oxidized amino acids as a tool for analysis of conformational events. The electroanalysis of catalytic activity of cytochromes P450 allowed developing system for screening of potential substrates, inhibitors or modulators of catalytic functions of this class of hemoproteins. The highly sensitive quartz crystal microbalance (QCM) immunosensor has been developed for analysis of bio affinity interactions of antibodies with troponin I in plasma. The QCM technique allowed real-time monitoring of the kinetic differences in specific interactions and nonspecific sorption, with out multiple labeling procedures and separation steps. The affinity binding process was characterized by the association (ka) and the dissociation (kd) kinetic constants and the equilibrium association (K) constant, calculated using experimental data. Based on the electroactivity of bacterial cells, the electrochemical system for determination of sensitivity of the microbial cells to antibiotics cefepime, ampicillin, amikacin, and erythromycin was proposed. It was shown that the minimally detectable cell number corresponds to 106 CFU per electrode. The electrochemical method allows estimating the degree of E.coli JM109 cells resistance to antibiotics within 2-5 h. Electrosynthesis of polymeric analogs of antibodies for myoglobin (molecularly imprinted polymer, MIP) on the surface of graphite screen-printed electrodes as sensor elements with o- phenylenediamine as the functional monomer was developed. Molecularly imprinted polymers demonstrate selective complementary binding of a template protein molecule (myoglobin) by the "key - lock" principle.
[Show abstract][Hide abstract] ABSTRACT: In order to surpass the problem of genetic variability of hepatitis C virus envelope proteins during vaccine development, we used the so-called "reverse vaccinology"approach - "from genome to vaccine". Database of HCV protein sequences was designed, viral genome analysis was performed, and several highly conserved sites were revealed in HCV envelope proteins in the framework of this approach. These sites demonstrated low antigenic activity in full-size proteins and HCV virions: antibodies against these sites were not found in all hepatitis C patients. However, two sites, which contained a wide set of potential T-helper epitope motifs, were revealed among these highly conserved ones. We constructed and prepared by solid-phase peptide synthesis several artificial peptide constructs composed of two linker-connected highly conserved HCV envelope E2 protein sites; one of these sites contained a set of T-helper epitope motifs. Experiments on laboratory animals demonstrated that the developed peptide constructs manifested immunogenicity compared with one of protein molecules and were able to raise antibodies, which specifically bound HCV envelope proteins. We succeeded in obtaining antibodies reactive with HCV from hepatitis C patient plasma upon the immunization with some constructs. An original preparation of a peptide vaccine against hepatitis C is under development on the basis of these peptide constructs.
[Show abstract][Hide abstract] ABSTRACT: The article summarizes the achievements of the pilot phase (2010-2014) of the Russian part of the international project "Human Proteome" and identifies the directions for further work on the study of the human chromosome 18 proteome in the framework of the project main phase (2015-2022). The pilot phase of the project was focused on the detection of at least one protein for each chromosome 18 protein-coding gene in three types of the biological material. The application of mass spectrometric detection of proteins by the methods of multiple reactions monitoring (MRM) and gene-centric approach made it possible to detect 95% of master forms of proteins, for 60% of which the quantitative assessment of the protein content was obtained in at least one type of the biological material. The task of the main phase of the project is to measure the proteome size of healthy individuals, taking into account the modified protein forms, providing for both the bioinformatics prediction of the quantity of proteins types and the selective experimental measurement of single proteoforms. Since the ranges of protein concentrations corresponding to the normal physiological state have not been identified, the work of the main phase of the project is focused on the study of clinically healthy individuals. The absence of these data complicates significantly the interpretation of the patients' blood proteomic profiles and prevents creating diagnostic tests. In the long term prospect, implementation of the project envisages development of a diagnostic test system based on multiple reactions monitoring (MRM) for quantitative measurement of the protein forms associated with some diseases. Development of such test systems will allow predicting the extent of risk of different diseases, diagnosing a disease at its early stage and monitoring the effectiveness of the treatment.