Alexander I Archakov

Orekhovich Institute of Biomedical Chemistry of Russian Academy of Sciences, Moskva, Moscow, Russia

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Publications (62)185.71 Total impact

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    ABSTRACT: Post-translational modifications of proteins play a key role in the regulation of various cellular processes. The analysis and identification of post-translational modifications are probably the most versatile and difficult, but also most frequently studied area of interest in proteomics research. This review focuses on the electroactivity of amino acids as a tool for analysis of post-translational modifications of proteins. The most attention is paid to the electrochemical detection of phosphorylation/dephosphorylation and glycosylation of proteins, to the best-studied and functionally-significant modifications, and, also, to the electrochemical analysis of activity of enzymes responsible for carrying out phosphorylation/dephosphorylation of proteins. Recent advances in electrochemistry with special references to proteomics are outlined and innovative technologies for protein detection are highlighted.
    Biosensors & bioelectronics. 05/2014; 61C:131-139.
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    ABSTRACT: Post-translational modifications of proteins play a key role in the regulation of various cellular processes. The analysis and identification of post-translational modifications are probably the most versatile and difficult, but also most frequently studied area of interest in proteomics research. This review focuses on the electroactivity of amino acids as a tool for analysis of post-translational modifications of proteins. The most attention is paid to the electrochemical detection of phosphorylation/dephosphorylation and glycosylation of proteins, to the best-studied and functionally-significant modifications, and, also, to the electrochemical analysis of activity of enzymes responsible for carrying out phosphorylation/dephosphorylation of proteins. Recent advances in electrochemistry with special references to proteomics are outlined and innovative technologies for protein detection are highlighted.
    Biosensors and Bioelectronics. 01/2014; 61:131–139.
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    ABSTRACT: The goal of this study was to evaluate the capacity for mass spectrometry of blood plasma to diagnose impaired glucose tolerance (IGT). For this study, blood plasma samples from control subjects (n = 30) and patients with IGT (n = 20) were treated with methanol and low molecular weight fraction were then analyzed by direct infusion mass spectrometry. A total of 51 metabolite ions strongly associated with IGT were detected. The area under a receiver operating characteristic (ROC) curve (AUC) for diagnosing IGT that was based on an analysis of all these metabolites was 0.93 (accuracy 90%, specificity 90%, and sensitivity 90%). The associated reproducibility was 85%. The metabolites identified were also consistent with risk factors previously associated with the development of diabetes. Thus, direct infusion mass spectrometry of blood plasma metabolites represents a rapid, single-step, and reproducible method for the analysis of metabolites. Moreover, this method has the potential to serve as a prototype for clinical analyses that could replace the currently used glucose tolerance test with a more patient-friendly assay.
    PLoS ONE 01/2014; 9(9):e105343. · 3.53 Impact Factor
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    ABSTRACT: The review describes a new method of therapeutic drug monitoring (TDM) based on direct infusion of low-molecular fraction of blood into an electrospray ionization source of mass spectrometer. This technique allows performing TDM of almost all drugs used in clinical practice. Universality and a high-throughput mode of the method significantly simplify wide application of this method. Moreover, the possibility of method application in most cases of drug therapy has been argued as a tool for control of drug doses, rationality of drug therapy, and quality of drugs used. In conclusion, prospects for application of the method as primary means of improving the quality and personalization of drug therapy have been discussed.
    Biochemistry (Moscow) Supplement Series B Biomedical Chemistry 01/2014; 8(1):1-10.
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    Dataset: elps4957
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    ABSTRACT: We report the results obtained in 2012-2013 by the Russian Consortium for the Chromosome-centric Human Proteome Project (C-HPP). The main scope of this work was the transcriptome profiling of genes on human chromosome 18 (Chr 18), as well as their encoded proteome, from three types of biomaterials: liver tissue, the hepatocellular carcinoma-derived cell line HepG2, and blood plasma. The transcriptome profiling for liver tissue was independently performed using two RNaseq platforms (SOLiD and Illumina) and also by droplet digital PCR (ddPCR) and quantitative RT-PCR. The proteome profiling of Chr 18 was accomplished by quantitatively measuring protein copy numbers in the three types of biomaterial (at a sensitivity of 10(-13) M) using selected reaction monitoring (SRM). In total, protein copy numbers were estimated for 228 master proteins, including quantitative data on 164 proteins in plasma, 171 in the HepG2 cell line, and 186 in liver tissue. Most proteins were present in plasma at 10(8) copies/μL, while the median abundance was 10(4) and 10(5) protein copies per cell in HepG2 cells and liver tissue, respectively. In summary, for liver tissue and HepG2 cells a "transcriptoproteome" was produced that reflects the relationship between transcript and protein copy numbers of the genes on Chr 18. The quantitative data acquired by RNaseq, PCR, and SRM were uploaded into the "Update_2013" data set of our knowledgebase ( www.kb18.ru ) and investigated for linear correlations.
    Journal of Proteome Research 12/2013; · 5.06 Impact Factor
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    ABSTRACT: The Chromosome-centric Human Proteome Project (C-HPP) is aimed to identify the variety of protein products and transcripts of the number of chromosomes. The Russian part of C-HPP is devoted to the study of the human chromosome 18. Using widely accepted Tophat and SpliceGrapher, a tool for accurate splice sites and alternative mRNA isoforms prediction, we performed the extensive mining of the splice variants of chromosome 18 transcripts and encoded protein products in liver, brain, lung, kidney, blood, testis, derma, and skeletal muscles. About 6.1 billion of the reads represented by 450 billion of the bases have been analyzed. The relative frequencies of splice events as well as gene expression profiles in normal tissues are evaluated. Using ExPASy PROSITE, the novel features and possible functional sites of previously unknown splice variants were highlighted. A set of unique proteotypic peptides enabling the identification of novel alternative protein species using mass-spectrometry is constructed. The revealed data will be integrated into the gene-centric knowledgebase of the Russian part of C-HPP available at http://kb18.ru and http://www.splicing.zz.mu/ .
    Journal of Proteome Research 12/2013; · 5.06 Impact Factor
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    ABSTRACT: Insufficient sensitivity of methods for detection of proteins at a single molecule level does not yet allow obtaining the whole image of human proteome. But to go further, we need at least to know the proteome size, or how many different protein species compose this proteome. This is the task that could be at least partially realized by the method described in this paper. The approach used in our study is based on detection of protein spots in 2DE after staining by protein dyes with various sensitivities. As the different protein spots contain different protein species, counting spots open way for estimation of number of protein species. The function representing the dependence of the number of protein spots on sensitivity or limit of detection (LOD) of protein dyes was generated. And extrapolation of this function curve to theoretical point of the maximum sensitivity (detection of a single smallest polypeptide) allowed to counting the number of different molecules (polypeptide species) at the concentration level of a single polypeptide per proteome. Using this approach, it was estimated that the minimal numbers of protein species for model objects, E.coli and P.furiosus, are 6200 and 3400, respectively. A single human cell (HepG2) we expect to contain minimum 70,000 protein species.
    Electrophoresis 11/2013; · 3.26 Impact Factor
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    ABSTRACT: Direct redox activity of different proteins was investigated on the surface of carbon screen printed electrodes (SPE). The signal attributed to the electrochemical oxidation of amino acid residues (cysteine (Cys), tryptophan (Trp) and tyrosine (Tyr)) was registered at Emax from 0.6 to 0.7 V (vs. Ag/AgCl). Based on the difference in the redox behavior of L-tyrosine and 3-nitro-L-tyrosine, the selective electrochemical detection of native and nitrated albumins was demonstrated. It was shown that the electrochemical signal correlated with the surface density of electroactive amino acid residues on the protein molecule. A simple electrochemical method for the total protein analysis was proposed.
    Electroanalysis 09/2013; 25(9). · 2.82 Impact Factor
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    ABSTRACT: The Human Proteome Project (HPP) was started two years ago and theinternational consortia have elaborated a number of informational resources to harbor the HPP data. Selected informational resources are currently used to elaborate the HPP baseline metrics, which were introduced to estimate future contribution of HPP to the knowledge domain. We developed a Web-based tool Gene-centric Content Management System (GenoCMS) for comparing public resources to proprietary results by using the representation of proteins as color-coded catalog. Within our CMS, the features of protein-coding genes are uploaded from the public domain and then appended by additional features derived from original experimental workflows. We describe the heat-map/traffic light representation of our proteomic experiments as the background of data taken from NextProt, MS/MS repositories, the Human Protein Atlas and the RNAseqAtlas. The system presented at www.kb18.ru comprises a collaborative knowledge base for annotating the gene sets and disseminating these annotations through the Web.
    Biochimica et Biophysica Acta 08/2013; · 4.66 Impact Factor
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    Oxana P Trifonova, Petr G Lokhov, Alexander I Archakov
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    ABSTRACT: Metabolomics, the youngest branch of “omics” sciences, intended for studying a comprehensive set of low molecular weight substances (metabolites) of various biological objects. Metabolite profiles represent a molecular phenotype of biological systems and reflect information encoded at the genome level and realized at the transcriptome and proteome levels. Analysis of the human blood metabolic profile is a universal and promising tool for clinical applications because it is a sensitive measure of both endogenous and exogenous (environmental) factors influencing the human body. In this review we discuss technical implementation of blood metabolic profiling methods and statistic analysis of metabolite profiles for effective diagnostics and risk assessments of diseases.
    Biochemistry (Moscow) Supplement Series B Biomedical Chemistry 07/2013; 7(3):179-186.
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    ABSTRACT: Cytochromes P450 comprise a large superfamily and several of their isoforms play a crucial role in metabolism of xenobiotics, including drugs. Although these enzymes demonstrate broad and cross-substrate specificity, different cytochrome P450 subfamilies exhibit certain selectivity for some types of substrates. Analysis of amino acid residues of the active sites of six cytochrome subfamilies (CYP1А, CYP2А, CYP2С, CYP2D, CYP2E and CYP3А) enables to define subfamily-specific patterns that consist of four residues. These residues are located on the periphery of the active sites of these cytochromes. We suggest that they can form a primary binding site at the entrance to the active site, defining cytochrome substrate recognition. Copyright © 2013 John Wiley & Sons, Ltd.
    Journal of Molecular Recognition 02/2013; 26(2):86-91. · 3.01 Impact Factor
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    ABSTRACT: A highly sensitive reverse sandwich immunoassay for the detection of human cardiac myoglobin (cMb) in serum was designed utilizing a gold nanoparticle (AuNP)-enhanced surface plasmon resonance (SPR) biosensor. First, a monoclonal anti-cMb antibody (Mab1) was covalently immobilized on the sensor surface. AuNPs were covalently conjugated to the second monoclonal anti-cMb antibody (Mab2) to form an immuno-gold reagent (Mab2-AuNP). The reverse sandwich immunoassay consists of two steps: (1) mixing the serum sample with Mab2-AuNP and incubation for the formation of cMb/Mab2-AuNP complexes and (2) sample injection over the sensor surface and evaluation of the Mab1/cMb/Mab2-AuNP complex formation, with the subsequent calculation of the cMb concentration in the serum. The biosensor signal was amplified approximately 30-fold compared with the direct reaction of cMb with Mab1 on the sensor surface. The limit of detection of cMb in a human blood serum sample was found to be as low as 10pM (approx. 0.18ngmL(-1)), and the inter-assay coefficient of variation was less than 3%. Thus, the developed SPR-based reverse sandwich immunoassay has a sensitivity that is sufficient to measure cMb across a wide range of normal and pathological concentrations, allowing an adequate estimation of the disease severity and the monitoring of treatment.
    Analytica chimica acta 01/2013; 759C:105-109. · 4.31 Impact Factor
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    ABSTRACT: In this paper, we present a method for the determination of low- and ultra-low copy-number proteins in biomaterials based on a combination of concentrating the protein from the sample onto CNBr-activated Sepharose 4B (via non-specific binding of free amino groups) and multiple reaction monitoring (MRM). The detection limit and the dependence of the MRM peak areas on the concentration of protein in the sample were determined using the proteins CYP 102 and BSA, as a model system, both in solution and after their addition to human plasma. Non-specific protein enrichment of proteins from diluted sample volumes of 10 to 50 mL was found to increase the range of linear dependence of the chromatographic peak area on concentration by more than three orders of magnitude, allowing a lower limit of detection limit (LLOD) of as low as 10(-18) M. At this LLOD, at least two tryptic peptides of CYP102 and BSA could be detected with signal-to-noise ratios of ≥7.0. The results were equally good for samples containing pure protein mixtures and proteins spiked into diluted depleted human blood plasma.
    Proteomics 12/2012; · 4.43 Impact Factor
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    ABSTRACT: Lung cancer is one of the most common types of cancer in men, and is a leading cause of cancer-related deaths. Therefore, the identification of specific markers associated with a risk of lung cancer development, particularly metabolites that are more easily assayed, would be very valuable. To this end, а comparative metabolomics study of blood plasma samples collected from patients with lung cancer (n=100) and controls (n=100) recruited in Moscow was carried out. After the extraction of blood plasma proteins with methanol, the remaining plasma metabolite fractions were analyzed directly using mass spectrometry. Hundreds of cancer-associated metabolites were detected, and at least 70 metabolite ions with odds ratio values of 10-288 were found to be associated with the presence of cancer. Although these metabolites were present at higher levels in cancer patients, particularly in the early stages of disease, they did not correlate positively with cancer progression. On the basis of these findings, this metabolomics study of blood plasma samples from cancer patients shows that numerous cancer-associated metabolites were present in the studied population, and these could be used as factors for calculating the risk of lung cancer development in addition to currently used risk factors.
    European journal of cancer prevention: the official journal of the European Cancer Prevention Organisation (ECP) 12/2012; · 2.21 Impact Factor
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    ABSTRACT: Silicon-on-isolator-nanowires (SOI-NWs) were used for the label-free, real-time biospecific detection of the hepatitis B marker HBsAg and cancer marker α-fetoprotein (AFP). Specific protein-protein recognition was carried out using individual NWs that were functionalized with antibodies. To solve the problem of non-specific binding of target protein molecules to the sensor element the use of a reference NW with immobilized antibodies against non-target proteins was proposed. Using individual SOI-NW surface functionalization allowed the fabrication of a NW array, containing working NWs and reference NWs within one chip. It was shown that this approach allows us to reach a detection limit up to 10(-14) and 10(-15) M for HBsAg and AFP, respectively. Our investigations also allowed us to reveal the influence of the charged state of the target protein molecules and antibodies in solutions with various pH values on the target protein detection limit. A high sensitivity NW-detector is of interest for the creation of diagnosticums for hepatitis B and for the early stages of cancer diseases.
    Lab on a Chip 10/2012; · 5.70 Impact Factor
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    ABSTRACT: Atomic force microscopy (AFM) study of the oligomeric state of CYP102A1 was carried out in three different environmental conditions – liquid, air and vacuum. It was shown that usage of a standard probe with a radius of curvature of 10 nm allowed determination of the monomers-to-oligomers ratio, α ≈ 0.5:0.5, in these three media, but it wasn't possible to estimate the oligomerization degree more precisely. Application of a supersharp probe with a radius of curvature of 2 nm made it possible not only to obtain data on the ratio of monomers-to-oligomers but also to obtain the approximate ratio between dimers, trimers and tetramers 0.3:0.1:0.1, but only in vacuum.
    Soft Matter 04/2012; 8(17):4602-4608. · 4.15 Impact Factor