Publications by authors named "Alexander I Archakov"

85 Publications

Non-immunogenic recombinant staphylokinase versus alteplase for patients with acute ischaemic stroke 4·5 h after symptom onset in Russia (FRIDA): a randomised, open label, multicentre, parallel-group, non-inferiority trial.

Lancet Neurol 2021 09;20(9):721-728

Institute of Biomedical Chemistry, Moscow, Russia; SuperGene, Moscow, Russia.

Background: Non-immunogenic staphylokinase is modified recombinant staphylokinase with low immunogenicity, high thrombolytic activity, and selectivity to fibrin. We aimed to assess the safety and efficacy of a single intravenous bolus of non-immunogenic staphylokinase compared with alteplase in patients with acute ischaemic stroke within 4·5 h after symptom onset.

Methods: We did a randomised, open-label, multicentre, parallel-group, non-inferiority trial in 18 clinical sites in Russia. We included patients aged 18 years and older with a diagnosis of acute ischaemic stroke (up to 25 points on the National Institutes of Health Stroke Scale). The study drug had to be administered within 4·5 h after the onset of symptoms. Patients were randomly assigned to receive either non-immunogenic staphylokinase (10 mg) or alteplase (0·9 mg/kg, maximum 90 mg), both administered intravenously. The randomisation sequence was created by an independent biostatistician using computer-generated random numbers. 84 blocks (block size of four) of opaque sealed envelopes were numbered sequentially from 1 to 336 and were opened in numerical order. Patients were unaware of their assigned treatment and were assessed by the study investigators who were also unaware of the treatment assignment on all trial days. Emergency department staff, who administered the assigned drug and opened the envelopes, were not masked to treatment. The primary efficacy endpoint was a favourable outcome, defined as a modified Rankin scale (mRS) score of 0-1 on day 90. The margin of non-inferiority was established as 16% for the difference in mRS score of 0-1 on day 90. Non-inferiority was tested using Welch's t-test for the primary outcome only. Endpoints were analysed in the per-protocol population, which comprised all randomly assigned patients who completed treatment without any protocol violations; this population was identical to the intention-to-treat population. This trial is completed and registered at ClinicalTrials.gov, NCT03151993.

Findings: Of 385 patients recruited from March 18, 2017, to March 23, 2019, 336 (87%) were included in the trial. 168 (50%) patients were randomly assigned to receive non-immunogenic staphylokinase and 168 (50%) to receive alteplase. The median duration of follow-up was 89 days (IQR 89-89). 84 (50%) of 168 patients in the non-immunogenic staphylokinase group had a favourable outcome at day 90 compared with 68 (40%) of 168 patients in the alteplase group (odds ratio [OR] 1·47, 95% CI 0·93 to 2·32; p=0·10). The difference in the rate of favourable outcome at day 90 was 9·5% (95% CI -1·7 to 20·7) and the lower limit did not cross the margin of non-inferiority (p <0·0001). Symptomatic intracranial haemorrhage occurred in five (3%) patients in the non-immunogenic staphylokinase group and in 13 (8%) patients in the alteplase group (p=0·087). On day 90, 17 (10%) patients in the non-immunogenic staphylokinase group and 24 (14%) patients in the alteplase group had died (p=0·32). 22 (13%) patients in the non-immunogenic staphylokinase group had serious adverse events, compared with 37 (22%) patients in the alteplase group (p=0·044).

Interpretation: Non-immunogenic staphylokinase was non-inferior to alteplase for patients with acute ischaemic stroke. Mortality, symptomatic intracranial haemorrhage, and serious adverse events did not differ significantly between groups. Future studies are needed to continue to assess the safety and efficacy of non-immunogenic staphylokinase in patients with acute ischaemic stroke within the 4·5 h time window, and to assess the drug in patients with acute ischaemic stroke outside this time window with reperfusion CT or magnetic resonance angiography followed by thrombectomy if necessary.

Funding: The Russian Academy of Sciences.
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http://dx.doi.org/10.1016/S1474-4422(21)00210-6DOI Listing
September 2021

Nanoribbon-Based Electronic Detection of a Glioma-Associated Circular miRNA.

Biosensors (Basel) 2021 Jul 13;11(7). Epub 2021 Jul 13.

Laboratory of Nanobiotechnology, Institute of Biomedical Chemistry, 119121 Moscow, Russia.

Nanoribbon chips, based on "silicon-on-insulator" structures (SOI-NR chips), have been fabricated. These SOI-NR chips, whose surface was sensitized with covalently immobilized oligonucleotide molecular probes (oDNA probes), have been employed for the nanoribbon biosensor-based detection of a circular ribonucleic acid (circRNA) molecular marker of glioma in humans. The nucleotide sequence of the oDNA probes was complimentary to the sequence of the target oDNA. The latter represents a synthetic analogue of a glioma marker-NFIX circular RNA. In this way, the detection of target oDNA molecules in a pure buffer has been performed. The lowest concentration of the target biomolecules, detectable in our experiments, was of the order of ~10 M. The SOI-NR sensor chips proposed herein have allowed us to reveal an elevated level of the NFIX circular RNA in the blood of a glioma patient.
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http://dx.doi.org/10.3390/bios11070237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8301916PMC
July 2021

Use of Silicon Nanowire Sensors for Early Cancer Diagnosis.

Molecules 2021 Jun 18;26(12). Epub 2021 Jun 18.

Institute of Biomedical Chemistry, 119121 Moscow, Russia.

The review covers some research conducted in the field of medical and biomedical application of devices based on silicon sensor elements (Si-NW-sensors). The use of Si-NW-sensors is one of the key methods used in a whole range of healthcare fields. Their biomedical use is among the most important ones as they offer opportunities for early diagnosis of oncological pathologies, for monitoring the prescribed therapy and for improving the people's quality of life.
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http://dx.doi.org/10.3390/molecules26123734DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8234636PMC
June 2021

Micro-Raman Characterization of Structural Features of High-k Stack Layer of SOI Nanowire Chip, Designed to Detect Circular RNA Associated with the Development of Glioma.

Molecules 2021 Jun 18;26(12). Epub 2021 Jun 18.

Laboratory of Nanobiotechnology, Institute of Biomedical Chemistry, 119121 Moscow, Russia.

The application of micro-Raman spectroscopy was used for characterization of structural features of the high-k stack (h-k) layer of "silicon-on-insulator" (SOI) nanowire (NW) chip (h-k-SOI-NW chip), including AlO and HfO in various combinations after heat treatment from 425 to 1000 °C. After that, the NW structures h-k-SOI-NW chip was created using gas plasma etching optical lithography. The stability of the signals from the monocrine phase of HfO was shown. Significant differences were found in the elastic stresses of the silicon layers for very thick (>200 nm) AlO layers. In the UV spectra of SOI layers of a silicon substrate with HfO, shoulders in the Raman spectrum were observed at 480-490 cm of single-phonon scattering. The h-k-SOI-NW chip created in this way has been used for the detection of DNA-oligonucleotide sequences (oDNA), that became a synthetic analog of circular RNA-circ-SHKBP1 associated with the development of glioma at a concentration of 1.1 × 10 M. The possibility of using such h-k-SOI NW chips for the detection of circ-SHKBP1 in blood plasma of patients diagnosed with neoplasm of uncertain nature of the brain and central nervous system was shown.
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http://dx.doi.org/10.3390/molecules26123715DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8234461PMC
June 2021

Detection of Influenza Virus Using a SOI-Nanoribbon Chip, Based on an N-Type Field-Effect Transistor.

Biosensors (Basel) 2021 Apr 12;11(4). Epub 2021 Apr 12.

Laboratory of Nanobiotechnology, Institute of Biomedical Chemistry, 119121 Moscow, Russia.

The detection of influenza A virions with a nanoribbon detector (NR detector) has been demonstrated. Chips for the detector have been fabricated based on silicon-on-insulator nanoribbon structures (SOI nanoribbon chip), using a complementary metal-oxide-semiconductor (CMOS)-compatible technology-by means of gas-phase etching and standard optical photolithography. The surface of the SOI nanoribbon chip contains a matrix of 10 nanoribbon (NR) sensor elements. SOI nanoribbon chips of n-type conductance have been used for this study. For biospecific detection of target particles, antibodies against influenza virus have been covalently immobilized onto NRs. Influenza A virus detection was performed by real-time registration of the source-drain current through the NRs. The detection of the target viral particles was carried out in buffer solutions at the target particles concentration within the range from 10 to 10 viral particles per milliliter (VP/mL). The lowest detectable concentration of the target viral particles was 6 × 10 M (corresponding to 10 VP/mL). The use of solutions containing ~10 to 10 VP/mL resulted in saturation of the sensor surface with the target virions. In the saturation mode, detection was impossible.
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http://dx.doi.org/10.3390/bios11040119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8069153PMC
April 2021

Raman Spectroscopy-Based Quality Control of "Silicon-On-Insulator" Nanowire Chips for the Detection of Brain Cancer-Associated MicroRNA in Plasma.

Sensors (Basel) 2021 Feb 13;21(4). Epub 2021 Feb 13.

Laboratory of Nanobiotechnology, Institute of Biomedical Chemistry, 119121 Moscow, Russia.

Application of micro-Raman spectroscopy for the monitoring of quality of nanowire sensor chips fabrication has been demonstrated. Nanowire chips have been fabricated on the basis of «silicon-on-insulator» (SOI) structures (SOI-NW chips). The fabrication of SOI-NW chips was performed by optical litography with gas-phase etching. The so-fabricated SOI-NW chips are intended for highly sensitive detection of brain cancer biomarkers in humans. In our present study, two series of experiments have been conducted. In the first experimental series, detection of a synthetic DNA oligonucleotide (oDNA) analogue of brain cancer-associated microRNA miRNA-363 in purified buffer solution has been performed in order to demonstrate the high detection sensitivity. The second experimental series has been performed in order to reveal miRNA-363 itself in real human plasma samples. To provide detection biospecificity, the SOI-NW chip surface was modified by covalent immobilization of probe oligonucleotides (oDNA probes) complementary to the target biomolecules. Using the SOI-NW sensor chips proposed herein, the concentration detection limit of the target biomolecules at the level of 3.3 × 10 M has been demonstrated. Thus, the approach employing the SOI-NW chips proposed herein represents an attractive tool in biomedical practice, aimed at the early revelation of oncological diseases in humans.
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http://dx.doi.org/10.3390/s21041333DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7918486PMC
February 2021

Optical Monitoring of the Production Quality of Si-Nanoribbon Chips Intended for the Detection of ASD-Associated Oligonucleotides.

Micromachines (Basel) 2021 Feb 3;12(2). Epub 2021 Feb 3.

Laboratory of Nanobiotechnology, Institute of Biomedical Chemistry, 119121 Moscow, Russia.

Gas-phase etching and optical lithography were employed for the fabrication of a silicon nanoribbon chip (Si-NR chip). The quality of the so-fabricated silicon nanoribbons (Si-NRs) was monitored by optical Raman scattering spectroscopy. It was demonstrated that the structures of the Si-NRs were virtually defect-free, meaning they could be used for highly sensitive detection of biological macromolecules. The Si-NR chips were then used for the highly sensitive nanoelectronics detection of DNA oligonucleotides (oDNAs), which represent synthetic analogs of 106a-5p microRNA (miR-106a-5p), associated with the development of autism spectrum disorders in children. The specificity of the analysis was attained by the sensitization of the Si-NR chip sur-face by covalent immobilization of oDNA probes, whose nucleotide sequence was complementary to the known sequence of miR-106a-5p. The use of the Si-NR chip was demonstrated to al-low for the rapid label-free real-time detection of oDNA at ultra-low (~10 M) concentrations.
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http://dx.doi.org/10.3390/mi12020147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7913754PMC
February 2021

Does Proteomic Mirror Reflect Clinical Characteristics of Obesity?

J Pers Med 2021 Jan 21;11(2). Epub 2021 Jan 21.

Institute of Biomedical Chemistry, Pogodinskaya Street 10/8, 119121 Moscow, Russia.

Obesity is a frightening chronic disease, which has tripled since 1975. It is not expected to slow down staying one of the leading cases of preventable death and resulting in an increased clinical and economic burden. Poor lifestyle choices and excessive intake of "cheap calories" are major contributors to obesity, triggering type 2 diabetes, cardiovascular diseases, and other comorbidities. Understanding the molecular mechanisms responsible for development of obesity is essential as it might result in the introducing of anti-obesity targets and early-stage obesity biomarkers, allowing the distinction between metabolic syndromes. The complex nature of this disease, coupled with the phenomenon of metabolically healthy obesity, inspired us to perform data-centric, hypothesis-generating pilot research, aimed to find correlations between parameters of classic clinical blood tests and proteomic profiles of 104 lean and obese subjects. As the result, we assembled patterns of proteins, which presence or absence allows predicting the weight of the patient fairly well. We believe that such proteomic patterns with high prediction power should facilitate the translation of potential candidates into biomarkers of clinical use for early-stage stratification of obesity therapy.
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http://dx.doi.org/10.3390/jpm11020064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7912072PMC
January 2021

Mass Spectrometric Identification of Proteins Enhanced by the Atomic Force Microscopy Immobilization Surface.

Int J Mol Sci 2021 Jan 4;22(1). Epub 2021 Jan 4.

Institute of Biomedical Chemistry, 119121 Moscow, Russia.

An approach to highly-sensitive mass spectrometry detection of proteins after surface-enhanced concentrating has been elaborated. The approach is based on a combination of mass spectrometry and atomic force microscopy to detect target proteins. (1) Background: For this purpose, a technique for preliminary preparation of molecular relief surfaces formed as a result of a chemical or biospecific concentration of proteins from solution was developed and tested on several types of chip surfaces. (2) Methods: mass spectrometric identification of proteins using trailing detectors: ion trap, time of flight, orbital trap, and triple quadrupole. We used the electrospray type of ionization and matrix-assisted laser desorption/ionization. (3) Results: It is shown that when using locally functionalized atomically smooth surfaces, the sensitivity of the mass spectrometric method increases by two orders of magnitude as compared with measurements in solution. Conclusions: It has been demonstrated that the effective concentration of target proteins on specially prepared surfaces increases the concentration sensitivity of mass spectrometric detectors-time-of-flight, ion trap, triple quadrupole, and orbital ion trap in the concentration range from up to 10 M.
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http://dx.doi.org/10.3390/ijms22010431DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7795915PMC
January 2021

Highly Sensitive Detection of CA 125 Protein with the Use of an n-Type Nanowire Biosensor.

Biosensors (Basel) 2020 Dec 18;10(12). Epub 2020 Dec 18.

Laboratory of nanotechnology, Institute of Biomedical Chemistry, 119121 Moscow, Russia.

The detection of CA 125 protein in a solution using a silicon-on-insulator (SOI)-nanowire biosensor with n-type chip has been experimentally demonstrated. The surface of nanowires was modified by covalent immobilization of antibodies against CA 125 in order to provide the biospecificity of the target protein detection. We have demonstrated that the biosensor signal, which results from the biospecific interaction between CA 125 and the covalently immobilized antibodies, increases with the increase in the protein concentration. At that, the minimum concentration, at which the target protein was detectable with the SOI-nanowire biosensor, amounted to 1.5 × 10 M.
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http://dx.doi.org/10.3390/bios10120210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7766891PMC
December 2020

Proteomic Analysis of Chr 18 Proteins Using 2D Fractionation.

J Proteome Res 2020 12 17;19(12):4901-4906. Epub 2020 Nov 17.

Institute of Biomedical Chemistry, Pogodinskaya 10, Moscow 119121, Russia.

One of the main goals of the Chromosome-Centric Human Proteome Project (C-HPP) is detection of "missing proteins" (PE2-PE4). Using the UPS2 (Universal proteomics standard 2) set as a model to simulate the range of protein concentrations in the cell, we have previously shown that 2D fractionation enables the detection of more than 95% of UPS2 proteins in a complex biological mixture. In this study, we propose a novel experimental workflow for protein detection during the analysis of biological samples. This approach is extremely important in the context of the C-HPP and the neXt-MP50 Challenge, which can be solved by increasing the sensitivity and the coverage of the proteome encoded by a particular human chromosome. In this study, we used 2D fractionation for in-depth analysis of the proteins encoded by human chromosome 18 (Chr 18) in the HepG2 cell line. Use of 2D fractionation increased the sensitivity of the SRM SIS method by 1.3-fold (68 and 88 proteins were identified by 1D fractionation and 2D fractionation, respectively) and the shotgun MS/MS method by 2.5-fold (21 and 53 proteins encoded by Chr 18 were detected by 1D fractionation and 2D fractionation, respectively). The results of all experiments indicate that 111 proteins encoded by human Chr 18 have been identified; this list includes 42% of the Chr 18 protein-coding genes and 67% of the Chr 18 transcriptome species (Illumina RNaseq) in the HepG2 cell line obtained using a single sample. Corresponding mRNAs were not registered for 13 of the detected proteins. The combination of 2D fractionation technology with SRM SIS and shotgun mass spectrometric analysis did not achieve full coverage, i.e., identification of at least one protein product for each of the 265 protein-coding genes of the selected chromosome. To further increase the sensitivity of the method, we plan to use 5-10 crude synthetic peptides for each protein to identify the proteins and select one of the peptides based on the obtained mass spectra for the synthesis of an isotopically labeled standard for subsequent quantitative analysis. Data are available via ProteomeXchange with the identifier PXD019263.
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http://dx.doi.org/10.1021/acs.jproteome.0c00856DOI Listing
December 2020

Mass Spectrometry-Based Metabolomics Analysis of Obese Patients' Blood Plasma.

Int J Mol Sci 2020 Jan 15;21(2). Epub 2020 Jan 15.

Institute of Biomedical Chemistry, 10 building 8, Pogodinskaya street, 119121 Moscow, Russia.

Scientists currently use only a small portion of the information contained in the blood metabolome. The identification of metabolites is a huge challenge because only highly abundant and well-separated compounds can be easily identified in complex samples. However, new approaches that enhance the identification of compounds have emerged; among them, the identification of compounds based on their involvement in a particular biological context is a recent development. In this work, this approach was first applied to identify metabolites in complex samples and, together with metabolite set enrichment analysis, was used for the evaluation of blood plasma from obese patients. The proposed approach was found to provide a statistically sound overview of the biochemical pathways, thus presenting additional information on obesity. Obesity progression was demonstrated to be accompanied by marked alterations in steroidogenesis, androstenedione metabolism, and androgen and estrogen metabolism. The findings of this study suggest that the workflow used for blood analysis is sufficient to demonstrate obesity at the biochemical pathway level as well as to monitor the response to treatment. This workflow is also expected to be suitable for studying other metabolic diseases.
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http://dx.doi.org/10.3390/ijms21020568DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7014187PMC
January 2020

The "Missing" Proteome: Undetected Proteins, Not-Translated Transcripts, and Untranscribed Genes.

J Proteome Res 2019 12 18;18(12):4273-4276. Epub 2019 Oct 18.

Institute of Biomedical Chemistry , 119121 Moscow , Russia.

The Chromosome-centric Human Proteome Project aims at characterizing the expression of proteins encoded in each chromosome at the tissue, cell, and subcellular levels. The proteomic profiling of a particular tissue or cell line commonly results in a substantial portion of proteins that are not observed (the "missing" proteome). The concurrent transcriptome profiling of the analyzed tissue/cells samples may help define the set of untranscribed genes in a given type of tissue or cell, thus narrowing the size of the "missing" proteome and allowing us to focus on defining the reasons behind undetected proteins, namely, whether they are technical (insufficient sensitivity of protein detection) or biological (correspond to not-translated transcripts). We believe that the quantitative polymerase chain reaction (qPCR) can provide an efficient approach to studying low-abundant transcripts related to undetected proteins due to its high sensitivity and the possibility of ensuring the specificity of detection via the simple Sanger sequencing of PCR products. Here we illustrated the feasibility of such an approach on a set of low-abundant transcripts. Although inapplicable to the analysis of whole transcriptome, qPCR can successfully be utilized to profile a limited cohort of transcripts encoded on a particular chromosome, as we previously demonstrated for human chromosome 18.
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http://dx.doi.org/10.1021/acs.jproteome.9b00383DOI Listing
December 2019

Challenges of the Human Proteome Project: 10-Year Experience of the Russian Consortium.

J Proteome Res 2019 12 28;18(12):4206-4214. Epub 2019 Oct 28.

Institute of Biomedical Chemistry , Moscow 119435 , Russia.

This manuscript collects all the efforts of the Russian Consortium, bottlenecks revealed in the course of the C-HPP realization, and ways of their overcoming. One of the main bottlenecks in the C-HPP is the insufficient sensitivity of proteomic technologies, hampering the detection of low- and ultralow-copy number proteins forming the "dark part" of the human proteome. In the frame of MP-Challenge, to increase proteome coverage we suggest an experimental workflow based on a combination of shotgun technology and selected reaction monitoring with two-dimensional alkaline fractionation. Further, to detect proteins that cannot be identified by such technologies, nanotechnologies such as combined atomic force microscopy with molecular fishing and/or nanowire detection may be useful. These technologies provide a powerful tool for single molecule analysis, by analogy with nanopore sequencing during genome analysis. To systematically analyze the functional features of some proteins (CP50 Challenge), we created a mathematical model that predicts the number of proteins differing in amino acid sequence: proteoforms. According to our data, we should expect about 100 000 different proteoforms in the liver tissue and a little more in the HepG2 cell line. The variety of proteins forming the whole human proteome significantly exceeds these results due to post-translational modifications (PTMs). As PTMs determine the functional specificity of the protein, we propose using a combination of gene-centric transcriptome-proteomic analysis with preliminary fractionation by two-dimensional electrophoresis to identify chemically modified proteoforms. Despite the complexity of the proposed solutions, such integrative approaches could be fruitful for MP50 and CP50 Challenges in the framework of the C-HPP.
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http://dx.doi.org/10.1021/acs.jproteome.9b00358DOI Listing
December 2019

Immuno-MALDI MS dataset for improved detection of HCVcoreAg in sera.

Data Brief 2019 Aug 8;25:104240. Epub 2019 Jul 8.

Institute of Biomedical Chemistry, Pogodinskaya St. 10, Moscow, 119121 Russia.

Complicated and large-scale challenge the contemporary biomedical community faces are development of highly-sensitive analytical methods for detection of protein markers associated with development of pathogenic mechanisms [2]. The atomic force microscopy (AFM) method in combination with specific fishing is unique among other analytical protein detection approaches; it allows visualization and counting of single protein molecules [3-6]. The present dataset focus on mass spectrometry method for detection of human hepatitis C virus core antigen (HCV core Ag) taking into account the potential modification with cations in blood serum samples, using mica chips for the atomic force microscopy (AFM-chips). To conduct specific protein fishing, we used flat AFM-chips preliminary sensibilized with molecular probes - aptamers, which are single-stranded DNA sequences. In our study we used four types of aptamers up to 85 nucleotides specific against the target protein - HCVcoreAg [3,4]. Working (n = 19) and control (n = 11) AFM-chips with aptamers were preliminarily immobilized on the surface in four zones and incubated in blood serum samples (See Supplementary fig. 1). Analysis of MS data regarding modification of marker protein peptides with Na+, K+, K2Cl+, and Na2Cl + ions enables to enhance the reliability of target proteins detection in the serum thereby demonstrating a high diagnostic potential.
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http://dx.doi.org/10.1016/j.dib.2019.104240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6656991PMC
August 2019

In vitro interactions of abiraterone, erythromycin, and CYP3A4: implications for drug-drug interactions.

Fundam Clin Pharmacol 2020 Feb 5;34(1):120-130. Epub 2019 Aug 5.

Institute of Biomedical Chemistry, Pogodinskaya Street, 10, Build 8, Moscow, 119121, Russia.

Potential drug-drug interactions of the antitumor drug abiraterone and the macrolide antibiotic erythromycin were studied at the stage of cytochrome P450 3A4 (CYP3A4) biotransformation. Using differential spectroscopy, we have shown that abiraterone is a type II ligand of CYP3A4. The dependence of CYP3A4 spectral changes on the concentration of abiraterone is sigmoidal, which indicates cooperative interactions of CYP3A4 with abiraterone; these interactions were confirmed by molecular docking. The dissociation constant (K ) and Hill coefficient (h) values for the CYP3A4-abiraterone complex were calculated as 3.8 ± 0.1 μM and 2.3 ± 0.2, respectively. An electrochemical enzymatic system based on CYP3A4 immobilized on a screen-printed electrode was used to show that abiraterone acts as a competitive inhibitor toward erythromycin N-demethylase activity of CYP3A4 (apparent K  = 8.1 ± 1.2 μM), while erythromycin and its products of enzymatic metabolism do not affect abiraterone N-oxidation by CYP3A4. In conclusion, the inhibition properties of abiraterone toward CYP3A4-dependent N-demethylation of erythromycin and the biologically inert behavior of erythromycin toward abiraterone hydroxylation were demonstrated.
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http://dx.doi.org/10.1111/fcp.12497DOI Listing
February 2020

Detection of Hepatitis C Virus Core Protein in Serum Using Aptamer-Functionalized AFM Chips.

Micromachines (Basel) 2019 Feb 15;10(2). Epub 2019 Feb 15.

Institute of Biomedical Chemistry, Moscow 119121, Russia.

In the present study, we demonstrate atomic force microscopy (AFM)-based detection of hepatitis C virus (HCV) particles in serum samples using a chip with aptamer-functionalized surface (apta-based AFM chip). The target particles, containing core antigen of HCV (HCVcoreAg protein), were biospecifically captured onto the chip surface from 1 mL of test solution containing 10 µL of serum collected from a hepatitis C patient. The registration of aptamer/antigen complexes on the chip surface was performed by AFM. The aptamers used in the present study were initially developed for therapeutic purposes; herein, these aptamers have been successfully utilized as probe molecules for HCVcoreAg detection in the presence of a complex protein matrix (human serum). The results obtained herein can be used for the development of detection systems that employ affine enrichment for protein detection.
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http://dx.doi.org/10.3390/mi10020129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6413090PMC
February 2019

Metabolomic diagnostics and human digital image.

Per Med 2019 03 15;16(2):133-144. Epub 2019 Feb 15.

Department of Proteomics & Mass Spectrometry, Institute of Biomedical Chemistry, Pogodinskaya st 10, 119121, Moscow, Russia.

The existing clinical laboratory practice has limitations in terms of specificity and sensitivity of diagnosis, making the introduction of new methods in medicine more topical. Application of 'omics' technologies, especially metabolomics, allows overcoming these limitations. The composition of blood metabolites reflects the physical state of an organism at the molecular level. The analysis of blood metabolome can serve as effective means of diagnosis, implementation of which in healthcare is timely and relevant. This paper demonstrates the versatility of metabolomic diagnostics, its applicability to various diseases. We discussed the standard of human digital image, which includes the metabolomic data sufficient to make an accurate assessment of general health and carry out precision diagnostics of a wide range of diseases.
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http://dx.doi.org/10.2217/pme-2018-0066DOI Listing
March 2019

200+ Protein Concentrations in Healthy Human Blood Plasma: Targeted Quantitative SRM SIS Screening of Chromosomes 18, 13, Y, and the Mitochondrial Chromosome Encoded Proteome.

J Proteome Res 2019 01 11;18(1):120-129. Epub 2018 Dec 11.

Institute of Biomedical Chemistry , Moscow 119435 , Russia.

This work continues the series of the quantitative measurements of the proteins encoded by different chromosomes in the blood plasma of a healthy person. Selected Reaction Monitoring with Stable Isotope-labeled peptide Standards (SRM SIS) and a gene-centric approach, which is the basis for the implementation of the international Chromosome-centric Human Proteome Project (C-HPP), were applied for the quantitative measurement of proteins in human blood plasma. Analyses were carried out in the frame of C-HPP for each protein-coding gene of the four human chromosomes: 18, 13, Y, and mitochondrial. Concentrations of proteins encoded by 667 genes were measured in 54 blood plasma samples of the volunteers, whose health conditions were consistent with requirements for astronauts. The gene list included 276, 329, 47, and 15 genes of chromosomes 18, 13, Y, and the mitochondrial chromosome, respectively. This paper does not make claims about the detection of missing proteins. Only 205 proteins (30.7%) were detected in the samples. Of them, 84, 106, 10, and 5 belonged to chromosomes 18, 13, and Y and the mitochondrial chromosome, respectively. Each detected protein was found in at least one of the samples analyzed. The SRM SIS raw data are available in the ProteomeXchange repository (PXD004374, PASS01192).
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http://dx.doi.org/10.1021/acs.jproteome.8b00391DOI Listing
January 2019

Increased Sensitivity of Mass Spectrometry by Alkaline Two-Dimensional Liquid Chromatography: Deep Cover of the Human Proteome in Gene-Centric Mode.

J Proteome Res 2018 12 19;17(12):4258-4266. Epub 2018 Nov 19.

Institute of Biomedical Chemistry, RAS , Moscow , Russia.

Currently, great interest is paid to the identification of "missing" proteins that have not been detected in any biological material at the protein level (PE1). In this paper, using the Universal Proteomic Standard sets 1 and 2 (UPS1 and UPS2, respectively) as an example, we characterized mass spectrometric approaches from the point of view of sensitivity (Sn), specificity (Sp), and accuracy (Ac). The aim of the paper was to show the utility of a mass spectra approach for protein detection. This sets consists of 48 high-purity human proteins without single aminoacid polymorphism (SAP) or post translational modification (PTM). The UPS1 set consists of the same 48 proteins at 5 pmols each, and in UPS2, proteins were grouped into 5 groups in accordance with their molar concentration, ranging from 10 to 10 M. Single peptides from the 92% and 96% of all sets of proteins could be detected in a pure solution of UPS2 and UPS1, respectively, by selected reaction monitoring with stable isotope-labeled standards (SRM-SIS). We also found that, in the presence of a biological matrix such as Escherichia coli extract or human blood plasma (HBP), SRM-SIS makes it possible to detect from 63% to 79% of proteins in the UPS2 set (sensitivity) with the highest specificity (∼100%) and an accuracy of 80% by increasing the sensitivity of shotgun and selected reaction monitoring combined with a stable-isotope-labeled peptide standard (SRM-SIS technology) by fractionating samples using reverse-phase liquid chromatography under alkaline conditions (2D-LC_alk). It is shown that this technique of sample fractionation allows the SRM-SIS to detect 98% of the single peptides from the proteins present in the pure solution of UPS2 (47 out of 48 proteins). When the extracts of E. coli or Pichia pastoris are added as biological matrixes to the UPS2, 46, and 45 out of 48 proteins (∼95%) can be detected, respectively, using the SRM-SIS combined with 2D-LC_alk. The combination of the 2D-LC_alk SRM-SIS and shotgun technologies allows us to increase the sensitivity up to 100% in the case of the proteins of the UPS2 set. The usage of that technology can be a solution for identifying the so-called "missing" proteins and, eventually, creating the deep proteome of a particular chromosome of tissue or organs. Experimental data have been deposited in the PeptideAtlas SRM Experiment Library with the dataset identifier PASS01192 and the PRIDE repository with the dataset identifier PXD007643.
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http://dx.doi.org/10.1021/acs.jproteome.8b00754DOI Listing
December 2018

Next Steps on in Silico 2DE Analyses of Chromosome 18 Proteoforms.

J Proteome Res 2018 12 2;17(12):4085-4096. Epub 2018 Oct 2.

Institute of Biomedical Chemistry of Russian Academy of Medical Sciences , Pogodinskaya 10 , Moscow 119121 , Russia.

In the boundaries of the chromosome-centric Human Proteome Project (c-HPP) to obtain information about proteoforms coded by chromosome 18, several cell lines (HepG2, glioblastoma, LEH), normal liver, and plasma were analyzed. In our study, we have been using proteoform separation by two-dimensional electrophoresis (2DE) (a sectional analysis) and a semivirtual 2DE with following shotgun mass spectrometry using LC-ESI-MS/MS. Previously, we published a first draft of this research, where only HepG2 cells were tested. Here, we present the next step using more detailed analysis and more samples. Altogether, confident (2 significant sequences minimum) information about proteoforms of 117 isoforms coded by 104 genes of chromosome 18 was obtained. The 3D-graphs showing distribution of different proteoforms from the same gene in the 2D map were generated. Additionally, a semivirtual 2DE approach has allowed for detecting more proteoforms and estimating their pI more precisely. Data are available via ProteomeXchange with identifier PXD010142.
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http://dx.doi.org/10.1021/acs.jproteome.8b00386DOI Listing
December 2018

From electrochemistry to enzyme kinetics of cytochrome P450.

Biosens Bioelectron 2018 Dec 23;121:192-204. Epub 2018 Aug 23.

Institute of Biomedical Chemistry, 119121 Moscow, Russia; N.I. Pirogov Russian National Research Medical University, 117997 Moscow, Russia.

This review is an attempt to describe advancements in the electrochemistry of cytochrome P450 enzymes (EC 1.14.14.1) and to study molecular aspects and catalytic behavior of enzymatic electrocatalysis. Electroanalysis of cytochrome P450 demonstrates how to translate theoretical laws and equations of classical electrochemistry for the calculation of the kinetic parameters of enzymatic reactions and then translation of kinetic parameters to interpretation of drug-drug interactions. The functional significance of cytochrome P450s (CYPs) includes the metabolism of drugs, foreign chemicals, and endogenic compounds. The pharmaceutical industry needs sensitive and cost-effective systems for screening new drugs and investigation of drug-drug interactions. The development of different types of CYP-based biosensors is now in great demand. This review also highlights the characteristics of electrode processes and electrode properties for optimization of the cytochrome P450 electroanalysis. Electrochemical cytochrome P450-biosensors are the most studied. In this review, we analyzed electrode/cytochrome P450 systems in terms of the mechanisms underlying P450-catalyzed reactions. Screening of potential substrates or inhibitors of cytochromes P450 by means of electrodes were described.
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http://dx.doi.org/10.1016/j.bios.2018.08.040DOI Listing
December 2018

Micro-Raman Spectroscopy for Monitoring of Deposition Quality of High-k Stack Protective Layer onto Nanowire FET Chips for Highly Sensitive miRNA Detection.

Biosensors (Basel) 2018 Jul 27;8(3). Epub 2018 Jul 27.

Institute of Biomedical Chemistry (IBMC), Moscow 119121, Russia.

Application of micro-Raman spectroscopy for the monitoring of quality of high-k (h-k) dielectric protective layer deposition onto the surface of a nanowire (NW) chip has been demonstrated. A NW chip based on silicon-on-insulator (SOI) structures, protected with a layer of high-k dielectric ((h-k)-SOI-NW chip), has been employed for highly sensitive detection of microRNA (miRNA) associated with oncological diseases. The protective dielectric included a 2-nm-thick Al₂O₃ surface layer and a 8-nm-thick HfO₂ layer, deposited onto a silicon SOI-NW chip. Such a chip had increased time stability upon operation in solution, as compared with an unprotected SOI-NW chip with native oxide. The (h-k)-SOI-NW biosensor has been employed for the detection of DNA oligonucleotide (oDNA), which is a synthetic analogue of miRNA-21 associated with oncological diseases. To provide biospecificity of the detection, the surface of (h-k)-SOI-NW chip was modified with oligonucleotide probe molecules (oDVA probes) complementary to the sequence of the target biomolecule. Concentration sensitivity of the (h-k)-SOI-NW biosensor at the level of ~10 M has been demonstrated.
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http://dx.doi.org/10.3390/bios8030072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6164057PMC
July 2018

Atomic Force Microscopy for Protein Detection and Their Physicoсhemical Characterization.

Int J Mol Sci 2018 Apr 10;19(4). Epub 2018 Apr 10.

Institute of Biomedical Chemistry, 10, Pogodinskaya St., 119121 Moscow, Russia.

This review is focused on the atomic force microscopy (AFM) capabilities to study the properties of protein biomolecules and to detect the proteins in solution. The possibilities of application of a wide range of measuring techniques and modes for visualization of proteins, determination of their stoichiometric characteristics and physicochemical properties, are analyzed. Particular attention is paid to the use of AFM as a molecular detector for detection of proteins in solutions at low concentrations, and also for determination of functional properties of single biomolecules, including the activity of individual molecules of enzymes. Prospects for the development of AFM in combination with other methods for studying biomacromolecules are discussed.
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http://dx.doi.org/10.3390/ijms19041142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5979402PMC
April 2018

Relative Abundance of Proteins in Blood Plasma Samples from Patients with Chronic Cerebral Ischemia.

J Mol Neurosci 2018 Mar 5;64(3):440-448. Epub 2018 Mar 5.

V.-N. Orekhovich Research Institute of Biomedical Chemistry, 10, Pogodinskaya St, Moscow, Russian Federation, 119121.

A comparative protein profile analysis of 17 blood plasma samples from patients with ischemia and 20 samples from healthy volunteers was carried out using ultra-high resolution mass spectrometry. The analysis of measurements was performed using the proteomics search engine OMSSA. Normalized spectrum abundance factor (NSAF) in the biological samples was assessed using SearchGUI. The findings of mass spectrometry analysis of the protein composition of blood plasma samples demonstrate that the depleted samples are quite similar in protein composition and relative abundance of proteins. By comparing them with the control samples, we have found a small group of 44 proteins characteristic of the blood plasma samples from patients with chronic cerebral ischemia. These proteins contribute to the processes of homeostasis maintenance, including innate immune response unfolding, the response of a body to stress, and contribution to the blood clotting cascade.
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http://dx.doi.org/10.1007/s12031-018-1040-3DOI Listing
March 2018

Spatial features of proteins related to their phosphorylation and associated structural changes.

Proteins 2018 Jan 17;86(1):13-20. Epub 2017 Oct 17.

Department of Bioinformatics, Institute of Biomedical Chemistry (IBMC), Moscow, Russia.

Protein phosphorylation is widely used in biological regulatory processes. The study of spatial features related to phosphorylation sites is necessary to increase the efficacy of recognition of phosphorylation patterns in protein sequences. Using the data on phosphosites found in amino acid sequences, we mapped these sites onto 3D structures and studied the structural variability of the same sites in different PDB entries related to the same proteins. Solvent accessibility was calculated for the residues known to be phosphorylated. A significant change in accessibility was shown for many sites, but several ones were determined as buried in all the structures considered. Most phosphosites were found in coil regions. However, a significant portion was located in the structurally stable ordered regions. Comparison of structures with the same sites in modified and unmodified states showed that the region surrounding a site could be significantly shifted due to phosphorylation. Comparison between non-modified structures (as well as between the modified ones) suggested that phosphorylation stabilizes one of the possible conformations. The local structure around the site could be changed due to phosphorylation, but often the initial conformation of the site surrounding is not altered within bounds of a rather large substructure. In this case, we can observe an extensive displacement within a protein domain. Phosphorylation without structural alteration seems to provide the interface for domain-domain or protein-protein interactions. Accounting for structural features is important for revealing more specific patterns of phosphorylation. It is also necessary for explaining structural changes as a basis for regulatory processes.
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http://dx.doi.org/10.1002/prot.25397DOI Listing
January 2018

Why Are the Correlations between mRNA and Protein Levels so Low among the 275 Predicted Protein-Coding Genes on Human Chromosome 18?

J Proteome Res 2017 12 27;16(12):4311-4318. Epub 2017 Oct 27.

Institute of Biomedical Chemistry RAS , 119121 Moscow, Russia.

In this work targeted (selected reaction monitoring, SRM, PASSEL: PASS00697) and panoramic (shotgun LC-MS/MS, PRIDE: PXD00244) mass-spectrometric methods as well as transcriptomic analysis of the same samples using RNA-Seq and PCR methods (SRA experiment IDs: SRX341198, SRX267708, SRX395473, SRX390071) were applied for quantification of chromosome 18 encoded transcripts and proteins in human liver and HepG2 cells. The obtained data was used for the estimation of quantitative mRNA-protein ratios for the 275 genes of the selected chromosome in the selected tissues. The impact of methodological limitations of existing analytical proteomic methods on gene-specific mRNA-protein ratios and possible ways of overcoming these limitations for detection of missing proteins are also discussed.
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http://dx.doi.org/10.1021/acs.jproteome.7b00348DOI Listing
December 2017

Molecular imprinting coupled with electrochemical analysis for plasma samples classification in acute myocardial infarction diagnostic.

Biosens Bioelectron 2018 Jan 20;99:216-222. Epub 2017 Jul 20.

Institute of Biomedical Chemistry, 119121 Moscow, Russia; N.I. Pirogov Russian National Research Medical University, 117997 Moscow, Russia.

Electroanalysis of myoglobin (Mb) in 10 plasma samples of healthy donors (HDs) and 14 plasma samples of patients with acute myocardial infarction (AMI) was carried out with screen-printed electrodes modified first with multi-walled carbon nanotubes (MWCNT) and then with a molecularly imprinted polymer film (MIP), viz., myoglobin-imprinted electropolymerized poly(o-phenylenediamine). The differential pulse voltammetry (DPV) parameters, such as a maximum amplitude of reduction peak current (A, nA), a reduction peak area (S, nA × V), and a peak potential (P, V), were measured for the MWCNT/MIP-sensors after their incubation with non-diluted plasma. The relevance of the multi-parameter electrochemical data for accurate discrimination between HDs and patients with AMI was assessed on the basis of electrochemical threshold values (this requires the reference standard method (RAMP® immunoassay)) or alternatively on the basis of the computational cluster assay (this does not require any reference standard method). The multi-parameter electrochemical analysis of biosamples combined with computational cluster assay was found to provide better accuracy in classification of plasma samples to the groups of HDs or AMI patients.
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http://dx.doi.org/10.1016/j.bios.2017.07.026DOI Listing
January 2018

Label-free data standardization for clinical metabolomics.

BioData Min 2017 28;10:10. Epub 2017 Feb 28.

Institute of Biomedical Chemistry, Pogodinskaya st.10, 119121 Moscow, Russia.

Background: In metabolomics, thousands of substances can be detected in a single assay. This capacity motivates the development of metabolomics testing, which is currently a very promising option for improving laboratory diagnostics. However, the simultaneous measurement of an enormous number of substances leads to metabolomics data often representing concentrations only in conditional units, while laboratory diagnostics generally require actual concentrations. To convert metabolomics data to actual concentrations, calibration curves need to be generated for each substance, and this process represents a significant challenge due to the number of substances that are present in the metabolomics data. To overcome this limitation, a label-free standardization algorithm for metabolomics data is required.

Results: It was discovered that blood plasma has a set of stable internal standards. The appropriate usage of these newly discovered internal standards provides a background for the label-free standardization of metabolomics data that underlies the SantaOmics (Standardization algorithm for nonlinearly transformed arrays in Omics) algorithm. In this study, using the knee point, it was shown that the metabolomics data can be converted by SantaOmics into a standardized scale that can substitute actual concentration measurements, thus making the metabolomics data directly comparable with each other as well as with reference data presented in the same scale.

Conclusion: The developed algorithm sufficiently facilitates the usage of metabolomics data in laboratory diagnostics.
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http://dx.doi.org/10.1186/s13040-017-0132-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5329969PMC
February 2017

State of the Art of Chromosome 18-Centric HPP in 2016: Transcriptome and Proteome Profiling of Liver Tissue and HepG2 Cells.

J Proteome Res 2016 11 29;15(11):4030-4038. Epub 2016 Aug 29.

Institute of Biomedical Chemistry , Pogodinskaya Street, 10, Moscow 119121, Russia.

A gene-centric approach was applied for a large-scale study of expression products of a single chromosome. Transcriptome profiling of liver tissue and HepG2 cell line was independently performed using two RNA-Seq platforms (SOLiD and Illumina) and also by Droplet Digital PCR (ddPCR) and quantitative RT-PCR. Proteome profiling was performed using shotgun LC-MS/MS as well as selected reaction monitoring with stable isotope-labeled standards (SRM/SIS) for liver tissue and HepG2 cells. On the basis of SRM/SIS measurements, protein copy numbers were estimated for the Chromosome 18 (Chr 18) encoded proteins in the selected types of biological material. These values were compared with expression levels of corresponding mRNA. As a result, we obtained information about 158 and 142 transcripts for HepG2 cell line and liver tissue, respectively. SRM/SIS measurements and shotgun LC-MS/MS allowed us to detect 91 Chr 18-encoded proteins in total, while an intersection between the HepG2 cell line and liver tissue proteomes was ∼66%. In total, there were 16 proteins specifically observed in HepG2 cell line, while 15 proteins were found solely in the liver tissue. Comparison between proteome and transcriptome revealed a poor correlation (R ≈ 0.1) between corresponding mRNA and protein expression levels. The SRM and shotgun data sets (obtained during 2015-2016) are available in PASSEL (PASS00697) and ProteomeExchange/PRIDE (PXD004407). All measurements were also uploaded into the in-house Chr 18 Knowledgebase at http://kb18.ru/protein/matrix/416126 .
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http://dx.doi.org/10.1021/acs.jproteome.6b00380DOI Listing
November 2016
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