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.
•Amino acids generate protein electroactivity.•Post-translational modifications of protein amino acids are important for medicine.•Electrochemistry is sensitive to the post-translational modifications of proteins.•Oxidation, phosphorylation and glycosylation of proteins are best-studied.•Activity of enzymes responsible for carrying out post-translational modifications is measured.
The three-dimensional structure of monomers and homodimers of CYP102A1/WT (wild-type) proteins and their A83F and A83I mutant forms was predicted using the AlphaFold2 (AF2) and AlphaFold Multimer ...(AFMultimer) programs, which were compared with the rate constants of hydroxylation reactions of these enzyme forms to determine the efficiency of intra- and interprotein electron transport in the CYP102A1 hydroxylase system. The electron transfer rate constants (
), which determine the rate of indole hydroxylation by the CYP102A1 system, were calculated based on the distances (R) between donor-acceptor prosthetic groups (PG) FAD→FMN→HEME of these proteins using factor β, which describes an exponential decay from R the speed of electron transport (ET) according to the tunnelling mechanism. It was shown that the structure of monomers in the homodimer, calculated using the AlpfaFold Multimer program, is in good agreement with the experimental structures of globular domains (HEME-, FMN-, and FAD-domains) in CYP102A1/WT obtained by X-ray structural analysis, and the structure of isolated monomers predicted in AF2 does not coincide with the structure of monomers in the homodimer, although a high level of similarity in individual domains remains. The structures of monomers and homodimers of A83F and A83I mutants were also calculated, and their structures were compared with the wild-type protein. Significant differences in the structure of all isolated monomers with respect to the structures of monomers in homodimers were also found for them, and at the same time, insignificant differences were revealed for all homodimers. Comparative analysis for CYP102A1/WT between the calculated intra- and interprotein distances FAD→FMN→HEME and the rate constants of hydroxylation in these proteins showed that the distance between prosthetic groups both in the monomer and in the dimer allows the implementation of electron transfer between PGs, which is consistent with experimental literature data about
. For the mutant form of monomer A83I, an increase in the distance between PGs was obtained, which can restrict electron transportation compared to WT; however, for the dimer of this protein, a decrease in the distance between PGs was observed compared to the WT form, which can lead to an increase in the electron transfer rate constant and, accordingly,
. For the monomer and homodimer of the A83F mutant, the calculations showed an increase in the distance between the PGs compared to the WT form, which should have led to a decrease in the electron transfer rate, but at the same time, for the homodimer, the approach of the aromatic group F262 with heme can speed up transportation for this form and, accordingly, the rate of hydroxylation.
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.
•Electrochemistry of CYP as a model system for investigation of drug metabolism.•Translation of electrochemical parameters to kinetic constants.•Enzymes as recognition elements in the electrochemical biosensor analysis.
In metabolomics, many metabolites are measured simultaneously in a single run. Such analytical performance opens up prospects for clinical laboratory diagnostics. In this work, a mass spectrometric ...metabogram was developed as a simplified and clinically applicable way of measuring the blood plasma metabolome. To develop the metabogram, blood plasma samples from healthy male volunteers (n = 48) of approximately the same age, direct infusion mass spectrometry (DIMS) of the low molecular fraction of samples, and principal component analysis (PCA) of the mass spectra were used. The seven components of the metabogram defined by PCA, which cover ~70% of blood plasma metabolome variability, were characterized using a metabolite set enrichment analysis (MSEA) and clinical test results of participating volunteers. It has been established that the components of the metabogram are functionally related groups of the blood metabolome associated with regulation, lipid-carbohydrate, and lipid-amine blood components, eicosanoids, lipid intake into the organism, and liver function thereby providing a lot of clinically relevant information. Therefore, metabogram provides the possibility to apply the metabolomics performance in the clinic. The features of the metabogram are also discussed in comparison with the thin-layer chromatography and with the analysis of blood metabolome by liquid chromatography combined with mass spectrometry.
Currently, nanopore-based technology for the determination of the functional activity of single enzyme molecules continues its development. The use of natural nanopores for studying single enzyme ...molecules is known. At that, the approach utilizing artificial solid-state nanopores is also promising but still understudied. Herein, we demonstrate the use of a nanotechnology-based approach for the investigation of the enzymatic activity of a single molecule of horseradish peroxidase with a solid-state nanopore. The artificial 5 nm solid-state nanopore has been formed in a 40 nm thick silicon nitride structure. A single molecule of HRP has been entrapped into the nanopore. The activity of the horseradish peroxidase (HRP) enzyme molecule inserted in the nanopore has been monitored by recording the time dependence of the ion current through the nanopore in the course of the reaction of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) oxidation reaction. We have found that in the process of ABTS oxidation in the presence of 2.5 mM hydrogen peroxide, individual HRP enzyme molecules are able to retain activity for approximately 700 s before a decrease in the ion current through the nanopore, which can be explained by structural changes of the enzyme.
The Size of the Human Proteome: The Width and Depth Ponomarenko, Elena A.; Poverennaya, Ekaterina V.; Ilgisonis, Ekaterina V. ...
International Journal of Analytical Chemistry,
01/2016, Letnik:
2016
Journal Article
Recenzirano
Odprti dostop
This work discusses bioinformatics and experimental approaches to explore the human proteome, a constellation of proteins expressed in different tissues and organs. As the human proteome is not a ...static entity, it seems necessary to estimate the number of different protein species (proteoforms) and measure the number of copies of the same protein in a specific tissue. Here, meta-analysis of neXtProt knowledge base is proposed for theoretical prediction of the number of different proteoforms that arise from alternative splicing (AS), single amino acid polymorphisms (SAPs), and posttranslational modifications (PTMs). Three possible cases are considered: ( 1 ) PTMs and SAPs appear exclusively in the canonical sequences of proteins, but not in splice variants; ( 2 ) PTMs and SAPs can occur in both proteins encoded by canonical sequences and in splice variants; ( 3 ) all modification types (AS, SAP, and PTM) occur as independent events. Experimental validation of proteoforms is limited by the analytical sensitivity of proteomic technology. A bell-shaped distribution histogram was generated for proteins encoded by a single chromosome, with the estimation of copy numbers in plasma, liver, and HepG2 cell line. The proposed metabioinformatics approaches can be used for estimation of the number of different proteoforms for any group of protein-coding genes.
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.
Electrosynthesis of molecularly imprinted polymer (MIP) templated with myoglobin (Mb) and the reference non-imprinted polymer (NIP) was examined with o-phenylenediamine (o-PD) as a monomer. ...Mass-sensitive quartz crystal microbalance with dissipation monitoring supplied by an electrochemical module (EQCM-D) was applied to characterize and optimize MIP/NIP electrosynthesis. Mb rebinding was detected by direct electrocatalytic reduction of Mb by square wave voltammetry (SWV) or differential pulse voltammetry (DPV). The results obtained showed high specificity of polymeric antibodies to template Mb, with an imprinting factor determined as a ratio Imax(MIP)/Imax(NIP) of 2–4. The prepared MIP sensor is characterized by an apparent dissociation constant of (3.3±0.5)×10−9M and has a broad range of working concentrations of 1nM–1μМ, with the detection limit of 0.5nM (9ng/ml). Mb rebinding was examined in Mb-free diluted human serum spiked with Mb as well as in plasma samples of patients with acute myocardial infarction (AMI) and in control plasma of healthy donors in order to demonstrate the potential medical application of developed MIP sensors.
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•Electrosynthesis of molecularly imprinted polymer with template myoglobin was examined with o-phenylenediamine as monomer.•Myoglobin rebinding was detected by direct electrocatalytic reduction Fe+3.•Myoglobin was registered in plasma samples of patients with acute myocardial infarction.
In omics sciences, many compounds are measured simultaneously in a sample in a single run. Such analytical performance opens up prospects for improving cellular cancer vaccines and other cell-based ...immunotherapeutics. This article provides an overview of proteomics technology, known as cell proteomic footprinting. The molecular phenotype of cells is highly variable, and their antigenic profile is affected by many factors, including cell isolation from the tissue, cell cultivation conditions, and storage procedures. This makes the therapeutic properties of cells, including those used in vaccines, unpredictable. Cell proteomic footprinting makes it possible to obtain controlled cell products. Namely, this technology facilitates the cell authentication and quality control of cells regarding their molecular phenotype, which is directly connected with the antigenic properties of cell products. Protocols for cell proteomic footprinting with their crucial moments, footprint processing, and recommendations for the implementation of this technology are described in this paper. The provided footprints in this paper and program source code for their processing contribute to the fast implementation of this technology in the development and manufacturing of cell-based immunotherapeutics.
•intrinsic electroactivity of proteins is generated by their prosthetic groups and amino acids.•medical application of direct protein electrochemistry is discussed.•mediator-free and label-free ...protein detection is considered.•protein molecule modification, unfolding and aggregation are analyzed.•a search for new enzyme substrates and inhibitors is described.
This mini-review outlines the current state of knowledge in field of protein electrochemistry and its application in medicine. Protein electrochemistry is considered according to (i) its intrinsic redox activity as generated by prosthetic groups and/or amino acid residues as well as (ii) charge transfer or adsorption at interfaces between two immiscible electrolyte solutions (liquid-liquid interfaces). Despite protein electrochemistry already has experimental and theoretical basis, its potential applications in medicine–where proteins serve as the most valuable biomarkers–is not yet attended. Mediator-free and label-free protein detection, analysis of its denaturation and aggregation, measurement and regulation of enzyme activity, a search for new substrates and inhibitors–i.e. the major problems of protein research–are treated in this mini-review from the electrochemical viewpoint. Significance of protein electrochemistry for clinical, pharmacological and scientific medicine is discussed.
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