Identification of protein components of the transformation system in the cell line of immortalized human keratinocytes HaCaT exposed to surfactants.

Using the method of shotgun mass spectrometry, we have evaluated changes in the proteomic profile of HaCat cells in response to the treatment with sodium dodecyl sulfate (anionic surfactant) and Triton-X100 (non-ionic surfactant) in two concentrations (12.5 µg/ml and 25.0 µg/ml). The study revealed induction of orphan CYP2S1 (biotransformation phase I) in response to Triton-X100. We have identified proteins of II (glutathione-S-transferases, GSTs) and III (solute carrier proteins, SLCs) biotransformation phases, as well as antioxidant proteins (peroxiredoxins, PRDXs; catalase, CAT; thioredoxin, TXN). Thus, proteins of all three xenobiotic detoxification phases were detected. The presented results suggest a new prospect of using HaCaT keratinocytes as a model of human epidermis for studying the metabolism of drugs/toxicants in human skin in vitro.

[Comparative study of the human keratinocytes proteome of the HaCaT line: identification of proteins encoded by genes of 18 chromosomes under the influence of detergents]. / Sravnitel'noe issledovanie proteoma kletok HaCaT keratinotsitov cheloveka: identifikatsiia belkov, kodiruemykh genami 18 khromosomy pri vozdeistvii detergentov.

Using electrospray ionization tandem mass spectrometry, a comparative analysis of the HaCaT keratinocyte proteins encoded by the 18th chromosome was performed before and after exposure to sodium dodecyl sulfate (25 mg/ml) and to Triton X-100 (12.5 mg/ml) in a subtoxic dose for 48 hours. Proteins were identified using the SearchGUI platform (X!Tandem and MS-GF+ search engines). In total, 1284 proteins were found in immortalized human HaCaT keratinocytes and about 75% of them were identified by two or more peptides. Were identified, that 26 proteins were encoded by genes of chromosome 18. Among these proteins, 17 were common for control cells and HaCaT cells treated with SDS. Proteins MARE2 and CTIF were identified only in control keratinocytes. Seven identified proteins encoded by genes of chromosome 18 were found only in detergent-treated keratinocytes: LMAN1, NDUV2, SPB3, VPS4B, KDSR, ROCK1 and RHG28.

[Comparative analysis of post-translational modifications in plasma proteome of patients with cerebral ischemia based on HPLC-MS/MS method]. / Sravnitel'nyi analiz modifikatsii proteoma plazmy krovi bol'nykh tserebral'noi ishemiei na osnove metoda VÉZhKh-MS/MS.

The relative differences between post-translational modifications (PTM) of proteins in blood plasma samples of patients with cerebral ischemia (CI) and healthy people were investigated using of the method of label-free comparative proteomic analysis based on the technology of tandem HPLC-MS/MS. For PTM detection we used multiple MS/MS search in the database Mascot for variable PTM and Progenesis LS-MS software. In the CI plasma samples, we observed an increase in the proportion of peptides with such PTM as phosphorylation of serine, threonine, and tyrosine, acetylation of lysine and protein N-term, ubiquitination of lysine and deamidation of glutamine related to clinically significant processes were revealed.

Analysis of Blood Plasma Protein Composition in Patients with Cerebral Ischemia.

Blood plasma proteome in patients with cerebral ischemia and healthy individuals was studied using comparative proteomic analysis based on tandem HPLC-MS/MS. Mass spectra were analysed in an automated mode using Progenesis LS-MS software and 256 proteins were identified. Significant quantitative differences were revealed for 20 proteins. It was found that changes in the blood plasma proteome in subjects with cerebral ischemia involved a wide range of proteins: molecular chaperones, fibrinolysis, angiogenesis, and immune system proteins, proteins involved in homeostasis maintenance, cell differentiation and proliferation, regulators of apoptosis, and cytoskeleton proteins.

[Comparative proteome analysis of blood plasma of patients with early-stage chronic cerebral ischemia]. / Sravnitel'nyi analiz proteoma plazmy krovi bol'nykh na rannei stadii khronicheskoi tserebral'noi ishemii.

In the present study, we explored the technology of liquid chromatography-mass spectrometry (HPLC-MS/MS) for the proteome analysis of blood plasma of patients with early chronic cerebral ischemia. Analysis of mass-spectrometer data carried out in automatic mode using the software Progenesis LS-MS. As a result of this study identified 43 proteins. The differences identified in the study group compared with the control in 7 proteins. It was found that in the early stages of chronic cerebral ischemia proteome changes in blood plasma affect proteins related to the immune system, the system for the maintenance of hemostasis and lipid metabolism.

[Analysis of proteomic profile changes of zebrafish embryos during exposure to doxorubicin, built-in the phospholipid transport nanosystem].

The proteome profile of Danio rerio embryos grown in the medium containing doxorubicin, included in the phospholipid transport nanosystem (doxolip) has been investigated using combination of 1D-electrophoresis with subsequent MALDI-TOF-PMF mass spectrometry. Cultivation of growing of D. rerio embryos in the medium with doxolip caused a substantial increase in expression of the cytoskeletal proteins, a decrease in the number of nuclear proteins involved in DNA and RNA synthesis and disappearance of vitellogenin 2 in comparison with control (the cultivation medium containing the phospholipid transport nanosystem). Analysis of the proteomic profiles of doxolip-treated embryos suggests lower toxicity of doxorubicin incorporated in the phospholipid nanosystem.

Effects of fullerene C60 on proteomic profile of Danio rerio fish embryos.

The effects of phosphatidylcholine-based phospholipid nanoparticles containing fullerene C60 on Danio rerio fish embryos were studied. Exposure of the embryos with the nanoparticles for 48 h did not lead to appreciable changes in the number of protein bands in SDS-PAGE in comparison with the control (exposure in medium with phosphatidylcholine). Mass spectrometric identification of proteins showed differences in the proteomic profiles of the samples. The content of vitellogenins changed after exposure with phosphatidylcholine-based nanoparticles with C60 fullerenes. This could indicate low toxicity of the nanoparticles towards D. rerio embryos under experimental conditions.

The study of the proteome of healthy human blood plasma under conditions of long-term confinement in an isolation chamber.

We identified changes in the proteome of healthy human blood plasma caused by exposure to 105-day confinement in an isolation chamber. After removal of major proteins and concentration of minor proteins, plasma fractions were analyzed by two-dimensional electrophoresis followed by identification of significantly different protein spots by mass spectrometric analysis of the peptide fragments. The levels of α- and ß-chains of fibrinogen, a fragment of complement factor C4, apolipoproteins AI and E, plasminogen factor C1 complement, and immunoglobulin M changed in participants during the isolation period. These changes probably reflect the adaptive response to altered conditions of life.

[Study of proteomic profile of Danio rerio embryos using one-dimensional electrophoresis and mass spectrometry].

In the present study, a proteomic technology combining one-dimensional gel electrophoresis (1DE) with subsequent mass spectrometry (MALDI-TOF-PMF) has been successfully applied for revelation of changes in the protein profile of zebrafish (Danio rerio) 52 hpf embryos. Prior to 1DE separation of zebrafish embryonic proteins, the procedure for obtaining embryos homogenate was optimized by ultrasonic treatment. A total of 84 proteins, including 15 vitellogenins, were identified. It was shown that growing ofzebrafish embryos in the medium with doxorubicin (DOX) stimulated Caspase-3 induction and promoted the disappearance of cardiac troponins, both these findings being consistent with literature data on doxorubicin-induced cardiotoxicity. The 1DE-based proteomic mapping approach proposed herein enabled not only to identify proteins but also to register those changes in embryos' proteomic profile that were caused by doxorubicin.

[Fluorescence-based determination of enzyme activity of recombinant CYPS1B1 (sterol 14alpha-demethylase) with coumarin derivatives].

The current investigation was undertaken with the aim to carry out an in vitro evaluation of the ability of coumarin derivatives as probe substrates to predict the activity of CYP51b1. The results obtained indicate that 7-aminocoumarin-4-acetic acid (ACAC) can be used to determine the recombinant CYP51b1 activity. Determination of CYP51b1 activity with ACAC is based on the direct registration of fluorescence increasing at 30 degrees C. It was found also that BMR in a simple soluble model system can be used as an electron donor for CYP51B1. Fluorescence-based assay is highly sensitive and can be used for the screening of sterol 14alpha-demethylase inhibitors.

One-dimensional proteomic mapping of human liver cytochromes p450.

A method for constructing one-dimensional proteomic maps (1D-PM) based on mass spectrometric identification of proteins from adjacent slices of one-dimensional electrophoregram has been developed. For the proteomic mapping, gel lanes were sectioned into slices less than 0.2 mm thick and each slice was subjected to enzymatic hydrolysis. The resultant mixture of peptide fragments was analyzed by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF) and liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS). Proteins were identified by the mass spectra obtained. Data on peptide fragments and corresponding identified proteins were presented as a 1D-PM. Proteomic maps were constructed by assigning individual proteins to gel slices based on number of matching peptides in a corresponding MS-data. On 1D-PM of human liver microsomal fraction, 18 proteins were identified in the region of 40-65 kDa. These included 12 membrane proteins belonging to the superfamily of cytochromes P450. Pooling of mass spectrometric data, obtained from several adjacent gel slices (molecular zooming) increased sequence coverage of CYP2A (cytochrome P450 family 2A). The maximal coverage of 66% significantly exceeded the level of 48% that could be obtained using one (even the most informative) slice. This method can be applied to the proteomic profiling of membrane-bound proteins.

[Cytochromes P450, drug disease and personified medicine. Part 2. Therapeutic drug monitoring as a method of monooxygenase activity assessment].

In part 2 of the review the authors consider factors, having influence on the state of monooxygenase system (MOS): cytochrome P-450 gene polymorphism, induction or inhibition of these systems under effect of drugs, crossed substance specificity of cytochrome P-450 forms. Various methodical approaches (genomic, proteomic, bioelectrical technologies, therapeutic drug monitoring) to receive full information about a profile of cytochrome P-450 for every specific person, are compared. Necessity of MOS individual features assessment for optimization of drug therapy is proved.

[Cytochromes P-450, drug disease, and personified medicine. Part I].

The review summarizes data on the molecular basis of medication metabolism, factors forming the personal profile of cytochrome P-450, methods to estimate the activity of the monooxygenase system, and problems of medication interference and therapeutic pharmacomonitoring, forming the basis of personified medicine. Special attention is paid to the cytochrome-P-450-containing hepatic system, responsible for inter-individual differences after administration of medications in therapeutic doses. Factors influencing the condition of the monooxygenase system: cytochrome P-450 genetic polymorphism, inducing of inhibition of these systems by medications, and crisscross substrate specificity of cytochrome P-450 are considered. Different methodical approaches (genomic and proteomic ones, bioelectrochemical nanotechnologies, and therapeutic pharmacomonitoring) to obtaining full information on cytochrome P-450 for each individual are compared. The article substantiates the necessity to assess individual features of the monooxygenase system to optimize medication therapy.

[Identification of cytochromes P450 in the human liver microsomes by mass spectrometry].

Proteomic approaches have been used for detection and identification of cytochromes P450 from highly-purified membrane preparations of human liver. These included the protein separation by 2D- and/or 1D-electrophoresis and molecular scanning of a SDS-PAGE gel fragment in the range of 45-66 kD (this area corresponds molecular weights of cytochromes P450). The analysis of protein content was statistically evaluated by means of original 1D-ZOOMER software package which allowed to carry out processing of mass spectra mixture instead of individual mass spectra used by standard techniques. In the range of 45-66 kDa we identified 13 microsomal membrane proteins including 11 cytochromes P450, namely CYPs 1A2, 1B1, 2A6, 2E1, 2C8, 2C9, 2C10, 2D6, 3A4, 4A11, 4F2. The microsomal samples were characterized by the enzymatic assays using the marker substrates for CYP1A, 2B, 3A4, 2C and 2E1. The 7-methoxy- and 7-ethoxyresorufin-O-dealkylase activities (i.e. the marker activities for cytochromes P450 1A1/1A2, respectively) and the erythromycin-N-demethylase activity (i.e. the marker activity for cytochrome P450 3A4) are lowered in pathology compared to these activities in norm. At the same time the benzyloxyresorufin-O-debenzylase activity (which characterizes the total activity of CYP2B and CYP2C), the activities of CYP2E1 (methanol), 7-pentoxyresorufin-O-dealkylation (CYP2B), 7-ethoxy- and 7-methoxycoumarin-O-dealkylases (CYP2B1) did not change. On the basis of the results obtained efficiency of a combination proteomic and biochemical analyses for inventory cytochromes P450 and revealing of their level expression is shown, and opportunities of mass spectrometry for a quantitative estimation of proteins are discussed.

Electrochemical reduction of sterol-14alpha-demethylase from Mycobacterium tuberculosis (CYP51b1).

The electrochemical reduction of the heme protein sterol-14alpha-demethylase from Mycobacterium tuberculosis (CYP51b1, or further CYP51) was investigated. Direct electron transfer was demonstrated between CYP51 and graphite screen-printed electrodes modified with gold nanoparticles and with the membrane-like synthetic surfactant didodecyl dimethylammonium bromide. The formal potential of the Fe3+/Fe2+ pair, E(1/2), is equal to -273 mV (vs. Ag/AgCl). The cathodic current corresponding to the reduction of oxygen by immobilized heme protein was registered in the presence of oxygen. Addition of lanosterol, one of the substrates of the CYP51 family, to the oxygenated solution caused a concentration-dependent increase in the reduction current in voltammetric and amperometric experiments. Ketoconazole, an inhibitor of CYP51, inhibited the catalytic cathodic current in the presence of lanosterol. Electrochemical reduction of CYP51 may serve as an adequate alternative to the reconstituted system, which requires additional redox partners for the exhibition of catalytic activity of heme proteins of the cytochrome P450 superfamily.

Electrochemical properties of cytochroms P450 using nanostructured electrodes: direct electron transfer and electro catalysis.

The present study demonstrates direct electron transfer between cytochromes P450 2B4 (CYP2B4), P450 1A2 (CYP1A2), sterol 14alpha-demethylase (CYP51b1) on the one hand and screen-printed graphite electrodes, modified with gold nanoparticles and didodecyldimethylammonium bromide (DDAB) on the other. Electro detection of heme proteins was possible when 2-200 pmol P450/electrode were adsorbed on the surface of nanostructured electrochemical interfaces. Electron transfer, direct electrochemical reduction and interaction with P450 substrates (oxygen, benzphetamine, and lanosterol) and with P450 inhibitor (ketoconazole) were analyzed using cyclic voltammetry (CV), square wave voltammetry (SWV) differential pulse voltammetry (DPV), and amperometry.

[Nanoelectrochemistry of cytochrome P450s: direct electron transfer and electrocatalysis].

The present study demonstrates the direct electron transfer between cytochrome P450 2B4 (CYP2B4), P450 1A2 (CYP1A2), sterol 14alpha-demethylase (CYP51MT) and screen printed graphite electrodes, modified with gold nanoparticles and didodecyldimethylammonium bromide (DDAB). Electrodetection of heme proteins is possible when 2-200 pmol P450/electrode were adsorbed on the surface of nanostructured electrochemical interfaces. Electron transfer, direct electrochemical reduction and interaction with P450 substrates (oxygen, benzphetamine, lanosterol) and inhibitor ketoconazole were analyzed using cyclic voltammetry (CV), square wave (SWV) or differential pulse (DPV) voltammetry, amperometry.

Characterization of human liver cytochromes P450 by combining the biochemical and proteomic approaches.

Highly purified human liver microsomes were processed by a combination of the biochemical and proteomic methods. Microsomes were purified from the morphologically normal liver tissue obtained from the resected and discarded masses of surrounding liver upon surgical treatment for hemangioma (control) or hepatic metastases arising from colon cancer (pathology). Proteins of each sample were separated by two-dimensional (2-DE) and one-dimensional electrophoresis (1-DE); selected gel regions were excised, in-gel digested and analyzed by matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry. Analysis of collected fingerprints has revealed a total of 13 microsomal membrane proteins involved in the biotransformation of xenobiotics. These were disulfide isomerase, flavine monooxygenase, NADPH-cytochrome P450 reductase and 10 cytochrome P450 forms, namely: CYPs 1B1, 2A6, 2E1, 2C8, 2C9, 2C10, 2D6, 3A4, 4A11, 4F2. These same samples were characterized by the enzymatic assays using the marker substrates for CYPs 1A, 2B, 3A4, 2C and 2E1. Correlations between mass spectrometric data and enzymatic activities were investigated to demonstrate the manner in which the functional and structural aspects of proteomics meet each other in the field of cytochromes P450.

Proteomic and biochemical analysis of the mouse liver microsomes.

The efficiency of the proteomic approach for the revelation of proteins, including components of the liver microsomal monooxygenase system (cytochromes b5 and P450) was demonstrated. The liver microsomes and their ghosts (i.e. membranes devoid of "ballast" proteins) were prepared from the control and phenobarbital-treated mice. Microsomes and their ghosts were characterized using the conventional biochemical assay and analysed by one- and two-dimensional electrophoresis (1-DE and 2-DE, respectively) coupled with MALDI-TOF peptide mass fingerprinting procedure. Catalytic activity of cytochromes P450 was measured using specific fluorogenic substrates for CYP1A, CYP2A, CYP2B and CYP2C families. The protein composition of control and phenobarbital-induced ghosts was analysed. The proteomic 2D-based protein separation method enabled us to reveal up to 1005 proteins, the majority of them being soluble. Among the 34 identified proteins, the cytochrome b5-like protein was revealed; however, cytochromes P450 appeared to be undetectable under 2-DE separation conditions. The separation of microsomal ghosts proteins by 1-DE, followed by mass-spectrometric analysis of bands from the 45 to 66 kDa gel range made it possible to identify hydrophobic proteins including cytochromes P450 (CYP2A4 and CYP2A5) and dimethylaniline monooxygenase. The high O-deethylation rate of 7-ethoxycoumarin-a substrate for rodent CYPs 2A and 2B, in particular for CYP2A5-was observed, in agreement with the results of mass-spectrometric identification. Collectively, the data obtained indicate that a combination of enzyme activity assays and various protein separation techniques coupled with mass-spectrometric protein identification allows a more comprehensive insight into the machinery of the cellular detoxifying system.

[Study of the mouse liver microsomes by the methods of proteome analysis]. / Issledovanie mikrosom kletok pecheni myshi metodami poreomnogo analiza.

Proteome maps of microsomes and their ghosts (i.e. membranes purified from "ballast" proteins) were obtained using highly purified mouse liver microsomes. Comparative analysis of protein composition of ghosts without and after the induction with phenobarbital (cytochromes P450 inducer) by using 1D- and 2D-electrophoresis and MALDI-TOF-mass-spectrometry revealed more than 30 new proteins, in the course of induction in the 45-60 kDa range (corresponding to the mol. weights of cytochromes P450). In the 17 kDa range (corresponding to the mol. wt. of cytochrome b5) there were 4 additional protein stains about 20 proteins disappeared over the entire electrophoregram). Separation of microsomal ghosts proteins by 1D electrophoresis followed by mass-spectrometric analysis allowed to identify cytochromes P450. The present investigation demonstrates the efficiency of different proteomic methods combination (1D- and 2D-electrophoresis, mass-spectrometry, bioinformatics and determination of the enzyme activities) for cytochromes P450 identification and elucidation of their functioning in different animal tissues and then extrapolating this approach to humans.