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The search for minimally invasive methods for diagnostics of colorectal cancer (CRC) is the most important task for early diagnostics of the disease and subsequent successful treatment. Human plasma represents the main type of biological material used in the clinical practice; however, the complex dynamic range of substances circulating in it complicates determination of CRC protein markers by the mass spectrometric (MS) method. Studying the proteome of extracellular vesicles (EVs) isolated from human plasma represents an attractive approach for the discovery of tissue-secreted CRC markers. We performed shotgun mass spectrometry analysis of EV samples obtained from plasma of CRC patients and healthy volunteers. This MS analysis resulted in identification of 370 proteins (which were registered by at least two peptides). Stable isotope-free relative quantitation identified 55 proteins with altered abundance in EV samples obtained from plasma samples of CRC patients as compared to healthy controls. Among the EV proteins isolated from blood plasma we found components involved in cell adhesion and the VEGFA-VEGFR2 signaling pathway (TLN1, HSPA8, VCL, MYH9, and others), as well as proteins expressed predominantly by gastrointestinal tissues (polymeric immunoglobulin receptor, PIGR). The data obtained using the shotgun proteomic profiling may be added to the panel for targeted MS analysis of EV-associated protein markers, previously developed using CRC cell models.

Detection of low-copy proteins in complex biological samples is one of the most important issues of modern proteomics. The main reason for inefficient detection of low protein concentrations is the insufficient sensitivity of mass spectrometric detectors and the high dynamic range of protein concentrations. In this study we have investigated the possibilities and limitations of a targeted mass spectrometric analysis using the reconstructed system of standard proteins UPS1 (Universal Proteomic Standard 1) as an example. The study has shown that the sensitivity of the method is affected by the concentration of target proteins of the UPS1 system, as well as by a high level of biological noise modelled by proteins of whole E. coli cell lysate. The limitations of the method have been overcome by concentrating and pre-fractionating the sample peptides in a reversed phase chromatographic system under alkaline elution conditions. Proteomic analysis of the biological sample (proteins of the human hepatocellular carcinoma cell line HepG2 encoded by genes of human chromosome 18) showed an increase in the sensitivity of the method as compared to the standard targeted mass spectrometric analysis. This culminated in registration of 94 proteins encoded by genes located on human chromosome18.

Parkinsonism in rats induced by the pesticide rotenone is one of the most adequate models of Parkinson's disease (PD). Isatin (indole-2,3-dione) is an endogenous regulator found in mammals and humans and exhibiting a wide range of biological activities mediated by numerous isatin-binding proteins, including those associated with neurodegenerative pathology. A course of rotenone administration to rats caused behavioral impairments and changes in the profile and relative content of isatin-binding proteins in the brain. In this study, we have investigated the delayed neuroprotective effect of isatin (5 days after completion of the course of rotenone administration) on behavioral reactions and the relative content of isatin-binding proteins in the brain of rats with rotenone-induced experimental parkinsonism. Although during this period the rats retained locomotor dysfunction, the proteomic analysis data (profile of isatin-binding proteins in the brain and changes in their relative content) differed from the results obtained immediately after completion of the course of rotenone administration. Moreover, all isatin-binding proteins with altered relative content changed during this period are associated to varying degrees with neurodegeneration (many with Parkinson's and Alzheimer's diseases).

Renalase (RNLS) is a recently discovered protein that plays an important role in the regulation of blood pressure by acting inside and outside cells. Intracellular RNLS is a FAD-dependent oxidoreductase that oxidizes isomeric forms of β-NAD(P)H. Extracellular renalase lacking its N-terminal peptide and cofactor FAD exerts various protective effects via non-catalytic mechanisms. Certain experimental evidence exists in the literature that the RP220 peptide (a 20-mer peptide corresponding to the amino acid sequence RNLS 220-239) reproduces a number of non-catalytic effects of this protein, acting on receptor proteins of the plasma membrane. The possibility of interaction of this peptide with intracellular proteins has not been studied. Taking into consideration the known role of RNLS as a possible antihypertensive factor, the aim of this study was to perform proteomic profiling of the kidneys of normotensive and hypertensive rats using RP220 as an affinity ligand. Proteomic (semi-quantitative) identification revealed changes in the relative content of about 200 individual proteins in the kidneys of hypertensive rats bound to the affinity sorbent as compared to the kidneys of normotensive animals. Increased binding of SHR renal proteins to RP220 over the normotensive control was found for proteins involved in the development of cardiovascular pathology. Decreased binding of the kidney proteins from hypertensive animals to RP220 was noted for components of the ubiquitin-proteasome system, ribosomes, and cytoskeleton.

Comparative proteomic analysis of kidney tissue from normotensive (WKY) and spontaneously hypertensive (SHR) rats revealed quantitative and qualitative changes in renal proteins. The number of renal proteins specific for WKY rats (blood pressure 110-120 mm Hg) was 13-16. There were 20-24 renal proteins specific for SHR (blood pressure 180 mm Hg and more). The total number of identified renal proteins common for both rat strains included 972-975 proteins. A pairwise comparison of all possible (SHR-WKY) variants identified 8 proteins specific only for normotensive (WKY) animals, and 7 proteins specific only for hypertensive ones (SHR). Taking into consideration their biological roles, the lack of some enzyme proteins in hypertensive rats (for example, biliverdin reductase A) reduces the production of molecules exhibiting antihypertensive properties, while the appearance of others (e.g. betaine-homocysteine S-methyltransferase 2, septin 2, etc.) can be interpreted as a compensatory reaction. Renal proteins with altered relative content (with more than 2.5-fold change) accounted for no more than 5% of all identified proteins. Among the proteins with an increased relative content in hypertensive animals, the largest group consisted of proteins involved in the processes of energy generation and carbohydrate metabolism, as well as antioxidant and protective proteins. In the context of the development of hypertension, the identified relative changes can apparently be considered compensatory. Among the proteins with the most pronounced decrease in the relative content in hypertensive rats, the dramatic reduction in acyl-CoA medium-chain synthetase-3 (ACSM3) appears to make an important contribution to the development of renal pathology in these animals.

Isatin (indoldione-2,3) is an endogenous biological regulator found in the brain, peripheral tissues, and biological fluids of humans and animals. Its biological activity is realized via isatin-binding proteins, many of which were identified during proteomic profiling of the brain of mice and rats. A number of these proteins are related to the development of neurodegenerative diseases. Previously, using a model of experimental Parkinsonism induced by a seven-day course of rotenone injections, we have observed behavioral disturbances, as well as changes in the profile and relative content of brain isatin-binding proteins. In this study, we have investigated behavioral responses and the relative content of brain isatin-binding proteins in rats with rotenone-induced Parkinsonism 5 days after the last administration of this neurotoxin. Despite the elimination of rotenone, animals exhibited motor and coordination impairments. Proteomic profiling of isatin-binding proteins revealed changes in the relative content of 120 proteins (the relative content of 83 proteins increased and that of 37 proteins decreased). Comparison of isatin-binding proteins characterized by the changes in the relative content observed in the brain right after the last injection of rotenone (n=16) and 5 days later (n=11) revealed only two common proteins (glyceraldehyde-3-phosphate dehydrogenase and subunit B of V-type proton ATPase). However, most of these proteins are associated with neurodegeneration, including Parkinson's and Alzheimer's diseases.

Plasma membrane proteins with extracellular-exposed domains are responsible for transduction of extracellular signals into intracellular responses, and their accessibility to therapeutic molecules makes them attractive targets for drug development. In this work, using omics technologies and immunochemical methods, we have studied changes in the content of markers of clusters of differentiation (CD markers) of neutrophils (CD33, CD97, CD54, CD38, CD18, CD11b, CD44, and CD71) at the level of transcripts and proteins in NB4, HL-60 and K562 cell lines, induced by the treatment with all-trans-retinoic acid (ATRA). Transcriptomic analysis revealed the induction of CD38, CD54, CD11b, and CD18 markers as early as 3 h after the addition of the inducer in the ATRA-responsive cell lines HL-60 and NB4. After 24 h, a line-specific expression pattern of CD markers could be observed in all cell lines. Studies of changes in the content of CD antigens by means of flow cytometry and targeted mass spectrometry (MS) gave similar results. The proteomic profile of the surface markers (CD38, CD54, CD11b, and CD18), characteristic of the NB4 and HL-60 lines, reflects different molecular pathways for the implementation of ATRA-induced differentiation of leukemic cells into mature neutrophils.

Effects of the endogenous neuroprotector isatin and the pharmacological drug afobazole (exhibiting neuroprotective properties) on behavioral reactions and quantitative changes in the brain proteomic profile have been investigated in rats with experimental rotenone Parkinsonism. A single dose of isatin (100 mg/kg subcutaneously on the last day of a 7-day course of rotenone administration) improved the motor activity of rats with rotenone-induced Parkinsonism in the open field test (horizontal movements) and the rotating rod test. Afobazole (10 mg/kg intraperitoneally, daily during the 7-day course of rotenone administration) reduced the manifestations of rigidity and postural instability. Proteomic analysis, performed using brain samples obtained the day after the last administration of rotenone and neuroprotectors, revealed similar quantitative changes in the brain of rats with rotenone Parkinsonism. An increase in the relative content of 65 proteins and a decrease in the relative content of 21 proteins were detected. The most pronounced changes - an almost ninety-fold increase in the alpha-synuclein content - were found in the brains of rats treated with isatin. In animals of the experimental groups treated with "Rotenone + Isatin", as well as "Rotenone + Afobazole", the increase in the relative content of this protein in the brain was almost 60 and 50 times higher than the control values. Taking into consideration the known data on the physiological role of alpha-synuclein, an increase in the content of this protein in the brain upon administration of neuroprotectors to animals with rotenone Parkinsonism may represent a compensatory reaction, at least in the early stages of this disease and the beginning of its treatment.

Isatin (indoldione-2,3) is an endogenous regulator found in humans and animals. It exhibits a broad range of biological activity mediated by numerous isatin-binding proteins. Isatin produces neuroprotective effects in several experimental models of diseases, including Parkinsonism induced by the neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine).Rotenone (a neurotoxin used to modeling Parkinson's disease in rodents) causes significant changes in the profile of isatin-binding proteins of rat brain. Comparative proteomic identification of brain proteins of control rats and the rats with the rotenone-induced Parkinsonian syndrome (PS) revealed significant quantitative changes of 86 proteins under the influence of rotenone. This neurotoxin mainly caused the increase of the quantity of proteins involved in signal transduction and regulation of enzyme activity (24), proteins involved in cytoskeleton formation and exocytosis (23), and enzymes involved in energy generation and carbohydrate metabolism (19). However, only 11 of these proteins referred to isatin-binding proteins; the content of eight of them increased while the content of three proteins decreased. This suggests that the dramatic change of the profile of isatin-binding proteins, found in the development of the rotenone-induced PS, comes from changes in the state of the pre-existing molecules of proteins, rather than altered expression of corresponding genes.

The neurotoxins rotenone and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (МPTP) are used for modeling Parkinson's disease in animals (PD). They induce the mitochondrial respiratory chain dysfunction, which leads to the dopaminergic (DA) neuron degeneration. The advantage of the rotenone model consists in ability of rotenone to cause neurodegeneration showing symptoms and molecular biological characteristics similar to those of PD. Isatin (indoldione-2,3) is an endogenous regulator found in tissues and biological fluids of humans and animals. It exhibits a broad range of biological activity mediated by numerous isatin-binding proteins. In this work we have investigated behavioral reactions and profiles of brain isatin-binding proteins of rats with Parkinson's syndrome (PS) in comparison with the corresponding parameters of MPTP-induced Parkinsonism in mice. Systemic injection of rotenone caused severe PS comparable with the effect of MPTP injection. It was accompanied by significant body weight loss, death, oligokinesia, muscular rigidity, and postural instability of animals. In spite of the same pathogenic basis of PS caused by rotenone and MPTP, the molecular mechanisms of their action differ. In the case of rotenone-induced PS, the pool of isatin-binding proteins common of the control rats and the rats with PS (146) significantly exceeded the pool of the common proteins of control mice and mice with PS induced by MPTP, whether right after neurotoxin injection (27), or (all the more) in a week after the MPTP injection (14). The comparison of isatin-binding proteins specific of the animals with MPTP-induced PS and with the rotenone-induced PS (as compared with the control animals) revealed total absence of proteins common of these two models of PD. It is to be noted that both neurotoxins particularly affected the proteins participating in the signal transmission and enzyme activity regulation. The changes of the profile of isatin-binding proteins in response to the injection of rotenone suggest that the neuroprotector isatin could also influence positively in the case of the rotenone model of PD.

Antibodies represent an essential component of humoral immunity; therefore their study is important for molecular biology and medicine. The unique property of antibodies to specifically recognize and bind a certain molecular target (an antigen) determines their widespread application in treatment and diagnostics of diseases, as well as in laboratory and biotechnological practices. High specificity and affinity of antibodies is determined by the presence of primary structure variable regions, which are not encoded in the human genome and are unique for each antibody-producing B cell clone. Hence, there is little or no information about amino acid sequences of the variable regions in the databases. This differs identification of antibody primary structure from most of the proteomic studies because it requires either B cell genome sequencing or de novo amino acid sequencing of the antibody. The present review demonstrates some examples of proteomic and proteogenomic approaches and the methodological arsenal that proteomics can offer for studying antibodies, in particular, for identification of primary structure, evaluation of posttranslational modifications and application of bioinformatics tools for their decoding.

We have isolated fractions of 26S and 20S proteasomes were from the rabbit liver and the brain. According to mass spectrometric (MS) analysis, the 26S proteasome fractions from these organs contained catalytic and regulatory subunits characteristic of the proteasome core and regulatory subunits. The 20S fractions of brain and liver proteasomes contained only catalytic proteasome subunits. In addition to proteasome subunits, the isolated fractions contained components of the ubiquitin-proteasome system, ubiquitinated proteins, enzymes that play an important role in metabolic processes, cytoskeletal components, signaling, regulatory, and protective proteins, as well as proteins regulating gene expression, cell division, and differentiation. The abundance of a number of proteasome-associated proteins was comparable or exceeded the abundance of intrinsic proteasome components. About a third of the proteins common to all studied fractions (26S and 20S of brain and liver proteasomes) belong to the group of multifunctional proteins. Selective biosensor validation confirmed the affinity binding of proteins (aldolase, phosphoglycerate kinase) identified during MS analysis to the brain 20S proteasome. Comparison of the subproteomes of the 26S and 20S brain proteasomes showed that removal of components of the regulatory (19S) subparticles caused almost two-fold increase in the total number of individual proteins associated with the core part of the proteasome (20S). In the liver, the number of proteins associated with the core part of the proteasome remained basically unchanged after the removal of the components of the regulatory (19S) subparticles. This indicates that in the brain and, possibly, in other organs, proteins of the regulatory (19S) subunit play an important role in the formation of the proteasome interactome.

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.

The proteomic composition of a biological sample serves as the most important feature of a biological object, and it allows discriminating normal and pathological conditions. Targeted mass spectrometric analysis, namely, multiple reaction monitoring (MRM) using synthetic isotopically-labeled internal standard (SIS), is the main alternative to the ELISA method for the analysis of diagnostically significant proteins. Based on the MRM results, a prototype test system has been developed; it employs the targeted mass spectrometric method for multiplex, quantitative analysis of FDA-verified proteins in whole blood plasma. Using this approach, it was possible to measure the content of 42 proteins in 31 samples in a concentration range spanning five orders of magnitude. The interindividual variability for 30 of the 42 registered proteins was less than 40%. The largest scatter was observed for haptoglobin (68%), immunoglobulin heavy constant delta IGHD (90%), angiotensin (72%), sex hormone-binding globulin SHBG (100%) and lipoprotein-(a) (136%). The obtained results on the concentration of proteins correlate with published data (Hortin et al., 2008, Clinical Chemistry, 54, 1608) with R2=0.84. The developed prototype test system based on targeted mass spectrometric analysis of proteins can be considered as an alternative to methods using monoclonal antibodies.

The main problems in the diagnostics and treatment of malignant tumors are early detection of the disease, prediction of the course of the disease and response to therapy. The solution may be associated with identification of biomarkers secreted by tumor cells within extracellular vesicles, known as exosomes. The study of exosome proteins attracts special attention, because their molecular composition can have information about tumor identity, and also represent a set of signaling molecules that regulate the processes of tumor progression and growth. In addition, the analysis of exosomes secreted into the extracellular space corresponds to the promising concept of a liquid biopsy. In this review, we have summarized the current experience in the molecular study of exosomes in various types of malignant tumors, including colorectal cancer, lung cancer, ovaries, prostate and breast cancer, with special emphasis on omics methods and outlined the prospects for their use in diagnosis.

Isatin (indol-2,3-dione), an endogenous biofactor found in the brain, peripheral tissues and biological body fluids of humans and animals, exhibits a wide range of biological and pharmacological activities. They are realized via interaction with numerous isatin-binding proteins. Some of these proteins identified during proteomic profiling of the brain are involved in the development of neurodegenerative pathology. In the context of the neuroprotective effect, the effect of isatin is comparable to the effects of deprenyl (selegiline), a pharmacological agent used for treatment of Parkinson's disease. In this study, we have investigated the effect of a single dose administration of isatin (100 mg/kg) and deprenyl (10 mg/kg) to mice on the profile of the brain isatin-binding proteins. Comparative proteomic analysis of brain isatin-binding proteins of mice treated with isatin or deprenyl resulted in identification of a representative group of proteins (n=200) sensitive to the administration of these substances. The change in the profile of isatin-binding proteins may be obviously attributed to accumulation of isatin and deprenyl in the brain and their interaction with target proteins; this prevents protein binding to the affinity sorbent. Thus identified brain isatin-binding proteins of the control animals obviously represent specific targets that interact directly with isatin (and also with deprenyl) in vivo . Isatin or deprenyl administered to animals interact with these proteins and thus inhibit their binding to the affinity sorbent (immobilized isatin analogue).

HL-60 promyelocytic cells are a widely used as a model for studying induced granulocytic differentiation. Investigation of proteins of the nuclear fraction, particularly transcription factors, is necessary for a better understanding of molecular mechanisms of cell maturation. Mass spectrometry is a powerful tool for analyzing a proteome due to its high sensitivity, specificity and performance. In this paper, using the selected reaction monitoring (SRM) method, we have assessed the levels of RBPJ, STAT1, CEBPB, CASP3, VAV1, PRKDC, PARP1 and UBC9 nuclear proteins isolated using hypertonic buffer, detergents (sodium dodecyl sulfate (SDS), sodium deoxycholate (DOC) and fissionable detergent ProteaseMAX™) and using centrifugation in a sucrose density gradient. The minimum and maximum protein content was 1.13±0.28 and 14.34±1.63 fmol/mkg of total protein for the transcription factor RBPJ and ubiquitin-protein ligase type I UBC9, respectively. According to the results of shotgun mass spectrometric analysis of nuclear fractions, 2356 proteins were identified, of which 106 proteins were annotated as transcription factors. 37 transcription factors were uniquely identified in the fraction obtained by centrifugation in a sucrose density gradient, while only 9 and 8 transcription factors were uniquely identified in the nuclear fractions obtained using hypertonic buffer and detergents, respectively. The transcription factors identified in the HL-60 cell line represent regulatory molecules; their directed profiling under the influence of differentiation inducers, will shed light on the mechanism of granulocyte maturation.

It becomes increasingly clear that ubiquitination of cellular proteins is not an indispensable prerequisite of their degradation in proteasomes. There are a number of proteins to be eliminated which are not pre-ubiquitinated for their recognition by regulatory subcomplex of 26S proteasome, but which directly interact with the 20S proteasome core particle (20S proteasome). The obligatory precondition for such interaction consists in existence of disordered (hydrophobic) fragments in the target protein. In this study we have investigated the interaction of a number of multifunctional (moonlighting) proteins (glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldolase, pyruvate kinase) and neurodegeneration-related proteins (a-synuclein, myelin basic protein) with 20S proteasome immobilized on the SPR-biosensor chip and stabilized by means of a bifunctional agent dimethyl pimelimidate (in order to prevent possible dissociation of this subcomplex). Only two of all investigated proteins (aldolase and pyruvate kinase) interacted with the immobilized 20S proteasome (Kd of 8.17´10-7 M and 5.56´10-7 M, respectively). In addition to earlier detected GAPDH ubiquitination, mass spectrometric analysis of the studied proteins revealed the presence of the ubiquitin signature (Lys-e-Gly-Gly) only in aldolase. Oxidation of aldolase and pyruvate kinase, which promotes elimination of proteins via their direct interaction with 20S proteasome, caused a 2-3-fold decrease in their Kd values as comparison with this parameter obtained for the intact proteins. The results of this study provide further evidence for direct interaction of both ubiquitinated proteins (aldolase), and non-ubiquitinated proteins (pyruvate kinase) with the 20S proteasome core particle (20S proteasome). The effectiveness of this interaction is basically equal for the ubiquitinated proteins and non-ubiquitinated proteins.

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.

Exosomes are extracellular membrane vesicles secreted by cells into biological fluids. The outer membrane of exosomes protects their content from degradation and contains markers of the parent cell. Almost all cells of the body produce exosomes, however, tumor cells secrete them more intensively. Due to fact that exosomes contain proteins of cells secreting them, these vesicles could be a valuable source for biomarkers discovery. Currently, a number of studies prove the participation of exosomes in carcinogenesis. However, there is a problem of isolating pure and characterized exosomes for further use in investigation of functions or identification of tumor protein biomarkers. In this work, we have performed experiments on exosomes isolation from human plasma by three methods: differential ultracentrifugation, ultracentrifugation in sucrose cushion, sedimentation of the exosomal fraction from serum by using a commercial kit. The protein composition of the obtained samples was determined by mass spectrometric methods of selected reactions monitoring (SRM) and shotgun proteomic analysis. The obtained exosomal samples were searched for the presence of exosomal markers (CD9, CD82, HSPA8, CD63). In the samples of exosomes isolated by ultracentrifugation with the sucrose cushion, the content of the above markers was determined as 32.85, 15.59, 6.07 fmol/mg of total protein, correspondently. It was shown that the centrifugation method with the sucrose cushion was optimal for the isolation of exosomes.

Isatin (indol-2,3-dione) is an endogenous indole found in the brain, peripheral tissues and biological body fluids of humans and animals. Its wide spectrum of biological activity is realized via interaction with numerous isatin-binding proteins; these include proteins playing an important role in the development of neurodegenerative pathology. In the context of the neuroprotective effect, the effect of isatin is comparable to the effects of deprenyl, a pharmacological agent used for treatment of Parkinson's disease. In this study, the effects of the course of deprenyl (1 mg/kg) and isatin (20 mg/kg) administration for 21 days on the profile of the isatin-binding proteins of the liver of mice have been investigated. Proteomic profiling of liver isatin-binding proteins of control mice by means of 5-aminocaproylisatin as an affinity ligand resulted in identification of 105 proteins. Treatment of animals with a low dose of isatin slightly decreased (up to 91), while injections of deprenyl slightly increased (up to 120) the total number of isatin-binding proteins. 75 proteins were common for all three groups; they represented from 62.5% (in deprenyl treated mice) and 71% (in control mice), to 82% (isatin treated mice) of the total number of identified liver isatin-binding proteins. Proteomic analysis of the isatin-binding proteins of mice treated with isatin (20 mg/kg) or deprenyl (1 mg/kg) for 21 days revealed a representative group of proteins (n=30) that were sensitive to the administration of these substances. Taking into account the previously obtained results, it is reasonable to suggest that the change in the profile of isatin-binding proteins may be attributed to accumulation of isatin and deprenyl in the liver and interaction with target proteins prevents their subsequent binding to the affinity sorbent. In this context, the identified isatin-binding liver proteins of control animals that do not bind to the affinity sorbent (immobilized isatin analogue) after treatment of animals with either deprenyl or isatin appear to be specific targets directly interacting with isatin in vivo.

In the present study, we applied selected reaction monitoring (SRM) to a group of proteins that were previously reported to be associated with lung cancer (Novikova S.E. et al. (2017) Biomeditsinskaya khimiya, 63, 181-210. [1]). Measurements were performed on 59 plasma samples. These samples included: 23 samples of plasma of patients diagnosed with lung adenocarcinoma (LAC), 11 samples of plasma of patients diagnosed with squamous cell lung carcinoma (SqCC), 25 samples of donors with no previous history of oncological diseases, and one pooled sample from each of the above group. As a result of the SRM measurements 52 proteins were detected at least in one individual plasma sample. Statistical analysis showed that there were two groups confidently differentiated by the concentration value of 8 proteins wherein 5 proteins displayed increased level (P00738, P26639, P21926, P08603, P51149) in LAC group and 3 proteins (P51884, O15162, Q8N2K0) indicated diminishing the concentration level towards the control level. Data on protein concentrations obtained for LAC and SqCC did not distinguish the samples by statistical clustering analysis. These potential biomarkers can be used for further development of methods for early diagnostics of lung cancer.

It becomes increasingly clear that most proteins of living systems exist as components of various protein complexes rather than individual molecules. The use of various proteomic techniques significantly extended our knowledge not only about functioning of individual complexes but also formed a basis for systemic analysis of protein-protein interactions. In this study gel-filtration chromatography accompanied by mass-spectrometry was used for the interactome analysis of human liver proteins. In six fractions (with average molecular masses of 45 kDa, 60 kDa, 85 kDa, 150 kDa, 250 kDa, and 440 kDa) 797 proteins were identified. In dependence of their distribution profiles in the fractions, these proteins could be subdivided into four groups: (1) single monomeric proteins that are not involved in formation of stable protein complexes; (2) proteins existing as homodimers or heterodimers with comparable partners; (3) proteins that are partially exist as monomers and partially as components of protein complexes; (4) proteins that do not exist in the monomolecular state, but also exist within protein complexes containing three or more subunits. Application of this approach to known isatin-binding proteins resulted in identification of proteins involved in formation of the homo- and heterodimers and mixed protein complexes.

The relationship between the amount of a target protein in a complex biological sample and its amount measured by selected reaction monitoring (SRM) mass spectrometry upon the affinity enrichment of target protein with aptamers immobilized on a solid phase was studied. Human thrombin added in known concentrations to cellular extracts derived from bacterial cells was used as model target protein. It has been demonstrated that the affinity enrichment of thrombin in cellular extracts by means of the thrombin-binding aptamer immobilized on the surface of magnetic microbeads results in an approximately 10-fold increase of the concentration of target protein and a 100-fold decrease of the low limit of a target protein concentration range where its quantitative detection by SRM is possible without an interference from other peptides present in a tryptic digest.

Current proteomic studies are generally focused on the most abundant proteoforms encoded by canonical nucleic sequences. Transcriptomic and proteomic data, accumulated in a variety of postgenome sources and coupled with state-of-art analytical technologies, allow to start the identification of aberrant (non-canonical) proteoforms. The main sources of aberrant proteoforms are alternative splicing, single nucleotide polymorphism, and post-translational modifications. The aim of this work was to estimate the heterogeneity of HepG2 proteome. We suggested multiomics approach, which combines transcriptomic (RNAseq) and proteomic (2DE-MS/MS) methods, as a promising strategy to explore the proteome.

Isatin (indole-2,3-dione) is an endogenous indole found in the mammalian brain, peripheral organs and body fluids. It acts as a neuroprotector, which decreases manifestation of locomotor impairments in animal models of Parkinson's disease. A wide range of biological activity of isatin is associated with interaction of this regulator with numerous isatin-binding proteins. The aim of this study was to investigate the profile of brain isatin-binding proteins in mice with MPTP-induced Parkinsonism (90 min and seven days after administration of this neurotoxin). A single dose administration of MPTP (30 mg/kg, ip.) was accompanied by locomotor impairments in the open field test 90 min after administration; seven days after MPTP administration locomotor activity of mice significantly improved but did not reach the control level. Five independent experiments on proteomic profiling of isatin-binding proteins resulted in confident identification of 96±12 proteins. Development of MPTP-induced locomotor impairments was accompanied by a significant decrease in the number of isatin-binding proteins (63±6; n=5; p<0.01). Seven days after MPTP administration the total number of identified proteins increased and reached the control level (132±34; n=4). The profiles of isatin-binding proteins were rather specific for each group of mice: in the control group these proteins (which were not found in both groups of MPTP-treated mice) represented more than 70% of total proteins. In the case of MPTP treated mice this parameter was 60% (90 min after MPTP administration) and >82% (seven days after MPTP administration). The major changes were found in the groups of isatin-binding proteins involved into cytoskeleton formation and exocytosis, regulation of gene expression, cell division and differentiation and also proteins involved in signal transduction.

To date lung adenocarcinoma (LAC) is the most common type of lung cancer. Numerous studies on LAC biology resulted in identification of crucial mutations in protooncogenes and activating neoplastic transformation pathways. Therapeutic approaches that significantly increase the survival rate of patients with LAC of different etiology have been developed and introduced into clinical practice. However, the main problem in the treatment of LAC is early diagnosis, taking into account both factors and mechanisms responsible in tumor initiation and progression. Identification of a wide biomarker repertoire with high specificity and reliability of detection appears to be a solution to this problem. In this context, proteins with differential expression in normal and pathological condition, suitable for detection in biological fluids are the most promising biomarkers. In this review we have analyzed literature data on studies aimed at search of LAC biomarkers. The major attention has been paid to protein biomarkers as the most promising and convenient subject of clinical diagnosis. The review also summarizes existing knowledge on posttranslational modifications, splice variants, isoforms, as well as model systems and transcriptome changes in LAC.

Using reverse transcription in conjunction with the quantitative real-time PCR or digital droplet PCR, the transcriptome profiling of human chromosome 18 has been carried out in liver hepatocytes and hepatoblastoma cells (HepG2 cell line) in terms of the absolute number of each transcript per cell. The transcript abundance varies within the range of 0.006 to 9635 and 0.011 to 4819 copies per cell for HepG2 cell line and hepatocytes, respectively. The expression profiles for genes of chromosome 18 in hepatocytes and HepG2 cells were found to significantly correlate: the Spearman's correlation coefficient was equal to 0.81. The distribution of frequency of transcripts over their abundance was bimodal for HepG2 cells and unimodal for liver hepatocytes. Bioinformatic analysis of the differential gene expression has revealed that genes of chromosome 18, overexpressed in HepG2 cells compared to hepatocytes, are associated with cell division and cell adhesion processes. It is assumed that the enhanced expression of those genes in HepG2 cells is related to the proliferation activity of cultured cells. The differences in transcriptome profiles have to be taken into account when modelling liver hepatocytes with cultured HepG2 cells.

In recent decades, studies in the molecular origins of socially significant diseases have made a big step forward with the development and using of high-performance methods in genomics and proteomics. Numerous studies in the framework of the global program "Human Proteome" were aimed at the identification of all possible proteins in various cell cultures and tissues, including cancer. One of the objectives was to identify biomarkers - proteins with high specificity to certain pathologies. However, in many cases, it is shown that the development of the disease is not associated with the appearance of new proteins, but depends on the level of gene expression or forming of proteoforms - splice variants, single amino acid substitutions (SAP variants), and post-translational modifications (PTM) of proteins. PTM may play a key role in the development of pathology because they activate a variety of regulatory or structural proteins in the majority of cell physiological processes. Phosphorylation is among the most significant of these protein modifications.This review will describe methods for analysis of protein phosphorylation used in the studies of such diseases as cancer and neurodegenerative diseases, as well as examples of cases when the modified proteins are involved directly to their development, and screening such significant PTM is used for the diagnosis and choice of treatment.

Accuracy of the microarray technology results is raised by using the multi-stage normalization of results. One of the principal requirements of such normalization is usage of internal standards. The routine Agilent microarray-based gene expression analysis protocol utilizes a Spike-In Kit during preparation of the samples representing a mixture of RNA fragments in different ratios. RNA probes which were synthesized in vitro conditions could be also used to establish how the magnitude of the fluorescent signal reflects the presence of RNA in the sample. A significant disadvantage of this type of standards is a difficulty of their production and the low RNA stability. In accordance with the Agilent protocol, the presence of the T7 promoter is necessary for the synthesis of labeled cRNA during sample preparation procedure. We hypothesized that we can successfully synthesize any RNA sequence having such type of promoter in its start position. Moreover, DNA sequence would serve as a matrix in this case. Using a set of different genes attached downstream of the T7-promoter in the plasmid DNA we have demonstrated in this study that such system can serve as a reliable template for the fluorescent labeled RNA sequence synthesis. In comparison with the routinely used internal RNA based controls, this template is stable, easy to manufacture and can be easily obtained in large quantities.

MicroRNA is a special type of regulatory molecules governing gene expression. Circulating microRNAs found in blood and other biological fluids are considered today as potential biomarkers of human pathology. Presently, quantitative alterations of particular microRNAs are revealed for a large number of oncological diseases and other disorders. The recently emerged method of digital droplet PCR (ddPCR) possesses a number of advantages making this method the most suitable for verification and validation of perspective microRNA markers of human pathologies. Among these advantages are the high accuracy and reproducibility of microRNA quantification as well as the capability to directly measure the absolute number of microRNA copies with the large dynamic range and a high throughput. The paper reviews microRNA biogenesis, the origin of circulating microRNAs, and methods used for their quantification. The special technical features of ddPCR, which make it an attractive method both for studying microRNAs as biomarkers of human pathologies and for basic research devoted to aspects of gene regulation by microRNA molecules, are also discussed.

Isatin-binding activity of mice liver proteins has been investigated in the samples from the control and flight groups by using the methods of biosensor and proteomic analysis. It was found the higher isatin-binding activity in mice of flight group. The content of a number of individual isatin-binding proteins in the samples of the flight groups differ slightly from the ground control. However, in samples from animals which have weekly post-flight adaptation, the level of certain proteins was significantly increased. The latter allows us to assume that the main events in the proteome of mice (at least in subproteome of isatin-binding proteins), occurs in early post-flight period.

Induced differentiation of leukemia cells is in the focus of basic and applied biomedical studies medicine and biology for more than 30 years. During this period specific regulatory molecules involved in the maturation process have been identified by biochemical and molecular biological methods. Recent developments of high-throughput transcriptomic and proteomic techniques made it possible to analyze large sets of mRNA and proteins; this resulted in identification of functionally important signal transduction pathways and networks of molecular interactions, and thus extent existing knowledge on the molecular mechanisms of induced differentiation. Despite significant advances in mechanisms of induced differentiation, many problems related to the molecular mechanism of cell maturation, a phenomenon of therapeutic resistance of leukemic cells need better understanding and thus require further detailed study. Transcriptomics and proteomics methods provide a suitable methodological platform for the implementation of such studies. This review highlights the use of transcriptomic and proteomic methods in studies aimed at various aspects of the induced differentiation. Special attention is paid to the employment of the systems approach for investigation of various aspects of cell maturation. The use of the systems approach in studies of induced differentiation is an important step for the transition from the formal data accumulation on expression of mRNA and proteins towards creating models of biological processes in silico.

Huge range of concentrations of different protein and insufficient sensitivity of methods for detection of proteins at a single molecule level does not yet allow obtaining the whole image of human proteome. In our investigations, we tried to evaluate the size of different proteomes (cells and plasma). The approach used is based on detection of protein spots in 2-DE after staining by protein dyes with different sensitivities. The function representing the dependence of the number of protein spots on sensitivity of protein dyes was generated. Next, by extrapolation of this function curve to theoretical point of the maximum sensitivity (detection of a single smallest polypeptide) it was calculated that a single human cell (HepG2) may contain minimum 70,000 proteoforms, and plasma--1.5 mln. Utilization of this approach to other, smaller proteomes showed the competency of this extrapolation. For instance, the size of mycoplas ma (Acholeplasma laidlawii) was estimated in 1100 proteoforms, yeast (Saccharomyces cerevisiae)--40,000, E. coli--6200, P. furiosus--3400. In hepatocytes, the amount of proteoforms was the same as in HepG2--70,000. Significance of obtained data is in possibilities to estimating the proteome organization and planning next steps in its study.

Concentrations of 46 proteins have been determined in human blood plasma using PlasmaDeepDive™ MRM Panel ("Biognosys AG", Switzerland). 18 of them were included into the group of proteins with higher concentrations, also identified by the shotgun proteomic analysis. Based on literature data it is concluded that the PlasmaDeepDive™ MRM Panel is applicable for studies of human plasma samples for potential biomarkers of various nervous system disorders.

The article summarizes the achievements of the pilot phase (2010-2014) of the Russian part of the international project "Human Proteome" and identifies the directions for further work on the study of the human chromosome 18 proteome in the framework of the project main phase (2015-2022). The pilot phase of the project was focused on the detection of at least one protein for each chromosome 18 protein-coding gene in three types of the biological material. The application of mass spectrometric detection of proteins by the methods of multiple reactions monitoring (MRM) and gene-centric approach made it possible to detect 95% of master forms of proteins, for 60% of which the quantitative assessment of the protein content was obtained in at least one type of the biological material. The task of the main phase of the project is to measure the proteome size of healthy individuals, taking into account the modified protein forms, providing for both the bioinformatics prediction of the quantity of proteins types and the selective experimental measurement of single proteoforms. Since the ranges of protein concentrations corresponding to the normal physiological state have not been identified, the work of the main phase of the project is focused on the study of clinically healthy individuals. The absence of these data complicates significantly the interpretation of the patients' blood proteomic profiles and prevents creating diagnostic tests. In the long term prospect, implementation of the project envisages development of a diagnostic test system based on multiple reactions monitoring (MRM) for quantitative measurement of the protein forms associated with some diseases. Development of such test systems will allow predicting the extent of risk of different diseases, diagnosing a disease at its early stage and monitoring the effectiveness of the treatment.

Phosphorylation is one of the most common posttranslational modification (PTM) of proteins. Main challenge of phosphoprotein detection is their low abundance comparing to abundance of unmodified proteins. The method of selected reactions monitoring (SRM) allows to perform very sensitive and selective analysis of desired PTMs. Using myelin basic protein (MBP) as a model we have developed a method for phosphoprotein detection by SRM. The method is based on obtaining of phosphoproteins in a reconstituted kinase system and following usage these phosphorylated protein as a template for the development of the SRM method. The developed method was successfully applied for detection of phosphopeptides of myelin basic protein in the samples of human brain glioma.

A new method for screening of essential peptides for protein detection and quantification analysis in the direct positive electrospray mass spectrometry has been proposed. Our method is based on the prediction of the normalized abundance of the mass spectrometric peaks using a linear regression model. This method has the following limitations: (i) selected peptides should be taken so that at pH 2.5 the tested peptides must be presented mainly as the 2+ and 3+ ions; (ii) only peptides having C-terminal lysine or arginine residues are considered. The amino acid composition of the peptide, the peptide concentration, the ratio of the polar surface of peptide to common surface and ratio of the polar volume to common volume are used as independent variables in equation. Several combinations of variables were considered and the best linear regression model had a determination coefficient in leave-one-out validation procedure equal 0.54. This model confidently discriminates peptides with high response ability and peptides with low response ability, and therefore it allows to select only the most promising peptides. This screening method, a plain and fast, can be successfully applied to reduce the list of observed peptides.

Current review describes recent approaches of cytochrome P450 concentration and activity evaluation. Special attention paid to modem methods of proteomic analysis such as electrophoresis and chromato-mass-spectrometry. Methods of targeted proteomic applicable for quantitative and qualitative study of P450s in biological samples as well as methods for the enzyme activity measurements are reviewed. Finally, data on correlation between certain P450 isoform content and its specific enzymatic activities were described and discussed in the review.

Renalase is a recently discovered secretory protein, which is suggested to play a role (which still remains elusive) in regulation of blood pressure. Earlier it was purified from urine of healthy volunteers by means of ammonium sulfate fractionation and subsequent affinity chromatography (Xu et al. (2005) J. Clin. Invest., 115, 1275). The resultant purified preparation of renalase contained 2 proteins with molecular masses of 35 and 67-75 kDa. The authors believed that the latter represents a dimerization (aggregation) product of the 35 kDa protein. In this study we have detected relanase in urinary samples of 2 of 6 volunteers only after immunoaffinity enrichment of urinary samples subjected to ammonium sulfate precipitation. Electrophoresis of the purified preparation also demonstrated the presence of 2 proteins with molecular masses of 35 and 66 kDa, respectively. Mass spectrometry analysis of these proteins identified 35 and 66 kDa proteins as renalase and serum albumin, respectively. Thus, our results do not support suggestion on formation of renalase dimers and they indicate that urinary renalase excretion significantly varies in humans.

Possibility of detection and identification of hepatitis C viral particles with mass spectrometry (MS) in combination with atomic force microscopy (AFM) had been investigated. AFM/MS approach is based on two technologies: (1) AFM-biospecific fishing that allows to detect, concentrate from solution and to count protein complexes on a surface of AFM-nanochip; (2) mass spectrometric identification of these complexes. AFM-biospecific fishing of HCVcoreAg from solution was carried onto surface of AFM-nanochips with immobilized anti-HCVcoreAg. It was shown that HCVcoreAg/anti-HCVcore(im) complexes were formed onto AFM-nanochips in quantity sufficient for mass spectrometric identification. Thus, AFM/MS approach allows to identify fragments of hepatitis C virus fished onto a surface of AFM-nanochip from serum.

We determined the interaction sites of the cytochrome P450's protein-partners: 2B4 (d-2B4) and NADPH-cytochrome P450 of reductase (d-Fp). While in operation, these proteins are forming the complexes. We used 4-4'-dithio(bisphenyl)azide linker for non-specific covalent coupling of d-2B4 complexes with d-Fp in Emulgen-913-monomerized system. Covalently-linked peptides in this complex were identified with ESI-MS/MS. Several sites of these proteins' binding with each other were revealed. Based on them, a model of intermolecular protein interactions was created. The model includes 5 cross-linker-stabilized contact sites of d-2B4 with d-Fp involving the following peptides of d-2B4 and d-Fp: (1) d-2B4423-433 and d-Fp 102-109; (2) d-2B4324-336 and d-Fp570-585; (3) d-2B4327-336 and d-Fp452-464; (4) d-2B4 192-197 and d-Fp456-464; (5) d-2B4 134-139 and d-Fp406-425. Herein, in the latter two cases, the peptides of d-Fp are located in their inter-domain slit and stabilize protein-protein complex via nanoprobe cross-linker; therefore, the formation of d-2B4/d-Fp complexes in these sites may involve aminoacid residues d-Fp456-464 and d-Fp406-425 surrounding inter-domain slit.

The equilibrium dissociation constants K(D), the complex association / dissociation rate constants (k(on)/ k(off)) and the lifetimes of redox partners' complexes were measured for three cytochrome P450-containing monooxygenase systems (P450cam, P450 2B4 and P450scc). To estimate the productivity of complexes formed within the systems studied, the Q parameter--i.e. the ratio of protein-protein complex lifetime (T(LT)) to the time required for a single hydroxylation cycle (tau(cat))--was determined. It was shown that Q was changed (albeit insignificantly) upon transition from the oxidation to hydroxylation conditions in all the three P450 monooxygenase systems studied. It was shown that the binary complexes formed within the P450cam and the P450scc systems requiring an intermediate electron-transfer protein between the reductase and cytochrome P450 were non-productive while the binary complexes formed within the P450 2B4 system, not requiring such an intermediate electron-transfer protein, proved to be productive. Formation of ternary complexes within the three systems was demonstrated under hydroxylation conditions. Analysis of Q values led to the conclusion that the ternary complexes formed within the P450cam and the P450scc systems were virtually 100% productive. Within the P450 2B4 system, more than half (about 60%) ternary complexes were also found to be productive.

In the present paper we highlight the dependence of normalized mass spectrometry intensity on protein concentration. We have defined dynamic range of concentrations from 1 nM to 1 mcM. It has been shown that the dependence holds linearity along the whole concentration range as for pure proteins as for proteins mixtures. Our observation revealed that the correlation curve possess protein-specific tangent between 1 nM and 1 mcM. The efficacy of the method evaluated by AQUA and 18O-labelling techniques of quantitative proteomics.

Lipids represent 20% of the total weight of dried pool of medicinal leech salivary gland secretion (SGS) received from about 50 individual animals. There were detected steroids, but not pospholipids in SGS lipid fraction. Immunochemiluminescent analysis identified free steroid hormones in SGS: cortisol, progesterone, testosterone, estradiol and dehydroepiandrosterone. Microchromatografic-mass-spectrometric analysis of SGS and of low-molecular weight fraction (LMW) (molecular masses ranged from 220 to 850 Da) has shown the multicomponent nature of the LMW fraction. Using standart preparations as the reference steroid hormones (cortisol, dehydroepiandrosterone, androstenedione and testosterone) and histamine and serotonine have been identified in SGS.

In modern science proteomic analysis is inseparable from other fields of systemic biology. Possessing huge resources quantitative proteomics operates colossal information on molecular mechanisms of life. Advances in proteomics help researchers to solve complex problems of cell signaling, posttranslational modification, structure and functional homology of proteins, molecular diagnostics etc. More than 40 various methods have been developed in proteomics for quantitative analysis of proteins. Although each method is unique and has certain advantages and disadvantages all these use various isotope labels (tags). In this review we will consider the most popular and effective methods employing both chemical modifications of proteins and also metabolic and enzymatic methods of isotope labeling.

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.

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.

Mycobacterium tuberculosis sterol 14 alpha-demethylase CYP51 is currently considered a promising target for a new generation of antitubercular drugs. Similarly to eucaryotic sterol 14 alpha-demethylases, candidate selective inhibitors of M. tuberculosis CYP51 belong to azoles, the N-substituted derivatives of imidazole and triazole. We have constructed a highly effective system for heterologous expression of MT CYP51 in E. coli. The recombinant protein was purified by metalloaffinity chromatography for primary in vitro screening of novel antitubercular azole compounds for their high affinity to the molecular target. Yield of the purified recombinant MT CYP51 was about 1.5 micromoles of the purified native protein per 1 l of E. coli cell culture. The recombinant protein interacted with ketoconazole with a Kd of 7.7 microM. MT CYP51 produced by our system for heterologous expression in E. coli may be used for initial testing of novel antimycobacterial azole drugs.

Possible mechanisms of cytochrome P4502B4 modification by H2O2 formed during catalytic turnover have been studied. Oxidative self-inactivation of cytochrome P4502B4 in monooxygenase system, reconstituted from highly purified membrane proteins: cytochrome P4502B4, NADDPH-cytochrome P450 reductase and cytochrome b5 in the presence of detergent Emulgen 913, involves the heme destruction and apoenzyme modification. The cytochrome P450 self-inactivation is accompanied by protein aggregation, oxidation of SH-groups and changes of the surface charge. H2O2 and non-specific radical reactions may be responsible for the intermolecular cross-linking. Oxidative modification of cytochrome P4502B4 may be initial stage of the protein decay in the cell.