This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Exogenous bioactive peptides are considered promising for the wound healing therapy in humans. In... more Exogenous bioactive peptides are considered promising for the wound healing therapy in humans. In this regard, parasitic trematodes proteins may potentially become a new perspective agents. Foodborne trematode Opisthorchis felineus is widespread in Europe and has the ability to stimulate proliferation of bile duct epithelium. In this study, we investigated skin wound healing potential of O. felineus proteins in mouse model. C57Bl/6 mice were inflicted with superficial wounds with 8 mm diameter. Experimental groups included several non-specific controls and specific treatment groups (excretory-secretory product and lysate). After 10 days of the experiment, the percentage of wound healing in the specific treatment groups significantly exceeded the control values. We also found that wound treatment with excretory-secretory product and worm lysate resulted in: (i) inflammation reducing, (ii) vascular response modulating, (iii) type 1 collagen deposition promoting dermal ECM remodeling. An additional proteomic analysis of excretory-secretory product and worm lysate samples was revealed 111 common proteins. The obtained data indicate a high wound-healing potential of liver fluke proteins and open prospects for further research as new therapeutic approaches. Chronic non-healing wounds or abnormal scarring is a major public health problem worldwide. There are numerous causes of such pathological conditions: severe mechanical and burn injuries, obesity and physical inactivity, diseases of cardiovascular system and autoimmune diseases, including diabetes mellitus, as well as a number of other diseases 1-3. Normally, skin regeneration is a natural homeostatic process that includes four successive stages: (1) hemostasis (platelet activation, clot formation to prevent blood loss and fibrin matrix formation, up to 48 h); (2) inflammation (immune response, activation of inflammatory cells, up to 96 h); (3) proliferation (modulated M2 to M1 polarization of macrophages, keratinocyte migration, angiogenesis activation, tissue granulation, epithelial folds and wet crust/eschar, up to 10-14 days); (4) remodeling (maturation of epidermis, overall reduction of wound area, regression of blood vessels, structuring of extracellular matrix, more than 10 days) 4-6. In case of pathological no overgrowth, wounds do not undergo normal stages of healing, linger at stage of inflammation or proliferation and remain incurable despite intense therapeutic treatment 7,8. Promising therapeutic approaches to wound healing correction include various gels, growth factors, herbal preparations, natural polysaccharides (chitosan) and bioactive peptides of various origins 9-12. Due to this, proteins of parasitic trematodes may potentially become novel perspective agents for wound healing, as well as treatment of autoimmune diseases 13-15. Currently, helminth therapy is considered as an experimental method for treatment of certain autoimmune diseases: Crohn's disease, celiac disease, multiple sclerosis, asthma and etc. 16-18. Given the role in a regulation of host's immune response-bioactive and regulatory molecules of parasites may be considered a relevant therapeutic method. These regulatory molecules allow parasite to control host's local immune response and exist in mammalian organism for a long time. The foodborne liver trematode Opisthorchis felineus is one of the most well-known and harmful Opisthorchiidae parasites. This parasite is widespread in Eurasia, including Western Europe. Prevalence of human infection is highest in Russia, where up to 40,000 cases of O. felineus infection are diagnosed annually 19. Disease caused by this parasite is characterized by a long course and may be asymptomatic for a long time. O. felineus is able to reduce acute inflammation caused by invasion and stimulate repair of damaged liver epidermis and tissue remodeling 20,21. It becomes well documented that parasite-derived bioactive substances and regulatory molecules may actively improve wound healing in mammals 14. In db/db mice model of diabetes mellitus, treatment
In vitro models are often used for studying macrophage functions, including the process of phagoc... more In vitro models are often used for studying macrophage functions, including the process of phagocytosis. The application of primary macrophages has limitations associated with the individual characteristics of animals, which can lead to insufficient standardization and higher variability of the obtained results. Immortalized cell lines do not have these disadvantages, but their responses to various signals can differ from those of the living organism. In the present study, a comparative proteomic analysis of immortalized PMJ2-R cell line and primary peritoneal macrophages isolated from C57BL/6 mice was performed. A total of 4005 proteins were identified, of which 797 were quantified. Obtained results indicate significant differences in the abundances of many proteins, including essential proteins associated with the process of phagocytosis, such as Elmo1, Gsn, Hspa8, Itgb1, Ncf2, Rac2, Rack1, Sirpa, Sod1, C3, and Msr1. These findings indicate that outcomes of studies utilizing PMJ2-...
CD133 is an extensively studied marker of the most malignant tumor cell population, designated as... more CD133 is an extensively studied marker of the most malignant tumor cell population, designated as cancer stem cells (CSCs). However, the function of this glycoprotein and its involvement in cell regulatory cascades are still poorly understood. Here we show a positive correlation between the level of CD133 plasma membrane expression and the proliferative activity of cells of the Caco-2, HT-29, and HUH7 cancer cell lines. Despite a substantial difference in the proliferative activities of cell populations with different levels of CD133 expression, transcriptomic and proteomic profiling revealed only minor distinctions between them. Nonetheless, a further in silico assessment of the differentially expressed transcripts and proteins revealed 16 proteins that could be involved in the regulation of CD133 expression; these were assigned ranks reflecting the apparent extent of their involvement. Among them, the TRIM28 transcription factor had the highest rank. The prominent role of TRIM28 i...
The main goal of the Russian part of C-HPP is to detect and functionally annotate missing protein... more The main goal of the Russian part of C-HPP is to detect and functionally annotate missing proteins (PE2-PE4) encoded by human chromosome 18. However, identifying such proteins in a complex biological mixture using mass spectrometry (MS)-based methods is difficult due to the insufficient sensitivity of proteomic analysis methods. In this study, we determined the proteomic technology sensitivity using a standard set of UPS1 proteins as an example. The results revealed that 100% of proteins in a mixture could only be identified at a concentration of at least 10−9 M. The decrease in concentration leads to protein losses associated with technology sensitivity, and no UPS1 protein is detected at a concentration of 10−13 M. Therefore, two-dimensional fractionation of samples was applied to improve sensitivity. The human liver tissue was examined by selected reaction monitoring and shotgun methods of MS analysis using one-dimensional and two-dimensional fractionation to identify the protein...
Isatin (indole-2,3-dione) is an endogenous regulator, exhibiting a wide range of biological and p... more Isatin (indole-2,3-dione) is an endogenous regulator, exhibiting a wide range of biological and pharmacological activities. At doses of 100 mg/kg and above, isatin is neuroprotective in different experimental models of neurodegeneration. Good evidence exists that its effects are realized via interaction with numerous isatin-binding proteins identified in the brain and peripheral tissues studied. In this study, we investigated the effect of a single dose administration of isatin to mice (100 mg/kg, 24 h) on differentially expressed proteins and a profile of the isatin-binding proteins in brain hemispheres. Isatin administration to mice caused downregulation of 31 proteins. However, these changes cannot be attributed to altered expression of corresponding genes. Although at this time point isatin influenced the expression of more than 850 genes in brain hemispheres (including 433 upregulated and 418 downregulated genes), none of them could account for the changes in the differentially...
LC-MS/MS allows identification of thousands of proteins in the complex proteomes. However, a sign... more LC-MS/MS allows identification of thousands of proteins in the complex proteomes. However, a significant part of a proteome remains inaccessible for identification due to the absence or poor quality of MS/MS spectra. The method described herein allows identifying the desired proteins of human blood plasma by comparing aligned chromatographic data of digested by trypsin sample and the same sample with spikedin synthetic peptides. Identification of human blood plasma proteins is archived by assigning tandem mass spectra of spiked-in peptides to the corresponding aligned chromatographic peaks of proteolytic peptides. Using the described approach we have identified 19 low abundant proteins in human blood plasma, which corresponded to 19 synthetic peptides used in the study. SRM verification of the identifications with isotopically labelled standards (SIS) confirmed the presence in the plasma of above 17 proteins.
Blue G 250, and deprenyl were purchased from Sigma (USA), [ 14 C]2 phenylethylamine hydrochloride... more Blue G 250, and deprenyl were purchased from Sigma (USA), [ 14 C]2 phenylethylamine hydrochloride was from Amersham (UK), acetonitrile was from Fisher Chemical (UK), formic acid, trifluoroacetic acid (TFA), and 2 propanol were from Fluka (USA), TCEP (tris (2 car boxyethyl) phosphine) was from Pierce (USA), sequenc ing grade modified trypsin was from Promega (USA), and 10 kDa acetate cellulose filters were from Sartorius Stedium Biotech (Germany). Proteasome 19S Rpn10/ S5a subunit (human), (recombinant) (GST tag) (agarose immobilized) was from Enzo Life Sciences (USA). C 18 spin columns were from Pierce, Amicon Ultracel 10K centrifugal filter units were from Millipore (USA), and Acclaim PepMap® RSLC C18 column (150 mm × 75 μm, 2 μm particle size, 100 Å pore size) was from Dionex (Rockford). Preparation of brain mitochondrial fraction and assay of MAO B activity. After analysis of behavioral reactions, animals were decapitated. Mouse brains were dissected and homogenized in the isolation medium containing 0.32 M sucrose, 1 mM EDTA, 10 mM Tris HCl buffer, pH 7.5, using an Ultra Turrax T 10 homogenizer at a low speed, to obtain 30% w/v homogenate. Brain mitochon drial fraction was isolated by differential centrifugation as described in [27]. MAO B activity was assayed radiomet rically using 0.005 mM [ 14 C]phenylethylamine as sub strate [28]. Mitochondrial MAO B activity assayed with this substrate concentration demonstrated sensitivity to inhibition by "diagnostic" MAO B inhibitor deprenyl and also to isatin. Affinity fractionation of Rpn10 binding proteins. Agarose resin with the immobilized proteasome Rpn10 subunit (0.5 ml) was washed five times with TBS (dis solved in 10 ml and centrifuged). The mitochondrial preparation (5 mg/ml protein, TBS, 1% Triton X 100) was added to the agarose slurry up to suspension (1 : 1), taking into consideration the additions of glycerol, PMSF, and bacitracin (final concentrations 5% (v/v), 1 mM, and 0.2 mg/ml, consequently). The resulting sus pension was incubated overnight at 4°C with gentle stir
This work continues the series of the quantitative measurements of the proteins encoded by differ... more This work continues the series of the quantitative measurements of the proteins encoded by different chromosomes in the blood plasma of a healthy person. Selected Reaction Monitoring with Stable Isotope-labeled peptide Standards (SRM SIS) and a gene-centric approach, which is the basis for the implementation of the international Chromosome-centric Human Proteome Project (C-HPP), were applied for the quantitative measurement of proteins in human blood plasma. Analyses were carried out in the frame of C-HPP for each protein-coding gene of the four human chromosomes: 18, 13, Y, and mitochondrial. Concentrations of proteins encoded by 667 genes were measured in 54 blood plasma samples of the volunteers, whose health conditions were consistent with requirements for astronauts. The gene list included 276, 329, 47, and 15 genes of chromosomes 18, 13, Y, and the mitochondrial chromosome, respectively. This paper does not make claims about the detection of missing proteins. Only 205 proteins (30.7%) were detected in the samples. Of them, 84, 106, 10, and 5 belonged to chromosomes 18, 13, and Y and the mitochondrial chromosome, respectively. Each detected protein was found in at least one of the samples analyzed. The SRM SIS raw data are available in the ProteomeXchange repository (PXD004374, PASS01192).
Vestibular schwannomas are relatively rare intracranial tumors compared to other brain tumors. Da... more Vestibular schwannomas are relatively rare intracranial tumors compared to other brain tumors. Data on the molecular features, especially on schwannoma proteome, are scarce. The 41 cerebrospinal fluid (liquor) samples were obtained during the surgical removal of vestibular schwannoma. Obtained peptide samples were analyzed by shotgun LC-MS/MS high-resolution mass spectrometry. The same peptide samples were spiked with 148 stable isotopically labeled peptide standards (SIS) followed by alkaline fractionation and scheduled multiple reaction monitoring (MRM) for quantitative analysis. The natural counterparts of SIS peptides were mapped onto 111 proteins that were Food and Drug Administration (FDA)-approved for diagnostic use. As a result, 525 proteins were identified by shotgun LC-MS/MS with high confidence (at least two peptides per protein, FDR < 1%) in liquor samples. Absolute quantitative concentrations were obtained for 54 FDA-approved proteins detected in at least five experi...
Human pluripotent stem cells are promising for a wide range of research and therapeutic purposes.... more Human pluripotent stem cells are promising for a wide range of research and therapeutic purposes. Their maintenance in culture requires the deep control of their pluripotent and clonal status. A non-invasive method for such control involves day-to-day observation of the morphological changes, along with imaging colonies, with the subsequent automatic assessment of colony phenotype using image analysis by machine learning methods. We developed a classifier using a convolutional neural network and applied it to discriminate between images of human embryonic stem cell (hESC) colonies with “good” and “bad” morphological phenotypes associated with a high and low potential for pluripotency and clonality maintenance, respectively. The training dataset included the phase-contrast images of hESC line H9, in which the morphological phenotype of each colony was assessed through visual analysis. The classifier showed a high level of accuracy (89%) in phenotype prediction. By training the classi...
Studies of induced granulocytic differentiation help to reveal molecular mechanisms of cell matur... more Studies of induced granulocytic differentiation help to reveal molecular mechanisms of cell maturation. The nuclear proteome represents a rich source of regulatory molecules, including transcription factors (TFs). It is important to have an understanding of molecular perturbations at the early stages of the differentiation processes. By applying the proteomic quantitative profiling using isobaric labeling, we found that the contents of 214, 319, 376, 426, and 391 proteins were altered at 3, 6, 9, 12, and 72 h, respectively, compared to 0 h in the HL-60 cell nuclear fraction under all-trans-retinoid acid (ATRA) treatment. From 1860 identified nuclear proteins, 231 proteins were annotated as proteins with transcription factor (TF) activity. Six TFs (RREB1, SRCAP, CCDC124, TRIM24, BRD7, and BUD31) were downregulated and three TFs EWSR1, ENO1, and FUS were upregulated at early time points (3–12 h) after ATRA treatment. Bioinformatic annotation indicates involvement of the HL-60 nuclear ...
Induced granulocytic differentiation of human leukemic cells under all-trans-retinoid acid (ATRA)... more Induced granulocytic differentiation of human leukemic cells under all-trans-retinoid acid (ATRA) treatment underlies differentiation therapy of acute myeloid leukemia. Knowing the regulation of this process it is possible to identify potential targets for antileukemic drugs and develop novel approaches to differentiation therapy. In this study, we have performed transcriptomic and proteomic profiling to reveal up- and down-regulated transcripts and proteins during time-course experiments. Using data on differentially expressed transcripts and proteins we have applied upstream regulator search and obtained transcriptome- and proteome-based regulatory networks of induced granulocytic differentiation that cover both up-regulated (HIC1, NFKBIA, and CASP9) and down-regulated (PARP1, VDR, and RXRA) elements. To verify the designed network we measured HIC1 and PARP1 protein abundance during granulocytic differentiation by selected reaction monitoring (SRM) using stable isotopically labele...
Cold shock domain proteins (CSDPs) participate in plant development and resistance, but the under... more Cold shock domain proteins (CSDPs) participate in plant development and resistance, but the underlying molecular mechanisms are poorly understood. In this study, we demonstrated that the CSDPs, including EsCSDP1, EsCSDP2, and EsCSDP3, from the extremophyte Eutrema salsugineum possess all basic properties of RNA chaperones. EsCSDP1-3 melt secondary structures in RNAs with various nucleotide sequences and exhibit RNA chaperone activity in vitro. EsCSDP1 and EsCSDP3 were shown to have higher RNA melting activity, whereasile EsCSDP2 had higher RNA chaperone activity. We demonstrated that higher RNA melting activity correlates with the longer C-terminal fragment in many zinc finger motifs, whereas increased RNA chaperone activity was most likely due to the higher glycine content and RGG repeat number in the C-terminal fragment.
A gene-centric approach was applied for a large-scale study of expression products of a single ch... more A gene-centric approach was applied for a large-scale study of expression products of a single chromosome. Transcriptome profiling of liver tissue and HepG2 cell line was independently performed using two RNA-Seq platforms (SOLiD and Illumina) and also by Droplet Digital PCR (ddPCR) and quantitative RT-PCR. Proteome profiling was performed using shotgun LC-MS/MS as well as selected reaction monitoring with stable isotope-labeled standards (SRM/SIS) for liver tissue and HepG2 cells. On the basis of SRM/SIS measurements, protein copy numbers were estimated for the Chromosome 18 (Chr 18) encoded proteins in the selected types of biological material. These values were compared with expression levels of corresponding mRNA. As a result, we obtained information about 158 and 142 transcripts for HepG2 cell line and liver tissue, respectively. SRM/SIS measurements and shotgun LC-MS/MS allowed us to detect 91 Chr 18-encoded proteins in total, while an intersection between the HepG2 cell line ...
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Exogenous bioactive peptides are considered promising for the wound healing therapy in humans. In... more Exogenous bioactive peptides are considered promising for the wound healing therapy in humans. In this regard, parasitic trematodes proteins may potentially become a new perspective agents. Foodborne trematode Opisthorchis felineus is widespread in Europe and has the ability to stimulate proliferation of bile duct epithelium. In this study, we investigated skin wound healing potential of O. felineus proteins in mouse model. C57Bl/6 mice were inflicted with superficial wounds with 8 mm diameter. Experimental groups included several non-specific controls and specific treatment groups (excretory-secretory product and lysate). After 10 days of the experiment, the percentage of wound healing in the specific treatment groups significantly exceeded the control values. We also found that wound treatment with excretory-secretory product and worm lysate resulted in: (i) inflammation reducing, (ii) vascular response modulating, (iii) type 1 collagen deposition promoting dermal ECM remodeling. An additional proteomic analysis of excretory-secretory product and worm lysate samples was revealed 111 common proteins. The obtained data indicate a high wound-healing potential of liver fluke proteins and open prospects for further research as new therapeutic approaches. Chronic non-healing wounds or abnormal scarring is a major public health problem worldwide. There are numerous causes of such pathological conditions: severe mechanical and burn injuries, obesity and physical inactivity, diseases of cardiovascular system and autoimmune diseases, including diabetes mellitus, as well as a number of other diseases 1-3. Normally, skin regeneration is a natural homeostatic process that includes four successive stages: (1) hemostasis (platelet activation, clot formation to prevent blood loss and fibrin matrix formation, up to 48 h); (2) inflammation (immune response, activation of inflammatory cells, up to 96 h); (3) proliferation (modulated M2 to M1 polarization of macrophages, keratinocyte migration, angiogenesis activation, tissue granulation, epithelial folds and wet crust/eschar, up to 10-14 days); (4) remodeling (maturation of epidermis, overall reduction of wound area, regression of blood vessels, structuring of extracellular matrix, more than 10 days) 4-6. In case of pathological no overgrowth, wounds do not undergo normal stages of healing, linger at stage of inflammation or proliferation and remain incurable despite intense therapeutic treatment 7,8. Promising therapeutic approaches to wound healing correction include various gels, growth factors, herbal preparations, natural polysaccharides (chitosan) and bioactive peptides of various origins 9-12. Due to this, proteins of parasitic trematodes may potentially become novel perspective agents for wound healing, as well as treatment of autoimmune diseases 13-15. Currently, helminth therapy is considered as an experimental method for treatment of certain autoimmune diseases: Crohn's disease, celiac disease, multiple sclerosis, asthma and etc. 16-18. Given the role in a regulation of host's immune response-bioactive and regulatory molecules of parasites may be considered a relevant therapeutic method. These regulatory molecules allow parasite to control host's local immune response and exist in mammalian organism for a long time. The foodborne liver trematode Opisthorchis felineus is one of the most well-known and harmful Opisthorchiidae parasites. This parasite is widespread in Eurasia, including Western Europe. Prevalence of human infection is highest in Russia, where up to 40,000 cases of O. felineus infection are diagnosed annually 19. Disease caused by this parasite is characterized by a long course and may be asymptomatic for a long time. O. felineus is able to reduce acute inflammation caused by invasion and stimulate repair of damaged liver epidermis and tissue remodeling 20,21. It becomes well documented that parasite-derived bioactive substances and regulatory molecules may actively improve wound healing in mammals 14. In db/db mice model of diabetes mellitus, treatment
In vitro models are often used for studying macrophage functions, including the process of phagoc... more In vitro models are often used for studying macrophage functions, including the process of phagocytosis. The application of primary macrophages has limitations associated with the individual characteristics of animals, which can lead to insufficient standardization and higher variability of the obtained results. Immortalized cell lines do not have these disadvantages, but their responses to various signals can differ from those of the living organism. In the present study, a comparative proteomic analysis of immortalized PMJ2-R cell line and primary peritoneal macrophages isolated from C57BL/6 mice was performed. A total of 4005 proteins were identified, of which 797 were quantified. Obtained results indicate significant differences in the abundances of many proteins, including essential proteins associated with the process of phagocytosis, such as Elmo1, Gsn, Hspa8, Itgb1, Ncf2, Rac2, Rack1, Sirpa, Sod1, C3, and Msr1. These findings indicate that outcomes of studies utilizing PMJ2-...
CD133 is an extensively studied marker of the most malignant tumor cell population, designated as... more CD133 is an extensively studied marker of the most malignant tumor cell population, designated as cancer stem cells (CSCs). However, the function of this glycoprotein and its involvement in cell regulatory cascades are still poorly understood. Here we show a positive correlation between the level of CD133 plasma membrane expression and the proliferative activity of cells of the Caco-2, HT-29, and HUH7 cancer cell lines. Despite a substantial difference in the proliferative activities of cell populations with different levels of CD133 expression, transcriptomic and proteomic profiling revealed only minor distinctions between them. Nonetheless, a further in silico assessment of the differentially expressed transcripts and proteins revealed 16 proteins that could be involved in the regulation of CD133 expression; these were assigned ranks reflecting the apparent extent of their involvement. Among them, the TRIM28 transcription factor had the highest rank. The prominent role of TRIM28 i...
The main goal of the Russian part of C-HPP is to detect and functionally annotate missing protein... more The main goal of the Russian part of C-HPP is to detect and functionally annotate missing proteins (PE2-PE4) encoded by human chromosome 18. However, identifying such proteins in a complex biological mixture using mass spectrometry (MS)-based methods is difficult due to the insufficient sensitivity of proteomic analysis methods. In this study, we determined the proteomic technology sensitivity using a standard set of UPS1 proteins as an example. The results revealed that 100% of proteins in a mixture could only be identified at a concentration of at least 10−9 M. The decrease in concentration leads to protein losses associated with technology sensitivity, and no UPS1 protein is detected at a concentration of 10−13 M. Therefore, two-dimensional fractionation of samples was applied to improve sensitivity. The human liver tissue was examined by selected reaction monitoring and shotgun methods of MS analysis using one-dimensional and two-dimensional fractionation to identify the protein...
Isatin (indole-2,3-dione) is an endogenous regulator, exhibiting a wide range of biological and p... more Isatin (indole-2,3-dione) is an endogenous regulator, exhibiting a wide range of biological and pharmacological activities. At doses of 100 mg/kg and above, isatin is neuroprotective in different experimental models of neurodegeneration. Good evidence exists that its effects are realized via interaction with numerous isatin-binding proteins identified in the brain and peripheral tissues studied. In this study, we investigated the effect of a single dose administration of isatin to mice (100 mg/kg, 24 h) on differentially expressed proteins and a profile of the isatin-binding proteins in brain hemispheres. Isatin administration to mice caused downregulation of 31 proteins. However, these changes cannot be attributed to altered expression of corresponding genes. Although at this time point isatin influenced the expression of more than 850 genes in brain hemispheres (including 433 upregulated and 418 downregulated genes), none of them could account for the changes in the differentially...
LC-MS/MS allows identification of thousands of proteins in the complex proteomes. However, a sign... more LC-MS/MS allows identification of thousands of proteins in the complex proteomes. However, a significant part of a proteome remains inaccessible for identification due to the absence or poor quality of MS/MS spectra. The method described herein allows identifying the desired proteins of human blood plasma by comparing aligned chromatographic data of digested by trypsin sample and the same sample with spikedin synthetic peptides. Identification of human blood plasma proteins is archived by assigning tandem mass spectra of spiked-in peptides to the corresponding aligned chromatographic peaks of proteolytic peptides. Using the described approach we have identified 19 low abundant proteins in human blood plasma, which corresponded to 19 synthetic peptides used in the study. SRM verification of the identifications with isotopically labelled standards (SIS) confirmed the presence in the plasma of above 17 proteins.
Blue G 250, and deprenyl were purchased from Sigma (USA), [ 14 C]2 phenylethylamine hydrochloride... more Blue G 250, and deprenyl were purchased from Sigma (USA), [ 14 C]2 phenylethylamine hydrochloride was from Amersham (UK), acetonitrile was from Fisher Chemical (UK), formic acid, trifluoroacetic acid (TFA), and 2 propanol were from Fluka (USA), TCEP (tris (2 car boxyethyl) phosphine) was from Pierce (USA), sequenc ing grade modified trypsin was from Promega (USA), and 10 kDa acetate cellulose filters were from Sartorius Stedium Biotech (Germany). Proteasome 19S Rpn10/ S5a subunit (human), (recombinant) (GST tag) (agarose immobilized) was from Enzo Life Sciences (USA). C 18 spin columns were from Pierce, Amicon Ultracel 10K centrifugal filter units were from Millipore (USA), and Acclaim PepMap® RSLC C18 column (150 mm × 75 μm, 2 μm particle size, 100 Å pore size) was from Dionex (Rockford). Preparation of brain mitochondrial fraction and assay of MAO B activity. After analysis of behavioral reactions, animals were decapitated. Mouse brains were dissected and homogenized in the isolation medium containing 0.32 M sucrose, 1 mM EDTA, 10 mM Tris HCl buffer, pH 7.5, using an Ultra Turrax T 10 homogenizer at a low speed, to obtain 30% w/v homogenate. Brain mitochon drial fraction was isolated by differential centrifugation as described in [27]. MAO B activity was assayed radiomet rically using 0.005 mM [ 14 C]phenylethylamine as sub strate [28]. Mitochondrial MAO B activity assayed with this substrate concentration demonstrated sensitivity to inhibition by "diagnostic" MAO B inhibitor deprenyl and also to isatin. Affinity fractionation of Rpn10 binding proteins. Agarose resin with the immobilized proteasome Rpn10 subunit (0.5 ml) was washed five times with TBS (dis solved in 10 ml and centrifuged). The mitochondrial preparation (5 mg/ml protein, TBS, 1% Triton X 100) was added to the agarose slurry up to suspension (1 : 1), taking into consideration the additions of glycerol, PMSF, and bacitracin (final concentrations 5% (v/v), 1 mM, and 0.2 mg/ml, consequently). The resulting sus pension was incubated overnight at 4°C with gentle stir
This work continues the series of the quantitative measurements of the proteins encoded by differ... more This work continues the series of the quantitative measurements of the proteins encoded by different chromosomes in the blood plasma of a healthy person. Selected Reaction Monitoring with Stable Isotope-labeled peptide Standards (SRM SIS) and a gene-centric approach, which is the basis for the implementation of the international Chromosome-centric Human Proteome Project (C-HPP), were applied for the quantitative measurement of proteins in human blood plasma. Analyses were carried out in the frame of C-HPP for each protein-coding gene of the four human chromosomes: 18, 13, Y, and mitochondrial. Concentrations of proteins encoded by 667 genes were measured in 54 blood plasma samples of the volunteers, whose health conditions were consistent with requirements for astronauts. The gene list included 276, 329, 47, and 15 genes of chromosomes 18, 13, Y, and the mitochondrial chromosome, respectively. This paper does not make claims about the detection of missing proteins. Only 205 proteins (30.7%) were detected in the samples. Of them, 84, 106, 10, and 5 belonged to chromosomes 18, 13, and Y and the mitochondrial chromosome, respectively. Each detected protein was found in at least one of the samples analyzed. The SRM SIS raw data are available in the ProteomeXchange repository (PXD004374, PASS01192).
Vestibular schwannomas are relatively rare intracranial tumors compared to other brain tumors. Da... more Vestibular schwannomas are relatively rare intracranial tumors compared to other brain tumors. Data on the molecular features, especially on schwannoma proteome, are scarce. The 41 cerebrospinal fluid (liquor) samples were obtained during the surgical removal of vestibular schwannoma. Obtained peptide samples were analyzed by shotgun LC-MS/MS high-resolution mass spectrometry. The same peptide samples were spiked with 148 stable isotopically labeled peptide standards (SIS) followed by alkaline fractionation and scheduled multiple reaction monitoring (MRM) for quantitative analysis. The natural counterparts of SIS peptides were mapped onto 111 proteins that were Food and Drug Administration (FDA)-approved for diagnostic use. As a result, 525 proteins were identified by shotgun LC-MS/MS with high confidence (at least two peptides per protein, FDR < 1%) in liquor samples. Absolute quantitative concentrations were obtained for 54 FDA-approved proteins detected in at least five experi...
Human pluripotent stem cells are promising for a wide range of research and therapeutic purposes.... more Human pluripotent stem cells are promising for a wide range of research and therapeutic purposes. Their maintenance in culture requires the deep control of their pluripotent and clonal status. A non-invasive method for such control involves day-to-day observation of the morphological changes, along with imaging colonies, with the subsequent automatic assessment of colony phenotype using image analysis by machine learning methods. We developed a classifier using a convolutional neural network and applied it to discriminate between images of human embryonic stem cell (hESC) colonies with “good” and “bad” morphological phenotypes associated with a high and low potential for pluripotency and clonality maintenance, respectively. The training dataset included the phase-contrast images of hESC line H9, in which the morphological phenotype of each colony was assessed through visual analysis. The classifier showed a high level of accuracy (89%) in phenotype prediction. By training the classi...
Studies of induced granulocytic differentiation help to reveal molecular mechanisms of cell matur... more Studies of induced granulocytic differentiation help to reveal molecular mechanisms of cell maturation. The nuclear proteome represents a rich source of regulatory molecules, including transcription factors (TFs). It is important to have an understanding of molecular perturbations at the early stages of the differentiation processes. By applying the proteomic quantitative profiling using isobaric labeling, we found that the contents of 214, 319, 376, 426, and 391 proteins were altered at 3, 6, 9, 12, and 72 h, respectively, compared to 0 h in the HL-60 cell nuclear fraction under all-trans-retinoid acid (ATRA) treatment. From 1860 identified nuclear proteins, 231 proteins were annotated as proteins with transcription factor (TF) activity. Six TFs (RREB1, SRCAP, CCDC124, TRIM24, BRD7, and BUD31) were downregulated and three TFs EWSR1, ENO1, and FUS were upregulated at early time points (3–12 h) after ATRA treatment. Bioinformatic annotation indicates involvement of the HL-60 nuclear ...
Induced granulocytic differentiation of human leukemic cells under all-trans-retinoid acid (ATRA)... more Induced granulocytic differentiation of human leukemic cells under all-trans-retinoid acid (ATRA) treatment underlies differentiation therapy of acute myeloid leukemia. Knowing the regulation of this process it is possible to identify potential targets for antileukemic drugs and develop novel approaches to differentiation therapy. In this study, we have performed transcriptomic and proteomic profiling to reveal up- and down-regulated transcripts and proteins during time-course experiments. Using data on differentially expressed transcripts and proteins we have applied upstream regulator search and obtained transcriptome- and proteome-based regulatory networks of induced granulocytic differentiation that cover both up-regulated (HIC1, NFKBIA, and CASP9) and down-regulated (PARP1, VDR, and RXRA) elements. To verify the designed network we measured HIC1 and PARP1 protein abundance during granulocytic differentiation by selected reaction monitoring (SRM) using stable isotopically labele...
Cold shock domain proteins (CSDPs) participate in plant development and resistance, but the under... more Cold shock domain proteins (CSDPs) participate in plant development and resistance, but the underlying molecular mechanisms are poorly understood. In this study, we demonstrated that the CSDPs, including EsCSDP1, EsCSDP2, and EsCSDP3, from the extremophyte Eutrema salsugineum possess all basic properties of RNA chaperones. EsCSDP1-3 melt secondary structures in RNAs with various nucleotide sequences and exhibit RNA chaperone activity in vitro. EsCSDP1 and EsCSDP3 were shown to have higher RNA melting activity, whereasile EsCSDP2 had higher RNA chaperone activity. We demonstrated that higher RNA melting activity correlates with the longer C-terminal fragment in many zinc finger motifs, whereas increased RNA chaperone activity was most likely due to the higher glycine content and RGG repeat number in the C-terminal fragment.
A gene-centric approach was applied for a large-scale study of expression products of a single ch... more A gene-centric approach was applied for a large-scale study of expression products of a single chromosome. Transcriptome profiling of liver tissue and HepG2 cell line was independently performed using two RNA-Seq platforms (SOLiD and Illumina) and also by Droplet Digital PCR (ddPCR) and quantitative RT-PCR. Proteome profiling was performed using shotgun LC-MS/MS as well as selected reaction monitoring with stable isotope-labeled standards (SRM/SIS) for liver tissue and HepG2 cells. On the basis of SRM/SIS measurements, protein copy numbers were estimated for the Chromosome 18 (Chr 18) encoded proteins in the selected types of biological material. These values were compared with expression levels of corresponding mRNA. As a result, we obtained information about 158 and 142 transcripts for HepG2 cell line and liver tissue, respectively. SRM/SIS measurements and shotgun LC-MS/MS allowed us to detect 91 Chr 18-encoded proteins in total, while an intersection between the HepG2 cell line ...
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Papers by Olga Tikhonova