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Although helminth parasites have different life cycles, their hosts share similar immune responses involving Th2 cell-type. Here, we extracted proteins from the larvae of Anisakis simplex complex and Trichinella spiralis to identify common and specific antigens (or allergens) associated with the Th2 immune response. We performed two-dimensional electrophoresis analysis and Matrix-assisted laser desorption ionization-time of flight/time of flight (MALDI-TOF/TOF) experiments. We found 13 potentially immunogenic proteins, which included 5 spots specific to T. spiralis and 8 common to T. spiralis and A. simplex, by tandem mass spectrometry. These molecules were identified structurally as actin, tropomyosin, col cuticle N domain-containing protein, and heat shock proteins. We also identified molecules related to parasite-host immune modulation and interactions. Our results may contribute to reveal potential roles of immunological proteins in parasite-derived immune modulation.

The gastrointestinal nematode Heligmosomoides polygyrus bakeri shows enhanced survival in mice with colitis. As the antibody response plays an important role in antiparasitic immunity, antibodies against male and female L4 H. polygyrus were examined in mice with and without colitis. Levels of specific antibodies in the mucosa and serum were determined by enzyme-linked immunosorbent assay and immunogenic proteins of male and female parasites were identified using 2D electrophoresis and mass spectrometry. The function of identified proteins was explored with Blast2Go. Nematodes in mice with colitis induced higher levels of specific immunoglobulin G (IgG1) and IgA, a lower level of IgE in the small intestine and a higher level of IgE in serum against female L4. Infected mice with colitis recognized 12 proteins in male L4 and 10 in female L4. Most of the recognized proteins from male L4 were intermediate filament proteins, whereas the proteins from female L4 were primarily actins and galectins. Nematodes from mice with colitis were immunogenically different from nematodes from control mice. This phenomenon gives new insights into helminth therapy as well as host-parasite interactions.

Drought is the most important abiotic stress limiting wheat production worldwide. Triticum boeoticum, as wild wheat, is a rich gene pool for breeding for drought stress tolerance. In this study, to identify the most drought-tolerant and susceptible genotypes, ten T. boeoticum accessions were evaluated under non-stress and drought-stress conditions for two years. Among the studied traits, water-use efficiency (WUE) was suggested as the most important trait to identify drought-tolerant genotypes. According to the desirable and undesirable areas of the bi-plot, Tb5 and Tb6 genotypes were less and more affected by drought stress, respectively. Therefore, their flag-leaves proteins were used for two-dimensional gel electrophoresis. While, Tb5 contained a high amount of yield, yield components, and WUE, Tb6 had higher levels of water use, phenological related traits, and root related characters. Of the 235 spots found in the studied accessions, 14 spots (11 and 3 spots of Tb5 and Tb6, respectively) were selected for sequencing. Of these 14 spots, 9 and 5 spots were upregulated and downregulated, respectively. The identified proteins were grouped into six functional protein clusters, which were mainly involved in photosynthesis (36%), carbohydrate metabolism (29%), chaperone (7%), oxidation and reduction (7%), lipid metabolism and biological properties of the membrane (7%) and unknown function (14%). We report for the first time that MICP, in the group of lipid metabolism proteins, was significantly changed into wild wheat in response to drought stress. Maybe, the present-identified proteins could play an important role to understand the molecular pathways of wheat drought tolerance. We believe comparing and evaluating the similarity-identified proteins of T. boeoticum with the previously identified proteins of Aegilops tauschii, can provide a new direction to improve wheat tolerance to drought stress., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Jacquinia macrocarpa , a plant native to northwestern Mexico, has an inhibitory effect against phytopathogenic fungi. Previous studies have shown that the butanolic extract of J. macrocarpa causes retardation and atrophy in mycelial growth of Fusarium verticillioides . However, the action mechanism of this extract is unknown. We used a proteomics approach to understand the inhibitory effect of J. macrocarpa butanolic extract, based on differential protein accumulation in F. verticillioides . Proteins were extracted from F. verticillioides cultured in Czapek broth with and without 202.12 μg/mL (IC 50 ) of butanolic extract of J. macrocarpa . Thirty-eight protein spots showing statistically significant changes (ANOVA, p < 0.01) and at least a 2-fold change in abundance between experimental conditions were analyzed by mass spectrometry. Identified proteins were grouped into different biological processes according to Gene Ontology, among them were amino acid metabolism, protein folding and stabilization, protein degradation, protein transport, carbohydrate metabolism, oxidative stress response, and miscellaneous. This work is the first report of changes in the proteomic profile of F. verticillioides exposed to the J. macrocarpa extract. This information provides new insights into the inhibitory mechanism of the extract and represents a starting point for dissection of the fungal response against the J. macrocarpa extract components.

The aim of the study was to evaluate the influence of dietary supplementation with inulin extract from chicory root and dried chicory root on the protein profile of the renal cortex and medulla of growing pigs. The experiment was carried out on renal cortex and medulla tissue collected from 24 50-day-old PIC x Penarlan P76 crossbred piglets (males). Animals were divided into three dietary groups (n = 8) and fed with a control diet, diet supplemented with 2% inulin extract from chicory root and a diet supplemented with 4% dried chicory root. Kidney samples were collected after 40 days of feeding, and renal cortex and medulla proteins were separated by two-dimensional electrophoresis. Protein identification was performed using MALDI-TOF mass spectrometry. The diet supplemented with 2% chicory inulin induced significant expression changes of 20 and 26 protein spots in the renal cortex and medulla respectively. Supplementation with 4% dried chicory root triggered changes in the expression of 44 and 24 proteins in the renal cortex and medulla respectively. Both forms of chicory inulin-type fructans effectively affected the expression of proteins involved in energy metabolism, heat shock proteins and other chaperones, cytoskeletal and cytoskeleton-related proteins, as well as other proteins. Additionally, changes in transferrin abundance in both experimental groups suggested the significance of chicory fructan supplementation for iron absorption and bioavailability. In conclusion, 2% inulin extract from chicory root and 4% dried chicory root exerted a similar effect on changes in renal protein expression; however, more pronounced alterations were induced by dried chicory root. Nevertheless, further studies are needed for better understanding the mechanism underlying the effect of chicory inulin-type fructans and their fermentation end products on the kidneys of growing pigs., (© 2019 Blackwell Verlag GmbH.)

Obstructive sleep apnea (OSA) affects an estimated 20% of adults worldwide and has been associated with electrical and structural abnormalities of the atria, although the molecular mechanisms are not well understood. Here, we used two-dimensional polyacrylamide gel electrophoresis (2D PAGE) coupled with nanoliquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) to investigate the proteins that are dysregulated in the atria from severe and moderate apnea when compared to control. We found enzymes involved in the glycolysis, beta-oxidation, electron transport chain and Krebs cycle to be down-regulated. The data suggested that the dysregulated proteins may play a role in atrial pathology developing via chronic obstructive apnea and hypoxia. Our results are consistent with our previous 1D-PAGE and nanoLC-MS/MS study (Channaveerappa et al, J Cell Mol Med. 2017), where we found that some aerobic and anaerobic glycolytic and Krebs cycle enzymes were down-regulated, suggesting that apnea may be a result of paucity of oxygen and production of ATP and reducing equivalents (NADH). The 2D-PAGE study not only complements our current study, but also advances our understanding of the OSA. The complete mass spectrometry data are available via ProteomeXchange with identifier PXD011181., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

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

Olive is one of the most important fruit crop trees in the history of Mediterranean because of the high quality oil. Olive oil has a well-balanced fatty acid composition along with biophenols, which make it exceptional in human diet and provide an exceptional value to the olive oil. Leaf non-glandular peltate trichomes are specialized cell types representing a protective barrier against acute environmental conditions. To characterize the proteome of this highly differentiated cell type, we performed a comparative proteomic analysis among isolated trichomes and trichome-less leaves. Proteins were separated and identified using the 2-DE MALDI-TOF/MS method. A number of enzymes involved in abiotic and biotic stress responses are present and may be responsible for the adaptation to prolonged adverse environmental conditions. The results show that this highly differentiated cell type is physiologically active fulfilling the demands of the trichomes in furnishing the leaf with a highly protective mechanism., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Catechin exhibits antimicrobial activity against various microorganisms, such as EHEC O157:H7. This study reports the bactericidal effect of catechin on EHEC O157:H7 in simulated human gastrointestinal environment and the underlying antibacterial mechanism. Bacteriostasis test results showed that the minimum bactericidal concentration of catechin for EHEC O157:H7 was 5 g/L. The bactericidal effect of catechin in the gastrointestinal juice became more significant with increased culture time, and catechin exhibited a synergistic effect with bile salt in inhibiting EHEC O157:H7. Changes in the profile of protein expression in EHEC O157:H7 in response to catechin intervention were investigated. Two-dimensional electrophoresis identified 34 proteins with significantly altered expression. A total of 2 and 12 proteins were upregulated and downregulated, respectively. However, 20 proteins disappeared. No new protein was expressed compared with the control. Hence, catechin intervention resulted in diverse changes in the expression of proteins associated with cell structure and genetic information processing. Catechin could cause the disappearance of certain proteins or the destruction of certain peptides. These processes lead to the inhibition of EHEC O157: H7 cells., (Copyright © 2018. Published by Elsevier Ltd.)

Sclerotinia sclerotiorum (Lib.) de Bary is a devastating necrotrophic fungal pathogen attacking a broad range of agricultural crops. In this study, although the transcript accumulation of SsNsd1, a GATA-type IVb transcription factor, was much lower during the vegetative hyphae stage, its mutants completely abolished the development of compound appressoria. To further elucidate how SsNsd1 influenced the appressorium formation, we conducted proteomics-based analysis of the wild-type and Δ SsNsd1 mutant by two-dimensional electrophoresis (2-DE). A total number of 43 differentially expressed proteins (≥3-fold change) were observed. Of them, 77% were downregulated, whereas 14% were upregulated. Four protein spots fully disappeared in the mutants. Further, we evaluated these protein sequences by mass spectrometry analysis of the peptide mass and obtained functionally annotated 40 proteins, among which only 17 proteins (38%) were identified to have known functions including energy production, metabolism, protein fate, stress response, cellular organization, and cell growth and division. However, the remaining 23 proteins (56%) were characterized as hypothetical proteins among which four proteins (17%) were predicted to contain the signal peptides. In conclusion, the differentially expressed proteins identified in this study shed light on the Δ SsNsd1 mutant-mediated appressorium deficiency and can be used in future investigations to better understand the signaling mechanisms of SsNsd1 in S. sclerotiorum .

Proteomic analyses are rapid and powerful tools that are used to increase the understanding of cancer pathogenesis, discover cancer biomarkers and predictive markers, and select and monitor novel targets for cancer therapy. Feline injection-site sarcomas (FISS) are aggressive skin tumours with high recurrence rates, despite treatment with surgery, radiotherapy, and chemotherapy. Doxorubicin is a drug of choice for soft tissue sarcomas, including FISS. However, multidrug resistance is one of the major causes of chemotherapy failure. The main aim of the present study was to identify proteins that differentiate doxorubicin-resistant from doxorubicin-sensitive FISS using two-dimensional gel electrophoresis (2DE), followed by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) analysis. Using the three-dimensional (3D) preclinical in ovo model, which resembles features of spontaneous fibrosarcomas, three significantly ( p ≤ 0.05) differentially expressed proteins were identified in tumours grown from doxorubicin-resistant fibrosarcoma cell lines (FFS1 and FFS3) in comparison to the doxorubicin-sensitive one (FFS5): Annexin A5 (ANXA5), Annexin A3 (ANXA3), and meiosis-specific nuclear structural protein 1 (MNS1). Moreover, nine other proteins were significantly differentially expressed in tumours grown from the high doxorubicin-resistant cell line (FFS1) in comparison to sensitive one (FFS5). This study may be the first proteomic fingerprinting of FISS reported, identifying potential candidates for specific predictive biomarkers and research targets for doxorubicin-resistant FISS., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results. According to Polish law (The Act on the protection of animals used for scientific and educational purposes, which was passed in January 2015 and transposed Directive 2010/63/EU into current Polish legislation) the chick embryo studies do not need the approval of the Animal Ethic Commission. Nevertheless, all experiments were performed according to the animal welfare, minimizing the stress of chicken embryos according to American Veterinary Medical Association and Animal Care and Use Committee guidelines on the use of chicken embryos for scientific purposes.

The main objectives of the study were to: (1) deeply analyse the serum protein composition of Equus caballus, (2) assess the effectiveness of the high-abundant protein depletion and improve the concentration of medium- and low-abundant proteins. The analysis were performed on the blood plasma of three healthy part-Arabian mares. The implementation of two-dimensional electrophoresis and matrix-assisted laser desorption/ionisation - time of flight mass spectrometry allowed us to establish a horse plasma proteome map. Serum proteins were resolved at pH 4 to 7, followed by 12% SDS-PAGE. As a result 136 spots were successfully identified, representing the products of 46 unique genes. Of these, 22 gene products have not been previously identified in horse serum/plasma samples using proteomic tools. Gene ontology analysis showed that almost 30% of all identified gene products belong to the coagulation and complement cascades. These results can undoubtedly serve as a useful and prospective prerequisite for the future analysis of horse plasma proteome changes in different physiological and pathophysiological conditions. The use of the medium- and low-abundant protein enrichment tool increased their abundance and allowed us to identify a higher number of protein gene products. The highest depletion efficiency was observed for the most abundant plasma proteins, that is albumin, IgG heavy chains and serotransferrin.

The functions and properties of fungal sheath, an extracellular polysaccharide produced by many white-rot fungi, have been studied. However, the strong adherence of the sheath to fungal hyphae had been a major impediment in preparing intracellular proteins from the fungi and analyzing their cellular responses. To overcome this issue, we developed a rapid and easy method to remove the polysaccharide sheath using a selective lignin degrader, Ceriporiopsis subvermispora, which produces large sheath amounts in the presence of a lignin-derived aromatic compound. Using this approach, we achieved thorough removal of sheath and cell disruption using beads and a solution with a high protein-solubilizing power, which enabled the efficient extraction of intracellular proteins from C. subvermispora surrounded by sheath. In addition, for proteomic analysis, we investigated whether these extracted proteins were compatible with two-dimensional electrophoresis. By efficiently concentrating on protein solubilization in the first dimension and using a stacking gel in the second dimension, we successfully obtained a high-resolution proteome map of C. subvermispora. We also used the same proteins for fluorescence two-dimensional difference gel electrophoresis to obtain the quantitative protein expression profiles. These steps demonstrated that two-dimensional electrophoresis-based proteomics can be used to clarify the composition of intracellular proteins from sheath-producing white-rot fungi., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Nine geographically diverse Moniliophthora perniciosa (witches' broom disease pathogen) isolates were cultured in vitro. They included six C-biotypes differing in virulence on cacao (Theobroma cacao), two S-biotypes (solanaceous hosts), and an L-biotype (liana hosts). Mycelial growth rates and morphologies differed considerably, but no characters were observed to correlate with virulence or biotype. In plant inoculations using basidiospores, one C-biotype caused symptoms on tomato (an S-biotype host), adding to evidence of limited host adaptation in these biotypes. Mycelial proteomes were analysed by two-dimensional gel electrophoresis (2-DE), and 619 gel spots were indexed on all replicate gels of at least one strain. Multivariate analysis of gel spots discriminated the L-biotype, but not the S-biotypes, from the remaining strains. The proteomic similarity of the S- and C-biotypes is consistent with their reported lack of phylogenetic distinction. Sequences from tandem mass spectrometry of tryptic peptides from major 2-DE spots were matched with Moniliophthora genome and transcript sequences on NCBI and WBD Transcriptome Atlas databases. Protein-spot identifications indicated that M. perniciosa saprotrophic mycelial proteomes expressed functions potentially connected with a 'virulence life-style', including peroxiredoxin, heat-shock proteins, nitrilase, formate dehydrogenase, a prominent complement of aldo-keto reductases, mannitol-1-phosphate dehydrogenase, and central metabolism enzymes with proposed pathogenesis functions., (Copyright © 2017 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Cochliobolus lunatus abundantly produces four-celled conidia at high temperatures (>30 °C) and under suitable conditions; the fungus colonizes potato (Solanum tuberosum L.) cultivars by adopting different invasion strategies at the microscopic level. Long-lasting defence during infection requires an upsurge in proteome changes particularly pathogenesis-related proteins chiefly under the control of nonexpresser of pathogenesis-related proteins. In order to gain molecular insights, we profiled the changes in proteome and potato nonexpresser of pathogenesis-related proteins (StNPR1) during the infection process. It is found that C. lunatus significantly (P < 0.05) suppressed the host functional proteome by 96 h after infection (hai), principally, affecting the expression of ribulose bisphosphate carboxylase enzyme, plastidic aldolase enzyme, alcohol dehydrogenase 2 and photosystem II protein prior to the formation of brown-to-black leaf spot disease. Strongest host response was observed at 24 hai hallmarked by 307 differentially expressed peptide spots concurring with the active phase of production of penetrating hyphae. Additionally, C. lunatus differentially down-regulated StNPR1 transcript by 8.19 fold by 24 hai. This study is the first to elucidate that C. lunatus transiently down-regulates the expression of StNPR1 at the onset of infection, and as a whole, infection negatively affects the expression of proteome components involved in photosynthesis, carbon fixation and light assimilation. This study contributes towards better understanding of the mechanism underlining the invasion strategies of C. lunatus.

Certain clinical isolates of Tritrichomonas foetus infect the urogenital tract of cattle while others infect the gastrointestinal tract of cats. Previous studies have identified subtle genetic differences between these isolates with the term "genotype" adopted to reflect host origin. The aim of this work was to seek evidence of host-specific adaptation and to clarify the relationship between T. foetus genotypes. To do this we characterised the proteomes of both genotypes using two-dimensional gel electrophoresis (2DE) coupled with LC-MS/MS. Our comparative analysis of the data revealed that both genotypes exhibited largely similar proteoform profiles; however differentiation was possible with 24 spots identified as having a four-fold or greater change. Deeper analysis using 2DE zymography and protease-specific fluorogenic substrates revealed marked differences in cysteine protease (CP) expression profiles between the two genotypes. These variances in CP activities could also account for the pathogenic and histopathological differences previously observed between T. foetus genotypes in cross-infection studies. Our findings highlight the importance of CPs as major determinants of parasite virulence and provide a foundation for future host-parasite interaction studies, with direct implications for the development of vaccines or drugs targeting T. foetus., (Copyright © 2017 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Background: The cell nucleus is a highly dynamic subcellular compartment performing crucial processes for functioning and administration of the cell. Proteomic analysis of isolated nuclear fraction enables in-depth insight into these processes leading to better understanding of physiological and pathological state of the brain. There is no universal method for nuclear proteome investigation and each biological material needs individual optimization. An additional difficulty is the large amount of nucleic acids, which impairs isoelectrofocusing of proteins and shotgun mass spectrometry analysis of complex peptide samples., New Method: We performed the first comprehensive optimization of intact nuclei isolation from mouse brain in combination with nuclear protein purification prior to two-dimensional gel electrophoresis (2DE) and gel-free proteomic analysis., Results: Application of sonication, digestion with nuclease and protein precipitation with acetone allowed to obtain high quality 2DE gels. Shotgun mass spectrometry analysis of isolated proteins proved an enrichment in nuclear proteins. The 66.4% of 265 identified proteins had assigned nucleus localization in UniProt database. Gene Ontology analysis using DAVID platform revealed the highest fold enrichment in spliceosome (24.5), nuclear periphery (12.4) and nuclear matrix (11.3)., Comparison With Existing Methods: The proposed procedure is tailored to mouse brain tissue nuclear subproteome investigation. The quality of isolated nuclei, the effectiveness of the protein purification, efficiency of protein recovery after precipitation and overall method reproducibility was taken into detailed consideration., Conclusions: The elaborated procedure could be further applied for in-depth proteomic analysis of molecular processes occurring in the mouse brain nucleus., (Copyright © 2015 Elsevier B.V. All rights reserved.)

To obtain more information about human proteome, especially about proteoforms (protein species) coded by 18th chromosome, we separated proteins from human cancer cell line (HepG2) by two-dimensional gel electrophoresis (2DE). Initially, proteins in major spots were identified by MALDI-MS peptide mass fingerprinting. According to parameters (pI/Mw) of identified proteins the gel was calibrated. Using this calibrated gel, a virtual 2D map of proteoforms coded by Chromosome 18 was constructed. Next, the produced gel was divided into 96 sections with determined coordinates. Each section was cut, shredded, and treated by trypsin according to mass-spectrometry protocol. After protein identification by shotgun mass spectrometry using ESI LC-MS/MS, a list of 20 462 proteoforms (product of 3774 genes) was generated. Among them, 165 proteoforms are representing 39 genes of 18th chromosome. The 3D graphs showing the distribution of different proteoforms from the same gene in 2D map were generated. This is a first step in creation of 2DE-based knowledge database of proteins coded by 18th chromosome.

The effect of daily intragastric administration of an aqueous dispersion of silicon nanoparticles (NPs) (the dose range from 1.0 mg/kg to 100 mg/kg body weight for 28 days) to rats on the proteomic profile of liver microsomes has been investigated by 2D-electrophoresis followed by subsequent mass spectrometry identification. The liver microsomal fraction was isolated by differential centrifugation and its protein composition was analyzed by 2D-polyacrylamide gel electrophoresis. Identification of protein spots was carried out using MALDI-TOF mass spectrometric analysis. The mass spectrometry analysis revealed the protein GRP78 (78 kD glucose-regulated protein precursor), belonging to the family of heat shock proteins. This protein present in animals of the control group was not detected in NP-treated rats of group 2 (1 mg/kg body weight/day) and group 3 (10 mg/kg body weight/day). This protein predominantly localized in the liver cell endoplasmic reticulum and plasma membrane has the chaperone biological activity. Possible mechanisms of the effects of engineered nanoparticles on biosynthetic processes in the body are discussed.

Objective: Understanding the molecular basis of complex adaptive traits, such as snake venom, demands qualitative and quantitative comparisons of the temporal and spatial patterns of venom variation. Here, we assessed the proof-of-concept that locus-resolved reference venom proteome maps can be achieved through efficient pre-MS venom proteome decomplexation, peptide-centric MS/MS analysis and species-specific database searching., Methods: Venom proteome components were fractionated and quantified by RP-HPLC, SDS-PAGE and 2DE prior to LC-MS/MS matching against a species-specific transcriptomic dataset., Results: Combination of RP-HPLC/SDS-PAGE and 2DE followed by LC-MS/MS showed the existence of ∼178-180 venom protein species generated from ∼48 unique transcripts., Conclusions: Our results underscore that if sufficient pre-MS and MS efforts are applied, comprehensive venom maps can be achieved. And - equally important - dissociating the venom decomplexing steps from the protein identification process represents the key to achieving a quantitative and locus-resolved insight of the venom proteome.

Alkaloids are one of the most attractive sources for obtaining active natural products. However, alkaloids exist in the plants as the secondary metabolites with tracing amount, and there is an enormous demand for a large production. In the present study, we aimed to profile the modification of benzylisoquinoline alkaloids in Mahonia bealei seedlings under the binary stress of ultraviolet-B irradiation and dark incubation. Comparative proteomics analysis was carried out to address the underlying proteome variations that accounted for the alkaloid induction under treatment. Thirteen differential proteins were identified in the leaves under binary stress. Of note, the abundance of S-adenosyl-L-methionine synthetase was highly increased to sustain a high concentration of S-adenosyl-L-methionine for the enhanced biosynthesis of alkaloids. Additionally, we presented the application of CPLL to M. bealei leaf proteins. Three new secondary metabolism proteins and 12 additional differential proteins were identified only after CPLL treatment. Six genes in the benzylisoquinoline alkaloid biosynthesis pathway were selected to verify their variable expression using quantitative real time polymerase chain reaction. The results suggest that the benzylisoquinoline alkaloids in M. bealei leaf were increased to eliminate the adverse effect of UV-B exposure. The suppression of photosynthesis and respiratory rate may save an extra energy for the secondary metabolites, and the enhanced N-metabolism may supply sufficient primary metabolite precursors. To our best knowledge, this is the first work aimed at the secondary metabolism proteomic characterization of M. bealei using the CPLL technique. It also presented an effective and innovative process to improve the contents of alkaloids in medicinal plants for industrial production., Biological Significance: Besides the effective and innovative process to improve the contents of alkaloids in M. bealei leaves for industrial production, the presented combinatorial hexapeptide ligand library technology was applied for the study of low-abundance protein in medicinal plant. It may be an available tool for the analysis of the secondary proteins., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Urine is a source of potential markers of disease. In the context of renal disease, urine is particularly important as it may directly reflect kidney injury. Current markers of renal dysfunction lack both optimal specificity and sensitivity, and improved technologies and approaches are needed. There is no clear consensus about the best sample pretreatment procedure for 2DE analysis of the urine proteome. Sample pretreatment conditions spots resolution and detection sensitivity, critically. As a first goal, we exhaustively compared eight different sample cleaning and protein purification methodologies for 2DE analysis of urine from healthy individuals. Oasis® HLB cartridges allowed the detection of the highest number of low molecular weight proteins; while PD10 desalting columns resulted in the highest number of detected spots in the high molecular weight area. Sample pretreatment strategies were also explored in the context of proteinuria, a clinical condition often associated to renal damage. Testing of urine samples from 13 patients with hypertension or kidney disease and different levels of proteinuria identified Oasis® HLB cartridge purification in combination with albumin depletion by ProteoPrep kit as the best option for urine proteome profiling from patients with proteinuric (> 30 mg/L albumin in urine) renal disease., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Oenococcus oeni is the main lactic acid bacterium that carries out the malolactic fermentation in virtually all red wines and in some white and sparkling wines. Oenococcus oeni possesses an array of metabolic activities that can modify the taste and aromatic properties of wine. There is, therefore, industrial interest in the proteins involved in these metabolic pathways and related transport systems of this bacterium. In this work, we report the characterization of the O. oeni ATCC BAA-1163 proteome. Total and membrane protein preparations from O. oeni were standardized and analysed by two-dimensional gel electrophoresis. Using tandem mass spectrometry, we identified 224 different spots corresponding to 152 unique proteins, which have been classified by their putative function and subjected to bioinformatics analysis.

Green fluorescent protein (GFP) is the most commonly used reporter of expression in cell biology despite evidence that it affects the cell physiology. The molecular mechanism of GFP-associated modifications has been largely unexplored. In this paper we investigated the proteome modifications following stable expression of GFP in breast cancer cells (MDA-MB-231). A combination of three different proteome analysis methods (2-DE, iTRAQ, label-free) was used to maximise proteome coverage. We found that GFP expression induces changes in expression of proteins that are associated with protein folding, cytoskeletal organisation and cellular immune response. In view of these findings, the use of GFP as a cell reporter should be carefully monitored., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Urine is a suitable biological fluid to look for markers of physiological and pathological processes, including renal and nonrenal diseases. In addition, it is an optimal body sample for diagnosis, because it is easily obtained without invasive procedures and can be sampled in large quantities at almost any time. Rats are frequently used as a model to study human diseases, and rat urine has been analyzed to search for disease biomarkers. The normal human urinary proteome has been studied extensively, but the normal rat urinary proteome has not been studied in such depth. In light of this, we were prompted to analyze the normal rat urinary proteome using three complementary proteomics platforms: SDS-PAGE separation, followed by LC-ESI-MS/MS; 2DE, followed by MALDI-TOF-TOF and 2D-liquid chromatography-chromatofocusing, followed by LC-ESI-Q-TOF. A total of 366 unique proteins were identified, of which only 5.2% of unique proteins were identified jointly by the three proteomics platforms used. This suggests that simultaneous proteomics techniques provide complementary and nonredundant information. Our analysis affords the most extensive rat urinary protein database currently available and this may be useful in the study of renal physiology and in the search for biomarkers related to renal and nonrenal diseases., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Proteomic analysis by two-dimensional electrophoresis (2D)-mass spectrometry was used to identify differentially expressed proteins in the Clostridium sp. native strain (IBUN 158B) in two phases of the 1,3-propanediol (1,3-PD) production (lag phase and exponential growth phase). Intracellular protein fraction extraction conditions were standardised, as well as the 2D electrophoresis. Differences were found between both of the growth phases evaluated here. Thirty-two of the differentially expressed proteins were chosen to be identified by tandem mass spectrometry (MALDI TOF/TOF). The presence of four enzymes implicated in the 1,3-PD metabolic pathway was recorded: one from the reductive route (1,3-propanediol dehydrogenase) and three from the oxidative route (3-hydroxybutyryl-CoA dehydrogenase, NADPH-dependent butanol dehydrogenase and phosphate butyryl transferase). The following enzymes which have not been previously reported for Clostridium sp., were also identified: phosphoglycerate kinase, glucose 6-phosphate isomerase, deoxyribose phosphate aldolase, transketolase, cysteine synthetase, O-acetylhomoserine sulphhydrylase, glycyl-tRNA ligase, aspartate-β-semialdehyde dehydrogenase, inosine-5-monophosphate dehydrogenase, aconitate hydratase and the PrsA protein. The foregoing provides a novel contribution towards knowledge of the native strain for the purpose of designing genetic manipulation strategies to obtain strains with high production of 1,3-PD., Biological Significance: The article "Protein identification in two phases of 1,3-propanediol production by proteomic analysis" provides a novel contribution towards knowledge regarding the Colombian Clostridium sp. native strain (IBUN 158B) because this is a new approximation in comparative proteomics in two phases of the bacterial growth and 1,3-propanediol (1,3-PD) production conditions. The proteomic studies are very important to identify the enzymes that are expressed at different stages of production and therefore genes of interest in the genetic manipulation strategies; the results can be taken into account in future studies in metabolic engineering when optimising 1,3-PD production, in a cost-effective process having direct industrial applications., (© 2013 Elsevier B.V. All rights reserved.)

This study involves the comparison between the exoproteomes of two different strains of Corynebacterium pseudotuberculosis, the etiologic agent of caseous lymphadenitis in small ruminants. In a previous study, based on a gel-free system (TPP-LC/MS(E)), 70 exoproteins for the strain 1002 and 67 for the strain C231, totaling 93 different extracellular proteins for C. pseudotuberculosis, were identified. In the present work, we have used 2D gel electrophoresis to resolve the extracellular proteins of both strains, which were then digested with trypsin, analyzed by MALDI-TOF/TOF and identified with the software MASCOT(®). A total of 45 extracellular proteins of C. pseudotuberculosis were identified by this approach. The comparative analysis between the strains 1002 and C231 identified 13 and 3 strain-specific proteins, respectively, 11 of which are novel. These newly identified proteins may play an important role in the physiology and virulence of C. pseudotuberculosis., (Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.)

Many foods including edible oils contain 2-monochloropropane-1,3-diol (2-MCPD), a processing-induced chemical contaminant. Cardiotoxic effects have been shown to result from oral 2-MCPD exposure in rodents, but the underlying mechanisms of action remain poorly understood. We undertook a comprehensive multi-omics approach to assess changes at the transcriptomic, proteomic, and oxylipin levels in heart tissues from male F344 rats that were exposed to 0 or 40 mg/kg BW/day of 2-MCPD in the diet for 90 days, in a regulatory compliant rodent bioassay. Heart tissues were collected for RNA sequencing, quantitative PCR analysis, proteomic analysis via two-dimensional gel electrophoresis and mass spectrometry, and targeted lipidomic profiling by high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS). Transcriptomic and proteomic data analyses revealed upregulation of immune/inflammatory response processes and downregulation of energy metabolism and cardiac structure and functions. Among differentially expressed gene–protein pairs, coronin-1A, a key leukocyte-regulating protein, emerged as markedly up-regulated. Oxylipin profiling highlighted a selective suppression of docosahexaenoic acid-derived metabolites, suggesting a disruption in cardioprotective lipid pathways. These findings suggest that 2-MCPD disrupts homeostasis through inflammatory activation and suppression of metabolic and cardiac function. This research provides insights into 2-MCPD's cardiotoxicity, emphasizing the need for further studies to support hazard characterization. [ABSTRACT FROM AUTHOR]

Viral diseases of potatoes are among the main problems causing deterioration in the quality of tubers and loss of yield. The growth and development of potato plants largely depend on soil moisture. Prevention strategies require comprehensive protection against pathogens and abiotic stresses, including modeling the beneficial microbiome of agroecosystems combining microorganisms and immunostimulants. Chitosan and its derivatives have great potential for use in agricultural engineering due to their ability to induce plant immune responses. The effect of chitosan conjugate with caffeic acid (ChCA) in combination with Bacillus subtilis 47 on the transcriptional activity of PR protein genes and changes in the proteome of potato plants during potato virus Y (PVY) infection and drought was studied. The mechanisms of increasing the resistance of potato plants to PVY and lack of moisture are associated with the activation of transcription of genes encoding PR proteins: the main protective protein (PR-1), chitinase (PR-3), thaumatin-like protein (PR-5), protease inhibitor (PR-6), peroxidase (PR-9), and ribonuclease (PR-10), as well as qualitative and quantitative changes in the plant proteome. The revealed activation of the expression of marker genes of systemic acquired resistance and induced systemic resistance under the influence of combined treatment with B. subtilis and chitosan conjugate indicate that, in potato plants, the formation of resistance to viral infection in drought conditions proceeds synergistically. By two-dimensional electrophoresis of S. tuberosum leaf proteins followed by MALDI-TOF analysis, 10 proteins were identified, the content and composition of which differed depending on the experiment variant. In infected plants treated with ChCA, the synthesis of proteinaceous RNase P 1 and oxygen-evolving enhancer protein 2 was enhanced in conditions of normal humidity, and 20 kDa chaperonin and TMV resistance protein N-like was enhanced in conditions of lack of moisture. The virus coat proteins were detected, which intensively accumulated in the leaves of plants infected with potato Y-virus. ChCA treatment reduced the content of these proteins in the leaves, and in plants treated with ChCA in combination with Bacillus subtilis, viral proteins were not detected at all, both in conditions of normal humidity and lack of moisture, which suggests the promising use of chitosan derivatives in combination with B. subtilis bacteria in the regulation of plant resistance. [ABSTRACT FROM AUTHOR]

The fungus Ustilaginoidea virens, the causative agent of false smut in rice (Oryza sativa L.), is responsible for one of the severe grain diseases that lead to significant losses worldwide. In this research, microscopic and proteomic analyses were performed by comparing U. virens infected and non-infected grains of the susceptible and resistant rice varieties to provide insights into the molecular and ultrastructural factors involved in false smut formation. Prominent differentially expressed peptide bands and spots were detected due to false smut formation as revealed by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis (2-DE) SDS-PAGE profiles and were identified using liquid chromatography-mass spectrometry (LC-MS/MS). The proteins identified from the resistant grains were involved in diverse biological processes such as cell redox homeostasis, energy, stress tolerance, enzymatic activities, and metabolic pathways. It was found that U. virens produces diverse degrading enzymes such as β-1, 3-endoglucanase, subtilisin-like protease, putative nuclease S1, transaldolase, putative palmitoyl-protein thioesterase, adenosine kinase, and DNase 1 that could discretely alter the host morphophysiology resulting in false smut. The fungus also produced superoxide dismutase, small secreted proteins, and peroxidases during the smut formation. This study revealed that the dimension of rice grain spikes, their elemental composition, moisture content, and the specific peptides produced by the grains and the fungi U. virens play a vital role in the formation of false smut. [ABSTRACT FROM AUTHOR]

Context: Systemic events resulting from mother-embryo relation at the peri-implantation period may result in specific changes to the protein composition of serum and thus provide a source of biomarkers for early detection of pregnancy. Aims: We set out to use two different quantitative proteomic approaches to test this hypothesis by comparing heifer serum at the peri-implantation period to that from cycling heifers. Methods: Two-dimensional electrophoresis (2-DE) and isobaric tags were used for relative and absolute quantitation (iTRAQ) of proteins. Key results: These methods yielded complementary data indicating biomarker candidate proteins. Conclusions: Different proteomic methods provide different and complementary information that needs to be analysed in order to consider proteins as potential biomarkers. Implications: In order to characterise the proteome under specific conditions, the use of complementary techniques is advisable. Early pregnancy detection is essential to cattle reproductive management and production efficiency in farms, and proper methods to this aim require biomarkers which may be identified by proteomic methods. We describe the use of two proteomic techniques that give complementary information to perform a primary screening of proteins which may undergo systemic changes related to pregnancy establishment. The way to combine these methods may become of great use to detect changes related to a specific event such as early pregnancy. [ABSTRACT FROM AUTHOR]

Proteins play an essential regulatory role in the innate immune response of host plants following elicitation by either biotic or abiotic stresses. Isonitrosoacetophenone (INAP), an unusual oxime-containing stress metabolite, has been investigated as a chemical inducer of plant defence responses. Both transcriptomic and metabolomic studies of various INAP-treated plant systems have provided substantial insight into this compound's defence-inducing and priming capabilities. To complement previous 'omics' work in this regard, a proteomic approach of time-dependent responses to INAP was followed. As such, Nicotiana tabacum (N. tabacum) cell suspensions were induced with INAP and changes monitored over a 24-h period. Protein isolation and proteome analysis at 0, 8, 16 and 24 h post-treatment were performed using two-dimensional electrophoresis followed by the gel-free eight-plex isobaric tags for relative and absolute quantitation (iTRAQ) based on liquid chromatography and mass spectrometry. Of the identified differentially abundant proteins, 125 were determined to be significant and further investigated. INAP treatment elicited changes to the proteome that affected proteins from a wide range of functional categories: defence, biosynthesis, transport, DNA and transcription, metabolism and energy, translation and signalling and response regulation. The possible roles of the differentially synthesised proteins in these functional classes are discussed. Results indicate up-regulated defence-related activity within the investigated time period, further highlighting a role for proteomic changes in priming as induced by INAP treatment. [ABSTRACT FROM AUTHOR]

Vibrio cholerae can cause pandemic cholera in humans. The waterborne bacterium is frequently isolated from aquatic products worldwide. However, current literature on the impact of aquatic product matrices on the survival and pathogenicity of cholerae is rare. In this study, the growth of eleven non-O1/0O139 V. cholerae isolates recovered from eight species of commonly consumed fish and shellfish was for the first time determined in the eight aquatic animal matrices, most of which highly increased the bacterial biomass when compared with routine trypsin soybean broth (TSB) medium. Secretomes of the V. cholerae isolates (draft genome size: 3,852,021–4,144,013 bp) were determined using two-dimensional gel electrophoresis (2DE-GE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques. Comparative secretomic analyses revealed 74 differential extracellular proteins, including several virulence- and resistance-associated proteins secreted by the V. cholerae isolates when grown in the eight matrices. Meanwhile, a total of 8,119 intracellular proteins were identified, including 83 virulence- and 8 resistance-associated proteins, of which 61 virulence-associated proteins were absent from proteomes of these isolates when grown in the TSB medium. Additionally, comparative genomic and proteomic analyses also revealed several strain-specific proteins with unknown functions in the V. cholerae isolates. Taken, the results in this study demonstrate that distinct secretomes and proteomes induced by the aquatic animal matrices facilitate V. cholerae resistance in the edible aquatic animals and enhance the pathogenicity of the leading waterborne pathogen worldwide. [ABSTRACT FROM AUTHOR]

The present study was aimed to investigate the effect of docosanol on the protein expression profile of methicillin-resistant Staphylococcus aureus (MRSA). Thus, two-dimensional gel electrophoresis coupled with MALDI-TOF MS technique was utilized to identify the differentially regulated proteins in the presence of docosanol. A total of 947 protein spots were identified from the intracellular proteome of both control and docosanol treated samples among which 40 spots were differentially regulated with a fold change greater than 1.0. Prominently, the thiol-dependent antioxidant system and stress response proteins are downregulated in MRSA, which are critical for survival during oxidative stress. In particular, docosanol downregulated the expression of Tpx, AhpC, BshC, BrxA, and YceI with a fold change of 1.4 (p = 0.02), 1.4 (p = 0.01), 1.6 (p = 0.002), 4.9 (p = 0.02), and 1.4 (p = 0.02), respectively. In addition, docosanol reduced the expression of proteins involved in purine metabolic pathways, biofilm growth cycle, and virulence factor production. Altogether, these findings suggest that docosanol could efficiently target the antioxidant pathway by reducing the expression of bacillithiol and stress-associated proteins. [ABSTRACT FROM AUTHOR]

We aimed to identify proteins that were differentially regulated in spermatozoal samples collected from fertile healthy men (FHM) and infertile patients with varicocele (IFPV) before and after varicocelectomy. Seminal samples were collected from 20 IFPV before and after varicocelectomy and from 14 FHM as controls. Samples underwent seminal examination and proteomic analysis. Extracted spermatozoal proteins were analysed using two‐dimensional gel electrophoresis, and differentially regulated spermatozoal proteins (DRSPs) were identified. In particular, attention was placed on those DRSPs in which the concentration changed after varicocelectomy and corrected to approximate levels observed in FHM. Varicocelectomy significantly improved the sperm count and concentration in IFPV (p < 0.05). Proteomic analysis showed that 11 DRSPs were identified when comparisons were made among the three groups. Among these 11 proteins, change in the SERPIN A5 concentrations was notable because it was 100‐fold downregulated in pre‐operative IFPV samples and nearly resembled to control concentrations following varicocelectomy. Western blot analysis using an anti‐SERPIN antibody validated the changes observed in SERPIN A5 levels before and after varicocelectomy operation. Increase in SERPIN A5 after varicocelectomy may be due to improvement in semen quality, suggesting that SERPIN A5 is a potential seminal biomarker for assessment of semen quality in varicocele‐related infertility. [ABSTRACT FROM AUTHOR]

Objectives Proteomics principles and techniques were used to detect and screen out the expression of differential proteins in urine of athletes after exercise with different intensities; the changes of urinary protein components and the correlations among the changes, human immune function and exercise fatigue were investigated, which provided scientific basis and practical methods for exercise biochemical monitoring. Methods Based on Two-dimensional electrophoresis (2-DE), differential expression of urine proteome of 8 male athletes was analyzed after exercises of 55%, 75%, 85% and 95% VO2max intensity, respectively; the protein expressed levels which were up-regulated by at least 5-fold and with repeatability were screened and analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Results A total of 275 differentially expressed protein points was detected after different exercise intensities, down-regulated protein points were 85 and up-regulated 190. After screening expressed levels which were up-regulated by at least 5-fold protein, a total of 29 protein was identified, showing a close relation to the biological processes of immune regulation and inflammatory response. Conclusions Proteomics better explains what happens to the urine proteome after different exercises, among which, apolipoprotein and zinc-a2-glycoprotein are related to lipid metabolism after exercise; immunoglobulin, albumin, complement component C3, mannose-binding lectin protein 19 and Vitamin D binding protein are related to immune regulation. The theoretical basis and application method are provided for the evaluation of the effects of exercise training on human immune function and fatigue. [ABSTRACT FROM AUTHOR]

Brassinosteroids (BRs) represent a unique class of steroidal plant hormones that display pronounced growth‐promoting activity at very low concentrations. Although many efforts have been made to characterize the molecular basis of BR action, little is known about the mechanisms behind the growth‐promoting effect of BRs at protein level.Proteomic analysis of response to the steroid plant hormone 24‐epibrassinolide (EBR) in wheat seedling shoots (Triticum aestivum L.) was performed using two‐dimensional electrophoresis (2‐DE) and immunoblotting with highly specific antibodies (PY20) to phosphotyrosine. EBR‐modulated proteins and phosphotyrosine polypeptides were identified using MALDI‐TOF mass spectrometry.The study revealed that EBR‐stimulated growth of wheat seedlings was accompanied by changes in the content of multiple proteins as well as in tyrosine phosphorylation of numerous polypeptides. Among them, 22 differentially accumulated proteins and 13 phosphotyrosine proteins were identified. Based on their performed functions, the identified proteins are involved in physiological processes (photosynthesis, growth, energy and amino acid metabolism) closely associated with intensification of plant metabolism.The EBR‐induced changes in protein abundance and tyrosine phosphorylation profile may contribute to growth stimulation of wheat seedlings under the action of EBR. The obtained data suggest an important role for EBR in the activation of protein metabolism underlying fundamental physiological processes, including growth promotion. [ABSTRACT FROM AUTHOR]

The main objectives of the study were to: (1) deeply analyse the serum protein composition of Equus caballus, (2) assess the effectiveness of the high-abundant protein depletion and improve the concentration of medium- and low-abundant proteins. The analysis were performed on the blood plasma of three healthy part-Arabian mares. The implementation of two-dimensional electrophoresis and matrix-assisted laser desorption/ionisation – time of flight mass spectrometry allowed us to establish a horse plasma proteome map. Serum proteins were resolved at pH 4 to 7, followed by 12% SDS-PAGE. As a result 136 spots were successfully identified, representing the products of 46 unique genes. Of these, 22 gene products have not been previously identified in horse serum/plasma samples using proteomic tools. Gene ontology analysis showed that almost 30% of all identified gene products belong to the coagulation and complement cascades. These results can undoubtedly serve as a useful and prospective prerequisite for the future analysis of horse plasma proteome changes in different physiological and pathophysiological conditions. The use of the medium- and low-abundant protein enrichment tool increased their abundance and allowed us to identify a higher number of protein gene products. The highest depletion efficiency was observed for the most abundant plasma proteins, that is albumin, IgG heavy chains and serotransferrin.

The aim of this study was to analyze the effects of the cryopreservation process on the protein profile of ram sperm using two-dimensional electrophoresis (2-DE) coupled with mass spectroscopy. Semen was collected from five rams and cryopreserved in a Tris-based extender supplemented with glycerol and egg yolk as the main cryoprotectants. The fresh and post-thaw sperm total proteins were extracted and purified, followed by the 2-DE. The differential proteins in the stained gel were determined by mass spectrometry. The results indicated that there were 39 differential proteins between fresh sperm and frozen-thawed sperm. Among these proteins, the abundance of 28 proteins in fresh sperm was higher than those in post-thaw sperm (P < 0.05). However, 11 proteins in post-thaw sperm were up-regulated instead. The gene ontology (GO) analysis showed that most of differential proteins were implicated in cellular process, metabolism and regulation of the biological process. The networks of protein-protein interaction indicated a strong interaction among these differential proteins, which may be involved in sperm metabolism, acrosomal function, sperm motility, and reducing ROS level. In conclusion, the cryopreservation process modifies the proteome of ram sperm, which may be directly associated with ram sperm cryodamage, consequently influencing their fertility. Additionally, these differential proteins can be used as biomarkers for evaluation of frozen ram semen quality. • Cryopreservation alters the ram sperm proteome. • Comparison of the spermatozoa proteomes between fresh and frozen in rams. • ATP5B and PRDX5 may be used as biomarkers to evaluate the quality of ram semen [ABSTRACT FROM AUTHOR]

Objectives: The larval stage of Echinococcus granulosus in the internal organs of a human causes cystic hydatidosis. Thus, the identification of excretory/secretory (E/S) proteins from E. granulosus protoscoleces may help to discover new candidates for drug targets, along with immunodiagnostic and vaccine products. The purpose of this study was to assess the efficacy of albendazole and mebendazole on protein spots of E/S products of hydatid cyst protoscolices which can be helpful for detecting some target proteins for therapeutic purposes. Materials and Methods: In this experimental study, protoscoleces were divided into three groups in order to assess the effects of the drug. The first and the second groups were treated with albendazole/mebendazole, respectively, and the third group was considered as control. To determine the proteome spots, E/S proteins were precipitated with trichloroacetic acid/acetone and loaded on the isoelectric focusing gel, resulting in gel differentiation on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the 20 mA constant current. In addition, the gels were stained with Coomassie Brilliant Blue. Finally, the protein spots resulted from 2-DE gels were analyzed using the Progenesis SameSpots software. Results: The comparison between the proteome gels of the treated groups and the control group showed that 35 protein spots are paired among which, 11 spots had significant differences in their expressions (P < 0.05). Conclusions: In general, the comparison between the expression levels of protein gel spots indicated the increasing expression of some protein spots while the suppression of the others. This suppression can be considered as a specific effect of the drugs on the E/S product of the protoscoleces of hydatid cyst. [ABSTRACT FROM AUTHOR]

Background: Malignant pleural mesothelioma (MPM) a rare neoplasm linked to asbestos exposure is characterized by a poor prognosis. Soluble mesothelin is currently considered the most specific diagnostic biomarker. The aim of the study was to identify novel biomarkers by proteomic analysis of two MPM cell lines secretome. Materials and Methods: The protein patterns of MPM cells secretome were examined and compared to a non-malignant mesothelial cell line using two-dimensional gel electrophoresis coupled to mass spectrometry. Serum levels of candidate biomarkers were determined in MPM patients and control subjects. Results: Two up-regulated proteins involved in cancer biology, prosaposin and quiescin Q6 sulfhydryl oxidase 1, were considered candidate biomarkers. Serum levels of both proteins were significantly higher in MPM patients than control subjects. Combining the data of each receiveroperating characteristic analysis predicted a good diagnostic accuracy. Conclusion: A panel of the putative biomarkers represents a promising tool for MPM diagnosis. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) is a ubiquitous bimolecule that regulates various physiological processes in plants. Despite the widespread study of NO in plants, the involvement of NO in proteomic regulation has been poorly investigated compared to the physiological processes. To explore the NO-induced proteomic regulation in rice leaves, 2-week-old leaves were exposed to sodium nitroprusside for 12 h. Protein profiles were analyzed by two-dimensional gel electrophoresis. A total of 53 NO-regulated proteins were identified by the matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis. Out of these proteins, 41 were up-regulated, 7 were down-regulated, and 5 were newly induced. The identified proteins were involved in photosynthesis, carbohydrate and energy metabolism, growth and development, metabolite biosynthesis, signal transduction, lignin modification, and defense response. Importantly, several key proteins including glutathione reductase, monodehydroascorbate reductase, dehydroascorbate reductase, ascorbate peroxidase, superoxide dismutase, catalase, 2-cysteine peroxiredoxin, peroxidase (newly induced), and glutathione S-transferase were significantly up-regulated in rice leaves and these proteins were associated with the ascorbate–glutathione cycle and ROS homeostasis system in plants. Thus, the identification of several key and newly induced proteins has provided a new insight into NO-mediated overall molecular response in plants. [ABSTRACT FROM AUTHOR]

The main objective of the study was to create a reproducible protein map of the liver in healthy growing piglets. The analysis was performed on liver homogenates obtained from 8 castrated male piglets (PIC x Penerlan P76) at the 50 days. Two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry allowed to determine the proteomic profile of the liver. Liver proteins were separated at pH 4 - 7, followed by 12% SDS-PAGE. As a result, 470 ± 44 spots were present on the 2-D maps, of which 265 were successfully identified, representing products of 142 unique genes. Of these, 26 gene products have not been previously observed on the protein maps of porcine liver. Gene ontology analysis showed that the most of identified gene products belonged to the known metabolic pathways: protein processing in endoplasmic reticulum, arginine and proline metabolism, microbial metabolism in diverse environments, carbon metabolism, Epstein-Barr virus infection, propionate metabolism, biosynthesis of amino acids, proteasome. These results can undoubtedly serve as a useful and prospective prerequisite for the future analysis of the liver proteome changes in different physiological and pathophysiological conditions. [ABSTRACT FROM AUTHOR]

Abstract The main intricacy in the human proteome is that it is tremendously complex and composed from diverse and heterogeneous gene products. These products are called protein species or proteoforms and are the smallest units of the proteome. In pursuit of the comprehensive profiling of the human proteome, significant advances in the technology of so called "Top-Down" mass spectrometry based proteomics, have been made. However, the scale of performance of this approach is still far behind the "Bottom-Up", peptide-centric techniques. The classical two-dimensional electrophoresis (2-DE) as the most powerful and convenient method for separation of proteoforms remains as a superior method in "Top-Down" proteomics. Here, some aspects of approaches for establishing an inventory of proteoforms based on 2-DE and mass spectrometry are discussed. Biological significance The systematic efforts in the Human Proteome project to map the entire human proteome greatly depend on currently available and emerging techniques and approaches. Here, the possibilities of a visual representation of the human proteome by combination of virtual/experimental 2-DE with protein identification by mass spectrometry or immunologically is discussed. By application of this approach on several profiles of gene products we show its convenience in informative representation of the whole proteome and single gene products, proteoforms (protein species). This approach could be very helpful in the emerging global inventory of all human proteoforms. Graphical abstract Unlabelled Image Highlights • There is need of development of a knowledge base for the inventory of human proteoforms • Some approaches for obtaining and establishing an inventory of information about proteoforms are discussed. • It is shown that 2-DE methodology together with mass spectrometry can be a good complement for proteoforms atlas [ABSTRACT FROM AUTHOR]

Abstract: Using the sectional analysis of two-dimensional electrophoretic gels with liquid chromatography-mass spectrometry, proteoform profiles for individual genes expressed in cancer (glioblastoma) and normal (FLEH) cells were obtained. Profiles of more than 5000 genes were analyzed. It turned out that many genes encoding potential biomarkers of glioblastoma are characterized by sets of proteoforms that are different in normal and cancer cells. These proteoforms could be sources of highly specific markers and targets for therapy. Using a section analysis of two-dimensional electrophoretic gels with liquid chromatography by mass spectrometry, proteoform profiles were obtained for individual genes expressed in cancer (glioblastoma) and normal (FLEH) cells. Profiles of more than 5000 genes were analyzed. It turned out that many genes encoding potential biomarkers of glioblastoma are characterized by sets of proteoforms, which are different in normal and cancer cells. These proteoforms could be sources of highly specific markers and targets for therapy. [ABSTRACT FROM AUTHOR]

Abstract: Here, we developed an integrated course based on two dimensional‐electrophoresis and spectrometry mass (2DE‐MS) technique for undergraduate students to help them learn proteomic techniques. The soluble proteins in wild type and gene knockout bacteria were separated by 2DE and the differently expressed proteins were identified by MS analysis. The proteomic data was finally confirmed by RT‐PCR detection. The separated experiments of 2DE, MS, RNA isolation, RT‐PCR, as well as essential bioinformatic analysis, were integrated into a one‐week course, which provided students an opportunity to systematically understand the proteomic techniques and their applications in current scientific research. © 2018 by The International Union of Biochemistry and Molecular Biology, 46:354–360, 2018. [ABSTRACT FROM AUTHOR]

Currently, a wide array of plant preparations exerting health-promoting properties are commonly used as feed additives. Among them, Cichorium intybus L. have gained considerable attention as a source of compounds showing prebiotic character. Large body of evidence suggests that products of prebiotic fermentation (short-chain fatty acids) may influence the expression of genes encoding liver enzymes involved in the regulation of energetic metabolism. Given the above, the present study was aimed at estimating the influence of a diet supplemented with chicory root or water extract of chicory inulin on liver proteome in growing pigs. The study was performed on 24 castrated male piglets ( PIC × Penarlan P76). Animals were assigned to three equal groups ( n = 8) and fed cereal-based isoenergetic diets: control and supplemented with 2% of inulin extract from chicory root or 4% of dried chicory root. Liver proteins were separated using two-dimensional electrophoresis, followed by the identification of statistically valid protein spots with the aid of MALDI- TOF mass spectrometry. Both experimental factors significantly modulated the expression of liver proteins associated with energetic metabolism, particularly those involved in cholesterol and triglyceride metabolism. Additionally, both dietary additives induced increased expression of proteins involved in hepatocyte protection against oxidative stress. In the present study, we have shown for the first time that diet supplementation with dried chicory root or inulin caused significant changes in the expression of liver cytoskeletal proteins. Close attention should be paid to the downregulation of cytokeratin 18, hepatic acute phase protein that can enhance the anti-inflammatory properties of inulin-type fructans. [ABSTRACT FROM AUTHOR]

Although cell phones have been used worldwide, some adverse and toxic effects were reported for this communication technology apparatus. To analyze in vivo effects of exposure to radiofrequency-electromagnetic field (RF-EMF) on protein expression in rat testicular proteome, 20 Sprague-Dawley rats were exposed to 900 MHz RF-EMF for 0, 1, 2, or 4 h/day for 30 consecutive days. Protein content of rat testes was separated by high-resolution two-dimensional electrophoresis using immobilized pH gradient (pI 4-7, 7 cm) and 12% acrylamide and identified by MALDI-TOF/TOF-MS. Two protein spots were found differentially overexpressed ( P < 0.05) in intensity and volume with induction factors 1.7 times greater after RF-EMF exposure. After 4 h of daily exposure for 30 consecutive days, ATP synthase beta subunit (ASBS) and hypoxia up-regulated protein 1 precursor (HYOU1) were found to be significantly up-regulated. These proteins affect signaling pathways in rat testes and spermatogenesis and play a critical role in protein folding and secretion in the endoplasmic reticulum. Our results indicate that exposure to RF-EMF produces increases in testicular proteins in adults that are related to carcinogenic risk and reproductive damage. In light of the widespread practice of men carrying phones in their pockets near their gonads, where exposures can exceed as-tested guidelines, further study of these effects should be a high priority. [ABSTRACT FROM AUTHOR]