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Some health disorders, such as obesity and type 2 diabetes, are associated with a poor diet and low quality of the fat in it. The type and duration of the diet have an impact on the liver. This investigation uses the proteomic approach to identify changes in the mouse liver protein profile in adaptation to high-fat diets with different saturated fatty acid contents and linoleic acid (18:2n-6) to α-linolenic acid (18:3n-3) fatty acid ratios.

The aim of the present study was to determine the proteins in canine seminal plasma with direct effect on specific sperm parameters during fertilisation. The influence of seminal plasma proteins was fo-cused on three processes: capacitation, hyperactivation and decapacitation. Size exclusion chroma-tographic fractional separation of proteins from seminal plasma based on their molecular weights was performed. In addition, two-dimensional electrophoresis of two seminal protein fractions was done. The effect of protein molecules on the motion and kinetic parameters of the male gametes in in vitro conditions was monitored by computer-assisted sperm analysis. The activity of alkaline phosphatase, creatine kinase and alanine aminotransferase in whole seminal plasma and each chromatography fraction was measured spectrophotometrically using the BA-88A semi-automatic chemistry analyzer. The protein fractions with different molecular weights affected the in vitro motility and kinetic characteristics (curvilinear velocity, straightness, linearity and amplitude of lateral head displacement) of spermatozoa, leading to hyperactivation or initiating decapacitation kinetics changes. The detailed study of the seminal plasma proteome would add fundamental information about the processes associated with physiological changes occurring in spermatozoa before fertilisation. The study of the canine seminal plasma proteome could add relevant information about its effects on the fertilising ability of the male gametes and the changes occurring in them before fertilisation.

To explore the effect of a chitosan-based preservative solution (10 mg/mL chitosan + 5 mg/mL tea polyphenol + 2 000 U/mL lysozyme) on the change in the muscle proteome of grass carp during cold storage, differential proteomic analysis of fresh fish and nine-day-stored fish with or without (control) preservative treatment was conducted by two-dimensional electrophoresis-mass spectrometry (2DE-MS). The results showed that the 2DE map of the preservative-treated group was not obviously different from that of the fresh group, except for a decrease in the abundance of fast troponin T and an increase in the abundance of slow troponin T and adenylate kinase isoenzyme. However, the 2DE profile of the control group differed greatly from that of the fresh group in both the number and signal intensity of protein spots. Further analysis showed that the abundance of fast troponin T, tropomodulin 4, synaptopodin 2 and pyruvate kinase type M were down-regulated, while the abundance of myosin heavy chain, myosin light chain 1, α-actin, α-tropomyosin and intermediate light meromyosin were up-regulated. Moreover, compared with the preservative-treated group, the abundance of three troponins T were decreased, while the abundance of myosin heavy chain, intermediate light meromyosin, α-actin, α-tropomyosin and creatine kinase M-type were increased in the control group, suggesting that creatine kinase could serve as an indicator for evaluating grass carp muscle spoilage. This study demonstrates that the chitosan-based composite preservative can maintain the stability of grass carp muscle protein during cold storage.

In the gastropod mollusk Lymnaea stagnalis, insulin-like peptides in the central nervous system (CNS) control behavioral changes associated with associative learning. Insulin administration to the Lymnaea CNS enhances the synaptic plasticity involved in this type of learning, but it has remained unclear which molecules in the insulin response cascade are involved. Here, to advance a comprehensive analysis, we used two-dimensional electrophoresis and comparative quantitative mass spectrometry to perform a protein analysis investigating the CNS molecules that respond to insulin administration. Our results revealed increased phosphorylation of AKT and RICTOR in the PI3K/AKT/mTOR signaling cascade and cytoskeleton-related proteins. Although it was expected that the molecules in the PI3K/AKT/mTOR signaling cascade were phosphorylated by insulin administration, our findings confirmed the correlation between insulin-induced phosphorylation of cytoskeleton-related proteins strongly involved in the synaptic changes and learning and memory mechanisms. These results contribute to elucidate the relationship between the insulin response and learning and memory mechanisms not only in Lymnaea but also in various invertebrates and vertebrates.

Abstract Background Tear proteomic analysis has become an important tool in medical and veterinary research. The tear collection method could influence the tear protein profile. This study aims to evaluate the protein profiles of dog tears collected using microcapillary tubes (MT), Schirmer tear strips (ST), and ophthalmic sponges (OS). Methods The tear samples were collected using MT, ST, and OS. Tear protein profiles were analyzed using two-dimensional electrophoresis (2-DE) and the different protein spots’ expression was compared. Fourteen protein spots were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results Tear protein concentrations ranged from 2.80 to 4.03 μg/μL, with no statistically significant differences among collection methods. Protein expression in each collection method differed in terms of both the number and intensity of the spots. There were 249, 327, and 330 protein spots found from tears collected with MT, ST, and OS, respectively. The proteins albumin, haptoglobin, and lactoferrin identified from OS were found to have higher spot intensities than other methods of collection. The use of MT demonstrated the downregulation of nine proteins. Conclusions The recent study supported that tear protein analysis is affected by different tear collection methods. Although ST is commonly used for tear collection, it provides insufficient information to study particular tear proteins.

In this study, we aimed to characterize the liver protein profile of Chaohu ducks using two-dimensional electrophoresis and proteomics. The livers were quickly collected from 120 healthy, 84-day-old Chaohu ducks. The intramuscular fat (IMF) content of the left pectoralis muscle was determined using the Soxhlet extraction method. The total protein of liver tissues from the high and low IMF groups was extracted for proteomics. Functional enrichment analysis of the differentially expressed proteins (DEPs) was conducted using gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG). In total, 43 DEPs were identified. Functional enrichment analysis indicated that these DEPs were significantly related to four lipid metabolic processes: carboxylic acid metabolic process, ATP metabolic process, oxoacid metabolic process, and organic acid metabolic process. Three pathways correlated with lipid metabolism were identified using KEGG analysis: glycolysis/gluconeogenesis, pentose phosphate pathway, fructose, and mannose metabolism. Eight key proteins associated with lipid metabolism were identified: ALDOB, GAPDH, ENO1, RGN, TPI1, HSPA9, PRDX1, and GPX1. Protein–protein interaction analysis revealed that the glycolysis/gluconeogenesis pathway mediated the interaction relationship. Key proteins and metabolic pathways were closely related to lipid metabolism and showed a strong interaction in Chaohu ducks.

Adipose-derived mesenchymal stem cells (ASCs) have the potential to differentiate into bone, cartilage, fat, and neural cells and promote tissue regeneration and healing. It is known that they can have variable responses to hypoxic conditions. In the present study, we aimed to explore diverse changes in the cells and secretome of ASCs under a hypoxic environment over time and to present the possibility of ASCs as therapeutic agents from a different perspective. The expression differences of proteins between normoxic and hypoxic conditions (6, 12, or 24 h) were specifically investigated in human ASCs using 2-DE combined with MALDI-TOF MS analysis, and secreted proteins in ASC-derived conditioned media (ASC-derived CM) were examined by an adipokine array. In addition, genetic and/or proteomic interactions were assessed using a DAVID and miRNet functional annotation bioinformatics analysis. We found that 64 and 5 proteins were differentially expressed in hypoxic ASCs and in hypoxic ASC-derived CM, respectively. Moreover, 7 proteins among the 64 markedly changed spots in hypoxic ASCs were associated with bone-related diseases. We found that two proteins, cathepsin D (CTSD) and cathepsin L (CTSL), identified through an adipokine array independently exhibited significant efficacy in promoting osteocyte differentiation in bone-marrow-derived mesenchymal stem cells (BM-MSCs). This finding introduces a promising avenue for utilizing hypoxia-preconditioned ASC-derived CM as a potential therapeutic approach for bone-related diseases.

The production of high-quality pork is closely related to the growth and development of muscle tissue. The present article provides a comparative proteomic research of l. dorsi, b. femoris, m. brachiocephalicus during the pigs’ growth and development (at age of 60 days and 180 days). This work was supported by data of electrophoretic methods: one-dimensional electrophoresis according to Laemmli with densitometric assessment in the ImageJ software and two-dimensional electrophoresis according to O’Farrell method with its further processing on the software ImageMaster. The mass spectrometric identification was conducted with the help of the high-performance liquid chromatography (HPLC) system connected to a mass spectrometer; further the data were interpreted by search algorithm Andromeda. When comparing frequency diagrams of one-dimensional electrophoregrams of all three muscle tissues of weaned pigs, the greatest difference was observed for the muscle sample l. dorsi. Comparison of diagrams of muscle tissue samples taken for mature pigs showed a great similarity of all three studied muscles samples. Within the framework of the research, the Fold indicator was calculated. The exceeding its value by more than 2 units is generally considered to be a statistically significant difference. When analyzing two-dimensional electrophoretograms of weaned pigs’ muscles, 18 protein fractions were revealed with Fold > 2. When examining the muscle tissue of mature pigs, 15 of those proteins were found; the differences were mostly detected in the minor protein fractions. The mass spectrometric analysis of the cut bands with well-pronounced differences from the onedimensional electrophoretogram revealed 214 proteins involved to a greater extent in cellular and metabolic processes, physical activity and localization. Growth and development protein — semaphorin‑6B (96.78 kDa) — was revealed in muscle tissue of l. dorsi, a. Also in l. dorsi and b. femoris the growth and development proteins were found: cadherin‑13 (78.23 kDa), cadherin‑7 (87.01 kDa), the F‑actin-cap protein beta subunit (30.66 kDa), and two uncharacterized proteins at 65.60 kDa and 63.88 kDa.

Protein adsorption is essential for determining material biocompatibility and promoting adherent cell growth. In this study, we focused on the a-plane structure of hydroxyapatite (HAp). This a-plane structure closely resembles the crystal plane where apatite is exposed in long bones. We conducted protein adsorption experiments using HAp ceramics with a preferred orientation to a-planes (aHAp), employing bovine serum albumin (BSA), lysozyme, and fetal bovine serum (FBS) as protein models to mimic the in vivo environment. Higher zeta potential and contact angle values were found in aHAp than in HAp ceramics fabricated from commercial HAp powder (iHAp). Bradford-quantified protein adsorption revealed BSA adsorption of 212 ng·mm−2 in aHAp and 28.4 ng mm−2 in iHAp. Furthermore, the Bradford-quantified protein adsorption values for FBS were 2.07 μg mm−2 in aHAp and 1.28 µg mm−2 in iHAp. Two-dimensional electrophoresis (2D-PAGE) showed a higher number of protein-derived major spots in aHAp (37 spots) than in iHAp (12 spots). Mass spectrometry analysis of the resulting 2D-PAGE gels revealed proteins adsorbed on aHAp, including secreted frizzled-related protein 3 and vitamin K epoxide reductase complex 1, which are involved in cellular bone differentiation. Overall, these proteins are expected to promote bone differentiation, representing a characteristic property of aHAp.

Aim. To isolate and purify protein complexes – ATP synthase and RuBisCO – from pea leaf chloroplasts and study the effect of a microbiological fertilizer “Extracon” and sulfonamide inhibitors acetazolamide and ethoxyzolamide on the enzymatic activity of these proteins. Materials and methods. Chloroplasts were isolated from the leaves of two-week-old pea sprouts, protein complexes of purified thylakoid membranes were solubilized with digitonin (10 mg of digitonin per 1 mg of protein), the protein concentration was determined according to Lowry. Native electrophoresis with displacement of the charge of the soluble protein fraction from the chloroplast stroma, as well as membrane proteins, was carried out in the modified system of Anderson et al., Kolisnichenko et al. A modified Lemmley system was applied to the protein electrophoresis in the polyacrylamide gel in the presence of sodium dodecyl sulfate. The methods of Alain and Hintsik, as well as Gomorrah were used to determine the ATPase activity in the polyacrylamide gel. Visualization of the carbonic anhydrase activity in the polyacrylamide gel was performed by the method of Edwards and Petton. Results and discussion. Using physicochemical methods of potentiometry, spectrophotometry the ATPase, carbonic anhydrase and esterase activities of the enzymes were studied. The results obtained indicate that specific carbonic anhydrase inhibitors (acetazolamide and ethoxyzolamide) also block the esterase and ATPase activity of the enzyme complexes. “Extracon” (a multifunctional microbiological preparation) almost 1.5 times increases the activity of the enzymes, showing a complex activating effect of the fertilizer on both light and dark reactions of photosynthesis. Conclusions. The method of identification and isolation of RuBisCO and ATP synthase on the basis of two-dimensional electrophoresis and electrophoretic elution has been proposed. It allows determining the presence of certain enzyme activity of complexes at first in SDS plates (express analysis) and further to study the effect of various factors of endogenous and exogenous origin on the enzymatic properties of electrophoretically pure enzymes. The use of two-dimensional electrophoresis as a tool for assessing the impact of various factors of endogenous and exogenous origin on the plant cell and the plant as a whole through constant monitoring of the work and activity of enzyme systems of the plant cell is promising.

Abstract Background Both the highly pathogenic avian influenza (HPAI) H5N1 and low pathogenic avian influenza (LPAI) H9N2 viruses have been reported to cross species barriers to infect humans. H5N1 viruses can cause severe damage and are associated with a high mortality rate, but H9N2 viruses do not cause such outcomes. Our purpose was to use proteomics technology to study the differential expression of mitochondrial-related proteins related to H5N1 and H9N2 virus infections. Methods According to the determined viral infection titer, A549 cells were infected with 1 multiplicity of infection virus, and the mitochondria were extracted after 24 h of incubation. The protein from lysed mitochondria was analyzed by the BCA method to determine the protein concentration, as well as SDS-PAGE (preliminary analysis), two-dimensional gel electrophoresis, and mass spectrometry. Differential protein spots were selected, and Western blotting was performed to verify the proteomics results. The identified proteins were subjected to GO analysis for subcellular localization, KEGG analysis for functional classification and signaling pathways assessment, and STRING analysis for functional protein association network construction. Results In the 2-D gel electrophoresis analysis, 227 protein spots were detected in the H5N1-infected group, and 169 protein spots were detected in the H9N2-infected group. Protein spots were further subjected to mass spectrometry identification and removal of redundancy, and 32 differentially expressed proteins were identified. Compared with the H9N2 group, the H5N1-infected group had 16 upregulated mitochondrial proteins and 16 downregulated proteins. The differential expression of 70-kDa heat shock protein analogs, short-chain enoyl-CoA hydratase, malate dehydrogenase, and ATP synthase was verified by Western blot, and the results were consistent with the proteomics findings. Functional analysis indicated that these differentially expressed proteins were primarily involved in apoptosis and metabolism. Conclusions Compared with their expression in the H9N2 group, the differential expression of eight mitochondrial proteins in the H5N1 group led to host T cell activation, antigen presentation, stress response, ATP synthesis and cell apoptosis reduction, leading to higher pathogenicity of H5N1 than H9N2.

The proteostasis system of animals, including various types of protein modification during the growth stage, leads to an almost incomprehensible number of possible forms of protein, and each can regulate numerous functions. In the presented work, the composition of muscle tissue protein from different portions of piglets was studied to understand the main muscle protein formation. Comparative analysis of weaned piglets' main muscle protein from l. dorsi, biceps femoris, and brachiocephalicus were analyzed using two-dimensional electrophoresis. Changes in the staining intensity of protein fractions inherent in different muscles were revealed. As part of this work, candidate groups of pig muscle proteins have been selected. Eleven protein spots were revealed for the longest muscle of the back, and seven for the biceps; the muscles of the neck are characterized by indicators of low protein fraction volume. Among the proteins found, myosin light chains, phosphoglycerate mutase, troponins, and adenylate kinase is most likely present. The obtained results of protein identification in muscle tissues, obtained during the intensive growth period, will allow a more detailed understanding of protein regulation, function, and interactions in complex biological systems, which will subsequently be significantly important for biomonitoring health and predicting farm animals productivity.

It is important to understand the molecular mechanisms that take place in muscle tissues and to predict meat quality characteristics. One of the most popular methods is two-dimensional electrophoresis, which allows us to visualize, share and identify different molecules, including meat proteins. However, the standard conditions of this method are not universal for all types of raw material, so the authors suggest a new variation of two-dimensional electrophoresis for muscle tissue analysis. Samples were tested by the classical version of isoelectric focusing (cathode buffer in the top and anode buffer in the bottom chamber of the electrophoresis cell) and its variation (anode buffer in the top and cathode buffer in the bottom chamber of the electrophoresis cell). Next, extruded gels were incubated in two different buffer systems: the first was equilibration buffer I (6 M urea, 20% w/v glycerol, 2% w/v SDS and 1% w/v Ditiothreitol in 375 mM Tris-HCl buffer, pH 8.8) followed by equilibration buffer II (6 M urea, 20% w/v glycerol, 2% w/v SDS and 4% w/v iodoacetamide in 375 mM Tris-HCl buffer pH 8.8 and the second, buffer А, consisting of 5 M urea, 2% w/v SDS, 5% v/v mercaptoethanol, 62.5 mM Tris-HCl buffer, pH 6.8 and 0.01% w/v bromophenol blue. Electrophoretic studies of muscle tissue revealed the best protein separation after changing the direction of the current (authors' variation), while no differences were detected after changing incubation buffers.

In this study, two bread wheat (Triticum aestivum L.) cultivars, Pishgam (drought-tolerant) and Shahryar (drought-sensitive), were grown in the greenhouse under control and moisture stress conditions. Based on phenological and morpho-physiological results, Pishgam was confirmed as a moisture stress tolerant cultivar. In the fallowing, at the start of heading time, its treated and untreated flag leaves were sampled for two-dimensional electrophoresis (2-DE) based on proteomics approach. Among approximately 263 protein spots appearing in two-dimensional gels, 23 and 10 protein spots were up- and down-regulated, respectively. Among these differentially expressed proteins, 11 proteins with more differences were identified by MALDI TOF/TOF MS which allocated to six functional protein groups involved in photosynthesis or respiration, carbohydrate metabolism, energy metabolism, chaperon, lipid metabolism and unknown function. We report this for the first time that chloroplastic acyl carrier protein synthase I and chloroplastic 20 kDa chaperonin proteins were significantly changed in wheat in response to moisture stress.

Background: Our aim was to study changes in the serum proteomic profile in coronary atherosclerosis. Methods: The study involved two groups of patients: 1) men with coronary heart disease and coronary atherosclerosis (n = 15); 2) control (n = 15): men without coronary heart disease. The object of this study was blood serum. Separation of proteins for the investigation of differences in serum protein components was performed by two-dimensional electrophoresis. Identification of protein fractions was carried out using peptide mass maps by the matrix-assisted laser desorption ionization method. Results: In blood serum samples from patients with coronary atherosclerosis, protein separation in two-dimensional gels with mass-spectrometric identification revealed an increase of some proteins: hemopexin, transthyretin (monomeric form), retinol-binding protein 4, and components of the complement system: C3 (chain B) and C9. There was a decrease of some proteins: kininogen, zinc finger protein 133, and B-cell CLL/lymphoma 6 member B protein. Comparisons between the experimental and control group were carried out in protein fractions where the protein amount differed more than 1.5-fold (p < 0.05). Conclusions: Proteome profiling of serum revealed a change in the content of kininogen, hemopexin, transthyretin, retinol-binding protein, and proteins of the complement system (C9, and C3) in coronary atherosclerosis. The contribution to the differential expression of a protein was often made by isoforms of the protein, particularly transthyretin. The change in the concentrations of functionally interacting proteins, such as transthyretin and retinol-binding protein, were noted.

The importance of examining environmental stresses and their role in predicting and evaluating the growth and yield of crops is very evident. Environmental stresses are the most important factor in reducing agricultural yields worldwide. In order to evaluate the environmental impact of water loss on the amount of proteins affected in sunflower, an experiment was conducted in randomized complete block design with three replications at Karaj Oil Seeds Research Institute. In order to study the response of susceptible line (BGK221) and tolerant line (RGK46) under drought stress conditions, extraction of protein by acetone deposition method was performed in 8 leaves and of 3 seedlings in each replication. The amount of extracted protein was determined by Bradford method using two-dimensional electrophoresis and existence of significant difference between the bands in sensitive and tolerant lines was investigated. A total of 467 repeatable bands were found in the tolerant line and 417 repeatable bands appeared in the sensitive line. Among these proteins, 6 bands in tolerant line (No 503, 1901, 904, 3301, 7011, 9005) and 6 bands in sensitive line (No 704, 811, 3205, 4108, 7307, and 9207) were significantly affected by drought stress. In both sensitive and tolerate lines the main consequence is increase in amount of protein. The results showed that the most important factor of tolerant line adaptive for environmental stress conditions is maintaining normal cell metabolism, keeping moisture in the cell, strengthening cellular structure and antioxidant defense. The study also showed that drought stress had the greatest effect on cytoplasmic and nucleus proteins, metabolism and energy of proteins

Abstract Background Two-dimensional electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) are widely used in plant proteomics research. However, these two techniques cannot be simultaneously satisfied by traditional protein extraction methods when investigate cotton leaf proteome. Results Here, we evaluated the efficiency of three different protein extraction methods for 2-DE and LC-MS/MS analyses of total proteins obtained from cotton leaves. The protein yield of the borax/PVPP/phenol (BPP) method (0.14%) was significantly lower than the yields of the trichloroacetic acid/acetone (TCA) precipitation method (1.42%) and optimized TCA combined with BPP (TCA-B) method (0.47%). The BPP method was failed to get a clear 2-DE electrophoretogram. Fifty pairs of protein spots were randomly selected from the 2-DE gels of TCA- and TCA-B-extracted proteins for identification by MALDI TOF/TOF, and the results of 42 pairs were consistent. High-throughput proteomic analysis showed that 6339, 9282 and 9697 unique proteins were identified from the total cotton leaf proteins extracted by the TCA, BPP and TCA-B methods, respectively. Gene Ontology (GO) analysis revealed that the proteins specifically identified by TCA method were primarily distributed in the plasma membrane, while BPP and TCA-B methods specific proteins distributed in the cytosol, indicating the sub-cellular preference of different protein extraction methods. Further, ATP-dependent zinc metalloprotease FTSH 8 could be observed in the 2-DE gels of TCA and TCA-B methods, and could only be detected in the LC-MS/MS results of the BPP and TCA-B methods, showing that TCA-B method might be the optimized choice for both 2-DE and LC-MS/MS. Conclusion Our data provided an improved TCA-B method for protein extraction that is compatible with 2-DE and LC-MS/MS for cotton leaves and similar plant tissues which is rich in polysaccharides and polyphenols.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a major pathogen causing severe economic loss. However, the molecular mechanism of silkworm resistance to BmNPV and the interactions of this virus with the host during infection remain largely unclear. To explore the virus-binding proteins of silkworms, the midgut subcellular component proteins that may interact with BmNPV were analyzed in vitro based on one- and two-dimensional electrophoresis and far-western blotting combined with mass spectrometry (MS). A total of 24 proteins were determined to be specifically bound to budded viruses (BVs) in two subcellular fractions (mitochondria and microsomes). These proteins were involved in viral transportation, energy metabolism, apoptosis and viral propagation, and they responded to BmNPV infection with different expression profiles in different resistant strains. In particular, almost all the identified proteins were downregulated in the A35 strain following BmNPV infection. Interestingly, there were no virus-binding proteins identified in the cytosolic fraction of the silkworm midgut. Two candidate proteins, RACK1 and VDAC2, interacted with BVs, as determined with far-western blotting and reverse far-western blotting. We speculated that the proteins interacting with the virus could either enhance or inhibit the infection of the virus. The data provide comprehensive useful information for further research on the interaction of the host with BmNPV.

Eight protein substances that undergo changes during autolysis were found using the electrophoretic methods with the following mass spectrometric identification. The revealed protein substances have different origin: structural proteins (fragments of troponins T and myosin light chains), and metabolic proteins (creatine kinase, pyruvate kinase and alpha-enolase). The decomposition of the fractions of fast skeletal muscle troponin T in 28.0 kDa, 27 kDa and 26.5 kDa was most pronounced.Identification of constitutive proteins and detection of the products of post — mortem degradation of protein molecules make them suitable candidates for meat quality markers and the following study of these specific fragments will lead to better understanding of the proteolytic activities that take part in the post mortem muscle transformation into meat.

One of the ways to address the food quality issues facing the industry is the development of standardized and certified methods related to the conduct of in-depth studies of biochemical indicators of quality and safety of meat and meat products. The world laboratory practice in the field of food quality and safety shows a constant expansion of the list of controlled indicators of food raw materials and products. An important feature of the modern period in the development of biomedical and biotechnological research is the introduction of a whole complex of postgenomic technologies, which are based on a systematic approach to the study of the functioning of the mammalian proteome in various physiological and pathological conditions, including the formation and development of alimentary-dependent pathologies. In this regard, the problem of multilateral study of food products, in particular their identification, is the most relevant, because the modern technology of their production has undergone significant changes and requires the development of “gentle “ processing modes. They concern raw materials and auxiliary materials used at all stages of production. This and new technologies of production of protein products from plant raw materials, as well as the introduction of food raw materials and food additives of artificial origin and the excess introduction of additives of plant and animal origin can cause falsification of products, as well as affect the health of the consumer. Food quality assessment includes the control of components of finished products. It is most difficult to determine the proportion of muscle protein in multi-component meat products that have undergone heat treatment. Therefore, in practice, when assessing the quality of food products, there is a need to identify its real composition in accordance with the declared normative documents. Currently, a promising area of research in the field of determining the composition of finished food is the selection of biomarkers of various components. Therefore, it is important to develop a methodology for the identification of biochemical changes in food raw materials under the influence of technological factors using modern research methods. This paper provides an overview of the protein and peptide analysis methodology, including the latest technologies that are becoming increasingly important.

Abstract The aim of study was to isolate, characterize and analyse the protein profiles of Myoviridae-like bacteriophages obtained from M. haemolytica using MALDI TOF mass spectrometry. The material consisted of the M. haemolytica reference strain ATCC® BAA410, reference serotypes A1, A2, A5, A6, A7, A9, and A11, and wild-type isolates of serotype A1. Bacteriophage morphology was examined with a transmission electron microscope. The proteins were separated in SDS-PAGE and two-dimensional electrophoresis and characterized by MALDI-TOF. Among the phages obtained, seven were specific for strains A1, A2, A5, A6, A7 and 25, and PHL-1 was specific for the BAA410 strain. The protein profiles for the phages were very similar to one another, but differed from the reference phage in that they lacked protein fractions with molecular weights of 22.9, 56.3 and 73.1 kDa. 2D electrophoresis revealed significant differences in the size of proteins and their localization in the pH gradient. The most similar profiles were observed in phages specific for strains BAA-410 and A6. In all profiles two main spots were observed in the molecular weight range from 44 to 70 kDa at pH

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.

At present, different methods are used to accumulate functional peptides in meat raw materials, including the use of spontaneous microflora during autolysis, the use of the microbial enzymes (the application of starter cultures) and the use of the non-microbial enzymes (enzymes of animals and plant origin). Each method has its own specific characteristics of an impact on raw materials, which requires their detail study. This paper examines an effect of spontaneous microflora of fermented meat products from horsemeat on formation of biologically active peptides. Using the T-RFLP analysis, it was established that in air dried and uncooked smoked sausages produced with the use of the muscle tissue of horsemeat as a raw material, a significant proportion of microflora was presented by lactic acid microorganisms. The highest content of lactic acid microflora was observed in sample 1 (52.45 %), and the least in sample 3 (29.62 %). Sample 2 had the medium percent content of microflora compared to samples 1 and 3 — 38.82 %. It is necessary to note that about 25 % of microflora was unculturable; i.e., it had metabolic processes but did not grow on culture media. In the samples, the representatives of Actinobacteria and Pseudomonadales were found. Pathogenic and conditionally pathogenic microflora was not detected. Not only quantitative but also qualitative changes were observed in the studied samples. For example, in samples 1 and 2, the fractions of amilo-1,6-glucosidase, fast-type muscle myosin-binding-protein C; glucose-6-phosphate isomerase; fast skeletal muscle troponin I, phosphoglycerate kinase, pyruvate kinase and skeletal muscle actin were found, which were absent or reduced in sample 3. Therefore, in the studied product, good preservation of the main spectra of muscle proteins was observed, and the identified fractions, apparently, can be sources of new functional peptides. Not only quantitative but also qualitative changes were observed in the studied samples. For example, in samples 1 and 2, the C-terminal fragments of the myosin heavy chain were found, which were absent in sample 3. Also, the significant content of myoglobin was revealed in samples 2 and 3, and the myosin light chain was found in sample 1. Therefore, in the studied product, good preservation of muscle proteins myosin and myoglobin, which can be a source of new functional peptides, was observed. Based on the results of tandem mass-spectrometry, the proteins and natural short peptides present in the analyzed extracts were identified by the obtained masses. They belonged mainly to different peptides of equine myoglobin. Also, we identified several fragments, among which fast skeletal muscle troponin T and muscle creatine kinase were found. The obtained materials can be regarded as an experimental basis for the directed impact of starter cultures with a possibility to predict the protein and peptide composition of a finished product including with the aim of obtaining biologically active peptides.

The results of studying the effect of various temperatures on the protein composition of minced meat from porcine m. longissimus dorsi by two-dimensional electrophoresis are presented. The most complete distribution of protein fractions was observed in fresh raw minced meat, and when it was exposed to negative temperature, there was a sharp decrease in protein components (carbonic anhydrase 3, αβ-crystallin), as well as a decrease in the staining intensity of protein spots of the main constitutive fractions (tropomyosin alpha 1, myosin light chain 1). In the case of heat treatment, structural muscle proteins were retained with some changes in high molecular weight fractions, namely, protein molecules degraded to compounds with a simpler structure. It was noted that fractions of tropomyosin β-chain, triosephosphate isomerase 1, myosin light chains 2 were not detected after minced meat was frozen, while tropomyosin alpha 1 was retained in all samples.

Liver is responsible for 90% of lipid synthesis in poultry; thus, it plays an important role in the growth of Muscovy ducks, which have a high fat deposition ability in a time-dependent manner. Therefore, male Muscovy ducks at 14, 28, 42, and 56 days were selected for body weight (BW), carcass weight (CW), subcutaneous fat thickness (SFT), abdominal fat weight (AFW), intramuscular fat content (IMF), and breast muscle fiber (BMF) diameter and density determination. Two-dimensional electrophoresis (2-DE) combining liquid chromatography linked to tandem mass spectrometry (LC-MS/MS) was used to analyze proteomic changes in liver at each stage. The BW, CW, AFW, SFT, and BMF diameter and density were significantly increased, while IMF content was significantly decreased at 28 to 42 days of age (p < 0.05). There were 57 differentially abundant protein (DEP) spots representing 40 proteins identified among the ages, in which 17, 41 and 4 spots were differentially abundant at 14 vs. 28, 28 vs. 42, and 42 vs. 56, respectively. Gene Ontology enrichment analysis found that DEPs were mostly enriched in the oxidation-reduction process, carboxylic acid metabolism, etc. Protein–protein interaction showed that catalase (CAT), triosephosphate isomerase (TPI), and protein disulfide-isomerase (PDI) were the key proteins responsible for the growth of Muscovy duck. In conclusion, 28 to 42 days of age is the crucial period for Muscovy ducks, and the ability of metabolism and antioxidants were significantly enhanced in liver.

Abstract Background A wide range of molecules are used by tapeworm metacestodes to establish successful infection in the hostile environment of the host. Reports indicating the proteins in the cestode-host interactions are limited predominantly to taeniids, with no previous data available for non-taeniid species. A non-taeniid, Hymenolepis diminuta, represents one of the most important model species in cestode biology and exhibits an exceptional developmental plasticity in its life-cycle, which involves two phylogenetically distant hosts, arthropod and vertebrate. Results We identified H. diminuta cysticercoid proteins that were recognized by sera of H. diminuta-infected rats using two-dimensional gel electrophoresis (2DE), 2D-immunoblotting, and LC-MS/MS mass spectrometry. Proteomic analysis of 42 antigenic spots revealed 70 proteins. The largest number belonged to structural proteins and to the heat-shock protein (HSP) family. These results show a number of the antigenic proteins of the cysticercoid stage, which were present already in the insect host prior to contact with the mammal host. These are the first parasite antigens that the mammal host encounters after the infection, therefore they may represent some of the molecules important in host-parasite interactions at the early stage of infection. Conclusions These results could help in understanding how H. diminuta and other cestodes adapt to their diverse and complex parasitic life-cycles and show universal molecules used among diverse groups of cestodes to escape the host response to infection.

Abstract Background Alfalfa (Medicago sativa) is the most extensively cultivated forage legume in the world, and salinity stress is the most problematic environmental factors limiting alfalfa production. To evaluate alfalfa tissue variations in response to salt stress, comparative physiological and proteomic analyses were made of salt responses in the roots and shoots of the alfalfa. Method A two-dimensional gel electrophoresis (2-DE)-based proteomic technique was employed to identify the differentially abundant proteins (DAPs) from salt-treated alfalfa roots and shoots of the salt tolerance cultivars Zhongmu No 1 cultivar, which was subjected to a range of salt stress concentrations for 9 days. In parallel, REL, MAD and H2O2 contents, and the activities of antioxidant enzymes of shoots and roots were determinand. Result Twenty-seven spots in the shoots and 36 spots in the roots that exhibited showed significant abundance variations were identified by MALDI-TOF-TOF MS. These DAPs are mainly involved in the biological processes of photosynthesis, stress and defense, carbohydrate and energy metabolism, second metabolism, protein metabolism, transcriptional regulation, cell wall and cytoskeleton metabolism, ion transpor, signal transduction. In parallel, physiological data were correlated well with our proteomic results. It is worth emphasizing that some novel salt-responsive proteins were identified, such as CP12, pathogenesis-related protein 2, harvest-induced protein, isoliquiritigenin 2′-O-methyltransferase. qRT-PCR was used to study the gene expression levels of the four above-mentioned proteins; four patterns are consistent with those of induced protein. Conclusion The primary mechanisms underlying the ability of alfalfa seedlings to tolerate salt stress were photosynthesis, detoxifying and antioxidant, secondary metabolism, and ion transport. And it also suggests that the different tissues responded to salt-stress in different ways.

In the study of proteomic profiles of proteins, many scientists stop at the stage of obtaining the final data of the experiment in the form of gels. They have got no information on the possibilities and prospects concerning the application of modern computer and bioinformatics resources that allow to convert the result from qualitative to quantitative form. The use of computer technology allowed to save the recorded images and carry out the calculations with chromatograms using digital video images.Densitometry with the use of video technology is characterized by high calculation speed and low cost of consumables. Digitally archived chromatograms may be used at any time for a number of applications including calculation.Thus, the “manual” bioinformatics analysis allows not only to use different densitometer software for conversion and storage of gels in digital form, but also to quantitatively interpret the results obtained.This paper presents the methods for practical application of bioinformatics tools in the interpretation of protein profiles obtained by one-dimensional and two-dimensional electrophoresis and converted into digital image. The aspects of the quantitative interpretation of electrophoretograms from one-dimensional electrophoresis (1DE) and two-dimensional electrophoresis (2DE) resulting from the studies of muscle tissue of farm animals are reviewed. Examples of various calculation software usage are given. The work in this direction will allow to considerably expand approaches for identification and quantification of protein markers related to quality, functionality and safety of food raw materials and finished products and to carry out metrological examination of the results for confirmation of product compliance.

The use of resistant cultivars is one of the safest and most profitable methods of controlling post including alfalfa weevil. After farm and greenhouse evaluating, Tafresh 42 genotype for molecular studies selected and evaluated to examine the pattern of protein expression in stress and non-stress conditions to alfalfa weevil. The Changes in protein expression were investigated using two-dimensional electrophoresis gels. Clustering analysis, principal component analysis and discriminant analysis were used in statistic studies. The results showed that 11 protein spots had expression changes among 218 protein spots. The most expression pattern of proteins with a similar increase in Tafresh 42, was related to a specific group of proteins which had 45.46% variations in the expression response to the stress. From the point of protein expression, cluster analysis caused a dividing of proteins into three main clusters indicating the presence of proteins with a similar performance in each cluster. Therefore, the presence of these proteins in a common biologic pathway is confirmed. The analysis of discriminant function confirmed 100% the results from clustering that indicated protein conformity with the conditions of the experiment. It is concluded that by using statistical analysis and software, it is possible to evaluate significant expression changes induced damage alfalfa weevil in proteome level and determine the index changes, and after that genetic engineering is utilized for enhancing the level of resistance in the plant under studying.

This work is devoted to pig spleen proteome study. Spleens were taken from Duroc pigs (females, 145 – 160 days old) and typical two-dimensional electrophoregrams were obtained. On proteomic maps after visualization and image analysis there were detected 600 fractions, including organ-specific proteins – 3 62 fractions. Among the identified constitutive fractions, the highest expression was observed (Vol spots more than 3.0E + 07) four protein spots S1, S9, S12 and S21, which are supposedly Annexin A1 (MW 38.76 kDa), Ectonucleoside triphosphate diphosphohydrolase 1 (MW 57.75 kDa) Pro-cathepsin H CD59 (MW 37.45 kDa) and glycoprotein (MW 13.79 kDa), respectively. Obtained electrophoregrams analysis using information resources made it possible to identify different active compounds in spleen with various functions, mainly immunoregulatory – glycoprotein CD59 (Mm 13.79 kDa) and ATP-dependent RNA helicase (Mm 107.58 kDa); the intensely expressed LIM-domain of the actin-binding protein (Mm 83.99 kDa). The results obtained are a prospect for immunomodulating biologic development based on animal raw materials for farm animals.

This study investigated the changes in Weissella koreensis (WK) protein expression levels during fermentation in MRS medium supplemented with garlic of WK. WK was first discovered as lactic acid bacteria (LAB) in a Korean fermented cabbage dish known as kimchi. The number of WK cells in MRS medium with garlic (MBCG) and without (MB) after 7 days was 3.55 × 1010 and 2.55 × 1010 CFU/mL, respectively. To observe the changes in the carbon sources in the media, we measured the glucose, sucrose, lactic acid, and acetic acid levels in each medium (MB and MBCG). Thus, 67.2 ± 2.4 (MB) and 64.2 ± 4.7 (MBCG) mmol−1 of glucose were consumed. For sucrose, the level was 3.5 ± 2.2 (MB), and 3.4 ± 2.5 (MBCG) mmol−1. There was not much difference in the lactic acid and acetic acid levels at 160.8 ± 0.4 (MB) and 159.2 ± 0.2 (MBCG) and 2.4 ± 0.4 (MB) and 2.2 ± 8.1 (MBCG) mmol−1, respectively. After the 7-day fermentation period, two-dimensional electrophoresis (2DE) was used to confirm the protein expression pattern in the WK strain. The results show that the fusA and ssb1 proteins were reduced, and the clpP protein was increased. Afterwards, the expression patterns of the above proteins were confirmed through qRT-PCR. Thus, this study confirms the changes in protein expression levels in Weissella koreensis when garlic was added to the media. This study provides basic data for future studies on the major biosynthetic pathways of WK.

The fungal metabolite sporidesmin is responsible for severe necrotizing inflammation of biliary tract and liver of livestock grazing on pasture containing spores of Pithomyces chartarum that synthesizes the toxin. The toxin is secreted into bile causing the erosion of the biliary epithelium accompanied by inflammation and damage to surrounding tissues. Toxicity has been suggested to be due to cycles of reduction and oxidation of sporidesmin leading to oxidative damage from the formation of reactive oxygen species. The current work is the first test of the oxidative stress hypothesis using cultured cells. Oxidative stress could not be detected in HepG2 cells incubated with sporidesmin using a dichlorodihydrofluorescein diacetate assay or by use of two-dimensional electrophoresis to search for oxidized peroxiredoxins. There was also no evidence for necrosis or apoptosis, although there was a loss of cell adhesion that was accompanied by the disruption of intracellular actin microfilaments that have known roles in cell adhesion. The results are consistent with a model in which altered contact between cells in situ leads to altered permeability and subsequent inflammation and necrosis, potentially from the leakage of toxic bile into surrounding tissues. There is now a need for the further characterization of the damage processes in vivo, including the investigation of altered permeability and mechanisms of cell death in the biliary tract and other affected organs.

Background/Aims: We previously showed that a hypoxic environment modulates the antiarrhythmic potential of mesenchymal stem cells. Methods: To investigate the mechanism by which secreted proteins contribute to the pathogenesis of antiarrhythmic potential in mesenchymal stem cells, we used two-dimensional electrophoresis combined with MALDI-TOF-MS to perform a proteomic analysis to compare the paracrine media produced by normoxic and hypoxic cells. Results: The proteomic analysis revealed that 66 protein spots out of a total of 231 matched spots indicated differential expression between the normoxic and hypoxic conditioned media of mesenchymal stem cells. Interestingly, two tropomyosin isoforms were dramatically increased in the hypoxic conditioned medium of mesenchymal stem cells. An increase in tropomyosin was confirmed using Western blot to analyze the conditioned media between normoxic and hypoxic cells. In a network analysis based on gene ontology (GO) Molecular Function by GeneMANIA analysis, most of the identified proteins were found to be involved in the regulation of heart processes. Conclusion: Our results show that hypoxia up-regulates tropomyosin and other secreted proteins which suggests that tropomyosin may be involved in regulating proarrhythmic and antiarrhythmic functions.

Background/Aims: It is known that mesenchymal stem cells (MSCs) can have variable responses to hypoxic conditions and that hypoxia may specifically stimulate differentiation into osteogenic, chondrogenic, or adipogenic cells. Based on our previous study, we hypothesized that hypoxia may also induce MSC differentiation into cardiomyocytes and/or cells with comparable phenotypes. Methods: The differences in the proteomes were specifically investigated in bone marrow-derived rat MSCs (BM-rMSCs) under normoxic and hypoxic conditions using 2-DE combined with a MALDI-TOF-MS analysis and western blot analysis. In addition, genetic and/or proteomic interactions were assessed using a String network analysis. Results: Among the 35 markedly changed spots from a total of 393 matched spots, 24 were highly up-regulated and 11 were significantly down-regulated in hypoxic rMSCs based on a proteomic analysis. Although hypoxia failed to induce the direct differentiation of rMSCs into cardiomyocytes, several cardiomyocyte differentiation-related genes and proteins were significantly increased by hypoxic stress. Conclusion: We found that BM-rMSCs alter their expression of several cardiomyocyte differentiation-related genes and proteins under hypoxic conditions, and we examined the interactions between these genes and/or proteins, providing new insights for the applicability of MSCs preconditioned by hypoxic stimulation for use in cardiac diseases.

In the present work crude Agaricus bisporus extract (ABE) has been prepared and characterized by its tyrosinase activity, protein composition and substrate specificity. The presence of mushroom tyrosinase (PPO3) in ABE has been confirmed using two-dimensional electrophoresis, followed by MALDI TOF/TOF MS-based analysis. GH27 alpha-glucosidases, GH47 alpha-mannosidases, GH20 hexosaminidases, and alkaline phosphatases have been also detected in ABE. ABE substrate specificity has been studied using 19 phenolic compounds: polyphenols (catechol, gallic, caffeic, chlorogenic, and ferulic acids, quercetin, rutin, dihydroquercetin, l-dihydroxyphenylalanine, resorcinol, propyl gallate) and monophenols (l-tyrosine, phenol, p-nitrophenol, o-nitrophenol, guaiacol, o-cresol, m-cresol, p-cresol). The comparison of ABE substrate specificity and affinity to the corresponding parameters of purified A. bisporus tyrosinase has revealed no major differences. The conditions for spectrophotometric determination have been chosen and the analytical procedures for determination of 1.4 × 10−4–1.0 × 10−3 M l-tyrosine, 3.1 × 10−6–1.0 × 10−4 M phenol, 5.4 × 10−5–1.0 × 10−3 M catechol, 8.5 × 10−5–1.0 × 10−3 M caffeic acid, 1.5 × 10−4–7.5 × 10−4 M chlorogenic acid, 6.8 × 10−5–1.0 × 10−3 M l-DOPA have been proposed. The procedures have been applied for the determination of l-tyrosine in food supplements, l-DOPA in synthetic serum, and phenol in waste water from the food manufacturing plant. Thus, we have demonstrated the possibility of using ABE as a substitute for tyrosinase in such analytical applications, as food supplements, medical and environmental analysis.

Blue light is an important environmental factor that induces mushroom growth and morphological changes. In this study, after confirming the morphological difference between Lentinula edodes (LE) under blue light condition (BL) and lightless condition (LL), the increase and decrease in LE protein and the expression of RNA of each protein were confirmed under each condition. LE specimens grown in BL and LL were identified by 253 spots in BL through 2D electrophoresis and LC-MSMS analysis, and 22 types of proteins were identified. It was confirmed that 14 types of proteins showed reduced expression in BL compared to LL. On the other hand, eight kinds of proteins with increased expression in blue light compared to LL were identified. As a result of confirming the difference from the expression pattern in 2D electrophoresis through Quantitative Real-Time PCR, it was confirmed that the expression pattern of the two proteins showed a difference. Therefore, this study will be a key study on the changes in mushroom morphology induced by blue light and the proteins that induce it.

Urine is a biological diagnostic material suitable not only for the analysis of kidney and urinary tract functions but also the function of other tissues and organs. The urine proteome of adult mammals differs from the urine proteome of neonatal ones. The establishment of urinary protein maps of healthy newborn calves is important for diagnosing and monitoring the progression of various diseases. The experiment was carried out on a Polish-Friesian var. of Black-and-White male calves in the sixth day of postnatal life. The two proteomics approaches used for separation and identification of urinary proteins were: 2-DE with MALDI-TOF-TOF-MS/MS and 1-DE with LC-MS/MS. This resulted in the identification of 692 urinary proteins. The majority of them were classified as extracellular proteins (40.32%), as well as proteins involved in regulation of major cellular processes (31.07%). We have observed the presence of unique proteins associated with embryonic (ameloblastin, alpha-fetoprotein, Delta-like protein, embryo-specific fibronectin 1 transcript variant, Indian hedgehog homolog) and kidney development (angiotensin-converting enzyme, angiotensinogen, aquaporin-1, calbindin, glypican 3, nidogen 1, pro-cathepsin H). Additionally, proteins involved in the renal regulation of water and electrolyte balance (angiotensinogen, angiotensin-converting enzyme, aquaporin-1, ezrin, uromodulin) were detected. Presented in the current study 1-D and 2-D urinary proteomic maps are the basis for the identification and detection of prognostic biomarkers important for defining a calf’s health status.

To investigate proteome pattern of wheat cultivars, young leaves were collected from tillering stage of seedlings two weeks after development of the salinity stress. The extraction of proteins from leaf tissue was done and two dimensional electrophoresis using IPG strips and SDS-PAGE in the control and salinity treatments were performed. In total, 198 and 203 protein spots were identified in tolerant (‘Moghan3’) and susceptible (‘Pishtaz’) cultivars respectively. Also, among these, spots number 21 and 22 were detected with significant IF in ‘Moghan3’ and ‘Pishtaz’ respectively. Two-stage mass spectrometry (MS/MS) was used to identify protein spots. Common identified proteins, including proteins involved in removal of oxidants, Calvin cycle proteins, proteins involved in light reaction of photosynthesis and proton transfer, and heat shock protein were identified on basis of the functional groups and their frequency. In total, ‘Moghan3’ maintained the stability of the structure and performance of carbon metabolism under stress better than susceptible cultivar. In addition, defense against oxidative stress induced by salinity stress was performed by 2-cys peroxiredoxin BAS1 and Cu-Zn SOD proteins that tolerant cultivar defended against oxidative stress better than the susceptible cultivar. The greatest strength of ‘Moghan3’ and major weakness in ‘Pishtaz’ are relying on the unique proteins formed under salinity stress for the removal of oxidants and to maintain the activity of the photosynthetic light reactions, respectively.

Proteome changes in the longissimus thoracis bovine muscle in response to pre-slaughter stress were assessed on the basis of two-dimensional electrophoresis (2-DE) data. In this study, the bootstrap resampling statistical technique and a new measure of relative change of the volume of 2-DE protein spots are shown to be more efficient than commonly used statistics to reliably quantify changes in protein abundance in stress response. The data are supplied in this article and are related to “Tackling proteome changes in the longissimus thoracis bovine muscle in response to pre-slaughter stress” by Franco et al. [1].

The chitosan-induced coacervation of milk proteins was investigated using a proteomic approach. The addition of 0.8% chitosan to milk caused the milk proteins to coacervate after a 1 h incubation period. Approximately 86% of the milk proteins were present in the milk pellet fraction (MPF), and the protein concentration of the milk supernatant fraction (MSF) decreased from 29.4 ± 0.2 to 4.2 ± 0.6 mg/mL. SDS-PAGE analysis showed that the total intensities of serum albumin (BSA), αS-casein (αS-CN), β-casein (β-CN), κ-casein (κ-CN) and β-lactoglobulin (β-LG) in the MSF decreased to 8.5% ± 0.2%, 0.9% ± 0.3%, 0.7% ± 0.3%, 0.5% ± 0.2% and 15.0% ± 0.5%, respectively. Two-dimensional electrophoresis analysis indicated that αS1-, αS2-, β- and κ-CN and a fraction of the β-LG and BSA were found in the MSF following incubation with 0.8% chitosan. Isothermal titration calorimetry analysis indicated that binding of chitosan to milk proteins is an exothermic reaction based on binding titration curves of milk proteins dispersions with chitosan, and the enthalpy of binding (ΔH) and binding constant (Ka) were −7.85 × 104 cal/mol and 1.06 × 105/mol, respectively. These results suggested that the addition of 0.8% chitosan causes milk proteins to coacervate due to polysaccharide-protein interactions.

Ovarian cancer (OC) is one of the most common malignant tumors of the reproductive system. The five-year survival rate is extremely low. This disease is difficult to verify due to lack of pathognomonic symptoms and timely diagnosis. The key issue to increase survival in ovarian cancer is finding new methods for early diagnosis. Thus, the development of new methods for diagnosis of ovarian cancer is one of the most urgent problems in modern oncology. The basic approach in this matter is searching for new biomarkers of ovarian cancer, which will be characterized by such concepts as sensitivity and specificity. Biomarkers present in modern oncology, including CA-125, have insufficient specificity and have low sensitivity. The advantage of using a combination of several diagnostic biomarkers instead of one or panels of markers has been proved. Modern proteomic technologies such as two-dimensional electrophoresis, mass spectrometric methods are a valuable tool for finding new biomarkers for various malignancies, particularly ovarian cancer. The best results are achieved by using SELDI-TOF technology (Surface-enhanced laser desorption/ionization time-of-flight), combining the use of chromatographic protein chips with mass spectrometric detection. Serum amyloid A1 deserves the greatest attention between all of the biomarkers identified using mass spectrometric methods, as its ability to have a direct influence on the development of the tumor has been proved. Also its increase by hundreds of times during the disease has been found compared to the other candidate biomarkers identified by mass spectrometry. The main characteristics of serum amyloid 1A have been analyzed and found as one of the most promising markers for the combined determination for prompt diagnosis of ovarian cancer.

Parkinson's disease (PD), similar with other neurodegenerative disorders, would benefit from the identification of early biomarkers for differential diagnosis and prognosis to address prompt clinical treatments. Together with hypothesis driven approaches, PD has been investigated by high-throughput differential proteomic analysis of cerebrospinal fluid (CSF) protein content. The principal methodologies and techniques utilized in the proteomics field for PD biomarker discovery from CSF are presented in this mini review. The positive aspects and challenges in proteome-based biomarker research are also discussed.

Abstract Background Aedes albopictus is widely distributed across tropical and sub-tropical regions and is associated with the transmission of several arboviruses. Although this species is increasingly relevant to public health due its ability to successfully colonize both urban and rural habitats, favoring the dispersion of viral infections, little is known about its biochemical traits, with all assumptions made based on studies of A. aegypti. In previous studies we characterized the peptidase profile of pre-imaginal stages of A. albopictus and we reported the first proteomic analysis of the midgut from sugar-fed females of this insect species. Methods In the present work, we further analyzed the peptidase expression in the midgut of sugar-fed females using 1DE-substrate gel zymography, two-dimensional electrophoresis (2DE), mass spectrometry (MS), and protein identification based on similarity. Results The combination of zymography, in solution assays using fluorescent substrates and 2DE-MS/MS allowed us to identify the active serine peptidase “fingerprint” in the midgut of A. albopictus females. Zymographic analysis revealed a proteolytic profile composed of at least 13 bands ranging from ~25 to 250 kDa, which were identified as trypsin-like serine peptidases by using specific inhibitors of this class of enzymes. Concomitant use of the fluorogenic substrate Z-Phe-Arg-AMC and trypsin-like serine protease inhibitors corroborated the zymographic findings. Our proteomic approach allowed the identification of two different trypsin-like serine peptidases and one chymotrypsin in protein spots of the alkaline region in 2DE map of the A. albopictus female midgut. Identification of these protein coding genes was achieved by similarity to the A. aegypti genome sequences using Mascot and OMSSA search engines. Conclusion These results allowed us to detect, identify and characterize the expression of active trypsin-like serine peptidases in the midgut of sugar-fed A. albopictus females. In addition, proteomic analysis allowed us to confidently assign the expression of two trypsin genes and one chymotrypsin gene to the midgut of this mosquito. These results contribute to the gene annotation in this species of unknown genome and represent a small but important step toward the protein-level functional and localization assignment of trypsin-like serine peptidase genes in the Aedes genus.

AIM: To find the significant altered proteins in age-related macular degeneration (AMD) patients as potential biomarkers of AMD.METHODS: A comparative analysis of the protein pattern of AMD patients versus healthy controls was performed by means of proteomic analysis using two-dimensional gel electrophoresis followed by protein identification with MALDI TOF/TOF mass spectrometry.RESULTS: We identified 28 proteins that were significantly altered with clinical relevance in AMD patients. These proteins were involved in a wide range of biological functions including immune responses, growth cytokines, cell fate determination, wound healing, metabolism, and anti-oxidance.CONCLUSION: These results demonstrate the capacity of proteomic analysis of AMD patient plasma. In addition to the utility of this approach for biomarker discovery, identification of alterations in endogenous proteins in the plasma of AMD patient could improve our understanding of the disease pathogenesis.

Proteins from leaves of Rubus chamaemorus propagated in vitro were subjected to miniaturized 2-D electrophoresis. The 2-DE patterns of proteins showed qualitative differences between plants propagated in vitro and control seedlings. More proteins of a high molecular weight were observed in leaves of plants from in vitro culture. A two-dimensional map of proteins from leaves provides detailed data concerning both polymorphism and protein patterns of this species. This makes it possible to start constructing a protein map of R. chamaemorus. The reasons for qualitative differences are discussed.

Metal-containing drugs have long been used in anticancer therapies. The mechansims of action of platinum-based drugs are now well-understood, which cannot be said of drugs containing other metals, such as gold or copper. To gain further insights into such mechanisms, we used a classical proteomic approach based on two-dimensional elelctrophoresis to investigate the mechanisms of action of a hydroxyquinoline-copper complex, which shows promising anticancer activities, using the leukemic cell line RAW264.7 as the biological target. Pathway analysis of the modulated proteins highlighted changes in the ubiquitin/proteasome pathway, the mitochondrion, the cell adhesion-cytoskeleton pathway, and carbon metabolism or oxido-reduction. In line with these prteomic-derived hypotheses, targeted validation experiments showed that the hydroxyquinoline-copper complex induces a massive reduction in free glutathione and a strong alteration in the actin cytoskeleton, suggesting a multi-target action of the hydroxyquinoline-copper complex on cancer cells.

Clinical mastitis is still an intractable problem for sheep breeding. The natural immunologic mechanisms of the mammary gland against infections are not yet understood. For a better understanding of the disease-associated proteins during clinical mastitis in meat sheep, we performed two-dimensional electrophoresis (2-DE)-based comparative proteomic analyses of mammary tissues, including from healthy mammary tissues (HMTs) and from mammary tissues with clinical mastitis (CMMTs). The 2-DE results showed that a total of 10 up-regulated and 16 down-regulated proteins were identified in CMMTs when compared to HMTs. Of these, Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment analyses revealed that most proteins were associated with immune responses or metabolisms. The results of qRT-PCR and Western blot for randomly selected four differentially expressed proteins (DEPs) including superoxide dismutase [Mn] (SOD2), annexin A2 (ANAX2), keratin 10 (KRT10) and endoplasmic reticulum resident protein 29 (ERP29) showed that their expression trends were consistent with 2-DE results except ANXA2 mRNA levels. This is an initial report describing the 2-DE-based proteomics study of the meat sheep mammary gland with clinical mastitis caused by natural infection, which provides additional insight into the immune and metabolic mechanisms during sheep mastitis.

Seeds are the most important plant storage organ and play a central role in the life cycle of plants. Since little is known about the protein composition of rice (Oryza sativa) seeds, in this work we used proteomic methods to obtain a reference map of rice seed proteins and identify important molecules. Overall, 480 reproducible protein spots were detected by two-dimensional electrophoresis on pH 4-7 gels and 302 proteins were identified by MALDI-TOF MS and database searches. Together, these proteins represented 252 gene products and were classified into 12 functional categories, most of which were involved in metabolic pathways. Database searches combined with hydropathy plots and gene ontology analysis showed that most rice seed proteins were hydrophilic and were related to binding, catalytic, cellular or metabolic processes. These results expand our knowledge of the rice proteome and improve our understanding of the cellular biology of rice seeds.

In this study, differentially expressed proteins in A549 cells (human lung adenocarcinoma epithelial cell line) infected with H9N2 avian influenza virus (AIV) were investigated by two-dimensional electrophoresis (2-DE). Sixteen different spots between the groups (ratio>2, p

In vitro mimicking conditions are thought to reflect the environment experienced by M. tuberculosis inside the host granuloma. The majority of the in vitro dormancy experimental models used laboratory adapted strain H37Rv or Erdman strain over the prevalent clinical strains involved during disease outbreaks. Thus, we included the most prevalent clinical strains (S7 and S10) of M. tuberculosis from south India in addition to H37Rv for our in vitro oxygen depletion (hypoxia) experimental model. Cytosolic proteins were prepared from the hypoxic cultures, resolved by two-dimensional electrophoresis (2-DE) and protein spots were characterized by mass spectrometry. Totally 49 spots were characterized as over-expressed or newly appeared between the 3 strains. Two antigens (ESAT-6, Lpd) out of the 49 characterized spots were readily available in recombinant form in our lab. Hence, these 2 genes were overexpressed, purified and used for in vitro stimulation of whole blood collected from healthy household contacts (HHC) and active pulmonary tuberculosis patients (PTB). Multicolour flow cytometry analysis showed high levels of antigen specific CD4+ central memory T cells in circulation of HHC when compared to PTB (p