Two-dimensional Electrophoresis Method Research Articles

A novel 2D-electrophoresis method for the simultaneous visualization of phosphorylated and O-GlcNAcylated proteoforms of a protein.

Post-translational modifications (PTMs), such as phosphorylation and O-N-acetyl-β-d-glucosaminylation (O-GlcNAcylation), are involved in the fine spatiotemporal regulation of protein functions, and their dynamic interplay is at the heart of protein language. The coexistence of phosphorylation and O-GlcNAcylation on a protein leads to the diversification of proteoforms. It is therefore essential to decipher the phosphorylation/O-GlcNAcylation interplay on protein species that orchestrates cellular processes in a specific physiological or pathophysiological context. However, simultaneous visualization of phosphorylation and O-GlcNAcylation patterns on a protein of interest remains a challenge. To map the proteoforms of a protein, we have developed an easy-to-use two-dimensional electrophoresis method with a single sample processing permitting simultaneous visualization of the phosphorylated and the O-GlcNAcylated forms of the protein of interest. This method, we termed 2D-WGA-Phos-tag-PAGE relies on proteoforms retardation by affinity gel electrophoresis. With this novel approach, we established the cartography of phospho- and glycoforms of αB-crystallin and desmin in the whole extract and the cytoskeleton protein subfraction in skeletal muscle cells. Interestingly, we have shown that the pattern of phosphorylation and O-GlcNAcylation depends of the subcellular subfraction. Moreover, we have also shown that proteotoxic stress condition increased the complexity of the pattern of PTMs on αB-crystallin.

The study of proteotomic profile of mutton with consideration to the influence of feed combinatorics

The present article is devoted to the search for markers that attest the changes in the protein profile when fattening the lambs with enriched diets, taking into account tissue specificity. The purpose of scientific research was to expand scientific knowledge about the influence of essential trace elements of organic origin in the diet of lambs on the marker-proteins of skeletal muscle tissue, of myocardium and renal cortical substance. The objects of research were the tissues of the m.L.dorsi skeletal muscle, myocardium and cortical substance of the kidneys in lambs of the Edilbaev breed. These lambs received a diet with organic trace elements (iodine, selenium, silicon). To study the effect of microelements in the diets of lambs on change in marker-proteins, we ran the studies of the skeletal muscle, myocardium and cortical substance of the kidneys of lambs, using the method of two-dimensional electrophoresis. When searching for proteins which mark the action of organic additives in the fattening diet of lambs within the tissue of the myocardium and the cortical substance of the kidneys, a number of differences were noted; no pronounced effects were observed in the skeletal muscle. Enrichment of the diet of lambs with selenium derivatives led to a change in the protein composition in the kidneys cortical substance for some isoforms of selenium-containing proteins and related enzymes of the glutathione cycle. Reaction in this process of thermal shock proteins, utilization of aflatoxins and changes in proteins of energy metabolism within mitochondria of kidney cells was also revealed. The determination of functional activity, or immunofermental analysis of glutathione peroxidase 3 count in the blood, can be a promising biomarker for controlling the level of selenium digestion and the count of selenium in blood as a more rapid and cheap method in comparison with the proteomic technologies. And for assessing the rate of digestion and fixation of iodine, it seems promising to determine it via the activity of mitochondrial superoxide dismutase [Mn]. In the myocardium, the effect of digested feed additives mainly affected the mitochondrial apparatus, which changed its functional orientation, with the activation of several other metabolic cycles. These results are prerequisites for obtaining more fundamental data on the effect of diets enriched with essential micronutrients on the molecular mechanisms that take place within the myocardial and kidney tissue of lambs. New data have been obtained on the effect of diets enriched with essential microelements on the molecular mechanisms occurring in the myocardial tissue and cortical substance of the kidneys of goats and sheep. The research results obtained by proteomics methods will be used for modeling and targeted adjustment of diets in order to obtain raw materials with the necessary technological characteristics.

Adaptation of the two-dimensional electrophoresis method for canned meat

Studies of the qualitative indicators of canned meat in accordance with regulatory documents are carried out on average samples of specimens, but when studying by proteomic methods, such sampling does not allow high-quality separation of protein components due to the high fat content in the product. When two-dimensional electrophoresis was carried out on an average sample, fragments of the main muscle and connective tissue proteins of beef were found in small quantities, but the electrophoretogram was not very informative. A significantly better separation was achieved after removing the fat fraction from the product. When studying broth from canned meat, the largest amount of intensely coloured high-molecular-weight protein fractions with a mass of more than 50 kDa was revealed. The electrophoretogram of the meat pieces showed a wide range of proteins across the entire molecular weight range of the polyacrylamide gel, including major muscle proteins. The study of broth together with meat pieces but after fat removal is optimal for the primary screening of the protein component of canned meat.

Integrated physiological and chloroplast proteome analysis of wheat seedling leaves under salt and osmotic stresses

In this study, we performed an integrated physiological and chloroplast proteome analysis of wheat seedling leaves under salt and osmotic stresses by label-free based quantitative proteomic approach. Both salt and osmotic stresses significantly increased the levels of abscisic acid and methyl jasmonate and led to damages of chloroplast ultrastructure. Main parameters of chlorophyll fluorescence and gas exchange showed a significant decline under both stresses. Quantitative proteomic analysis identified 194 and 169 chloroplast-localized differentially accumulated proteins (DAPs) responsive to salt and osmotic stresses, respectively. The abundance of main DAPs involved in light-dependent reaction were increased under salt stress, but decreased in response to osmotic stress. On the contrary, salt stress induced a significant upregulation of the DAPs associated with Calvin cycle, transcription and translation, amino acid metabolism, carbon and nitrogen metabolism, and some of them exhibited a downregulation under osmotic stress. In particular, both treatments significantly upregulated the DAPs involved in plastoglobule development, protein folding and proteolysis, hormone and vitamin synthesis. Finally, we proposed a putative synergistic responsive network of wheat chloroplast proteome under salt and osmotic stresses, aiming to provide new insights into the underlying response and defense mechanisms of wheat chloroplast proteome in response to abiotic stresses. SignificanceSalt and osmotic stresses are the two most common abiotic stresses that severely affect crop growth and productivity. As the main site of photosynthesis of plant cells, the chloroplast also plays important role in plant tolerance to abiotic stress. However, the response of chloroplast proteome to salt and osmotic is still poorly understood by using the traditional two-dimensional electrophoresis (2-DE) method due to a poor resolution of chloroplast protein separation and low throughput identification of differentially accumulated proteins (DAPs). In this study, we employed label-free based quantitative proteomic approach to perform an integrated physiological and large-scale chloroplast proteome analysis of wheat seedling leaves under salt and osmotic stresses, which laid a solid foundation for future studies into the response and defense mechanisms of wheat chloroplast in response to abiotic stresses.

Proteomics analysis of erythrocyte membrane in rats with high altitude polycythemia before and after intervention with salidroside

To investigate the effect of salidroside on the proteomics of erythrocyte membrane in high altitude erythrocytosis(HAPC) rats, in order to explore the mechanism of salidroside in improving HAPC based on the proteomics analysis. First, HPAC rat models were established, and 16 rats were randomly divided into HAPC model group and salidroside(100 mg·kg~(-1)) treatment group(8 rats per group). Saline was administered to the HAPC model group, while salidroside treatment group was given 100 mg·kg~(-1) salidroside once a day. After continuous oral administration with salidroside for 40 days(once a day), blood was collected from the femoral artery to obtain total red blood cell membrane proteins. Two-dimensional electrophoresis was used to separate total proteins. The two-dimensional electrophoresis of erythrocyte membrane proteins was analyzed before and after salidroside intervention, and the proteins with significant differences were identified by mass spectrometry. Finally, biological functions were analyzed using bioinformatics. A two-dimensional electrophoresis method was used to establish a protein expression profile with a high resolution and reproducibility of erythrocyte membranes in HAPC rats. Salidroside treatment significantly changed 18 protein spots in the 2-DE map of erythrocyte membranes, of which 13 proteins were up-regulated and 5 proteins were down-regulated. Eight differential proteins were successfully identified by mass spectrometry. Moreover, bioinformatics analysis found that these differential proteins were involved in such biological processes as oxidative stress, redox, and peroxisome pathway, which are mainly associated with peroxisome and MAPK signaling pathways. Therefore, salidroside could significantly change the expressions of erythrocyte membrane proteins in HAPC rats. Eight differential proteins were identified by a proteomic-based approach. The differential proteins were involved in such biological processes as oxidative stress, redox, peroxisome pathway.

GENOMIC, TRANSCRIPTOMIC AND PROTEOMIC TECHNOLOGIES AS A MODERN TOOL FOR HEALTH DISORDERS DIAGNOSTICS, ASSOCIATED WITH THE IMPACT OF ENVIRONMENTAL FACTORS

Introduction. Today, it is relevant to use modern critical technologies for identifying and evaluating the negative effects associated with the effects of chemicals at the stages of pre-nosological changes. This improves the efficiency of the early detection of progress in pre-pathological conditions prior to the onset of pronounced functional changes and the aggravation of the disease. The use of molecular diagnostic methods based on genomic, transcriptomic, and proteomic analysis technologies is one of the most promising approaches. Aim of the work is an analysis of both aspects and practical use of the modern critical technologies capabilities (genomic, transcriptomic and proteomic technologies) in the implementation of biomedical and experimental studies for the tasks of the detection biomarkers of negative effects of chemical risk factors on the example of exposure conditions with aluminum compounds. Material and methods. The proteomic analysis was carried out by the method of two-dimensional electrophoresis, polymorphism of alleles and genotypes of candidate genes by a real-time polymerase chain reaction. The transcriptome state was assessed based on the results of gene expression studies. The expression of membrane and serum proteins was studied by biochemical and immunological methods analysis. Statistical processing of the results was carried out in the systems “Gencalculator,” “Gene Expert” and online program “SNPStats”. Results. The results of using proteomic analysis technologies made it possible to identify proteins annexin-13, SH3-domain protein-RF3, cathepsin L1 and, accordingly, genes CTSL, SH3RF3, THO complex subunit 2 as Ohmic markers of aerogenic exposure of inorganic compounds. The results of the analysis of gene polymorphism in the population exposed to environmental pollution allowed establishing the changed frequency of variant alleles and genotypes of genes: immune control - TLR4 (toll-like receptor); vascular factors - eNOS rs1799983 (endothelial NOsintase); detoxification - coproporphyrinogen oxidase CPOX (rs1131857), cytochrome P450 CYP1A1 (rs 1048943); neuro-humoral regulation of ANKK1 rs1800497 (dopamine receptor gene) and HTR2A rs7997012 (serotonin receptor gene). The results of gene expression analysis made it possible to establish negative transcriptomic effects induced by exposure to amphoteric metals due to the isolation of specific CD4+, CD8+, CD16+ cell phenotypes expressing the proteomic profile gene of blood plasma lipoprotein A (LPA gene). Discussion. The obtained results correspond data of a number of scientific studies, noting the importance of identifying polymorphic deviations of genes determining the individual risk of health problems in a variety of stressful environmental factors affecting humans. Minor genotypes of candidate genes under conditions of excessive contamination with amphoteric metal compounds significantly increase the risk of deviations in immune regulation indices, which modifies apoptosis mechanisms, which are crucial for the formation of atopy and onco-proliferation. Conclusion. The use of genome, transcriptomic and proteomic technologies as a modern tool for the diagnostics of health disorders allowed justifying the set of priority exposition and effect Ohmic-markers, associated with aerogenic effect of amphoteric metals, which have a modifying effect on the pathogenetic mechanisms of the formation of disorders of nervous and immune systems, the 1st and 2nd phase of detoxification, the likelihood of vascular disorders and onco-proliferative processes.

Применение метода двумерного электрофореза для оценки состава мясных продуктов на примере модельных фаршей

Применение метода двумерного электрофореза для оценки состава мясных продуктов на примере модельных фаршей

Altered Proteins in the Hippocampus of Patients with Mesial Temporal Lobe Epilepsy.

Mesial temporal lobe epilepsy (MTLE) is usually associated with drug-resistant seizures and cognitive deficits. Efforts have been made to improve the understanding of the pathophysiology of MTLE for new therapies. In this study, we used proteomics to determine the differential expression of proteins in the hippocampus of patients with MTLE compared to control samples. By using the two-dimensional electrophoresis method (2-DE), the proteins were separated into spots and analyzed by LC-MS/MS. Spots that had different densitometric values for patients and controls were selected for the study. The following proteins were found to be up-regulated in patients: isoform 1 of serum albumin (ALB), proton ATPase catalytic subunit A (ATP6V1A), heat shock protein 70 (HSP70), dihydropyrimidinase-related protein 2 (DPYSL2), isoform 1 of myelin basic protein (MBP), and dihydrolipoamide S-acethyltransferase (DLAT). The protein isoform 3 of the spectrin alpha chain (SPTAN1) was down-regulated while glutathione S-transferase P (GSTP1) and protein DJ-1 (PARK7) were found only in the hippocampus of patients with MTLE. Interactome analysis of the nine proteins of interest revealed interactions with 20 other proteins, most of them involved with metabolic processes (37%), presenting catalytic activity (37%) and working as hydrolyses (25%), among others. Our results provide evidence supporting a direct link between synaptic plasticity, metabolic disturbance, oxidative stress with mitochondrial damage, the disruption of the blood–brain barrier and changes in CNS structural proteins with cell death and epileptogenesis in MTLE. Besides this, the presence of markers of cell survival indicated a compensatory mechanism. The over-expression of GSTP1 in MTLE could be related to drug-resistance.

Two-Dimensional Capillary Electrophoresis with On-Line Sample Preparation and Cyclodextrin Separation Environment for Direct Determination of Serotonin in Human Urine.

An advanced two-dimensional capillary electrophoresis method, based on on-line combination of capillary isotachophoresis and capillary zone electrophoresis with cyclodextrin additive in background electrolyte, was developed for effective determination of serotonin in human urine. Hydrodynamically closed separation system and large bore capillaries (300–800 µm) were chosen for the possibility to enhance the sample load capacity, and, by that, to decrease limit of detection. Isotachophoresis served for the sample preseparation, defined elimination of sample matrix constituents (sample clean up), and preconcentration of the analyte. Cyclodextrin separation environment enhanced separation selectivity of capillary zone electrophoresis. In this way, serotonin could be successfully separated from the rest of the sample matrix constituents migrating in capillary zone electrophoresis step so that human urine could be directly (i.e., without any external sample preparation) injected into the analyzer. The proposed method was successfully validated, showing favorable parameters of sensitivity (limit of detection for serotonin was 2.32 ng·mL−1), linearity (regression coefficient higher than 0.99), precision (repeatability of the migration time and peak area were in the range of 0.02–1.17% and 5.25–7.88%, respectively), and recovery (ranging in the interval of 90.0–93.6%). The developed method was applied for the assay of the human urine samples obtained from healthy volunteers. The determined concentrations of serotonin in such samples were in the range of 12.4–491.2 ng·mL−1 that was in good agreement with literature data. This advanced method represents a highly effective, reliable, and low-cost alternative for the routine determination of serotonin as a biomarker in human urine.

Dissecting Pistil Responses to Incompatible and Compatible Pollen in Self-Incompatibility Brassica oleracea Using Comparative Proteomics.

Angiosperms have developed self-incompatibility (SI) systems to reject self-pollen, thereby promoting outcrossing. The Brassicaceae belongs to typical sporophytic system, having a single S-locus controlled SI response, and was chosen as a model system to study SI-related intercellular signal transduction. In this regard, the downstream factor of EXO70A1 was unknown. Here, protein two-dimensional electrophoresis (2-DE) method and coupled with matrix-assisted laser desorption ionization/time of flight of flight mass spectrometry (MALDI-TOF -MS) and peptide mass fingerprinting (PMF) was used to further explore the mechanism of SI responses in Brassica oleracea L. var. capitata L. at protein level. To further confirm the time point of protein profile change, total proteins were collected from B. oleracea pistils at 0min, 1h, and 2h after self-pollination. In total 902, 1088 and 1023 protein spots were separated in 0min, 1h and 2h 2-DE maps, respectively. Our analyses of self-pollination profiles indicated that proteins mainly changed at 1h post-pollination in B. oleracea. Moreover, 1077 protein spots were separated in cross-pollinated 1h (CP) pistil 2-DE map. MALDI-TOF-MS and PMF successfully identified 34 differentially-expressed proteins (DEPs) in SP and CP 1h 2-DE maps. Gene ontology and KEGG analysis revealed an array of proteins grouped in the following categories: stress and defense response (35%), protein metabolism (18%), carbohydrate and energy metabolism (12%), regulation of translation (9%), pollen tube development (12%), transport (9%) and cytoskeletal (6%). Sets of DEPs identified specifically in SP or only up-regulated expressed in CP pistils were chosen for funther investigating in floral organs and during the process of self- and cross-pollination. The function of these DEPs in terms of their potential involvement in SI in B. oleracea is discussed.

Analysis of Telomere-Homologous DNA with Different Conformations Using 2D Agarose Electrophoresis and In-Gel Hybridization.

In mammalian cells, in addition to double-stranded telomeric DNA at chromosome ends, extra telomere-homologous DNA is present that adopts different conformations, including single-stranded G- or C-rich DNA, extrachromosomal circular DNA (T-circle), and telomeric complex (T-complex) with an unidentified structure. The formation of such telomere-homologous DNA is closely related to telomeric DNA metabolism and chromosome end protection by telomeres. Conventional agarose gel electrophoresis is unable to separate DNA based on conformation. Here, we introduce the method of two-dimensional (2D) agarose electrophoresis in combination with in-gel native/denatured hybridization to determine different conformations formed by telomere-homologous DNA.

Native structure of rat liver immune proteasomes.

Native structure of active forms of rat liver immune proteasomes has been studied by two-dimensional electrophoresis method modified for analysis of unpurified protein fractions. The developed method allowed revealing the proteasome immune subunits LMP7 and LMP2 in 20S subparticles and in the structures bound to one or two PA28αβ activators, but not to the PA700 activator, which is involved in the hydrolysis of ubiquitinated proteins. The results obtained indicate the participation of the immune proteasomes in delicate regulatory mechanisms based on the production of biologically active peptides and exclude their participation in processes of crude degradation of "rotated" ubiquitinated proteins.

Proteome responses of Citrobacter werkmanii BF-6 planktonic cells and biofilms to calcium chloride

Calcium ions are well-known as intracellular second messengers that also have an important extracellular structural role for bacteria. Recently, we found that denser biofilms were formed by Citrobacter werkmanii BF-6 in the presence of 400mM Ca2+ than that of 12.5mM Ca2+. Therefore, we employed two-dimensional (2-D) electrophoresis methods to investigate the proteome profiles of planktonic cells and biofilms in BF-6 under different concentrations of Ca2+. Meanwhile, BF-6 biofilm architecture was also visualized with confocal laser scanning microscopy (CLSM). The results demonstrated that BF-6 biofilms formed at the bottom of microtiter plates when grown in the presence of 400mM Ca2+. A total of 151 proteins from planktonic cells and biofilms after exposure of BF-6 cells to 12.5 and 400mM Ca2+ were successfully identified. Different gene ontology (GO) and KEGG pathways were categorized and enriched for the above proteins. Growth in the presence of 400mM Ca2+ induced more complex signal pathways in BF-6 than 12.5mM Ca2+. In addition, the biofilm architectures were also affected by Ca2+. Our results show two different modes of biofilm enhancement for C. werkmanii in the presence of excess Ca2+ and provide a preliminary expression of these differences based on proteomic assays.

Engineering Streptavidin and a Streptavidin-Binding Peptide with Infinite Binding Affinity and Reversible Binding Capability: Purification of a Tagged Recombinant Protein to High Purity via Affinity-Driven Thiol Coupling.

To extend and improve the utility of the streptavidin-binding peptide tag (SBP-tag) in applications ranging from affinity purification to the reversible immobilization of recombinant proteins, a cysteine residue was introduced to the streptavidin mutein SAVSBPM18 and the SBP-tag to generate SAVSBPM32 and SBP(A18C), respectively. This pair of derivatives is capable of forming a disulfide bond through the newly introduced cysteine residues. SAVSBPM32 binds SBP-tag and biotin with binding affinities (Kd ~ 10-8M) that are similar to SAVSBPM18. Although SBP(A18C) binds to SAVSBPM32 more weakly than SBP-tag, the binding affinity is sufficient to bring the two binding partners together efficiently before they are locked together via disulfide bond formation–a phenomenon we have named affinity-driven thiol coupling. Under the condition with SBP(A18C) tags in excess, two SBP(A18C) tags can be captured by a tetrameric SAVSBPM32. The stoichiometry of the disulfide-bonded SAVSBPM32-SBP(A18C) complex was determined using a novel two-dimensional electrophoresis method which has general applications for analyzing the composition of disulfide-bonded protein complexes. To illustrate the application of this reversible immobilization technology, optimized conditions were established to use the SAVSBPM32-affinity matrix for the purification of a SBP(A18C)-tagged reporter protein to high purity. Furthermore, we show that the SAVSBPM32-affinity matrix can also be applied to purify a biotinylated protein and a reporter protein tagged with the unmodified SBP-tag. The dual (covalent and non-covalent) binding modes possible in this system offer great flexibility to many different applications which need reversible immobilization capability.

Proteomic analysis of Daphnia magna exposed to glyphosate and methidathion

Proteomic analysis was performed to identify proteins involved in the stress responses of Daphnia magna to glyphosate and methidathion. Lethal concentration of chemicals causing 50% mortality (LC50) determined by acute toxicity assays were used to expose to daphnias from 24 h to 21 days prior to total protein extraction. The proteomic profile of tested organisms was analyzed by the two-dimensional electrophoresis method. The average intensity of each spots was then analyzed to determine the differently expressed proteins (DEPs). The DEPs was considered as down- or up-regulations only if their expression level in control samples are linearly higher than LC50 or lower than LC50, respectively. The result showed that there are 22 and 27 DEPs responding to the exposure of glyphosate and methidathion, respectively. The expression pattern of established DEPs is helpful to understand the molecular response of D. magna to glyphosate and methidathion, and discover potential candidates as novel biomarkers.

E2F8 promotes hepatic steatosis through FABP3 expression in diet-induced obesity in zebrafish.

BackgroundDiet-induced hepatic steatosis is highly associated with nonalcoholic fatty liver disease, which is related to the development of metabolic syndrome. While advanced stage nonalcoholic hepatic steatosis and steatohepatitis (NASH) result ultimately in fibrosis and cirrhosis, the molecular basis for lipid droplet formation is poorly understood. Common pathways underlie the pathology of mammalian obesity and the zebrafish diet-induced obesity model (DIO-zebrafish) used in this study.MethodsOur analysis involved a combination of transcriptome (DNA microarray) and proteome (two-dimensional electrophoresis) methods using liver tissue from DIO-zebrafish to find candidate genes involved in hepatic steatosis. We conducted intraperitoneal injection (i.p.) of morpholino antisense oligonucleotides (MOs) for each gene into DIO-zebrafish. We also conducted in vitro overexpression in human cells. Additionally, we examined gene expression during feeding experiments involving anti-obesity compounds, creatine and anserine.ResultsWe found that fatty acid binding protein 3 (fabp3) and E2F transcription factors were upregulated in hepatic steatosis. E2f8 MO i.p. suppressed fabp3 expression in liver, and ameliorated hepatic steatosis. In human cells (HepG2), E2F8 overexpression promoted FABP3 expression. Additionally, co-administration of creatine and anserine suppressed obesity associated phenotypes including hepatic steatosis as indicated by e2f8 and fabp3 down regulation.ConclusionWe discovered that the e2f8–fabp3 axis is important in the promotion of hepatic steatosis in DIO-zebrafish. The combination of transcriptome and proteome analyses using the disease model zebrafish allow identification of novel pathways involved in human diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12986-015-0012-7) contains supplementary material, which is available to authorized users.

Protein Drug Targets ofLavandula angustifoliaon treatment of Rat Alzheimer's Disease.

Different treatment strategies of Alzheimer's disease (AD) are being studied for treating or slowing the progression of AD. Many pharmaceutically important regulation systems operate through proteins as drug targets. Here, we investigate the drug target proteins in beta-amyloid (Aβ) injected rat hippocampus treated with Lavandula angustifolia (LA) by proteomics techniques. The reported study showed that lavender extract (LE) improves the spatial performance in AD animal model by diminishing Aβ production in histopathology of hippocampus, so in this study neuroprotective proteins expressed in Aβ injected rats treated with LE were scrutinized. Rats were divided into three groups including normal, Aβ injected, and Aβ injected that was treated with LE. Protein expression profiles of hippocampus tissue were determined by two-dimensional electrophoresis (2DE) method and dysregulated proteins such as Snca, NF-L, Hspa5, Prdx2, Apoa1, and Atp5a1were identified by MALDI-TOF/TOF. KEGG pathway and gene ontology (GO) categories were used by searching DAVID Bioinformatics Resources. All detected protein spots were used to determine predictedinteractions with other proteins in STRING online database. Different isoforms of important protein, Snca that exhibited neuroprotective effects by anti-apoptotic properties were expressed. NF-L involved in the maintenance of neuronal caliber. Hspa5 likewise Prdx2 displays as anti-apoptotic protein that Prdx2 also involved in the neurotrophic effects. Apoa1 has anti-inflammatory activity and Atp5a1, produces ATP from ADP. To sum up, these proteins as potential drug targets were expressed in hippocampus in response to effective components in LA may have therapeutic properties for the treatment of AD and other neurodegenerative diseases.

Changes in ribosomal proteins in wheat embryos in the course of grain development and maturation

It was found, by comparing the densitometric profiles of ribosomal proteins of wheat embryos in milk and full grain ripeness, that in the process of development and ripening of caryopses the percentual proportion of low molecular weight proteins increases at the cost of those of high molecular weight. This concerns both acidic and basic proteins. In electrophoretic separation of ribosomal proteins from embryos of fully ripe seeds by the method of two-dimensional electrophoresis the appearance of three new low molecular weight proteins - an acidic one and two basic ones - was observed. These proteins were not found in the embryos of caryopses of milk ripeness. These results indicate that with development and ripening of wheat caryopses new low molecular weight ribosomal proteins are built into the ribosomes in the embryo. These changes are both quantitative and qualitative.

Proteomics for discovery of candidate colorectal cancer biomarkers.

Colorectal cancer (CRC) is the second most common cause of cancer-related deaths in Europe and other Western countries, mainly due to the lack of well-validated clinically useful biomarkers with enough sensitivity and specificity to detect this disease at early stages. Although it is well known that the pathogenesis of CRC is a progressive accumulation of mutations in multiple genes, much less is known at the proteome level. Therefore, in the last years many proteomic studies have been conducted to find new candidate protein biomarkers for diagnosis, prognosis and as therapeutic targets for this malignancy, as well as to elucidate the molecular mechanisms of colorectal carcinogenesis. An important advantage of the proteomic approaches is the capacity to look for multiple differentially expressed proteins in a single study. This review provides an overview of the recent reports describing the different proteomic tools used for the discovery of new protein markers for CRC such as two-dimensional electrophoresis methods, quantitative mass spectrometry-based techniques or protein microarrays. Additionally, we will also focus on the diverse biological samples used for CRC biomarker discovery such as tissue, serum and faeces, besides cell lines and murine models, discussing their advantages and disadvantages, and summarize the most frequently identified candidate CRC markers.

A Proteomic Analysis of Placental Trophoblastic Cells in Preeclampsia–Eclampsia

To explore the proteomic changes of placental trophoblastic cells in preeclampsia-eclampsia (PE), placental trophoblastic cells from normally pregnant women and women with hypertension during gestational period were prepared by laser capture microdissection (LCM), and proteins isolated from these cells were subjected to labeling and proteolysis with isotope-coded affinity tag reagent. A qualitative and quantitative analysis of the proteome expression of placental trophoblastic cells was made using two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS). A total of 831 proteins in placental trophoblastic cells were identified by combined use of LCM technique and 2D LC-MS/MS. The result was superior to that of conventional two-dimensional electrophoresis method. There were marked differences in 169 proteins of placental trophoblastic cells between normally pregnant women and women with PE. Of 70 (41.4 %) proteins with more than twofold differences, 31 proteins were down-regulated, and 39 were up-regulated in placental trophoblastic cells of the woman with PE. Laminin expression in placenta trophoblastic cells of women with PE was significantly down-regulated as confirmed by Western blot analysis. These findings provide insights into the proteomic changes in placental trophoblastic cells in response to PE and may identify novel protein targets associated with the pathogenesis of PE.