Comparative proteomic analysis of human placenta derived from assisted reproductive technology

The aim of this study was to use proteomics-based approach to examine differences in protein expression in placenta derived from assisted reproductive technology (ART) and normal pregnancy. Using 2-DE we found that, compared with the control group, 12 spots in standard in vitro fertilization group and 18 spots in intracytoplasmic sperm injection group were identified as significantly differentially expressed proteins. Among them, six spots were differentially expressed in both standard IVF and ICSI groups with the same change tendency. Totally, 20 proteins were successfully identified by MALDI TOF/TOF MS, including proteins involved in the membrane traffic, metabolism, nucleic acid processing, stress response and cytoskeleton. Notably, five proteins detected to be differentially expressed in both ART groups were identified as annexin A3, hnRNP C1/C2, alpha-SNAP, FTL and ATP5A. Some of the proteins were confirmed by Western blot and immunohistochemistry analysis. Our study allowed for the initial identification of these proteins related to various functions in placentation with significantly altered abundance in ART groups. The present results reveal that abnormal protein profiles are involved in ART placenta and these differentially expressed proteins may be valuable for the evaluation of potential association between ART treatment and offspring outcome.     

Proteome characterization of human NK-92 cells identifies novel IFN-alpha and IL-15 target genes

Natural killer (NK) cells are important components of innate immune defense. NK cells kill virus-infected cells and secrete cytokines that are involved in activation of other immune cells. Macrophage-derived cytokines interferon-alpha (IFN-alpha) and interleukin-15 (IL-15) are in turn important activators of NK cells, but the receptors and intracellular pathways that are involved in NK cell functions are still incompletely known. Here we have used expression proteomics to find new IFN-alpha and IL-15 regulated proteins in human NK-92 cells, which have the characteristics of activated NK cells. Cells were stimulated with cytokines for 20 h, lysed, and soluble proteins were separated by two-dimensional electrophoresis, and differentially expressed protein spots were identified with mass spectrometry and database searches. A total of 57 protein spots were found to be reproducibly differentially expressed between control and cytokine stimulated gel pairs, 26 spots being more than 2-fold upregulated and 3 spots being at least 2-fold downregulated. The rest 28 spots showed minor, less than 2-fold changes in their expression levels after quantification. From the differentially expressed protein spots we identified 47 different proteins, most of which are new IFN-alpha and IL-15 target proteins. Interestingly, we show that e.g., adenylate kinase 2 is highly upregulated by IFN-alpha and IL-15 stimulation in NK-92 cells. The expression of selected genes with high expression level differences after cytokine stimulation were further studied at mRNA level. Northern blot analysis showed that the genes studied were induced by IFN-alpha, IL-15, and IL-2 already at 3 h time point, suggesting that they are primary target genes of these cytokines.     

Proteomic analysis of cellular protein alterations using a hepatitis B virus-producing cellular model

Hepatitis B virus (HBV) is one of the major etiological factors responsible for acute and chronic liver disease and for the development of hepatocellular carcinoma (HCC). To determine the effects of HBV replication on host cell-protein expression, we utilized 2-DE and MS/MS analysis to compare and identify differentially expressed proteins between an HBV-producing cell line HepG2.2.15 and its parental cell line HepG2. Of the 66 spots identified as differentially expressed (+/- over twofold, p <0.05) between the two cell lines, 62 spots (corresponding to 61 unique proteins) were positively identified by MS/MS analysis. These proteins could be clearly divided into three major groups by cluster and metabolic/signaling pathway analysis: proteins involved in retinol metabolism pathway, calcium ion-binding proteins, and proteins associated with protein degradation pathways. Other proteins identified include those that function in diverse biological processes such as signal transduction, immune regulation, molecular chaperone, electron transport/redox regulation, cell proliferation/differentiation, and mRNA splicing. In summary, we profiled proteome alterations between HepG2.2.15 and HepG2 cells. The proteins identified in this study would be useful in revealing the mechanisms underlying HBV-host cell interactions and the development of HCC. This study can also provide some useful clues for antiviral research.     

Protein alteration of HepG2.2.15 cells induced by iron overload

Hepatitis B can progress into hepatocellular carcinoma. Body irons may interfere with the clearance of hepatitis B virus (HBV) and contribute to genesis of tumor. To investigate the role of iron played in HBV-related pathogenesis, here we studied the effect of iron with different concentrations and valence states on growth of HepG2.2.15 cells and secretion of virus proteins. A strong tolerance of HepG2.2.15 cells to iron challenge was found. The concentration of hepatitis B surface antigen in cell culture medium was decreased after iron stimulation. Lower concentrations of iron facilitated hepatitis B e-antigen (HBeAg) secretion. Fe(2+) appeared more effective on HBeAg secretion than Fe(3+) did. In parallel, the differential protein profiles in HepG2.2.15 cells were studied by iTRAQ and LC-MS/MS. The differentially expressed proteins were mainly involved in stress response, signal transduction, apoptosis, etc. Four proteins (14-3-3 β/α, VCP, migration inhibitory factor, and Nup153) were verified by Western-blotting and found to be consistent with the iTRAQ data. Interestingly, nuclear import of Nuclear factor kappa B (NFκB) and its activity were found to be affected by the decreased Nup153 in iron stimulated HepG2.2.15 cells. The results may indicate possible molecular mechanism how the synergism of HBV and iron stimulation damages host liver cells.     

Comparative proteomics analysis of serum proteins in ulcerative colitis patients

In the present study, we investigated the differentially expressed proteins associated with ulcerative colitis (UC) using proteomic methods. Two-dimensional electrophoresis (2-DE) technology was performed to separate the total proteins of ulcerative tissues from those of the normal tissues of UC patients. PDQuest software was applied to analyze the obtained 2-DE images. Candidate protein spots between the two groups were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and bioinformatics analysis. The well resolution and reproducible 2-DE patterns of UC and normal tissues were established. Of the 12 differentially expressed proteins, 9 were successfully identified, of which 6 proteins were up-regulated including apolipoprotein C-III, haptoglobin, receptor tyrosine kinase, aldehyde reductase, pericentriolar material 1, and heat shock factor protein 2, and 3 were down-regulated including keratin, filamin A-interacting protein 1, and tropomyosin 3. These identified proteins were related to hormonal modulation, immune response, oxidative stress, and signal conduction. The 2-DE protein expression profile of the UC tissues displays an obvious difference from that of the normal controls. Various proteins may be involved in the occurrence of UC.     

Proteomic analysis of differentially expressed proteins in Lactobacillus brevis NCL912 under acid stress

Protein expression of Lactobacillus brevis NCL912 under acid stress was analysed by two-dimensional gel electrophoresis and MS. Twenty-five proteins were differentially expressed under acid stress. Among them, eight protein spots were identified by matrix-assisted laser desorption/ionization time-of-flight MS, of which seven were upregulated and one was downregulated. The function of the downregulated protein was unknown and the putative functions of the upregulated proteins were categorized as stress response, DNA repair, protein synthesis and glycolysis. Quantitative real-time PCR was used to further validate these differentially expressed proteins at the mRNA level and a positive correlation between the content of the proteins and their mRNA levels was found. The results suggest that these proteins are involved in the acid stress response mechanisms of this bacterium.     

Response to nickel in the proteome of the metal accumulator plant Brassica juncea

To gain a comprehensive understanding of the molecular mechanism of heavy metal accumulation in Brassica juncea, comparative proteomic approaches were used to analysis protein profiles in leaf tissues of 6-week-old B. juncea after exposure to 100 µM Ni. Proteomic analysis revealed that 61 protein spots showed 1.5-fold change in protein abundance after Ni exposure as compared to that of corresponding spots in control. Out of the 61 differentially expressed protein spots, 37 protein spots were ambiguously identified by matrix assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS). The majority of these identified proteins were found to be involved in sulphur metabolism, protection against oxidative stress, clearly indicated that heavy metal sequestration and antioxidant system were activated by Ni treatment. The induced expression of photosynthesis and ATP generation-related proteins were also observed in plants exposed to metals, suggesting the tolerance and accumulation is an energy-demanding process. The identification of these proteins in response to Ni can lead a deep understanding of heavy metal accumulation and tolerance in B. juncea.     

Protein profiling the effects of in vitro hyperoxic exposure on fetal rabbit lung

The aim of this study was to test the hypothesis that acute in vitro exposure of prematurely delivered fetal rabbit lungs to hyperoxic conditions will induce the expression of an adaptive cassette of proteins that mediates antioxidant and inflammatory processes. To test this hypothesis, ex situ fetal rabbit lung explants were prepared from New Zealand white rabbits delivered by cesarean section on day 29 of gestation and incubated under air (21% O2; 5% CO2) or hyperoxic (95% O2; 5% CO2) atmospheres. Total tissue protein was extracted following incubation and subjected to 2-DE. Using this technique, 1500-2000 protein spots were resolved per gel. Treatment-dependent, differentially expressed proteins were identified by image analysis (Melanie II) and MALDI-TOF MS and MALDI-MS/MS. The analysis identified 12 protein spots that were differentially expressed by 1.5-fold or more (p<0.05) by exposure to hyperoxic conditions. Six of these differentially expressed proteins were identified as vimentin, annexin I, inorganic pyrophosphatase, prohibitin, an N-terminal fragment of ATP synthase and heat shock protein 27. The data obtained are consistent with the roles of these proteins in mediating cellular response to oxidative stress and in regulating cell proliferation.     

Oxidative stress-induced proteome alterations target different cellular pathways in human myoblasts

Although increased oxidative stress has been associated with the impairment of proliferation and function of adult human muscle stem cells, proteins either involved in the stress response or damaged by oxidation have not been identified. A parallel proteomics approach was performed for analyzing the protein expression profile as well as proteins preferentially oxidized upon hydrogen peroxide-induced oxidative stress. Fifteen proteins involved in the oxidative stress response were identified. Among them, protein spots identified as peroxiredoxins 1 and 6, glyceraldehyde-3-phosphate dehydrogenase, and α-enolase were shifted to a more acidic isoelectric point upon oxidative stress, indicating posttranslational modifications. Oxidized proteins were evidenced by immunodetection of derivatized carbonyl groups followed by identification by mass spectrometry. The carbonylated proteins identified are mainly cytosolic and involved in carbohydrate metabolism, cellular assembly, cellular homeostasis, and protein synthesis and degradation. Pathway analysis revealed skeletal and muscular disorders, cell death, and cancer-related as the main molecular networks altered. Interestingly, these pathways were focused on two distinct proteins: p53 for altered protein expression and huntingtin for increased protein carbonylation. This study emphasizes the importance of performing analysis addressing different aspects of the cellular proteome to have a more accurate view of their changes upon stress.     

Genetic, proteomic and metabolic analysis of the regulation of energy storage in rice seedlings in response to drought

We used proteomic analysis to determine the response of rice plant seedlings to drought-induced stress. The expression of 71 protein spots was significantly altered, and 60 spots were successfully identified. The greatest down-regulated protein functional category was translation. Up-regulated proteins were mainly related to protein folding and assembly. Additionally, many proteins involved in metabolism (e.g. carbohydrate metabolism) also showed differences in expression. cDNA microarray and GC-MS analysis showed 4756 differentially expressed mRNAs and 37 differentially expressed metabolites. Once these data were integrated with the proteomic analysis, we were able to elucidate the metabolic pathways affected by drought-induced stress. These results suggest that increased energy consumption from storage substances occurred during drought. In addition, increased expression of the enzymes involved in anabolic pathways corresponded with an increase in the content of six amino acids. We speculated that energy conversion from carbohydrates and/or fatty acids to amino acids was increased. Analysis of basic metabolism networks allowed us to understand how rice plants adjust to drought conditions.     

Comparative proteomics analysis reveals an intimate protein network provoked by hydrogen peroxide stress in rice seedling leaves

Hydrogen peroxide (H2O2) plays a dual role in plants as the toxic by-product of normal cell metabolism and as a regulatory molecule in stress perception and signal transduction. However, a clear inventory as to how this dual function is regulated in plants is far from complete. In particular, how plants maintain survival under oxidative stress via adjustments of the intercellular metabolic network and antioxidative system is largely unknown. To investigate the responses of rice seedlings to H2O2 stress, changes in protein expression were analyzed using a comparative proteomics approach. Treatments with different concentrations of H2O2 for 6 h on 12-day-old rice seedlings resulted in several stressful phenotypes such as rolling leaves, decreased photosynthetic and photorespiratory rates, and elevated H2O2 accumulation. Analysis of approximately 2000 protein spots on each two-dimensional electrophoresis gel revealed 144 differentially expressed proteins. Of them, 65 protein spots were up-regulated, and 79 were down-regulated under at least one of the H2O2 treatment concentrations. Furthermore 129 differentially expressed protein spots were identified by mass spectrometry to match 89 diverse protein species. These identified proteins are involved in different cellular responses and metabolic processes with obvious functional tendencies toward cell defense, redox homeostasis, signal transduction, protein synthesis and degradation, photosynthesis and photorespiration, and carbohydrate/energy metabolism, indicating a good correlation between oxidative stress-responsive proteins and leaf physiological changes. The abundance changes of these proteins, together with their putative functions and participation in physiological reactions, produce an oxidative stress-responsive network at the protein level in H2O2-treated rice seedling leaves. Such a protein network allows us to further understand the possible management strategy of cellular activities occurring in the H2O2-treated rice seedling leaves and provides new insights into oxidative stress responses in plants.     

Crucial role of antioxidant proteins and hydrolytic enzymes in pathogenicity of Penicillium expansum: analysis based on proteomics approach

Penicillium expansum, a widespread filamentous fungus, is a major causative agent of fruit decay and may lead to the production of mycotoxin that causes harmful effects on human health. In this study, we compared the cellular and extracellular proteomes of P. expansum in the absence and presence of borate, which affects the virulence of the fungal pathogen. The differentially expressed proteins were identified using ESI-Q-TOF-MS/MS. Several proteins related to stress response (glutathione S-transferase, catalase, and heat shock protein 60) and basic metabolism (glyceraldehyde-3-phosphate dehydrogenase, dihydroxy-acid dehydratase, and arginase) were identified in the cellular proteome. Catalase and glutathione S-transferase, the two antioxidant enzymes, exhibited reduced levels of expression upon exposure to borate. Because catalase and glutathione S-transferase are related to oxidative stress response, we further investigated the reactive oxygen species (ROS) levels and oxidative protein carbonylation (damaged proteins) in P. expansum. Higher amounts of ROS and carbonylated proteins were observed after borate treatment, indicating that catalase and glutathione S-transferase are important in scavenging ROS and protecting cellular proteins from oxidative damage. Additionally to find secretory proteins that contribute to the virulence, we studied the extracellular proteome of P. expansum under stress condition with reduced virulence. The expression of three protein spots were repressed in the presence of borate and identified as the same hydrolytic enzyme, polygalacturonase.     

Proteomic analysis of Arabidopsis thaliana ecotypes with contrasted root architecture in response to phosphate deficiency

Owing to a weak availability in soil, plants have developed numerous morphological, physiological and biochemical adaptations to acquire phosphate (Pi). Identification and characterisation of key genes involved in the initial steps of Pi-signalling might provide clues about the regulation of the complex Pi deficiency adaptation mechanism. A two-dimensional gel electrophoresis approach was performed to investigate proteome responses to Pi starvation in Arabidopsis. Two ecotypes were selected according to contrasting responses of their root system architecture to low availability of Pi. Thirty protein spots were shown to be affected by Pi deficiency. Fourteen proteins appeared to be up-regulated and ten down-regulated with ecotype Be-0, wheras only thirteen proteins were observed as down-regulated for ecotype Ll-0. Furthermore, systematic and opposite responses to Pi deficiency were observed between the two ecotypes. The sequences of these 30 differentially expressed protein spots were identified using mass spectrometry, and most of the proteins were involved in oxidative stress, carbohydrate and proteins metabolism. The results suggested that the modulation of alcohol dehydrogenase, malic enzyme and aconitate hydratase may contribute to the contrasted adaptation strategy to Pi deficiency of Be-0 and Ll-0 ecotypes. A focus on aconitate hydratase highlighted a complex reverse response of the pattern of corresponding spots between the two ecotypes. This protein, also potentially involved in iron homeostasis, was speculated to contribute, at least indirectly, to the root architecture response of these ecotypes.     

Role of curcuminoids in ameliorating oxidative modification in β-thalassemia/Hb E plasma proteome

Thalassemic patients often exhibit high levels of oxidative stress and iron overload, which can lead to hazardous complications. Curcuminoids, extracted from the spice turmeric, are known to have antioxidant and iron-chelating properties and have been proposed as a potential upstream therapy of thalassemia. Here we have applied proteomic techniques to study the protein profile and oxidative damage in the plasma of β-thalassemia/Hb E patients before and after treatment with curcuminoids. In this study, 10 β-thalassemia/Hb E patients were treated with 500 mg curcuminoids daily for 12 months. The plasma protein profile and protein carbonyl content were determined at baseline, 6 and 12 months using two-dimensional fluorescence difference gel electrophoresis and carbonyl immunoblotting, respectively. Other hematological, clinical, and biochemical parameters were also analyzed. Twenty-six spots, identified as coagulation factors and proteins involved in iron homeostasis, showed significantly decreased intensity in thalassemic plasma, compared to those of normal subjects. Treatment with curcuminoids up-regulated the plasma levels of these proteins and reduced their oxidative damage. Serum non-transferrin bound iron, platelet factor-3 like activity, oxidative stress parameters and antioxidant enzymes were also improved after curcuminoids treatment. This study is the first proteomic study of plasma in the thalassemic state and also shows the ameliorating role of curcuminoids towards oxidative stress and iron overload in the plasma proteome.     

Proteomics reveals new insights into the role of light in cadmium response in Arabidopsis cell suspension cultures

Light plays an important role in plant growth, development, and response to environmental stresses. To investigate the effects of light on the plant responses to cadmium (Cd) stress, we performed a comparative physiological and proteomic analysis of light‐ and dark‐grown Arabidopsis cells after exposure to Cd. Treatment with different concentrations of Cd resulted in stress‐related phenotypes such as cell growth inhibition and decline of cell viability. Notably, light‐grown cells were more sensitive to heavy metal toxicity than dark‐grown cells, and the basis for this appears to be the elevated Cd accumulation, which is twice as much under light than dark growth conditions. Protein profiles analyzed by 2D DIGE revealed a total of 162 protein spots significantly changing in abundance in response to Cd under at least one of these two growing conditions. One hundred and ten of these differentially expressed protein spots were positively identified by MS/MS and they are involved in multiple cellular responses and metabolic pathways. Sulfur metabolism‐related proteins increased in relative abundance both in light‐ and dark‐grown cells after exposure to Cd. Proteins involved in carbohydrate metabolism, redox homeostasis, and anti‐oxidative processes were decreased both in light‐ and dark‐grown cells, with the decrease being lower in the latter case. Remarkably, proteins associated with cell wall biosynthesis, protein folding, and degradation showed a light‐dependent response to Cd stress, with the expression level increased in darkness but suppressed in light. The possible biological importance of these changes is discussed.     

Proteomic identification of differentially expressed proteins in Arabidopsis in response to methyl jasmonate

Jasmonates (JAs) are the well characterized fatty acid-derived cyclopentanone signals involved in the plant response to biotic and abiotic stresses. JAs have been shown to regulate many aspects of plant metabolism, including glucosinolate biosynthesis. Glucosinolates are natural plant products that function in defense against herbivores and pathogens. In this study, we applied a proteomic approach to gain insight into the physiological processes, including glucosinolate metabolism, in response to methyl jasmonate (MeJA). We identified 194 differentially expressed protein spots that contained proteins that participated in a wide range of physiological processes. Functional classification analysis showed that photosynthesis and carbohydrate anabolism were repressed after MeJA treatment, while carbohydrate catabolism was up-regulated. Additionally, proteins related to the JA biosynthesis pathway, stress and defense, and secondary metabolism were up-regulated. Among the differentially expressed proteins, many were involved in oxidative tolerance. The results indicate that MeJA elicited a defense response at the proteome level through a mechanism of redirecting growth-related metabolism to defense-related metabolism.     

Proteome analysis of cortical neuronal cultures following cycloheximide, heat stress and MK801 preconditioning

Studying endogenous neuroprotective mechanisms induced by preconditioning may provide drug leads to reduce ischemic neuronal death. In this study, we used 2-DE to examine protein expression following cycloheximide, heat stress, and MK801 preconditioning in rat cortical neuronal cultures. Of 150 differentially expressed protein spots selected for identification the protein or tentative protein(s) were identified in 84 cases, representing 50 different proteins. Different protein spots representing the same protein or closely related protein(s) occurred for 21 of the identified proteins and are likely to represent PTMs or proteolytic fragments of the protein. Six protein spots (actin, elongation factor 1-alpha 1, peptidyl-prolyl cis-transisomerase A, Cu/Zn superoxide dismutase, stathmin, tropomyosin) were differentially expressed in all three preconditioning treatments. Twenty-seven protein spots were differentially expressed in two preconditioning treatments, while 51 spots were differentially expressed in one treatment. Three proteins heterogeneous nuclear ribonucleoproteins A2/B1, mitochondrial stress-70 protein, and tropomyosin were detected in control neuronal cultures, but not following one or more preconditioning treatments, while a posttranslational modified form of the voltage dependent anion channel 1 was only detected following cycloheximide preconditioning. In summary, this study has revealed multiple protein changes potentially involved in neuroprotective and neurodamaging pathways, which require further characterization.