Proteomic analysis of methyl parathion-responsive proteins in zebrafish (Danio rerio) brain

Methyl parathion (MP), an organophosphorus pesticide used worldwide, has been associated with a wide spectrum of toxic effects on organisms in the environment. This study set out to analyze the alteration of protein profiles in MP-exposed zebrafish (Danio rerio) brain and find the proteins responsive to MP toxicity. Zebrafish were subjected to 1, 3 and 5 mg/L MP and the proteomic changes in their brains were revealed using two-dimensional gel electrophoresis. Six protein spots were observed to be significantly changed by MP exposure. Among these, 4 spots were down-regulated, while 2 spots were up-regulated. These altered spots were excised from the gels and identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry and database searching. The results indicate that these proteins were involved in binding, catalysis, regulation of energy metabolism and cell structure. These data may provide novel biomarkers for the evaluation of MP contamination and useful insights for understanding the mechanisms of MP toxicity.     

Proteomic characterization of the effects of clofibrate on protein expression in rat liver

Clofibrate is a peroxisome proliferator known to induce liver tumours in rats. A proteomics study was conducted to provide new insights into the molecular mechanisms of clofibrate-induced non-genotoxic hepatocarcinogenesis. Rats were treated with 250 mg/kg day clofibrate orally and sacrificed after 7 days. Proteins extracted from the liver were analysed by 2-DE using DIGE technology. The protein identification performed by MS showed that clofibrate induced up-regulation of 77 proteins and down-regulation of 27 proteins. The highest expression ratios corresponded to proteins involved in a series of biochemical pathways such as lipid metabolism, fatty acid metabolism, amino acid metabolism, protein metabolism, citric acid cycle, xenobiotic detoxification and oxidative stress. Proteins implicated in cell proliferation and apoptosis, such as prohibitin, 10-formyl tetrahydrofolate dehydrogenase, senescence marker protein-30, pyridoxine 5'-phosphate oxidase and vimentin, were also identified as being regulated. These results provide leads for further investigations into the molecular mechanisms of liver tumours induced by clofibrate. In addition, MS results showed that a series of regulated proteins were detected as several spots corresponding to different pI and/or M(r). Differential effects on those variants could result from specific PTM and could be a specific molecular signature of the clofibrate-induced protein expression modulation in rat liver.     

DSA affinity glycoproteome of human liver tissue

Due to the critical roles of glycoproteins in the activities of cells to tissues, mapping of liver glycoproteome may provide valuable basic information for finding disease marker proteins. In this study, Datura Stramonium Agglutinin (DSA) was chosen to enrich N-linked glycoproteins for its broader specificity with tri- or tetra-antennary complex type. DSA affinity glycoproteins’ profiles of human liver tissue were generated by two-dimensional electrophoresis (2-DE) followed by glycoprotein staining based on multiplexed proteomics (MP) technology. 64 ± 3 (n = 3) protein spots were detected and 41 of glycoproteins were identified via peptide mass fingerprinting (PMF) using MALDI-TOF-MS/MS and annotated to IPI databases. Identified glycoproteins definitely take part in the regulation of cell cycle and metabolic processes. The detailed carbohydrate moiety of some glycoproteins might be concluded according to the literatures. The construction of DSA affinity glycoprotein database would contribute to the subsequent research.     

Analysis of the budding yeast pH 4–7 proteome in meiosis

Meiosis, the developmental programme generating haploid gametes from diploid precursors, requires two cell divisions and many innovations. In budding yeast, a large number of genes are expressed exclusively during meiosis while others are repressed compared to vegetative growth. Microarray analysis has shown that gene expression during meiosis is highly regulated, and has been used to classify yeast genes according to meiotic temporal expression pattern. In this study, we have begun to investigate the kinetics of meiotic protein expression using a proteomics approach. 2‐D DIGE was used to characterise the temporal protein expression patterns of the budding yeast pH 4–7 proteome in meiosis. More than 1400 meiotic protein spots were visualised and at least 63 spots were temporally regulated during meiosis in a statistically significant manner. Gel spots with significant expression changes were excised and 26 unique proteins were identified using LC‐MS/MS. The identified proteins could be classified into functional categories and the genes encoding a number of these were previously shown to be involved in yeast sporulation and meiosis. This data set was used to assemble the first differential 2‐D PAGE map of budding yeast meiosis, which can be accessed through a web server. This work represents one of the first quantitative proteomic analyses of meiosis in yeast and will provide a valuable resource for future investigations.     

Con A affinity glycoproteomics of normal human liver tissue

In order to establish the novel high throughput, high efficiency and low cost technological platform for the research of N-glycoproteomics, to resolve the significance of characteristic expression profile of glycoprotein and to find the proteins with biological functional importance, the glycoproteins with high-mannose core and the two antennary types were purified and enriched by the Con A affinity chromatography. Con A affinity protein expression profiles of normal human liver tissue were generated by using SDS-PAGE, two-dimensional electrophoresis (2-DE) followed by fast fluorescence staining based on multiplexed proteomics (MP) technology. 301 visible protein spots on the gel were detected and 85 of glycoproteins were further successfully identified via peptide mass fingerprinting (PMF) by a matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS/MS) and annotated to IPI databases. Identified glycoproteins definitely take part in the regulation of cell cycle and metabolic processes. The glycosylation sites were predicted with NetNGlyc 1.0 and NetOGlyc 3.1 software, meanwhile they were classified according to the geneontology methods. The construction of Con A affinity glycoprotein database of normal human liver tissue would contribute to the subsequent research.     

Investigation on glycosylation patterns of proteins from human liver cancer cell lines based on the multiplexed proteomics technology

Glycosylation, a very important post-translational modification of proteins, is increasingly coming into notice. However, large-scale, throughput investigations on glycosylated proteins are few. We applied a sensitive and fast fluorescence-based multiplexed proteomics (MP) technology which included two-dimensional gel electrophoresis (2-DE) followed by the fluorescence staining of glycoprotein and mass spectrometry identification for the purpose of constructing glycoprotein databases of the typical human hepatocellular carcinoma cell lines including Hep3B cell line without metastasis and MHCC97H with highly metastatic potential as well as the control non-tumor Chang liver cell. 74+/-2 (n=3), 78+/-3 (n=3) and 72+/-5 (n=3) glycoprotein spots were detected on 2-DE gels from Chang liver, Hep3B and MHCC97H cell sample using this MP technique, respectively. In all, 80 glycoproteins from three cell lines were successfully identified via peptide mass profiling using MALDI-TOF-MS/MS and the identified glycoproteins were annotated to our databases. In addition, we also found the glycosylation pattern differences among these three cell lines. The protein glycosylation alteration would be have great significance for the diagnosis of HCC and prediction of its metastasis. This study described the construction of glycosylation patterns of proteins and glycoproteome databases of human liver cells by the novel technological platform. The glycoproteome databases also provide essential basis for following study.     

Comparison of SYPRO Ruby and Deep Purple using commonly available UV transilluminator: wide-scale application in proteomic research

Fluorescent stains are becoming increasingly useful in proteomics research involving protein expression as well as post-translational modification studies and are particularly useful for samples which are expensive and scarce. The fluorescent dyes Deep Purple and SYPRO Ruby are widely used in protein expression studies. Using UV transillumination and Charged Coupled Device (CCD) based imaging system, their relative sensitivity to detect proteins separated by two-dimensional polyacrylamide gel electrophoresis and downstream protein identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was compared. Using mouse liver homogenate, we detected a greater number of spots using SYPRO Ruby over Deep Purple stain. However, the number of matched peptides and the percentage of amino acid residues identified for 21 different proteins were comparable suggesting their equivalency for LC-MS/MS identification. In spite of comparable MS compatibility, we recommend the use of SYPRO Ruby for expression proteomics due to its higher sensitivity in detecting protein spots.     

Comparative proteomic analysis of differentiation of mouse F9 embryonic carcinoma cells induced by retinoic acid

The multipotent mouse F9 embryonic carcinoma cell is an ideal model system to investigate the mechanism of retinoic acid (RA) in cell differentiation and cell growth control and the biochemical basis of early embryonic development. We reported here a proteomics approach to study protein expression changes during the differentiation of F9 cells into the visceral endoderm. F9 cells were incubated with or without RA at 0, 24, 48, and 72 h. Total proteins extracted were separated by two‐dimensional electrophoresis (2‐DE) and the protein patterns on the gels were comparatively analyzed by computer. Approximately 1,100 protein spots were detected in the F9 proteome, within the pH 3–10 range. Fourteen protein spots which the levels of expression were found to be altered dramatically during the F9 cells differentiating, and were identified by MALDI‐TOF MS or ESI‐MS/MS. These proteins included metabolism enzymes, HSP60s, RAN, hnRNP K, FUBP1, VDAC1, STI1, and prohibitin. These proteins are involved in cellar metabolism, gene expression regulation, stress response, and apoptosis, respectively. The data from proteomic analyze are consistent with the result obtained from Western blot analysis. This study increases our understanding of the proteomics changes during F9 cells differentiation induced by RA. J. Cell. Biochem. 113: 1811–1819, 2012. © 2012 Wiley Periodicals, Inc.     

Hepatic proteome sensitivity in rainbow trout after chronically exposed to a human pharmaceutical verapamil

Verapamil (VRP), a cardiovascular pharmaceutical widely distributed and persistent in the aquatic environment, has potential toxicity to fish and other aquatic organisms. However, the molecular mechanisms that lead to these toxic effects are not well known. In the present study, proteomic analysis has been performed to investigate the protein patterns that are differentially expressed in liver of rainbow trout exposed to sublethal concentrations of VRP (0.5, 27.0, and 270 μg/liter) for 42 days. Two-dimensional electrophoresis coupled with MALDI-TOF/TOF mass spectrometry was employed to detect and identify the protein profiles. The analysis revealed that the expression of six hepatic acidic proteins were markedly altered in the treatment groups compared with the control group; three proteins especially were significantly down-regulated in fish exposed to VRP at environmental related concentration (0.5 μg/liter). These results suggested that the VRP induce mechanisms against oxidative stress (glucose-regulated protein 78 and 94 and protein disulfide-isomerase A3) and adaptive changes in ion transference regulation (calreticulin, hyperosmotic glycine-rich protein). Furthermore, for the first time, protein Canopy-1 was found to be significantly down-regulated in fish by chronic exposure to VRP at environmental related levels. Overall, our work supports that fish hepatic proteomics analysis serves as an in vivo model for monitoring the residual pharmaceuticals in aquatic environment and can provide valuable insight into the molecular events in VRP-induced toxicity in fish and other organisms.     

Proteomic analysis and identification of aqueous humor proteins with a pathophysiological role in diabetic retinopathy

Diabetic retinopathy (DR) can cause irreversible blindness and is the severest microvascular complication in the eyes of patients with diabetic mellitus (DM). The identification of susceptibility factors contributing to development of DR is helpful for identifying predisposed patients and improving treatment efficacy. Although proteomics analysis is useful for identifying protein markers related to diseases, it has never been used to explore DR-associated susceptibility factors in the aqueous humor (AH). To better understand the pathophysiology of DR and to identify DR-associated risk factors, a gel-based proteomics analysis was performed to compare AH protein profiles of DM patients with and without development of DR. MALDI-TOF MS was then performed to identify protein spots that were differentially expressed between the two groups and western blot analysis was used to validate the expressional change of protein demonstrated by proteomics. Our proteomics and bioinformatics analysis identified 11 proteins differentially expressed between DR and control groups. These proteins are linked to biological networks associated with nutrition transport, microstructure reorganization, angiogenesis, anti-oxidation, and neuroprotection. The data may provide potential AH biomarkers and susceptibility factors for predicting DR development, and provide an insight into the underlying pathophysiological mechanisms of DR. This article is part of a Special Issue entitled: Proteomics: The clinical link.     

Con A affinity glycoproteomics of normal human liver tissue

In order to establish the novel high throughput, high efficiency and Iow cost technological platform for the research of N-glycoproteomics, to resolve the significance of characteristic expression profile of glycoprotein and to find the proteins with biological functional importance, the glycoproteins with high-mannose core and the two antennary types were purified and enriched by the Con A affinity chromatography. Con A affinity protein expression profiles of normal human liver tissue were generated by using SDS-PAGE, two-dimensional electrophoresis (2-DE) followed by fast fluorescence staining based on multiplexed proteomics (MP) technology. 301 visible protein spots on the gel were detected and 85 of glycoproteins were further successfully identified via peptide mass fingerprinting (PMF) by a matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS/MS) and annotated to IPI databases. Identified glycoproteins definitely take part in the regulation of cell cycle and metabolic processes. The glycosylation sites were predicted with NetNGlyc 1.0 and NetOGlyc 3.1 software, meanwhile they were classified according to the geneontology methods. The construction of Con A affinity glycoprotein database of normal human liver tissue would contribute to the subsequent research.     

Proteomic analysis of seed development in Chinese fir (<Emphasis Type="Italic">Cunninghamia lanceolata</Emphasis>)

Seed development is a complex process governed by highly coordinated changes in the expression of a large protein set. DIGE (Difference Gel Electrophoresis)-based proteomics was used to study developing Chinese fir seeds. 153 spots were obtained by using the analysis of DeCyder software (v. 6.5). Cluster analysis showed that they could be joined into three main groups. Eleven spots, more actively expressed at early cotyledonary stage of developing seeds, were identified by LC/MS/MS (tandem MS). Ten spots were identified by searching NCBInr or EST databases. They included two legumin-like storage proteins, LEA protein, small heat-shock protein, PR10-1.13, a protein similar to eukaryotic translation initiation factor, a protein similar to maternal effect embryo arrest 51, ORF115, a protein similar to monodehydroascorbate reductase, and unknown proteins. The potential function of these proteins during the precotyledonary stage of seed development was discussed.     

Comparison of two anoxia models in rainbow trout cells by a 2-DE and MS/MS-based proteome approach

In the literature, a variety of ways have been used to obtain anoxia, and most often results are compared between studies without taking into consideration how anoxia has been obtained. Here, we provide a comprehensive study of two types of anoxia, using a proteomics approach to compare changes in protein expression. The two investigated situations were 30 min of chemical anoxia (10 mM NaN(3)) followed by reoxygenation overnight (CR) and 2 h of N(2)-induced anoxia (achieved by flushing with N(2)) followed by reoxygenation overnight (NR), after which samples were resolved by 2-DE. Forty-five protein spots changed their abundance in response to CR and 35 protein spots changed their abundance in response to NR, but only six proteins changed their abundance in response to both stimuli. By the means of MS/MS, 40 protein spots were identified including proteins involved in processes like cell protection and protein synthesis. It was also revealed that the level of a number of keratins was down-regulated. This study therefore provides a valuable comparison of two different anoxia models and shows that great care should be taken when comparing the effects of anoxia in studies that have used different types and durations of anoxia.     

Proteomic response to sublethal cadmium exposure in a sentinel fish species, Cottus gobio

The present study aimed at evaluating the toxicity of short-term cadmium (Cd) exposure in the European bullhead Cottus gobio, a candidate sentinel species. Several enzymatic activity assays (citrate synthase, cytochrome c oxidase, and lactate dehydrogenase) were carried out in liver and gills of fish exposed to 0.01, 0.05, 0.25, and 1 mg Cd/L for 4 days. Exposure to high Cd concentrations significantly altered the activity of these enzymes either in liver and/or in gills. Second, 2D-DIGE technique was used to identify proteins differentially expressed in tissues of fish exposed to either 0.01 or 1 mg Cd/L. Fifty-four hepatic protein spots and 37 branchial protein spots displayed significant changes in abundance in response to Cd exposure. A total of 26 and 12 different proteins were identified using nano LC-MS/MS in liver and gills, respectively. The identified differentially expressed proteins can be categorized into diverse functional classes, related to metabolic process, general stress response, protein fate, and cell structure for instance. This work provides new insights into the biochemical and molecular events in Cd-induced toxicity in fish and suggests that further studies on the identified proteins could provide crucial information to better understand the mechanisms of Cd toxicity in fish.     

Influence of acute cadmium exposure on the liver proteome of a teleost fish,ayu (<Emphasis Type="Italic">Plecoglossus altivelis</Emphasis>)

Cadmium (Cd) is a toxic heavy metal that causes the disruption of a variety of physiological processes. In this study, the effect of Cd on liver proteome of ayu, Plecoglossus altivelis, was investigated by two-dimensional gel electrophoresis (2-DE) and matrix assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF–MS/MS). Twenty-three altered protein spots were successfully identified. They were involved in oxidative stress response, metal metabolism, methylation, and so on. The mRNA expression of 60S acidic ribosomal protein P0, heat shock protein 70, apolipoprotein A-I, betaine-homocysteine S-methyltransferase, parahox cluster neighbor, and transferrin was subsequently determined by real-time PCR. The mRNA expression of these genes was consistent with proteomic results. These findings enrich our knowledge on the influence of Cd toxicity to teleost fish, and may be worthy of further investigation to develop biomarkers.     

Proteome Changes in Thai Indigenous Chicken Muscle during Growth Period

Proteomic profiling of the pectoralis muscle of Thai indigenous chickens during growth period was analyzed using two-dimensional gel electrophoresis (2-DE) and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF/MS). A total of 259, 161, 120 and 107 protein spots were found to be expressed in the chicken pectoralis muscles at 0, 3, 6 and 18 weeks of age, respectively. From these expressed proteins, five distinct protein spots were significantly associated with chicken age. These protein spots were characterized and showed homology with phosphoglycerate mutase 1 (PGAM1), apolipoprotein A1 (APOA1), triosephosphate isomerase 1 (TPI1), heat shock protein 25 kDa (HSP25) and fatty acid binding protein 3 (FABP3). These five protein spots were categorized as follows: (i) the expression levels of PGAM1 and TPI1 proteins were positively correlated with chicken aging (p<0.05), (ii) the expression levels of APOA1 and FABP3 proteins were negatively correlated with chicken aging (p<0.05) and (iii) the expression levels of the HSP25 protein were up- and down-regulated during growth period. Moreover, the mRNA expression levels of the FABP3 and HSP25 genes were significantly decreased in muscle during the growth period (p<0.05), whereas no significant changes of the PGAM1, TPI1 and APOA1 gene expression from the chicken muscle was observed. The identified proteins were classified as metabolic and stress proteins. This demonstrates a difference in energy metabolism and stress proteins between age groups and shows that proteomics is a useful tool to uncover the molecular basis of physiological differences in muscle during the growth period.     

Proteome modifications of juvenile beluga (Huso huso) brain as an effect of dietary methylmercury

Methylmercury (MeHg) is the most toxic form of mercury which is bioaccumulated in the aquatic food chain. It has been shown that one of the main targets of MeHg toxicity is the brain, but there is little knowledge of the molecular mechanisms of its toxic effects. In this work we used a proteomics analysis to determine the changes in the brain proteome of juvenile beluga (Huso huso) exposed to dietary MeHg. The juvenile beluga were fed the diet containing 0.8 ppm MeHg for 70 days. Proteins of the brain tissue were analyzed using two-dimensional electrophoresis and MALDI-TOF/TOF mass spectrometry. We found eight proteins with significant altered expression level in the fish brain exposed to MeHg. These proteins are involved in different cell functions including cell metabolism, protein folding, cell division, and signal transduction. Our results support the idea that MeHg exerts its toxicity through oxidative stress induction and apoptotic effects. They also suggest that chronic MeHg exposure would induce an important metabolic deficiency in the brain. These findings provide basic information to understand possible mechanisms of MeHg toxicity in aquatic ecosystems.