Proteomic analysis of altered proteins in lymphoid organ of yellow head virus infected Penaeus monodon

A comparative proteomic analysis was employed to identify altered proteins in the yellow head virus (YHV) infected lymphoid organ (LO) of Penaeus monodon. At 24 h post-infection, the infected shrimps showed obvious signs of infection, while the control shrimps remained healthy. Two-dimensional electrophoresis of proteins extracted from the LO revealed significant alterations in abundance of several proteins in the infected group. Protein identification by MALDI-TOF MS and nanoLC-ESI-MS/MS revealed significant increase of transglutaminase, protein disulfide isomerase, ATP synthase beta subunit, V-ATPase subunit A, and hemocyanin fragments. A significant decrease was also identified for Rab GDP-dissociation inhibitor, 6-phosphogluconate dehydrogenase, actin, fast tropomyosin isoform, and hemolymph clottable protein. Some of these altered proteins were further investigated at the mRNA level using real-time RT-PCR, which confirmed the proteomic data. Identification of these altered proteins in the YHV-infected shrimps may provide novel insights into the molecular responses of P. monodon to YHV infection.     

Comparative proteomic approach to identify proteins involved in flooding combined with salinity stress in soybean

Salinity together with waterlogging or flooding, a condition that occurs frequently in the field, can cause severe damage to crops. Combined flooding and salinity decreases the growth and survival of plants more than either stress alone. We report here the first proteomic analysis to investigate the global effects of saline flooding on multiple metabolic pathways. Soybean seedlings at the emergence (VE) stage were treated with 100 mM NaCl and flooded with water or 100 mM sodium chloride solution for 2 days. Proteins were extracted from hypocotyl and root samples and analyzed by two-dimensional gel electrophoresis followed by MALDI-TOF, MALDI-TOF/TOF mass spectrometry or immunoblotting. A total of 43 reproducibly resolved, differentially expressed protein spots visualized by Coomassie brilliant blue staining were identified by MALDI-TOF MS. Identities of several proteins were also validated by MS/MS analysis or immunoblot analysis. Twenty-nine proteins were upregulated, eight proteins were downregulated and six spots were newly induced. The identified proteins include well-known salt and flooding induced proteins as well as novel proteins expressed by the salinity-flooding combined stress. The comparative analysis identified changes at the proteome level that are both specific and part of a common or shared response. The identification of such differentially expressed proteins provides new targets for future studies that will allow assessment of their physiological roles and significance in the response of glycophytes to a combination of flooding and salinity.     

Proteomic Analysis of Cd-Responsive Proteins in Solanum torvum

Proteomic changes induced by Cd have been described in plants in different scenarios. However, there has been no proteomic study on Cd toxicity, including any low Cd-accumulating species. Here, we investigate the response of a low Cd-accumulating species, Solanum torvum, to Cd toxicity at the root proteomic level using two-dimensional gel electrophoresis (2-DGE). The root 2-DGE map consisted of at least 927 reproducible protein spots, of which 45 were classified as differentially expressed proteins based on three replicated separations. MALDI-TOF MS analysis identified 19 of these spots, and MALDI-TOF/TOF MS analysis identified 8 of the spots. The eight proteins identified were two S-adenosylmethionine (SAM) synthetases, actin, an ATP synthase subunit, two tubulin proteins, alcohol dehydrogenase (ADH), and 14-3-3 protein 4. These proteins are involved in phytohormone synthesis, defense responses, energy metabolism, and cytoskeleton construction. Thus, our proteomic analysis revealed that Cd stress promotes an increase in the abundance of proteins involved in antioxidant defenses and anti-stress protection.     

Proteomic understanding of intracellular responses of recombinant chinese hamster ovary cells adapted to grow in serum‐free suspension culture

To understand the intracellular responses in recombinant Chinese hamster ovary (rCHO) cells adapted to grow in serum‐free suspension culture, a proteomic approach was employed. After rCHO cells producing erythropoietin were adapted to grow in suspension culture with the two different serum‐free media (SFM4CHO? and SF‐L1), proteome analyses were carried out using 2‐D PAGE and based on spot intensities, 58 high‐intensity protein spots were selected. Of the 58 protein spots, which represented 34 different kinds of proteins, 55 were identified by MALDI‐TOF‐MS, and MS/MS. Compared with the results in serum‐containing medium, six proteins, four de novo synthesis of nucleotides‐related proteins (dihydrolipoamide S‐acetyltransferase, transaldolase, inosine‐5′‐monophosphate dehydrogenase 2, and lymphoid‐restricted membrane protein) and two molecular chaperones (heat shock protein 70 kDa and 60 kDa [HSC70, HSP60]) were significantly increased in SFM4CHO?. From the results of proteomic analysis, HSP60 and HSC70, which were increased in both SFM, were selected as candidate proteins for engineering and rCHO cell lines overexpressing these genes were constructed. Cells overexpressing HSP60 and/or HSC70 showed 10–15% enhanced cell concentration during serum‐free adaptation and 15–33% reduction in adaptation time. Taken together, identification of differentially expressed proteins in rCHO cells by a proteomic study can provide insights into understanding the intracellular events and clues to find candidate genes for cell engineering for improved performance of rCHO cells during adaptation to serum‐free suspension culture. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Physical training improves thermogenesis and insulin pathway,and induces remodeling in white and brown adipose tissues

Physical training (PT) has been considered as a treatment in metabolic syndrome (MS), since it induces thermogenic activity in brown (BAT) and white (WAT) adipose tissues. We evaluated the therapeutic effect of PT on activity of WAT and BAT in rats with MS induced by high-fat diet (30% lard) for 13 weeks and submitted, for the last 6 weeks, to swimming or kept sedentary (SED) rats. MS-SED rats compared to control diet (CT-SED) rats showed low physical fitness and high levels of glucose, insulin, homeostasis evaluation of insulin resistance (HOMA-IR), homeostasis evaluation of the functional capacity of β-cells (HOMA-β), and blood pressure. The gastrocnemius muscle decreased in peroxisome proliferator-activated receptor gamma coactivator 1-alpha and beta (PGC-1α, PGC-1β), and uncoupled protein 2 and 3 (UCP2 and UCP3) expressions. Both WAT and BAT increased in the adipocyte area and decreased in blood vessels and fibroblast numbers. WAT increased in expression of pro-inflammatory adipokines and decreased in anti-inflammatory adipokine and adiponectin. WAT and gastrocnemius showed impairment in the insulin signaling pathway. In response to PT, MS rats showed increased physical fitness and restoration of certain biometric and biochemical parameters and blood pressure. PT also induced thermogenic modulations in skeletal muscle, WAT and BAT, and also improved the insulin signaling pathway. Collectively, PT was effective in treating MS by inducing improvement in physical fitness and interchangeable effects between skeletal muscle, WAT and BAT, suggesting a development of brown-like adipocyte cells.     

Proteome changes in tomato fruits prior to visible symptoms of chilling injury are linked to defensive mechanisms, uncoupling of photosynthetic processes and protein degradation machinery

A comparative proteomic analysis between tomato fruits stored at chilling and non-chilling temperatures was carried out just before the appearance of visible symptoms of chilling injury. At this stage of the stress period it was possible to discriminate between proteins involved in symptoms and proteins implicated in response. To investigate the changes in the tomato fruit proteome under this specific stressful condition, two-dimensional differential in-gel electrophoresis coupled with spot identification by mass spectrometry was applied. This proteomic approach allowed the identification of differentially expressed proteins which are involved in two main biological functions: (i) defensive mechanisms represented by small heat shock and late embryogenesis proteins; and (ii) reaction to the uncoupling of photosynthetic processes and the protein degradation machinery. One of the first changes observed in chilled fruits is the down-regulation of ATP synthase, 26S proteasome subunit RPN11 and aspartic proteinase, whereas the first responses in order to deal with the stress are mainly multifunctional proteins involved not only in metabolism but also in stress regulation such as glyceraldehyde phosphate dehydrogenase, 2-oxoglutarate dehydrogenase and invertase. In addition, our data seem to indicate a possible candidate to be used as a protein marker for further studies on cold stress: aldose-1-epimerase, which seems to have an important role in low temperature tolerance.     

Antibacterial effect and proteomic analysis of graphene-based silver nanoparticles on a pathogenic bacterium Pseudomonas aeruginosa

Graphene-based silver nanoparticles (Ag NPs–GE) material has been developed and demonstrated antibacterial effect against Escherichia coli and Pseudomonas aeruginosa. In this study, the antibacterial activity and mechanism on P. aeruginosa were investigated. The experiments results showed the minimum bactericidal concentration of Ag NPs–GE to P. aeruginosa is 20 μg/ml. When P. aeruginosa were exposed to 20 μg/ml Ag NPs–GE for 1 h, the cell wall was breakdown. In order to study the mechanism of antibacterial effect of Ag NPs–GE, two-dimensional electrophoresis was carried out to compare the protein expressional profiles of P. aeruginosa exposed to 5 μg/ml Ag NPs–GE or 5 μg/ml AgNO₃ with the untreated bacteria. Identification of differentially expressed protein was performed by MALDI–TOF/TOF MS. The change of proteomic profile induced by Ag NPs–GE was distinct from that induced by AgNO₃. Seven identified proteins were found induced and nine proteins were suppressed by Ag NPs–GE. Five identified proteins were found induced and twenty proteins were suppressed by AgNO₃. In addition, either Ag NPs–GE or AgNO₃ suppressed the expression of eight proteins, amidotransferase, 30S ribosomal protein S6, bifunctional proline dehydrogenase/pyrroline-5-carboxylate dehydrogenase, arginyl-tRNA synthetase, nitroreductase, acetolactate synthase 3, methionyl-tRNA synthetase and periplasmic tail-specific protease. Furthermore, gene ontology analysis and KEGG pathway analysis were used to characterize the functions of those proteins.     

Lipid synthesis is promoted by hypoxic adipocyte-derived exosomes in 3T3-L1 cells

Hypoxia occurs within adipose tissues as a result of adipocyte hypertrophy and is associated with adipocyte dysfunction in obesity. Here, we examined whether hypoxia affects the characteristics of adipocyte-derived exosomes. Exosomes are nanovesicles secreted from most cell types as an information carrier between donor and recipient cells, containing a variety of proteins as well as genetic materials. Cultured differentiated 3T3-L1 adipocytes were exposed to hypoxic conditions and the protein content of the exosomes produced from these cells was compared by quantitative proteomic analysis. A total of 231 proteins were identified in the adipocyte-derived exosomes. Some of these proteins showed altered expression levels under hypoxic conditions. These results were confirmed by immunoblot analysis. Especially, hypoxic adipocyte-released exosomes were enriched in enzymes related to de novo lipogenesis such as acetyl-CoA carboxylase, glucose-6-phosphate dehydrogenase, and fatty acid synthase (FASN). The total amount of proteins secreted from exosomes increased by 3–4-fold under hypoxic conditions. Moreover, hypoxia-derived exosomes promoted lipid accumulation in recipient 3T3-L1 adipocytes, compared with those produced under normoxic conditions. FASN levels were increased in undifferentiated 3T3-L1 cells treated with FASN-containing hypoxic adipocytes-derived exosomes. This is a study to characterize the proteomic profiles of adipocyte-derived exosomes. Exosomal proteins derived from hypoxic adipocytes may affect lipogenic activity in neighboring preadipocytes and adipocytes.     

Proteomics profiling of fiber development and domestication in upland cotton (Gossypium hirsutum L.)

Comparative proteomic analyses were performed to detail the evolutionary consequences of strong directional selection for enhanced fiber traits in modern upland cotton (Gossypium hirsutum L.). Using two complementary proteomic approaches, 2-DE and iTRAQ LC–MS/MS, fiber proteomes were examined for four representative stages of fiber development. Approximately 1,000 protein features were characterized using each strategy, collectively resulting in the identification and functional categorization of 1,223 proteins. Unequal contributions of homoeologous proteins were detected for over a third of the fiber proteome, but overall expression was balanced with respect to the genome-of-origin in the allopolyploid G. hirsutum. About 30 % of the proteins were differentially expressed during fiber development within wild and domesticated cotton. Notably, domestication was accompanied by a doubling of protein developmental dynamics for the period between 10 and 20 days following pollination. Expression levels of 240 iTRAQ proteins and 293 2-DE spots were altered by domestication, collectively representing multiple cellular and metabolic processes, including metabolism, energy, protein synthesis and destination, defense and stress response. Analyses of homoeolog-specific expression indicate that duplicated gene products in cotton fibers can be differently regulated in response to selection. These results demonstrate the power of proteomics for the analysis of crop domestication and phenotypic evolution.     

Comparative proteomics analysis of global cellular stress responses to hydroxyurea-induced DNA damage in HeLa cells

Both environmental agents and spontaneous cellular events cause serious DNA damage, threatening the integrity of the genome. In response to replication stress or genotoxic agents triggered DNA damage, degradation of p12 subunit of DNA polymerase delta (Pol δ) results in an inter-conversion between heterotetramer (Pol δ4) and heterotrimer (Pol δ3) forms and plays a significant role in DNA damage response in eukaryotic cells. In this work, we used mass spectrometry-based proteomic approach to identify those cellular stress response protein changes corresponding to the degradation of p12 in DNA-damaged HeLa cells by the treatment with hydroxyurea (HU). A total of 736 ± 13 proteins in non-treated control group and 741 ± 19 protein spots in HU-treated cells were detected, of which 34 proteins (17 up-regulated and 17 down-regulated) exhibited significantly altered protein expression levels. Their physiological roles are mainly associated with cellular components, molecular functions, and biological processes by gene ontology analysis, among which 21 proteins were mapped to KEGG pathways. They are involved in 5 primary pathways with the subsets involving 16 secondary pathways by further KEGG analysis. More interestingly, the up-regulation of translationally controlled tumor protein was further identified to be associated with p12 degradation by Western blot analysis. Our works may enlarge and broaden our view for deeply understanding how global cellular stress responds to DNA damage, which could contribute to the etiology of human cancer or other diseases that can result from loss of genomic stability.     

Evaluation of three different protocols of protein extraction for Arabidopsis thaliana leaf proteome analysis by two-dimensional electrophoresis

This work was performed to compare three precipitation protocols of protein extraction for 2-DE proteomic analysis using Arabidopsis leaf tissue: TCA-acetone, phenol, and TCA-acetone-phenol. There were no statistically significant differences in protein yield between the three methods. Samples were subjected to 2-DE in the 5 to 8 pH and 14-80 kDa ranges. The TCA-acetone-phenol protocol provided the best results in terms of spot focusing, resolved spots, spot intensity, unique spots detected, and reproducibility. In all, 93 qualitative or quantitative statistically significant differential spots were found between the three protocols. The 2-DE map of TCA-acetone-phenol extracts presented more resolved spots above 40 kDa, with no pI-dependent differences observed between the three protocols. 54 spots were selected for trypsin digestion, and the peptides were analyzed by MALDI-TOF-TOF MS. After database search using peptide mass fingerprinting, and MS/MS combined search, 30 proteins were identified, the proteins from chloroplastic photosynthetic and carbohydrate metabolism being those most highly represented. From these data, we were able to conclude that each extraction protocol had its main features. Considering this, the workflow of any standard comparative proteomic experiment should include the optimization and adaptation of the protein extraction protocol to the plant tissue and to the particular objective pursued.     

Host plant adaptability and proteomic differences of diverse Rhopalosiphum maidis (Fitch) lineages

Corn leaf aphid Rhopalosiphum maidis (Fitch) can feed on various cereal crops and transmit viruses that may cause serious economic losses. To test the impact of both host plant species and age on R. maidis, as well as the proteomic difference of diverse populations, we first investigated the survival and reproduction of six R. maidis populations (i.e., LF, HF, GZ, DY, BJ, and MS) via a direct observation method in the laboratory on 10 and 50 cm high maize seedlings, and 10 cm high barley seedlings. Then a proteomic approach was implemented to identify the differentially expressed proteins from both aphids and endosymbionts of BJ and MS populations. Results indicated that the BJ population performed significantly better than the others on both barley and 50 cm high maize seedlings, while no population could survive on 10 cm high maize seedlings. The proteomic results demonstrated that the expression levels of myosin heavy chain (muscle isoform X12) (spot 781) and peroxidase (spot 1383) were upregulated, while ATP-dependent protease Hsp 100 (spot 2137) from Hamiltonella defensa and protein SYMBAF (spot 2703) from Serratia symbiotica were downregulated in the BJ population when compared to expression levels of the MS population. We hypothesize that the fatalness observed on 10 cm high maize seedlings may be caused by secondary metabolites that are synthesized by the seedlings and the MS population of R. maidis should be more stress-resistant than the BJ population. Our results also provide insights for understanding the interaction between host plants and aphids.     

Serum depletion induces changes in protein expression in the trophoblast-derived cell line HTR-8/SVneo

Background

How nutrition and growth factor restriction due to serum depletion affect trophoblast function remains poorly understood. We performed a proteomic differential study of the effects of serum depletion on a first trimester human immortalized trophoblast cell line.

Methods

The viability of HTR-8/SVneo trophoblast cells in culture with 0, 0.5 and 10 % fetal bovine serum (FBS) were assayed via MTT at 24, 48 and 64 h. A comparative proteomic analysis of the cells grown with those FBS levels for 24 h was performed using two-dimensional electrophoresis (2DE), followed by mass spectrometry for protein spot identification, and a database search and bioinformatics analysis of the expressed proteins. Differential spots were identified using the Kolmogorov-Smirnov test (n?=?3, significance level 0.10, D?>?0.642) and/or ANOVA (n?=?3, p?<?0.05).

Results

The results showed that low serum doses or serum depletion differentially affect cell growth and protein expression. Differential expression was seen in 25 % of the protein spots grown with 0.5 % FBS and in 84 % of those grown with 0 % FBS, using 10 % serum as the physiological control. In 0.5 % FBS, this difference was related with biological processes typically affected by the serum, such as cell cycle, regulation of apoptosis and proliferation. In addition to these changes, in the serum-depleted proteome we observed downregulation of keratin 8, and upregulation of vimentin, the glycolytic enzymes enolase and pyruvate kinase (PKM2) and tumor progression-related inosine-5’-monophosphate dehydrogenase 2 (IMPDH2) enzyme. The proteins regulated by total serum depletion, but not affected by growth in 0.5 % serum, are members of the glycolytic and nucleotide metabolic pathways and the epithelial-to-mesenchymal transition (EMT), suggesting an adaptive switch characteristic of malignant cells.

Conclusions

This comparative proteomic analysis and the identified proteins are the first evidence of a protein expression response to serum depletion in a trophoblast cell model. Our results show that serum depletion induces specific changes in protein expression concordant with main cell metabolic adaptations and EMT, resembling the progression to a malignant phenotype.

     

Proteomic adaptation to chronic high intensity swimming training in the rat heart

Cardiac hypertrophy induced by exercise is associated with less cardiac fibrosis and better systolic and diastolic function, suggesting that the adaptive mechanisms may exist in exercise-induced hypertrophy. To identify molecular mechanisms by which exercise training stimulates this favorable phenotype, a proteomic approach was employed to detect rat cardiac proteins that were differentially expressed or modified after exercise training. Sixteen male Sprague–Dawley rats were divided into trained (T) and control(C). T rats underwent eight weeks of swimming training seven days/week, using a high intensity protocol. Hearts were used to generate 2-D electrophoretic proteome maps. Training significantly altered 23 protein spot intensities (P < 0.05), including proteins associated with the mitochondria oxidative metabolism, such as prohibitin, malate dehydrogenase, short-chain acyl-CoA dehydrogenase, triosephosphate isomerase, electron transfer flavoprotein subunit beta, ndufa10 protein, ATP synthase subunit alpha and isocitrate dehydrogenase [NAD] subunit. Additionally, Prohibitin was increased in the exercise-induced hearts. Cytoskeletal, signal pathway, stress and oxidative proteins also increased within T groups. These results strongly support the notion that the observed changes in the expression of energy metabolism proteins resulted in a potential increase in the capacity to synthesise ATP, probably via mitochondrial oxidative metabolism. The observed changes in the expression of these metabolic and structural proteins induced by training may beneficially influence heart metabolism, stress response and signalling paths, and therefore improve the overall cardiac function.     

Exposure to an extremely low-frequency electromagnetic field only slightly modifies the proteome of Chromobacterium violaceumATCC 12472

Several studies of the physiological responses of different organisms exposed to extremely low-frequency electromagnetic fields (ELF-EMF) have been described. In this work, we report the minimal effects of in situ exposure to ELF-EMF on the global protein expression of Chromobacterium violaceum using a gel-based proteomic approach. The protein expression profile was only slightly altered, with five differentially expressed proteins detected in the exposed cultures; two of these proteins (DNA-binding stress protein, Dps, and alcohol dehydrogenase) were identified by MS/MS. The enhanced expression of Dps possibly helped to prevent physical damage to DNA. Although small, the changes in protein expression observed here were probably beneficial in helping the bacteria to adapt to the stress generated by the electromagnetic field.     

Evaluation of sample extraction methods for proteomic analysis of coniferous seeds

In this study, three methods of protein extraction from the seeds of the Chinese fir were compared by examining the quality (including the number of protein spots observed) in the two-dimensional gel electrophoresis (2-DE), obtained by isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis of the total released protein. Three protein extraction methods were: TCA-acetone precipitation, SDS extraction/acetone precipitation, and phenol extraction methanol/ammonium acetate precipitation. The results showed that TCA-acetone precipitation was the most effective method for protein extraction; it gave the highest yield of total protein (8.9 mg protein per g seed weight) and the greatest number of proteins spots (1,034 spots) on the 2-DE gel. Further, several proteins were identified by liquid chromatography mass spectrometry (LC MS/MS), which are legumin-like storage protein, similar to AMP binding/acetate-CoA ligase, similar to 40S ribosomal protein S20, actin, ascorbate peroxidase, Similar to cysteine synthase, and unknown protein. These data demonstrates that TCA-acetone precipitation followed by 2-DE and LC MS/MS is a suitable method for proteomic analysis of coniferous species, such as Chinese fir and provides a valuable starting point for similar proteomic analysis of other coniferous tree species.     

Identification of HSP90 as Potential Biomarker of Biliary Atresia Using Two-Dimensional Electrophoresis and Mass Spectrometry

Biliary atresia (BA) is a devastating cholestatic liver disease targeting infants. Current diagnosis depends on surgical exploration of the biliary tree. The aim of the present study was to identify potential biomarkers for the diagnosis of biliary atresia (BA). Two-dimensional electrophoresis was utilized for the identification of proteins that were differentially expressed in liver biopsies of 20 BA patients and 12 infants with non-BA neonatal cholestasis (NC) as controls. Using mass spectrometry, we identified 15 proteins with expressions significantly altered. Out of the 15 proteins identified, heat shock protein (HSP) 90 was the most significantly altered and was down-regulated in BA samples compared to NC samples using immunoblotting analysis. Our findings suggest that HSP90 might be a potential biomarker for the diagnosis of BA and may be used for monitoring further development and therapy for BA. This study demonstrated that a comprehensive strategy of proteomic identification combined with further validation should be adopted in biomarker discovery.