2D Gel Research Articles

A robust protocol for the isolation of cellular proteins from Xanthomonas campestris to analyze the methionine effect in 2D-gel experiments.

Two-dimensional PAGE (2D-PAGE) is a key technique for the separation of complex protein samples to survey the protein inventory of prokaryotic microorganisms. Although the preparation of proteins is a critical step in 2D gel experiments, its effect on the outcome of proteome data is often underestimated. In this work, we show that the choice of protein isolation method can have a profound impact on the quality of 2D gels of protein extracts from prokaryotes. Based on Xanthomonas campestris, a commercially relevant producer of the thickening agent xanthan, we validated a phenol extraction protocol for the purification of bacterial proteins that provides excellent 2D gel separation. As a proof of concept, this method was used to study the effect of methionine-a medium compound that reduces the xanthan output of industrial fermentations-on the cellular proteome of Xanthomonas. The detection of nine regulated proteins associated with sulfur metabolism (Cgl, CysI, CysJ, CysK, MetH1, MetY) and sugar nucleotide biosynthesis (Pgi, Ugd, XanA) proved the efficiency of phenol extraction for the screening of statistically significant abundance changes in 2D gel spots and MALDI-TOF-MS based identification in bacteria. Since this method is very robust, it may be useful for the study of other prokaryotes that are relevant in industrial biotechnology.

Reduction and Alkylation of Proteins in 2D Gel Electrophoresis: Before or after Isoelectric Focusing?

Reduction and Alkylation of Proteins in 2D Gel Electrophoresis: Before or after Isoelectric Focusing?

Proteomic Profile of Mycobacterium Tuberculosis after Eupomatenoid-5 Induction Reveals Potential Drug Targets

We investigated a proteome profile, protein-protein interaction and morphological changes of Mycobacterium tuberculosis after different times of eupomatenoid-5 (EUP-5) induction to evaluate the cellular response to the drug-induced damages. The bacillus was induced to sub-minimal inhibitory concentration of EUP-5 at 12 h, 24 h and 48 h. The proteins were separated by 2D gel electrophoresis, identified by LC/MS-MS. Scanning electron microscopy and Search Tool for the Retrieval of Interacting Genes/Proteins analyses were performed. EUP-5 impacts mainly in M. tuberculosis proteins of intermediary metabolism and interactome suggests a multisite disturbance that contributes to bacilli death. Scanning electron microscopy revealed the loss of bacillary form. Some of the differentially expressed proteins have the potential to be drug targets such as citrate synthase (Rv0896), phosphoglycerate kinase (Rv1437), ketol-acid reductoisomerase (Rv3001c) and ATP synthase alpha chain (Rv1308).

Inhibition of 17beta-hydroxysteroid dehydrogenase type 7 modulates breast cancer protein profile and enhances apoptosis by down-regulating GRP78

17beta-hydroxysteroid dehydrogenase type 7 (17β-HSD7) promotes breast cancer cell growth via dual-catalytic activity by modulating estradiol and DHT. Here, we clarified the expression pattern of 17β-HSD7 in postmenopausal luminal A type breast cancer with The Cancer Genome Atlas (TCGA) cohort. The impact of 17β-HSD7 inhibition on the proteome of MCF-7 cells was investigated and on cell apoptosis was revealed. MCF-7 cells were treated with an efficient inhibitor of 17β-HSD7 (INH7) or with vehicle, and a differential proteomics study was performed using two-dimensional (2D) gel electrophoresis followed by mass spectrometry and ingenuity pathway analysis (IPA). Cell apoptosis was analyzed by flow cytometry, followed by reverse transcription quantitative real-time PCR (RT-qPCR) and Western blot to investigate the expression of apoptosis-related genes. Our data showed 17β-HSD7 is amplified in primary and progressive breast cancer, inhibition of 17β-HSD7 in MCF-7 cells modulated 104 proteins primarily involved in cell death/survival, cell growth and DNA processing. The expression of 78kDa glucose-regulated protein (GRP78) and anti-apoptosis factor Bcl-2 were significantly suppressed via 17β-HSD7 inhibition with INH7, consequently induced MCF-7 cell apoptosis. However, INH7 treatment of T47D, another widely used epithelial ER+ breast cancer cell line, led to an up-regulation of GRP78 expression, resulting in a limited increase in apoptosis. These results suggest cell-specific effects of INH7 in the breast cancer, which is interesting for further study. An combinatory effect on apoptosis by INH7 and Letrozole (aromatase inhibitor) was further demonstrated in MCF-7. Down-regulation of GRP78 via 17β-HSD7 inhibition enhances cell apoptosis in response to Letrozole. This study highlights GRP78 as a key regulator related to 17β-HSD7 inhibition and effect. Taken together, results from the present study suggest a hypothesis that inhibition of 17β-HSD7 would be a complementary strategy to Letrozole by suppression of GRP78 in ER+ breast cancer. However, from a research perspective, further studies have to be carried out with more breast cancer cell lines as well as in vivo model to assess the efficacy of inhibitor combination.

Conditioned medium from the three-dimensional culture of human umbilical cord perivascular cells accelerate the migration and proliferation of human keratinocyte and fibroblast

Previous studies have reported that the conditioned medium (CM) of bone marrow-mesenchymal stem cells (BM-MSCs) stimulate the migration and proliferation of cell types involved in the wound healing process. However, these studies only show MSC-CM effects that were obtained using a two-dimensional (2D) culture. Recently, a three-dimensional (3D) culture has been considered to be a more physiologically appropriate system than the 2D culture. In addition, it has been shown that the procurement of BM-MSC is invasive, and other sources of MSC are thus being explored. Recently, perivascular cells (PVCs) have been considered as an alternative source of cells for dermal wound healing. Therefore, in this study, a PVC-conditioned medium (CM) was collected from a 3D culture (PVC-CM-3D) using highly porous polystyrene-based membranes and compared with PVC-CM from a 2D culture (PVC-CM-2D) to investigate the effects on the migration and proliferation of human keratinocytes and fibroblasts. Moreover, the PVC-CM components from the 2D and 3D cultures were identified using 2D gel electrophoresis. The migrations of the keratinocytes cells and fibroblasts were significantly higher with PVC-CM-3D than with the 2D culture; similarly, the proliferation of keratinocytes was also highly stimulated by PVC-CM-3D. Proteomic analyses of the PVC-CM revealed that type I collagen was highly expressed in the 3D-culture system. Microtubule-actin cross-linked factor 1 (KIAA0465), nebulin-related anchoring protein, and thioredoxin were specifically expressed only in PVC-CM-3D. In addition, more EVs could be isolated from the PVC-CM-3D, and EVs were found to stimulate keratinocyte migration. Taken together, 3D-culture using a polystyrene scaffold is demonstrated to be a better system for providing better physiological conditions; therefore, PVC-CM-3D could be a promising option for skin-wound healing.

Metaproteomics approaches and techniques: A review

Extensive studies have been done on metagenomics for various microbial communities. The advancements in metagenomics level led to the need of metaproteomics approaches. Metaproteomics involve the identification, function and expression of various proteins present in microbial community, it also involves the identification and expression analysis of stress related proteins. The concepts of metaproteomics come with advancement in proteomics techniques which includes 2D gel electrophoresis for the identification of proteins and peptides in a particular microbial community. Mass spectrometry which is used to separate the proteins by desorption and ionization using a gas on a liquid medium. MALDI use for protein identification and separation, connected with TOF to give better results. The metaproteomics approaches become more advanced when HPLC and LC were used for peptides and protein with computational tools to sequence the peptide and protein. It is concluded that there is a requirement of research in metaproteomics. Many scientists have done research on these approaches but there is lack of better quality and desirable results.

Keeping good friends close - The surface and secreted proteomes of a probiotic bacterium provide candidate proteins for intestinal attachment and communication with the host.

Bacteria use cell surface proteins and secreted proteins to interact with host tissues. Several dozen previously published proteomics studies have identified cell surface proteins for pathogens. In this issue, Celebioglu and Svensson (Proteomics 2017, 17, 1700019) use 2D gel electrophoresis and mass spectrometry to identify secreted and cell surface proteins of a commensual gut bacterium, Lactobacillus acidophilus NCFM. Some of the proteins are known to have functions in the cytoplasm, and their presence on the cell surface suggests they might be moonlighting proteins. In addition, comparisons of proteins used by pathogenic and probiotic species to interact with their hosts could lead to improved treatments of infections and chronic diseases that are associated with an imbalance of pathogenic and probiotic gut bacteria.

Heparin/Collagen 3D Scaffold Accelerates Hepatocyte Differentiation of Wharton's Jelly-Derived Mesenchymal Stem Cells.

Both mature and stem cell-derived hepatocytes lost their phenotype and functionality under conventional culture conditions. However, the 3D scaffolds containing the main extracellular matrix constitutions, such as heparin, may provide appropriate microenvironment for hepatocytes to be functional. The current study aimed to investigate the efficacy of the differentiation capability of hepatocytes derived from human Wharton's jelly mesenchymal stem cells (WJ-MSCs) in 3D heparinized scaffold. In this case, the human WJ-MSCs were cultured on the heparinized and non-heparinized 2D collagen gels or within 3D scaffolds in the presence of hepatogenic medium. Immunostaining was performed for anti-alpha fetoprotein, cytokeratin-18 and -19 antibodies. RT-PCR was performed for detection of hepatic nuclear factor-4 (HNF-4), albumin, cytokeratin-18 and -19, glucose-6-phosphatase (G6P), c-met and Cyp2B. The results indicated that hepatogenic media induced the cells to express early liver-specific markers including HNF4, albumin, cytokeratin-18 and 19 in all conditions. The cells cultured on both heparinized culture conditions expressed late liver-specific markers such as G6P and Cyp2B as well. Besides, the hepatocytes differentiated in 3D heparinized scaffolds stored more glycogen that indicated they were more functional. Non-heparinized 2D gel was the superior condition for cholangiocyte differentiation as indicated by higher levels of cytokeratin 19 expression. In conclusion, the heparinized 3D scaffolds provided a microenvironment to mimic Disse space. Therefore, 3D heparinized collagen scaffold can be suggested as a good vehicle for hepatocyte differentiation.

Chromatin-modifying agents convert fibroblasts to OCT4+ and VEGFR-2+ capillary tube-forming cells.

RationaleThe human epigenome is plastic. The goal of this study was to address if fibroblast cells can be epigenetically modified to promote neovessel formation.Methods and resultsHere, we used highly abundant human adult dermal fibroblast cells (hADFCs) that were treated with the chromatin-modifying agents 5-aza-2'-deoxycytidine and trichostatin A, and subsequently subjected to differentiation by activating Wnt signaling. Our results show that these epigenetically modified hADFCs increasingly expressed β-catenin, pluripotency factor octamer-binding transcription factor-4 (OCT4, also known as POU5F1), and endothelial cell (EC) marker called vascular endothelial growth factor receptor-2 (VEGFR-2, also known as Fetal Liver Kinase-1). In microscopic analysis, β-catenin localized to cell-cell contact points, while OCT4 was found to be localized primarily to the nucleus of these cells. Furthermore, in a chromatin immunoprecipitation experiment, OCT4 bound to the VEGFR-2/FLK1 promoter. Finally, these modified hADFCs also transduced Wnt signaling. Importantly, on a two-dimensional (2D) gel substrate, a subset of the converted cells formed vascular network-like structures in the presence of VEGF.ConclusionChromatin-modifying agents converted hADFCs to OCT4+ and VEGFR-2+ capillary tube-forming cells in a 2D matrix in VEGF-dependent manner.

Physiological and proteomic response of Limonium bicolor to salinity

Medicinal halophyte Limonium bicolor (Bag.) Kuntze was treated with 200 mM NaCl, the physiological parameters including fresh weight, dry weight, salt gland number, MDA levels and root activity were investigated, comparative proteomic analyses of leaf and root were carried out using 2D-PAGE combined with MALDI-TOF/TOF-MS. The results showed that L. bicolor performed a positive effect on its growth under NaCl stress since increase in different extent of fr wt, dry wt, salt gland number, MDA level and SOD activity of leaves and roots, as well as root activity. More than 460 protein spots in leaves and 600 protein spots in roots were visualized and matched in 2D gels, the results of proteomic analyses showed that 45 proteins were identified in leaves and 49 proteins in roots to be differentially expressed. Based on their functions, these proteins were grouped into categories covering a wide range of molecular processes, including carbohydrate, energy, lipid, nucleotide and amino acid metabolism, glycan biosynthesis and metabolism, metabolism of cofactors and vitamins, biosynthesis of secondary metabolites, redox homeostasis, transcription, cytoskeleton and transport, as well as folding, sorting and degradation. The upregulating and downregulating of differentially- expressed proteins with different physiological function were also reflected in the physiological parameters. These results are helpful for further research work on salt response mechanisms and medicinal utilization of L. bicolor.

Allergenome characterization of the mosquito Aedes aegypti.

Saliva and muscle-derived mosquito allergens have been purified and characterized. However, the complete set of allergens remains to be elucidated. In this study, we identified and characterized IgE-binding proteins from the mosquito species Aedes aegypti. Serum was obtained from 15 allergic individuals with asthma and/or rhinitis and sensitized to mosquito. IgE binding was determined by ELISA. Total proteins from freeze-dried bodies of A. aegypti were extracted and IgE-reactive proteins were identified by 2D gel electrophoresis, followed by Western blot with pooled or individual sera. IgE-reactive spots were further characterized by mass spectrometry. Twenty-five IgE-reactive spots were identified, corresponding to 10 different proteins, some of which appeared as different variants or isoforms. Heat-shock cognate 70 (HSC-70) and tropomyosin showed IgE reactivity with 60% of the sera, lysosomal aspartic protease, and "AAEL006070-PA" (Uniprot: Q177P3) with 40% and the other proteins with <33.3% of the sera. Different variants or isoforms of tropomyosin, arginine or creatine kinase, glyceraldehyde-3-phosphate dehydrogenase (GPDH), calcium-binding protein, and phosphoglycerate mutase were also identified. The mixture of three allergens (Aed a 6, Aed a 8, and Aed a 10) seems to identify more than 80% of A. aegypti-sensitized individuals, indicating that these allergens should be considered when designing of improved mosquito allergy diagnostic tools. The newly identified allergens may play a role in the pathophysiology of mosquito allergy in the tropics, and some of them might be important arthropod-related proteins involved in cross-reactivity between A. aegypti and other allergenic arthropods.

Identification of a host cell protein impurity in therapeutic protein, P1

Residual host cell proteins (HCPs) are process-related impurities present in biotherapeutics that can pose safety health risks to patients. An adequate control of HCP levels in the final product, and demonstration of HCP clearance throughout a product manufacturing process is critical for all biotherapeutic products. Developing effective downstream purification processes can be challenging as HCPs and product proteins may possess an affinity for each other or have similar physicochemical properties, resulting in co-purification. In the current study, we identified the presence of CHO-catalase subunit protein as an impurity present in purified P1 protein. This previously unreported HCP impurity, was detected in P1 protein generated in Chinese hamster ovary (CHO) cells. Purified drug substance samples contained elevated CHO HCP levels when measured using a commercial anti-CHO HCP Enzyme-Linked Immunosorbent Assay (ELISA) kit. This finding, prompted further characterization of the HCP profile using 1D and 2D gels/ western blots using an anti-human IgG antibody as well as a commercial anti-CHO HCP antibody (Cygnus 813) for the detection of host cell proteins. The CHO-catalase protein has been characterized using a combination approach of one-dimensional (1D) and two-dimensional (2D) gels and western blotting techniques, and the identity confirmed using liquid chromatography-mass spectrometry (LC-MS/MS) analysis. Western blot analyses using the anti-CHO HCP antibody detected a potential HCP band at ∼60 kDa and a pI of ∼8 in the purified P1 sample. The 60 kDa HCP band was excised from 1D SDS-PAGE gels and LC-MS/MS analysis identified it to be CHO-catalase subunit. The identity of catalase monomer was further confirmed by western blot analysis using a specific anti-catalase antibody.

Screening, Purification and Characterization of Anionic Antimicrobial Proteins from Foeniculum Vulgare.

Foeniculum vulgare Mill., commonly called fennel, is a medicinal plant belonging to the Umbelliferae (Apiaceae) family, and is used in traditional medicine. Antibacterial peptides were isolated using sodium phosphate citrate buffer and, for extraction, cetyltrimethyl ammonium bromide (CTAB) buffer with pH 6, have been employed and antimicrobial activity tested against four reference strains. The extracted protein was subjected to 3 kDa dialysis and separation was carried out by DEAE-ion exchange chromatography and further proteins were identified by 2D gel electrophoresis. The results of Foeniculum vulgare elutes obtained from DEAE-ion exchange chromatography were tested for antibacterial activity. Elute 3 shows the highest antibacterial activity on Pseudomonas aeruginosa with a diameter of a zone of inhibition of 16 mm and IC50 value 25.02 (mcg/mL). Based on the findings of the wide usage in treatment of various ailments and day-to-day life, Foeniculum vulgare seeds were used in the present research and have shown promising antibacterial effects, which requires further proteomic research to authenticate the role of the anticipated proteins.

The use of high hydrostatic pressure to generate folate-enriched extracts from the granule fraction of hen’s egg yolk

The present work characterized the effects of pre-treatments on the protein profile and microstructure of granule fractions from egg yolk. The granule microstructure was studied using confocal laser scanning microscopy (CLSM). The protein profiles of the pre-treated granule and the corresponding plasma fraction were studied using 2D gel electrophoresis techniques. Further, we explored the potential for using high hydrostatic pressure (HHP) to promote disintegration of the granule structure. The CLSM micrographs provided evidence of the substantial disintegration of granules due to HHP (600MPa/5min). Results from high performance liquid chromatography (HPLC) analysis indicated high concentrations of folate in the plasma fractions (230µg/g dry matter) separated from the HHP-treated granule. Sodium Dodecyl Sulphate–Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis revealed the localization of phosvitin in the plasma fraction, which correlated with higher folate concentrations. The results demonstrate that phosvitin and folate were stable under the HHP conditions applied in this study. These findings provide evidence of a putative interaction between phosvitin and folate, and offer an improved model for the structure of granule.

Transcriptome and Functional Genomics Reveal the Participation of Adenine Phosphoribosyltransferase in Trypanosoma cruzi Resistance to Benznidazole.

Currently, the only available treatments for Trypanosoma cruzi are benznidazole (Bz) and nifurtimox (Nfx). The mechanisms of action and resistance to these drugs in this parasite are not complete known. In order to identify differentially expressed transcripts between sensitive and resistant parasites, a massive pyrosequencing of the T. cruzi transcriptome was carried out. Additionally, the 2D gel electrophoresis profile of sensitive and resistant parasites was analyzed and the data were supported with functional genomics. The results showed 133 differentially expressed genes in resistant parasites. The transcriptome analysis revealed the regulation of different genes with several functions and metabolic pathways, which could suggest that resistance in T. cruzi is a multigenic process. Additionally, using transcriptomics, one gene, adenine phosphoribosyltransferase (APRT), was found to be down-regulated in the resistant parasites and its expression profile was confirmed by 2D electrophoresis analysis. The role of this gene in the resistance to Bz was confirmed overexpressing it in sensitive and resistant parasites. Interestingly, both parasites became more sensitive to Bz and H2 O2 . This is the first RNA-seq study to identify regulated genes in T. cruzi associated with Bz resistance and to show the role of APRT in T. cruzi resistance. Although T. cruzi regulation is mainly post-transcriptional, the transcriptome analysis, supported by 2D gel analysis and functional genomic, provides an overall idea of the expression profiles of genes under resistance conditions. These results contribute essential information to further the understanding of the mechanisms of action and resistance to Bz in T. cruzi. J. Cell. Biochem. 118: 1936-1945, 2017. © 2017 Wiley Periodicals, Inc.

Diurnal expression of proteins in the retina of the blind cone-rod homeobox (Crx-/- ) mouse and the 129/Sv mouse: a proteomic study.

The vertebrate retina contains a circadian clock participating in adaptations to day and night vision. This peripheral clock is independent of the master clock in the suprachiasmatic nucleus (SCN). The retinal clock is located in several cell types, including the photoreceptors. To investigate the role of the circadian clock of the photoreceptor cells in regulation of retinal protein rhythms, we analysed diurnal protein expression in the photoreceptor-deficient cone-rod homeobox knockout mouse (Crx-/- ) and the 129/Sv mouse. 2D gels were made from retinal homogenates of 129/Sv and Crx-/- mice killed at midday and midnight. Stained gels were analysed by use of PDQuest 2D gel analysis software. After trypsin digestion of differential expressed spots, the proteins were identified by LC-MS/MS using a nano-liquid chromatograph connected to a Q-TOF Premier mass spectrometer. These data were used to search the SWISS-PROT database. Both the retinae of the control and the Crx-/- mice exhibited diurnal proteins rhythms. As expected, proteins involved in phototransduction were not detected in the Crx-/- mouse; in this phenotype, however, proteins from spots showing diurnal rhythms were specifically identified as enzymes involved in glucose metabolism, Krebs cycle, and mitochondrial enzymes. Data are available via ProteomeXchange with identifier PXD005556. We show diurnal protein rhythms in the retina of a mouse lacking the rods and cones. The diurnal protein rhythms in this genotype, lacking the circadian clock of the photoreceptors, might be caused by a circadian clock in other retinal cell types or a direct light input to the retina.

Proteomic characterization of canine seminal plasma

The present study was conducted to identify the major proteome of the sperm-rich fraction and prostatic fraction of canine seminal plasma. Three semen samples from four healthy dogs were obtained by digital manipulation. The pre-sperm fraction, sperm-rich fraction and prostatic fraction were separated from each ejaculate. Immediately after sperm analysis, a protease inhibitor was added to the sperm-rich fraction and prostatic fraction, and the fractions were separately centrifuged and frozen at −80 °C. The samples were thawed, re-centrifuged, and the total protein concentration was determined. Samples were subjected to 1D SDS-PAGE and Coomassie-blue stained gels, were analyzed by Quantity One 1D Analysis Software. Bands detected in the gels were excised and proteins subjected to digestion with trypsin. Proteins were identified by nano-HPLC-MS and tools of bioinformatics. Tandem mass spectrometry allowed the detection of 268 proteins in the gels of sperm-rich fraction and prostatic fraction of canine ejaculate. A total of 251 proteins were common to the sperm-rich and prostatic fractions, while 17 proteins were present in the sperm-rich fraction and absent in the prostatic fraction. The intensity of the bands detected in range 1 and 2 represented 46.5% of all of the band intensities detected in the 1D gels for proteins of the sperm-rich fraction and 53.0% of all bands in the prostatic fraction. Arginine esterase and lactotransferrin precursor were the protein with the highest intensity observed in the both fractions. Among the proteins present only in the sperm-rich fraction, the proteins UPF0764 protein C16orf89 homolog and epididymal-specific lipocalin-9 were the most abundant. In conclusion, canine sperm-rich fraction and prostatic fraction express a very diverse set of proteins, with unique biochemical properties and functions. Moreover, although most proteins are common to both sperm-rich fraction and prostatic fraction, there are some exclusive proteins in sperm-rich fraction.

Identification of candidate protein markers of Bovine Parainfluenza Virus Type 3 infection using an in vitro model

Bovine Parainfluenza Virus Type 3 (BPI3V) infections are often asymptomatic, causing respiratory tissue damage and immunosuppression, predisposing animals to severe bacterial pneumonia, the leading cause of Bovine Respiratory Disease (BRD) mortality. As with many pathogens, routine BPI3V serology does not indicate the presence of damaged respiratory tissue or active infection. In vitro proteomic marker screening using disease relevant cell models could help identify markers of infection and tissue damage that are also detectable during in vivo infections. This study utilised a proteomic approach to investigate in vitro cellular responses during BPI3V infection to enhance the current understanding of intracellular host-virus interactions and identify putative markers of in vivo infection. Through 2D gel electrophoresis proteomic analysis, BPI3V Phosphoprotein P and host T-complex Protein 1 subunit theta were found to be accumulated at the latter stages of infection within bovine fibroblasts. These proteins were subsequently detected using targeted multiple reaction monitoring (MRM) mass spectrometry in the plasma of animals challenged with BPI3V, with differential protein level profiles observed dependant on animal vaccination status. Potential mechanisms by which BPI3V overcomes host cellular immune response mechanisms allowing for replication and production of viral proteins were also revealed. Assessment of circulating protein marker levels identified through an in vitro approach as described may enable more effective diagnosis of active viral infection and diseased or damaged respiratory tissue in animals and allow for more effective utilisation of preventative therapeutic interventions prior to bacterial disease onset and significantly aid the management and control of BRD.

Dioscorea alata tuber proteome analysis shows over thirty dioscorin isoforms and novel tuber proteins

In Dioscorea, dioscorin (31 kDa) is the major storage protein constituting 85% of the total tuber proteins. An integrated proteomic and biochemical approach was used to understand the physiological role of dioscorin in the two contrasting growth stages (germinating and mature tuber). HPLC analysis showed 3 fold reduction in mannitol and 12.88 and 1.24 fold increase in sucrose and maltose in the germinating tuber. A 1.8 and 3 fold increase in sucrose phosphate synthase and mannitol dehydrogenase activity respectively was observed in the germinating tuber while a 2 fold higher invertase probably lowers the sucrose accumulation in the mature tuber. SDS-PAGE and 2-D maps of the mature and germinating tubers confirmed depletion (more than 50%) of dioscorin on germination. Dioscorin was purified using ion exchange and gel filtration chromatography with 43.32 fold purification and 38.16 yield. Out of a trail of 35 spots at 31 kDa only 12 spots (identified as dioscorin isoforms) were present in the 2D gel of the purified fraction. To search for other tuber proteins besides dioscorin, the unbound fractions of DEAE column were analysed by 2DGE. DREB 1A, caffeic acid 3-O-methyltransferase and Rab-1 small GTP binding protein were identified perhaps for the first time in the Dioscorea proteome. The interactome analysis revealed these to be involved in oxidative stress, carotenoid synthesis and vesicular transport. This is perhaps the first attempt to identify tuber proteome (although limited) and to understand the physiological significance of these proteins.

Proteomic profiling of mature leaves from oil palm (Elaeis guineensis Jacq.).

Oil palm is one of the most productive oil bearing crops grown in Southeast Asia. Due to the dwindling availability of agricultural land and increasing demand for high yielding oil palm seedlings, clonal propagation is vital to the oil palm industry. Most commonly, leaf explants are used for in vitro micropropagation of oil palm and to optimize this process it is important to unravel the physiological and molecular mechanisms underlying somatic embryo production from leaves. In this study, a proteomic approach was used to determine protein abundance of mature oil palm leaves. To do this, leaf proteins were extracted using TCA/acetone precipitation protocol and separated by 2DE. A total of 191 protein spots were observed on the 2D gels and 67 of the most abundant protein spots that were consistently observed were selected for further analysis with 35 successfully identified using MALDI TOF/TOF MS. The majority of proteins were classified as being involved in photosynthesis, metabolism, cellular biogenesis, stress response, and transport. This study provides the first proteomic assessment of oil palm leaves in this important oil crop and demonstrates the successful identification of selected proteins spots using the Malaysian Palm Oil Board (MPOB) Elaeis guineensis EST and NCBI-protein databases. The MS data have been deposited in the ProteomeXchange Consortium database with the data set identifier PXD001307.