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Two-dimensional Gel Electrophoresis (two-dimensional + gel_electrophoresis)
Kinds of Two-dimensional Gel Electrophoresis Selected AbstractsMolecular responses of Campylobacter jejuni to cadmium stressFEBS JOURNAL, Issue 20 2008Nadeem O. Kaakoush Cadmium ions are a potent carcinogen in animals, and cadmium is a toxic metal of significant environmental importance for humans. Response curves were used to investigate the effects of cadmium chloride on the growth of Camplyobacter jejuni. In vitro, the bacterium showed reduced growth in the presence of 0.1 mm cadmium chloride, and the metal ions were lethal at 1 mm concentration. Two-dimensional gel electrophoresis combined with tandem mass spectrometry analysis enabled identification of 67 proteins differentially expressed in cells grown without and with 0.1 mm cadmium chloride. Cellular processes and pathways regulated under cadmium stress included fatty acid biosynthesis, protein biosynthesis, chemotaxis and mobility, the tricarboxylic acid cycle, protein modification, redox processes and the heat-shock response. Disulfide reductases and their substrates play many roles in cellular processes, including protection against reactive oxygen species and detoxification of xenobiotics, such as cadmium. The effects of cadmium on thioredoxin reductase and disulfide reductases using glutathione as a substrate were studied in bacterial lysates by spectrophotometry and nuclear magnetic resonance spectroscopy, respectively. The presence of 0.1 mm cadmium ions modulated the activities of both enzymes. The interactions of cadmium ions with oxidized glutathione and reduced glutathione were investigated using nuclear magnetic resonance spectroscopy. The data suggested that, unlike other organisms, C. jejuni downregulates thioredoxin reductase and upregulates other disulfide reductases involved in metal detoxification in the presence of cadmium. [source] Endochitinase activity in the apoplastic fluid of Phellinus weirii -infected Douglas-fir and its association with over wintering and antifreeze activityFOREST PATHOLOGY, Issue 5 2003A. Zamani Summary Extracellular proteins were extracted from Phellinus weirii infected Douglas-fir (Pseudotsuga menziesii var. menziesii) roots and needles to examine endochitinase activity. Chitinases have been associated with the plant's defence response against fungal attack because they hydrolyse chitin, a structural component of fungal cell walls. Protein separation using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western immunoblot analysis using a polyclonal antibody specific to an endochitinase-like protein (ECP) resulted in the detection of up to three polypeptides between 27 and 30 kDa in size. Two-dimensional gel electrophoresis (2-D PAGE) followed by Western immunoblot analysis revealed that the apoplastic fluid contained multiple ECP isoforms with isoelectric points (pIs) ranging from 5.3 to 5.8 and molecular masses of 27,30 kDa. Chitinase activity in needle and root tissues was measured spectrophotometrically using a colorimetric assay. A gel overlay technique using glycol chitin as a substrate for endochitinase was applied to confirm that the ECP antibody detected an enzymatically active protein. The apoplastic fluid collected from P. weirii -infected winter Douglas-fir needles showed anti-freeze activity and seasonal analysis of needle tissue showed some evidence of ECP accumulation in winter months. ECP was distributed systemically throughout the tree. Increased levels of endochitinase activity in the region of P. weirii infection supports a physiological role for ECP in the plant defence response. Résumé Les protéines extra-cellulaires ont été extraites des racines et aiguilles de douglas (Pseudotsuga menziesii var menziesii) infectés par Phellinus weirii (Murr.) Gilbn., pour étudier l'activité endochitinase. Les chitinases ont été associées aux réactions de défense des plantes contre les attaques fongiques parce-qu'elles hydrolysent la chitine, un composant de la paroi des cellules fongiques. La séparation des protéines, réalisée par électrophorèse en gel de polyacrylamide avec sodium dodecyl sulfate (SDS-PAGE), suivie par une analyse par Western immunoblot en utilisant un anticorps polyclonal spécifique d'une protéine de type endochitinase (ECP), a permis la détection de 3 polypeptides de taille comprise entre 27 et 30 kDa. Une électrophorèse sur gel en 2-dimensions (2-D PAGE) suivie par une analyse par Western immunoblot a révélé que le fluide apoplastique contient de multiples isoformes d'ECP avec des pI dans une gamme de 5.3 à 5.8 et des masses moléculaires de 27 à 30 kDa. L'activité chitinase dans les aiguilles et tissus racinaires a été mesurée par spectrophotométrie par une méthode colorimétrique. Une technique d'overlay utilisant de la chitine glycol comme substrat de l'endochitinase a été appliquée pour confirmer que l'anticorps ECP avait détecté une protéine active du point de vue enzymatique. Le fluide apoplastique d'aiguilles récoltées en hiver sur des douglas infectés par P. weirii a montré une activité antigel et l'analyse saisonnière des tissus foliaires a montré une certaine accumulation d'ECP pendant l'hiver. L'ECP est répartie de façon systémique dans l'ensemble de l'arbre. Les niveaux accrus d'activité endochitinase dans la zone infectée par P. weirii suggère un rôle physiologique de l'ECP dans les réactions de défense de la plante. Zusammenfassung Aus Wurzeln und Nadeln von mit Phellinus weirii infizierten Douglasien (Pseudotsuga menziesii var. menziesii) wurden extrazelluläre Proteine extrahiert, um die Endochitinase-Aktivität zu bestimmen. Chitinasen werden mit der pflanzlichen Abwehrreaktion auf Pilzinfektionen in Verbindung gebracht, da sie Chitin, eine Strukturkomponente der pilzlichen Zellwand, hydrolysieren. Die Proteine wurden mit Natrium-Dodecyl-Sulfat-Polyacrylamid-Gelelektrophorese (SDS-PAGE) getrennt, gefolgt von einer Western Immunoblot-Analyse mit einem gegen ein Endochitinase-ähnliches Protein (ECP) spezifischen polyklonalen Antikörper. Hiermit liessen sich bis zu drei Polypeptide zwischen 27-30 kDa nachweisen. Eine zweidimensionale Gelelektrophorese (2-D PAGE) mit anschliessender Western Immunoblot-Analyse ergab, dass die Apoplastenflüssigkeit multiple ECP-Isoformen enthielt (mit pIs von 5,3 bis 5,8 und Molekularmassen von 27 bis 30 kDa). Die Chitinase-Aktivität wurde auch im Nadel- und Wurzelgewebe spektrophotometrisch mit einer Farbreaktion gemessen. Um sicher zu stellen, dass der ECP-Antikörper ein enzymatisch aktives Protein nachwies, wurde eine Gel-Overlay-Methode verwendet, mit Glycolchitin als Substrat für die Endochitinase. Die Apoplastenflüssigkeit der Nadeln von mit P. weirii infizierten Douglasien zeigte in Winterzustand eine Antifrost-Aktivität, ihre Analyse während des gesamten Jahres ergab aber keine Hinweise auf eine ECP-Anreicherung während der Wintermonate. ECP war systemisch im gesamten Baum enthalten. Die erhöhte Endochitinase-Aktivität in Bereichen mit P. weirii -Infektion lässt auf eine physiologische Rolle von ECP in der Pflanzenabwehr schliessen. [source] Proteomic identification of peroxiredoxin 6 for host defence against Opisthorchis viverrini infectionPARASITE IMMUNOLOGY, Issue 5 2010J. KHOONTAWAD Summary Opisthorchis viverrini infection causes opisthorchiasis and is a risk factor for cholangiocarcinoma via chronic inflammation. To investigate the mechanism of O. viverrini -induced liver disease, we applied a proteomic approach to examine alterations in hepatic protein levels in O. viverrini -infected hamsters. Two-dimensional gel electrophoresis (2DE) revealed that O. viverrini infection induced upregulation (1·5- to 4·3-fold) of 25 proteins and downregulation (1·5 to 2·5-fold) of 24 proteins compared with uninfected animals. Expression of proteins related to stress response, DNA replication and repair, and cell structure was significantly increased, whereas that of proteins associated with normal liver function, such as metabolism, blood volume maintenance and fatty acid cycle was decreased. Among the upregulated proteins, a 2·7-fold increase in peroxiredoxin 6 (Prdx6), an antioxidant protein, was confirmed by 2DE and immunoblot analysis, Western blot and quantitative PCR. Immunohistochemical analysis showed that Prdx6 expression was observed mainly in the cytoplasm of inflammatory cells. These results suggest that Prdx6 is important for host defence against O. viverrini infection. This study provides basic information for Prdx6 as a potential biomarker and therapeutic target for opisthorchiasis. [source] Characterisation of Zea mays L. plastidial transglutaminase: interactions with thylakoid membrane proteinsPLANT BIOLOGY, Issue 5 2010A. Campos Abstract Chloroplast transglutaminase (chlTGase) activity is considered to play a significant role in response to a light stimulus and photo-adaptation of plants, but its precise function in the chloroplast is unclear. The characterisation, at the proteomic level, of the chlTGase interaction with thylakoid proteins and demonstration of its association with photosystem II (PSII) protein complexes was accomplished with experiments using maize thylakoid protein extracts. By means of a specific antibody designed against the C-terminal sequence of the maize TGase gene product, different chlTGase forms were immunodetected in thylakoid membrane extracts from three different stages of maize chloroplast differentiation. These bands co-localised with those of lhcb 1, 2 and 3 antenna proteins. The most significant, a 58 kDa form present in mature chloroplasts, was characterised using biochemical and proteomic approaches. Sequential fractionation of thylakoid proteins from light-induced mature chloroplasts showed that the 58 kDa form was associated with the thylakoid membrane, behaving as a soluble or peripheral membrane protein. Two-dimensional gel electrophoresis discriminated, for the first time, the 58-kDa band in two different forms, probably corresponding to the two different TGase cDNAs previously cloned. Electrophoretic separation of thylakoid proteins in native gels, followed by LC-MS mass spectrometry identification of protein complexes indicated that maize chlTGase forms part of a specific PSII protein complex, which includes LHCII, ATPase and pSbS proteins. The results are discussed in relation to the interaction between these proteins and the suggested role of the enzyme in thylakoid membrane organisation and photoprotection. [source] Enhanced resolution of glycosylphosphatidylinositol-anchored and transmembrane proteins from the lipid-rich myelin membrane by two-dimensional gel electrophoresisPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 7 2003Christopher M. Taylor Abstract Two-dimensional gel electrophoresis (2-DE) has become a powerful and widely used technique for proteomic analyses. However, the limited ability of 2-DE to resolve transmembrane and glycosylphosphatidylinositol (GPI)-anchored proteins has slowed the identification of proteins from membrane-rich biological samples. Myelin is an unusually lipid-rich membrane with relatively few major proteins but many quantitatively minor proteins, most of which have an unknown identity and/or function. The goal of this study was to identify the optimal conditions of 2-DE for the separation of myelin proteins. We have identified two detergents, the nonionic n -dodecyl ,- D -maltoside and the zwitterionic amidosulfobetaine ASB-14, that are more effective in solubilizing myelin proteins than the commonly used zwitterionic detergent 3-[(3-cholamidopropyl)- dimethylammonio]-1-propanesulfonate (CHAPS). These detergents significantly enhance the solubility of both transmembrane (e.g., the highly hydrophobic and multiply acylated myelin proteolipid protein) and GPI-anchored (e.g., contactin and neuronal cell adhesion molecule) myelin proteins and enable their resolution by 2-DE. We conclude that these detergents are effective tools for the 2-DE analysis of myelin, and that they may be more generally useful for the analysis of membrane-rich biological samples. [source] Comparative Proteomics Analysis of the Proteins Associated With Laryngeal Carcinoma-Related Gene 1,THE LARYNGOSCOPE, Issue 2 2006Xiaopeng Zhang PhD Abstract Objectives: A novel gene, laryngeal carcinoma-related gene 1 (LCRG1), had the characteristics of tumor-suppressor genes. It was cloned in our laboratory. The objective was to find and characterize the proteins related to LCRG1 and to elucidate the molecular mechanism of LCRG1. Study Design: We used the established cell lines of Hep-2/LCRG1 (Hep-2 cells transfected by recombinant plasmid pcDNA3.1[+]/LCRG1) and Hep-2/pcDNA3.1(+) (Hep-2 cells transfected by control vector pcDNA3.1[+]) as cell models. Methods: Two-dimensional gel electrophoresis (2-DE) technology was performed to separate the proteins of Hep-2/LCRG1 and Hep-2/pcDNA3.1(+) cell lines, respectively. The differential protein spots were analyzed by software analysis, subject to in-gel digestion, and identified by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and electrospray ionization,quadruple time-of-flight MS/MS (ESI-Q-TOF MS/MS). Then the differential expression levels of partial identified proteins were determined by Western blotting analysis and quantitative real-time reverse transcriptase,polymerase chain reaction. Results: The results showed the attained 2-DE patterns of the two cell lines were well-resolved and reproducible. There were 1075 ± 43 and 1027 ± 23 protein spots observed in Hep-2/LCRG1 and Hep-2/pcDNA3.1(+) cell lines, respectively. The average matching rate of the two cell lines was 91%. Twenty-six differentially expressed protein spots were identified (twenty spots for MALDI-TOF-MS, six spots for ESI-Q-TOF MS/MS). Most of the characterized proteins were characterized as the members of enzymes (phosphoglycerate mutase, manganese superoxide dismutase, and so on), transcription proteins (rho gdp dissociation inhibitor), and so on. Those identified proteins might contribute to the tumor-suppressive function of LCRG1. The differential expression levels of the partial proteins were confirmed by real-time polymerase chain reaction and Western blotting. Conclusions: We tentatively proposed those differentially expressed proteins were involved in the tumor-suppressive process related to LCRG1. These data will be helpful to elucidate the molecular mechanism of LCRG1. [source] Two-dimensional gel electrophoresis-based proteomic analysis of the Medicago truncatula,rust (Uromyces striatus) interactionANNALS OF APPLIED BIOLOGY, Issue 2 2010M.Á. Castillejo A two-dimensional gel electrophoresis (2-DE) based proteomic approach has been used to study the Medicago truncatula,Uromyces striatus interaction. The 2-DE leaf protein profile of three M. truncatula genotypes displaying different phenotypes (susceptible and showing prehaustorial and posthaustorial resistance) in both noninoculated and inoculated plants have been compared. Multivariate statistical analysis identified 63 differential protein spots under the experimental conditions (genotypes/treatments). Variable spots were subjected to tandem mass spectrometry (MS, matrix-assisted laser desorption ionisation time of flight, MALDI-TOF/TOF) analysis to identify their possible functions. A total of 27 proteins were identified using a combination of peptide mass fingerprinting (PMF) and MSMS fragmentation. Most of these observed changes correspond to enzymes involved in photosynthesis, energy metabolic pathways and stress related, whose pattern expression was different in relation to susceptibility/resistance of the genotypes studied. Results obtained in this work suggest that differences observed could be related to efficiency in energy utilisation and the induction of proteins involved in defence mechanism operating during early stages of infection. [source] Synovial fluid proteins differentiate between the subtypes of juvenile idiopathic arthritisARTHRITIS & RHEUMATISM, Issue 6 2010Margalit E. Rosenkranz Objective Juvenile idiopathic arthritis (JIA) is a heterogeneous group of inflammatory diseases, and no clinically useful prognostic markers to predict disease outcome in children with JIA are currently available. Synovial fluid likely reflects the proteins present in the inflamed synovium. The purpose of this study was to delineate the synovial fluid proteome and determine whether protein expression differs in the different subtypes of JIA. Methods Synovial fluid samples obtained from children with oligoarticular JIA, polyarticular JIA, or systemic JIA were compared. Two-dimensional gel electrophoresis for protein separation and matrix-assisted laser desorption ionization,time-of-flight mass spectrometry and quadripole time-of-flight mass spectrometry for protein identification were used for this study. Synovial fluid cells were analyzed by polymerase chain reaction (PCR) for the presence of haptoglobin messenger RNA (mRNA). Results The synovial fluid proteome of the samples was delineated. The majority of proteins showed overexpression in JIA synovial fluid as compared with noninflammatory control samples. There were 24 statistically significantly differentially expressed spots (>2-fold change; P < 0.05) between the subtypes of JIA. PCR analysis revealed haptoglobin mRNA, suggesting that haptoglobin is locally produced in an inflamed joint in JIA. Conclusion Despite the similar histologic appearance of inflamed joints in patients with different subtypes of JIA, there are differences in protein expression according to the subtype of JIA. Haptoglobin is differentially expressed between the subtypes of JIA and is locally produced in an inflamed joint in JIA. Haptoglobin and other differentially expressed proteins may be potential biomarkers in JIA. [source] Introducing proteomics in the undergraduate curriculum: A simple 2D gel electrophoresis exercise with serum proteinsBIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 1 2010Thomas D. Kim Abstract Two-dimensional gel electrophoresis (2DGE) remains an important tool in the study of biological systems by proteomics. While the use of 2DGE is commonplace in research publications, there are few instructional laboratories that address the use of 2DGE for analyzing complex protein samples. One reason for this lack is the fact that the preparation of samples for 2DGE is a complex and difficult process that can commonly yield gels of poor quality and resolution. In this experiment, we use a serum-based sample to mitigate many of the sample preparation issues that occur in cell-based sample preparations and incorporate a protein precipitation method that was developed to address the problem of high-abundance proteins and dynamic range in serum proteomics research. By focusing on 2DGE apart from many other facets of proteomic experimental design, students have the opportunity to gain fruitful experience in the use of this workhorse proteomics technique. This simplified focus also makes this exercise accessible to biochemistry instructors who are not active in proteomics; the requisite techniques may require some new equipment (i.e. an isoelectric focusing apparatus), but this exercise focuses on using familiar techniques (primarily electrophoresis) to cross the threshold of a new field, proteomics. [source] Proteome analysis of antibody-producing CHO cell lines with different metabolic profilesBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2007Deborah E. Pascoe Abstract Two-dimensional gel electrophoresis and tandem mass spectrometry were used to identify proteins associated with a metabolic shift during fed-batch cultures of two recombinant antibody-producing CHO cell lines. The first cell line underwent a marked change in lactate metabolism during culture, initially producing lactate and then consuming it, while the second cell line produced lactate for a similar duration but did not later consume it. The first cell line displayed a declining specific antibody productivity during culture, correlating to the 2-D gel results and the intracellular antibody concentration determined by HPLC. Several statistical analysis methods were compared during this work, including a fixed fold-change criterion and t -tests using standard deviations determined in several ways from the raw data and mathematically transformed data. Application of a variance-stabilizing transformation enabled the use of a global empirical standard deviation in the t -tests. Most of the protein spots changing in each cell line did not change significantly in the other cell line. A substantial fraction of the changing proteins were glycolytic enzymes; others included proteins related to antibody production, protein processing, and cell structure. Enolase, pyruvate kinase, BiP/GRP78, and protein disulfide isomerase were found in spots that changed over time in both cell lines, and some protein changes differed from previous reports. These data provide a foundation for future investigation of metabolism in industrially relevant mammalian cell culture processes, and suggest that along with differences between cell types, the proteins expressed in cultures with low lactate concentrations may depend on how those conditions were generated. Biotechnol. Bioeng. 2007;98: 391,410. © 2007 Wiley Periodicals, Inc. [source] Proteome analysis to assess physiological changes in Escherichia coli grown under glucose-limited fed-batch conditionsBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2005Babu Raman Abstract Proteome analysis was used to compare global protein expression changes in Escherichia coli fermentation between exponential and glucose-limited fed-batch phase. Two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry were used to separate and identify 49 proteins showing >2-fold difference in expression. Proteins upregulated during exponential phase include ribonucleotide biosynthesis enzymes and ribosomal recycling factor. Proteins upregulated during fed-batch phase include those involved in high-affinity glucose uptake, transport and degradation of alternate carbon sources and TCA cycle, suggesting an enhanced role of the cycle under glucose- and energy-limited conditions. We report the upregulation of several putative proteins (ytfQ, ygiS, ynaF, yggX, yfeX), not identified in any previous study under carbon-limited conditions. © 2005 Wiley Periodicals, Inc. [source] Separation of product associating E. coli host cell proteins OppA and DppA from recombinant apolipoprotein A-IMilano in an industrial HIC unit operationBIOTECHNOLOGY PROGRESS, Issue 2 2009Alan K. Hunter Abstract We have shown how product associating E. coli host cell proteins (HCPs) OppA and DppA can be substantially separated from apolipoprotein A-IMilano (apo A-IM) using Butyl Sepharose hydrophobic interaction chromatography (HIC). This work illustrates the complex problems that frequently arise during development and scale-up of biopharmaceutical manufacturing processes. Product association of the HCPs is confirmed using co-immunoprecipitation and Western blotting techniques. Two-dimensional gel electrophoresis and mass spectrometry techniques are used to confirm the identity of OppA and DppA. In this example, clearance of these difficult to separate HCPs decreased significantly when the process was scaled to a 1.4 m diameter column. Laboratory-scale experimentation and trouble shooting identified several key parameters that could be further optimized to improve HCP clearance. The key parameters included resin loading, peak cut point on the ascending side, wash volume, and wash salt concentration. By implementing all of the process improvements that were identified, it was possible to obtain adequate HCP clearance so as to meet the final specification. Although it remains speculative, it is believed that viscosity effects may have contributed to the lower HCP clearance observed early in the manufacturing campaign. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Comparative proteomic analysis between normal skin and keloid scarBRITISH JOURNAL OF DERMATOLOGY, Issue 6 2010C.T. Ong Summary Background, Keloids are pathological scars and, despite numerous available treatment modalities, continue to plague physicians and patients. Objectives, Identification of molecular mediators that contribute to this fibrotic phenotype. Methods, Two-dimensional gel electrophoresis, MALDI-TOF, Mascot online database searching algorithm and Melanie 5 gel analysis software were employed for comparative proteomic analysis between normal skin (NS) and keloid scar (KS) tissue extracts. Results, Seventy-nine protein spots corresponding to 23 and 32 differentially expressed proteins were identified in NS and KS, respectively. Isoforms of heat shock proteins, gelsolin, carbonic anhydrase and notably keratin 10 were strongly expressed in NS along with manganese superoxide dismutase, immune components, antitrypsin, prostatic binding protein and crystalline. Various classes of proteins were found either to be present or to be upregulated in keloid tissue: (i) inflammatory/differentiated keratinocyte markers: S100 proteins, peroxiredoxin I; (ii) wound healing proteins: gelsolin-like capping protein; (iii) fibrogenetic proteins: mast cell ,-tryptase, macrophage migration inhibitory factor (MIF); (iv) antifibrotic proteins: asporin; (v) tumour suppressor proteins: stratifin, galectin-1, maspin; and (vi) antiangiogenic proteins: pigment epithelium-derived factor. Significant increases in expression of asporin, stratifin, galectin-1 and MIF were observed by Western blot analysis in KS. Conclusions, This work has identified differentially expressed proteins specific to KS tissue extracts which can potentially be used as specific targets for therapeutic intervention. [source] Measurement of specific radioactivity in proteins separated by two-dimensional gel electrophoresisELECTROPHORESIS, Issue 5-6 2006Shaobo Zhou Abstract We report a method to quantify the specific radioactivity of proteins that have been separated by 2-DE. Gels are stained with SyproRuby, and protein spots are excised. The SyproRuby dye is extracted from each spot using DMSO, and the fluorescence is quantified automatically using a plate reader. The extracted gel piece is then dissolved in hydrogen peroxide and radioactivity is quantified by liquid scintillation counting. Gentle agitation with DMSO for 24,h was found to extract all the SyproRuby dye from gel fragments. The fluorescence of the extract was linearly related to the amount of BSA loaded onto a series of 1-D gels. When rat muscle samples were run on 2-DE gels, the fluorescence extracted from 54,protein spots showed a good correlation (r = 0.79, p < 0.001) with the corresponding spot intensity measured by conventional scanning and image analysis. DMSO extraction was found not to affect the amount of radioactive protein left in the gel. When a series of BSA solutions of known specific radioactivity were run on 2-DE gels, the specific radioactivity measured by the new method showed a good correlation (r = 0.98, p < 0.01, n = 5) with the specific radioactivity measured directly before loading. Reproducibility of the method was measured in a series of 2-DE gels containing proteins from the livers of rats and mice that had been injected with [35S]methionine. Variability tended to increase when the amount of radioactivity in the protein spot was low, but for samples containing at least 10,dpm above background the CV was around 30%, which is comparable to that obtained when measuring protein expression by conventional image analysis of SyproRuby-stained 2-DE gels. Similar results were obtained whether spots were excised manually or using a spot excision robot. This method offers a high-throughput, cost-effective and reliable method of quantifying the specific radioactivity of proteins from metabolic labelling experiments carried out in,vivo, so long as sufficient quantities of radioactive tracer are used. [source] An effective skeletal muscle prefractionation method to remove abundant structural proteins for optimized two-dimensional gel electrophoresisELECTROPHORESIS, Issue 11 2005Bradley Jarrold Abstract Proteomic analysis of biological samples in disease models or therapeutic intervention studies requires the ability to detect and identify biologically relevant proteins present in relatively low concentrations. The detection and analysis of these low-level proteins is hindered by the presence of a few proteins that are expressed in relatively high concentrations. In the case of muscle tissue, highly abundant structural proteins, such as actin, myosin, and tropomyosin, compromise the detection and analysis of more biologically relevant proteins. We have developed a practical protocol which exploits high-pH extraction to reduce or remove abundant structural proteins from skeletal muscle crude membrane preparations in a manner suitable for two dimensional gel electrophoresis. An initial whole-cell muscle lysate is generated by homogenization of powdered tissue in Tris-base. This lysate is subsequently partitioned into a supernatant and pellet containing the majority of structural proteins. Treatment of the pellet with high-pH conditions effectively releases structural proteins from membrane compartments which are then removed through ultracentrifugation. Mass spectrometric identification shows that the majority of protein spots reduced or removed by high-pH treatment were contractile proteins or contractile-related proteins. Removal of these proteins enabled successful detection and identification of minor proteins. Structural protein removal also results in significant improvement of gel quality and the ability to load higher amounts of total protein for the detection of lower abundant protein classes. [source] Quantitative evaluation of sample application methods for semipreparative separations of basic proteins by two-dimensional gel electrophoresisELECTROPHORESIS, Issue 19-20 2003Richard C. Barry Abstract The use of cup-loading for sample application has become widely used in two-dimensional electrophoresis (2-DE) for resolution of basic proteins, but no side-by-side quantitative study has been published which compares cup-loading with the alternative passive and active rehydration methods to fully promote one type of loading method over another. Replicate 2-D gels from each loading method were quantitatively evaluated for gel-to-gel reproducibility using IPG 6,11 strips and semipreparative protein loads (300 ,g). Gels were stained with SYPRO Ruby and analyzed with PDQuest. An inexpensive home-made assembly for cup-loading was used with the Protean IEF Cell for separation of whole cell extracts from the archaeon, Sulfolobus solfataricus. Cup-loading was determined to be far superior for IPG 6,11 separations than active or passive rehydration methods. Cup-loading consistently produced the greatest number of detectable spots, the best spot matching efficiency (56%), lowest spot quantity variations (28% coefficient of variation, CV), and the best-looking gels qualitatively. The least satisfactory results were obtained with active rehydration, followed closely by passive rehydration in off-line tubes. Passive rehydration experiments, performed using an on-line isoelectric focusing (IEF) tray, produced comparable spot numbers to cup-loading (84%), with 55% of the spots having higher intensity but 10% more spot quantity variance than cup-loading. [source] Casein phosphoproteome: Identification of phosphoproteins by combined mass spectrometry and two-dimensional gel electrophoresisELECTROPHORESIS, Issue 16 2003Gianfranco Mamone Abstract We report a fast and easy-to-use procedure that combines polyacrylamide gel electrophoresis with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF) and nanoelectrospray-tandem mass spectrometry (nES-MS/MS) analysis for the identification of casein components and defined phosphorylated sites. This methodology ensured identification of more than 30 phosphorylated proteins, five ,-, fifteen ,s1 -, ten ,s2 -, and four ,-casein (CN) components, including nonallelic, differently phosphorylated, and glycosylated forms. The sugar motif covalently bound to ,-CN was identified as chains, trisaccharide GalNAc, Gal, NeuGc, and tetrasaccharide 1GalNAc, 1Gal, 2NeuGc. Also identified was a biantennary chain made up of both chains of trisaccharide 1GalNAc, 1Gal, 1NeuGc, and tetrasaccharide 1GalNAc, 1Gal, 2NeuGc moiety on a single ,-CN component. The phosphate group on site Ser12 of tryptic peptide 8,22 of most phosphorylated ,s1 -CN (11 phosphate groups) was localized and the oligosaccharide sequence of the main tryptic glycopeptides of two ,-CN components was determined by means of MS/MS analysis. [source] Proteome analysis of human liver tumor tissue by two-dimensional gel electrophoresis and matrixassisted laser desorption/ionization-mass spectrometry for identification of disease-related proteinsELECTROPHORESIS, Issue 24 2002Jina Kim Abstract Hepatocellular carcinoma (HCC) is a common malignancy worldwide and is a leading cause of death. To contribute to the development and improvement of molecular markers for diagnostics and prognostics and of therapeutic targets for the disease, we have largely expanded the currently available human liver tissue maps and studied the differential expression of proteins in normal and cancer tissues. Reference two-dimensional electrophoresis (2-DE) maps of human liver tumor tissue include labeled 2-DE images for total homogenate and soluble fraction separated on pH 3,10 gels, and also images for soluble fraction separated on pH 4,7 and pH 6,9 gels for a more detailed map. Proteins were separated in the first dimension by isoelectric focusing on immobilized pH gradient (IPG) strips, and by 7.5,17.5% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels in the second dimension. Protein identification was done by peptide mass fingerprinting with delayed extraction-matrix assisted laser desorption/ionization-time of flight-mass spectrometry (DE-MALDI-TOF-MS). In total, 212 protein spots (117 spots in pH 4,7 map and 95 spots in pH 6,9) corresponding to 127 different polypeptide chains were identified. In the next step, we analyzed the differential protein expression of liver tumor samples, to find out candidates for liver cancer-associated proteins. Matched pairs of tissues from 11 liver cancer patients were analyzed for their 2-DE profiles. Protein expression was comparatively analyzed by use of image analysis software. Proteins whose expression levels were different by more than three-fold in at least 30% (four) of the patients were further analyzed. Numbers of protein spots overexpressed or underexpressed in tumor tissues as compared with nontumorous regions were 9 and 28, respectively. Among these 37 spots, 1 overexpressed and 15 underexpressed spots, corresponding to 11 proteins, were identified. The physiological significance of the differential expressions is discussed. [source] Proteomic analysis of cellular responses to low concentration N -methyl- N,-nitro- N -nitrosoguanidine in human amnion FL cellsENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 2 2004Jinghua Jin Abstract We have shown previously that exposure to a low concentration of N -methyl- N,-nitro- N -nitrosoguanidine (MNNG) induces comprehensive changes in the protein expression profile of human amnion FL cells, including the induction, suppression, upregulation, and downregulation of various proteins. In addition, by proteomic analysis combining two-dimensional gel electrophoresis (2-DE) and mass spectrometry, some of the induced and suppressed proteins were identified. In this study, we identified an additional 18 proteins among those that were either up- or downregulated by MNNG treatment. The proteins identified were a heterogeneous group that included several zinc finger proteins, proteins involved in signal transduction, cytoskeletal proteins, cell-cycle regulation proteins, and proteins with unknown functions. The involvement of these proteins in the cellular responses to alkylating agents has not been reported before and their physiological relevance is not clear. Therefore, our findings may help better understand the global cellular stress responses to chemical carcinogens, and may lead to new studies on the functions of these MNNG-responsive proteins. Furthermore, some of these proteins may serve as biomarkers for detecting exposure of human populations to environmental carcinogens. Environ. Mol. Mutagen. 43:93,99, 2004. © 2004 Wiley-Liss, Inc. [source] Identification of quorum-sensing regulated proteins in the opportunistic pathogen Pseudomonas aeruginosa by proteomicsENVIRONMENTAL MICROBIOLOGY, Issue 12 2003Catalina Arevalo-Ferro Summary The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic human pathogen which is responsible for severe nosocomial infections in immunocompromised patients and is the major pathogen in cystic fibrosis. The bacterium utilizes two interrelated quorum-sensing (QS) systems, which rely on N -acyl-homoserine lactone (AHL) signal molecules, to control the expression of virulence factors and biofilm development. In this study, we compared the protein patterns of the intracellular, extracellular and surface protein fractions of the PAO1 parent strain with those of an isogenic lasI rhlI double mutant by means of two-dimensional gel electrophoresis (2-DE). This analysis showed that the intensities of 23.7% of all detected protein spots differed more than 2.5-fold between the two strains. We only considered those protein spots truly QS regulated that were changed in the mutant in the absence of signal molecules but were rescued to the wild-type situation when the medium was supplemented with AHLs. These protein spots were characterized by MALDI-TOF peptide mapping. Twenty-seven proteins were identified that were previously reported to be AHL controlled, among them several well-characterized virulence factors. For one of the identified proteins, the serine protease PrpL, a biochemical assay was established to verify that expression of this factor is indeed QS regulated. Furthermore, it is shown that the quorum-sensing blocker C-30 specifically interferes with the expression of 67% of the AHL-controlled protein spots of the surface fraction, confirming the high specificity of the compound. Importantly, 20 novel QS-regulated proteins were identified, many of which are involved in iron utilization, suggesting a link between quorum sensing and the iron regulatory system. Two of these proteins, PhuR and HasAp, are components of the two distinct haem-uptake systems present in P. aeruginosa. In agreement with the finding that both proteins are positively regulated by the QS cascade, we show that the lasI rhlI double mutant grows poorly with haemoglobin as the only iron source when compared with the wild type. These results add haemoglobin utilization to the list of phenotypes controlled through QS in P. aeruginosa. The surprisingly high number of AHL-regulated proteins relative to the number of regulated genes suggests that quorum-sensing control also operates via post-transcriptional mechanisms. To strengthen this hypothesis we investigated the role of quorum sensing in the post-translational modification of HasAp, an extracellular protein required for the uptake of free and haemoglobin-bound haem. [source] Towards second-generation proteome analysis of murine enamel-forming cellsEUROPEAN JOURNAL OF ORAL SCIENCES, Issue 2006Jonathan E. Mangum Proteome analysis of rat enamel-forming cells, initiated over a decade ago, has provided valuable insights to enamel biology. In preparation for a more comprehensive, second-generation proteomic exploration, we evaluated an updated microsample-profiling strategy that comprises sequential extraction of enamel epithelium, parallel one- and two-dimensional gel electrophoresis, and mass spectrometric sequence analysis. The results indicated that several hundred proteins, representing various cellular compartments (including membranes), are amenable to identification with a starting tissue volume of <,10 µl. With its increased proteomic depth and breadth, this straightforward approach constitutes a major advance from the first-generation work (10-fold increased proteome coverage), although care was needed to ensure a comparably high stringency of protein identification. Expression proteomics has an exciting potential to elucidate the inner workings of murine enamel epithelial cells, leading to an improved understanding of enamel in health and disease. [source] Identification of the rat adrenal zona fasciculata/reticularis specific protein, inner zone antigen (IZAg), as the putative membrane progesterone receptorFEBS JOURNAL, Issue 7 2001Farah S. Raza Using immunological methods, a protein specific to the inner zones of the rat adrenal cortex, and called inner zone antigen (IZAg), was previously shown to have two interrelated forms of 26 kDa (IZAg1) and 55,60 kDa (IZAg2), and to have an action on steroid hydroxylation. After two-dimensional gel electrophoresis, and immunoaffinity column purification, N-terminal amino-acid analysis showed that the first 12 amino acids were identical to those of a recently described putative membrane located progesterone receptor (PPMR). RT-PCR was then used to generate the cDNA of this protein, using RNA extracted from rat adrenals. A glutathione S-transferase (GST)-fusion construct was expressed in Escherichia coli, and shown to generate an immunoreactive product of molecular mass consistent with its identification as IZAg1. More detailed examination of the distribution of this protein, not only in the zona fasciculata/reticularis of the adrenal cortex, but also in the Leydig cell, kidney and liver, suggest it may have a role in steroid hormone synthesis and/or metabolism. [source] Apparent growth phase-dependent phosphorylation of malonyl coenzyme A:acyl carrier protein transacylase (MCAT), a major fatty acid synthase II component in Mycobacterium bovis BCGFEMS MICROBIOLOGY LETTERS, Issue 1 2003Indrajit Sinha Abstract Probing protein extracts from exponentially growing and stationary phase cultures of Mycobacterium bovis BCG with anti-phospho amino acid antibodies revealed a 31-kDa anti-phospho threonine antibody-reactive protein specific to growing culture. The corresponding protein was purified via two-dimensional gel electrophoresis and identified via mass spectrometry to be malonyl coenzyme A:acyl carrier protein transacylase (MCAT), a component of the fatty acid biosynthetic pathway. MCAT tagged with histidine reacted with anti-phospho threonine antibody and was positive in an in-gel chemical assay for phospho proteins. Analysis of the growth phase dependence of MCAT-His phosphorylation and protein levels showed that phosphorylated MCAT-His can be detected only in growing culture. In contrast, MCAT-His protein level was growth phase-independent. These results suggest that MCAT may be a substrate of a protein kinase and phosphatase, and that aspects of fatty acid synthesis in tubercle bacilli are regulated by protein phosphorylation. [source] Strain-dependent regulation of neurotransmission and actin-remodelling proteins in the mouse hippocampusGENES, BRAIN AND BEHAVIOR, Issue 2 2006D. D. Pollak Individual mouse strains differ significantly in terms of behaviour, cognitive function and long-term potentiation. Hippocampal gene expression profiling of eight different mouse strains points towards strain-specific regulation of genes involved in neuronal information storage. Protein expression with regard to strain- dependent expression of structures related to neuronal information storage has not been investigated yet. Herein, a proteomic approach based on two-dimensional gel electrophoresis coupled with mass spectrometry (MALDI-TOF/TOF) has been chosen to address this question by determining strain-dependent expression of proteins involved in neurotransmission and activity-induced actin remodelling in hippocampal tissue of five mouse strains. Of 31 spots representing 16 different gene products analysed and quantified, N -ethylmaleimide-sensitive fusion protein, N -ethylmaleimide-sensitive factor attachment protein-,, actin-like protein 3, profilin and cofilin were expressed in a strain-dependent manner. By treating protein expression as a phenotype, we have shown significant genetic variation in brain protein expression. Further experiments in this direction may provide an indication of the genetic elements that contribute to the phenotypic differences between the selected strains through the expressional level of the translated protein. In view of this, we propose that proteomic analysis enabling to concomitantly survey the expression of a large number of proteins could serve as a valuable tool for genetic and physiological studies of central nervous system function. [source] Stage-specific alterations of the genome, transcriptome, and proteome during colorectal carcinogenesis,GENES, CHROMOSOMES AND CANCER, Issue 1 2007Jens K. Habermann To identify sequential alterations of the genome, transcriptome, and proteome during colorectal cancer progression, we have analyzed tissue samples from 36 patients, including the complete mucosa-adenoma-carcinoma sequence from 8 patients. Comparative genomic hybridization (CGH) revealed patterns of stage specific, recurrent genomic imbalances. Gene expression analysis on 9K cDNA arrays identified 58 genes differentially expressed between normal mucosa and adenoma, 116 genes between adenoma and carcinoma, and 158 genes between primary carcinoma and liver metastasis (P < 0.001). Parallel analysis of our samples by CGH and expression profiling revealed a direct correlation of chromosomal copy number changes with chromosome-specific average gene expression levels. Protein expression was analyzed by two-dimensional gel electrophoresis and subsequent mass spectrometry. Although there was no direct match of differentially expressed proteins and genes, the majority of them belonged to identical pathways or networks. In conclusion, increasing genomic instability and a recurrent pattern of chromosomal imbalances as well as specific gene and protein expression changes correlate with distinct stages of colorectal cancer progression. Chromosomal aneuploidies directly affect average resident gene expression levels, thereby contributing to a massive deregulation of the cellular transcriptome. The identification of novel genes and proteins might deliver molecular targets for diagnostic and therapeutic interventions. © Wiley-Liss, Inc. [source] Changes in Drosophila melanogaster midgut proteins in response to dietary Bowman,Birk inhibitorINSECT MOLECULAR BIOLOGY, Issue 5 2007H.-M. Li Abstract The midgut proteome of Drosophila melanogaster was compared in larvae fed dietary Bowman,Birk inhibitor (BBI) vs. larvae fed a control diet. By using two-dimensional gel electrophoresis, nine differentially expressed proteins were observed, which were associated with enzymes or transport functions such as sterol carrier protein X (SCPX), ubiquitin-conjugating enzyme, endopeptidase, receptor signalling protein kinase, ATP-dependent RNA helicase and ,-tocopherol transport. Quantitative real-time PCR verified differential expression of transcripts coding for six of the proteins observed from the proteomic analysis. BBI evidently affects expression of proteins associated with protein degradation, transport and fatty acid catabolism. We then tested the hypothesis that SCPX was critical for the Drosophila third instars' response to BBI treatment. Inhibition of SCPX caused the third instars to become more susceptible to dietary BBI. [source] The effect of paternal heat stress on protein profiles of pre-implantation embryos in the mouseINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 3 2005B. ZHU Summary The study was undertaken to compare the protein profiles of [35S]-methionine-labelled control-sired embryos with heat-sired embryos at 7, 14 or 21 days after mature fertile B6CBF F1 male mice were kept at 36 ± 0.3 °C and 62 ± 2.7% relative humidity for 24 h. One-dimensional gel electrophoresis and autoradiographs were used to examine the protein profiles between the two-cell embryos and the blastocysts. The results obtained demonstrate that paternal heat stress 7 or 14 days earlier did not apparently affect protein patterns of two-cell embryos, four-cell to eight-cell embryos, morulae or blastocysts. However, 21 days earlier, there were changes in protein patterns of two-cell embryos and abnormal embryos, but not the morulae. To further support and extend these results, two-dimensional gel electrophoresis and phosphorimaging were employed and the results obtained show that paternal heat stress 21 days before mating affected protein profiles of two-cell embryos and morulae in the mouse. Together, these findings have indicated that paternal heat stress affects most but not all protein patterns of pre-implantation embryos, which strongly supports our previous results demonstrating that paternal heat stress significantly reduced the developmental proportion of pre-implantation embryos in the mouse. [source] Elucidation of a protein signature discriminating six common types of adenocarcinomaINTERNATIONAL JOURNAL OF CANCER, Issue 4 2007Gregory C. Bloom Abstract Pathologists are commonly facing the problem of attempting to identify the site of origin of a metastatic cancer when no primary tumor has been identified, yet few markers have been identified to date. Multitumor classifiers based on microarray based RNA expression have recently been described. Here we describe the first approximation of a tumor classifier based entirely on protein expression quantified by two-dimensional gel electrophoresis (2DE). The 2DE was used to analyze the proteomic expression pattern of 77 similarly appearing (using histomorphology) adenocarcinomas encompassing 6 types or sites of origin: ovary, colon, kidney, breast, lung and stomach. Discriminating sets of proteins were identified and used to train an artificial neural network (ANN). A leave-one-out cross validation (LOOCV) method was used to test the ability of the constructed network to predict the single held out sample from each iteration with a maximum predictive accuracy of 87% and an average predictive accuracy of 82% over the range of proteins chosen for its construction. These findings demonstrate the use of proteomics to construct a highly accurate ANN-based classifier for the detection of an individual tumor type, as well as distinguishing between 6 common tumor types in an unknown primary diagnosis setting. © 2006 Wiley-Liss, Inc. [source] Cover Picture: J. Basic Microbiol.JOURNAL OF BASIC MICROBIOLOGY, Issue 1 20091/200 Two dimensional gel electrophoresis of heparin-bound proteins from Chlamydomonas reinhardtii. Proteins were extracted from cells harvested during night-phase and subjected to heparin-affinity chromatography. Proteins with a high affinity to heparin were eluted and applied to two-dimensional gel electrophoresis. The resulting pattern can be compared to, e.g., protein abundance during day-phase (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Examination of membrane protein expression in Paracoccus denitrificans by two-dimensional gel electrophoresisJOURNAL OF BASIC MICROBIOLOGY, Issue 1 2004Pavel Bouchal The well-known metabolic versatility of the soil bacterium Paracoccus denitrificans poses a challenge for modern proteomic approaches. We describe here improved preparation conditions that allow good separation and quantitative analyses of hundreds of membrane or periplasmic proteins. To illustrate this optimized procedure, the results of a screening for membrane proteins associated predominantly with aerobic or anaerobic (denitrifying) modes of growth are presented. [source] | ||||||||||||||||||||||||||||||||||