Application Of Two-dimensional Gel Electrophoresis Research Articles

Two-Dimensional Gel Electrophoresis Combined with Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Analysis of Eye Lens to Identify Biomarkers of Age-Related Cataract.

This chapter describes the application of two-dimensional gel electrophoresis (2DGE) combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in the analysis of rat eye lens proteins. The main purpose was to identify proteins that may serve as potential biomarkers in age-related cataract formation. This includes the family of proteins known as the crystallins. Structural proteins and enzymes involved antioxidant activities. In addition, we also analyzed lenses from other species to illustrate the potential of using this technique in clinical and preclinical biomarker studies.

Advances in proteomic technologies and their scope of application in understanding plant–pathogen interactions

Proteomics, one of the major tools of ‘omics’ is evolving phenomenally since the development and application of two-dimensional gel electrophoresis coupled with mass spectrometry at the end of twentieth century. However, the adoption and application of advanced proteomic technologies in understanding plant–pathogen interactions are far less, when compared to their application in other related fields of systems biology. Hence, this review is diligently focused on the advances in various proteomic approaches and their gamut of applications in different facets of phyto-pathoproteomics. Especially, the scope and application of proteomics in understanding fundamental concepts of plant–pathogen interactions such as identification of pathogenicity determinants (effector proteins), disease resistance proteins (resistance and pathogenesis-related proteins) and their regulation by post-translational modifications have been portrayed. This review, for the first time, presents a critical appraisal of various proteomic applications by assessing all phyto-pathoproteomics-related research publications that were published in peer-reviewed journals, during the period 2000–2016. This assessment has revealed the present status and contribution of proteomic applications in different categories of phyto-pathoproteomics, namely, cellular components, host–pathogen interactions, model and non-model plants, and utilization of different proteomic approaches. Comprehensively, the analysis highlights the burgeoning application of global proteome approaches in various crop diseases, and demand for acceleration in deploying advanced proteomic technologies to thoroughly comprehend the intricacies of complex and rapidly evolving plant–pathogen interactions.

Comparative Skeletal Muscle Proteomics Using Two-Dimensional Gel Electrophoresis.

The pioneering work by Patrick H. O’Farrell established two-dimensional gel electrophoresis as one of the most important high-resolution protein separation techniques of modern biochemistry (Journal of Biological Chemistry 1975, 250, 4007–4021). The application of two-dimensional gel electrophoresis has played a key role in the systematic identification and detailed characterization of the protein constituents of skeletal muscles. Protein changes during myogenesis, muscle maturation, fibre type specification, physiological muscle adaptations and natural muscle aging were studied in depth by the original O’Farrell method or slightly modified gel electrophoretic techniques. Over the last 40 years, the combined usage of isoelectric focusing in the first dimension and sodium dodecyl sulfate polyacrylamide slab gel electrophoresis in the second dimension has been successfully employed in several hundred published studies on gel-based skeletal muscle biochemistry. This review focuses on normal and physiologically challenged skeletal muscle tissues and outlines key findings from mass spectrometry-based muscle proteomics, which was instrumental in the identification of several thousand individual protein isoforms following gel electrophoretic separation. These muscle-associated protein species belong to the diverse group of regulatory and contractile proteins of the acto-myosin apparatus that forms the sarcomere, cytoskeletal proteins, metabolic enzymes and transporters, signaling proteins, ion-handling proteins, molecular chaperones and extracellular matrix proteins.

Down-regulation of flavin reductase and alcohol dehydrogenase-1 (ADH1) in metronidazole-resistant isolates of Trichomonas vaginalis

The microaerophilic parasite Trichomonas vaginalis is a causative agent of painful vaginitis or urethritis, termed trichomoniasis, and can also cause preterm delivery or stillbirth. Treatment of trichomoniasis is almost exclusively based on the nitroimidazole drugs metronidazole and tinidazole. Metronidazole resistance in T. vaginalis does occur and is often associated with treatment failure. In most cases, metronidazole-resistant isolates remain susceptible to tinidazole, but cross resistance between the two closely related drugs can be a problem.In this study we measured activities of thioredoxin reductase and flavin reductase in four metronidazole-susceptible and five metronidazole-resistant isolates. These enzyme activities had been previously found to be downregulated in T. vaginalis with high-level metronidazole resistance induced in the laboratory. Further, we aimed at identifying factors causing metronidazole resistance and compared the protein expression profiles of all nine isolates by application of two-dimensional gel electrophoresis (2DE).Thioredoxin reductase activity was nearly equal in all strains assayed but flavin reductase activity was clearly down-regulated, or even absent, in metronidazole-resistant strains. Since flavin reductase has been shown to reduce oxygen to hydrogen peroxide, its down-regulation could significantly contribute to the impairment of oxygen scavenging as reported by others for metronidazole-resistant strains. Analysis by 2DE revealed down-regulation of alcohol dehydrogenase 1 (ADH1) in strains with reduced sensitivity to metronidazole, an enzyme that could be involved in detoxification of intracellular acetaldehyde.

Identification of the cross-reactive and species-specific antigens between Neospora caninum and Toxoplasma gondii tachyzoites by a proteomics approach

The characterization of the cross-reactive and species-specific antigens of Neospora caninum and Toxoplasma gondii is important in the exploration to determine the common mechanisms of parasite-host interaction and to improve the serological diagnosis; it is also useful for the selection of the cross-reactive antigens that could be used in the development of vaccines or drugs for controlling the diseases caused by these two parasites. In this study, cross-reactive and species-specific antigens between N. caninum and T. gondii tachyzoites were comprehensively investigated using a proteomics approach with the application of two-dimensional gel electrophoresis, immunoblot analysis, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS), and MALDI-TOF/TOF-MS analysis. Immunoblotting and mass spectrometry analysis revealed that at least 42 individual protein spots of N. caninum were reacted with the anti-N. caninum serum, among which at least 18 protein spots were cross-reacted with the anti-T. gondii serum. Moreover, at least 31 protein spots of T. gondii were reacted with the anti-T. gondii serum, among which at least 19 protein spots were cross-reacted with the anti-N. caninum serum. Furthermore, some new specific proteins were also identified in the N. caninum protein profile by searching Toxoplasma sequences or sequences from other organisms. This study substantiates the usefulness of proteomics in the immunoscreening of the cross-reactive or species-specific antigens of both parasites. In addition, the present study showed that there was significant homology in the antigenic proteome profiles between the two parasites. These observations have implications for the design of multicomponent common vaccines against both parasite infections.

Application of Two-Dimensional Gel Electrophoresis To Interrogate Alterations in the Proteome of Genetically Modified Crops. 1. Assessing Analytical Validation

Current tools used to assess the safety of food and feed derived from modern biotechnology emphasize the investigation of possible unintended effects caused directly by the expression of transgenes or indirectly by pleiotropy. These tools include extensive multisite and multiyear agronomic evaluations, compositional analyses, animal nutrition, and classical toxicology evaluations. Because analytical technologies are rapidly developing, proteome analysis based on two-dimensional gel electrophoresis (2DE) was investigated as a complementary tool to the existing technologies. A 2DE method was established for the qualitative and quantitative analysis of the seed proteome of Arabidopsis thaliana with the following validation parameters examined: (1) source and scope of variation; (2) repeatability; (3) sensitivity; and (4) linearity of the method. The 2DE method resolves proteins with isoelectric points between 4 and 9 and molecular masses (MM) of 6-120 kDa and is sensitive enough to detect protein levels in the low nanogram range. The separation of the proteins was demonstrated to be very reliable with relative position variations of 1.7 and 1.1% for the pI and MM directions, respectively. The mean coefficient of variation of 254 matched spot qualities was found to be 24.8% for the gel-to-gel and 26% for the overall variability. A linear relationship (R2 > 0.9) between protein amount and spot volume was demonstrated over a 100-fold range for the majority of selected proteins. Therefore, this method could be used to interrogate proteome alterations such as a novel protein, fusion protein, or any other change that affects molecular mass, isoelectric point, and/or quantity of a protein.

Application of Two-Dimensional Gel Electrophoresis To Interrogate Alterations in the Proteome of Genetically Modified Crops. 2. Assessing Natural Variability

Proteomics is currently tested as a complementary tool for the safety assessment of genetically modified (GM) crops. Understanding the natural variability of the proteome is crucial for the interpretation of biological differences between transgenic and nontransgenic parental lines. The natural variation of seed protein profiles among a set of 12 Arabidopsis thaliana ecotypes was determined by utilizing two-dimensional electrophoresis (2DE). The total number of different resolved protein spots found among the 12 ecotypes was 931 with a range of 573 (Mt-0) to 653 (Condara) in any one ecotype. Although the ecotypes were grown side-by-side in an environmentally controlled growth chamber, almost half of the resolved spots varied with respect to their presence/absence, and 95% of the spots present in all ecotypes varied in spot quantity (2-53-fold). In the evaluation of unintended effects of genetic modification, it is concluded that the experimental design must account for existing natural variability, which, in the case of the expressed proteome, can be substantial.

Application of Two-Dimensional Gel Electrophoresis To Interrogate Alterations in the Proteome of Gentically Modified Crops. 3. Assessing Unintended Effects

The current procedures to assess the safety of food and feed derived from modern biotechnology include the investigation of possible unintended effects. To improve the probability of detecting unintended effects, profiling techniques such as proteomics are currently tested as complementary analytical tools to the existing safety assessment. An optimized two-dimensional gel electrophoresis (2DE) method was used as a proteomics approach to investigate insertional and pleiotropic effects on the proteome due to genetic engineering. Twelve transgenic Arabidopsis thaliana lines were analyzed by 2DE, and their seed proteomes were compared to that of their parental line as well as to 12 Arabidopsis ecotype lines. The genetic modification of the Arabidopsis lines, using three different genes and three different promoters, did not cause unintended changes to the analyzed seed proteome. Differences in spot quantity between transgenic and nontransgenic lines fell in the range of values found in the 12 Arabidopsis ecotype lines or were related to the introduced gene.

Pregnancy-associated corticosteroid-binding globulin: high resolution separation of glycan isoforms.

Corticosteroid-binding globulin (CBG) is a glycoprotein that functions as a specific carrier of cortisol in the circulation. CBG contains six sites for N-glycosylation with, on average, five sites occupied by a mixture of biantennary and triantennary oligosaccharides with variable additional terminal sialic acid residues leading to glycoforms with significant heterogeneity in mass and isoelectric points. During pregnancy, a form of CBG possessing only triantennary oligosaccharides comprising approximately 10 % of total CBG appears specifically. We describe the first application of two-dimensional gel electrophoresis to the separation of human CBG glycoforms. This technique resolved a greater degree of charge heterogeneity than previous studies, and allowed simultaneous visualization of changes to the size and isoelectric points of CBG during pregnancy. Profiles of CBG glycoforms during pregnancy showed a general increase in size followed by a shift to lower pI in a large proportion of the glycoprotein. This may result from the enhancement of triantennary glycosylation, with the extent of incorporation of sialic acid increasing with the number of available sites for its addition. The pregnancy-specific CBG previously defined probably represents a subset of the acidic and high molecular weight glycoforms we have resolved by two-dimensional electrophoresis and now describe as pregnancy-associated CBG.

Application of two-dimensional gel electrophoresis to detect proteins associated with harvest maturity in stonefruit

The correct assessment of harvest maturity stage of stonefruit is an important determinant of their quality when they reach the consumer. However, the current indices of ripening used (e.g. skin colour, firmness and soluble solids concentration) vary with cultivar, rootstock, growing conditions and seasonal climatic factors. To develop an index that is independent of environmental factors, total proteins were extracted from fruit and separated by 2D-PAGE. Four proteins (Z1, Z2, Y and X) that were synthesised in fruit a few days before optimum harvest maturity were identified in Japanese-type plums (Prunus salicina), peaches and nectarines (Prunus persica), and European plums (Prunus domestica). These proteins are first expressed a few days before optimum harvest date and their expression increases as the fruit mature. The N-termini of the three most prominent proteins from plums (Z1, Z2 and Y) were sequenced. There appears to be close homology between these proteins and those from a range of species including almond, grape and rubber. It appears that these proteins belong to a family of allergens common to plants whose function may be to confer some form of protection to the plant during periods of stress. These proteins may be used for the development of a test kit that will assist growers to determine optimum harvest maturity for cultivars of plums, peaches and nectarines. © 2002 Elsevier Science B.V. All rights reserved.

The 72/74-kDa polypeptides of the 70‒110 S large heterogeneous nuclear ribonucleoprotein complex (LH-nRNP) represent a discrete subset of the hnRNP M protein family

Pre-mRNA processing in eukaryotes is thought to take place on a multitude of nuclear ribonucleoprotein (RNP) complexes, the most abundant of them being the heterogeneous nuclear (hn) RNP complexes. The identification in mammalian nuclear extracts of a novel, less-abundant 70–110S heterogeneous RNP, named large heterogeneous nuclear RNP (LH-nRNP), has previously been reported by Aidinis, Sekeris and Guialis (1995) Nucleic Acids Res. 23, 2742–2753. The structural composition of the LH-nRNP complex has been determined following the production of polyclonal antibodies against the major protein constituents of the complex, the pair of the 72/74-kDa polypeptides. In the present study evidence is shown to prove that the 72/74-kDa proteins are members of the hnRNP M protein family, hereafter referred to as 72/74(M) polypeptides. The extensive application of two-dimensional gel electrophoresis, combined with specific immunoprecipitation and immunoblotting assays, has allowed the assignment of the 72/74(M) proteins to a subset of the hnRNP M family, characteristic of the presence of the LH-nRNP complex and distinct from the hnRNP-associated M1–M4 components. Moreover, the immunoselection of the LH-nRNP complex from [32P]orthophosphate-labelled HeLa cells, with the parallel application of UV irradiation, has permitted the identification of the 72/74(M) polypeptides as the sole protein constituents of the complex in direct contact with the RNA. It is proposed that LH-nRNP constitutes a discrete subset of hnRNP complexes, having a possible role in establishing specific interactions between hnRNP and nuclear-matrix protein components.

The 72/74-kDa polypeptides of the 70–110S large heterogeneous nuclear ribonucleoprotein complex (LH-nRNP) represent a discrete subset of the hnRNP M protein family

Pre-mRNA processing in eukaryotes is thought to take place on a multitude of nuclear ribonucleoprotein (RNP) complexes, the most abundant of them being the heterogeneous nuclear (hn) RNP complexes. The identification in mammalian nuclear extracts of a novel, less-abundant 70-110 S heterogeneous RNP, named large heterogeneous nuclear RNP (LH-nRNP), has previously been reported by Aidinis, Sekeris and Guialis (1995) Nucleic Acids Res. 23, 2742-2753. The structural composition of the LH-nRNP complex has been determined following the production of polyclonal antibodies against the major protein constituents of the complex, the pair of the 72/74-kDa polypeptides. In the present study evidence is shown to prove that the 72/74-kDa proteins are members of the hnRNP M protein family, hereafter referred to as 72/74(M) polypeptides. The extensive application of two-dimensional gel electrophoresis, combined with specific immunoprecipitation and immunoblotting assays, has allowed the assignment of the 72/74(M) proteins to a subset of the hnRNP M family, characteristic of the presence of the LH-nRNP complex and distinct from the hnRNP-associated M1-M4 components. Moreover, the immunoselection of the LH-nRNP complex from [(32)P]orthophosphate-labelled HeLa cells, with the parallel application of UV irradiation, has permitted the identification of the 72/74(M) polypeptides as the sole protein constituents of the complex in direct contact with the RNA. It is proposed that LH-nRNP constitutes a discrete subset of hnRNP complexes, having a possible role in establishing specific interactions between hnRNP and nuclear-matrix protein components.

Effects of drugs on mammalian cells growing in vitro

The microaerophilic parasite Trichomonas vaginalis is a causative agent of painful vaginitis or urethritis, termed trichomoniasis, and can also cause preterm delivery or stillbirth. Treatment of trichomoniasis is almost exclusively based on the nitroimidazole drugs metronidazole and tinidazole. Metronidazole resistance in T. vaginalis does occur and is often associated with treatment failure. In most cases, metronidazole-resistant isolates remain susceptible to tinidazole, but cross resistance between the two closely related drugs can be a problem.In this study we measured activities of thioredoxin reductase and flavin reductase in four metronidazole-susceptible and five metronidazole-resistant isolates. These enzyme activities had been previously found to be downregulated in T. vaginalis with high-level metronidazole resistance induced in the laboratory. Further, we aimed at identifying factors causing metronidazole resistance and compared the protein expression profiles of all nine isolates by application of two-dimensional gel electrophoresis (2DE).Thioredoxin reductase activity was nearly equal in all strains assayed but flavin reductase activity was clearly down-regulated, or even absent, in metronidazole-resistant strains. Since flavin reductase has been shown to reduce oxygen to hydrogen peroxide, its down-regulation could significantly contribute to the impairment of oxygen scavenging as reported by others for metronidazole-resistant strains. Analysis by 2DE revealed down-regulation of alcohol dehydrogenase 1 (ADH1) in strains with reduced sensitivity to metronidazole, an enzyme that could be involved in detoxification of intracellular acetaldehyde.

Two-dimensional gel electrophoretic comparison of proteins from virulent and avirulent strains of Mycoplasma pneumoniae

The protein composition of the virulent M129 strain of Mycoplasma pneumoniae was compared to that of its homologous avirulent strain by the use of standard one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Forty-nine individual M. pneumoniae cell proteins were resolved by this method, and the virulent strain was shown to possess a single high-molecular-weight protein not present in avirulent cells. Variability in the resolution of this particular protein in one-dimensional gels prompted the application of two-dimensional gel electrophoresis to the analysis of M. pneumoniae cell proteins. The sequential use of isoelectric focusing in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension permitted the resolution of a least 142 individual M. pneumoniae cell proteins. Application of nonequilibrium pH gradient electrophoresis in the first dimension achieved the resolution of at least 20 additional basic proteins. Three proteins which are synthesized only by cells of the virulent strain, and not by the homologous avirulent strain, were identified by these two-dimensional gel electrophoresis techniques.