Genetic variability of pepper (Capsicum annuum L.) seed proteins studied by 2-D electrophoresis with immobilized pH gradients.

Ten pepper (Capsicum annuum L.) inbred lines were successfully differentiated by two-dimensional electrophoresis with immobilized pH gradients. Qualitative polymorphism of water-soluble and urea/detergent-soluble seed proteins, respectively, was investigated by computer analysis and used for establishing a dendrogram derived from maximum-parsimony analysis. The dendrogram calculated from urea/detergent-soluble proteins shows four types of distance indices, whereas water-soluble proteins show two sets of inbred lines with similar intraset distance indices. The validity of the dendrograms with respect to quantitative inherited traits, such as cold tolerance and earliness, will be tested by field trials.

Qualitative and quantitative changes in barley seed protein patterns during the malting process analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with respect to malting quality.

Seeds of two barley cultivars, similar in total protein content and malt extract yield but different in their final attenuation values, were malted. Samples taken at daily intervals during the malting process were extracted sequentially with Tris-HCl buffer, aqueous 2-propanol, aqueous 2-propanol containing 0.5% dithiothreitol, and 4 M urea, containing 0.5% dithiothreitol and 1% Nonidet P-40. The protein composition of these extracts was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and computer densitometry to determine whether differences observed in the rate or extent of protein modification are related to the malting quality character final attenuation. It was found that, common to both cultivars, the albumin and globulin proteins were relatively resistant to proteolysis, whereas the hordeins suffered a dramatic breakdown during malting, with the D hordein being degraded most rapidly, followed by the B and C hordeins. Besides these similarities, differences between both cultivars were observed in the relative rates of D hordein degradation, as this rate was considerably higher in the cultivar with high malting quality. Similar, but much less distinct kinetics were seen with certain B hordeins. Since a possible relationship might exist between the rate of proteolysis of the D hordeins and the character final attenuation, we analyzed a larger number of barley cultivars with different final attenuation values with a simplified technique. For the ten cultivars examined, differences during germination were again seen in the rates of modification of the D hordeins. However, significant correlations between the D hordein breakdown and final attenuation values were not obtained, so that we propose that there exists at best a loose correlation between the relative rate of proteolysis of these proteins and the malting quality character final attenuation.

Application of sequential extraction procedures and glycoprotein blotting for the characterization of the 2-D polypeptide patterns of barley seed proteins.

Barley (Hordeum vulgare L.) proteins were sequentially extracted from ground seeds with Tris-HCl buffer, 55% 2-propanol, 55% 2-propanol containing 1% dithiothreitol, and 6 M urea containing 2% Nonidet P-40 and 1% dithiothreitol. The protein composition of these solubility fractions was then analyzed by high resolution two-dimensional gel electrophoresis with immobilized pH gradient 4-9 in the first dimension, followed by silver staining and glycoprotein blotting, respectively, for a more detailed characterization of the two-dimensional polypeptide pattern of barley seed proteins.

Detection of polypeptides and amylase isoenzyme modifications related to malting quality during malting process of barley by two-dimensional electrophoresis and isoelectric focusing with immobilized pH gradients.

Two cultivars ("Alexis" and "Lenka") of contrasting final attenuation values were malted, and the protein and amylase isoenzyme composition, as well as the change in protein and amylase isoenzyme composition during malting, was investigated by two-dimensional polyacrylamide gel electrophoresis of total proteins, and isoelectric focusing of amylase isoenzymes, respectively. Isoelectric focusing demonstrated that significant differences exist between the amylase isoenzyme patterns of the two cultivars, suggesting a correlation between the presence of certain amylase isoenzyme bands and final attenuation. This finding was confirmed by analysis of 36 barley cultivars with a wide range of quality. It was shown that all cultivars which are of low or, at best, moderate final attenuation values exhibit the amylase band "B" (isoelectric point approximately 6.8), whereas those cultivars which are predominantly of high malting grade do not possess this "B" isoenzyme band, but exhibit the pronounced "A" isoenzyme band (isoelectric point approximately 6.5) instead, suggesting that these isoenzymes (which we suppose to be beta-amylases) can be utilized to predict the final attenuation values of unknown barley samples or new lines. However, "final attenuation" is a complex function. Preliminary results of two-dimensional gel electrophoresis indicate that other factors, such as total amount of amylases, or a 19 kDa A hordein-like polypeptide, which was degraded faster in the low malting grade cultivar "Lenka", may also have a role in determining quality.

Two-dimensional polyacrylamide gel electrophoresis, with immobilized pH gradients in the first dimension, of barley seed proteins: discrimination of cultivars with different malting grades.

The suitability of high-resolution two-dimensional gel electrophoresis for barley cultivar discrimination and for classification with respect to their malting properties was studied. Seed proteins of 14 barley cultivars with different malting qualities were extracted with urea/dithiothreitol/Nonidet P-40 buffer and subjected to two-dimensional gel electrophoresis with immobilized pH gradients in the first dimension (IPG-DALT). The results of IPG-DALT were compared to the protein patterns obtained by a standard technique, sodium dodecyl sulfate polyacrylamide gel electrophoresis of hordeins. Sodium dodecyl sulfate-gel electrophoresis yielded seven different "B" and four different "C" hordein patterns; "A" and "D" hordein patterns were uniform in all cultivars tested. Four cultivars could be distinguished unequivocally, the others were classified into three groups containing between two and five cultivars. In contrast to these findings. IPG-DALT yielded three different "A", eight different "B", four different "C" and two different "D" hordein patterns. When the "A", "B", "C" and "D" hordein patterns were combined, ten cultivars exhibited unique hordein patterns whereas the remaining ones were classified into two groups containing two cultivars each. Moreover, when albumin and globulin proteins were used for evaluation in addition to the hordeins, all cultivars could be discriminated by IPG-DALT. IPG-DALT, performed on small-scale and/or ready-made gels, proved to be an ideal complementary system to one-dimensional electrophoretic methods for routine seed testing purposes because of its speed, reliability, and simplicity. IPG-DALT was also applied to study the relationship between the different polypeptide patterns and the malting quality. Although cultivars with identical one-dimensional protein patterns but different malting quality could be successfully differentiated by IPG-DALT, a direct correlation between specific protein spots or protein patterns to the malting quality was not found within the cultivars tested.

Temperature-dependent spot positional variability in two-dimensional polypeptide patterns.

The effect of temperature, at which isoelectric focusing with immobilized pH gradients is performed, on spot positions and pattern quality in two-dimensional (2-D) electrophoresis was examined. Increased temperatures revealed improved 2-D patterns with respect to sample entry, resolution, and background staining. Focusing at 20 degrees C was superior to focusing at 10 and 15 degrees C. Even at 30 degrees C, a pattern of well-resolved polypeptide spots with a minimum amount of horizontal streaking at the basic end was observed. A computer-based analysis showed that a substantial proportion of polypeptides assumed altered positions in the 2-D pattern in relation to temperature. Mobility shifts of polypeptides were more variable on the neutral part than on the acidic or basic end. The mobility shifts were not restricted to one direction for all the spots whose migration was altered. However, for any given spot, the direction was the same with subsequent increments of temperature. The results clearly demonstrate that a defined temperature for first-dimensional isoelectric focusing is a requirement for the reproducibility of 2-D electrophoresis. After elimination of the cathodic drift, a major source of variability in 2-D patterns, associated with carrier ampholytes, temperature control becomes a critical parameter.

Barley cultivar discrimination: I. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and glycoprotein blotting.

Two different methods of detecting electroblotted glycoproteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Tris-buffer soluble barley seed proteins were examined for their applicability for barley cultivar discrimination. These are the highly specific, lectin-based concanavalin A/peroxidase method and the more general periodate/danyslhydrazine method. The results of the periodate/dansylhydrazine method enabled us to divide the 20 examined cultivars into three groups, whereas the more sensitive concanavalin A/peroxidase method revealed six different glycoprotein patterns. In comparison, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of the alcohol-soluble barley seed proteins (hordeins) gave nine different banding patterns. A combination of hordein electrophoresis together with glycoprotein staining by the concanavalin A/peroxidase method made it possible to classify the cultivars into twelve groups, the largest of which contained four cultivars. The qualitative expression of the glycoprotein patterns seemed to be independent of growth conditions, whereas the band intensities obviously were not. As a whole, glycoprotein blotting is a valuable supplement to sodium dodecyl sulfate-polyacrylamide gel electrophoresis of hordeins in barley cultivar discrimination.

Barley cultivar discrimination: II. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing with immobilized pH gradients.

Isoelectric focusing performed with immobilized pH gradients was found superior to other commonly used electrophoretic methods for discrimination of 55 European winter and spring barley cultivars. Hordeins, the alcohol-soluble proteins, yielded 32 different patterns, allowing identification of 22 cultivars and classification of the remaining ones into ten groups of two to eight cultivars each. Only 21 different hordein patterns were observed using horizontal sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by silver staining. Twelve cultivars exhibited unique hordein patterns, the remaining nine groups contained 2-11 cultivars. Resolution of isoelectric focusing with immobilized pH gradients was further enhanced in some cases when the patterns of urea/dithiothreitol-soluble proteins were used instead of the hordein patterns. However, evaluation was more complicated because of the larger number of protein bands detected.

Changes in protein expression during melanoma differentiation determined by computer analysis of 2-D gels.

Cytodifferentiation in many melanocytic cells is regulated through the adenylate cyclase-cAMP pathway. To analyse the molecular changes associated with this process we have compared the proteins produced by two closely related cell lines which, though derived from a single cell line, respond very differently to modulation of this signalling pathway. The human melanoma cell line DX3 shows little change in in vitro characteristics following treatment with cAMP elevating agents; in contrast the more malignant DX3 LT5.1 variant, derived from the DX3 parental line, shows pronounced dendrification, decreased proliferation and a reduction in metastatic capacity after similar treatment. The two cell lines were treated with phosphodiesterase inhibitors for 5 days and then processed for two-dimensional gel characterization using an immobilized pH gradient for the IEF dimension. Proteins were detected by silver staining the gels and protein intensities were digitized using a laser densitometer. Two-dimensional gel patterns were edited, matched and a melanoma protein database of 637 spots constructed using PDQUEST software on an Orion 1/05 computer. Eleven proteins were lost and four new proteins were detected in both cell lines following treatment. Twenty-two proteins were present in DX3 LT5.1 after treatment but not in untreated lines or treated DX3. These differentially expressed proteins may be associated with the observed changes in differentiation patterns and metastasis. Our results illustrate the resolving power of this technique and suggest potential applications to the study of cellular differentiation.

Serum proteins of rats exposed to organic solvents examined by horizontal two-dimensional electrophoresis with an immobilized pH gradient in the first dimension.

Serum proteins of rats, exposed to methoxyethylacetate and a combination of methoxyethylacetate and isobutylacetate, were analyzed by horizontal high resolution two-dimensional gel electrophoresis with immobilized pH gradients in the first dimension. A total of 410 polypeptides were detected with either increasing or decreasing spot intensities after rat exposure to the organic solvents.

Two-dimensional electrophoresis with immobilized pH gradients of leaf proteins from barley (Hordeum vulgare): method, reproducibility and genetic aspects.

Leaf proteins from 14 barley cultivars (Hordeum vulgare) were analyzed by two-dimensional electrophoresis with immobilized pH gradients (IPG 4-7 and IPG 6-10) in the first dimension. Highly reproducible two-dimensional patterns were obtained, owing to constant spot positions along the isoelectric focusing axis. A number of variety-specific protein spots were detected, allowing us to discriminate barley cultivars not only into main groups but into individual cultivars.

Dual-label autoradiographic analysis of human skin fibroblast and myoblast proteins by two-dimensional polyacrylamide gel electrophoresis using immobilised pH gradients in the first dimension.

Horizontal two-dimensional polyacrylamide gel electrophoresis with immobilised pH gradients in the first dimension has been applied to the analysis of human skin fibroblast and muscle myoblast total cell proteins. Excellent two-dimensional separations of skin fibroblast proteins were obtained using pH 4-10 immobilised pH gradient gels with a long interelectrode distance (16 cm), but resolution was degraded, particularly of the more acidic proteins, by the use of shorter (10 cm) gels. Improved resolution of acidic and basic proteins was obtained using separate pH 4-7 and pH 7-10 immobilised pH gradient gels respectively in the first dimension. Two-dimensional protein maps of skin fibroblast proteins were visualised both by silver staining and by autoradiography of samples labelled synthetically with [35S]methionine. Horizontal two-dimensional electrophoresis, using pH 4-7 and pH 7-10 immobilised pH gradient gels in the first dimension, was applied to the analysis of protein samples from skin fibroblasts and muscle myoblasts dual-labelled synthetically with [35S]methionine and [75Se]selenomethionine in an attempt to identify sets of proteins specific to each cell type. In addition, two-dimensional maps or protein samples derived from normal individuals and patients with Duchenne muscular dystrophy were compared to search for protein changes associated with the disease state. Although sets of qualitative protein spot differences were observed by visual inspection of the two-dimensional gels, more rigorous qualitative and quantitative analysis of the patterns using a computerised analysis system will be required to obtain the maximum amount of information from these data.

Acid phosphatase typing for breeding nematode-resistant tomatoes by isoelectric focusing with an ultranarrow immobilized pH gradient.

The genetic variants of tomato acid phosphatase (Aps-1) systems have been analyzed by isoelectric focusing in immobilized pH gradients (IPG). By using an ultranarrow pH 4.25-4.55 IPG gel, the two genotypes Aps-1(1) and Aps-1+, differentiating tomato variants into nematode-resistant or nematode-susceptible plants, are separated into two sharp zones over a distance of 2.5 cm with isoelectric points of 4.37 and 4.43, respectively. Under these conditions, silver staining of the Aps-1 variants proved to be superior to enzyme staining. By applying more than 50 samples on one IPG gel, this method proved to be a powerful tool for reliable tomato nematode resistance screening.

Stable storage conditions of Immobiline chemicals for isoelectric focusing.

Most of the problems connected with the use of the Immobiline chemicals (a set of six, non-amphoteric, acrylamido buffers having pK values in the pH 3.5-9.5 interval) can be attributed to the alkaline species (with pK values 6.2, 7.0, 8.5 and 9.3). These compounds, to varying degrees are subjected to two degradation pathways: (a) hydrolysis of the amido bond, producing free acrylic acid and a diamine, the latter unable to be incorporated into the polyacrylamide matrix; (b) spontaneous auto-polymerization, producing a number of oligomers up to n-mers, able to aggregate and precipitate large proteins. Storage of their water solutions as frozen aliquots, a method widely employed, only partially alleviates the problem. Addition of trace-amounts of inhibitors, as lately adopted by the manufacturer, could only reduce the problem of auto-polymerization, but not block the hydrolysis of the amido bond. A new solution has been found, which abolishes both phenomena: storage in n-propanol. As demonstrated by gas chromatography, HPLC analyses and two-dimensional separations of complex samples, storage in organic solvent completely abolishes both hydrolysis and auto-polymerization and allows production of highly reproducible focusing patterns.

Horizontal two-dimensional electrophoresis with immobilized pH gradients using PhastSystem.

Protocols for horizontal two-dimensional electrophoresis with immobilized pH gradients in the first dimension were modified for horizontal micro two-dimensional electrophoresis using PhastSystem. Different equilibration conditions of the first-dimensional immobilized pH gradient gel strip prior to second-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis were evaluated. Silver stained two-dimensional patterns were obtained within 3.5 h.

Approach to stationary two-dimensional pattern: influence of focusing time and immobiline/carrier ampholytes concentrations.

Horizontal two-dimensional (2-D) electrophoresis with immobilized pH gradients (IPG) in the first dimension for buffer soluble proteins and for complex proteins solubilized in the presence of Nonidet P-40 (Görg et al., Electrophoresis 1987, 8, 45-51), has been extended to analyze basic proteins of yeast cells focused under non-equilibrium and equilibrium conditions. Transient state isoelectric focusing (IEF) in IPG gels revealed sample smearing and background staining, displaying horizontal streaks in the resultant 2-D patterns. Inclusion of 0.5% carrier ampholytes (CA) to the IPG gel (IPG-CA), resulted in the formation of many sharp protein bands after transient state IEF with resultant distinct spots in the 2-D patterns; however, resolution was poor and the gel contained heavy background staining. With prolonged focusing time, background staining disappeared and there was less difference in the final steady state IEF patterns obtained with IPG and IPG-CA. Reduction of the Immobiline concentration to one third the manufacturer's recommended amount did not improve IEF resolution with respect to streaking and background staining under either transient state or equilibrium conditions. In general, spot intensities were less on 2-D gels using diluted IPG gels than with "standard" IPG gels. Optimization of 2-D electrophoresis with IPGs in the first dimension was strongly related to IEF conditions. The use of IPG gels focused to equilibrium should not only improve inter-gel reproducibility and resolution but also the quality of the final 2-D patterns with respect to background staining and horizontal streaking.

Improved resolution of PI (alpha 1-antitrypsin) phenotypes by a large-scale immobilized pH gradient.

PI variants and PI M subtypes were examined by isoelectric focusing in a preformed immobilized pH gradient. Conditions were found that permit reliable classification of PI variants and of most PI M subtypes: the pH gradient ranges from 4.3 to 4.8 on a polyacrylamide gel with a length of 20 cm and a separation distance of approximately 16-17 cm (delta pH = 0.025 U/cm). The PI phenotypes observed are presented.

pH, urea and substrate gradients for the optimization of ultrathin polyacrylamide gel zymograms.

The preparation of ultrathin polyacrylamide gels with different kinds of gradients (pH, substrates, inhibitors) is described. By using these gels for contact printing after isoelectric focusing with Ampholines or Immobilines and for diffusion tests, the influence of pH or increasing amounts of substrates or inhibitors on enzyme and isoenzyme activities is studied. These methods are successfully applied for the optimization of zymogram techniques and for the easy characterization of industrial microbial enzyme preparations for technological purposes. With buffer-generated pH gradient gels, the pH optimum of all isoenzyme activities is demonstrated by contact printing; the total amount of isoenzyme activities dependent on pH is determined by a diffusion test. Gels with a linear gradient between 0 and 8 M urea are used for isoelectric focusing, diffusion tests and contact printing in order to differentiate the unfolding and denaturing effects of urea on isoenzymes. Alterations in polygalacturonase isoenzyme patterns dependent on urea concentration are not caused by inhibition or denaturation but by the change of charges. In respect to band sharpness and straightness urea can be added advantageously up to 2 M without changing the isoelectric points or activities of the isoenzymes. For the reproducibility of zymograms it is interesting to see that different substrate concentrations reveal different isoenzyme patterns.

Transferrin subtypes and variants in Germany; further evidence for a Tf null allele.

Isoelectric focusing (IEF) with carrier ampholytes was used for the determination of transferrin C subtypes and transferrin B and D variants in a sample of 1125 unrelated individuals from Southern Germany. The observed TfC allele frequencies were Tf*C1 = 0.7872, Tf*C2 = 0.1365, and Tf*C3 = 0.0675. The rare C subtype C6 was observed twice. A new C subtype, called C10, was observed and identified by IEF with immobilized pH gradients. The rare C subtypes C4 and C8 were also studied by this method. TfB and TfD variants were found with a heterozygous frequency of 1.53%. One new TfD was found which is located between D1 and D2 and therefore named D1-2. Evidence for a Tf null allele was obtained in a child and the putative father; they were considered to be heterozygous for an allele Tf0. The theoretical exclusion rate for paternity examinations was calculated for the Tf system and found to be 17.95%.