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.     

Application of proteomics to hordein screening in the malting process

This study was undertaken to investigate the effect of the malting process on hordein composition. For this purpose, combination of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and the method of isotopic peptides labeling iTRAQ was used. Barley proteins are essential components determining the quality of both malt and beer. Since hordeins represent the most abundant proteins accounting for about 40-50% of total protein fraction of mature barley grain, our research was focused on them. In this respect, the proteins of interest were extracted from milled samples of barley grain, germinated barley grain (samples collected at different time intervals), green malt and malt, respectively. Particular hordein extracts were firstly fractionated via SDS- PAGE, which was used as a relatively rapid and reliable technique providing information about hordein profile of analyzed samples. Then, separated proteins were in-gel digested and resulting peptides were measured by mass spectrometry. In addition, the chosen proteins, after in-gel digestion, were subjected to the iTRAQ method and the screening of proteins during malting process was evaluated. Our results have revealed that most of the hordein components present in the barley grain can be found in all stages of the malting process as well as in the final malt. The amount of hordeins decreases during the malting process; in the case of C hordein, the protein decrease is approximately 65%. On the other hand, significant degradation of D hordein was detected. The suggested procedure can be used to follow the development of the hordein profile during germination, which is of great technological importance in beer production.     

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.     

MALDI‐TOF mass spectrometry of hordeins: rapid approach for identification of malting barley varieties

A procedure for identification of malting barley varieties using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) of ethanol‐soluble barley proteins (hordeins) is described. The hordeins were first extracted from milled barley grains by several extraction protocols (using different extraction agents and conditions). Hordein extracts were then analyzed directly via MALDI‐TOF MS without any preliminary purification or separation step, and the protein profiles of analyzed hordein extracts were compared in order to find out the most suitable extraction procedure for mass spectrometric analysis. The optimized procedure was successfully applied to identification of 13 malting barley varieties. Our results revealed that the proposed mass spectrometry‐based approach provides characteristic mass patterns of extracted hordeins, which can be advantageously used for barley variety identification. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

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.     

Fluctuation in the ergosterol and deoxynivalenol content in barley and malt during malting process

This paper describes determination of the deoxynivalenol and ergosterol in samples from different varieties of barley and, consequently, malt produced from this barley. In total, 20 samples of barley and 20 samples of barley malt were analyzed. The alkaline hydrolysis with consequent extraction into hexane was applied to obtain the ergosterol from cereals. Extraction to acetonitrile/water and subsequent solid-phase extraction (SPE) were used for deoxynivalenol. The determination of the samples was performed on high-performance liquid chromatography using UV detection (ergosterol) and mass spectrometric detection (deoxynivalenol). The influence of the malting process on the production of two compounds of interest was assessed from obtained results. Ergosterol concentration ranged 0.88–15.87 mg/kg in barley and 2.63–34.96 mg/kg in malt, where its content increased to 95% compared to samples before malting. The malting process was observed as having a significant effect on ergosterol concentration (P = 0.07). The maximum concentration of deoxynivalenol was found to be 641 μg/kg in barley and 499 μg/kg in malt. Its concentration was lower than the legislative limit for unprocessed cereals (1,250 μg/kg). The statistic effect of the malting process on deoxynivalenol production was not found. Linear correlation between ergosterol and deoxynivalenol content was found to be very low (barley R = 0.02, malt R = 0.01). The results revealed that it is not possible to consider the ergosterol content as the indicator of deoxynivalenol contamination of naturally molded samples.     

Separation of hordein proteins from european barley by high-performance liquid chromatography: Its application to the identification of barley cultivars

Summary High-performance liquid chromatography using either reversed phase or anion-exchange techniques was used for the fractionation of hordein proteins extracted from European barley. A reversed phase method is presented which utilises an Ultrapore column packed with a wide pore (30 nm) C-3 alkylbonded silica support. Using this method up to 20 components may be separated in 54 min. Elution profiles were found to be reproducible. A further method using rapid anion-exchange chromatography indicated that up to 13 components may be separated, a number which is comparable to that found with electrophoresis. The separation of proteins extracted from different barley cultivars indicated that on the basis of elution profiles high-performance liquid chromatography using either reversed phase or anion-exchange offers considerable potential as a method for barley cultivar identification.     

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.