Hordein accumulation in developing barley grains

The temporal pattern of accumulation of hordein storage proteins in developing barley grains was studied by enzyme-linked immunosorbent assay (ELISA), western blot and liquid chromatography tandem mass spectrometry (LC-MS/MS). Hordein accumulation was compared to the pattern seen for two abundant control proteins, serpin Z4 (an early accumulator) and lipid transferase protein (LTP1, a late accumulator). Hordeins were detected from 6 days post-anthesis (DPA) and peaked at 30 DPA. Changes in fresh weight indicate that desiccation begins at 20 DPA and by 37 DPA fresh weight had decreased by 35%. ELISA analysis of hordein content, expressed on a protein basis, increased to a maximum at 30 DPA followed by a 17% decrease by 37 DPA. The accumulation of 39 tryptic and 29 chymotryptic hordein peptides representing all classes of hordein was studied by LC-MS/MS. Most peptides increased to a maximum at 30 DPA, and either remained at the maximum or did not decrease significantly. Only five tryptic peptides, members of the related B1- and γ1-hordeins decreased significantly by 21–51% at 37 DPA. Thus, the concentration of some specific peptides was reduced while remaining members of the same family were not affected. The N-terminal signal region was removed by proteolysis during co-translation. In addition to a suite of previously characterized hordeins, two novel barley B-hordein isoforms mapping to wheat low molecular weight glutenins (LMW-GS-like B-hordeins), and two avenin-like proteins (ALPs) sharing homology with wheat ALPs, were identified. These identified isoforms have not previously been mapped in the barley genome. Cereal storage proteins provide significant nutritional content for human consumption and seed germination. In barley, the bulk of the storage proteins comprise the hordein family and the final hordein concentration affects the quality of baked and brewed products. It is therefore important to study the accumulation of hordeins as this knowledge may assist plant breeding for improved health outcomes (by minimizing triggering of detrimental immune responses), nutrition and food processing properties

Proteome changes resulting from malting in hordein-reduced barley lines

Hordeum vulgare L., commonly known as barley, is primarily used for animal feed and malting. The major storage proteins in barley are hordeins, known triggers of celiac disease (CD). Here, sequential window acquisition of all theoretical mass spectra (SWATH)-MS proteomics was employed to investigate the proteome profile of grain and malt samples from the malting barley cultivar Sloop and single-, double-, and triple hordein-reduced lines bred in a Sloop background. Using a discovery proteomics approach, 2688 and 3034 proteins were detected from the grain and malt samples, respectively. By utilizing label-free relative quantitation through SWATH-MS, a total of 2654 proteins have been quantified from grain and malt. The comparative analyses between the barley grain and malt samples revealed that the C-hordein-reduced lines have a more significant impact on proteome level changes due to malting than B- and D-hordein-reduced lines. Upregulated proteins in C-hordein-reduced lines were primarily involved in the tricarboxylic acid cycle and fatty acid peroxidation processes to provide more energy for seed germination during malting. By applying proteomics approaches after malting in hordein-reduced barley lines, we uncovered additional changes in the proteome driven by the genetic background that were not apparent in the sound grain. Our findings offer valuable insights for barley breeders and maltsters seeking to understand and optimize the performance of gluten-free grains in malt products

From grain to malt: Tracking changes of ultra-low-gluten barley storage proteins after malting

Barley (Hordeum vulgare L.) is a major cereal crop produced globally. Hordeins, the major storage proteins in barley, can trigger immune responses leading to celiac disease or symptoms associated with food allergy. Here, proteomics approaches were employed to investigate the proteome level changes of grain and malt from the malting barley cultivar, Sloop, and single-, double- and triple hordein-reduced lines. The triple hordein-reduced line is an ultra-low gluten barley cultivar, Kebari®. Using discovery proteomics, 2,688 and 3,034 proteins in the barley and malt samples were detected respectively. Through the application of targeted proteomics, a significant reduction in the quantity of B-, D-, and γ-hordeins, as well as avenin-like proteins, was observed in the ultra-low gluten malt sample. A compensation mechanism was observed evidenced by increased biosynthesis of seed storage globulins, specifically vicilin-like globulins. Overall, this study has provided insights into protein compositional changes after malting in celiac-friendly barley varieties

Proteome and nutritional shifts observed in hordein double-mutant barley lines

Lysine is the most limiting essential amino acid in cereals, and efforts have been made over the decades to improve the nutritional quality of these grains by limiting storage protein accumulation and increasing lysine content, while maintaining desired agronomic traits. The single lys3 mutation in barley has been shown to significantly increase lysine content but also reduces grain size. Herein, the regulatory effect of the lys3 mutation that controls storage protein accumulation as well as a plethora of critically important processes in cereal seeds was investigated in double mutant barley lines. This was enabled through the generation of three hordein double-mutants by inter-crossing three single hordein mutants, that had all been backcrossed three times to the malting barley cultivar Sloop. Proteome abundance measurements were integrated with their phenotype measurements; proteins were mapped to chromosomal locations and to their corresponding functional classes. These models enabled the prediction of previously unknown points of crosstalk that connect the impact of lys3 mutations to other signalling pathways. In combination, these results provide an improved understanding of how the mutation at the lys3 locus remodels cellular functions and impact phenotype that can be used in selective breeding to generate favourable agronomic traits

Hordein Accumulation in Developing Barley Grains

The temporal pattern of accumulation of hordein storage proteins in developing barley grains was studied by enzyme-linked immunosorbent assay (ELISA), western blot and liquid chromatography tandem mass spectrometry (LC-MS/MS). Hordein accumulation was compared to the pattern seen for two abundant control proteins, serpin Z4 (an early accumulator) and lipid transferase protein (LTP1, a late accumulator). Hordeins were detected from 6 days post-anthesis (DPA) and peaked at 30 DPA. Changes in fresh weight indicate that desiccation begins at 20 DPA and by 37 DPA fresh weight had decreased by 35%. ELISA analysis of hordein content, expressed on a protein basis, increased to a maximum at 30 DPA followed by a 17% decrease by 37 DPA. The accumulation of 39 tryptic and 29 chymotryptic hordein peptides representing all classes of hordein was studied by LC-MS/MS. Most peptides increased to a maximum at 30 DPA, and either remained at the maximum or did not decrease significantly. Only five tryptic peptides, members of the related B1- and γ1-hordeins decreased significantly by 21–51% at 37 DPA. Thus, the concentration of some specific peptides was reduced while remaining members of the same family were not affected. The N-terminal signal region was removed by proteolysis during co-translation. In addition to a suite of previously characterized hordeins, two novel barley B-hordein isoforms mapping to wheat low molecular weight glutenins (LMW-GS-like B-hordeins), and two avenin-like proteins (ALPs) sharing homology with wheat ALPs, were identified. These identified isoforms have not previously been mapped in the barley genome. Cereal storage proteins provide significant nutritional content for human consumption and seed germination. In barley, the bulk of the storage proteins comprise the hordein family and the final hordein concentration affects the quality of baked and brewed products. It is therefore important to study the accumulation of hordeins as this knowledge may assist plant breeding for improved health outcomes (by minimizing triggering of detrimental immune responses), nutrition and food processing properties

The effect of protein-precipitant interfaces and applied shear on the nucleation and growth of lysozyme crystals

This paper is concerned with the effect of protein–precipitant interfaces and externally applied shear on the nucleation and growth kinetics of hen egg-white lysozyme crystals. The early stages of microbatch crystallization of lysozyme were explored using both optical and confocal fluorescence microscopy imaging. Initially, an antisolvent (precipitant) was added to a protein drop and the optical development of the protein– precipitant interface was followed with time. In the presence of the water-soluble polymer poly(ethylene glycol) (PEG) a sharp interface was observed to form immediately within the drop, giving an initial clear separation between the lighter protein solution and the heavier precipitant. This interface subsequently became unstable and quickly developed within a few seconds into several unstable ‘fingers’ that represented regions of high concentration-gradient interfaces. Confocal microscopy demonstrated that the subsequent nucleation of protein crystals occurred preferentially in the region of these interfaces. Additional experiments using an optical shearing system demonstrated that oscillatory shear significantly decreased nucleation rates whilst extending the growth period of the lysozyme crystals. The experimental observations relating to both nucleation and growth have relevance in developing efficient and reliable protocols for general crystallization procedures and the controlled crystallization of single large high-quality protein crystals for use in X-ray crystallography

Quantification of Hordeins by ELISA: The Correct Standard Makes a Magnitude of Difference

<div><p>Background</p><p>Coeliacs require a life-long gluten-free diet supported by accurate measurement of gluten (hordein) in gluten-free food. The gluten-free food industry, with a value in excess of $6 billion in 2011, currently depends on two ELISA protocols calibrated against standards that may not be representative of the sample being assayed.</p> <p>Aim</p><p>The factors affecting the accuracy of ELISA analysis of hordeins in beer were examined.</p> <p>Results</p><p>A simple alcohol-dithiothreitol extraction protocol successfully extracts the majority of hordeins from barley flour and malt. Primary hordein standards were purified by FPLC. ELISA detected different classes of purified hordeins with vastly different sensitivity. The dissociation constant (Kd) for a given ELISA reaction with different hordeins varied by three orders of magnitude. The Kd of the same hordein determined by ELISA using different antibodies varied by up to two orders of magnitude. The choice of either ELISA kit or hordein standard may bias the results and confound interpretation.</p> <p>Conclusions</p><p>Accurate determination of hordein requires that the hordein standard used to calibrate the ELISA reaction be identical in composition to the hordeins present in the test substance. In practice it is not feasible to isolate a representative hordein standard from each test food. We suggest that mass spectrometry is more reliable than ELISA, as ELISA enumerates only the concentration of particular amino-acid epitopes which may vary between different hordeins and may not be related to the absolute hordein concentration. MS quantification is undertaken using peptides that are specific and unique enabling the quantification of individual hordein isoforms.</p> </div

Hordeins do not bleed off in aqueous washes.

<p>Duplicate 50 mg aliquots of Sloop flour were extracted in 1 mL of 50% IPA containing 1% (w/v) DTT (IPA/DTT) and an aliquot containing 20 µg protein dried in a SpeedyVac, dissolved in Urea/SDS and resolved on duplicate SDS-PAGE gels (lanes P) which were either stained in colloidal Coomassie Blue (A and C) or blotted to nitrocellulose (iBLOT Promega), blocked in PBST containing 5% skim milk powder, 1% (w/v) Tween overnight at 4°C. The blot was exposed to anti-gliadin-HRP (Sigma) diluted at 1/2000 for 30 min, then washed in PBST, and the signal produced by ECL reagent (Amersham) exposed to Hyperfilm (Amersham) (B and D). Similarly, 50 mg of duplicate flour samples were first washed in 1 mL of water, and centrifuged to give a supernatant and a pellet, which was dissolved in IPA/DTT. Aliquots containing 20 µg protein from both pellet (P1) and supernatant (S1) were dried, redissolved and subject to SDS-PAGE as above. The process was repeated with additional aqueous washing steps to produce supernatants S2–4 and pellets P2–4. Low molecular weight proteins were removed from pellets (A: arrowed) and extracted into supernatants (C: arrowed) after 2 washes. Proteins were calibrated with Benchmark protein ladder (St; Invitrogen).</p