Proteomic Determination of Low-Molecular-Weight Glutenin Subunit Composition in Aroona Near-Isogenic Lines and Standard Wheat Cultivars.

The low-molecular weight glutenin subunit (LMW-GS) composition of wheat (Triticum aestivum) flour has important effects on end-use quality. However, assessing the contributions of each LMW-GS to flour quality remains challenging because of the complex LMW-GS composition and allelic variation among wheat cultivars. Therefore, accurate and reliable determination of LMW-GS alleles in germplasm remains an important challenge for wheat breeding. In this study, we used an optimized reversed-phase HPLC method and proteomics approach comprising 2-D gels coupled with liquid chromatography-tandem mass spectrometry (MS/MS) to discriminate individual LMW-GSs corresponding to alleles encoded by the Glu-A3, Glu-B3, and Glu-D3 loci in the 'Aroona' cultivar and 12 'Aroona' near-isogenic lines (ARILs), which contain unique LMW-GS alleles in the same genetic background. The LMW-GS separation patterns for 'Aroona' and ARILs on chromatograms and 2-D gels were consistent with those from a set of 10 standard wheat cultivars for Glu-3. Furthermore, 12 previously uncharacterized spots in 'Aroona' and ARILs were excised from 2-D gels, digested with chymotrypsin, and subjected to MS/MS. We identified their gene haplotypes and created a 2-D gel map of LMW-GS alleles in the germplasm for breeding and screening for desirable LMW-GS alleles for wheat quality improvement.

High-throughput analysis of high-molecular weight glutenin subunits in 665 wheat genotypes using an optimized MALDI-TOF-MS method.

Gluten protein composition determines the rheological characteristics of wheat dough and is influenced by variable alleles with distinct effects on processing properties. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), we determined the high-molecular weight glutenin subunit (HMW-GS) composition of 665 wheat genotypes employed in breeding programs in South Korea. We identified 22 HMW-GS alleles, including 3 corresponding to the Glu-A1 locus, 14 to Glu-B1, and 5 to Glu-D1. The Glu-1 quality score, which is an important criterion for high-quality wheat development, was found to be 10 for 105/665 (15.79%) of the studied genotypes, and included the following combinations of HMW-GS: 2*, 7 + 8, 5 + 10; 2*, 17 + 18, 5 + 10; 1, 7 + 8, 5 + 10; and 1, 17 + 18, 5 + 10. To select wheat lines with the 1Bx7 overexpression (1Bx7OE) subunit, which is known to have a positive effect on wheat quality, we used a combination of MALDI-TOF-MS and published genotyping markers and identified 6 lines carrying 1Bx7OE out of the 217 showing a molecular weight of 83,400 Da, consistent with 1Bx7G2 and 1Bx7OE. This study demonstrates that the MALDI-TOF-MS method is fast, accurate, reliable, and effective in analyzing large numbers of wheat germplasms or breeding lines in a high-throughput manner. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-020-02637-z.

Comparison of MALDI-TOF-MS and RP-HPLC as Rapid Screening Methods for Wheat Lines With Altered Gliadin Compositions.

The wheat gliadins are a complex group of flour proteins that can trigger celiac disease and serious food allergies. As a result, mutation breeding and biotechnology approaches are being used to develop new wheat lines with reduced immunogenic potential. Key to these efforts is the development of rapid, high-throughput methods that can be used as a first step in selecting lines with altered gliadin contents. In this paper, we optimized matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and reversed-phase high-performance liquid chromatography (RP-HPLC) methods for the separation of gliadins from Triticum aestivum cv. Chinese Spring (CS). We evaluated the quality of the resulting profiles using the complete set of gliadin gene sequences recently obtained from this cultivar as well as a set of aneuploid lines in CS. The gliadins were resolved into 13 peaks by MALDI-TOF-MS. α- or γ-gliadins that contain abundant celiac disease epitopes and are likely targets for efforts to reduce the immunogenicity of flour were found in several peaks. However, other peaks contained multiple α- and γ-gliadins, including one peak with as many as 12 different gliadins. In comparison, separation of proteins by RP-HPLC yielded 28 gliadin peaks, including 13 peaks containing α-gliadins and eight peaks containing γ-gliadins. While the separation of α- and γ-gliadins gliadins achieved by RP-HPLC was better than that achieved by MALDI-TOF-MS, it was not possible to link peaks with individual protein sequences. Both MALDI-TOF-MS and RP-HPLC provided adequate separation of ω-gliadins. While MALDI-TOF-MS is faster and could prove useful in studies that target specific gliadins, RP-HPLC is an effective method that can be applied more broadly to detect changes in gliadin composition.

Development of an Optimized MALDI-TOF-MS Method for High-Throughput Identification of High-Molecular-Weight Glutenin Subunits in Wheat.

Because high-molecular-weight glutenin subunits (HMW-GS) are important contributors to wheat end-use quality, there is a need for high-throughput identification of HMW-GS in wheat genetic resources and breeding lines. We developed an optimized method using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to distinguish individual HMW-GS by considering the effects of the alkylating reagent in protein extraction, solvent components, dissolving volume, and matrix II components. Using the optimized method, 18 of 22 HMW-GS were successfully identified in standard wheat cultivars by differences in molecular weights or by their associations with other tightly linked subunits. Interestingly, 1Bx7 subunits were divided into 1Bx7 group 1 and 1Bx7 group 2 proteins with molecular weights of about 82,400 and 83,000 Da, respectively. Cultivars containing the 1Bx7 group 2 proteins were distinguished from those containing 1Bx7OE using well-known DNA markers. HMW-GS 1Ax2* and 1Bx6 and 1By8 and 1By8*, which are difficult to distinguish due to very similar molecular weights, were easily identified using RP-HPLC. To validate the method, HMW-GS from 38 Korean wheat varieties previously evaluated by SDS-PAGE combined with RP-HPLC were analyzed by MALDI-TOF-MS. The optimized MALDI-TOF-MS method will be a rapid, high-throughput tool for selecting lines containing desirable HMW-GS for breeding efforts.

Towards reducing the immunogenic potential of wheat flour: omega gliadins encoded by the D genome of hexaploid wheat may also harbor epitopes for the serious food allergy WDEIA.

BACKGROUND: Omega-5 gliadins are a group of highly repetitive gluten proteins in wheat flour encoded on the 1B chromosome of hexaploid wheat. These proteins are the major sensitizing allergens in a severe form of food allergy called wheat-dependent exercise-induced anaphylaxis (WDEIA). The elimination of omega-5 gliadins from wheat flour through biotechnology or breeding approaches could reduce the immunogenic potential and adverse health effects of the flour. RESULTS: A mutant line missing low-molecular weight glutenin subunits encoded at the Glu-B3 locus was selected previously from a doubled haploid population generated from two Korean wheat cultivars. Analysis of flour from the mutant line by 2-dimensional gel electrophoresis coupled with tandem mass spectrometry revealed that the omega-5 gliadins and several gamma gliadins encoded by the closely linked Gli-B1 locus were also missing as a result of a deletion of at least 5.8 Mb of chromosome 1B. Two-dimensional immunoblot analysis of flour proteins using sera from WDEIA patients showed reduced IgE reactivity in the mutant relative to the parental lines due to the absence of the major omega-5 gliadins. However, two minor proteins showed strong reactivity to patient sera in both the parental and the mutant lines and also reacted with a monoclonal antibody against omega-5 gliadin. Analysis of the two minor reactive proteins by mass spectrometry revealed that both proteins correspond to omega-5 gliadin genes encoded on chromosome 1D that were thought previously to be pseudogenes. CONCLUSIONS: While breeding approaches can be used to reduce the levels of the highly immunogenic omega-5 gliadins in wheat flour, these approaches are complicated by the genetic linkage of different classes of gluten protein genes and the finding that omega-5 gliadins may be encoded on more than one chromosome. The work illustrates the importance of detailed knowledge about the genomic regions harboring the major gluten protein genes in individual wheat cultivars for future efforts aimed at reducing the immunogenic potential of wheat flour.

Proteomic Profiling and Epitope Analysis of the Complex α-, γ-, and ω-Gliadin Families in a Commercial Bread Wheat.

Wheat gliadins are a complex group of proteins that contribute to the functional properties of wheat flour doughs and contain epitopes that are relevant for celiac disease (CD) and wheat-dependent exercise-induced anaphylaxis (WDEIA). In this study, we extracted ethanol-soluble gliadin fractions from flour of the Korean bread wheat cultivar Keumkang. Proteins were separated by 2-dimensional gel electrophoresis (2-DE) using a pI range of 6-11 in the first dimension and subjected to tandem mass spectrometry. α-, γ-, and ω-gliadins were identified as the predominant proteins in 31, 28, and one 2-DE spot, respectively. An additional six ω-gliadins were identified in a separate experiment in which a pI range of 3-11 was used for protein separation. We analyzed the composition of CD- and WDEIA-relevant epitopes in the gliadin sequences from Keumkang flour, demonstrating the immunogenic potential of this cultivar. Detailed knowledge about the complement of gliadins accumulated in Keumkang flour provides the background necessary to devise either breeding or biotechnology strategies to improve the functional properties and reduce the adverse health effects of the flour.

Proteomic analysis of low-molecular-weight glutenin subunits and relationship with their genes in a common wheat variety.

Although many studies on low-molecular-weight glutenin subunit (LMW-GS) function have been reported, a comprehensive comparison between specific genes and their protein product is still lacking. This study aimed to link the 43 genes isolated from the Korean wheat variety "Jokyoung" in the authors' previous study to their protein products. Proteins were separated using two-dimensional gel electrophoresis (2-DGE) and identified by tandem mass spectrometry (MS/MS) at the gene haplotype level. Using MS/MS analysis of 17 protein spots, two spots were identified in the Glu-A3 locus and the corresponding haplotype was GluA3-13(Glu-A3c). Six spots were identified in the Glu-B3 locus and the corresponding haplotypes were GluB3-33 and GluB3-43 (Glu-B3h). Eight spots were identified in the Glu-D3 locus and the corresponding haplotypes were GluD3-11, GluD3-21, GluD3-31, GluD3-5, and GluD3-6 (Glu-D3a), and one spot was contaminated with gamma gliadin. Phylogenetic analysis and alignment of nucleotide and amino acid sequences assigned 35 of the 43 genes to seven haplotypes: GluA3-13, GluB3-43, GluD3-11, GluD3-21, GluD3-31, GluD3-42, and GluD3-5. Taken together, except for GluB3-33 and GluD3-6, which were not isolated, linking of each gene to the corresponding protein products at the gene haplotype level was accomplished using proteomic tools and phylogenetic analysis.

Allelic analysis of low molecular weight glutenin subunits using 2-DGE in Korean wheat cultivars.

Two-dimensional gel electrophoresis (2-DGE) was used as a complement to SDS-PAGE to determine the allelic compositions of LMW-GS in 32 Korean wheat cultivars. Protein patterns generated by 2-DGE from each cultivar were compared to patterns from standard wheat cultivars for each allele. At the Glu-A3 locus, thirteen c, twelve d, three e (null), two g and two new alleles were identified. At the Glu-B3 locus, one b, nineteen d, four h, one i and five ad alleles were identified. At the Glu-D3 locus, twenty-three a, four b, four c and one l alleles were identified. When compared to results obtained previously using SDS-PAGE, there were discrepancies in the allelic designations of 10 of 32 cultivars (31%). While SDS-PAGE is a rapid and relatively simple method for assessing LMW-GS composition, the similar mobilities of the proteins makes it difficult to discriminate certain alleles. 2-DGE is a more complicated technique, but provides a more accurate picture of the complement of the LMW-GS in a given cultivar. In addition to providing essential information for wheat breeders, the 2-DGE reference maps generated in this study will make it possible to study the contributions of individual LMW-GS to flour quality.

Improved Method for Reliable HMW-GS Identification by RP-HPLC and SDS-PAGE in Common Wheat Cultivars.

The accurate identification of alleles for high-molecular weight glutenins (HMW-GS) is critical for wheat breeding programs targeting end-use quality. RP-HPLC methods were optimized for separation of HMW-GS, resulting in enhanced resolution of 1By and 1Dx subunits. Statistically significant differences in retention times (RTs) for subunits corresponding to HMW-GS alleles were determined using 16 standard wheat cultivars with known HMW-GS compositions. Subunits that were not identified unambiguously by RP-HPLC were distinguished by SDS-PAGE or inferred from association with linked subunits. The method was used to verify the allelic compositions of 32 Korean wheat cultivars previously determined using SDS-PAGE and to assess the compositions of six new Korean cultivars. Three cultivars contained subunits that were identified incorrectly in the earlier analysis. The improved RP-HPLC method combined with conventional SDS-PAGE provides for accurate, efficient and reliable identification of HMW-GS and will contribute to efforts to improve wheat end-use quality.