ANALYSIS OF IMMUNOGENIC PEPTIDES IN WHEAT

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1016711
• Project Start Date: Oct 1, 2018
• Project End Date: Sep 30, 2021
• Program Code: [(N/A)]- (N/A)

Recipient Organization
NORTH DAKOTA STATE UNIV
1310 BOLLEY DR
FARGO,ND 58105-5750

Performing Department
Plant Sciences

Non Technical Summary
The incidence of CD in the United States and worldwide has been increasing during the last decade. For the moment, the only medical solution for CD is a life-long gluten-free diet. However, being on such a diet can have negative effects on those with this disease. For example, wheat is a major source of carbohydrates for the majority of the population. In the human body, carbohydrates are used as an energy source. If one is deprived of carbohydrates or any suitable energy source, this could result in malnutrition. Additionally, being on a gluten-free diet can affect the quality of life of those with celiac disease and gluten-related allergenicity. In this context, if ultra-low gluten or celiac-safe wheat were produced, those with celiac disease would not have to refrain from consuming wheat. In order to create such ultra-low gluten or celiac-safe wheat, existing wheat cultivars that are low in celiac antigenicity need to be identified. For this purpose, the amount of immunogenic peptides and total gliadin need to be quantified as proposed in this study. Additionally, determining the celiac antigenicity of wheat may have economic importance as well, since it is a method of categorizing different cultivars grown in the region according to their potential to cause allergic reactions. This study will have economic importance in North Dakota since will help identify specific HRS and durum wheat cultivars with low celiac antigenicity that are adapted to the environmental conditions of the state, and further research can be conducted to make this cultivars celiac-safe as explained above. Additionally, further studies can be conducted to determine the association between reducing celiac disease causing peptides and the effects on wheat functionality in food systems.

Animal Health Component

30%

Research Effort Categories

Basic

40%

Applied

30%

Developmental

30%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
701	1541	2000	100%

Knowledge Area
701 - Nutrient Composition of Food;

Subject Of Investigation
1541 - Hard red spring wheat;

Field Of Science
2000 - Chemistry;

Keywords

wheat

immunogenic peptides

celiac disease

protein

Goals / Objectives
North Dakota is the largest producer of hard red spring wheat (Triticum aestivum L.) (HRSW) in the United States (Ag Statistics 2017). On average, 6.8 million metric tons of hard red spring wheat is produced annually in ND, which accounts for approximately half of the country's production. This type of wheat is known for its superior qualities, such as high protein content and strong gluten. Thus, some of the world's finest bakery products are made using HRSW. As for durum wheat (T. turgidum L var. durum Desf.), nearly two-thirds of the nation's durum crop is produced in North Dakota (Ag Statistics 2017). There has been renewed interest in ancient grains, such as einkorn, emmer and kamut. These grains are related to domesticated hexaploid wheat, which accounts for up to 95% of the wheat grown worldwide.For many years, breeders have produced many spring wheat cultivars which are better adapted to conditions in the region and with other advantageous traits. No research has been reported to date about how the protein chemistry of these cultivars are different regarding their celiac antigenicity. If less antigenic cultivars are identified, this could lead the way for breeding wheat cultivars that are safe for celiac disease (CD) subjects. Thus, it would be possible for such individuals to consume wheat-based products, as opposed to a lifelong wheat-free diet.The incidence of CD in the United States and worldwide has been increasing during the last decade. For the moment, the only medical solution for CD is a life-long gluten-free diet. However, being on such a diet can have negative effects on those with this disease. For example, wheat is a major source of carbohydrates for the majority of the population. In the human body, carbohydrates are used as an energy source. If one is deprived of carbohydrates or any suitable energy source, this could result in malnutrition. Additionally, being on a gluten-free diet can affect the quality of life of those with celiac disease and gluten-related allergenicity. In this context, if ultra-low gluten or celiac-safe wheat were produced, those with celiac disease would not have to refrain from consuming wheat. In order to create such ultra-low gluten or celiac-safe wheat, existing wheat cultivars that are low in celiac antigenicity need to be identified. For this purpose, the amount of immunogenic peptides and total gliadin need to be quantified as proposed in this study. Additionally, determining the celiac antigenicity of wheat may have economic importance as well, since it is a method of categorizing different cultivars grown in the region according to their potential to cause allergic reactions. This study will have economic importance in North Dakota since will help identify specific HRS and durum wheat cultivars with low celiac antigenicity that are adapted to the environmental conditions of the state, and further research can be conducted to make this cultivars celiac-safe as explained above. Additionally, further studies can be conducted to determine the association between reducing celiac disease causing peptides and the effects on wheat functionality in food systems.To determine the presence of immunogenic peptides causing celiac disease in hard red spring wheat cultivars, durum wheat and ancient wheat species.To quantify the immunogenic peptides using heavy labeled peptides.

Project Methods
HRS cultivars, Marquis, Ceres, Pilot, Rival, Vesta, Mida, Conley, Justin, Fortuna, Waldron, Olaf, Len, Stoa, Butte86, Grandin, Pioneer 2375, Reeder, Alsen, Granite, Freyer, Steele-ND, RB07, Barlow, and Velva will be tested. Additional testing will be completed on ancient wheat species such as einkorn, emmer, and kamut. The following durum wheat cultivars will be investigated: Joppa, Carpio, Tioga, Divide, Grenora, Alkabo, Dilse, Pierce, Plaza, Lebsock, Mountrail, Maier, Belzer, Ben, Munich, Renville, Monroe, Lloyd, Vic, Calvin, Edmore, Cando, Botno, Crosby, Rugby, Ward, Rolette, Leeds, Lakota, Wells, Langdon and Strongfield. The samples will be grown in several locations in North Dakota, namely, Casselton (HRS) and Carrington, Minot, and Prosper (durum). The ancient grain samples for this study will be obtained by commercial gluten-free retailers.Gliadin proteins will be extracted using a modified Osborne fractionation method as described by Malalgoda et al. (2017).The amount of protein in each extract will be quantified using a PierceTM BCA assay kit. Afterward, each sample will be mixed with sample buffer (0.5M Tris-HCl, pH 6.8, 40% (v/v) glycerol, 10% SDS, 2-mercaptoethanol, and 0.05% bromophenol blue) so that each sample would contain 40 μg of protein. These samples will be boiled for 5 min and allowed to cool before separation by gel electrophoresis.An 8% Tris-Tricine gel will be used to separate the samples via electrophoresis (Schägger and von Jagow 1987). The gel will be run for 15 min at 55 V so that the proteins are allowed to separate and become incorporated into the gel. For staining the gels, a protocol adapted from the European Molecular Biology Laboratory (EMBL) will be used. The gel will be washed briefly with distilled water, then fixed in a solution containing 45% methanol (v/v), 45% water (v/v) and 10% acetic acid (v/v) for 15 min. Afterward, the gel will be washed again with distilled water and fixed in the same solution for another 15 min, after which, the gel will be washed again with distilled water. The gel will be stained in Colloidal Coomassie blue overnight. The following day, the individual gel bands will be cut using a sterile razor blade and stored at -20°C in 1.5 ml Eppendorf tubes for mass spectrometric analysis. The gliadin gel bands will be subjected to in-gel digestion with chymotrypsin according to the method of Shevchenko et al. (1996). The digested samples will be stored at -80°C until mass spectrometric analysis is performed. Prior to analysis, the samples will desalted using the STAGE (Stop And Go Extraction) tip desalting procedure according to Rappsilber et al. (2003).For the untargeted analysis of immunogenic peptides, approximately 1.5 µg of the chymotrypsin digested peptide mixtures will be used for liquid chromatography-mass spectrometric (LC-MS) analysis on an Orbitrap Velos system (Thermo Fisher Scientific, Waltham, MA) as described by Lin-Moshier et al. (2013). Identical LC conditions will be used, but the following modifications will be made to the MS acquisition settings: the MS1 scan range (360 - 1800 m/z); the minimum abundance for MS/MS trigger (10000 counts); lock mass not selected; and dynamic exclusion settings (list size 200 values, duration 30 sec, exclusion mass tolerance +/- 10 ppm).The PEAKS 7.0 software (Bioinformatics Solutions, Waterloo, ON, Canada) will be used to analyze the RAW data files against the database "non-redundant triticinae (nr_triticinae)"and a database of common contaminants 1648030_20150930_cRAP123 downloaded from NCBI. Search parameters will be set as follows: enzyme: chymotrypsin; max missed cleavages: 2; non-specific cleavages: 2; fixed modifications: carbamidomethylation; variable modifications: oxidation; max variable PTM per peptide: 3; parent mass error tolerance: 50 ppm, fragment mass error tolerance: 0.1 Da. Results filter parameters will be as follows, peptide (-l0lgP): ≥ 15; protein (-l0lgP): ≥ 20. The data will be used to determine if immunogenic epitopes are present in the different wheat cultivars being investigated.For the relative quantification of immunogenic peptides, peptides proposed by Van den Broeck et al. (2015) will be used. Immunogenic epitopes glia-α9 (PFPQPQLPY), glia-α20 (FRPQQPYPQ), and the amount of total α-gliadin will be quantified in this manner.Peptides P1-P5, P6-P7 and P8-P9 will be used for the quantification of glia-α9, glia-α20 and total α-gliadin, respectively.These mass lists will be imported into Analyst software (Sciex) and used to generate unscheduled SRM methods on a Qtrap 5500 (Sciex) to optimize the LC gradient and select optimal transitions. The final optimized transitions will be built into a scheduled method. The QTRAP 5500 is equipped with an Agilent 1100 capillary LC system. Solvents A and B will be 98% water (v/v), 2% acetonitrile (ACN) (v/v) and 0.1% formic acid (v/v), and ACN and 0.1% formic acid (v/v) respectively. A three-step gradient will be used to separate the peptides. The gradient will start with 0% B, which will be increased to 5% B in the next minute. From min 1- 17.5 the percentage of B will be increased to 45%, and then from 17.5-18 min it will be increased to 85%, which will be held until min 21. From 21-21.5 min, the percentage of B will be reduced to zero, which will be held until 24 min. A flow rate of 8 µl/min will be used from zero - 19.99 min, which will be changed to 10 µl/min from 20 - 24 min. A HALO C18 column (100 × 0.3 mm, with 2.7µm HALO C18 particles with 90Å pore size) (Eksigent) will be heated to 55°C using a sleeve column heater (Analytical Sales & Services). The digested samples will be reconstituted to 2 µg/µl, and mixed with an equal volume of heavy-labeled standard peptides at various concentrations matching the endogenous signal of a pooled sample. SRM data will be imported into Skyline for quantitation by normalizing endogenous signals to that of the spiked heavy peptides.For the absolute quantification of peptides, calibration curves will be used. The amount of the immunogenic epitopes glia-α9 and glia-α20, and total α-gliadin will be quantified using this method. Heavy labeled peptides P1-P9 will be diluted to 0.1 nmol/µl solutions. The dilution solvent will be comprised of 98% water (v/v), 2% ACN (v/v) and 0.1% formic acid (v/v). These solute ions will be diluted to give solutions of the following concentrations (fmol/µl): 5, 10, 20, 50, 70, 100, 200, 500, 700, 1000, 2000, 5000 and 10000. To construct the 'blank standard curve', a solvent composed of 98% water, 2% ACN and 0.1% formic acid will be used. A 1:1 dilution of each of the heavy labeled peptides will be done with the solvent, and 2 µl of the resulting mixture will be injected into the QTRAP 5500 instrument. The conditions, the solvents, and the gradient will be the same as for the relative quantitation method. To construct the 'matrix standard curve', a 1:1 dilution will be done with a wheat sample that was reconstituted to 2 µg/µl. The area for the heavy ions in the 'blank standard curve runs' and the 'matrix standard curve runs' will plotted against each other to determine the matrix effect. When conducting this analysis, the sum of the highest-ranking transitions will be used as a measure of the area of each ion. Calibration curves will be created for the heavy transitions in 'matrix standard curve' runs and used in the quantification process.The SAS 9.3 software will be used to determine significant differences between cultivars for immunogenic peptides glia-α9, glia-α20 and total α-gliadin. The analysis of peptides both for the qualitative and quantitative determination of antigenicity will be performed in duplicate.

Progress 10/01/18 to 09/30/21

Outputs
Target Audience:Cereal and Food Science researchers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?The results of this project have been disseminated through publication of a peer reviewed journal article and online articles. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Celiac disease (CD) develops in genetically susceptible individuals as a result of ingesting gluten-forming proteins found in cereals, such as wheat (Triticum aestivum L.). There are claims that breeding practices have changed wheat protein chemistry over the years, and this has resulted in modern wheat being more antigenic in terms of CD as opposed to historical wheat. Our objective was to determine changes in the different gliadin protein components, and identify and quantify CD causing α-gliadin epitopes and total α-gliadin in historical and modern wheat cultivars grown in the state of North Dakota, historical wheat refers to cultivars released prior to 1966, and modern wheat refers to cultivars released after 1966. Antigenicity, in this context, refers to the number of immunogenic epitopes and total α-gliadin. Objective 1. To determine the presence of immunogenic peptides causing CD in hard red spring wheat cultivars, durum wheat and ancient wheat species. 1) Major activities completed / experiments conducted; Samples of Hard Red Spring wheat varieties with release dates ranging from 1910 to 2013 were utilized to determine the presence of immunogenic peptides causing CD. The gliadin proteins were extracted from the ground whole wheat samples before being analyzed with reverse phase high performance liquid chromatography (RPHPLC). Separation of gliadin proteins was done with an Agilent Zorbax 300SB-C18 column. 2) Data collected; The peaks identified in the RP-HPLC analysis were analyzed for correlation with release year to determine the fractions that may have changed in the last 100 years. We were able to elucidate six peaks of ω-gliadin and four peaks of γ-gliadin that were of interest. Significant (p < .001) differences between the different ancient grains were observed for all three gliadin protein types. For ω-gliadin proteins, rye had the highest area percent (21%) while Kumut had the lowest (4%). For α/β-gliadins the percent area was highest in einkorn (68%) while the lowest was seen in rye (24%). For the γ-gliadins the highest percent was found in rye (55%) and the lowest in einkorn (23%). 3) Summary statistics and discussion of results; The total areas of the α/β-gliadin and ω-gliadin did not have significant correlation with release year. There were specific peaks of the ω-gliadins and γ-gliadins that had highly significant (P<0.01) and very highly significant (P<0.001) correlations with release year. Overall, ω-gliadins and some fractions of γ-gliadins increased with release year. 4) Key outcomes or other accomplishments realized. Some of the gliadin proteins in wheat have not changed in varieties released over 100 years, but there have been increases in some fractions of the gliadin proteins related to release year in these varieties. The percent absorbance area of the ω-gliadin protein peaks for the wheat related ancient grains was less than half that of rye; yet, the area percent of the α/β-gliadin peaks of the wheat-related ancient grains were more than double compared to that of rye. Therefore, when considering the abundance of α-gliadin proteins, rye may have lower celiac antigenicity; however, to produce celiac-safe rye the antigenic epitopes need to be removed. Objective 2. To quantify the immunogenic peptides using non-radioactive heavy labeled peptides. 1) Major activities completed / experiments conducted; The digested gliadin fractions were analyzed with LC-MS and relative quantification of the immunogenic epitopes was determined. The number of immunogenic epitopes of glia-α9 and glia-α20 and total α-gliadin were quantified for ten samples across the cultivars from the 100 years that were investigated. Heavy labelled peptides P1-P9 were used for quantification. To determine the presence of celiac epitopes, an untargeted mass spectrometric approach was used. 2) Data collected; The immunogenic epitopes that trigger the immune reaction related to CD were found in both historical and modern spring wheat cultivars. All five of the immunogenic epitopes belonging to α/β-gliadin proteins were found in the majority of cultivars. The epitope PFPQPQLPY was detected in all cultivars except cultivar Alsen (released 2000). Cultivar Steele-ND released in 2004, had a relatively high number of immunogenic epitopes compared to the other cultivars that were analyzed. Cultivar Russ released in 1995, was of lower antigenicity in comparison to the others, since the ratios are low for this cultivar. Celiac epitopes were detected in einkorn, emmer, Kamut, and rye. As expected, celiac epitopes were not detected in teff and sorghum and the pseudocereals. A third of the epitopes evaluated were detected in einkorn and four epitopes were found in Kamut. Celiac epitopes can be found in ancient wheat species, which make them unsuitable for consumption by celiac subjects. In this analysis, proteomics data were used to determine the presence of two T-cell epitopes in rye secalin proteins, where one of the two peptides were detected. 3) Summary statistics and discussion of results; The results of the LC-MS analysis show that immunogenic epitopes are present in historical and modern spring wheat cultivars irrespective of release year and that the presence or absence of immunogenic epitopes is independent of release year. Our results suggest that historical wheat is antigenic for those with CD. The glia-α-20 epitope was the most prevalent epitope analyzed, whereas the glia-α9 epitope was the second most prevalent in historical and modern wheat cultivars. Three of the nine γ-gliadin epitopes were also found in the wheat species, although they were detected in a more random manner. The lack of ω-gliadin epitopes in the ancient wheat species could be explained by the distribution of ω-gliadin T-cell epitopes encoded by the A/D genomes in comparison to the B genome. In hexaploid bread wheat, celiac epitopes are distributed throughout the chromosomes encoding ω-gliadin in the A/D genomes, whereas the B-encoded ω-gliadins do not show T-cell epitopes. Thus, the lack of the D genome in diploid and tetraploid ancient wheat species could be associated with the non-detection of ω-gliadin celiac epitopes. In teff and sorghum, pseudocereals that were analyzed, none of the celiac epitopes were detected, further confirming that they are celiac safe. 4) Key outcomes or other accomplishments realized. RP-HPLC was used to determine how the gliadin profiles changed in relation to release year. Mass spectrometric analysis was performed to determine if immunogenic epitopes causing celiac disease are present in the wheat cultivars being investigated, and to quantify these epitopes. Overall, the results indicate that immunogenic epitopes causing CD are detected in historical and modern hard red spring wheat cultivars irrespective of release year, and that the quantity of these epitopes does not show a relationship with cultivar release year, indicating that modern wheat cultivars are not higher in CD antigenicity in comparison to historical wheat cultivars. From our results, cultivar Russ showed the least number of immunogenic epitopes and total α-gliadin, making it a suitable starting point for breeding wheat cultivars that are safe for CD patients. Additionally, this study shows that celiac epitopes can be found in wheat-related ancient grains, such as einkorn, emmer, and Kamut. The study also shows that such epitopes are present in rye, which is known to trigger the immune response associated with celiac disease. As expected, such epitopes were not found in the protein extractions from teff and sorghum, which are gluten free, as well as the pseudocereals.

Publications

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Cereal and food science researchers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?The results have been disseminated to the target audience of cereal and food science researchers through the publication of a manuscript. What do you plan to do during the next reporting period to accomplish the goals?There were no issues or problems during the current reporting period. During the next reporting period we will evaluate the immunogenic peptides of durum wheat in order to address the goals and objectives of this project.

Impacts
What was accomplished under these goals? Celiac disease is a genetically predisposed autoimmune disease, which is one of the most common food intolerances in the world. Gluten-forming proteins are composed of gliadin and glutenin proteins. These proteins act as antigens in genetically susceptible individuals and trigger an immune response. Ancient grains have gained renewed interest in the last few years due to their perceived nutritional benefits. The possibility of producing celiac safe wheat has been explored in many previous studies. The genetic variation amongst cultivars can be exploited to find accessions that are low in antigenicity, which can be used as parental varieties in breeding efforts. Objective 1. To determine the presence of immunogenic peptides causing celiac disease (CD) in hard red spring wheat cultivars, durum wheat and ancient wheat species. Major activities completed / experiments conducted; In this study, ancient grains, as well as pseudocereals were analyzed for celiac epitopes and gliadin protein composition. Gliadin proteins were extracted according to the previously described method and analyzed using an Agilent Zorbax 300SB-C18 reverse phase column. The HPLC data were processed using MATLAB and statistical analysis was preformed using SAS v 9.4. Data collected; Using the HPLC analysis, the area percent corresponding to the individual gliadin proteins was determined for each sample. Significant (p < .001) differences between the different ancient grains were observed for all three gliadin protein types. For ω-gliadin proteins, rye had the highest area percent (21%) while Kumut had the lowest (4%). For α/β-gliadins the percent area was highest in einkorn (68%) while the lowest was seen in rye (24%). The γ-gliadins showed a similar trend to the ω-gliadin proteins, with the highest percent being in rye (55%) and the lowest in einkorn (23%). Summary statistics and discussion of results The percent absorbance area of the ω-gliadin protein peaks for the wheat related ancient grains was less than half that of rye. On the other hand, the area percent of the α/β-gliadin peaks of the wheat-related ancient grains were more than double compared to that of rye. Therefore, when considering the abundance of α-gliadin proteins, rye may have lower celiac antigenicity. However, to produce celiac-safe rye, the antigenic epitopes need to be removed. Key outcomes or other accomplishments realized. The analysis of the gliadin protein profile of the different ancient grains shows that the abundance of the different gliadin types varies widely between species, which could be indicative of differences in celiac antigenicity and functionality in food systems. Objective 2. To quantify the immunogenic peptides using non-radioactive heavy labeled peptides. Major activities completed / experiments conducted; To determine the presence of celiac epitopes, an untargeted mass spectrometric approach was used. Gliadin proteins were extracted using a modified Osborne fractionation method. The samples were prepared with SDS-PAGE and the gel was then stained overnight with Colloidal Coomassie Blue. The following day, gel bands were excised using a sterile blade and stored at −20 °C until mass spectrometric analysis. The extracted proteins were digested (in-gel) with chymotrypsin, and desalted using STAGE (Stop And Go Extraction) tip prior to mass spectrometric analysis. Digested protein samples were analyzed on an Orbitrap Velos system. The obtained data were analyzed against the nr_triticinae database (except amaranth and quinoa) and RefSeq_pentapentalae (for amaranth and quinoa) database downloaded from NCBI using the PEAKS 8.0 software. Data collected; Celiac epitopes were detected in all three wheat-related ancient grains (einkorn, emmer, and Kamut) analyzed in addition to rye, which is known to elicit the immune response associated with celiac disease. As expected, celiac epitopes were not detected in teff and sorghum as well as the pseudocereals, which are considered gluten-free and safe for celiac subjects. A third of the epitopes evaluated were detected in einkorn and four epitopes were found in Kamut, which are diploid and tetraploid wheat species, respectively, consisting of the AA genome and the AABB genome. In emmer, which is a tetraploid species (AABB), only one epitope was detected. It is interesting to note that the FRPQQPYPQ (glia-α20) epitope was detected in all three ancient wheat species while the glia-α9 epitope (PFPQPQLPY) and epitope QGSFQPSQQ were detected in two of the three species. The epitopes in ω-gliadin proteins were not detected in any of the ancient wheat species. Celiac epitopes can be found in ancient wheat species, which make them unsuitable for consumption by celiac subjects. In this analysis, the proteomics data were used to determine the presence of two T-cell epitopes in rye secalin proteins, where one of the two peptides was detected. Summary statistics and discussion of results The glia-α-20 epitope was the most prevalent epitope analyzed, whereas the glia-α9 epitope was the second most prevalent in historical and modern wheat cultivars released in North Dakota between 1910 and 2013. Three of the nine γ-gliadin epitopes were also found in the wheat species, although they were detected in a more random manner. The lack of ω-gliadin epitopes in the ancient wheat species could be explained by the distribution of ω-gliadin T-cell epitopes encoded by the A/D genomes in comparison to the B genome. In hexaploid bread wheat, celiac epitopes are distributed throughout the chromosomes encoding ω-gliadin in the A/D genomes, whereas the B-encoded ω-gliadins do not show T-cell epitopes. Thus, the lack of the D genome in diploid and tetraploid ancient wheat species could be associated with the non-detection of ω-gliadin celiac epitopes. All three wheat-related ancient grain species and rye fall under the Poaceae family which may explain why they contain celiac epitopes. In a few of the ancient grains, namely teff and sorghum, and in the pseudocereals that were analyzed, as expected, none of the celiac epitopes were detected, further confirming that they are celiac safe. Key outcomes or other accomplishments realized. This study shows that celiac epitopes can be found in wheat-related ancient grains, such as einkorn, emmer, and Kamut. The study also shows that such epitopes are present in rye, which is known to trigger the immune response associated with celiac disease. As expected, such epitopes were not found in the protein extractions from teff and sorghum, which are gluten free, as well as the pseudocereals.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Malalgoda, M., Ohm, J. B., & Simsek, S. (2019). Celiac antigenicity of ancient wheat species. Foods, 8(12), 675.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Cereal and food science researchers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?The results of this project have been disseminated through publication of a peer reviewed journal article and online article. What do you plan to do during the next reporting period to accomplish the goals?There were no issues or problems during the current reporting period. During the next reporting period we will the immunogenic peptides of durum wheat in order to address the goals and objectives of this project.

Impacts
What was accomplished under these goals? Celiac disease (CD) develops in genetically susceptible individuals as a result of ingesting gluten-forming proteins found in cereals, such as wheat (Triticum aestivum L.). There are claims that breeding practices have changed wheat protein chemistry over the years and this has resulted in modern wheat being more antigenic in terms of CD as opposed to historical wheat. Our objective was to determine changes in the different gliadin protein components, and identify and quantify CD causing α-gliadin epitopes and total α-gliadin, in historical and modern wheat cultivars grown in the state of North Dakota, during the last 110 years, and thereby, compare the antigenicity of the different wheat cultivars. In this study, historical wheat refers to cultivars released prior to 1966, and modern wheat refers to cultivars released after 1966. Antigenicity, in this context, refers to the amount of immunogenic epitopes and total α-gliadin. Objective 1. To determine the presence of immunogenic peptides causing celiac disease (CD) in hard red spring wheat cultivars, durum wheat and ancient wheat species. 1) Major activities completed / experiments conducted; Samples of Hard Red Spring wheat varieties with release dates ranging from 1910 to 2013 were utilized to determine the presence of immunogenic peptides causing celiac disease. The gliadin proteins were extracted with 70% ethanol, from the ground whole wheat samples, before being analyzed with reverse phase high performance liquid chromatography (RP-HPLC). Separation of gliadin proteins was done with an Agilent Zorbax 300SB-C18 column. 2) Data collected; The peaks identified in the RP-HPLC analysis were analyzed for correlation with release year to determine the fractions that may have changed in the last 100 years. Averages of ω-gliadin, α/β-gliadin and γ-gliadin chromatogram areas were 1218, 2272 and 1930, respectively. ω-gliadin area ranged from 876 to 1666, α/β-gliadin area ranged from 1719 to 3128 and γ-gliadin area ranged from 1274 to 3049 for the HRS wheat samples released over 100 years. We were able to elucidate six peaks of ω-gliadin and four peaks of γ-gliadin that were of interest. 3) Summary statistics and discussion of results The total areas of the α/β-gliadin and ω-gliadin did not have significant correlation with release year. The ω-gliadin did have significant (P<0.05) positive correlation with release year. There were specific peaks of the ω-gliadins and γ-gliadins that had highly significant (P<0.01) and very highly significant (P<0.001) correlations with release year. Overall, ω-gliadins and some fractions of γ-gliadins increased with release year. 4) Key outcomes or other accomplishments realized. Some of the gliadin proteins in wheat have not changed in varieties released over 100 years, but there have been increases in some fractions of the gliadin proteins related to release year in these varieties. Objective 2. To quantify the immunogenic peptides using non-radioactive heavy labeled peptides. 1) Major activities completed / experiments conducted; The digested gliadin fractions were analyzed with LC-MS and relative quantification of the immunogenic epitopes was determined. The amount of immunogenic epitopes of glia-α9 and glia-α20 and total α-gliadin were quantified for ten samples across the cultivars from the 100 years that were investigated. Heavy labelled peptides P1-P9 were used for quantification. 2) Data collected; The immunogenic epitopes that trigger the immune reaction related to CD were found in both historical and modern spring wheat cultivars. All five of the immunogenic epitopes belonging to α/β-gliadin proteins were found in the majority of cultivars. The epitope PFPQPQLPY was detected in all cultivars except cultivar Alsen (released 2000). Cluster analysis was done and at cutting point 2.7, the cluster analysis produced 5 clusters. The release year of the cultivars in the different clusters is as follows, cluster A: 1942-2009, B: 1969-2002, C: 1910-2012, D: 1926-2013, E: 1939-2005. Cultivar Steele-ND released in 2004, had a relatively high amount of immunogenic epitopes compared to the other cultivars that were analyzed. Cultivar Russ released in 1995, was of lower antigenicity in comparison to the others, since the ratios are low for this cultivar. Comparatively higher amounts of the epitope glia-α9 were found in cultivars Pilot, Pioneer-2375 and Barlow, released in 1939, 1990 and 2009 respectively. Cultivar Russ (1995), which showed low values in the relative quantification experiment showed the lowest values in the absolute quantification experiment as well, for the glia-α9 immunogenic epitope and the amount of total α-gliadin. 3) Summary statistics and discussion of results The results of the LC-MS analysis show that immunogenic epitopes are present in both historical and modern spring wheat cultivars irrespective of release year. To analyze the relationship between the presence of these epitopes and release year, cluster analysis was performed according to Ward's method. In the dendrogram the cultivars did not cluster according to release year. Thus, the cultivars in any of the clusters formed do not represent only modern or historical wheat cultivars. The ANOVA test showed that there is no significant difference (P≥0.05) between clusters for release year. Thus, the results suggest that the presence or absence of immunogenic epitopes is independent of release year, indicating that historical and modern wheat may not be different in terms of their CD antigenicity. The results of the cluster analysis further support this view. That is, in terms of the presence of immunogenic epitopes, modern wheat is not higher in antigenicity in comparison to historical wheat. The results suggest that the amount of immunogenic epitopes and total α-gliadin varied randomly across the hundred years that were analyzed, indicating that there is no relation between release year of cultivar and CD antigenicity. Our results suggest that historical wheat is antigenic for those with CD. However, historical wheat was not found to be higher is antigenicity in comparison to modern wheat. 4) Key outcomes or other accomplishments realized. RP-HPLC was used to determine how the gliadin profiles changed in relation to release year. Mass spectrometric analysis was performed to determine if immunogenic epitopes causing celiac disease are present in the wheat cultivars being investigated, and to quantify these epitopes. Overall, the results indicate that immunogenic epitopes causing CD are detected in historical and modern hard red spring wheat cultivars irrespective of release year, and that the quantity of these epitopes does not show a relationship with cultivar release year, indicating that modern wheat cultivars are not higher in CD antigenicity in comparison to historical wheat cultivars. From our results, cultivar Russ showed the least amount of immunogenic epitopes and total α-gliadin, making it a suitable starting point for breeding wheat cultivars that are safe for CD patients.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Malalgoda, M., Meinhardt, S. W. & Simsek, S. (2018). Detection and quantitation of immunogenic epitopes related to celiac disease in historical and modern hard red spring wheat cultivars. Food chemistry, 264, 101-107.
• Type: Other Status: Published Year Published: 2019 Citation: Simsek, S. (2019) Dont blame wheat breeding for celiac disease. American Council on Science and Health. https://www.acsh.org/news/2019/09/18/dont-blame-wheat-breeding-celiac-disease-14292