Quantitation of Specific Barley, Rye, and Oat Marker Peptides by Targeted Liquid Chromatography–Mass Spectrometry To Determine Gluten Concentrations

Celiac disease is triggered by the ingestion of gluten from wheat, barley, rye, and possibly oats. Gluten is quantitated by DNA-based methods or enzyme-linked immunosorbent assays (ELISAs). ELISAs mostly detect the prolamin fraction and potentially over- or underestimate gluten contents. Therefore, a new independent method is required to comprehensively detect gluten. A targeted liquid chromatography–tandem mass spectrometry method was developed to quantitate seven barley, seven rye, and three oat marker peptides derived from each gluten protein fraction (prolamin and glutelin) and type (barley, B-, C-, D-, and γ-hordeins; rye, γ-75k-, γ-40k-, ω-, and HMW-secalins). The quantitation of each marker peptide in the chymotryptic digest of a defined amount of the respective reference gluten protein type resulted in peptide-specific yields, which enabled the conversion of peptide into protein concentrations. This method was applied to quantitate gluten in samples from the brewing process, in raw materials for sourdough fermentation, and in dried sourdoughs

Analytical characterization of the isolated gluten protein types (GPT).

Proportions of each GPT in wheat, rye, barley and oat flours, protein content of each isolated GPT, their N-terminal sequence(s), molecular weight ranges (Mr) determined by LC-ESI-QTOF-MS and the Mr of the most appropriate reference sequence found in the NCBI database given with its specific accession.</p

Overview of the preparative strategy.

<p>This strategy allows the isolation of well-defined gluten protein fractions and types from wheat, rye, barley and oat flours. HMW-GS, high-molecular-weight glutenin subunits, LMW-GS, low-molecular-weight glutenin subunits.</p

SDS-PAGE of flours, prolamin and glutelin fractions and isolated gluten protein types.

<p>(A) Wheat. M: marker, 1: wheat flour, 2: wheat prolamin fraction, 3: ω5-gliadins, 4: ω1,2-gliadins, 5: α-gliadins, 6: γ-gliadins, 7: wheat glutelin fraction, 8: high-molecular-weight glutenin subunits (HMW-GS), 9: low-molecular-weight glutenin subunits (LMW-GS). (B) Rye. M: marker, 10: rye flour, 11: rye prolamin fraction, 12: ω-secalins, 13: γ-75k-secalins, 14: γ-40k-secalins, 15: rye glutelin fraction, 16: HMW-secalins. (C) Barley. M: marker, 17: barley flour, 18: barley prolamin fraction, 19: γ/B-hordeins, 20: C-hordeins, 21: barley glutelins, 22: B/γ-hordeins, 23: D-hordeins. (D) Oats. 24: oat prolamin fraction (avenins), 25: oat flour.</p

RP-HPLC chromatograms of the glutelin fractions.

<p>(A) Wheat glutelins, (B) rye glutelins, (C) barley glutelins, all reduced with 1% (w/v) DTT. AU, absorbance units at 210 nm, ωb, ωb-gliadins, HMW-GS, high-molecular-weight glutenin subunits, LMW-GS, low-molecular-weight glutenin subunits, HMW-Sec, HMW-secalins, γ-75k, γ-75k-secalins, γ-40k, γ-40k-secalins, D, D-hordeins, B/γ, B-hordeins and γ-hordeins.</p

RP-HPLC chromatograms of the prolamin fractions.

<p>(A) Wheat prolamins, (B) oat prolamins, (C) rye prolamins, unreduced, (D) rye prolamins, reduced with 1% (w/v) DTT, (E) barley prolamins, unreduced, (F) barley prolamins, reduced with 1% (w/v) DTT. AU, absorbance units at 210 nm, ω5, ω5-gliadins, ω1,2, ω1,2-gliadins, α, α-gliadins, γ, γ-gliadins, ave, avenins, ωs, ω-secalins, ωs+H, ω- and high-molecular-weight (HMW)-secalins, γ-75k, γ-75k-secalins, γ-40k, γ-40k-secalins, C, C-hordeins, γ/B, γ-hordeins and B-hordeins.</p

Analytical characterization of the flours.

<p>Contents of water, ash, crude protein (CP) and the Osborne fractions albumins/globulins (ALGL), prolamins and glutelins of wheat, rye, barley and oat flours (mixture of four cultivars each).</p

Data_Sheet_1_Lipidomic insights into the reaction of baking lipases in cakes.PDF

Lipases are promising improvers of cake batter and baking properties. Their suitability for use in various cake formulations cannot be predicted yet, because the reactions that lead to macroscopic effects need to be unravelled. Therefore, the lipidome of three different cake recipes with and without lipase treatment was assessed by ultra high performance liquid chromatography-mass spectrometry before and after baking. By comparing the reaction patterns of seven different lipases in the recipes with known effects on texture, we show that lipase substrate specificity impacts baking quality. Key reactions for the recipes were identified with the help of principal component analysis. In the eggless basic cake, glyceroglycolipids are causal for baking improvement. In pound cake, lysoglycerophospholipids were linked to textural effects. Lipase substrate specificity was shown to be dependent on the recipe. Further research is needed to understand how recipes can be adjusted to achieve optimal lipase substrate specificity for desirable batter and baking properties.</p

Data_Sheet_1_Basophil Activation to Gluten and Non-Gluten Proteins in Wheat-Dependent Exercise-Induced Anaphylaxis.PDF

Wheat-dependent exercise-induced anaphylaxis (WDEIA) is a cofactor-induced wheat allergy. Gluten proteins, especially ω5-gliadins, are known as major allergens, but partially hydrolyzed wheat proteins (HWPs) also play a role. Our study investigated the link between the molecular composition of gluten or HWP and allergenicity. Saline extracts of gluten (G), gluten with reduced content of ω5-gliadins (G-ω5), slightly treated HWPs (sHWPs), and extensively treated HWPs (eHWPs) were prepared as allergen test solutions and their allergenicity assessed using the skin prick test and basophil activation test (BAT) on twelve patients with WDEIA and ten controls. Complementary sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), high-performance liquid chromatography (HPLC), and mass spectrometry (MS) analyses revealed that non-gluten proteins, mainly α-amylase/trypsin inhibitors (ATIs), were predominant in the allergen test solutions of G, G-ω5, and sHWPs. Only eHWPs contained gliadins and glutenins as major fraction. All allergen test solutions induced significantly higher �63+ basophils/anti-FcεRI ratios in patients compared with controls. BAT using sHWPs yielded 100% sensitivity and 83% specificity at optimal cut-off and may be useful as another tool in WDEIA diagnosis. Our findings indicate that non-gluten proteins carrying yet unidentified allergenic epitopes appear to be relevant in WDEIA. Further research is needed to clarify the role of nutritional ATIs in WDEIA and identify specific mechanisms of immune activation.</p