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, 12 (2), e0172819

Isolation and Characterization of Gluten Protein Types From Wheat, Rye, Barley and Oats for Use as Reference Materials


Isolation and Characterization of Gluten Protein Types From Wheat, Rye, Barley and Oats for Use as Reference Materials

Kathrin Schalk et al. PLoS One.


Gluten proteins from wheat, rye, barley and, in rare cases, oats, are responsible for triggering hypersensitivity reactions such as celiac disease, non-celiac gluten sensitivity and wheat allergy. Well-defined reference materials (RM) are essential for clinical studies, diagnostics, elucidation of disease mechanisms and food analyses to ensure the safety of gluten-free foods. Various RM are currently used, but a thorough characterization of the gluten source, content and composition is often missing. However, this characterization is essential due to the complexity and heterogeneity of gluten to avoid ambiguous results caused by differences in the RM used. A comprehensive strategy to isolate gluten protein fractions and gluten protein types (GPT) from wheat, rye, barley and oat flours was developed to obtain well-defined RM for clinical assays and gluten-free compliance testing. All isolated GPT (ω5-gliadins, ω1,2-gliadins, α-gliadins, γ-gliadins and high- and low-molecular-weight glutenin subunits from wheat, ω-secalins, γ-75k-secalins, γ-40k-secalins and high-molecular-weight secalins from rye, C-hordeins, γ-hordeins, B-hordeins and D-hordeins from barley and avenins from oats) were fully characterized using analytical reversed-phase high-performance liquid chromatography (RP-HPLC), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal sequencing, electrospray-ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF-MS) and untargeted LC-MS/MS of chymotryptic hydrolyzates of the single GPT. Taken together, the analytical methods confirmed that all GPT were reproducibly isolated in high purity from the flours and were suitable to be used as RM, e.g., for calibration of LC-MS/MS methods or enzyme-linked immunosorbent assays (ELISAs).

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.


Fig 1
Fig 1. RP-HPLC chromatograms of the prolamin fractions.
(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.
Fig 2
Fig 2. RP-HPLC chromatograms of the glutelin fractions.
(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.
Fig 3
Fig 3. Overview of the preparative strategy.
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.
Fig 4
Fig 4. SDS-PAGE of flours, prolamin and glutelin fractions and isolated gluten protein types.
(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.
Fig 5
Fig 5
Mass spectra of isolated (A) ω-secalins and (B) γ-hordeins. The spectra show the average of scans under the peak with retention times (A) 8.9 min and (B) 12.5 min from the respective base peak MS chromatograms after LC-ESI-QTOF-MS analysis of the isolated ω-secalins and γ-hordeins, respectively. The insets show the mass spectra simulated by maximum entropy deconvolution.

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    1. FAOSTAT. Food and Agriculture Organization of the United Nations, Statistics Division. Available: (accessed December 12, 2016).
    1. Sapone A, Bai JC, Ciacci C, Dolinsek J, Green PH, Hadjivassiliou M, et al. Spectrum of gluten-related disorders: consensus on new nomenclature and classification. BMC Med. 2012; 10: 13 10.1186/1741-7015-10-13 - DOI - PMC - PubMed
    1. Ludvigsson JF, Leffler DA, Bai JC, Biagi F, Fasano A, Green PH, et al. The Oslo definitions for coeliac disease and related terms. Gut. 2013; 62: 43–52. 10.1136/gutjnl-2011-301346 - DOI - PMC - PubMed
    1. Scherf KA, Koehler P, Wieser H. Gluten and wheat sensitivities–an overview. J Cereal Sci. 2016; 67: 2–11.
    1. Tatham AS, Shewry PR. Allergens in wheat and related cereals. Clin Exp Allergy. 2008; 38: 1712–1726. 10.1111/j.1365-2222.2008.03101.x - DOI - PubMed

Grant support

Katharina Anne Scherf wishes to acknowledge the German Celiac Society (Deutsche Zöliakie-Gesellschaft e.V.) for financial support (research grant 2014). Peter Koehler acknowledges funding by the German Federal Ministry of Education and Research via the VDI Technologiezentrum GmbH (Grant No. 13GW0042) and by the Deutsche Forschungsgemeinschaft (DFG, Project No. KO 1762/12-1). The publication of this article was funded by the Open Access Fund of the Leibniz Association. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.