Isolation and characterization of gluten protein types from wheat, rye, barley and oats for use as reference materials

PLoS One. 2017 Feb 24;12(2):e0172819. doi: 10.1371/journal.pone.0172819. eCollection 2017.


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).

MeSH terms

  • Avena / chemistry*
  • Calibration
  • Chromatography, High Pressure Liquid
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme-Linked Immunosorbent Assay
  • Flour / analysis
  • Food Analysis / methods*
  • Glutens / analysis*
  • Hordeum / chemistry*
  • Hydrolysis
  • Peptides / analysis
  • Protein Domains
  • Reference Values
  • Secale / chemistry*
  • Spectrometry, Mass, Electrospray Ionization
  • Triticum / chemistry*


  • Peptides
  • Glutens
  • glutenin

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.