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

TLDR

Gluten proteins from wheat, rye, barley, and occasionally oats trigger celiac disease, non‑celiac gluten sensitivity, and wheat allergy, and well‑defined reference materials are essential for clinical studies, diagnostics, and food analyses, yet current RM often lack thorough characterization of source, content, and composition, which is critical due to gluten’s complexity and heterogeneity. The study developed a comprehensive strategy to isolate gluten protein fractions and types from wheat, rye, barley, and oats to produce well‑defined reference materials for clinical assays and gluten‑free compliance testing. All isolated gluten protein types—including ω‑gliadins, α‑gliadins, γ‑gliadins, glutenin subunits from wheat; ω‑secalins, γ‑secalins, high‑molecular‑weight secalins from rye; C‑, γ‑, B‑, D‑hordeins from barley; and avenins from oats—were fully characterized by RP‑HPLC, SDS‑PAGE, N‑terminal sequencing, LC‑ESI‑QTOF‑MS, and untargeted LC‑MS/MS of chymotryptic hydrolyzates. The analyses confirmed that each gluten protein type was reproducibly isolated in high purity and is suitable as a reference material for calibrating LC‑MS/MS methods and ELISAs.

Abstract

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).