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

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).

A Review of Rice Starch Digestibility: Effect of Composition and Heat‐Moisture Processing

“…Albumin and globulin are concentrated in the bran . Glutelin, prolamin and globulin are storage proteins which are a reserve of nitrogen, carbon and oxygen, whereas albumins are metabolic proteins such as enzymes and enzyme inhibitors . Leucine, lysine, sulphur‐containing amino acid and threonine are the major amino acids in rice protein …”

“…[138] Glutelin, prolamin and globulin are storage proteins which are a reserve of nitrogen, carbon and oxygen, [145] whereas albumins are metabolic proteins such as enzymes and enzyme inhibitors. [146,147] Leucine, lysine, sulphurcontaining amino acid and threonine are the major amino acids in rice protein. [144] Rice protein may affect starch digestibility by forming a protective layer around rice starch or by altering starch properties.…”

Non‐Celiac Gluten Sensitivity: How Its Gut Immune Activation and Potential Dietary Management Differ from Celiac Disease

“…[57][58][59] However, although oats are generally considered safe for CD patients, their role in CD pathogenesis is still a matter of debate. [60] Furthermore, oats can be considered safe also for NCGS subjects due to their lower prolamin content [61] and for their lower ATI innate immune stimulatory activity. [52] Another alternative treatment option to the GFD in patients affected by GRD could include the use of gene-editing technology in cereals.…”

Adverse Reactions to Wheat or Wheat Components

“…One question that needs to be addressed is the precision of analytical measurements. It should be recognized that the analysis of gluten and gluten epitopes presents challenges due to the difficulty in solubilizing gluten protein/peptides, variation in the compositions of gluten hydrolysates and food matrices, the lack of appropriate reference materials, the specificity of antibodies, and the use of different methods for separation and quantification (Lexhaller, Tompos, & Scherf, ; Schalk, Lang, Wieser, Koehler, & Scherf, ; Schalk, Lexhaller, Koehler, & Scherf, ; Scherf, ; Scherf & Poms, ; http://www.wgpat.com/aims.html).…”

“…One question that needs to be addressed is the precision of analytical measurements. It should be recognized that the analysis of gluten and gluten epitopes presents challenges due to the difficulty in solubilizing gluten protein/peptides, variation in the compositions of gluten hydrolysates and food matrices, the lack of appropriate reference materials, the specificity of antibodies, and the use of different methods for separation and quantification (Lexhaller, Tompos, & Scherf, 2017;Schalk, Lang, Wieser, Koehler, & Scherf, 2017;Schalk, Lexhaller, Koehler, & Scherf, 2017;Scherf, 2017;Scherf & Poms, 2016; http://www.wgpat.com/aims.html). Schopf and Scherf (2018) reported that the relative proportions of CD-immunogenic and -toxic peptides in gluten protein fractions vary depending on genetic factors such as species and cultivar in combination with environmental factors such as climate, soil, fertilization, and agricultural practices (Ashraf, 2014;Hajas et al, 2018).…”