Characterization and Relative Quantitation of Wheat, Rye, and Barley Gluten Protein Types by Liquid Chromatography-Tandem Mass Spectrometry

Abstract

The consumption of wheat, rye, and barley may cause adverse reactions to wheat such as celiac disease, non-celiac gluten/wheat sensitivity, or wheat allergy. The storage proteins (gluten) are known as major triggers, but also other functional protein groups such as α-amylase/trypsin-inhibitors or enzymes are possibly harmful for people suffering of adverse reactions to wheat. Gluten is widely used as a collective term for the complex protein mixture of wheat, rye or barley and can be subdivided into the following gluten protein types (GPTs): α-gliadins, γ-gliadins, ω5-gliadins, ω1,2-gliadins, high- and low-molecular-weight glutenin subunits of wheat, ω-secalins, high-molecular-weight secalins, γ-75k-secalins and γ-40k-secalins of rye, and C-hordeins, γ-hordeins, B-hordeins, and D-hordeins of barley. GPTs isolated from the flours are useful as reference materials for clinical studies, diagnostics or in food analyses and to elucidate disease mechanisms. A combined strategy of protein separation according to solubility followed by preparative reversed-phase high-performance liquid chromatography was employed to purify the GPTs according to hydrophobicity. Due to the heterogeneity of gluten proteins and their partly polymeric nature, it is a challenge to obtain highly purified GPTs with only one protein group. Therefore, it is essential to characterize and identify the proteins and their proportions in each GPT. In this study, the complexity of gluten from wheat, rye, and barley was demonstrated by identification of the individual proteins employing an undirected proteomics strategy involving liquid chromatography-tandem mass spectrometry of tryptic and chymotryptic hydrolysates of the GPTs. Different protein groups were obtained and the relative composition of the GPTs was revealed. Multiple reaction monitoring liquid chromatography-tandem mass spectrometry was used for the relative quantitation of the most abundant gluten proteins. These analyses also allowed the identification of known wheat allergens and celiac disease-active peptides. Combined with functional assays, these findings may shed light on the mechanisms of gluten/wheat-related disorders and may be useful to characterize reference materials for analytical or diagnostic assays more precisely.

Keywords: allergy; amylase/trypsin-inhibitor; celiac disease; gliadin; gluten; mass spectrometry; non-celiac gluten sensitivity; proteomics.

Figure 1

Composition and proportions of proteins in each GPT. Classification of identified proteins into the following groups for wheat (A), rye (B), and barley (C) gluten protein types: gluten proteins, α-amylase/trypsin-inhibitors (ATIs), globulins, other enzymes, β-amylase, farinins, serpins, grain softness proteins, and puroindolines (GSPs+PINs), uncharacterized proteins, avenin-like proteins, other inhibitors, and others. When a group is missing in individual GPT, no proteins were identified. Groups without number represent less than 2%. GS, glutenin subunits; HMW, high-molecular-weight; LMW, low-molecular-weight.

Figure 2

Relative protein quantification in GPTs. The summed peak areas of selected tryptic and chymotryptic peptides of the most abundant proteins representing protein groups in individual GPTs: peak areas of peptides representing α-gliadins, γ-gliadins, ω-gliadins, HMW-GS, LMW-GS, and avenin-like proteins in the GPTs of wheat (A), peak areas of peptides representing γ-prolamins, ω-secalins, HMW-secalins, LMW-GS, γ-75k-secalins, and avenin-like proteins in the GPTs of rye (B), peak areas of peptides representing γ3-hordeins, HMW-GS, D-hordeins, B-hordeins, C-hordeins, and avenin-like proteins in the GPTs of barley (C). Data is plotted as the mean ± standard deviation (n = 3).