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 lowmolecular-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, Frontiers in Plant Science | www.frontiersin.org

Synthesis and accumulation of amylase-trypsin inhibitors and FODMAPs in bread wheat (Triticum aestivum L.) during grain development

“…2b) illustrated a typical gliadin pattern with a majority of α-/ß-gliadins from 30-40 kDa and minor abundance of ω-gliadins from 40-55 kDa. Besides gluten type proteins, the PWG spectrum revealed the presence of ATIs and avenin-like proteins, which was recently con rmed by Lexhaller et al [15]. As ATIs are rather small proteins that range from 13-18 kDa [13,16,17], sample peaks in this area were assigned predominately to ATIs and to other proteins from the prolamine superfamily (e.g.…”

Identification of Isopeptides Between Human Tissue Transglutaminase and Wheat, Rye, and Barley Gluten Peptides

“…To reduce complexity compared to a total gluten hydrolysate, our experimental approach to identify TG2-gluten isopeptides started with the preparation of the following GPTs: α-gliadins, γ-gliadins, ω5-gliadins, ω1,2-gliadins, high-(HMW-GS) and low-molecular-weight glutenin subunits (LMW-GS) of wheat, ω-secalins, HMW-secalins, γ-75k-secalins and γ-40k-secalins of rye and C-hordeins, γ-hordeins, B-hordeins and D-hordeins of barley ( Fig. 1a) 17,18 . The individual GPTs were hydrolysed using a combination of pepsin and chymotrypsin/trypsin to mimic the main enzymatic processes during gastrointestinal digestion 16,19 .…”

“…Therefore, the aim of our study was to apply our recently developed reciprocal mass spectrometric approach, including discovery-driven mass spectrometry 15 and additional targeted proteomics, to complex gluten hydrolysates that had been incubated with TG2 and identify TG2-gluten isopeptides. We used well-characterized gluten protein types (GPTs) of wheat, rye and barley 17 and extended our analysis strategy with additional confirmation of isopeptide identities by parallel reaction monitoring (PRM) LC-MS/MS as follow-up measurements.…”

CRISPR/Cas9 Gene Editing of Gluten in Wheat to Reduce Gluten Content and Exposure—Reviewing Methods to Screen for Coeliac Safety

“…However, in particular, gene editing may generate novel peptides that are not present in any library and can only be predicted by sequencing all gluten genes in each geneedited line. Most studies use a targeted approach, focusing on a small set of peptides as reference for a class of gluten proteins (76,77,80). The extraction procedure used is also relevant, as it has impact on proteins and peptides recovered (81,82).…”