Proteome Analysis and Epitope Mapping in a Commercial Reduced-Gluten Wheat Product

Abstract

Gluten related disorders, such as coeliac disease, wheat allergy and baker's asthma are triggered by proteins present in food products made from wheat and related cereal species. The only treatment of these medical illnesses is a strict gluten-free diet; however, gluten-free products that are currently available in the market can have lower nutritional quality and are more expensive than traditional gluten containing cereal products. These constraints have led to the development of gluten-free or gluten-reduced ingredients. In this vein, a non-GMO wheat flour that purports to contain "65% less allergenic gluten" was recently brought to market. The present study aims to understand the alteration of the proteome profile of this wheat flour material. Liquid chromatography-mass spectrometry was used to investigate the proteome profile of the novel wheat flour, which was contrasted to a wheat flour control. Using both trypsin and chymotrypsin digests and a combined database search, 564 unique proteins were identified with 99% confidence. These proteins and the specific peptides used to identify them were mapped to the wheat genome to reveal the associated chromosomal regions in the novel wheat flour and the mixed wheat control. Of note, several ω- and γ-gliadins, and low-molecular weight glutenins mapping to the short arm of chromosome 1, as well as α-gliadins from the chromosome 6 short arm were absent or expressed at lower levels in the novel wheat variety. In contrast, the high-molecular weight glutenins and α-amylase/trypsin inhibitors were notably more abundant in this variety. A targeted quantitation experiment was developed using multiple reaction monitoring assays to quantify 359 tryptic and chymotryptic peptides from gluten and related allergenic proteins revealing a 33% decrease of gluten protein content in the novel wheat flour sample in comparison to mixed wheat control. However, additional mapping of known allergenic epitopes showed the presence of 53% higher allergenic peptides. Overall, the current study highlights the importance of proteomic analyses especially when complemented by sequence analysis and epitope mapping for monitoring immunostimulatory proteins.

Keywords: allergy; celiac disease; food safety; gluten; mass spectrometry; proteomics; wheat.

Figure 1

Summary of total and gluten-like proteins identifications at 1% FDR and their corresponding chromosomal locations. (A) Venn diagram showing the total number of proteins detected within the GW and MW datasets. (B) Detected gluten proteins in MW and GW. (C) Locations of genes for detected proteins on wheat chromosomes 1A, 1B, and 1D.

Figure 2

Volcano plots showing quantified tryptic (A) and chymotryptic (B) peptides in GW and MW samples colored according to gluten group. A fold-change of 2 is indicated by the dashed vertical lines [Log₂(FC) = ±1]. Peptides above the horizontal dashed line have a significant change in abundance between GW and MW (p-value <0.01 [–log₁₀(p) >2].

Figure 3

Relative abundance of different gluten or ATI protein groups (A). Quantitation was performed based on all detectable peptides from proteins classified to these groups. Error bars indicate SEM, and significant differences are indicated by asterisk. Adding together the LMW and HMW glutenins, and α-, γ-, ω-gliadins gives the net gliadin and glutenin content (B) which equated to GW having an estimated 67% of the gluten content of MW (dotted line).

Figure 4

GO enrichment analysis of proteins showing ≥2-fold increase in: MW (A); or GW (B).

Figure 5

Relative abundance of immune reactive epitopes in peptides quantified in MRM assays, reported as mean and standard error. Epitope sequences are highlighted in bold within the peptide sequence: (A) Coeliac disease HLA-DQ reactive epitopes; (B) Bakers' asthma; (C) Wheat Allergy plus peptide QQQQQQQQILQQILQQQLIPCR which contains the epitope QILQQQLIPC antigenic for wheat-dependent exercise induced anaphylaxis. When calculating the total, duplicate peptides where multiple epitopes were detected were only counted once.