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.

LC-MS/MS Reveals Hordeins Are Enriched in Brewers’ Spent Grain

“…Proteins were extracted from the malt flour and BSG flour by an established method and protocol, using 8 M urea and 0.1 M tris-HCl, pH 8.4, as the extraction solvent. Protein concentration was estimated by the Bradford assay.…”

“…Discovery proteomics was performed on malt samples to detect and identify hordein proteins using LC-MS instrumentation, conditions, and settings as described previously. 11 Data were searched against a FASTA file consisting of the hordeum genus of proteins from UniProt-KB (accessed 08/2022 and supplemented with additional translated gene models from the IWGSC RefSeq v1 Assembly, 12 as well as those listed on the common Repository of Adventitious Proteins (thegpm.org/ crap)). The FASTA file contained 57,925 protein sequences.…”