Gluten refers to a class of seed storage proteins present in wheat and related species. They contribute nutritional and functional attributes to pasta, bread, and other cereal products, but provoke an immune reaction in people susceptible to wheat allergy, coeliac disease, and non-coeliac gluten sensitivity. The market for gluten-free flour is largely supplied by flours from plant species that do not express gluten proteins, though there is also a ‘low gluten’ market for consumers who experience less severe reactions to dietary wheat products. In this vein, a non-GMO wheat flour that purports to contain ‘65% less allergenic gluten’ was recently brought to market. We sought to understand this alteration in protein profile and test its claim.
We conducted a bottom-up proteomics approach on the low-gluten flour, as well as a mixed wheat flour as control, using independent chymotryptic and tryptic digests of gluten-enriched extracts. Liquid chromatography-mass spectrometry (LC-MS) was performed on a SCIEX TripleTOF 6600 with microflow LC to determine the identities of proteins present, altogether revealing 635 distinct proteins at a 1% global false discovery rate. Multiple reaction monitoring assays were developed for the targeted quantitation of tryptic and chymotryptic wheat gluten peptides on a SCIEX QTRAP 6500+ with ExionLC system. Of these, 359 peptides were used to measure the abundance of gluten and allergenic proteins. Due to a high degree of sequence homology and shared peptides, proteins were categorised into groups by the presence of known domains. Relative abundance of protein groups in the low-gluten wheat and control wheat were plotted, and proteins were mapped to the wheat genome to reveal the chromosome locations that were altered in the ‘low gluten’ wheat variety. These data were used to determine gluten proteins that were present or absent in the novel low-gluten wheat, and the genome locations selected against in this variety. Of note, several omega- and gamma-gliadins, and low-molecular weight glutenins mapping to the short arm of chromosome 1, as well as alpha-gliadins from the chromosome 6 short arm were absent or expressed at lower levels in the low-gluten variety. In contrast, the high-molecular weight glutenins and alpha-amylase/trypsin inhibitors were notably more abundant in this variety.