Non-coeliac gluten sensitivity (NCGS) is a less understood gastrointestinal-related disorder, which affects ~10% of the world population. Alpha-amylase/trypsin inhibitors (ATIs) from wheat, barley and rye can trigger the innate immune system through activation of toll-like receptor 4 (TLR 4) on myeloid cells to initiate NCGS. In this study, we aimed to identify and measure the relative quantities of ATIs in ultra-low gluten (ULG) barley varieties by using LC-MS-based untargeted and targeted proteomics. The SWATH-MS experiment revealed the presence of 6,138 peptides mapping to 1,907 proteins at a 1% false discovery rate. In total, 333 (17%) proteins were perturbed between wild type and ULG barley lines where 10 ATI-like proteins were downregulated (p < 0.05; FC< 2.0) in the ULG line.
To further monitor the ATIs in ULG lines, we selected 12 ATI-like proteins with allergy-related epitope domains in their sequence from the assessment of multiple barley varieties. Multiple reaction monitoring (MRM) mass spectrometry showed that levels of ATI peptides were significantly reduced in the triple null ULG line in comparison to wild-type barley cv Sloop. Notably, one of the barley dimeric ATIs (UniProt: P13691) that is closely related to wheat dimeric ATIs — and has been implicated in cell and murine models of NCGS — was reduced five-fold in the triple null ULG line. In addition, another barley alpha-amylase/trypsin inhibitor CMd (P11643) closely matched to alpha-amylase/trypsin inhibitor CM3 (P17314) from wheat was reduced to ~88% in comparison to WTs. Wheat-derived ATI CM3 (Q6S5B1) was previously found to one of the most prevalent activators of TLR4 in wheat and were highly resistant to intestinal proteolysis. Overall, our study reveals that several ATIs were downregulated in ULG lines and future clinical investigation is warranted for safety assessment regarding the suitability of ULG barley lines for NCGS patients.