The prevalence of hypersensitivities towards wheat has increased in the last decades. Apart from celiac disease these include allergic and other inflammatory reactions summarized under the term non-celiac wheat sensitivity. One suspected trigger is the family of amylase/trypsin-inhibitors (ATIs), non-gluten proteins that are prominent wheat allergens and that activate the toll-like receptor 4 on intestinal immune cells to promote intestinal and extra-intestinal inflammation. We therefore quantified 13 ATIs in 60 German hexaploid winter wheat cultivars originating from 1891 to 2010 and harvested in three years by targeted liquid chromatography-tandem mass spectrometry combined with stable isotope dilution assay using specific marker peptides as internal standards. The total ATI content and that of the two major ATIs 0.19 and CM3 did not change from old cultivars (first registered from 1891–1950) to modern cultivars (1951–2010). There were also no significant changes in ATI distribution.

Flour (50 mg) was extracted twice with ammonium bicarbonate (Abic) solution (0.5 mL, 50 mmol/L, pH 7.8) for 30 min at 22 °C. After centrifugation, the combined extracts were evaporated to dryness in a rotational vacuum concentrator (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode, Germany). The residue was dissolved in Tris-HCl (320 µL, 0.5 mol/L, pH 8.5) and 1-propanol (320 µL) and a mixture of the internal standards was added. Reduction was performed with tris(2-carboxyethyl)phosphine for 30 min at 60 °C and alkylation with chloroacetamide for 45 min at 37 °C in the dark. The solvent was removed by evaporation to dryness. Tryptic hydrolysis (0.5 mL, enzyme-to-substrate ratio 1:50, 0.04 mol/L urea in 0.1 mol/L Tris-HCl, pH 7.8) was performed for 18 h overnight at 37 °C in the dark. The reaction was stopped with 2 µL trifluoroacetic acid. The solution was diluted 1+1 with 0.5 mL of 0.1 % formic acid (FA) and filtered through a 0.45 µm membrane. An UltiMate 3000 HPLC system (Dionex, Idstein, Germany) was coupled to a triple-stage quadrupole mass spectrometer (TSQ Vantage, ThermoFisher Scientific, Bremen, Germany). An Aqua®-C18 column (50×2 mm, 5 µm, 12.5 nm, Phenomenex, Aschaffenburg, Germany) was used with FA (0.1%, v/v) in water as solvent A and FA (0.1%, v/v) in acetonitrile as solvent B. Scheduled single reaction monitoring (SRM) was used to analyze the transitions from precursor to product ions. For calibration different molar ratios n(peptide)/n(internal standard) between 9.1 and 0.1 (9+1, 4+1, 3+1, 1+1, 1+3, 1+4 and 1+9) were mixed.

Sixty hexaploid wheat (T. aestivum L.) cultivars were selected from the German Federal ex situ Genebank of crops at the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK, Gatersleben, Germany). The 60 wheat cultivars represent the five most widely grown cultivars in Germany, which were first registered in the respective decade between 1891 and 2010. The samples were cultivated with three biological replicates each at Gatersleben in 2015, 2017 and 2019. Grains from the three biological replications were pooled for each growing year. The grains from 2015 and 2017 were milled using a laboratory grinder (Bosch, Stuttgart, Germany) and the flours were sieved through a 0.2 mm sieve. The grains from 2019 were milled using an ultra-centrifugal mill ZM 200 (Retsch, Haan, Germany).