Amylase/trypsin-inhibitors (ATIs) are triggers for wheat-related disorders like baker’s asthma and nonceliac wheat sensitivity. With the rise ofwheat-related disordersamong the population, the hypothesis that breeding may have resulted in changes in the protein composition of wheat was put forward. The ATI content of 14 German common wheat landraces and six modern varieties harvested in three consecutive years was analyzed by liquid chromatography–tandem mass spectrometry, and the inhibitory activity against α-amylase was measured with an enzymatic assay. The mean ATI content and proportion of crude protein of both groups did not differ. There were also only small differences in the content and proportion of single ATIs. The mean values for the inhibitory activity of both groups were also similar. These results indicate that breeding might not have led to changes in the protein composition and landraces are unlikely to be better tolerated than modern varieties.

Flour (50 mg) was extracted twice with Abic solution (0.5 mL, 50 mmol/L, pH 7.8) for 30 min at 22 °C using a magnetic stirrer. After every extraction step, the suspensions were centrifuged (25 min, 22°C, 3750 rcf) and the supernatants were combined. Using a rotational vacuum concentrator (Christ, Osterode, Germany), the extracts were then evaporated to dryness. The residue was dissolved in 350 µL of Tris–HCl (0.5 mol/L, pH 8.5) and 350 µL of 1 propanol. Then 50µL of standard solution, containing IS1-22, was added. The concentration of each IS in this solution was adjusted to the expected peptide content in the samples. After that, 50 µL of TCEP were added (0.05 mol/L TCEP in 0.5 mol/L Tris–HCl, pH 8.5) and incubated for 30 min at 60°C in a thermal shaker (Hettich Lab Technology, Tuttlingen, Germany) to perform reduction of the disulfide bonds. Alkylation was performed in the same device by adding 100 µL of CAA (0.5 mol/L CAA in 0.5 mol/L Tris–HCl, pH 8.5) and incubating for 45 min at 37°C in the dark. The solvent was again removed by evaporation to dryness. Last, 0.5 mL of trypsin solution (enzyme-to-substrate ratio 1:30, 0.04 mol/L urea in 0.1 mol/L Tris–HCl, pH 7.8) was added for tryptic digestion for 18 h overnight at 37°C in the dark. After that, the reaction was stopped with 5 µL of TFA. The solution was removed by evaporation and the residue was then dissolved in 1 mL of water containing 2% acetonitrile and 0.1% formic acid. For calibration, two solutions (25 – 100 µg/mL of each peptide) were prepared from the stock solutions, solution 1 with P1-P22 and solution 2 with IS1-IS22. An aliquot of each solution 1 and 2 was reduced and alkylated similar to the samples and as described in Geisslitz et al. 2. Alkylated solutions 1 and 2 (0.3 – 1.3 µg/mL of each peptide) were mixed in molar ratios n(P)/n(IS) between 9.1 and 0.1 (9+1, 7+1, 5+1, 3+1, 1+1, 1+3, 1+5, 1+7 and 1+9) and used for quantitation. Limits of detection and quantitation were also determined according to Geisslitz et al.

14 German common wheat (Triticum aestivum L.) landraces and six modern varieties were cultivated under organic conditions (without fertilization) at the Bavarian State Research Center for Agriculture (Ruhstorf an der Rott, Germany) and harvested in three consecutive years (2021–2023), with the exception of one variety which was only available in two years (WIW) (Tab.S1). The varieties were grown in a randomized complete block design in triplicate, but harvested and milled together with a Quadrumat Junior mill (Brabender, Duisburg, Germany) to obtain type 550 flours (ash content of 0.51% to 0.63% based on dry matter) according to the German flour classification system.