Amylase/trypsin inhibitors (ATIs) are major wheat allergens and they are also implicated in causing non-celiac gluten sensitivity and worsening other inflammatory conditions. With only few studies on ATI contents in different Triticum species available so far, we developed a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method based on stable isotope dilution assays to quantitate the 13 most important ATIs in a well-defined sample set of eight cultivars of common wheat and durum wheat (modern species), as well as spelt, emmer and einkorn (ancient species) grown at three locations in Germany, respectively. Only few ATIs with low contents were detected in einkorn. In contrast, spelt had the highest total ATI contents. Emmer and common wheat had similar total ATI contents, with durum wheat having lower contents than common wheat. Due to the lack of correlation, it was not possible to estimate ATI contents based on crude protein contents. The wheat species had a higher influence on ATI contents than the growing location and the heritability of this trait was high. Despite comparatively low intra-species variability, some cultivars were identified that may be promising candidates for breeding for naturally low ATI contents.

A UniProtKB search with the keywords “amylase inhibitor” and the taxonomy Triticeae (taxon identifier 1648030) was performed and the amino acid sequences of twelve ATIs were downloaded on 28 February 2018. The amino acid sequence of the thirteenth ATI (WTI) was taken from literature. The amino acid sequences were loaded into the targeted proteomics software Skyline (version 4.2, MacCoss Lab Software, University of Washington, Seattle, WA, U.S.A.). The recorded MS raw files were imported in Skyline and the SRM transitions of each peptide were manually analyzed to assess the signal quality and overlapping transitions at the same chromatographic retention time. Peptides with ambiguous or nonexistent peaks were excluded. At least one peptide, but ideally two peptides per ATI was selected resulting in 20 marker peptides. The final LC-MS/MS was: An UltiMate 3000 HPLC system (Dionex, Idstein, Germany) coupled to a triple-stage quadrupole mass spectrometer (TSQ Vantage, ThermoFisher Scientific) equipped with an Aqua®-C18 column (50×2 mm, 5 µm, 12.5 nm, Phenomenex, Aschaffenburg, Germany) was used for peptide separation with the following LC conditions: Solvent A: FA (0.1%, v/v) in water; solvent B: FA (0.1%, v/v) in acetonitrile; gradient: 0-18 min 5-30% B, 18-20 min 30% B, 20-21 min 30-90% B, 21-24 min 90% B, 24-25 min 90-5% B, 25-35 min 5% B; flow rate: 0.2 mL/min; injection volume: 10 μL; column temperature: 22 °C. The ion source was operated in the positive electrospray ionization (ESI) mode and the following source parameters were set: Spray voltage: 4500 V; vaporizer temperature: 50 °C; sheath gas pressure: 40 arbitrary units (au); aux gas pressure: 5 au; capillary temperature: 300 °C.

Eight cultivars representative for actual agricultural production for each of common wheat, spelt, durum wheat, emmer and einkorn were cultivated by the State Plant Breeding Institute, University of Hohenheim (Stuttgart, Germany) at three locations in Germany (Seligenstadt, Oberer Lindenhof and Eckartsweiher), and harvested in 2013. The grains were milled into wholemeal flours using a cross-beater mill (Perten Instruments, Hamburg, Germany) and stored in closed bottles for at least two weeks before analysis. Flour (50 mg) was stirred twice with ammonium bicarbonate solution (0.5 mL, 50 mmol/L, pH 7.8) for 30 min at 22 °C. After every extraction step, the suspensions were centrifuged for 25 min at 3,750×g and the supernatants were combined. The extracts were evaporated to dryness in a rotational vacuum concentrator (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode, Germany) and the residue was dissolved in Tris-HCl (320 µL, 0.5 mol/L, pH 8.5) and 1-propanol (320 µL). A mixture of IS1-20 (50 µL) was added for SIDA. The concentration of each IS in this solution was adjusted to the expected peptide content in the samples Reduction was performed by adding tris(2-carboxyethyl)phosphine (TCEP) (50 µL, 0.05 mol/L TCEP in 0.5 mol/L Tris-HCl, pH 8.5) and incubating for 30 min at 60 °C. Cysteine residues were alkylated with chloroacetamide (CAA) (100 µL, 0.5 mol/L CAA in 0.5 mol/L Tris-HCl, pH 8.5) 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 by adding 2 µL trifluoroacetic acid. The solution was diluted 1+1 with 0.5 mL of 0.1 % formic acid and filtered through a 0.45 µm membrane.