Amylase/trypsin-inhibitors (ATIs) are known allergens and triggers of non-celiac wheat sensitivity. Until now, ATIs were only quantitated in wheat species. We developed and validated a targeted stable isotope dilution analysis LC-MS/MS method to quantitate ten barley-specific ATIs, including one monomeric and one dimeric amylase-inhibitor, four chloroform/methanol-soluble types, three subtilisin/chymotrypsin-inhibitors and one amylase/subtilisin-inhibitor. After successful validation in terms of precision, recovery and limits of detection and quantitation, the method was applied to 181 barley accessions from the EcoSeed panel, comprising 113 two-row and 68 six-row barleys of different genetic backgrounds. The overall ATI content was 1.1–5.2 mg/g, corresponding to 0.7–3.6% of the total protein content with no clear distinction between two-row and six-row barleys. This study is the first to provide insights on the ATI content and composition of barley, which can be used to make low-ATI foods for special dietary needs.

Barley wholegrain flour (50 mg) was extracted with ammonium bicarbonate buffer (1 mL, 50 mmol/L, pH 7.8) twice under stirring at 22 °C for 30 min. After each extraction, the samples were centrifuged for 25 min at 3550 rcf and the supernatants were combined. The extracts were evaporated to dryness and the residue was resolubilized in Tris-HCl (320 µL, 0.5 mol/L, pH 8.5) and 1-propanol (320 µL). The isotope labeled standards (50 µL of a mixed solution containing all labeled standards in a concentration of 20 µg/mL each) were then added. Using a tris(2-carboxyethyl)phosphine-solution (TCEP) (50 µL, 0.05 mol/L TCEP in 0.5 mol/L Tris-HCl, pH 8.5), the proteins were reduced for 30 min at 60 °C. Afterwards, the 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, followed again by evaporation until dryness. Tryptic digestion (1 mL trypsin solution, enzyme-to-substrate ratio 1:50, 0.04 mol/L urea in 0.1 mol/L Tris-HCl, pH 7.8) was carried out overnight for 18 h at 37 °C in the dark. The reaction was stopped with 4 µL trifluoroacetic acid. The solution was again evaporated to dryness. The residue was dissolved in 1 mL of 0.1% formic acid (FA) and filtered through a 0.45 µm regenerated cellulose membrane (WICOM, Heppenheim, Germany) prior to the LC-MS/MS measurement. For peptide separation and quantitation of the 10 different barley ATIs, an UltiMate 3000 HPLC (Dionex, Idstein, Germany) with an Aqua-C18 column (150 × 2 mm, 5 µm, 12.5 nm, Phenomenex, Aschaffenburg, Germany) coupled with a TripleQuadrupole mass spectrometer (MS) (TSQ Vantage, ThermoFischer Scientific, Bremen, Germany) was used. The following conditions were used for HPLC separation: solvent (A), FA (0.1%, v/v) in water; solvent (B), FA (0.1%, v/v) in acetonitrile; flow rate 0.2 mL/min; injection volume 10 µL; column temperature 22 °C; 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). For the TripleQuad MS the following parameters were used: electrospray ionization (ESI) in positive mode; spray voltage 4500 V; declustering voltage -10 V; vaporizing temperature 50 °C; capillary temperature 300 °C; sheath gas pressure 40 au; aux gas pressure 5 au. Selected reaction monitoring (SRM) was used to analyze the transitions from precursor ions to product ions.

Barley grain samples (5 g) were provided by the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK, Gatersleben, Germany). The sample set contained 181 barley accessions of the EcoSeed Panel grown under field conditions in Gatersleben, Germany, and harvested in 2018. The panel contains different genetic backgrounds of accessions comprising 113 two-row and 68 six-row barley accessions, including 103 cultivars, ten breeding lines and 65 landraces. The grains were milled into wholemeal flours using a coffee-mill (Robert Bosch GmbH, Stuttgart, Germany). For method development, the barley cultivar Sandra of harvest year 2013 was used.