Chrompass software

The chromatographic analyses of all prepared solutions and extracts were performed using an HPLC from Jasco that consisted of a quaternary piston pump (PU-2089 Plus), an autosampler (AS-2055 Plus), and an UV/Vis detector (UV-2070 Plus). The data treatment was performed using Chrompass software (Jasco, Tokyo, Japan, v.1.8.6.1). The elution conditions, being based on previous work, were as follows [21 (link)]: the stationary phase was a column Gemini C₁₈ from Phenomenex (150 mm × 4.6 mm, 3 μm), and the elution was performed in gradient mode using water and acetonitrile (0 min: 50% acetonitrile, 50% water; 20 min: 65% of acetonitrile, 35% water; 45 min: 100% of acetonitrile; 50 min: 50% of acetonitrile, 50% water; and 55 min: 50% of acetonitrile, 50% water); the mobile phase flow rate was 0.45 mL min⁻¹. The volume of the injected sample was 25 µL, and the wavelength of detection was set at 360 nm.
For the identification of carbonyl compounds, the extracts obtained using the fan assisted extraction procedure were analyzed using an HPLC-DAD-MS/MS system, model LTQ XL, from Thermo Scientific (Waltham, MA, USA). The same elution conditions as above-mentioned were used. The settings of the mass spectrum detector (see Supplementary Data S1) were adjusted via proper tuning with the hydrazones of the carbonyl compounds studied.

The quantitation of protein fractions was performed by RP-HPLC on a Jasco XLC instrument (Jasco, Gross-Umstadt, Germany) with a C₁₈ column (Acclaim^TM 300, 2.1 × 150 mm, 3 µm, 30 nm, Thermo Fisher Scientific, Braunschweig, Germany) at 60 °C. Elution solvents were 0.1% (v/v) trifluoroacetic acid (TFA) in water (A) and 0.1% (v/v) TFA in acetonitrile (B) at a flow rate of 0.2 mL/min with the following gradient: 0 min 0% B, 0.5 min 24% B, 20 min 56% B, 20.1–24.1 min 90% B, 24.2–30 min 0% B. The injection volumes were 20 µL (ALGL, GLUT) and 10 µL (ALGL + GLIA) and protein absorbance was detected at 210 nm. PWG-gliadin [23 (link)] dissolved in 60% (v/v) ethanol was used for external calibration. The contents of ωb-gliadins, HMW-GS and LMW-GS were calculated relative to the total area of GLUT as described earlier [27 (link)]. To obtain the GLIA content, the chromatogram of the ALGL fraction was subtracted from that of the ALGL + GLIA fraction using the Chrompass software (version 1.2, Jasco, Gross-Umstadt, Germany). Then, the contents of ω5-, ω1,2-, α- and γ-gliadins were calculated from the absorbance area of each type relative to the total area.