Pepsin Trypsin Digestion

The gliadin extract (120 mg) was dissolved in 20 mL of sodium acetate buffer (50 mM, pH 4). Pepsin (3200 U/mg; P-6887; Sigma-Aldrich, United States) and mixed with sample prior to incubation at 37°C under agitation for 2 h. Following the incubation, 142 mg of sodium phosphate was added, and the pH was adjusted to 7.0 using NaOH. Afterward, trypsin (2500 U/mg; T-7418; Sigma-Aldrich, St. Louis, MO, United States) was added and the solution was mixed and incubated for 2 h under agitation at 37°C. The resulting solution was heated at a temperature greater than 95°C for 10 min to stop the enzymatic reaction and the solution was lyophilized and stored at -20°C for further analyses.