Peptic-Tryptic Digestion of Wheat Gliadin

Gliadin from wheat (Sigma) was digested, as described [18 (link)]. In detail, whole gliadin (1 g/mL) was firstly dissolved in 500 mL 0.2 N HCl for two hours at 37 °C with 1 g pepsin (Sigma). The resultant peptic digest was further digested by addition of 1 g trypsin (Sigma), after pH adjusted to 7.4 using 2 N NaOH; next, the solution was incubated at 37 °C for four hours with a vigorous agitation. Finally, the mixture was boiled to inactivate enzymes for 30 min and was stored at −20 °C (hereafter referred as PT-gliadin). Ovalbumin (Sigma) at 1 g/mL was used as an additional external protein for internalization and cell toxicity studies. PT-gliadin or Ovalbumin were then administered directly to the cells. For PT-gliadin Alexa Fluor 488 or 555 labeling, purification of unlabeled dyes was performed using affinity chromatography-purification G50 columns (GE Healthcare, Little Chalfont, UK). The resulted proteins were resuspended in PBS and quantified through Qubit Fluorometer (Invitrogen, Carlsbad, CA, USA). Then, PT-gliadin or Ovalbumin (100 µg each) proteins were labeled with Alexa Fluor 488 or Alexa Fluor 555 by specific Alexa Fluor Microscale labelling kits (ThermoFisher, Waltham, MA, USA), according to manufacturer’s instructions.