Cleavage of the 33-mer peptide in vitro

Cleavage of the 33-mer peptide of wheat α-gliadin (LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF, 3911 Da) was monitored using an AutoFLEX III MALDI-TOF mass spectrometer. The peptide (from GenScript) was dissolved in water to a concentration of ~20 mg/mL and stored at –20 °C. The cleavage reaction was carried out with ~1 mg/mL (~250 μM) substrate in 100 mM glycine pH 3.0 at 37 °C by adding 0.5 μM neprosin or 10 μM pepsin. Reactions were stopped at different time points (0 min, 10 min, 20 min, 45 min, 1 h and overnight) and samples were then diluted 1:10 with water, mixed with an equal volume of the 2,5-dihydroxybenzoic acid matrix at 10 mg/mL in a solution containing 30% acetonitrile and 70% 0.1% trifluoroacetic acid, and spotted on a ground steel plate (Bruker). Mass spectra were acquired in positive reflectron mode at 21 kV total acceleration voltage.