Trypsin digestion, peptide extraction, and MALDI-TOF/TOF

For digestion of the proteins, the gel particles were incubated with 25 ng/L trypsin gold (Promega; Madison, WI, USA) in 25 mM NH₄HCO₃ at 37 °C overnight. The supernatants were recovered and stored at −   20 °C, whereas the gel particles were incubated a second time with 50 mM NH₄HCO₃ at 37 °C overnight and the supernatants were then stored at −   20 °C. Peptides were extracted in 50 μL trifluoroacetic acid 0.1%/acetonitrile (1:1) for 30 min at room temperature. All supernatants were mixed and dried in a vacuum centrifuge. Peptides were resuspended in trifluoroacetic acid and analyzed using a MALDI-TOF (MS/MS) Ultraflextreme mass spectrometer (Bruker; Billerica, MA, USA). An AnchorChip target and α-cyano-4-hydroxycinnamic acid (HCCA) matrix were used, according to the manufacturer’s instructions. The laser intensity was adjusted to 50% for the acquisition of masses, with three or four repetitions. For the analysis, spectra with 1 × 10³–1 × 10⁴ intensity peaks were considered, and the identity of the protein was matched using the Mascot search engine with the following parameters: enzyme, trypsin; missed cleavages, 1; fixed modification, carbamidomethyl C; variable modification, oxidation M; parent tolerance, 0.2 Da; fragment tolerance, 0.5 Da. Positive protein identifications were considered reliable with a Mascot score higher than 30.