Protein bands observed in SDS-PAGE were carefully excised and
subjected to in-gel trypsin digestion according to Shevchenko and
colleagues (Shevchenko et al., 2006). Nano-LC MS/MS analyses were
performed as already described (Pallotta et al., 2013). Data were
searched against the National Center for Biotechnology Information
nonredundant database (NCBInr, Version 20150127) using computer
software (Mascot, in-house version 2.2, Matrix Science, London,
UK) with the following constraints: taxonomy ¼ Viridiplantae;
enzyme ¼ trypsin; missed cleavage ¼ 1; peptide and fragment mass
tolerance ¼ ±0.3 Da; fixed modifications ¼ carbamidomethyl (Cys);
variable modifications ¼ Oxidation (Met); Gln- > pyro-Glu (N-term
Q), Glu- > pyro-Glu (N-term E). For positive identification, the score
of the result of [e10 Log(P)] had to be over the significance
threshold level (p < 0.05).