Maize Protein Identification by nLC-nESI-MS/MS

The bands excised from the non-denaturing Deriphat-PAGE were individually analyzed by tandem mass spectrometry (nLC-nESI-MS/MS) using an 6520 Q-TOF mass spectrometer with HPLC Chip Cube source driven by 1200 series nano/capillary LC system (Agilent Technologies) as previously described (Prinsi and Espen, 2015 (link)). The spectra interpretation was performed by Spectrum Mill MS Proteomics Workbench (Rev B.04.00.127; Agilent Technologies). Cysteine carbamidomethylation and methionine oxidation were set as fixed and variable modifications, respectively, accepting two missed cleavages per peptide. The search was conducted against the subset of Zea mays protein sequences (ID tax: 4577, Oct 2015, 212069 entries) downloaded from the National Center for Biotechnology Information¹ and oncatenated with the reverse one. The threshold used for peptide identification was Spectrum Mill score ≥9, Score Peak Intensity ≥50%, mass MH+ Error ≤±10 ppm, Database Fwd-Rev Score ≥2, and Local False Discovery Rate ≤5%. Protein identification was accepted if confirmed by at least two distinct peptides.