ABSTRACT: Mass Spectrometry with theoretical database. Tryptic digests of the samples were solubilized in 0.1% (v/v) formic acid (solution A) and subjected to nano-ESI-LCMS/MS analysis, using an Ultimate 3000 HPLC (Dionex), coupled to a Q Exactive Orbitrap TM mass spectrometer (Thermo Fisher Scientific). Peptides were loaded on a Trap Column with nanoViper Fitting (P/N 164649, C18, 5 mm x 30 um, Thermo Fisher Scientific) and eluted at a flow rate of 300 nL/min using an isocratic gradient of 4% solution B (100% (v/v) acetonitrile containing 0.1% (v/v) formic acid) for 3 minutes. Thereafter, peptides were loaded on a C18 PicoChip column (Reprosil-Pur, C18-AQ, 3 um, 120 A, 105mm, New Objective, Woburn, MA, USA) using a segmented concentration gradient from 4-55% B for 30 min, 55-90% B for 1 min, 90% B over 5 min and then returning to 4% B over 20 min at a flow rate of 300 nL/min. Ion polarity was set to positive ionization mode using data-dependent acquisition (DDA) mode. Mass spectra were acquired with a scan range of m/z 200-2000, resolution of 70,000 and injection time of 100 ms. The fragmentation chamber was conditioned with collision energy between 29 to 35% with a resolution of 17,500, 50 ms of injection time, 4.0 m/z of isolation window and dynamic exclusion of 10s. The spectrometric data were acquired through the Thermo Xcalibur TM software v.4.0.27.19 (Thermo Fisher Scientific).
All Raw Data were submitted to the PatternLab software v.4.0.0.84 and searched against the uniprot and SLIDER theoretical database. The following search parameters were used: semi-tryptic cleavage products (two tryptic missed cleavage allowed), carbamidomethylation of cysteine as fixed modification and oxidation of methionine as variable modification. Parent mass tolerance error was set at 40 ppm and fragment mass error at 0.02 ppm. Protein identifications were considered with a minimum of one fragment ion per peptide, five fragment ions per protein, two peptides per protein and a false discovery identification rate set to 1%, estimated by a simultaneous search against a reversed database.
Rafael Junqueira Borges 
