Protein identification by mass spectrometry

Spots from 2D-DIGE were triturated, washed with water, in-gel reduced with DTT, S-alkylated with iodoacetamide, and then digested with trypsin. Resulting peptide mixtures were desalted by μZip-TipC₁₈ using 50% v/v acetonitrile, 5% v/v formic acid as eluent. Recovered peptides were then analyzed for protein identification by nLC-ESI-LIT-MS/MS, using an LTQ XL mass spectrometer (Thermo Fisher Scientific, USA) equipped with a Proxeon nanospray source connected to an Easy-nanoLC (Proxeon, Denmark) [35]. Peptides were resolved on an Easy C18 column (100 mm × 0.075 mm, 3 μm) (Proxeon). Mobile phases were 0.1% v/v formic acid (solvent A) and 0.1% v/v formic acid in acetonitrile (solvent B), running at a total flow rate of 300 nL/min. Linear gradient was initiated 20 min after sample loading; solvent B ramped from 5 to 35% over 45 min, from 35 to 60% over 10 min, and from 60 to 95% over 20 min. Spectra were acquired in the range m/z 400–2000. Each peptide mixture was analyzed under collision-induced dissociation (CID)-MS/MS data-dependent product ion scanning procedure, enabling dynamic exclusion (repeat count 1 and exclusion duration 60 s), over the three most abundant ions. Mass isolation window and collision energy were set to m/z 3 and 35%, respectively.

Raw data from nLC-ESI-LIT-MS/MS analysis were searched by MASCOT search engine (version 2.2.06, Matrix Science, UK) against an updated (2014/05/06) NCBI non-redundant database (taxonomy Solanum lycopersicum) in order to identify proteins from gel spots. Database searching was performed by using Cys carbamidomethylation and Met oxidation as fixed and variable protein modifications, respectively, a mass tolerance value of 1.8 Da for precursor ion and 0.8 Da for MS/MS fragments, trypsin as proteolytic enzyme, and a missed cleavage maximum value of 2. Other MASCOT parameters were kept as default. Protein candidates assigned on the basis of at least two sequenced peptides with an individual peptide expectation value <0.05 (corresponding to a confidence level for peptide identification >95%) were considered confidently identified. Definitive peptide assignment was always associated with manual spectra visualization and verification.