Spots were manually excised from the gel and subjected to in-gel tryptic digestion.
The protein spots of interest were destained, reduced, alkalized, and then digested overnight with trypsin (Promega Corporation, Madison, WI).
The tryptic peptides were recovered and cleaned using the C-18 ZipTip system (Millipore, Billerica, MA) and eluted with 5 L of 50% ACN containing 0.2% formic acid.
The protonated peptides were analyzed using a Waters Micromass Q-TOF Ultima (Waters, Mississauga, ON) with nano-spray injection as the sample delivery method.
Peak lists were generated and processed using MassLynx software version 3.5 (Waters, Mississauga, ON).
The resulting .dta files from each analysis were analyzed by the PEAKS software 3.1 (Bioinformatics Solutions Inc., Waterloo, ON), which combines auto de novo sequencing and database searching (MSDB was downloaded on February 28th, 2007).
The parental and fragment mass error tolerances were set to 0.2 Da and 0.1 Da, respectively.
One missed cleavage site was allowed with the trypsin digestion and carbamidomethylation and methionine oxidation were set as the fixed and variable modifications, respectively.
The identification was further confirmed using MASCOT MS/MS ion search and/or peptide-fingerprinting algorithm.
The same database, enzyme, PTM, missed cleavage sites, and peptide tolerance were used as with the PEAKS software.
An identification score had to be less than the threshold used in MASCOT searches (p < 0.05) to be retained.
Spot identities were further verified by matching calculated pI and molecular mass to values estimated on the 2-D gel.
All identified proteins were confirmed to contain at least two specific and non-redundant peptides.