Protein digestion was carried out following the protocol described by Wisniewski et al24 and the resulting peptide mixture was labeled using the 8-plex iTRAQ reagent according to the manufacturer's instructions (Applied Biosystems). Briefly, a total 200 μg protein/sample prepared in 30 μL STD buffer was diluted in 200 μL of UA buffer (800 mM urea, 150 mM Tris-HCI pH 8.0). Detergent, DTT and other low-molecular-weight components were removed using UA buffer by repeated ultrafiltration using Microcon 30 (30 kD MW cutoff). Then 100 μL of UA buffer (contain 50 mM iodoacetamide) was added to each filtrate, followed by 20 min incubation in the dark. After 10 min of centrifugation under the above conditions, the filters were washed three times with 100 μL UA buffer. Then, 100 μL of DS buffer (50 mM triethylammonium bicarbonate, pH 8.5) was added to the filters and centrifuged twice at 14000 g, each for 10 min. Finally 2 μg trypsin (Promega) in 40 μL of DS buffer was added to each filtrate and incubated overnight at 37°C. The resulting peptides were harvested by centrifugation. The peptide content was measured by UV light spectral density at 280 nm using an extinction coefficient of l.1 in 0.l% (g/L) solution which was calculated based on the frequency of tryptophan and tyrosine.