Digested individual samples from the 6 groups were randomized for analysis to minimize technical artifacts. For each sample, 20 μg of the tryptic peptides were injected onto a C18 microbore column (i.d. = 1 mm; length = 5 cm). Peptides were eluted with a linear gradient from 5 to 45% acetonitrile over 120 min at a flow rate of 50 μl/min; the effluent was electro-sprayed into a LTQ linear ion-trap mass spectrometer (MS; Thermo-Finnigan).
