Tissue samples from individual rats were homogenized in 1 M urea and 10 mM DTT. The resulting protein extracts were reduced and alkylated by volatile reagents triethyphosphine and iodoethanol, as previously described (Hale et al., 2004); the protein extract was then digested with trypsin. The volatile reduction and alkylation steps allow all sample preparation steps to be carried out in one tube, which minimizes variations in sample preparation. The total peptide concentration was determined before and after Trypsin digestion with the Bradford Protein Assay (Bradford 1976). Values were similar for both assays. The peptide determination after digestion was done to ensure similar amounts of each sample were injected onto the column.
