2.3. Brain Tissue Homogenization and Digestion

The brains from morphine-treated (MOR; n = 9) and control (CON; n = 9) rats were rapidly removed, dissected and snap frozen in liquid nitrogen and stored at −80 °C until use. In order to obtain a reasonable number of samples for proteomic analyses and, simultaneously, to preserve the effect of the intrinsic biological variation, three pooled samples of prefrontal cortex (Ctx), hippocampus (Hp), striatum (Str) or cerebellum (Cb) were prepared for both MOR and CON groups by mixing equal amounts of respective brain tissues from three trios of randomly selected animals in each group. The pooled brain tissue samples were homogenized in 10 volumes of TMES buffer (20 mM Tris, 3 mM MgCl₂, 1 mM EDTA, 250 mM sucrose; pH 7.4) containing protease and phosphatase inhibitors (cOmplete and PhosSTOP) using a glass-Teflon homogenizer (1200 rpm, 10 strokes), mixed 1:1 with 2% SDC (sodium deoxycholate) in 100 mM TEAB (triethylammonium bicarbonate; pH 8.0), and sonicated for 3 × 10 s in 2.0 mL Eppendorf tubes using a Bandelin UW 2070 sonicator (40% amplitude), as described previously [46]. Samples were cleared (14,000× g, 10 min). Protein concentration was determined using the Pierce BCA protein assay kit with bovine serum albumin as calibration standard and 250 µg of protein per sample were used for MS sample preparation. Proteins were digested by 5 µg of trypsin per sample at 37 °C overnight. Phosphopeptides were enriched using TiO₂ according to [47].