chronic exposure to tramadol induces oxidative damage, inflammation based on mice serum proteome

Peptides were labeled with TMT reagents based on the manufacturer’s instructions (Thermo Fisher Scientifific). To quantify six samples, each aliquot (100 µg of peptide equivalent) was reacted with one tube of TMT reagent. After the sample was dissolved in 100 µL of 0.05 M tetraethylammonium bromide (TEAB) solution, pH 8.5, the TMT reagent was dissolved in 41 µL of anhydrous acetonitrile. The mixture was incubated at room temperature for 1 hour.Then, 8 µL of 5% hydroxylamine was added to the sample and incubated for 15 min to quench the reaction. The 6-plex labeled samples were pooled together and lyophilized. The TMT-labeled peptides mixture was fractionated using a Waters XBridge BEH130 column (C18, 3.5 µm, 2.1 × 150 mm) on an Agilent 1290 high-performance liquid chromatography(HPLC) operating at 0.3 mL/min.