Iodine acetamide

Add Bond-Breaker™ TCEP Solution (TCEP) reductant (ThermoFisher Scientific, USA) at a final concentration of 10 mmol/L and react for 60 min at 37°C. Add iodine acetamide (Sigma, Germany) at a final concentration of 40 mmol/L and react for 40 min at room temperature, protected from light acetamide (Sigma, Germany) at room temperature for 40 min. Add pre-chilled acetone (acetone: sample volume = 6:1) to each tube and precipitate for 4 h at −20°C. Centrifuge the precipitate for 20 min at 10,000 × g using a Centrifuge 5430R (Eppendorf, Germany). The samples were fully dissolved with 50 mmol/L Triethylammonium bicarbonate buffer (TEAB, Sigma, Germany), and Trypsin was added at a mass ratio of 1:50 (enzyme: protein) and digested overnight at 37°C. The samples were labeled with TMT and mixed, and the TMT reagent was removed at −20°C (ThermoFisher Scientific, USA) to room temperature, add acetonitrile (ThermoFisher Scientific, USA), vortex centrifuge and add one tube of TMT reagent per 100 μg of peptide (TMT10-126 labeling, 127N labeling A2, 127C labeling A3, and 128N labeling B1). Incubate for 2 h at room temperature; add hydroxylamine, react for 15 min at room temperature, mix equal amounts of labeled products in one tube and evacuate using LNG-T88 vacuum concentrator (Taicang Huamei Biochemical Instrument Factory, China).