Triethylammonium bicarbonate buffer teab

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).

Urea, sodium phosphate dibasic dihydrate, sodium phosphate monobasic dihydrate, 1,4-Dithiothreitol (DTT), iodoacetamide, N-ethylmaleimide, borane pyridine complex, 4-Vinylpyridine, triethylammonium bicarbonate buffer (TEAB), 3-buten-2-one (MVK), and formic acid (FA) were all purchased from Sigma-Aldrich. Trifluoroacetic acid (TFA) (Acros Organics), acetic acid (Chemsolute), DNPH (Tokyo Chemical Industry Co., Ltd.), dimethyl sulfoxide (DMSO) (Thermo Scientific), and acetonitrile (ACN) (Chemsolute).

Ketamine (Ceva, Paulínia, SP, Brazil), xylazine (Syntec, Tamboré, SP, Brazil), flunixin (Ucb/Vet, Jaboticabal, SP, Brazil) and enrofloxacin (Venco, Londrina, PR, Brazil) are specifically for veterinary use. Nicotine-free base, dithiothreitol (DTT), iodoacetamide (IAA), formic acid, sodium deoxycholate and triethylammonium bicarbonate buffer (TEAB) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Phencyclidine was purchased from Alomone Labs (Jerusalem, Israel). Osmotic minipumps came from Alzet (Cupertino, CA, USA). Promega Biotecnologia do Brasil (São Paulo, SP, Brazil) was the source of trypsin, and Thermo Fischer Scientific (Waltham, MA, USA) was the source of the resin column model POROS 20 R2 and Qubit protein assay kit. The nanoEase Symmetry C18 TRAP column (5 µm, 180 µm × 20 mm), the nanoAcquity BEH130 C18 reversed-phase column (1.7 µm, 75 µm × 100 mm), [Glu1]-Fibrinopeptide B human (GFP) and Total Recovery vials were purchased from Waters Corp (Milford, MA, USA). Acetonitrile came from Merck HGaA (Darmstadt, Germany).