Thiourea

Acrylamide, N,N-methylene bisacrylamide, glycine and tris base were purchased from Jintai Hongda Biotechnology Co., Ltd. (Beijing, China). 2-Thiobarbituric acid, dithiothreitol, thiourea, Coomassie Brilliant Blue R-250, sodium dodecyl sulphate (SDS), bromophenol blue and 2,4-dinitrophenylhydrazine were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). N,N,N,N-tetramethylethylenediamine and β-mercaptoethanol were acquired from Macleans Biochemical Technology Co., Ltd. (Shanghai, China). Coomassie Brilliant Blue kit was provided by Nanjing Jiancheng Institute of Bioengineering (Jiangsu, China). 1,1,3,3-Tetraethoxypropane and chromatography grade formic acid were supplied by Sigma-Aldrich Co., Ltd. (MO, USA). Mass spectrometry-grade trypsin, acetonitrile and water were obtained from Thermo Fisher Scientific (MA, USA). All other chemical reagents were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

The iridium complexes 1 [21 (link)], 2 [22 (link)] and 3 [23 (link)] were synthesized according to the published procedures. ¹H-NMR spectra were recorded on a Bruker Advance (400 MHz) (Bruker, Karlsruhe, Germany) at ambient temperature, and were consistent with the respective reported literature. Complexs 1, 2 and 3 were solubilized in dimethyl sulfoxide (DMSO). 2,2′-bipyridyl and thiourea were purchased from Sangon Biotech Co. (Shanghai, China), and norfloxacin was purchased from Sigma-Aldrich (St. Louis, MO, USA).

The liver tissue from each mouse was manually homogenized into powder in liquid nitrogen using a mortar and pestle. Samples with equal weight tissue (200 mg) from each group were pooled into three fractions. The powder was mixed with lysis buffer (8 M urea (Sangon Biotech Co., Ltd., Shanghai, China), 2 M thiourea (Sangon Biotech Co., Ltd., Shanghai, China), 4% CHAPS (Sangon Biotech Co., Ltd., Shanghai, China), 20 mM Tris-base (Sangon Biotech Co., Ltd., Shanghai, China), 30 mM dithiothreitol (Sangon Biotech Co., Ltd., Shanghai, China)) and periodically vortexed in ice for 30 min and then centrifuged at 10,000× g for 15 min at 4 °C. Then protein pellets were dissolved with 100 µL of 5 M urea and then mixed with four volumes of 40 mM NH₄HCO₃ (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) solution. The protein concentration was determined using a bicinchoninic acid (BCA) (Beyotime Biotechnology, Shanghai, China) assay. The protein samples were reduced with dithiothreitol solution (final concentration 10 mM) and then alkalized with iodoacetamide (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) solution (final concentration: 50 mM). Subsequently, protein samples were digested with sequencing-grade modified trypsin (Beyotime Biotechnology, Shanghai, China) at 37 °C overnight (protein:trypsin = 40:1) [18 (link)].