Acetone

After quantification by the Bradford (Bio-Rad, United States) method, protein digestion was performed according to the cold acetone precipitation procedure. Briefly, 100 μg of protein from each sample was reduced. DTT was added to the protein extract to 20 mM. The extract was then kept at 37°C for 1 h. Iodoacetamide (IAM) (Sigma, United States) was then added to a final concentration of 100 mM. The mixture was then left for 30 min at room temperature in the dark. More than five volumes of acetone (Thermo Scientific, United States) precooled at −20°C was added. The mixture was left overnight at −20°C and centrifuged at 20,000 × g for 20 min. The supernatant was removed, and the resulting pellet was air-dried to remove the residual acetone and dissolved in 50 mM TEAB (Sigma, United States). Finally, trypsin (Promega, United States) was added at a 1:100 enzyme: substrate ratio. The mixture was incubated overnight at 37°C, and the resulting peptide was collected.

Tris, octylphenoxypolyethoxyethanol (NP-40), urea, sulfourea, 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), sodium dodecyl sulphate (SDS), acrylamide, N,N′-methylenebisacrylamide, ammonium persulfate, N,N,N′,N′-tetramethylethylene diamine, CBB G-250 and trichloroacetic acid were obtained from Amresco (Solon, OH, USA); phenylmethylsulfonyl fluoride was obtained from Sigma (St. Louis, MO, USA); immobilized pH gradient strips (IPG strips, 17 cm, pH 4–7) and iodoacetamide were obtained from Bio-Rad Laboratories (Hercules, CA, USA); acetone, glycerol, phosphoric acid, carbinol and alcohol (analytical reagents) were obtained from manufacturers in China. All water used in this experiment was Milli-Q hyperpure water (Millipore, Billerica, MA, USA).