Proteome analysis of mouse liver at ad libitum feeding

The liver samples were collected from ad libitum feeding and Lys(6)-SILAC (Silantes, München, Germany) 10-week-old wild-type and ob/ob mice. Tissues were lysed with 0.5 mL of a solution containing 50 mM Tris-HCl pH8.8, 2% sodium dodecyl sulfate (SDS), and 7 M urea, and then homogenized by ultrasonication using a Bioruptor (Digaenode, Denville, NJ, USA). After dilution with water, the samples were centrifuged at 15,000 g for 15 min at 4°C to exclude the insoluble fractions. By calibrating to 1 mg/mL, bicinchoninic acid (BCA) assay (Thermo Fisher Scientific) was used to quantify the protein concentration of the lysates. Then, 50 µg of non-labeled and labeled protein extracts were combined in another tube for peptide preparation. We blocked cysteine residues using 2 mM Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) (Thermo Fisher Scientific) at 37 °C for 30 min followed by alkylation with 10 mM 2-iodoacetamide (IAA) at room temperature for 30 min. By ultrasonically treating the pellet three times for 30 s with intervals of 30 s with a Bioruptor (Diagenode), the proteins precipitated with acetone for 3 h at −30 °C were dispersed in 50 mM triethylammonium bicarbonate. Lysyl endopeptidase (Wako, Osaka, Japan) was added to the protein suspension and digested at 37 °C for 16 h. Prior to MS analysis, the resulting peptides were purified using C18-StageTip purification after centrifuging at 15,000 g for 15 min at 4°C. Before MS analysis, peptides were resolved in 50 µL 3% ACN-0.1% foromic acid.