Proteome analysis of mouse liver and skeletal muscle

The liver and skeletal muscle were collected from 10-week-old wild-type and ob/ob mice fasted for 16 hours. Tissues were frozen immediately and lysed with 0.5 mL of a solution containing 50 mM Tris-HCl pH8.8, 2%SDS, and 7 M urea, and then subjected to ultrasonic treatment with a Bioruptor (Digaenode). The samples were diluted with an equal volume of water and centrifuged at 15,000 g for 15 min at 4°C to remove insoluble fraction. The protein concentration of the lysates was determined with the bicinchoninic acid assay (Thermo Fisher Scientific) and adjusted to 1 mg/mL. Cysteine residues were blocked by incubation of the samples with 2 mM tris(2-carboxyethyl)phosphine hydrochloride (Thermo Fisher Scientific) for 30 min at 37 °C followed by alkylation with 10 mM 2-iodoacetamide for 30 min at room temperature. The proteins (200 µg) were precipitated with acetone for 3 h at −30 °C and the resulting pellet was dispersed in 50 mM Triethylammonium bicarbonate by ultrasonic treatment (three times for 30 s with intervals of 30 s) with a Bioruptor (Diagenode). The protein suspension was subjected to digestion with lysyl endopeptidase (Wako) for 16 h at 37 °C. Resulting peptides were centrifuged at 15,000 g for 15 min at 4°C and subjected to C18-StageTip purification prior to MS analysis