2.8. Sample preparation for LC-MS/MS
The liver was homogenized in lysis buffer containing 50 mM NH4HCO3, 8 M urea, 4% deoxycholic acid, 1% phosphatase inhibitor cocktail 2 (SIGMA, St. Louis, MO), 1% phosphatase inhibitor cocktail 3 (SIGMA, St. Louis, MO), and protease inhibitor cocktail (Roche, Penzberg, Germany). Lysate was obtained after centrifugation at 15000 rpm for 10 min at 4°C and precipitation with 4× volume of cold acetone, then reconstituted with an appropriate volume of lysis buffer. A total of 110 μg of proteins extracted from each liver sample was reduced with dithiothreitol (10 mM final concentration) and alkylated with iodoacetamide (25 mM final concentration). After dilution with 3× volume of 50 mM NH4HCO3, the proteins were digested with trypsin (protein-to-enzyme ratio of 20:1) (Promega) for 18 h at 37°C. The protein digests were desalted using C18 StageTips with C18 Empore disks (3 M, St. Paul, MN, USA) following the removal of SDC (Masuda, Tomita & Ishihama, 2008; Rappsilber, Mann & Ishihama, 2007). The eluted peptides were used after being completely lyophilized in a vacuum concentrator. For phosphopeptide enrichment, we used Titansphere Phos-TiO beads (GL Sciences, Tokyo, Japan) according to the manufacturer’s protocol. Enriched phosphopeptides were desalted using C18 StageTips and lyophilized in a vacuum concentrator.