Research Resource: Identification of Novel Growth Hormone-Regulated Phosphorylation Sites by Quantitative Phosphoproteomics

Ray, Bridgette N.; Kweon, Hye Kyong; Argetsinger, Lawrence S.; Fingar, Diane C.; Andrews, Philip C.; Carter-Su, Christin

SILAC (reagents obtained from Invitrogen, Carlsbad, CA) was achieved by growing 3T3-F442A preadipocytes (cell stock from H. Green, Harvard Medical School, Boston, MA) in DMEM containing 5% dialyzed fetal bovine serum, 100 U/ml penicillin, 100 g/ml streptomycin, 0.25 g/ml amphotericin, and either [ 12 C]Lys and [ 12 C]Arg or [ 13 C]Lys and [ 13 C, 15 N]Arg for at least seven cell doublings. For the cells grown in [ 13 C]Lys and [ 13 C, 15 N]Arg, heavy isotope incorporation was evaluated by running a pilot digestion of the heavy isotope [ 13 C, 15 N]labeled cell extract followed by LC-MSMS analysis. The heavy isotope-labeled amino acids were incorporated into more than 99% of the cellular protein. Cells were washed and incubated in DMEM (SILAC) containing 1% BSA, 100 U/ml penicillin, 100 g/ml streptomycin, 0.25 g/ml amphotericin, and the appro-priate (either [ 12 C], [ 13 C], or [ 13 C / 15 N]) Lys and Arg for 15 h before treatment. Cells were treated with vehicle or GH (500 ng/ml) for the indicated times. Cells were then washed with PBSV (10 mM sodium phosphate, 150 mM NaCl, 1 mM Na 3 VO 4 , pH 7.5), harvested, and centrifuged. The cell pellet was frozen and stored at Ϫ80 C until use. Lysis buffer [50 mM Tris-HCl (pH 8.2) and 8 M urea containing protease inhibitors (Roche, Indianapolis, IN; Complete Mini, EDTA-free tablet) and phosphatase inhibitors (1 mM sodium fluoride, 1 mM sodium vanadate, 10 mM sodium pyrophosphate, 25 mM ␤-glycerophosphate)] was added, and the cells were lysed on ice by sonication [Fisher sonic dismembrator (Thermo-Fisher Scientific, Pittsburgh, PA), model 100, three 30-sec bursts, power setting of 5 watts]. The protein content of the lysates was measured using the Bradford protein assay (37) . Equal amounts of protein labeled with light isotope and heavy isotope were mixed, reduced, alkylated, and digested with TPCK-treated-trypsin (Worthington Biochemical Corp., Lakewood, NJ) at 37 C overnight.