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

To expand our current view of GH-regulated proteins, we used a combined approach of SILAC, dual phospho-peptide enrichment using phosphoTyr antibodies, ZrO 2 , and LC-MSMS to quantify rapid GH-dependent changes in protein phosphorylation (Fig. 1A) . 3T3-F442A preadipocytes, a highly GH-sensitive cell line with a relatively high GH receptor number and robust GH activation of JAK2, were used as a model system. 3T3-F442A preadipocytes have previously been used to identify and/or characterize multiple GH signaling proteins and pathways, including JAK2, Stats 1, 3, 5a, and 5b (5, 40 -42) , Shc/ grb2/SOS/Ras/Raf/MEK/Erks1/2 (9, 43) , and insulin receptor substrate 1/2/PI3K/Akt (10, 11, 44, 45) . Lightlabeled control cells grown in normal growth medium and heavy-labeled cells grown in medium containing [ 13 C]Lys and [ 13 C, 15 N]Arg were deprived of serum overnight, treated with (heavy-labeled) or without (lightlabeled) GH for 5 or 15 min, and lysed. Equal (protein) amounts of cell lysates from GH-treated (ϩGH) and control (ϪGH) cell samples were mixed and digested with trypsin. Because of the low abundance of Tyr phosphorylation compared with Ser/Thr phosphorylation and its importance in post-GH receptor signaling pathways, Tyrphosphorylated tryptic peptides were isolated by immunoaffinity purification using phosphoTyr-specific antibodies. Peptides present in the flow through from the immunoaffinity purification were fractionated using strong cationic exchange (SCX) HPLC and then enriched for Ser and Thr phosphopeptides using a ZrO 2 column. The enriched phosphopeptides from the immunoaffinity purification/SCX/ZrO 2 -based purification were analyzed by LC-MSMS. The collected MSMS data were searched against a concatenated target-decoy database of IPI mouse database (version 3.63) using Mascot and processed with MaxQuant (version 1.0.13.13) (38) (www. maxquant.org) for identification and quantification of peptides and proteins. We set the threshold for a significant change at a significance B less than 0.05 (P value provided by MaxQuant) (38) . A false discovery rate of 1% was used for peptide and protein identification. B less than 0.05 corresponded to a GH-dependent increase of approximately 50% or decrease of about 20% (Supplemental Tables 3-5) .