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

In our first experiment, 3T3-F442A cells were treated without or with GH for 15 min. In two subsequent experiments, 3T3-F442A cells were treated without or with GH for 5 min because we wanted to increase the probability that very rapidly phosphorylated phosphosites were detected. In these three trials, we identified 428, 721, and 1406 phosphorylation sites, respectively (Fig. 1B) . The increase in the number of phosphosites identified in each successive trial reflects, at least in part, an increase in the amount of cell lysate analyzed. In the first trial, 1.2 mg/ condition were analyzed, 2.2 mg/condition were analyzed in the second trial, whereas 3.0 mg/condition were analyzed in the third trial. Localization probabilities and posttranslational modification scores were calculated by MaxQuant software. Phosphorylation sites with localiza- tion probabilities above 0.75 and a posttranslational modification score difference (between highest scoring site and second highest) larger than 5.0 (46) were considered to be localized phosphorylation sites (i.e. the specific phosphorylated amino acid(s) within the peptide could be identified with high confidence). In phosphopeptides with unlocalized phosphorylation sites, there is some ambiguity in the exact amino acid(s) that is (are) phosphorylated.