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

Of the 1790 phosphorylation sites, we confidently localized 294, 500, and 869 phosphorylated residues on 240, 394, and 769 phosphopeptides in the 15-min and two 5-min trials, respectively. When the results of the three screens were combined, a total of 230 unique GH-dependent phosphosites (localized and unlocalized) on 141 different proteins were observed; 132 sites exhibited statistically significant GH-dependent increases (Table 1 and Supplemental Table 1 ) whereas the phosphorylation at 96 sites decreased by more than 30% (Supplemental Table 2 ). Ninety seven proteins had one GH-regulated phosphosite (68 increase, 29 decrease), 21 proteins had more than one site that showed increased phosphorylation, 17 had more than one site that showed decreased phosphorylation, and six had a combination of sites showing increased and decreased phosphorylation. When the phosphorylation ratios (ϩGH/ϪGH) for the 327 phosphorylation sites present in both 5-min trials were compared, the ratios were very similar, with a correlation coefficient of 0.8766 (r 2 ϭ 76.84%) (Supplemental Tables 1-4 ). The consistency of the phosphorylation ratios for the sites present in both 5-min trials validates the SILAC results and underscores the fact that the relative peptide levels encoded in the isotopically labeled samples are relatively unaffected by variability introduced during sample preparation (SCX-fractionation, phosphopeptide enrichment, and multiple desalting steps) that precedes the analysis by MS.