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

The current study is the first to use SILAC and quantitative MS-based phosphoproteomics to study GH signaling. To our knowledge, it is also the first study to use SILAC and quantitative MS-based phosphoproteomics to investigate ligand-dependent signaling via any member of the cytokine receptor family. This approach allowed us to identify and quantify GH-dependent changes in more than 200 protein phosphorylation sites. Among the seven GH-stimulated sites identified by phosphoproteomics that were the focus of the validation portion of our anal-ysis, the GH response determined by phosphoproteomics varied from robust to subtle. However, immunoblotting confirmed all seven sites tested, even those with the most subtle increases, including four that were picked up in only one of the three screens, supporting our contention that for the GH up-regulated phosphosites, identification in even a single screen makes that site likely to be a bona fide GH-regulated site. Clearly, complete coverage of the phosphoproteome was not obtained in any of our screens, as indicated by the absence of known GH-dependent phosphosites (e.g. in Akt, GH receptor, JAK2, SH2B1, Raf1, MEK1/2) (13, 45, 69 -72) and the fact that many of the phosphosites were identified in only one of our three screens. We did not expect complete coverage for multiple reasons. First, a certain amount of variability originates in the multistep preparation that precedes the analysis by MS. Second, ZrO 2 selects for a particular subpopulation of phosphopeptides in the phosphoproteome. Studies using yeast revealed that different modes of enriching for phosphopeptides sample quite different subpopulations of the phosphoproteome with little overlap among them. Use of four different enrichment methods significantly increased the coverage of the phosphopeptide population (Kweon and Andrews, manuscript in preparation), suggesting that future studies would benefit from combining the results from multiple, complementary enrichment schemes. Third, in the modern LC-mass spectrometer, for each successive peak fraction selected by the nano LC system for analysis, two steps are performed: first, the LTQ-Orbitrap mass spectrometer obtains a single MS survey scan in the Orbitrap analyzer, then for the seven most intense precursor ions from each MS survey scan, sequential MSMS scans are obtained in the LTQ to determine their peptide sequence. Due to the highly complex samples obtained from the cell lysates and the limited sequencing speed at the MSMS sequencing step, modern mass spectrometers are only capable of sequencing approximately 16% of the precursor ions present (47) . Thus, the relative abundance of a phosphopeptide greatly influences the probability that the ion derived from a particular phosphopeptide will be selected for MSMS sequencing. It is not surprising that in analytical replicates, and certainly with biological replicates, the ions from less abundant phosphopeptides might not be consistently selected due to slight changes in chromatography at either the SCX, ZrO 2 , or LC-MS steps (73) (74) (75) . As evidence of variation in selecting different precursor ions for sequencing, in our study, the inclusion of a technical replicate increased the number of identified peptides by about 20% on average. Furthermore, for low-abundance precursor ions, the quality of the MSMS spectra deteriorates, thus making it more difficult to obtain protein sequence. The fraction of identified peptides following th