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

e MSMS sequencing step has been determined to be approximately 60% for abundant precursor ions, but drops to about 10% for low-abundance ions (47) . In our typical screen, of the approximately 10,000 spectra we obtained, about 30% were identified. Fourth, the low expression levels of regulatory proteins and low stoichiometry of their phosphorylation further increases the difficulty of detecting phosphorylation events (76) . It is well known that compared with many growth factors, stimulation of cells with GH does not produce a very robust signaling response. In 3T3-F442A cells, the signaling response to GH is much less than for platelet-derived growth factor or epidermal growth factor (77) . Therefore, identifying GH-dependent phosphosites is likely to be particularly challenging. Finally, many phosphorylation sites are not identifiable either because the tryptic fragments are too large or too small for identification or because their sequence-specific properties lead to low efficiency ionization or fragmentation. However, based on the validation that we did with our immunoblotting studies, even those phosphopeptides that were detected in only one screen have a high likelihood of being valid and certainly provide sufficient evidence to warrant further investigation of all the identified phosphosites.