Comparison of three quantitative phosphoproteomic strategies to study receptor tyrosine kinase signaling.
There are three quantitative phosphoproteomic strategies most commonly used to study receptor tyrosine kinase (RTK) signaling. These strategies quantify changes in: (1) all three forms of phosphosites (phosphoserine, phosphothreonine and phosphotyrosine) following enrichment of phosphopeptides by titanium dioxide or immobilized metal affinity chromatography; (2) phosphotyrosine sites following anti- phosphotyrosine antibody enrichment of phosphotyrosine peptides; or (3) phosphotyrosine proteins and their binding partners following anti-phosphotyrosine protein immunoprecipitation. However, it is not clear from literature which strategy is more effective. In this study, we assessed the utility of these three phosphoproteomic strategies in RTK signaling studies by using EphB receptor signaling as an example. We used all three strategies with stable isotope labeling with amino acids in cell culture (SILAC) to compare changes in phosphoproteomes upon EphB receptor activation. We used bioinformatic analysis to compare results from the three analyses. Our results show that the three strategies provide complementary information about RTK pathways.
Kimmel Center for Biology and Medicine at the Skirball Institute, New York University School of Medicine, New York, New York 10016, United States.
Guoan
G
Zhang
Thomas A
TA
Neubert
D000818:Animals; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity / Q000379:methods; D019295:Computational Biology; D036387:Ephrin-B1 / Q000378:metabolism / Q000494:pharmacology; D047468:Immunoprecipitation; D007553:Isotope Labeling / Q000379:methods; D051379:Mice; D010750:Phosphoproteins / Q000032:analysis / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D019000:Phosphotyrosine / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods; D060066:Protein Interaction Maps; D040901:Proteomics / Q000379:methods*; D051381:Rats; D036081:Receptors, Eph Family / Q000378:metabolism*; D015398:Signal Transduction; D014025:Titanium / Q000378:metabolism
C092571:Efnb1 protein, mouse; D036387:Ephrin-B1; D010750:Phosphoproteins; D010769:Phosphothreonine; C009495:titanium dioxide; D010768:Phosphoserine; D019000:Phosphotyrosine; D014025:Titanium; D036081:Receptors, Eph Family