biostudies-literatureHerring LENSFNIHNIGMS NIH HHS166-74https://www.ebi.ac.uk/biostudies/studies/S-EPMC4489695biostudies-literatureUnknown988Phosphorylation is an important post-translational modification that is involved in regulating many signaling pathways. Of particular interest are the growth factor mediated Ras and phosphoinositide 3-kinase (PI3K) signaling pathways which, if misregulated, can contribute to the progression of cancer. Phosphoproteomic methods have been developed to study regulation of signaling pathways; however, due to the low stoichiometry of phosphorylation, understanding these pathways is still a challenge. In this study, we have developed a multi-dimensional method incorporating electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) with tandem IMAC/TiO2 enrichment for subsequent phosphopeptide identification by LC/MS/MS. We applied this method to PDGF-stimulated NIH 3T3 cells to proviJournal of chromatography. B, Analytical technologies in the biomedical and life sciencesDevelopment of a tandem affinity phosphoproteomic method with motif selectivity and its application in analysis of signal transduction networks.PMC4489695DBI-1126244R01GM088987R01 GM088987Goshe MBGrant KGHaugh JMBlackburn KHerring LEfalseDevelopment of a tandem affinity phosphoproteomic method with motif selectivity and its application in analysis of signal transduction networks.Phosphorylation is an important post-translational modification that is involved in regulating many signaling pathways. Of particular interest are the growth factor mediated Ras and phosphoinositide 3-kinase (PI3K) signaling pathways which, if misregulated, can contribute to the progression of cancer. Phosphoproteomic methods have been developed to study regulation of signaling pathways; however, due to the low stoichiometry of phosphorylation, understanding these pathways is still a challenge. In this study, we have developed a multi-dimensional method incorporating electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) with tandem IMAC/TiO2 enrichment for subsequent phosphopeptide identification by LC/MS/MS. We applied this method to PDGF-stimulated NIH 3T3 cells to provi2015-01-01T00:00:00Z2015 Apr2025-04-04T02:42:59.486Z2019-03-27T01:54:24ZS-EPMC44896952577748010.1016/j.jchromb.2015.02.017