title: Development of a tandem affinity phosphoproteomic method with motif selectivity and its application in analysis of signal transduction networks.
abstract: 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 provide over 11,000 unique phosphopeptide identifications. Upon motif analysis, IMAC was found to enrich for basophilic kinase substrates while the subsequent TiO2 step enriched for acidophilic kinase substrates, suggesting that both enrichment methods are necessary to capture the full complement of kinase substrates. Biological functions that were over-represented at each PDGF stimulation time point, together with the phosphorylation dynamics of several phosphopeptides containing known kinase phosphorylation sites, illustrate the feasibility of this approach in quantitative phosphoproteomic studies.
authors.affiliation: Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695-7622, United States.
authors.forename: Laura E
authors.initials: LE
authors.lastname: Herring
authors.affiliation: Gene Therapy Center, University of North Carolina-Chapel Hill, Chapel Hill, NC 27599-73522, United States.
authors.forename: Kyle G
authors.initials: KG
authors.lastname: Grant
authors.affiliation: Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695-7622, United States.
authors.forename: Kevin
authors.initials: K
authors.lastname: Blackburn
authors.affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695-7905, United States.
authors.forename: Jason M
authors.initials: JM
authors.lastname: Haugh
authors.affiliation: Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695-7622, United States. Electronic address: michael_goshe@ncsu.edu.
authors.forename: Michael B
authors.initials: MB
authors.lastname: Goshe
mesh_terms: D000595:Amino Acid Sequence; D002853:Chromatography, Liquid / Q000379:methods*; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction / Q000502:physiology*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D010748:Phosphopeptides
keywords: Liquid chromatography; Mass spectrometry; Motif analysis; Pathway analysis; Phosphopeptide enrichment