Development of a tandem affinity phosphoproteomic method with motif selectivity and its application in analysis of signal transduction networks.
Development of a tandem affinity phosphoproteomic method with motif selectivity and its application in analysis of signal transduction networks.
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DOI：
10.1016/j.jchromb.2015.02.017
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发表时间：
2015-04-15
影响因子：
3
通讯作者：
Goshe, Michael B.
中科院分区：
文献类型：
--
作者：
Herring, Laura E.;Grant, Kyle G.;Blackburn, Kevin;Haugh, Jason M.;Goshe, Michael B.
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.
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影响因子：
7.3
作者：
Alexander J;Lim D;Joughin BA;Hegemann B;Hutchins JR;Ehrenberger T;Ivins F;Sessa F;Hudecz O;Nigg EA;Fry AM;Musacchio A;Stukenberg PT;Mechtler K;Peters JM;Smerdon SJ;Yaffe MB
通讯作者：
Yaffe MB
影响因子：
4.4
作者：
Chien, Ko-yi;Liu, Hsiao-Ching;Goshe, Michael B.
通讯作者：
Goshe, Michael B.
影响因子：
7.4
作者：
Alpert, Andrew J.
通讯作者：
Alpert, Andrew J.
影响因子：
7
作者：
Larsen, MR;Thingholm, TE;Jorgensen, TJD
通讯作者：
Jorgensen, TJD
影响因子：
46.9
作者：
Ficarro, SB;McCleland, ML;White, FM
通讯作者：
White, FM