title: Global Identification of ERK Substrates by Phosphoproteomics Based on IMAC and 2D-DIGE.
abstract: Extracellular signal-regulated kinase (ERK) regulates various cellular functions through phosphorylation of numerous downstream substrates, which have not yet been fully characterized. To date, several phosphoproteomic approaches have been employed to identify novel substrates for ERK. In this chapter, we describe a method to globally identify ERK substrates by combining immobilized metal affinity chromatography (IMAC) and two-dimensional difference gel electrophoresis (2D-DIGE) followed by mass spectrometry. Phosphoprotein enrichment by IMAC enables the subsequent detection of many protein spots with different fluorescence intensities between ERK-inhibited and -activated cells in 2D-DIGE analysis. Furthermore, the advanced sensitivity and resolution of liquid chromatography coupled with tandem mass spectrometry allow for a direct identification of proteins obtained from silver-stained 2D-DIGE gels. Validation experiments such as Phos-tag Western blotting are important steps to further elucidate the functional roles of ERK-mediated phosphorylation of these newly identified substrates.
authors.affiliation: Division of Cell Signaling, Fujii Memorial Institute of Medical Sciences, Tokushima University, 3-18-15 Kuramoto-cho, Tokushima, 770-8503, Japan. kosako@tokushima-u.ac.jp.
authors.forename: Hidetaka
authors.initials: H
authors.lastname: Kosako
authors.affiliation: Division of Cell Signaling, Fujii Memorial Institute of Medical Sciences, Tokushima University, 3-18-15 Kuramoto-cho, Tokushima, 770-8503, Japan.
authors.forename: Kou
authors.initials: K
authors.lastname: Motani
mesh_terms: D000818:Animals; D002352:Carrier Proteins; D002460:Cell Line; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D051379:Mice; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D020543:Proteome; D040901:Proteomics / Q000379:methods; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
chemical_list: D002352:Carrier Proteins; D008024:Ligands; D010750:Phosphoproteins; D020543:Proteome; D048049:Extracellular Signal-Regulated MAP Kinases
keywords: 2D-DIGE; ERK; Immobilized metal affinity chromatography; LC-MS/MS; MAP kinase; Phos-tag; Phosphoproteomics; Phosphorylation; Proteomics