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Published on: February 21, 2019
Global Identification of ERK Substrates by Phosphoproteomics Based on IMAC and 2D-DIGE
1Division 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.
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.
Insights
This study presents a novel phosphoproteomic method to identify novel extracellular signal-regulated kinase (ERK) substrates. The technique combines immobilized metal affinity chromatography (IMAC) and 2D-DIGE for comprehensive ERK substrate discovery.
Area of Science:
- Cellular signaling pathways
- Molecular biology
- Proteomics
Background:
- Extracellular signal-regulated kinase (ERK) is a key regulator of cellular functions.
- ERK exerts its effects through phosphorylation of downstream substrates.
- Many ERK substrates remain uncharacterized, necessitating advanced identification methods.
Purpose of the Study:
- To develop and describe a comprehensive phosphoproteomic method for global identification of ERK substrates.
- To characterize novel protein substrates regulated by ERK-mediated phosphorylation.
Main Methods:
- Combined immobilized metal affinity chromatography (IMAC) for phosphoprotein enrichment.
- Utilized two-dimensional difference gel electrophoresis (2D-DIGE) to detect differentially phosphorylated proteins.
- Employed liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for protein identification.
Main Results:
- The IMAC-2D-DIGE-MS/MS approach successfully enriched and identified numerous ERK substrates.
- Differential protein expression was observed between ERK-inhibited and ERK-activated cellular states.
- The method demonstrated high sensitivity and resolution for identifying novel phosphorylated proteins.
Conclusions:
- The described phosphoproteomic strategy is effective for large-scale ERK substrate discovery.
- This method facilitates the identification of previously unknown components of ERK signaling pathways.
- Further validation, such as Phos-tag Western blotting, is crucial for functional characterization of identified substrates.
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