Overview of Phosphoproteomic Analysis
A sample large-scale phosphoproteomic analysis includes
- Cultured cells undergo SILAC encoding.
- Cells are stimulated with factor of interest (e.g. growth factor, hormone).
- Stimulation can occur for various lengths of time for temporal analysis.
- Cells are lysed and enzymatically digested.
- Peptides are separated using ion exchange chromatography.
- Phosphopeptides are enriched using phosphospecific antibodies, immobilized metal affinity chromatography or titanium dioxide (TiO2) chromatography.
- Phosphopeptides are analyzed using mass spectrometry.
- Peptides are sequenced and analyzed.
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