Summary
Protein phosphorylation is central to most signaling events in eukaryotic cells. Large-scale analysis of protein phosphorylation in vivo is a highly challenging undertaking that requires powerful analytical and bioinformatics tools; numerous phosphoproteomic methodologies that use various combinations of these tools have been developed recently. This chapter describes an in-gel isoelectric focusing–liquid chromatography–tandem mass spectrometry (IEF–LC–MS/MS) analytical strategy for phosphoproteome mapping. The strategy encompasses seven steps: (1) extraction of proteins from the biological system under study (e.g., a tissue); (2) separation of the protein mixture by isoelectric focusing in an immobilized pH gradient (IPG) strip; (3) protein fixation followed by sectioning of the IPG strip; (4) digestion of the proteins in each gel section; (5) enrichment of phosphopeptides in the digests by immobilized metal ion affinity chromatography; (6) analysis of the enriched digests by LC–MS/MS; and (7) identification of the phosphopeptides/proteins through database searches, and assignment of the sites of phosphorylation in these proteins.
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Beranova-Giorgianni, S., Desiderio, D.M., Giorgianni, F. (2009). Phosphoproteome Analysis by In-Gel Isoelectric Focusing and Tandem Mass Spectrometry. In: Tyther, R., Sheehan, D. (eds) Two-Dimensional Electrophoresis Protocols. Methods in Molecular Biology, vol 519. Humana Press. https://doi.org/10.1007/978-1-59745-281-6_25
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DOI: https://doi.org/10.1007/978-1-59745-281-6_25
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