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.
