Phosphoproteome analysis by in-gel isoelectric focusing and tandem mass spectrometry.
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.
Beranova-Giorgianni
Sarka
S
Department of Pharmaceutical Sciences, The University of Tennessee Health Science Center, 5P Crowe Bldg., 874 Union Avenue, Memphis, TN 38163, USA.
Desiderio
Dominic M
DM
Giorgianni
Francesco
F
D002845:Chromatography / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000379:methods*; D010748:Phosphopeptides / Q000032:analysis*; D010766:Phosphorylation; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*
D010748:Phosphopeptides; D020543:Proteome