title: Enrichment of Phosphopeptides via Immobilized Metal Affinity Chromatography.
abstract: Immobilized metal affinity chromatography (IMAC) is a frequently used method for the enrichment of phosphorylated peptides from complex, cellular lysate-derived peptide mixtures. Here we outline an IMAC protocol that uses iron-chelated magnetic beads to selectively isolate phosphorylated peptides for mass spectrometry-based proteomic analysis. Under acidic conditions, negatively charged phosphoryl modifications preferentially bind to positively charged metal ions (e.g., Fe(3+), Ga(3+)) on the beads. After washing away nonphosphorylated peptides, a pH shift to basic conditions causes the elution of bound phosphopeptides from the metal ion. Under optimal conditions, very high specificity for phosphopeptides can be achieved.
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, Washington 98195.
authors.forename: Danielle L
authors.initials: DL
authors.lastname: Swaney
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, Washington 98195.
authors.forename: Judit
authors.initials: J
authors.lastname: Villén
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D008280:Magnetics; D008670:Metals / Q000378:metabolism*; D008863:Microspheres; D010748:Phosphopeptides / Q000302:isolation & purification* / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D008670:Metals; D010748:Phosphopeptides