title: Reproducible isolation of distinct, overlapping segments of the phosphoproteome.
abstract: The ability to routinely analyze and quantitatively measure changes in protein phosphorylation on a proteome-wide scale is essential for biological and clinical research. We assessed the ability of three common phosphopeptide isolation methods (phosphoramidate chemistry (PAC), immobilized metal affinity chromatography (IMAC) and titanium dioxide) to reproducibly, specifically and comprehensively isolate phosphopeptides from complex mixtures. Phosphopeptides were isolated from aliquots of a tryptic digest of the cytosolic fraction of Drosophila melanogaster Kc167 cells and analyzed by liquid chromatography-electrospray ionization tandem mass spectrometry. Each method reproducibly isolated phosphopeptides. The methods, however, differed in their specificity of isolation and, notably, in the set of phosphopeptides isolated. The results suggest that the three methods detect different, partially overlapping segments of the phosphoproteome and that, at present, no single method is sufficient for a comprehensive phosphoproteome analysis.
authors.affiliation: Institute for Molecular Systems Biology, Swiss Federal Institute of Technology, ETH Hönggerberg, Wolfgang-Pauli-Str. 16, HPT, CH-8093 Zürich, Switzerland.
authors.forename: Bernd
authors.initials: B
authors.lastname: Bodenmiller
authors.forename: Lukas N
authors.initials: LN
authors.lastname: Mueller
authors.forename: Markus
authors.initials: M
authors.lastname: Mueller
authors.forename: Bruno
authors.initials: B
authors.lastname: Domon
authors.forename: Ruedi
authors.initials: R
authors.lastname: Aebersold
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D010449:Peptide Mapping / Q000379:methods*; D010748:Phosphopeptides / Q000737:chemistry* / Q000302:isolation & purification*; D020543:Proteome / Q000737:chemistry* / Q000302:isolation & purification*; D020539:Sequence Analysis, Protein / Q000379:methods; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D010748:Phosphopeptides; D020543:Proteome