title: Purification of phosphoproteins by immobilized metal affinity chromatography and its application to phosphoproteome analysis.
abstract: Prefractionation procedures facilitate the identification of lower-abundance proteins in proteome analysis. Here we have optimized the conditions for immobilized metal affinity chromatography (IMAC) to enrich for phosphoproteins. The metal ions, Ga(III), Fe(III), Zn(II), and Al(III), were compared for their abilities to trap phosphoproteins; Ga(III) was the best. Detailed analyses of the pH and ionic strength for IMAC enabled us to determine the optimal conditions (pH 5.5 and 0.5 m NaCl). When whole cell lysates were fractionated in this way, about one-tenth of the total protein was recovered in the eluate, and the recovery of phosphorylated extracellular signal-regulated kinase (ERK) was more than 90%. Phosphorylated forms of ribosomal S6 kinase (RSK) and Akt were also enriched efficiently under the same conditions. Our Ga(III) IMAC and a commercially available purification kit for phosphoproteins performed similarly, with a slight difference in the spectrum of phosphoproteins. When phosphoproteins enriched from NIH3T3 cells in which ERK was either activated or suppressed were analyzed by two-dimensional fluorescence difference gel electrophoresis, phosphorylated ERK was detected as discrete spots unique to ERK-activated cells, which overlapped with surrounding spots in the absence of prefractionation. We applied the same technique to search for Akt substrates and identified Abelson interactor 1 as a novel potential target. These results demonstrate the efficacy of phosphoprotein enrichment by IMAC and suggest that this procedure will be of general use in phosphoproteome research.
authors.affiliation: Division of Cellular Proteomics (BML), Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-6639, Japan.
authors.forename: Mitsuyo
authors.initials: M
authors.lastname: Machida
authors.forename: Hidetaka
authors.initials: H
authors.lastname: Kosako
authors.forename: Kyoko
authors.initials: K
authors.lastname: Shirakabe
authors.forename: Michimoto
authors.initials: M
authors.lastname: Kobayashi
authors.forename: Masato
authors.initials: M
authors.lastname: Ushiyama
authors.forename: Junichi
authors.initials: J
authors.lastname: Inagawa
authors.forename: Joe
authors.initials: J
authors.lastname: Hirano
authors.forename: Tomoyo
authors.initials: T
authors.lastname: Nakano
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Bando
authors.forename: Eisuke
authors.initials: E
authors.lastname: Nishida
authors.forename: Seisuke
authors.initials: S
authors.lastname: Hattori
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D008670:Metals; D051379:Mice; D041681:NIH 3T3 Cells; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification*; D020543:Proteome; D019893:Ribosomal Protein S6 Kinases / Q000378:metabolism
chemical_list: D008670:Metals; D010750:Phosphoproteins; D020543:Proteome; D019893:Ribosomal Protein S6 Kinases; D048049:Extracellular Signal-Regulated MAP Kinases