TAILIEUCHUNG - Báo cáo khoa học: Purification of phosphoproteins by immobilized metal affinity chromatography and its application to phosphoproteome analysis

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 phos-phoproteins. 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. | ễFEBS Journal Purification of phosphoproteins by immobilized metal affinity chromatography and its application to phosphoproteome analysis Mitsuyo Machida1 Hidetaka Kosako1 Kyoko Shirakabe1 Michimoto Kobayashi1 Masato Ushiyama2 Junichi Inagawa2 Joe Hirano2 Tomoyo Nakano3 Yasuhiko Bando3 Eisuke Nishida4 and Seisuke Hattori1 5 1 Division of Cellular Proteomics BML Institute of MedicalScience University of Tokyo Japan 2 GE Healthcare Bio-Sciences KK Tokyo Japan 3 AMR Incorporated Tokyo Japan 4 Graduate Schoolof Biostudies Kyoto University Japan 5 Schoolof PharmaceuticalSciences Kitasato University Tokyo Japan Keywords Akt extracellular signal-regulated kinase ERK immobilized metal affinity chromatography phosphoproteome twodimensional gel electrophoresis Correspondence S. Hattori Division of Cellular Proteomics BML Institute of MedicalScience University of Tokyo 4-6-1 Shirokanedai Minato-ku Tokyo 108-8639 Japan Fax Tel 81 3 5449 5314 E-mail hattoris@ Received 1 August 2006 revised 6 January 2007 accepted 17 January 2007 doi 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 and 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 .

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