• Eiji Kinoshita, Emiko Kinoshita-Kikuta, Yasunori Sugiyama, Yoshitaka Fukada, Tomomitsu Ozeki, and Tohru Koike.
• Department of Functional Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan. kinoeiji@hiroshima-u.ac.jp
• Proteomics. 2012 Apr 1; 12 (7): 932-7.

AbstractWe have previously shown that the dinuclear zinc(II) complex Phos-tag and its derivatives act as phosphate-capture molecules in aqueous solution under conditions of neutral pH. In this study, our aim was to develop more-advanced applications for the detection of phosphopeptides and phosphoproteins by using several newly synthesized Phos-tag derivatives, including a bisbiotinylated Phos-tag (BTL-108), a tetrakisbiotinylated Phos-tag (BTL-109), and a monobiotinylated Phos-tag with a dodeca(ethylene glycol) spacer (BTL-111), as well as the commercially available product BTL-104. Among these complexes, BTL-111 showed the best performance in Western blotting by an ECL system using HRP conjugated streptavidin. In addition, in a quartz-crystal microbalance analysis of a phosphoprotein, the presence of the long hydrophilic dodeca(ethylene glycol) spacer in a novel Phos-tag sensor chip coated with BTL-111 resulted in a greater sensitivity than was achieved with a similar chip coated with BTL-104. Moreover, a peptide microarray technique using the ECL system and BTL-111 permitted high-throughput assays for the specific and highly sensitive detection of protein kinase activities in cell lysates.© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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