Detection and quantification of on-chip phosphorylated peptides by surface plasmon resonance imaging techniques using a phosphate capture molecule

Kazuki Inamori, Motoki Kyo, Yoshiaki Nishiya, Yusuke Inoue, Tatsuhiko Sonoda, Eiji Kinoshita, Tohru Koike, Yoshiki Katayama

Research output: Contribution to journalArticle

109 Citations (Scopus)

Abstract

We describe herein a detection and quantification system for on-chip phosphorylation of peptides by surface plasmon resonance (SPR) imaging techniques using a newly synthesized phosphate capture molecule (i.e., biotinylated zinc(II) complex). The biotinylated compound is a dinuclear zinc(II) complex that is suitable for accessing phosphate anions as a bridging ligand on the two zinc(II) ions. The compound was exposed on the peptide array and detected with streptavidin (SA) via a biotin-SA interaction by SPR imaging. In the conventional method using antibody, both anti-phosphoserine and anti-phosphotyrosine antibodies were required for phosphoserine and phosphotyrosine detection, respectively. Detection of the phosphate group by the zinc(II) complex, however, was independent of the phosphorylated amino acid residues. The calibration curve for the phosphorylation ratios was established with a calibration chip, on which phosphoserine-containing peptide probes were immobilized. The peptide probes, which were phosphorylated on the surface by protein kinase A, were detected and quantified by SPR imaging using the zinc(II) complex, SA, and anti-SA antibody. The reaction rate and the kinetics of on-chip phosphorylation were also evaluated with the peptide array. The phosphorylation ratio was saturated at ∼20% in 2 h in this study.

Original languageEnglish
Pages (from-to)3979-3985
Number of pages7
JournalAnalytical chemistry
Volume77
Issue number13
DOIs
Publication statusPublished - Jul 1 2005

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry

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