Detection and quantification of on-chip phosphorylated peptides by surface plasmon resonance imaging techniques using a phosphate capture molecule.
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 approximately 20% in 2 h in this study.
Inamori
Kazuki
K
Biotechnology Frontier Project, Toyobo Co., Ltd., 10-24 Toyo-cho, Tsuruga, Fukui 914-0047, Japan.
Kyo
Motoki
M
Nishiya
Yoshiaki
Y
Inoue
Yusuke
Y
Sonoda
Tatsuhiko
T
Kinoshita
Eiji
E
Koike
Tohru
T
Katayama
Yoshiki
Y
D046208:Microchip Analytical Procedures / Q000379:methods*; D015394:Molecular Structure; D010455:Peptides / Q000737:chemistry*; D010710:Phosphates / Q000737:chemistry*; D010766:Phosphorylation; D019809:Streptavidin / Q000737:chemistry; D020349:Surface Plasmon Resonance / Q000379:methods*; D015032:Zinc / Q000737:chemistry
D010455:Peptides; D010710:Phosphates; D019809:Streptavidin; D015032:Zinc