Evaluation of protein kinase activities of cell lysates using peptide microarrays based on surface plasmon resonance imaging

• Published in: Analytical biochemistry Vol. 375; no. 2; pp. 223 - 231
• Main Authors: Mori, Takeshi, Inamori, Kazuki, Inoue, Yusuke, Han, Xiaoming, Yamanouchi, Go, Niidome, Takuro, Katayama, Yoshiki
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.04.2008
• Subjects: Amino Acid Sequence; Cell Extracts - chemistry; Cell Line, Tumor; Cell lysate; Drug stimulation; Humans; Inhibitor; Kinase; Molecular Sequence Data; Peptide microarray; Phosphorylation; Protein Array Analysis; Protein Kinases - chemistry; Protein Kinases - isolation & purification; Protein Kinases - metabolism; SPR; Stimulation, Chemical; Surface Plasmon Resonance; Cell lysate; Inhibitor; SPR; Kinase; Drug stimulation; Peptide microarray
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2007.12.011

We developed a peptide microarray based on surface plasmon resonance (SPR) imaging for monitoring protein kinase activities in cell lysates. The substrate peptides of kinases were tethered to the microarray surface modified with a self-assembled monolayer of an alkanethiol with triethylene glycol terminus to create a low nonspecific binding surface. The phosphorylation of the substrate peptides immobilized on the surface was detected with the following phosphate specific binders by amplifying SPR signals: anti-phosphotyrosine antibody for tyrosine kinases and Phos-tag biotin (a phosphate-specific ligand with biotin tag) for serine/threonine kinases. Using the microarray, 9 kinds of protein kinases were evaluated as a pattern of phosphorylation of 26 kinds of substrate peptides. The pattern was unique for each protein kinase. The microarray could be used to evaluate the inhibitory activities of kinase inhibitors. The microarray was applied successfully for kinase activity monitoring of cell lysates. The chemical stimuli responsive activity changes of protein kinases in cell lysates could also be monitored by the peptide microarray. Thus, the peptide microarray based on SPR imaging would be applicable to cell-based drug discovery, diagnosis using tissue lysates, and biochemical studies to reveal signal transduction pathways.