Phospho-Proteomics

Emiko Kinoshita-Kikuta, Eiji Kinoshita, Tohru Koike

Louise von Stechow

Protein kinases are widely considered to be invaluable target enzymes for drug discovery and for diagnosing diseases and assessing their prognosis. Effective analytical techniques for measuring the activities of cellular protein kinases are therefore required for studies in the field of phosphoproteomics. We have recently developed a highly sensitive microarray-based technique for tracing the activities of protein kinases. A series of peptides that are specific substrates of various protein kinases are immobilized on a glass slide and subjected to phosphorylation by cell lysates. The resulting phosphorylated forms of the various peptides are then selectively and simultaneously detected by using a phosphate-binding tag molecule, biotin-labeled Phos-tag, bound to horseradish peroxidase-conjugated streptavidin. Enhanced chemiluminescence signals can then be readily detected by using an automatic image analyzer. In this chapter, we describe a standard protocol for detecting phosphopeptides by biotin-labeled Phos-tag. We also describe a microarray system for high-throughput profiling of intracellular protein kinase activities. The Phos-tag-based method is expected to be useful in the rapid detection of the complex range of phosphorylation reactions involved in cellular signaling events, and it has potential applications in high-throughput screening of kinase activators or inhibitors.