Methods in Molecular Biology Volume 264, 2004, pp 205-214

Simultaneous Monitoring of Multiple Kinase Activities by SELDI-TOF Mass Spectrometry

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Cellular response to the external environment is often controlled by one or more protein kinases. We report a methodology for simultaneously monitoring multiple kinase activities across multiple signal-transduction pathways using ProteinChip® Array technology. Based on the addition of specific peptide reporters, kinase activity is detected by the presence of a mass shift of 80 Da (or multiple thereof) corresponding to the addition of one or more phosphate groups. These phosphorylated peptide substrates are then enriched using an immobilized metal affinity capture (IMAC)-Ga array and detected directly by surface-enhanced laser desorption/ ionization time-of-flight mass spectrometry (SELDI-TOF MS). SELDI-TOF MS is sensitive, tagless (nonradioactive, nonfluorescent), can be easily multiplexed for the analysis of several different kinases in a single reaction mixture (limited only by the specificity of the kinase for its substrate peptides), and is directly scalable through the use of robotic sample processing. By multiplexing kinase assays, one can dramatically increase the amount of information obtained from rare or volume-limited samples. More important, results reflect closely the complex interrelationships between kinases and show high correlation with in vivo assays.