Simultaneous Monitoring of Multiple Kinase Activities by SELDI-TOF Mass Spectrometry
- Vanitha ThulasiramanAffiliated withDepartment of Biology Research, Ciphergen Biosystems Inc.
- , Zheng WangAffiliated withDepartment of Biology Research, Ciphergen Biosystems Inc.
- , Anjali KatrekarAffiliated withDepartment of Biology Research, Ciphergen Biosystems Inc.
- , Lee LomasAffiliated withDepartment of Biology Research, Ciphergen Biosystems Inc.
- , Tai-Tung YipAffiliated withDepartment of Biology Research, Ciphergen Biosystems Inc.
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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.
Key WordsSELDI-TOF MS ProteinChip® Arrays IMAC-Ga Array phosphorylation multiplexed kinase assay
- Simultaneous Monitoring of Multiple Kinase Activities by SELDI-TOF Mass Spectrometry
- Book Title
- Protein Arrays
- Book Subtitle
- Methods and Protocols
- pp 205-214
- Print ISBN
- Online ISBN
- Series Title
- Methods in Molecular Biology
- Series Volume
- Series ISSN
- Humana Press
- Copyright Holder
- Humana Press Inc., Totowa, NJ
- Additional Links
- SELDI-TOF MS
- ProteinChip® Arrays
- IMAC-Ga Array
- multiplexed kinase assay
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