Protein kinase substrate identification on functional protein arrays
1 Invitrogen Corp., Protein Array Center, 688 East Main Street, Branford, CT 06405, USA
2 Novartis Institutes for Biomedical Research, Inc., Developmental & Molecular Pathways, 250 Massachusetts Avenue, Cambridge, MA 02139, USA
3 Yale University, Department of Biology, Kline Biology Tower, PO BOX 208103, New Haven, CT 06520-8103, USA
4 Merck & Co., Inc., UG2C-24, 351 N. Sumneytown Pike, North Wales, PA 19454-2505, USA
5 UCLA, Department of Molecular and Medical Pharmacology, 23-231 CHS, 10833 Le Conte Avenue, Los Angeles, CA 9009, USA
BMC Biotechnology 2008, 8:22 doi:10.1186/1472-6750-8-22Published: 28 February 2008
Over the last decade, kinases have emerged as attractive therapeutic targets for a number of different diseases, and numerous high throughput screening efforts in the pharmaceutical community are directed towards discovery of compounds that regulate kinase function. The emerging utility of systems biology approaches has necessitated the development of multiplex tools suitable for proteomic-scale experiments to replace lower throughput technologies such as mass spectroscopy for the study of protein phosphorylation. Recently, a new approach for identifying substrates of protein kinases has applied the miniaturized format of functional protein arrays to characterize phosphorylation for thousands of candidate protein substrates in a single experiment. This method involves the addition of protein kinases in solution to arrays of immobilized proteins to identify substrates using highly sensitive radioactive detection and hit identification algorithms.
To date, the factors required for optimal performance of protein array-based kinase substrate identification have not been described. In the current study, we have carried out a detailed characterization of the protein array-based method for kinase substrate identification, including an examination of the effects of time, buffer compositions, and protein concentration on the results. The protein array approach was compared to standard solution-based assays for assessing substrate phosphorylation, and a correlation of greater than 80% was observed. The results presented here demonstrate how novel substrates for protein kinases can be quickly identified from arrays containing thousands of human proteins to provide new clues to protein kinase function. In addition, a pooling-deconvolution strategy was developed and applied that enhances characterization of specific kinase-substrate relationships and decreases reagent consumption.
Functional protein microarrays are an important new tool that enables multiplex analysis of protein phosphorylation, and thus can be utilized to identify novel kinase substrates. Integrating this technology with a systems biology approach to cell signalling will help uncover new layers in our understanding of this essential class of enzymes.