This Small Business Innovation Research (SBIR) Phase II project will develop a novel antibody microarray for high-throughput, multiplexed profiling of a large number of signaling proteins from multiple pathways by measuring protein phosphorylation. The antibody array will simultaneously measure kinase activities in Ras effector pathways including the Raf-MEK-ERK pathway, the P13K-Akt pathway, the p38 and JNK pathways. Current kinase profiling technologies such as Western blotting of flow cytometry are low throughput, not quantitative and difficult to multiplex and standardize. This novel technology (Proteome Epitope Tag or PET) creates antibodies with pre-defined specificity that can be multiplexed using standardized assays on antibody microarrays for measuring protein phosphorylation. The PET approach will be further developed to construct highly multiplexed antibody arrays for simultaneous measurement of a large number of kinase protein activities from multiple pathways. The ability to measure all signaling proteins from interconnected pathways will provide an unprecedented opportunity to decipher the complexity of cell signaling.
The commercial applications of this technology will be in large scale protein analysis relevant to basic biological research, drug discovery, and clinical medicine. Protein biochips hold great promise for biomarker discovery which is important in all these areas. Large-scale protein biochips capable of standardized and high-throughput protein measurement on differentially perturbed biological systems do not exist today. This is due primarily to the lack of highly specific antibodies for all human proteins predicted by gene sequences. The PET technology addresses this urgent, unmet need by generating antibodies for highly specific peptide tags of defined sequences in a proteome, representing a universal method for producing antibodies and standardized chip-based assays for any protein of interest. PET chips for profiling kinase signaling networks will have enormous utility for drug discovery by better characterizing drug efficacy, side effects and potential toxicity.
