Proteomics is set to have a profound impact on disease diagnosis, treatment and monitoring. Protein expression profiling is one of the important parameters that can be monitored in the course of neoplastic disease. While several fast and efficient proteomic tools have been developed for protein identification, such as two-dimensional gel electrophoresis and mass spectrometry, quantitative proteomics has many limitations and is not widely applicable. The recently introduced Istotope Coded Affinity Tags (ICAT) technique, which when used in conjunction with mass spectrometry allows changes in the abundance of specific proteins in a complex mixture to be identified and monitored. However, there are several existing limitations to the ICAT technology including limited accuracy and proteome coverage.
The aim of this proposal is to overcome these limitations by developing a new class of PhotoCleavable ICAT (PC-ICAT) reagents based on AmberGen's proprietary PC-biotins. These novel PC-ICATs are composed of four distinct elements: a biotin affinity tag, a photocleavable linker, which exhibits rapid and efficient photocleavage, a variable mass linker labeled with stable isotopes and a reactive group. During Phase I, several PC-ICAT reagents with various reactive groups will be synthesized and evaluated using model peptides, peptide mixtures, proteins and cell lysates. Performance of novel PC-ICAT reagents will be compared to conventional ICAT reagents. During evaluation, several critical parameters will be compared, including quantitation accuracy and overall sensitivity. During Phase II, the best performing PC-ICAT reagents will be further optimized and evaluated for the discovery of biomarkers in a variety of normal and malignant cell lysates. Quantitative proteomic kits for research and clinical markets will be developed. Dr. Steven Gygi, one of the developers of ICAT approach, will serve as a consultant on all Phases of this project. ? ?
