The long-term goal of the project is to develop and then commercialize stable isotope reagents for quantitative proteomics using fluorous enrichment techniques. Successful demonstration in proteomics will be followed by extension into metabolomics and glycomics using a similar tagging strategy. Fluorous Technologies, Inc. (FTI) as the small business concern, and The Genomics Institute of the Novartis Research Foundation (GNF), as a collaborator, will synthesize fluorous isotope reagent tags (FLIRTs), develop protocols, benchmark vs. existing techniques, and demonstrate the FLIRT approach in the quantification of protein function.
The specific aims of the grant are: 1) Synthesis of aniline based FLIRTs. Synthetic routes to 5 fluorous probes for chemical labeling of peptides and proteins will be synthesized based on a fluorous aniline core. A goal of 500 mg of each unlabeled compound and 150 mg of each labeled compound is targeted. 2) Synthesis of stable isotope labeled versions of the existing fluorous reagents. FLIRTs with the stable isotopes directly in the fluorous chain will be synthesized. The goal will be to obtain 100 mg of each compound. These compounds will be the direct stable isotope labeled analogs of fluorous peptide labeling reagents which FTI has previously produced. 3) Development of quantitative tagging protocols using the fluorous isotope labeled reagents and benchmarking vs. cICAT. Protocols for tagging and enrichment using FLIRTs will be developed using LC/MS with ESI detection. Quantification by FLIRT will then be benchmarked against cICAT. Performance measures will include enrichment efficiency, selectivity biases, selectivity enrichment, recovery of enriched species, and accuracy of quantitation. 4) Fluorous isotope reagent tagging (FLIRT) for relative quantification of a targeted subproteome. FLIRT technology will be applied to the quantification of specific classes of proteins within a complex sample, including the characterization of the redox state of the various cysteine residues within a protein. Successful enrichment and quantitation through a single label will demonstrate the flexibility of FLIRTs in analyzing targeted classes of proteins. FLIRTs will provide a novel method for quantitative proteomics which can be applied to numerous research areas including target discovery, biomarker discovery, and diagnostics development for various diseases affecting the human condition. Project Narrative: The goal of the project is to develop and commercialize reagents for quantitative proteomics. The successful commercialization of these products will enable researchers to elucidate biological pathways leading to new biomarkers and targets by which to develop novel diagnostics and therapeutics for various diseases that affect the human condition. ? ? ?
