The overall goal of Core C, the Protein Characterization Core is to provide mass spectrometry, HPLC, and peptide synthesis services for Superfund investigators. 1) Analysis of metal complexes. With Dr. Schroeder, phytochelatins will be measured, which traffic toxic heavy metals from roots to leaves in plants. Core C will provide fluorescence HPLC quantification of phytochelatins from plant extracts, peptide synthesis of the various phytochelatins and analysis of the binding affinities of various heavy metals (Cd, Hg, Ni, As). With Dr. Tebo, the investigators will study pyoverdins (siderophores) and their metal complexes. 2) Analysis of the binding of acetylcholine esterase (AChE) to various organophosphonate pesticides. They have developed a MALDI-TOF-based method for quantifying the amount of AChE that had been modified at the active site serine by various organophosphonates. This method will be used to analyze the 'aging'process, by which the alkylation becomes permanent. Amide H/D exchange studies will give information on the conformational properties of AChE-acrylodan adducts to rationally design biosensors for various organophosphonate compounds. 3) Proteomics and phosphorylation site analysis. Drs. Tebo, Russell, Karin and Evans all have need of protein identification, proteomics, and phosphorylation site analysis. Robust methods will be in place for identification of proteins from gels (either 1D or 2D). The applicants will develop 2D chromatography methods to analyze protein mixtures using nanoLC-MS/MS. They will identify and sequence phosphorylated peptides. Sub-cellular fractionation to isolate mitochondria will be performed and ICAT proteomics analysis will identify proteins for which the abundance has changed in cells from knock-out mice. 4) Metabolic profiling. Drs. Bahtia, Evans and Tukey require metabolic profile analyses. For Dr. Bahtia, the core will assess metabolic integrity of hepatocytes embedded in engineered polymeric supports. For Drs. Tukey and Evans, we will assess metabolic changes in hepatocytes from knock-out mice.
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