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University of California-San Diego

Superfund Research Program

Protein Characterization

Project Leader: Elizabeth A. Komives
Grant Number: P42ES010337
Funding Period: 2005-2010

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Project Summary (2005-2010)

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, Dr. Komives team is analyzing phytochelatins, which traffic toxic heavy metals from roots to leaves in plants. Core C provides 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, core researchers are studying pyoverdins (siderophores) and their metal complexes.
  2. Analysis of the binding of acetylcholine esterase (AChE) to various organophosphonate pesticides. The researchers 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. They are using this method to analyze the "aging" process, by which the alkylation becomes permanent. Amide H/D exchange studies 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. The core has robust methods in place for identification of proteins from gels (either ID or 2D). The core researchers are developing 2D chromatography methods to analyze protein mixtures using nanoLC-MS/MS. They are identifying and sequencing phosphorylated peptides. Sub-cellular fractionation to isolate mitochondria is being performed and ICAT proteomics analysis is being used to 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 researchers are assessing metabolic integrity of hepatocytes embedded in engineered polymeric supports. For Drs. Tukey and Evans, they are assessing metabolic changes in hepatocytes from knock-out mice.
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