New Methods for the Characterization of Proteomes: Initial Application to D. radiodurans

Richard D. Smith
Pacific Northwest National Laboratory
P.O. Box 999 (K8-98)
Richland, WA 99352
telephone: 509-376-0723
fax: 509-376-7722
email: rd_smith@pnl.gov
prestype: Platform
presenter: Richard D. Smith

Richard D. Smith, Gordon A. Anderson, Mary S. Lipton, Ljiljana Pasa-Tolic, Margaret F. Romine, Jim Fredrickson, Yufeng Shen, Timothy D. Veenstra, Thomas P. Conrads, and Harold R. Udseth

The patterns of gene expression, protein post-translational modifications, covalent and
non-covalent associations, and how these may be affected by changes in the environment, cannot be accurately predicted from DNA sequences. Approaches for proteome characterization are presently based upon mass spectrometric analysis of 2-D gel separated proteins. However, this approach remains constrained by the speed of the 2-D gel separations, the sensitivity needed for protein visualization, the speed and sensitivity of subsequent mass spectrometric analyses for identification, and the limitations of spot visualization for quantitation. Our objective is to circumvent the limitations of this approach by directly characterizing the cell's polypeptide constituents using a combination of high resolution separations and the mass accuracy and sensitivity obtainable with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. Protein identification is based upon global approaches for protein digestion and accurate peptide mass analysis; i.e. "Accurate Mass Tags" (AMTs). Our two-stage strategy exploits FTICR to based upon validation and subsequent routine measurement of peptide AMTs from "potential mass tags" initially identified using tandem mass spectrometry methods, and thus providing the basis for both extremely high confidence in identifications and subsequent high throughput proteome-wide measurements. A single high resolution capillary liquid chromatography separation combined with high sensitivity, high resolution and accurate FTICR measurements has been shown to be capable of characterizing peptide mixtures of more than 100,000 poly peptide components. Attractions of the approach include the capability for automated high-confidence protein identification, broader proteome coverage, and the capability for exploiting stable-isotope labeling methods to realize high precision for relative protein abundance measurements. Using this approach we have been able to identify >60% of the predicted ORFs for Deinococcus radiodurans, including ~50% of proteins previously designated only as "hypothetical". The status of our efforts will be presented in the context of results for D. radiodurans.

This work was supported by the Office of Biological and Environmental Research of the U.S. Department of Energy. Pacific Northwest National Laboratory is operated for the U.S. Department of Energy by Battelle Memorial Institute through Contract No. DE-AC06-76RLO 1830.