DOE Human Genome Program Contractor-Grantee
35. Rapid Quantitative Measurements of Proteomes
Richard D. Smith, Ljiljana Pasa Tolic, Mary S. Lipton, Pamela K. Jensen, Gordon A. Anderson, and Timothy D. Veenstra
Pacific Northwest National Laboratory, Richland, WA 99352
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. In addition, direct protein measurements now constitute the most effective method for determining open reading frames for small proteins. Therefore, proteome characterization is increasingly viewed as a necessary complement to complete sequencing of the genome. Approaches for proteome characterization are increasingly based upon mass spectrometric analysis of in-gel digested electrophoretically separated proteins, allowing relatively rapid protein identification compared to conventional approaches. However, this technique 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 by combining fast separations and the mass accuracy and sensitivty obtainable with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. Several approaches are presently being pursued; one based upon the analysis of intact proteins and the second upon global approaches for protein digestion and accurate peptide mass analysis (i.e. the use of "accurate nmass tags"). A key attraction of FTICR is the enhanced facility for protein identification based upon the use of genome sequence data. Alternative versions of proteomes using stable isotope labeling are applied for the purposes of accurate quantitation. We describe the status of our efforts towards the development of a high throughput proteomics capability.
We thank the Office of Biological and Environmental Research, U. S. Department of Energy, for support of this research under contract DE-AC06-76RLO 1830.
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