|Functional Genomics Section
DOE Human Genome Program Contractor-Grantee Workshop
149. Rapid and Sensitive Characterization of Proteomes; an Adjunct to the Genome
Richard D. Smith, Ljiljana Pasa
Tolic, Mary S. Lipton, Pamela K. Jensen, Gordon A. Anderson, and James
In contrast to an organism's virtually static and well defined genome, the proteome continually changes in response to external and internal events. 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, and the speed and sensitivity of subsequent mass spectrometric analyses for identification.
Our objective is to circumvent the limitations of this approach by directly characterizing the cell's polypeptide constituents by combining the speed of capillary isoelectric focusing (CIEF) and the mass accuracy and sensitivity obtainable with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. CIEF-FTICR MS studies require orders of magnitude smaller sample sizes than required by 2-D PAGE technology, and initial efforts have demonstrated sensitivities well into the attomole range, as well as the potential for further significant improvements. A key attraction of FTICR is the enhanced facility for protein identification based upon the use of genome sequence data. Isotopically depleted growth media allow highly accurate molecular mass determinations for larger proteins than otherwise possible, and further improves achievable sensitivity and detection limits. We describe our efforts aimed at developing on-line CIEF-FTICR techniques, their comparison with conventional methodologies, and their initial application to several prokaryotes for which complete genome sequences are available. We will also describe new approaches for the determination of precise expression levels for large numbers of proteins in the same measurement.
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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