ULTRA-SENSITIVE APPROACH FOR MONITORING GENE PRODUCTION


Year of Award:
1999
Award Type:
R21
Project Number:
CA081654
RFA Number:
PAR-98-067
Technology Track:
Molecular & Cellular Analysis Technologies
PI/Project Leader:
SMITH, RICHARD D
Other PI or Project Leader:
N/A
Institution:
BATTELLE PACIFIC NORTHWEST LABORATORIES
The capability for identifying proteins and measuring changes with good precision of proteomes (the complement of proteins produced by a given organism or cell) for model organisms would provide a powerful tool for understanding the interrelated roles of individual gene products underlying the molecular basis of cancer. In this four year two phase (R21/R33) project we will develop a new approach for obtaining such broad systems level views of differential protein expression. The approach would involve two-dimensional capillary electrophoretic separations for rapid proteome separations and advanced mass spectrometry for the rapid identification of proteins and their modifications. The approach will be at least 2 to 3 orders of magnitude more sensitive than existing 2-D PAGE methodologies and able to rapidly identify and measure relative expression levels for thousands of proteins in a single analysis. A component of our approach involves on-line analysis using Fourier transform ion cyclotron resonance mass spectrometry for protein identification based upon very high mass measurement accuracy and multiplexed MS/MS measurements for polypeptides. We will also develop novel isotopic labeling (e.g., 13C, isotopic depletion or enrichment) methods allowing comparison of two proteomes in the same separation, effectively providing an internal standard, and enabling precise determination of expression levels. Phase 1 will involve initial development and validation of the instrumental approach. Phase 2 efforts will involve its extension to an automated format, further extension of its sensitivity, expansion of its applicability to more complex proteomes and the determination of modifications, and provide for computer-based 'differential displays' of results. The eukaryotic yeast strain Saccharomyces cerevisiae will serve the model system for evaluation of the technology. The technology to be developed will enable rapid and sensitive differential proteome displays for the study complex mechanisms and pathways for research into the molecular basis of cancer.