The nervous system possesses extreme cellular heterogeneity and there is a need for new technologies that will effectively, and with very high sensitivity, provide direct measurements of the arrays of gene products (i.e. proteins) expressed by specific groups of cells, so as to advance understanding of how their expression may conelate with different developmental, pathological, or functional states. This project will develop, implement and validate a new high throughput approach for making precise measurements of relative abundances of large numbers of proteins from mouse neural cells. The project is a collaboration between investigators involved in development of crucial technical aspects of the approach. The methodology will be based upon a new approach for proteome-wide stable isotope and biotin labeling of cysteine-containing polypeptides being developed at the University of Washington (Aebersold Laboratory), combined with new approaches that use accurate mass tags (AMTS) and the high sensitivity of Fourier transform ion cyclotron resonance mass spectrometry technology being developed at PNNL. This project will refine and integrate the sample processing, improve high throughput accurate mass measurements used for AMT generation, and develop the essential computer-based analysis and data visualization capabilities. The technology will be tested and evaluated using astrocytes derived from wild-type and p53- deficient mice. The efforts under this project will integrate and validate the instrumental approach, and provide the basis for broad dissemination of a version of the technology based upon relatively inexpensive instrumentation. The technology to be developed will enable ultra-sensitive (attomole level) proteome-wide precise profiling of all neural cell types, and provide an abundance of new information on protein expression, and enable precise measurements of differences in relative protein expression levels as a function of cell type, disease states and development stage, etc. To facilitate the broader application of this technology, all data processing, analysis and visualization tools will be rapidly disseminated to the scientific community.
