) The Proteomics Core will undertake protein profiling of approximately 1,200 tumor specimens for the three component projects. To that effect, state-of-the-art proteomics tools will be utilized to identify proteins that are predictive of tumor behavior and that will contribute to a molecular based classification scheme. A facility for two-dimensional (2-D) protein analysis has been in operation in the PI?s laboratory for 15 years. The Core laboratory has pioneered the use of immobilized pH gradients (IPG) for 2-D separations. Only microdissected specimens will be utilized for 2-D separations of tumor proteins. In most cases, as has been our experience, sufficient protein is obtained by microdissection to produce excellent IPG-based 2-D patterns using our standard gel format. We have also developed a smaller gel format for use when the number of cells is limited to microgram quantities, as may occur with laser capture microdissection. The Proteomics Core can produce up to 100 gels weekly, in batches of 20 and has the necessary resources for the quantitative image analysis of silver stained 2-D gels. A combination of Mass Spectrometry and Edman sequencing will be relied upon for protein identification. The main strategy will involve in gel-digestion followed by MALDI-MS analysis. A PerSeptive Voyager system is located in the Proteomics Core Laboratory. Proteins that fail to be identified with the main strategy are subjected to additional types of Mass Spectrometric analyses in the laboratory of the co-PL, Dr. David Lubman. Proteins that fail to be identified by Mass Spectrometry and Edman sequencing are subjected to internal sequencing, which is done at the protein sequencing core of the University of Michigan Medical Center. The unprecedented ease with which proteins are currently being identified by the Proteomics Core leads to a projection that approximately 1,000 proteins will be identified in 2-D patterns of each of the three types of tumors, namely colon, ovarian and lung. Quantitative data pertaining to the abundance of these proteins will be correlated with quantitative data pertaining to the abundance of their corresponding RNA?s determined by the DNA array core. The integrated approach with which protein and RNA levels will be determined will contribute to a robust profiling of gene expression in which the contribution of transcriptional and post-transcriptional processes to tumor behavior will be investigated.
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