Proteomics
While a considerable amount of transcriptome data on prostate cancer has been amassed by our group and others, there is minimal proteomic data available for prostate cancer (or for other cancers). In order to begin to integrate genomic and proteomic data, much more proteomic data will need to be generated.
In the Proteomics Laboratory, we plan to embark on 2-D liquid phase fractionation as a proteomics platform to profile prostate cancer specimens. The method involves separating proteins from tumor extracts using chromatofocusing, which is a column-based liquid separation technique where proteins are fractionated according to pI. CF will be performed over a pH range of 4-9 in 0.1-pH unit intervals to obtain a high degree of fractionation. Anion exchange via a salt gradient also will be used for further fractionation to extend the pH and hydrophobicity range. Each pI fraction will then be separated in a second dimension by nonporous silica (NPS) reversed phase HPLC. The result is a 2-D separation of proteins from tumor extracts where relatively pure proteins in the liquid phase are obtained, which will be collected for spotting onto a microchip array.
Using this method, >2,500 proteins will be available for mass spectrometry analysis and/or spotting on protein microarrays, and additional stages of fractionation will be used to provide additional separation if required. We also will need to develop new bioinformatics software to compare the 2-D liquid phase proteomic maps. This will facilitate the identification of differential proteins and post-translational modifications.
This work will nicely complement our gene expression profiling studies and will allow for systematic comparisons of protein levels with mRNA.
