This R-21 application will involve the use of new mass spec methodology to rapidly monitor changes in protein expression in cells for identification of important factors in the development of metastatic processes. The methods to be used to monitor protein expression involve the use of nonporous reversed phase (NPRP) HPLC separations which can rapidly separate large numbers of proteins from whole cell lysates and provide efficient recovery of those proteins in the liquid phase for further analysis. A tandem NP column method will be used to achieve rapid separations of expressed proteins up to m/z90 kDa with automated collection of the fractions in the liquid phase. This method will provide high resolution separations of proteins from cancer cell lines with high loadability and high recovery. (NPRP)HPLC also provides purified proteins which are readily interfaced to mass spec methods such as Matrix-Assisted Laser Desorption/Ionization (MALDI) mass spectrometry which will be used to mass size each target protein to generate a mass map of the expressed proteins from the cell and for digestion by trypsin or Glu-C which will be used to generate peptide maps. The molecular weight and peptide maps can be used to identify each protein and search for modification of proteins compared to protein databases. Capillary Electrophoresis/Electrospray Ionization Tandem Mass Spectrometry (CE(ESI)-TOFMS-MS) will be used to pinpoint modifications such as phosphorylation that occur during the metastasis process. In particular, we plan to use these methods to monitor changes with progression of cell lines developed at the Karmanos Cancer Institute from human breast epithelial cells. The MCF 10 model line developed on a common genetic background includes mortal and immortal normal cells, variants transfected with activated ras which do and do not form xenograft lesions, premalignant variants able to form DCIS which sporadically progress to invasive cancers and both metastatic and nonmetastatic carcinoma lines. Because of well recognized problems in monitoring changes in these cells by differential display methods, it is proposed to look at relative levels of proteins rather than nucleic acids. MALDI-TOFMS will allow us to rapidly screen and identify proteins which are differentially expressed during progression to fully metastatic tumor cells and to look at detailed alterations in these proteins due to mutations and phosphorylation, for example, which are often key components of changes towards cancer progression. The use of data generated in relation to future gene knockout experiments to determine the role of various proteins in the cancer process will be discussed.
NIH ExPorter
NIH ExPorter