BROAD AIMS: It is the goal of this research to achieve comprehensive characterization of proteins immunoextracted from cells and tissues at levels heretofore not possible, down to the low fmole level and, if possible, even lower. By comprehensive characterization we mean high sequence coverage, identity and attachment sites for all significant phosphorylated peptides and the attachment sites and glycan structure classifications for all significant glycopeptides. To accomplish these goals, this research will develop a new LC-MS platform, Extended Range Proteomics Analysis (ERPA). The platform utilizes a new hybrid mass spectrometer - a linear ion trap - Fourier transform MS (LTQ-FT MS) which allows senstitive structure elucidation of peptide digest fragments up to 10 kDa. We will focus on membrane receptors such as epidermal growth factor receptor (EGFR). Initial Studies will involve analysis of immunoprecipitated EGFR from EGF stimulated cancer cell lines to develop the technology, especially pushing sensitivity to low fmole levels. Software to support the new platform will be written, and new ultra-narrow (10 um i.d., 20 um i.d.) LC columns developed for ultra trace analysis. The platform, once developed, will then be used to comprehensively characterize (phosphorylation and glycosylation) membrane receptors in cancer tissue. Glycosylation patterns in the extracellular domain of the receptor will be correlated with intracellular pathways as measured by reversed protein arrays on the same tissue by our collaborators. While the focus in this work will be membrane receptor proteins, the ERPA platform will apply to a broad range of proteins for ultratrace comprehensive characterization.
A new technology platform will be developed and applied to the structure elucidation of exceeding complex membrane receptor proteins involved in cancer. These proteins are currently targets for therapeutic intervention. Through an examination of diseased tissue, the research should provide insight into cancer disease mechanisms. ? ?
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