The overall goal of this R21/R33 application is to develop and apply comparative """"""""genome-wide"""""""" proteomic approaches to determine the extent to which protein/peptide signatures of malignant cells enhance information obtained from cytogenetic and histopathologic analyses. Specifically, we will evaluate the relationship between molecular cytogenetic aberrations and protein expression in myeloid leukemia and determine whether distinctive patterns of protein expression (e.g., protein/peptide signatures) predict treatment response. Two-dimensional (2-D) gel electrophoresis, time-of-flight mass spectrometry (MALDI-TOF-MS) and nanoliter/min flow rate electrospray mass spectromety (ESI-MS) will be combined in a novel approach to map low and high abundance proteins/peptides in cell and nuclear lysates from primary leukemic specimens. In the R21 application we will establish methodologies to optimize reproducible proteome sampling of clinical specimens. In the R33 application, we will pursue two parallel approaches, based on 2-D gel separation of proteins according to isoelectric points (pI) and mass, to investigate the proteome of clinical leukemic specimens. In one approach, we will quantify differentially expressed moderate-to high abundance proteins in cell and nuclei lysates from normal and leukemic specimens us sample processing and in gel protein detection optimized in the R21 application. In another approach we will create peptide maps of the entire 2-D gel of cell and nuclear lysates from clinical specimens to increase the dynamic range of protein measurements on 2-D gels by identifying low abundance proteins. Both of these approaches will be used to 1) establish the protein signature of primary t(15;17) APL specimens at diagnosis and relapse using low and high abundance protein/peptide maps and 2) determine the extent to which protein/peptide signatures of APL at diagnosis predict treatment response. Additionally, we will initiate studies to develop antibody-based reagents to test the clinical potential of the gel protein signature associated with poor prognoses APL. We anticipate that protein/peptide signatures will lend new insight into the physiologically active forms of proteins associated with molecular cytogenetic aberrations, increase understanding about regulators of tumor phenotype, and identify novel diagnostic and predictive tumor markers.
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