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The Center for Proteomics Research is a highly interactive, multidisciplinary
program designed to develop innovative proteomic technologies and
apply them to the disease of Cystic Fibrosis (CF). We have successfully
worked together to publish the first studies on the genomics of
CF, and to identify the components of a prototype CF Genomic Chip.
Based on this experience, we are able to focus our current efforts
on the testable hypothesis that the candidate genes we have deduced
from CF genomics will be predictive of candidates for the CF Proteomic
Chip.
The goal of the present project is to create innovative proteomic
technologies and to apply these to the disease of CF. To reach this
goal we will develop and deliver a novel, generally applicable technology,
3D Proteomics, to identify candidate proteins for a CF Proteomic
Chip. 3D Proteomics is a sensitive, quantitative and dynamic technology
for determining the complete CF proteome from CF lung cells and
CF patient biopsy tissue. The 3D Proteomic technology is based on
pulsing the cells or tissues with a radiolabeled amino acid or other
substrate. While conventional 2D information contains the pI and
molecular weight, the novel third dimension of this approach provides
a quantitative, biosynthetic rate for a given protein. The strength
of this approach is its focus on the actual mechanisms that underlie
the relevant pathophysiology, emphasizing rapidly turning-over and
low abundance proteins. We will use 3D Proteomics to identify CF-specific
changes in protein expression in epithelial cells.
To achieve these goals we propose the following Specific Objectives:
Specific Objective #1: To identify all proteins in cultured CF
lung epithelial cells which are affected by CF, using the technique
of 3D Proteomics. The 3D Proteomic technology will be developed
in this objective. This technology involves pulsing lines of CF
and isogenic control cells with 35[S]-methionine, separating proteins
in two dimensions, either by conventional 2D electrophoresis or
free flow electrophoresis followed by SDS-PAGE, and detecting proteins
in the third dimension by radioimaging. Preliminary data indicate
that when comparing CF with control cells, 3D Proteomics identifies
3-5 fold more differences than silver stain technology.
Specific Objective #2: To identify all proteins in patient samples
of CF lung epithelial cells which are affected by the disease, using
3D Proteomics. Epithelial cells from biopsies of CF lungs will be
labeled with 35[S]-methionine, using methods developed in Specific
Objective #1. This novel biosynthetic paradigm will clearly delineate
proteins in CF epithelial cells from contaminating serum or bronchoalveolar
lavage proteins.
Specific Objective #3: To identify candidate proteins for a CF
Proteomic Chip using the 3D Proteomic information from CF cells
and tissues. We will develop novel bioinformatic algorithms, based
on simultaneous measurements of multiple physiological parameters,
to identify CF-specific changes in protein expression.
3D Proteomics and the identification of candidate proteins for
a CF Proteomic Chip offer a profound contribution to the technology
underlying attempts to understand and treat CF. The identification
of candidate proteins for a CF Proteomic Chip will provide multiple
surrogate endpoints for both CF diagnosis and patient monitoring
at the bedside as well as a platform for discovery of novel CF therapeutics
at the bench.
Participants in Proteomics Initiative Meeting (March 11-12)
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