HIGH DENSITY PEPTIDE ARRAYS FOR CANCER-RELATED POST-TRANSLATIONAL MODIFICATIONS


Year of Award:
2013
Award Type:
R21
Project Number:
CA182335
RFA Number:
RFA-CA-13-001
Technology Track:
Molecular & Cellular Analysis Technologies
PI/Project Leader:
OZERS, MARY SZATKOWSKI
Other PI or Project Leader:
N/A
Institution:
PROTEOVISTA, LLC
Post-translational modifications of proteins play a pivotal role in cancer etiology and progression by altering protein-protein interactions, enzymaticactivity, and protein conformation. Peptide arrays have played a significant role in cancer-related discoveries, such as cancer biomarkers, point-of-care diagnostics, and therapeutics directed at protein-protein interactions. This application will develop an integrated technology, the SNAP-Tide array (Specificity and Affinity for PepTides), to synthesize one million unique peptides on a single glass slide using transcriptional and translational machinery. The integrity of the innovative SNAP-Tide arrays will be validated in a three step process, involving mass spectrometry, in vitro fluorescent labeling of amino acids, and antibody recognition to ensure the peptides are accurately synthesized in this novel process. The peptides on the SNAP-Tide array will then be modified by purified enzymes that confer the common cancer- related post-translational modifications of phosphorylation, sumoylation, and arginine methylation. Lysates prepared from cancer cells will be applied to the SNAP-Tide array to evaluate differences in their ability to confer post-translational modifications on the array peptides. The specific aims o this grant are to: 1. Develop and validate an in vitro method to synthesize high density peptide microarrays that display all peptides in the human proteome. 2. Modify the peptides on the array by conferring phosphorylation, sumoylation, and methylation marks using purified enzymes. 3. Quantitate differences in tyrosine phosphorylation activity between two cancer cell lines. Currently available lithographic or spotted peptide arrays display 100-10,000 peptides, while the SNAP-Tide strategy will synthesize a million unique peptides on a single glass slide offering substantially greater throughput. The SNAP-Tide array will be developed for the cost of a standard DNA microarray (