Post-translational modifications of proteins play a pivotal role in cancer etiology and progression by altering protein-protein interactions, enzymatic activity, 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 (<$1000 per array), which is significantly less than current peptide arrays displaying only a fraction of the human proteome. The SNAP-Tide array is synthesized by a series of carefully timed and precise steps of in vitro DNA replication, RNA transcription, and peptide translation. Once synthesized, the peptides are re-attached in a novel process to specific addresses on the glass slide, and any experiments can occur on the glass slide within the low- volume chamber. This design greatly simplifies peptide array experiments and avoids the need for expensive equipment or complicated procedures, such as mass spectrometry or phage display. Direct applications of the SNAP-Tide array include small molecule screening of pivotal protein drug targets, identification of novel cancer biomarkers, and development of point-of-care diagnostic devices to evaluate cancer patient biospecimens.
This application will develop an integrated technology to synthesize a high throughput peptide microarray (SNAP- Tide; Specificity and Affinity for PepTides) on a single glass slide, resulting in the unprecedented display of one million unique 17 amino acid peptides which will span the entire human proteome. These arrays will be used to comprehensively determine the cellular targets of common post-translational modifications, which play pivotal roles in aberrant cancer signaling, and evaluate the activity of enzymes conferring post-translational modifications from complex cancer cell proteomes. By nature of its high density and high throughput synthesis, the SNAP-Tide technology will broadly transform cancer research, biomarker identification, therapeutic development, and point-of-care diagnostics.
