Epitope-targeted proteome analysis: towards a large-scale automated protein–protein-interaction mapping utilizing synthetic peptide arrays
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We describe the development of a process for the genome-wide mapping of interactions between protein domains and peptide ligands entirely based on high-throughput biochip technologies. A phage library displaying protein domains from a randomly fragmented and cloned cDNA library will be "panned" on an array of synthetic peptide ligands. After multiplexed affinity enrichment, peptide-specific phage populations will be automatically eluted, propagated, labelled and identified by hybridisation to a DNA microarray. Peptide arrays are synthesized in situ by SPOT synthesis on a planar substrate. By utilizing a commercially available library of human brain cDNA plus a set of distinct model domains cloned into T7-phage, we could show that a single panning round on an array of known peptide ligands for these model domains synthesized on a cellulose membrane can yield an enrichment of better than a factor of 1,000. This is sufficient to detect peptide-specific enrichment of Cy3(post-panning) against Cy5(pre-panning)-labelled phage DNA inserts on a cDNA microarray. Thus, the proof-of-principle of our approach could be successfully demonstrated and first interaction data are being collected.
KeywordsPeptide arrays DNA microarrays Protein domains Epitope Phage display Automation Proteomics Functional genomics
We are thankful to Lesley Mühle for endless PCR and gel analyses and to Susanne Daenicke for expert help in peptide array synthesis. We want to thank G. Cesarini for donation of λDisplay1 vector and A. Zucconi for helpful advice with the λ display system. This work was supported by BMBF grant 031U102G in the frame of the National Genome Research Net (NGFN). The authors are solely responsible for the content of this publication.
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