Epitope-targeted proteome analysis: towards a large-scale automated protein-protein-interaction mapping utilizing synthetic peptide arrays

被引:15
作者
Bialek, K [1 ]
Swistowski, A [1 ]
Frank, R [1 ]
机构
[1] GBF German Res Ctr Biotechnol, Res Grp Mol Recognit, D-38124 Braunschweig, Germany
关键词
peptide arrays; DNA microarrays; protein domains; epitope; phage display; automation; proteomics; functional genomics;
D O I
10.1007/s00216-003-1876-3
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:1006 / 1013
页数:8
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