Epitope-targeted proteome analysis: towards a large-scale automated protein-protein-interaction mapping utilizing synthetic peptide arrays.
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.
Bialek
Krzysztof
K
Research Group for Molecular Recognition, GBF (German Research Centre for Biotechnology), Mascheroder Weg 1, 38124 Braunschweig, Germany.
Swistowski
Andrzej
A
Frank
Ronald
R
D000595:Amino Acid Sequence; D001331:Automation; D017123:Bacteriophage T7 / Q000235:genetics; D010582:Bacteriophage lambda / Q000235:genetics; D018604:Epitope Mapping / Q000379:methods; D000939:Epitopes / Q000032:analysis; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D036103:Nanotechnology; D020411:Oligonucleotide Array Sequence Analysis; D019151:Peptide Library; D040081:Protein Array Analysis / Q000379:methods; D020543:Proteome / Q000737:chemistry*; D015203:Reproducibility of Results
D000939:Epitopes; D019151:Peptide Library; D020543:Proteome