Development of an antibody proteomics system using a phage antibody library for efficient screening of biomarker proteins.

Proteomics-based analysis is currently the most promising approach for identifying biomarker proteins for use in drug development. However, many candidate biomarker proteins that are over- or under-expressed in diseased tissues are found by such a procedure. Thus, establishment of an efficient method for screening and validating the more valuable targets is urgently required. Here, we describe the development of an "antibody proteomics system" that facilitates the screening of biomarker proteins from many candidates by rapid preparation of cross-reacting antibodies using phage antibody library technology. Using two-dimensional differential in-gel electrophoresis analysis, 16 over-expressed proteins from breast cancer cells were identified. Specifically, proteins were recovered from the gel pieces and a portion of each sample was used for mass spectrometry analysis. The remainder was immobilized onto a nitrocellulose membrane for antibody-expressing phage enrichment and selection. Using this procedure, antibody-expressing phages against each protein were successfully isolated within two weeks. The expression profiles of the identified proteins were then acquired by immunostaining of breast tumor tissue microarrays with the antibody-expressing phages. Using this approach, expression of Eph receptor A10, TRAIL-R2 and Cytokeratin 8 in breast tumor tissues were successfully validated. These results demonstrate the antibody proteomics system is an efficient method for screening tumor-related biomarker proteins.