A targeted proteomic approach for the identification of tumor-associated membrane antigens using the ProteomeLab ™ PF-2D in tandem with mass spectrometry

• Published in: Biochemical and biophysical research communications 2006-09, Vol.348 (3), p.1055-1062
• Main Authors: Chahal, Francina C., Entwistle, Joycelyn, Glover, Nick, MacDonald, Glen C.
• Format: Article
• Language: English
• Subjects: 2D-PAGE; AFP; Antibodies, Monoclonal - metabolism; Antibodies, Neoplasm - metabolism; Antigens, Neoplasm - analysis; Antigens, Neoplasm - immunology; Antigens, Neoplasm - metabolism; Binding Sites, Antibody; Blotting, Western; CD44; Cell Line, Tumor; Cells, Cultured; Chromatofocusing; Chromatography, High Pressure Liquid; Electrophoresis, Gel, Two-Dimensional; Humans; Immunoblotting; Immunoprecipitation; Isoelectric points; Isoforms; LC–MS/MS; Mass Spectrometry; Membrane Proteins - analysis; Membrane Proteins - immunology; Membrane Proteins - metabolism; Membrane-antigens; Neoplasm Proteins - analysis; Neoplasm Proteins - immunology; Neoplasm Proteins - metabolism; Proteome - analysis; Proteome - immunology; Proteome - metabolism; Receptor, ErbB-2 - chemistry; Receptor, ErbB-2 - isolation & purification; Reversed-phase HPLC; Tumor-associated
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2006.07.187

Mapping differential expression of soluble proteins has become fairly routine using chromatofocusing in combination with the reversed-phase HPLC (ProteomeLab ™ PF-2D by Beckman Coulter Inc.); however, identification of membrane antigens has not been reported thus far. In this report, we demonstrate a targeted proteomic approach employing immunoprecipitation, prior to 2D-LC separation, in tandem with MS/MS that can be used to identify tumor-associated membrane antigens. This system is very sensitive and reproducible in that only 1/4th the amount of starting material is required for analysis as compared to gel-based analysis, and permits a focused environment for eliminating non-specific interactions leading to an accurate resolution of the cognate antigen. This system also circumvents the well-known limitations associated with gel-based approaches. This approach has been validated in the identification of ErB2/HER-2 and was subsequently used to identify CD44E as the cognate antigen for VB1-008, one of our fully human, tumor-specific, monoclonal antibodies.