ABSTRACT
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.
