title: A targeted proteomic approach for the identification of tumor-associated membrane antigens using the ProteomeLab PF-2D in tandem with mass spectrometry.
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.
authors.affiliation: Viventia Biotech Inc., 147 Hamelin street, Winnipeg, MB, Canada R3T3Z1. fchahal@viventia.com
authors.forename: Francina C
authors.initials: FC
authors.lastname: Chahal
authors.forename: Joycelyn
authors.initials: J
authors.lastname: Entwistle
authors.forename: Nick
authors.initials: N
authors.lastname: Glover
authors.forename: Glen C
authors.initials: GC
authors.lastname: Macdonald
mesh_terms: D000911:Antibodies, Monoclonal / Q000378:metabolism; D000912:Antibodies, Neoplasm / Q000378:metabolism; D000951:Antigens, Neoplasm / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D001666:Binding Sites, Antibody; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D009363:Neoplasm Proteins / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D020543:Proteome / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D020543:Proteome; D018719:Erb-b2 Receptor Tyrosine Kinases