title: Mapping of Fab-1:VEGF Interface Using Carboxyl Group Footprinting Mass Spectrometry.
abstract: A proof-of-concept study was performed to demonstrate that carboxyl group footprinting, a relatively simple, bench-top method, has utility for first-pass analysis to determine epitope regions of therapeutic mAb:antigen complexes. The binding interface of vascular endothelial growth factor (VEGF) and the Fab portion of a neutralizing antibody (Fab-1) was analyzed using carboxyl group footprinting with glycine ethyl ester (GEE) labeling. Tryptic peptides involved in the binding interface between VEGF and Fab-1 were identified by determining the specific GEE-labeled residues that exhibited a reduction in the rate of labeling after complex formation. A significant reduction in the rate of GEE labeling was observed for E93 in the VEGF tryptic peptide V5, and D28 and E57 in the Fab-1 tryptic peptides HC2 and HC4, respectively. Results from the carboxyl group footprinting were compared with the binding interface identified from a previously characterized crystal structure (PDB: 1BJ1). All of these residues are located at the Fab-1:VEGF interface according to the crystal structure, demonstrating the potential utility of carboxyl group footprinting with GEE labeling for mapping epitopes. Graphical Abstract ᅟ.
authors.affiliation: Protein Analytical Chemistry Department, Genentech Inc., 1 DNA Way, South San Francisco, CA, 94080, USA.
authors.forename: Aaron T
authors.initials: AT
authors.lastname: Wecksler
authors.affiliation: Protein Analytical Chemistry Department, Genentech Inc., 1 DNA Way, South San Francisco, CA, 94080, USA.
authors.forename: Matt S
authors.initials: MS
authors.lastname: Kalo
authors.affiliation: Protein Analytical Chemistry Department, Genentech Inc., 1 DNA Way, South San Francisco, CA, 94080, USA. deperalta.galahad@gene.com.
authors.forename: Galahad
authors.identifier: http://orcid.org/0000-0001-9119-6763
authors.initials: G
authors.lastname: Deperalta
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000737:chemistry / Q000276:immunology*; D001666:Binding Sites, Antibody; D018604:Epitope Mapping / Q000379:methods; D000939:Epitopes / Q000032:analysis / Q000276:immunology; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D008958:Models, Molecular; D042461:Vascular Endothelial Growth Factor A / Q000737:chemistry / Q000276:immunology*
chemical_list: D057134:Antibodies, Neutralizing; D000939:Epitopes; D042461:Vascular Endothelial Growth Factor A
keywords: Carboxyl group footprinting mass spectrometry; Epitope mapping; Glycine ethyl ester labeling; Vascular endothelial growth factor